AEK th International AEK Cancer Congress · e-mail:. [email protected] URL:.. ... D-99407 Weimar or via...

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AEKAEK16th International AEK Cancer CongressAbteilung Experimentelle Krebsforschung

www.aek-congress.org

March 16th – 18th, 2011 | Heinrich-Heine-University Düsseldorf

Congress Program and Abstracts

www.krebsgesellschaft.dewww.krebshilfe.de

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AEKTable of Contents

■ Welcome.................................................................................................................................................................................... 5..

■ General.Information.................................................................................................................................................................. 6

■ Information.for.Presentations.and.Posters............................................................................................................................... 8

■ AEK.Member.Page..................................................................................................................................................................... 9

■ Program.Wednesday.(March,.16th.2011)..................................................................................................................................... 10

■ Program.Thursday.(March,.17th.2011)......................................................................................................................................... 11

■ Program.Friday.(March,.18th.2011)............................................................................................................................................. 12

■ Abstracts.Short.Talks.and.Poster............................................................................................................................................. 13

■ Tumor.stem.cells.and.reprogramming................................................................................................................................... 13

■ Invasion.and.Metastasis........................................................................................................................................................ 17

■ Mouse.models...................................................................................................................................................................... 26

■ Carcinogenesis,.Genome.stability,.DNA.Damage.and.Repair,.Epigenetics............................................................................ 28

■ Oncogene.Addiction............................................................................................................................................................. 35

■ Tumor.Metabolism............................................................................................................................................................... 38

■ Molecular.Diagnostics.and.Targeted.Therapy........................................................................................................................ 41

■ Novel.Approaches.to.Therapy.............................................................................................................................................. 52

■ Tumor.Inflammation.and.Immunology...................................................................................................................................57

■ Free.Topics............................................................................................................................................................................ 61

■ List.of.Speakers.and.Poster.Authors....................................................................................................................................... 63

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AEKAEK AEK

AEK

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AEKWelcome

Dear AEK-Members, dear colleagues, ladies and gentlemen,

on.behalf.of. the.program.committee. I.cordially.welcome.you.to. the. 16th.AEK. International.Cancer.Congress.in.Düsseldorf,.which.for.the.second.time.will.be.jointly.subsidized.by.the.„Deutsche.Krebsgesellschaft”.and.the.„Deutsche.Krebshilfe“..

Renowned.international.scientists.will.present.and.discuss.latest.data.and.future.perspec-tives.of.different.hot.topics.in.cancer.research.such.as.

1..Tumor.stem.cells.and.Reprogramming.2..Invasion.and.Metastasis3..Mouse.models.4..Carcinogenesis,.Genome.stability,.DNA.Damage.and.Repair,.Epigenetics.5..Oncogene.Addiction.6..Tumor.metabolism7..Molecular.Diagnostics.and.Targeted.Therapy.8..Novel.approaches.to.Therapy.9..Tumor.Inflammation.and.Immunology..

Furthermore,.two.central.poster.sessions.as.well.as.9.plenary.sessions.including.selected.talks.from.abstract.submissions.will.provide.excellent.platforms.for.all.participants.and.young.scientists.in.particular.to.present.and.discuss.their.own.results..

As.the.support.of.young.scientists.is.a.major.aim.of.the.AEK,.we.are.pleased.that.due.to.the.financial.support.of.the.„Krebs-gesellschaft.Nordrhein-Westfalen.e.V.“.20.travel.bursaries.and.3.poster.prizes.can.be.awarded..

Moreover,.awardees.of.the.German.Cancer.Award.will.be.honored.at.the.occasion.of.the.conference.

All. AEK. members. are. kindly. invited. to. join. the. AEK. member. assembly,. which. is. scheduled. for. Thursday,. 17th. of. March,.6.00.–.7.00.p.m.

On.behalf.of.the.program.committee,.I.wish.you.all.a.successful.and.stimulating.meeting.

With.best.wishes

Prof..Dr..Rainer.EngersCongress.Chairman

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AEKAEK AEKGeneral Information

Chairman

Prof..Dr..Rainer.EngersInstitute.of.Pathology,.University.Hospital.Düsseldorf.andInstitute.of.Pathology,.Am.Hasenberg,.Neuss

Co-Chair(wo)man

Prof..Dr..Petra.BoukampUniversity.Hosptial.Heidelberg

Programm Board

Peter.Angel.(Heidelberg)Jürgen.Becker.(Graz)Klaus.Bosslet.(Berlin)Petra.Boukamp.(Heidelberg)Matthias.Dobbelstein.(Göttingen)Martin.Eilers.(Marburg)Rainer.Engers.(Düsseldorf/Neuss).Bernd.Groner.(Frankfurt)Jens.Hoffmann.(Berlin)Klaus-Peter.Janssen.(München)Roland.Moll.(Marburg)Peter.Lichter.(Heidelberg)Martin.Lipp.(Berlin)Ulf.Rapp.(München)Reinhold.Schäfer.(Berlin)Michael.Schwarz.(Tübingen)Winfried.Wels.(Frankfurt)Lisa.Wiesmüller.(Ulm)

Homepage


Congress Venue

Heinrich-Heine-University.Hörsaalgebäude.der.MedizinGebäude.23.01Universitätsstraße.1D-40225.Düsseldorf

Conference Service

P.O..Box.36.64D-99407.Weimar

Phone:. +49.3643.2468-0Fax:. +49.3643.2468-31

e-mail:. [email protected]:.. www.kukm.de

Continued Medical Education Credit Points

The.congress.has.been.certified.by.the.Medical.Association.of.North.Rhine.by.granting. 7. Points. in. category. A. for. every. day.. Please. observe. the. fowolling.guidelines.to.ensure.that.the.correct.number.of.further.education.points.may.be.credited.to.you:.

Attendee. lists. prepared. by. the. Medical. Association. of. North. Rhine. will. be.available.at. the. registration.desk. for. the.event. for.every.day..No.electronic.credit.of. the.points.may.be.granted.unless.you.enter.your.data. in. the. lists,.which.are.available.for.every.day..This.means.that.you.should.enter.your.data.in.a.total.of.three.lists.if.you.participate.in.the.entire.Congress..

Please.paste.your.barcode.into.the.corresponding.field,.and.sign.next.to.it..If.you.have.forgotten.your.barcode,.please.enter.your.EFN.number.in.the.barcode.field..No.further.education.points.may.be.credited.electronically.if.neither.the.barcode.has.been.pasted.into.the.list,.nor.an.EFN.number.has.been.entered.

You.receive.your.Certificate.of.Attendance.at.the.participant.registration.when.leaving.the.congress.

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AEKGeneral Information

Registration fees

Early.Registration.till13th.January.2011Permanent.Ticket

Late.Registration.till8th.March.2011.and.

registration.on-site.Permanent.Ticket

Member.AEK 150,00.e 190,00.e

Non-Member 240,00.e 280,00.e

Student.** ..70,00.e ..90,00.e

Get.together. . . 45,00.e /ticket

Opening hours registration desk

Wednesday,.March.16th. 08:00.–.19:00Thursday,.March.17th. 07:30. –.18:30Friday,.March.18th. 08:00.–.16:00

Terms and Conditions

The. fees. for. participating. in. the. scientific. program. are. charged. in. the. name. and. on. account. of. the. Deutsche. Krebsgesell-schaft.(DKG)..In.accordance.with.§.4/22a.of.the.German.sales.tax.act.the.above.fees.are.tax.exempt..The.tax.ID.of.the.DKG.is.DE258657754..

The.fees.for.the.social.get-together.and.the.handling.fees.are.charged. in.the.name.and.on.account.of.the.KONGRESS-.UND..KULTURMANAGEMENT.(KUKM)..The.tax.ID.number.is.DE158265638..

Once.you.have.sent.the.completed.registration.form,.your.registration.is.binding..Further.information.on.prices.and.deadlines.can.be.found.on.www.aek-congress.org..Reduced.fees.can.only.ne.applied,.after.an.adequate.certification.has.been.presen-ted. to.KUKM..This. can.be.submitted.by. fax.+49.3643.2468-31,.by.mail. to.P.O..Box.36.64,.D-99407.Weimar.or.via.e-mail. [email protected].

In.case.of.cancellation.or.non-participation,.there.will.be.no.money.refund..In.the.event.of.a.change.of.registration.with.KUKM.a.processing.fee.of.15.E.is.charged..The.charges.for.rebooking.are.15.E..

By.registering,.the.participant.agrees.that.he/she.will.not.make.any.claims.toward.the.host.if.the.conference.in.consequence.of.political,. commercial.or.natural. forces.will.be.cancelled.or.altered. in. its.program..Neither. the.host.nor.KONGRESS-.UND..KULTURMANAGEMENT.GMBH.can.be.made.liable.for.lost.items,.accidents.or.damages.to.persons.and.things,.whatever.the.cause..Participants.(and.the.person.accompanying).to.the.conference.and.the.social.events.attend.at.their.own.risk.and.responsibility.

** Reduced fees can only be applied, after an adequate certification has been presented to KONGRESS- UND KULTUR-MANAGEMENT GMBH. This can be submitted by fax +49 3643 2468-31, by mail to P.O. Box 36 64, D-99407 Weimar or via e-mail to [email protected].

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AEKAEK AEKInformation for Presentations and Poster

Presentations

Presentations.will.just.be.accepted.as.a.PowerPoint.presentation..Equipment.for.projection.and.computers.are.available.in.the.conference.room..Please.save.your.presentation.either.on.a.USB.stick.or.on.a.CD-ROM,.and.bring.it.to.the.conference..Your.presentation.will.be.copied.to.the.notebook.computer.two.hours.prior.to.the.session,.and.will.be.deleted.after.the.event..Please.be.aware.that.some.symposia.follow.each.other.without.a.break.inbetween..We.cannot.give.any.guarantee.for.the.successful.playback.of.presentations.which.include.embedded.videos..In.every.case,.please.contact.the.assistant.personnel.at.the.regis-tration.desk.15.minutes.before.the.start.of.your.session..The.use.of.own.computers.to.run.your.presentations.is.not.permitted.

Poster and Short Talks

There.will.be.a.poster.board.provided.for.each.poster..The.abstract.number.published.in.the.scientific.program.will.be.attached.to.the.upper.left.corner..Material.to.mount.your.poster.will.be.available.on.site,.using.your.own.material.to.mount.your.poster.is.not.permitted.

All.posters.have.to.be.mounted.by.10.00.am.on.Wednesday,.March.16th.and.are.expected.to.remain.in.place.at.least.until.5.00.pm.on.Thursday,.March.17th..Please.remove.your.poster.until.3.pm.on.Friday.March.18th.at.the.latest.

Posters.that.are.not.taken.down,.will.be.kept.until.the.end.of.the.conference.and.will.be.disposed.of.if.not.claimed.

There.will.be.two.different.poster.sessions:

1. Poster session (guided tour) – March 16th, 2011: 3 pm to 5 pm

Please.be.prepared.to.give.a.short.summary.(4.minutes.plus.4.minutes.discussion).in.front.of.your.poster!.

2. Poster session (free session – not guided!) - March 17th, 2011: 3 pm to 5 pm

AEK

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AEKAEK Member Page

AEK member assembly

The.AEK.member.assembly.is.scheduled.for.Thursday,.March.17th,.6.00.–.7.00.p.m.

Benefits of AEK Membership.·. Being.member.of.a.large.interdisciplinary.group.of.scientists.in.experimental.and.translational.cancer.research

·. Reduced.fees.for.future.AEK.Congresses.and.German.Cancer.Congresses.(forthcoming.DKK.2012)

·. Privileged.contact.to.more.than.900.members.via.www.krebsgesellschaft-aek.de

·. Corporate.membership.in.the.European.Association.for.Cancer.Research.(EACR).http://www.eacr.org/

·. Free.member.journal.FORUM

·. Opportunities.to.help.shaping.future.AEK.congresses.and.activities

·. All.AEK.members.are.eligible.to.apply.for.EACR.Fellowships.and.Awards

Application.for.membership.can.be.downloaded.from.http://krebsgesellschaft.de/wub_mitgliedschaft,78241.html

If.you.have.any.question.or.need.more.information.please.contact:.Katrin.HacklDeutsche.Krebsgesellschaft.e.V.Straße.des.17..Juni.106.–.108D-10623.Berlin.Phone:. +49.30.3229329-36Fax:. +49.30.3229329-22e-mail:. [email protected]

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AEKAEK AEKTime Table Wednesday, March, 16th 2011

08:00 – 10:00 Registration

10:00 – 11:00 Welcome Rainer.Engers.(Düsseldorf/Neuss)

Key Note LectureChanning.Der.(Chapel.Hill,.NC,.USA)

11:00 – 12:30 Symposium 1 - Tumor Stem Cells and ReprogrammingChair:.Petra.Boukamp.(Heidelberg),.Bernd.Groner.(Frankfurt/M.)

11:00 – 11:35 MicroRNAs, EMT AND CANCER STEM CELLSThomas.Brabletz.(Freiburg)

11:35 – 12:10 Stem cells, cancer stem cells and breast cancer heterogeneityMatthew.Smalley.(London)

12:10 – 12:20 Tumour stem cells and the microenvironmentBerit.Falkowska-Hansen.(Heidelberg).(short.talk.101)

12:20 – 12:30 Wnt/β-catenin drives MLL1 activity which locks salivary gland cancer stem cells in a specific epigenetic stateUlrike.Ziebold.(Berlin).(short.talk.102)

12:30 – 13:30 Lunch Break

13:30 – 15:00 Symposium 2 - Invasion and MetastasisChair:.Rainer.Engers.(Düsseldorf/Neuss),.Klaus-Peter.Janssen.(München)

13:30 – 14:05 Intravital microscopy of cancer invasion and experimental therapy response: how to over-come resistance nichesPeter.Friedl.(Nijmegen).

14:05 – 14:40 Unraveling Breast to Lung Metastasis Suppressor FunctionsRoger.Gomis.(Barcelona)

14:40 – 14:50 Role of breast cancer susceptibility gene 2 (BRCA2) in Tiam1-dependent adhesion, migration and invasionEva.Margarethe.Müller.(Düsseldorf).(short.talk.201)

14:50 – 15:00 Tumor-microenvironment interactions studied by zonal transcriptional profiling of squamous cell lung carcinomaHui.Wu.(Heidelberg).(short.talk.202)

15:00 – 17:00 Poster Session 1 and Coffee

17:00 – 18:30 Symposium 3 - Mouse ModelsChair:.Iduna.Fichtner.(Berlin),.Klaus-Peter.Janssen.(München)

17:00 – 17:35 Targeting DNA repair deficiencies in mouse models of hereditary breast cancerJos.Jonkers.(Amsterdam)

17:35 – 18:10 Patient-derived cancer xenografts - a preclinical model system for the identification of bio-markers and for the development of targeted therapiesIduna.Fichtner.(Berlin)

18:10 – 18:20 Mouse lessons on tumor drug resistance and on the mechanisms of side effectsJürgen.Thomale.(Essen).(short.talk.301)

18:20 – 18:30 Impaired liver tumor formation and oval cell activation in the RAGE knockout mouseTobias.Pusterla.(Heidelberg).(short.talk.302)

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AEKTime Table Thursday, March, 17th 2011

08:30 – 10:00 Symposium 4 – Carcinogenesis, Genome stability, DNA Damage and Repair, Epigenetics Chair:.Michael.Schwarz.(Tübingen),.Lisa.Wiesmüller.(Ulm)

08:30 – 09:05 Susceptibility Genes for Familial CancersHelmut.Hanenberg.(Indianepolis,.USA)

09:05 – 09:40 Loss of the tumor suppressor genes CHK2 and BRCA1 cause chromosomal instability in hu-man somatic cellsHolger.Bastians.(Göttingen)

09:40 – 09:50 An endogenous retrovirus drives hitherto unknown pro-apoptotic p63 isoforms in the male germline of humans and great apesUlrike.Beyer.(Göttingen).(short.talk.401)

09:50 – 10:00 A ROLE FOR TELOMERE LENGTH AND ORGANISATION IN HUMAN SKIN CARCINOGENESISChristine.Leufke.(Heidelberg).(short.talk.402)

10:00 - 10:15 Coffee break

10:15 - 11:45 Symposium 5 - Oncogene AddictionChair:.Martin.Eilers.(Marburg),.Reinhold.Schäfer.(Berlin)

10:15 – 10:50 Exploiting synthetic lethality and non-oncogene addiction for cancer therapyStefan.Fröhling.(Ulm).

10:50 – 11:25 Oncogene Addiction: Inside and OutDean.Felsher.(Stanford,.USA)

11:25 – 11:35 Regulation of EZH2 via MAPK signalling, c-MYC and miRNA 101 in colorectal cancer cell linesChristine.Sers.(Berlin).(short.talk.501)

11:35 – 11:45 Eliminating Cancer Stem Cells by Inhibition of the Wnt Pathway via Small Molecule Kinase InhibitorsHella.Kohlhof.(Martinsried).(short.talk.502)

11:45 - 12:30 Key Note Lecture 2Klaus.Pantel.(Hamburg)

12:30 - 13:30 Lunch Break

13:30 - 15:00 Symposium 6 - Tumor MetabolismChair:.Ivan.Dikic.(Frankfurt),.Matthias.Dobbelstein.(Göttingen)

13:30 – 14:05 Autophagy and ATP release: metabolism determines the chemotherapy-induced anticancer immune responseGuido.Kroemer.(Paris)

14:05 – 14:40 Ubiquitin networks in cancerIvan.Dikic.(Frankfurt)

14:40 – 14:50 The danger signaling protein HMGB1 induces a novel form of cell death accompanied by for-mation of giant mitochondriaWilfried.Roth.(Heidelberg).(short.talk.601)

14:50 – 15:00 Horizontal transfer of genetic information by mean of extracellular vesicles: the role in tumorigenesis and potential application in tumor diagnosticIrina.Nazarenko.(Eggenstein-Leopoldshafen).(short.talk.602)

15:00 - 17:00 Poster session 2 and Coffee

17:00 - 18:00 Cancer Awards

18:00 - 19:00from 20:00

AEK Member Assembly Social Get-together (45,00 E /.ticket)Hausbrauerei.„Zum.Schlüssel“.(Bolkerstraße.41.–.47.|.40213.Düsseldorf)

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AEKAEK AEKTime Table Friday, March, 18th 2011

08:30 - 10:00 Symposium 7 - Molecular Diagnostics and Targeted TherapyChair:.Rainer.Engers.(Düsseldorf/Neuss),.Roland.Moll.(Marburg)

08:30 – 09:05 Personalized Therapy of Lung CancerJürgen.Wolf.(Köln)

09:05 – 09:40 Predictive Biomarkers in the targeted therapy of colorectal cancerAndreas.Jung.(München)

09:40 – 09:50 Specific targeting of colorectal and pancreatic cancer with Shiga toxin B-subunitMatthias.Maak.(München).(short.talk.701)

09:50 – 10:00 Na+K+ATPase activity might be the crucial factor for selective inhibition of tumor stem cellsHelmut.Bühler.(Herne).(short.talk.702)

10:00 - 10:15 Coffee break

10:15 - 11:45 Symposium 8 - Novel Approaches to TherapyChair:.Klaus.Bosslet.(Berlin),.Jens.Hoffmann.(Berlin)

10:15 – 10:50 Vascular tumor targetingDario.Neri.(Zürich)

10:50 – 11:25 Cyclins and CDKs as targets for tumor therapiesNisar.Malek.(Hannover)

11:25 – 11:35 The radiosensitizing effect of Cetuximab is attenuated by activation of JNK2 signalingIris.Eke.(Dresden).(short.talk.801)

11:35 – 11:45 Radiotherapy stimulates pro-metastatic normal tissue responses, which are attenuated by the lipid-lowering drug lovastatinGerhard.Fritz.(Mainz).(short.talk.802)

11:45 - 12:30 Key note Lecture 3 Carlo.Croce.(Columbus,.OH,.USA)

12:30 - 13:30 Lunch Break

13:30 - 15:00 Symposium 9 - Tumor Inflammation and ImmunologyChair:.Martin.Lipp.(Berlin),.Winfried.Wels.(Frankfurt)

13:30 – 14:05 Intratumoral immune reaction, a novel paradigm for cancerJérôme.Galon.(Paris)

14:05 – 14:40 Novel functions of NF-kappaB in inflammation and cancer Florian.Greten.(München)

14:40 – 14:50 Activity of ER Aminopeptidase 1 in Malignant Melanoma Determines the Strength of the Overall CD8+ T Cell Response Against Autologous Tumor CellsChristin.Seifert.(Essen).(short.talk.901)

14:50 – 15:00 CXC-CHEMOKINES AS CRUCIAL IMMUNE MEDIATORS IN COLORECTAL CARCINOGENESISLarissa.Keller.(München).(short.talk.902)

15:00 - 15:30 Poster Prizes and concluding remarksRainer.Engers.(Düsseldorf/Neuss)

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AEKAbstracts Short TalksTumor stem cells and reprograming

short talk 101

Falkowska-Hansen, Berit (German Cancer Research Center, Heidel-berg); Kirschner,.Marc.(University.of.Würzburg,.Würzburg);.Kollar,.Jas-min.(German.Cancer.Research.Center,.Heidelberg);.Stark,.Hans-Jürgen.(German.Cancer.Research.Center,.Heidelberg).Boukamp,.Petra.(German.Cancer.Research.Center,.Heidelberg)

Tumour stem cells and the microenvironment

Human.epidermis.is.maintained.by.the.ability.of.the.stem.cells.to.self-renew.and.generate.a.hierarchy.of.proliferating.and.differentiating.cells..We.recently.established.a.method.to.detect.basal.epidermal.stem.cells.as.slow-cycling.label-retaining.cells.(LRCs).in.a.modified.long-term.scaf-fold-based.organotypic.culture.(OTC)..Introducing.a.human.tumorigenic.HaCaT-ras.cell.line.into.this.model,.we.asked.whether.these.tumour.cells.would.establish.a.similar.stem.cell.hierarchy.as.normal.keratinocytes..Also.HaCaT-ras.cells.formed.a.well.stratified,.differentiated.epithelium.with.slow-cycling.LRCs.in.the.basal.layer,.thus.establishing.cells.with.a.stem.cell.phenotype..As.these.cells.form.tumours.upon.s.c..injection.in.nude.mice.with.only.10.000.cells.in.matrigel,.we.further.asked.whether.LRCs.would. also.establish. in. tumours.. By. utilizing. HaCaT-ras.H2B-GF-PTetOFF. cells. we. performed. pulse-chase. experiments. in. developing.tumours.and.identified.LRCs..This.suggests.that.the.establishment.of.a.stem.cell.hierarchy.is.not.restricted.to.the.surface.epithelium.but.also.accounts.for.the.tumour.The.heterogeneity.of.solid.tumours,.especially.the.small.population.of.tumour.stem.cells.are.believed.to.be.resistant. to. therapy.and. induce.recurrences..We,.therefore,.asked.whether.basal.malignant.LRCs.could.resist.chemotherapeutic.treatment.and.cause.tumour.re-.growth..Tre-ating.HaCaT-ras.OTCs.with.Doxorubicine.demonstrated.that.the.bulk.of.the.tumour.cells.died.within.the.first.2.weeks.and.only.some.basal.epi-thelial.cells.survived.the.treatment,.which.all.were.LRCs..Within.the.next.4.weeks.a.new.tumour.tissue.developed.with.a.subsequent.dilution.of.the.basal.LRCs..Interestingly,.by.establishing.an.novel.OTC.model.which.allowed.HaCaT-ras.cells.to.invade.the.underlying.dermal.equivalent,.we.observed.an.increased.drug.resistance.as.compared.to.the.non-invasive.growing.conditions..These.results.not.only.confirm.the.common.hypo-thesis.that.the.tumour.stem.cells.are.resistant.to.chemotherapeutics.and. are. responsible. for. tumour. recurrences. but. adds. that. also. the.microenvironment. can. modulate. the. tumour. cells’. sensitivity,. making.tumour. (genetic. and. epigenetic). heterogeneity. equally. important. for.treatment.Taken. together,.we.have.established.a.novel.human.model. for. study-ing.malignant.tumour.cells.in.both.non-invasive.and.invasive.conditions.which.now.allow.detailed.analysis.on.the.role.of.the.microenvironment/niche.in.establishing.and.maintaining.tumour.growth.

short talk 102

Ziebold Ulrike, Max-Delbrück Centrum, Berlin); Wend,.Peter.(Max-Del-brück.Centrum,.Berlin);. Schipper,. Jörg. (University,.Düsseldorf);.Kahn,.Michael. (University. of. Southern. California,. Los. Angeles);. Birchmeier,.Walter.(Max-Delbrück.Centrum,.Berlin)

Wnt / β - catenin drives MLL1 activity which locks salivary gland can-cer stem cells in a specific epigenetic state

Wnt/β-catenin.is.a.commonly.deregulated.signaling.pathway.in.epithe-lial. cancers,. which. also. regulates. leukemic. stem. cells.. Less. is. known.about.cancer.stem.cells.in.solid.tumors,.hindering.efficient.strategies.for. tumor. therapies.. Here. we. show. that. deregulated. Wnt/β-catenin.and.Bmp.signaling. in.a.genetic.mouse.model.of.salivary.gland.cancer.transform.normal.stem.cells. into.cancer.stem.cells.(K14-Cre;.β-catein.GOF;.BMPR.LOF)..The.CD24/CD29.stem.cell.marker.combination.proved.successful. to. characterize. the. fast-growing,. easily. transplantable. tu-mors,.using.500.cells.per.injection..The.cancer.stem.retained.characte-ristics.of.normal.stem.cells,.i.e.,.relentless.ability.for.self-renewal.and.expression.of.a.subset.of.ES.cell-associated.genes..We.found.that.in.the.cancer.stem.cells,.a.β-catenin/CBP/MLL1-addicted.activity.converts.the.chromatin. into. a. permissive. state. characterized. by. heightened. H3K4.trimethylation..Consequently,.stemness.markers,.chromatin.remodelers.and.self-renewal.are.increased..Chemically.blocking.β-catenin/CBP.in-teraction.with.ICG-001.forces.the.cancer.stem.cells.to.differentiate.into.organotypic.structures..Also,.treating.cancer.stem.cell-initiated.tumors.in.immune-suppressed.mice.with.ICG-001.resulted.in.tumor.remission..Knowing. that. a. specific. Wnt/β-catenin. inhibitor,. ICG-001,. reverts. the.chromatin.state,.leading.to.differentiation.and.tumor.remission.provi-des.a.step.to.safely.eradicate.cancer.stem.cells.in.vivo..Human.head.and.neck.cancers.also.exhibited.high.Wnt/β-catenin.and.low.BMP.signaling.We. provide. a. molecular. explanation. of. how. nuclear. β-catenin. remo-dels.the.chromatin.state.in.the.cancer.stem.cells,.requiring.the.action.of.both.CBP.and.the.H3K4.methyltransferase.MLL1.that.results. in.the.profound.changes.of.stem.cell.fates..MLL1.(mixed.lineage.leukaemia).is.implicated. with. conferring. stem-like. properties. in. hematopoietic. ma-lignancies.. Our. study. now. links. MLL1. activity,. propelled. by. β-catenin,.to. an. epigenetic. program,. which. creates. and. maintains. cancer. stem.cells.in.solid.cancers..The.differentiation.of.the.cancer.stem.cells.could.also.be.chemically.reversed.using.either.VPA.or.HDAC-inhibitors,.which.argues. that.also.here.chromatin. remodeling. is.used.. It. is.known.that.displacement.of.β-catenin.from.Wnt-responsive.elements.could.induce.the.recruitment.of.Groucho,.which.may.directly.tether.the.histone.de-acetylase.HDAC1.

Notes:

14

AEKAEK AEKAbstracts PosterTumor stem cells and reprograming

Notes:

P-101

Zimmerer, Rüdiger (Medizinische Hochschule, Hannover); Barbero,.Andrea.(University.Hospital,.Basel);.Kampmann,.Andreas.(Medizinische.Hochschule,. Hannover);. Tavassol,. Frank. (Medizinische. Hochschule,.Hannover);.Spagnoli,.Giulio. (University.Hospital,.Basel);.Gellrich,.Nils-Claudius. (Medizinische. Hochschule,. Hannover);. Eckardt,. André. (Med-izinische.Hochschule,.Hannover)

Cancer Stem Cells in Melanoma Cell Lines and Head and Neck Squa-mous Cell Carcinomas

Objective:.Subpopulations.of.cancer.cells.are.capable.of.reproducing.tu-mours.in.immunocompromised.mice..Cancer.stem.cells.(CSC).are.usually.rare.in.clinical.specimens.and.hardly.amenable.to.functional.studies.or.to.analysis.of.gene.expression.profiles..We.screened.a.panel.of.melano-ma.cell.lines.and.biopsies.from.head.and.neck.squamous.cell.carcino-mas.(HNSCC).to.identify.cellular.reagents.sharing.typical.CSC.features,.including.expression.of.specific.surface.markers.and.genes,.capacity.to.grow.in.spheroids.and.high.clonogenic.capacity.Methods:.Nine.heterogenic.and.well.established.melanoma.cell.lines.and.biopsies.from.seven.HNSCC.were.investigated.for.the.expression.of.stem.cell.associated.surface.markers.using.flow.cytometry.(FACS)..Clonoge-nic.assays.were.performed.by.limiting.dilution.analysis.(LDA).on.cells.sorted.according.to.expression.of.selected.markers..Gene.expression.profiles.were.assessed.by.using. real-time.reverse-transcriptase.poly-merase.chain.reaction.and.DNA.Microarrays.(Affymetrix.GeneChip®).Results:.Only.D10.cell.line.expressed.the.classical.CSC.marker.CD133..CD44.was.found.to.be.expressed.by.all.HNSCC.investigated..CD117,.a.known.diffe-rentiation.marker,.was.only.expressed.by.HBL.cell.line.and.cells.from.one.HNSCC.biopsy..In.D10.cell.line,.CD133pos.cells.displayed.a.significantly.high-er.clonogenic.capacity.as.compared.to.CD133neg.cells..D10.cells.expressed.at.least.two.of.the.three.embryonic.transcription.factors.SOX2,.NANOG.and.OCT4..Na8,.D10.and.HBL.cells. formed.spheroids.when.cultured.on.plastic.coated.with.poly-HEMA,.preventing.cell.attachment..Gene.profiling.of.CD-133pos.and.CD133neg.D10.cells.showed.68.up-.and.47.down-regulated.genes.(+/-.1.3.fold,.p.<.0.01).as.compared.to.CD133neg.cells..Two.genes,.MGP.and.PROM1.(CD133),.were.outstandingly.up-regulated..A.number.of.other.genes.up-regulated.in.CD133+.D10.cells.encode.proteins.involved.in.cell.cycle.regu-lation.and.cell.proliferation,.including.IGF.1.(insulin-like.growth.factor-1),.its.binding.protein.and.PDGFc.(platelet.derived.growth.factor.c)..Down-regula-ted.genes.included.those.encoding.Tenascin.C.and.TIMP-1.Conclusions:.Established.melanoma.cell.lines.and.cells.from.HNSCC.bi-opsies.exhibit.to.variable.extents.typical.features.of.CSC..D10.cell.line,.growing.in.spheroids.and.expressing.CD133.might.qualify.as.a.potential.„in.vitro“.model.of.melanoma.stem.cells.

P-102

Olszewski, Ulrike; Boltzmann, Ludwig (Cluster, Vienna); Liedauer,.Richard. (Medical. University,. Vienna);. Thalhammer,. Theresia. (Medical.University,. Vienna);. Gerhard,. Hamilton;. Boltzmann,. Ludwig. (Cluster,..Vienna)

Overexpression of CYP3A4 in a COLO 205 colon cancer stem cell mo-del in vitro

Advances.have.been.made.in.the.treatment.of.colon.cancer,.but.despite.apparently. curative. surgery. and. adjuvant. chemotherapy. a. significant.number.of.patients.succumb.to.recurrence.of.the.disease..Cancer.stem.cells. (CSCs). seem. to. constitute. a. subpopulation. of. tumor. cells. that.escape. chemotherapy. and. cause. recurrent. disease.. Low. proliferation.rates,. protection. in. a. stem.cell. niche.and.overexpression.of.drug. re-sistance. proteins. are. considered. to. confer. chemoresistance. to. these.cells..We.established.an. in.vitro.colon.CSC-like.model.using. the.COLO.205.colon.cancer.cell.line,.which.revealed.transiently.increased.expres-sion.of.CD133.when.transfered.to.serum-free.stem.cell.culture.medium.supplemented. with. EGF,. FGF-2,. BSA. and. insulin/transferring/selenite..Although. colon. cancer. specimens. display. a. high. variability. in. their.patterns.and.levels.of.expression.of.some.CSC.markers,.CD133.at.least.correlates. with. tumor. aggressiveness. and. poorer. clinical. outcome..COLO.205.cells.precultivated.in.serum-free.stem.cell.medium.revealed.a.significant.increase.in.resistance.to.most.cytotoxic.drugs,.except.for.satraplatin.with.comparable.sensitivity.for.standard.and.stem.cell-like.cells.and. titanocene.Y,. that. showed.higher.activity.against. the. latter.cells..Assessment.of.global.gene.expression.(Applied.Biosystems.Human.Genome.Survey,.32K).of.COLO.205.cells.cultured.under.these.conditions.identified.a.set.of.upregulated.genes.including.cytochrome.P450.3A4.(CYP3A4).and.aldehyde.dehydrogenase.1A1.(ALDH1A1).as.confirmed.by.real-time.QPCR..ALDH1A1.is.a.CSC.marker.for.distinct.tumor.entities.and.confers.resistance.to.drugs.like.cyclophosphamide..CYP3A4.is.expressed.in.liver.and.colon,.and.overexpression.seems.to.be.particularly.relevant.in.colon.cancer,. since. it. inactivates. irinotecan.and.other.xenobiotics,.such.as.taxols.and.Vinca.alkaloids..Other.anticancer.drugs.affected.in-clude.docetaxel,.paclitaxel,.gefitinib.and.erlotinib..Additionally.human.tumors. like.breast,. lung,. liver,.kidney.and.prostate.cancer.are.known.to. express. CYP. isoforms. including. 3A. and. 1A. subfamily. members.. In.conclusion,.this.COLO.205.model.provides.evidence.for.transient.CD133.induction.with.concomitant.overexpression.of.CYP3A4,.which,.together.with.ATP-binding.cassette,.subfamily.G,.member.2.(ABCG2).and.others,.may.have.a.role.in.chemoresistant.colon.CSCs,.and.a.negative.impact.on.disease-free.survival.in.colon.cancer.patients.

AEK

15

AEKAbstracts Poster Tumor stem cells and reprograming

Notes:

P-103

Kollar, Jasmin (German Cancer Research Center, Heidelberg); Buschke,Susanne. (German. Cancer. Research. Center,. Heidelberg);. Cerezo,. Ana.(Centro. Nacional. de. Investigaciones. Cardiovasculares,. Madrid);. Stark,.Hans-Jürgen.(German.Cancer.Research.Center,.Heidelberg);.Boukamp,.Petra.(German.Cancer.Research.Center,.Heidelberg)

The multifactorial role of TGFß/Smad signaling in normal and patho-logical growth

TGFß.is.known.to.cause.growth.inhibition.in.keratinocytes.and.is.belie-ved.to.regulate.differentiation..In.tumor.cells.TGFß-dependent.growth.inhibition.is.frequently.abrogated..Different.from.keratinocytes,.in.der-mal. fibroblasts. TGFß. stimulates. growth. and. the. expression. of. matrix.components. and. matrix. modulating. enzymes. thus. modulates. the. mi-croenvironment,. i.e.. the.stem.cell.niche..This.dual. role.makes.TGFß.a.prime.candidate.for.regulating.stem.cell.activity.in.human.skin.and.its.alterations.being.required.for.tumorigenesis..We.hypothesize.that.TGFß.is.regulating.the.slow.growth.of.stem.cells.via.the.Smad.pathway..Ab-rogating.this.pathway.should.perturb.stem.cell.quiescence..To.test.this.we.used.HaCaT.variants.that.had.obtained.a.siRNA.construct.for.Smad2,.3,.and.4.(H-S234KD),.HaCaT.cells.overexpressing.Smad7.(H-Smad7),.and.HaCaT.cells.transfected.with.a.dominant-negative.TGFβ.receptor.type.II.(H-TßRIIdn),.all.of.which.were.abrogated.in.TGFß-dependent.growth.in-hibition.but.varied.in.the.molecular.profile.of.pathway.abrogation..Stem.cell.behavior.was. tested. in.organotypic.cultures.which.allowed. these.cells.to.form.in.vivo-like.tissues.with.stem.cells.that.could.be.identified.as.slowly.cycling.label.retaining.cells.(LRCs)..Irrespective.of.the.mole-cular.differences,.all.variants.established.LRCs.which.could.be.seen.for.>10.weeks.in.the.absence.and.presence.of.TGFß,.demonstrating.that.the.Smad.pathway.was.not.involved.in.stem.cell.quiescence.and.TGFß.was.not.the.regulatory.trigger.for.this.stem.cell.characteristics..Instead.we.found. that. Smad. pathway. abrogation. disturbed. tissue. homeostasis. –.proliferating. cells. and. stem. cells. were. located. throughout. the. entire.epithelium.instead.of.being.restricted.to.the.basal. layer..Additionally,.Smad.pathway.abrogation.caused.a.switch.to.an.alternative.differenti-ation.program,.demonstrating.that.Smad.signaling.is.essential.for.ter-minal.epidermal.differentiation..Finally,.abrogation.of.Smads.2,3,4.but.not.overexpression.of.Smad7.provided.the.cells.with.an.invasive.poten-tial.showing.that.abrogating.all.Smads.and.thus.hindering.interaction/interference.with.other. regulatory.pathways.or. components.provides.an.additional.different.phenotype.than.blocking.pathway.activation.in.a. “natural”. manner. by. overexpressing. the. endogenous. inhibitor. and.leaving.the.pathway.intact..This.suggests.that.invasion.is.not.a.conse-quence.of.canonical.Smad.pathway.abrogation.but.abrogation.of.one.of.the.Smads.with.other,.as.yet.unknown.cellular.targets.

P-104

Bünger, Stefanie; Vollbrandt,.Tillman;.Bruch,.Hans-Peter;.Kruse,.Charli;.Roblick,.Uwe;.Habermann,.Jens.(University,.Lübeck)

Frequency and Variability of cancer stem cell contents in colorectal carcinoma cell lines reflects heterogeneity of clinical tissue

Introduction.Cancer.stem.cells.(CSCs).are.thought.to.be.responsible.for.tumor.progression.and.therapy.resistance..They.have.been.identified.in.a.variety.of.human.solid.tumors.and.cancer.cell.lines..Cell.lines.might.therefore.serve.as.an.attractive.source. for.CSC. in.vitro. research..We.investigated.to.which.extent.colorectal.cancer.cell. lines.contain.CSC-like. cell. clones.and. if. their. expression.profiles. correlate.with. clinical.measures.Material.&.Methods.Altogether,. 12.colorectal. cancer.cell. lines.and.30.primary.colorectal.carcinomas.and.adjacent.normal.colon.mucosa.were.analyzed.by.flow.cytometry.using.a.panel.of.six.cell.surface.markers.of.CSC-like.phenotype..Markers.comprised.CD326,.CD133,.CD44,.CD166,.Msi-1.and.Gpr49..Expression.frequency.of.CSC-like.markers.was.divided.into.four.categories.with.high.(>.70%.of.cells),.moderate.(<.70%.and.>.30%),.low.(<.30%.and.>.1%),.and.absent.(<.1%).expression..Results.All.cell.lines.but.one.(HT29).showed.a.stable.expression.pattern.throughout. all. four. replicates.. HT29. showed. an. increased. expression.for. CD133. and. CD166. over. time. and. was. thus. excluded. from. further.analyses..The.majority.(91%).of.cell. lines.showed.high.expression.for.CD326..About.one. third.of. the.cell. lines.expressed.at.high. frequency.CD44,.CD166.and.CD133..In.contrast,.most.cell.lines.expressed.Msi-1.and.Gpr49.at.low.frequency..Comparing.primary.colorectal.carcinomas.with.the.cell.lines,.we.found.the.expression.of.cancer.stem.cell.markers.to.be.fairly.similar.in.both.sources..Comparison.of.adjacent.normal.mucosa.and.primary.carcinoma.detected.the.most.prominent.expression.diffe-rences.for.CD326,.CD133,.CD44.and.CD166..Markers.were.subsequently.evaluated.by.immunhistochemistry.on.a.tissue.micro.array.consisting.of.78.primary.colorectal.carcinoma.and.normal.mucosa.and.correlated.re-sults.to.clinical.parameters..Conclusion.Established.cell.lines.do.harbour.to.a.substantial.amount.cells.with.CSC-like.properties..A.comparison.of.cell.lines.with.clinical.tissue.shows.similar.pattern.of.CSC-markers.ex-pression..Some.cell.lines.present.higher.variability.of.cancer.stem.cell.marker. expression. than. others.. Overall. we. conclude. that. commercial.cell.lines.do.reflect.CSC.properties.of.clinical.tissue.and.could.serve.for.CSC.in-vitro.research.to.different.extents.

16

AEKAEK AEKAbstracts Poster Tumor stem cells and reprograming

Notes:

P-105

Vafaizadeh, Vida; Brendel,.Christian;.Graab,.Ulrike;.Engelbrecht,.Chris-tian;.Chiba,.Tomohiro;.Groner,.Bernd.(Georg-Speyer-Haus,.Frankfurt/M.)

Mammary Gland Reconstitution with Gene-Modified Stem Cells: Ana-lysis of Stem Cell Signaling Pathways in the Mammary Gland Develop-ment and Tumorigenesis

The.mammary.gland.develops.mainly.postnatally.and.contains.a.small.fraction.of.mammary.stem.cells. (MaSCs),. required. for. the.generation.and.maintenance.of. its.epithelium..The.mouse.mammary.gland.repre-sents.a.unique.model.system.to.study.gene.functions.in.adult.stem.cells..Transformations. in. MaSCs. and. progenitors. with. acquired. self-renewal.are. probably. responsible. for. the. initiation,. progression. and. mainte-nance.of.breast.tumors..The.molecular.mechanisms.that.govern.the.cell.fate.decision.in.MaSCs.and.regulate.the.proliferation.and.differentiation.of.progenitor.cells.provide.crucial.insights.into.mammary.gland.deve-lopment.and.advance.our.understanding.of.the.molecular.pathology.of.breast.cancer..The.presence.of.MaSCs.in.the.mammary.epithelium,.the.efficient.gene.transfer.potential.of. lentiviral.vectors.and. the.possibi-lity.to.transplant.these.cells.into.cleared.mammary.fat.pads.to.gauge.their.functional.potential.opens.up.an.alternative.strategy.for.functio-nal.genomics..We.optimized.lentiviral.transduction.of.adherent.primary.mammary. epithelial. cells. and. genetically. modified. non-enriched. Ma-SCs.with.multi-color.lentiviral.vectors..Loss.and.gain.of.Stat5.function.studies.using.this.technique.reveal.new.insights.into.its.roles.in.gland.development.and.breast. cancer..Persistent.activation.of.Stat5.during.the.postlactational.involution.stage.causes.the.formation.of.adenocar-cinomas.which. resemble. the.human. luminal-A.breast.cancer. subtype..They.express.estrogen.and.progesterone.receptor.(ER+PR+).and.acti-vated.Stat3.and.Stat5..The.tumors.provide.a.valuable.mouse.model.to.investigate.the.therapeutic.potential.of.anti-estrogens.and.aromatase.inhibitors.in.ER+PR+.tumors.and.the.consequences.of.Stat3.and.Stat5.inhibition.on.mammary.tumor.growth.

P-106

Welte, Yvette; Saint-Paul,. Janne;. Regenbrecht,. Christian. (Charité,.Berlin)

Application of in vitro cytotoxicity assays to investigate functional differences between cancer stem cells and bulk tumor cells in me-lanoma

During.the.past.years.in.vivo.transplantation.experiments.and.in.vitro.colony-forming.assays.indicated.that.tumors.arise.only.from.rare.cells..These.cells.were.shown.to.bear.self-renewal.capacities.and.the.ability.to. recapitulate. all. cell. types. within. an. individual. tumor.. Due. to. their.phenotypic.resemblance.to.normal.stem.cells,.the.term.“cancer.stem.cells”.is.used..However,.important.pieces.of.the.puzzle.are.missing:.(a).a.stringent.definition.and.characterization.of.cancer.stem.cells.in.solid.tumors.(b).specific.markers.that.only.target.cancer.stem.cells.in.a.cer-tain.type.of.tumor..We.analyzed. low.passage.melanoma.cell. lines.by.RT-PCR,.cell. culture.conditions. for. embryonic. stem. cells. and. dye-exclusion. experiments..These.experiments.revealed.the.existence.of.a.small.subpopulation.of.melanoma. cells. with. stem. cell. characteristics.. Therefore. we. then. fo-cused.on.key.stem.cell.properties. like.the.ability.to.self-renew.in.or-der.to.identify.further.common.characteristics.between.stem.cells.and.cancer. stem.cells..We. found.pathways. like. the.FGF. signaling.cascade.only.active.in.melanoma.cells.cultivated.in.embryonic.stem.cell.medi-um.or. in. sorted.CD133+.putative.cancer. stem.cells.. Furthermore,. the.self.renewal.factor.OCT4.is.only.expressed.in.cancer.stem.cells.but.not.in.bulk.tumor.cells..To.determine.the.underlying.mechanisms.of.these.functional.differences.between.cancer.stem.cells.and.bulk.tumor.cells.we. performed. genome-wide. expression. profiling. and. found. genes. in-volved. in.angiogenesis,. inhibition.of.apoptosis,.adhesion.and. invasion.differentially.expressed..Whole.transcriptome.mRNA.sequencing.revealed.that.these.differences.are.not.due.to.mutations.at.the.sequence.level..The.tremendous.vari-eties.among.gene.expression.patterns.between.cancer.stem.cells.and.non-cancer.stem.cells.in.melanoma.with.no.differences.in.coding.muta-tions.at.the.genome.or.transcriptome.level.argues.in.favor.of.epigenetic.regulation.and.maintenance.of.the.phenotype.or.alternative.splicing.To. further. investigate. the. main. signaling. pathways. which. are. mostly.altered.in.cancer.cells.like.Ras.and.PI3K.signaling.pathway,.we.evalua-ted.the.chemotoxic.effects.of.several.kinase.inhibitors.on.CD133+.and.CD133-.cells.by.viability.and.apoptosis.assays.By.combining.different.approaches.from.stem.cell.and.cancer.research,.it.may.become.possible.to.define.cancer.stem.cells. in.melanoma.and.therapeutic.strategies.could.be.redirected.towards.these.cells. to.cir-cumvent.the.failure.of.conventional.therapies.

AEK

17

AEKAbstracts Short Talks Invasion and Metastasis

Notes:

short talk 201

Müller, Eva Margarethe (University Medical Center, Düsseldorf);Hoffmann,. Michèle. Janine. (University. Medical. Center,. Düsseldorf);.Friedl,.Peter.(Radboud.University,.Nijmegen);.Hanenberg,.Helmut.(Uni-versity.Medical.Center,.Düsseldorf);.Gabbert,.Helmut.Erich.(University.Medical.Center,.Düsseldorf);.Engers,.Rainer.(University.Medical.Center,.Düsseldorf)

Role of breast cancer susceptibility gene 2 (BRCA2) in Tiam1-depen-dent adhesion, migration and invasion

Objective:. Tiam1. specifically. activates. the. Rho-like. GTPase. Rac,. and.Tiam1/Rac.signalling.affects.important.biologic.functions.such.as.onco-genic.transformation,.cell.adhesion,.migration,.invasion.and.metastasis..As.the.molecular.mechanisms,.through.which.Tiam1/Rac.exert.their.ef-fects,.are.only.partly.understood,.we.searched.for.new.target.genes.of.Tiam1.and.investigated.their.functional.role..Methods:.Stable.transfection.of.different.human.epithelial.cell.lines.with.cDNAs.of.empty.vector,.constitutively.active.Tiam1.(C1199-Tiam1).and/or.BRCA2.by.the.Fugene.method.or.by.retroviral.transduction..Compara-tive.gene.expression.analysis.by.cDNA.microarrays.and.immunoblotting..Functional.in.vitro.assays.for.cell.proliferation,.transformation,.adhesi-on,.migration.and.invasion.Results:.By.comparative.gene.expression.analysis.of.mock-.and.C1199-Tiam1-transfected.cancer.cells.BRCA2.was.found.to.be.markedly.down-regulated. by. C1199-Tiam1.. This. was. confirmed. by. immunoblotting. of.different. mock-. and. C1199-Tiam1-transfected. benign. and. malignant.epithelial. cell. lines.. Moreover,. increased. Tiam1. expression. correlated.with. decreased. BRCA2. expression. in. vivo.. C1199-Tiam1-induced. down-regulation.of.BRCA2.appears.to.be.predominantly.mediated.by.the.JNK.pathway.but.in.a.cell.type-dependent.manner.other.pathways.might.be.implicated.as.well..In.colon.cancer.cells.C1199-Tiam1.stimulated.pheno-typic.alterations,.cell.proliferation,.colony.formation.as.well.as.cell-cell.and.cell-substrate.adhesion..Further,.C1199-Tiam1.inhibited.cell.migrati-on.and.affected.invasion.depending.on.the.extracellular.matrix.(ECM).used..C1199-Tiam1-induced.effects.on.invasion.were.mediated.by.chan-ging.the.balance.between.matix-metalloproteinases.(MMPs).and.their.specific.inhibitors.(TIMPs).in.an.ECM-dependent.manner..Re-expression.of.BRCA2.in.C1199-Tiam1-transfected.cells.reverted.C1199-Tiam1-induced.effects. on. adhesion,. migration. and. invasion,. but. had. no. influence. on.C1199-Tiam1-induced. effects. on. cell. morphology,. proliferation. and. co-lony.formation..Conclusions:.Several.C1199-Tiam1-induced.biological. functions.are.me-diated.by.downregulation.of.BRCA2..Our.results. identify.yet.unknown.functions.of.BRCA2.in.cell.adhesion,.migration.and.invasion.aside.from.its.established.role.in.DNA.repair.and.tumorigenesis..Moreover,.our.re-sults.might.provide.new.clues.to.the.role.of.Tiam1.in.tumor.development.

short talk 202

Hui Wu (German Cancer Research Center, Heidelberg); Haag,.Daniel.(German.Cancer.Research.Center,.Heidelberg);.Muley,.Thomas.(Univer-sitätklinikum,.Heidelberg);.Warth,.Arne.(University.of.Heidelberg,.Hei-delberg);.Zapatka,.Marc.(German.Cancer.Research.Center,.Heidelberg);Tödt,.Grishna.(German.Cancer.Research.Center,.Heidelberg);.Pscherer,.Armin. (BioRN. Cluster. Management,. Heidelberg);. Rieker,. Ralf. (Univer-sity,. Erlangen);. Meister,. Michael. (Thoraxklinik,. Heidelberg);. Schnabel,.Philipp.(University,.Heidelberg);.Müller-Decker,.Karen.(German.Cancer.Research. Center,. Heidelberg);. Hoffmann,. Hans. (Thoraxklinik,. Heidel-berg);. Lichter,. Peter. (German. Cancer. Research. Center,. Heidelberg);.Rogers,.Michael.A..(German.Cancer.Research.Center,.Heidelberg)

Tumor-microenvironment interactions studied by zonal transcriptio-nal profiling of squamous cell lung carcinoma

Using. “Punch-aided. laser. capture. microdissection”,. total. RNA. from.inner-tumor,. tumor. invasion. front-,. adjacent. lung-. and. normal. lung.tissue. from. 18. patients. with. squamous. cell. lung. carcinoma. were. iso-lated.and.used.to.analyze.by.matched-pair.microarray.the.expression.of.genes.found.on.the.Operon.Human.Genome.Oligo.set. (version.2.1)..Nine.of. these.samples.were.also.analyzed.using.Taqman.Low.Density.Arrays.(version.1.0).in.order.to.evaluate.the.regional.expression.of.365.different.microRNAs..Statistical.analysis.of.the.expression.microarray.data.from.the.tumor.invasion.front.showed.strong.changes.in.gene.ex-pression.between.the.normal.lung.and.adjacent.lung,.but.only.13.genes.were.differentially.expressed.between.the.tumor.invasion.front.and.in-ner.tumor..Ingenuity.Pathway.Analysis.placed.a.majority.of.these.genes.in.a.network.associated.with.cell-to-cell.signalling,.cellular.interaction,.inflammation,.cell.migration,.gene.expression.and.cell.cycle.arrest..The.adjacent.lung.tissue.specific.expression.of.three.genes.(GIMAP7,.APLNR.and.CCL19).was.validated.by.immunohistochemistry..Ingenuity.analysis.of.genes.differentially.expressed.between.the.inner.tumor.and.the.adja-cent.lung/normal.lung.tissue.showed,.in.addition.to.squamous.cell.car-cinoma-associated.keratin.gene.expression,.strong.expression.related.to.arachidonic.acid.metabolism.and.eicosanoid.signalling..Evaluation.of.the.microRNA.data.pointed.to.expression.changes.in.nearly.a.fourth.of.the.microRNAs.analyzed..Of.particular.interest.were.the.expression.of.mir-196a.in.the.inner.tumor,.of.mir-650.in.adjacent.lung.tissue,.and.the.expression.of.mir-224.in.the.inner.tumor.and.tumor.invasion.front,.all.of.which.were.validated.by.in.situ.hybridization.

18

AEKAEK AEKAbstracts Poster Invasion and Metastasis

Notes:

P-201

Domaschke, Christoph (University of Heidelberg, Heidelberg); Diel,.Ingo.(CGG.Clinic,.Mannheim);.Rom,.Joachim.(University.of.Heidelberg,.Heidelberg);. Schütz,. Christof. (University. of. Heidelberg,. Heidelberg);.Schütz,.Florian.(University.of.Heidelberg,.Heidelberg)

Disseminated tumor cells as mediators of distant metastases and overall survival in primary breast cancer patients

Background:.The.detection.of.disseminated.tumor.cells.(DTC).in.primary.breast.cancer.(BC).patients`.bone.marrow.(BM).seems.to.be.a.surro-gate.marker.of.tumor.spread.and.an.independent.prognostic.factor.for.disease-free.and.overall. survival..Altogether,. in.BC.patients.with.DTC.in. BM. shorter. progression-free. and. overall. survival. rates. have. been.shown.Patients.and.methods:.Here.we.present.the.largest.single-centre.cohort.of.patients.(n=.1378).with.the.longest.follow-up.time.(median.122.months)..Immunocytoche-mical. staining.was.performed.using.murine.monoclonal.antibody.2E11.with.the.avidin-biotin.complex.technique.Results:.At. primary. surgery,. 49%. of. patients. showed. MUC-1. positive. cells. in-side. their. BM.. Patients. without. BM. DTC. had. significantly. more. often.T1-tumors. (p=0.042). with. less. often. affected. axillary. lymph. nodes.(p=0.045)..We.observed.a.significantly.higher.incidence.of.distant.me-tastases. in. DTC. positive. patients. (p<0.0001).. This. leads. to. a. reduced.disease-free.survival. (p=0.031)..Furthermore,. in.DTC.positive.patients.there.was.a.higher.mortality. rate.and,.accordingly,.a. reduced.overall.survival.(p=0.020)..Conclusions:.Due.to.the.presence.of.BM.DTC,.patients.with.a.clinically.poorer.out-come.can.be.identified.at.primary.surgery..We,.therefore,.suggest.that.DTC.analysis.can.be.used.as.a.prognostic.factor.and.predictive.marker.in.clinical.trials..Future.study.concepts.relating.to.DTC.should.aim.at.iden-tification.of.BC.patients.who.may.profit.from.adjuvant.systemic.therapy.

P-202

Hirschfeld, Marc (University Medical Center, Freiburg); Jäger,.Markus.(University.Medical.Center,.Freiburg);.Buratti,.Emanuele.(ICGEB.Trieste,.Trieste);.Cristiana.(ICGEB.Trieste,.Trieste);.Grueneisen,.Johannes.(Uni-versity. Medical. Center,. Freiburg);. Gitsch,. Gerold. (University. Medical.Center,.Freiburg);.Stickeler,.Elmar.(University.Medical.Center,.Freiburg)

Alternative splicing of tumor-promoting Cyr61 is regulated by hTRA2-beta1 and acidosis

Cysteine. rich.61. (Cyr61). is. a.matricellular.protein.displaying.a. remar-kable. diversity. of. multiple. cellular. activities. and. biological. functions.involved.in.significant.physiologic.and.pathologic.processes..Cyr61.is.an.important. player. in. tumor. progression,. promoting. neovascularisation.and.metastasis..Recently,.we.showed.that.Cyr61.pre-mRNA.undergoes.a.specifically.regulated.retention.of.intron.3.during.its.alternative.splicing.process.as.an.on/off.switch.for.its.biological.function..Hypoxia.leads.to.increased.expression.of.the.intron.free.splice.variant,.the.solely.tran-script.generating.active.Cyr61.protein..In.this.work,.we.now.identify.extracellular.acidosis.as.a.potent.modula-tor.of.Cyr61.alternative.splicing.pattern..In.our.functional.experiments,.we.observed. that.acidosis.was.capable.of. inducing.similar.effects.on.Cyr61. splicing. like. hypoxia.. Intriguingly,. splicing. factor. hTRA2-beta1.displayed.an.opposite.effect.on.Cyr61.alternative.splicing.pattern.than.hypoxia.or.acidosis..Considering.that,.nuclear.hTRA2-beta1.protein.ex-pression. was. markedly. reduced. under. hypoxic. and. acidic. conditions..Our.findings.strongly.support.the.hypothesis.of.a.specific.regulation.of.Cyr61.alternative.splicing.by.the.splicing.factor.hTRA2-beta1..In.keeping.with.these.conclusions,.we.show.that.hTRA2-beta1.can.specifically.bind.a. ‘GAAG’.motif. in.Cyr61.exon.3.RNA,. that. the. splicing. factor.displays.acidosis-dependent.protein. localization. in.cellular.compartments,.and.that. shRNA-mediated. hTRA2-beta1. knock-down. triggers. the. same. ef-fects.on.Cyr61.alternative.splicing.like.acidosis.or.hypoxia,.respectively.

AEK

19

AEK

Notes:

Abstracts Poster Invasion and Metastasis

P-203

Georges, Rania; Adwan,. Hassan;. Hamdi,. Hadjar;. Hielscher,. Thomas;.Linnemann,.Ulrich;.Berger,.Martin.(DKFZ,.Heidelberg)

The insulin-like growth factor binding proteins 3 and 7 are associa-ted with colorectal cancer and liver metastasis

Over.the.last.few.decades,.a.great.deal.of.attention.has.been.directed.to.the.IGF.system.for.its.vital.role.in.regulating.cell.and.tissue.survival,.growth. and. differentiation.. The. main. constituents. of. this. system. are.IGF-I.and.–II.as.well.as.their.receptors.IGF-IR.and.-IIR,.IGF.binding.prote-ins.(IGFBPs).and.IGFBP.proteases..The.growth.promoting.effects.of.IGFs.are.mediated.through.the.type.I.receptor,.and.modulated.by.6.binding.proteins. (IGFBPs. 1-6). with. high. affinity. for. IGFs. as. well. as. at. least. 4.IGFBPs.with.low.affinity.also.known.as.IGFBP-related.proteins.(IGFBP-rp-1-4)..IGFBPs.have.been.implicated.in.the.tumorigenesis.of.colorectal.cancer..Intrigued.by.the.functional.properties.of.IGFBP3.and.7,.which.apparently.have.activities.supporting,.both.the.inhibition.and.stimulation.of.CRC,.we.intended.to.study.these.two.genes.in.more.detail..To.that.purpose,.we.selected.human.and.rat.CRC.cell. lines,.which.are.known. to. express. IGFBP3. or/and. IGFBP7.. Following. silencing. of. their.mRNA.expression,.the.effects.on.proliferation,.migration.and.colony.for-mation.were.recorded..In.addition,.the.pattern.of.IGFBP3.and.7.expres-sions.was.related.to.the.staging.of.human.CRC.samples.to.determine.an.association.between.the.two.genes.and.human.cancer.progression..Fi-nally,.an.experimental.study.in.rats.was.performed.in.which.CC531.cells.homing.to.rat.liver.were.re-isolated.after.defined.periods.and.evaluated.for.alterations.in.IGFBP3.and.7.gene.expression.Our.experiments.on.silencing.IGFBP3.or.IGFBP7.in.the.two.human.CRC.cell.lines.SW480,.Caco2,.and.in.the.rat.CRC.cell.line.CC531.show.reduced.proliferation,.colony.formation,.and.for. IGFBP3,.also.reduced.migrati-on..The.expression.of.both.genes.in.68.human.CRC.samples.was.higher.in. UICC. stages. II. and. III. than. in. stages. I. and. IV.. Additionally,. IGFBP3.was.negatively.correlated.with.age.(p=0.05).and.positively.related.to.IGFBP7.expression. (p=0.0001).. Further,. the.expression.of.both.genes.was.drastically.increased.in.response.to.early.metastatic.growth.in.vivo..Since.these.high.levels.returned.gradually.to.normal.thereafter,.it.could.be.assumed.that.the.up-regulation.of.IGFBPs.is.vital.during.the.process.of.homing.into.the.liver.and.early.metastatic.dissemination..Our.results.indicate.that.IGFBP3.and.7.cannot.be.simply.considered.as.tumor.suppressors.but.have.additional.properties,.which.become.evi-dent.only.during.cancer.progression.and.metastasis.formation.

P-204

Müller, Annett; Linnig,.Monika;.Galle,.Peter.R.;.Moehler,.Markus. (Uni-versity,.Mainz)

New strategies of targeted agents in human colorectal cancer- multi targeting in vitro and in vivo

Recently,.new.targeted.agents.like.monoclonal.antibodies.and.small.mo-lecules.clearly.expanded.standard.chemotherapy.for.advanced.human.colorectal.cancer..Many.of.these.target.not.only.growth.receptors.like.EGFR.on.tumour.cells.and.the.adjacent.vasculature.through.VEGFR.but.also.disturb.their.signalling.pathways.inside.the.cells,.inhibiting.tumour.progression.and.angiogenesis..Thus,.we.analysed.new.combinations.of.cetuximab,.vandetanib.and/or.sunitinib.with.or.without.classical.cytoto-xic.agents.in.vitro.and.in.vivo.in.human.colorectal.cancer.models.Four.human.colon.cancer.cell.lines.(HT-29,.SW480,.HCT-116,.DLD-1).were.analysed.for.their.KRAS,.B-RAF,.PI3Kand.PTEN.status.and.intracellular.expression.levels.of.signalling.pathways.(RT-PCR,.Western.blot,.FACS)..Different. viability. and.apoptosis. assays.were.performed.during.diffe-rent. combination. therapies. with. or. without. targeted. inhibitors,. such.as.cetuximab,.vandetanib.and.sunitinib..Additionally,. the.promising. in.vitro.data.were.verified.in.human.xenograft.NOD/SCID.mouse.models,.in.which.human.colon.cells.were.subcutaneously.injected.In.the.in.vitro.experiments,.combination.treatment.of.vandetanib.with.sunitinib. had. strongly. synergistic,. pro-apoptotic. and. anti-angiogenic.effects. in. both. KRAS. mutated. as. well. as. non-mutated. cells,. which.could.also.be.verified.by.a. intracellular.blockade.of. typical. signalling.pathways..Caspase.stimulation.could.be.detected.in.all.cell.lines.which.reacted. with. apoptosis. induction. after. treatment.. Similarly,. additive.effects. were. detected. when. both. targeted. agents. were. additionally.combined.with.cytotoxic.drugs,.such.as.irinotecan.or.5-FU..In.contrast,.cetuximab.showed.no.additional.effect.alone.or.combined.with.standard.chemotherapeutic.agents. in.KRAS.mutated.colon..Furthermore,.the. in.vivo. model. showed. comparable. results. with. the. combination. of. both.small. molecules. synergistically. leading. to. significant. tumour. volume.and.tumour.weight.reduction.in.the.mice,.a.clear.reduction.of.Ki-67.stai-ning.and.a.loss.of.blood.vessel.formation.in.the.tumours..Also.the.com-bination.of.irinotecan.and.sunitinib.showed.a.decrease.in.tumour.size.Our.data.clearly.support.significant.pro-apoptotic.effects.of.vandetanib.and.sunitinib.in.human.colon.cancer,.independently.of.the.mutation.sta-tus..Combination.of.standard.chemotherapies.with.this.double.targeting.may.be.an.effective.treatment.option.for.a.wide.range.of.advanced.or.metastatic.colon.cancer.patients.

20


Notes:

P-205

Faethe, Christina (University Medical Center, Mainz); Goetze,.Kristina(University.Medical.Center,.Mainz);.Scholz,.Michael.(GSI.Helmholtz.Cen-tre. for. Heavy. Ion. Research,. Darmstadt);. Taucher-Scholz,. Gisela. (GSI.Helmholtz.Centre.for.Heavy.Ion.Research,.Darmstadt);.Mueller-Klieser,.Wolfgang.(University.Medical.Center,.Mainz)

Impact of X-Ray and heavy ion irradiation on tumor cell migration in vitro and related intracellular signaling pathways

Heavy-charged.particles.are.applied.to.deep-seated.tumors.or.tumors.located.close.to.radiosensitive.structures..Previous.results.of.our.group.suggest.that.X-ray.irradiation.of.tumor.cells.can.induce.migration.and.invasion.in.vitro.under.certain.conditions..In.contrast,.12C.ions.invari-antly.showed.a.reduction.of.tumor.cell.motility.. In.the.present.study,.effects.of.X-ray.and.heavy. ion.radiation.on.migration.were.compared.including. approaches. to. deciphering. molecular. mechanisms,. such. as.the.activation.status.of.related.signaling.proteins,.e..g..Akt,.Erk1/2.and.PLCgamma1..We.hypothesize.that.there.is.a.causal.link.between.the.type.of.irradiation.applied,.the.signaling.protein.modulation,.and.changes.in.cell.migration.in.vitro..To.challenge.this,.we.used.the.human.glioblastoma.cell. lines.U87.MG,.A172.and.LN229.exposed.to.either.high.energy.(500.MeV/u).40Ar.ions..or.X-ray.irradiation.at.doses.of.2.and.6.Gy..Boyden.chamber.assays.were.used.to.test.for.the.cellular.migratory.behavior.directly.after.irradiati-on..Furthermore,.the.expression.and.activation.of.the.signaling.proteins.Akt,.Erk1/2.and.PLCgamma1.were.analyzed.using.Western.Blot..Our.results.show.a.radiation-induced.variation.of.tumor.cell.migration.depending.on.the.cell. line.and.type.of. irradiation..While.X-rays.at. low.doses.decrease.the.migration.of.the.cell.lines.A172.and.LN229,.U87.MG.respond.with.an. increase. in.migratory.activity..Higher.doses.of.X-ray.lead.to.a.reduced.migration.for.all.cell.lines..In.contrast,.heavy.ion.irra-diation.cause.a.reduction.of.migration.in.all.cell.lines.and.at.all.doses..Analysis.of.the.signaling.proteins.Akt,.Erk1/2.and.PLCgamma1.also. in-dicate.a.cell.line.and.radiation.type.specific.dependency..For.U87.MG,.an.activation.of.Akt.is.detected.after.exposure.to.X-ray.which.may.be.mechanistically. involved. in. the. concomitant. increase. in. migration.. In.contrast,.A172.show.an.Akt.induction.after.heavy.ion.irradiation.and.a.reduced. migration.. Erk1/2. undergoes. an. inactivation. after. both. X-ray.and.heavy.ion.irradiation.and.the.expression.of.PLCgamma1.is.only.af-fected.by.conventional.X-ray.irradiation.Altogether,.alteration.of.migration.of.glioblastoma.cell. lines.seems.to.involve.different.signaling.pathways.after.exposure.to.X-ray.or.heavy.ion.radiation.with.pronounced.cell.line.specificity..Supported.by.DFG.grants.Mu-90831

P-206

Schmid, Felicitas (Max-Delbrück Centrum, Berlin); Klockmeier,.Konrad.(Freie. Universität,. Berlin);. Osterland,. Marc. (Freie. Universität,. Berlin);.Schlag,.Peter.M..(Charité,.Berlin);.Stein,.Ulrike.(Max-Delbrück-Centrum,.Berlin)

Single nucleotide polymorphisms of the metastasis-inducing gene MACC1 and association with colon cancer metastasis

Colorectal. cancer. is.one.of. the.most.common.cancer.diseases. in. the.Western.world..90%.of.the.cancer.deaths.arise.from.tumor.cells.that.spread.to.distant.organs..Recently,. the.new.gene.MACC1. (metastasis-associated.in.colorectal.cancer.1).was.identified..The.MACC1.expression.in. colon. tumors. is.an. independent.prognostic. indicator.of.metastasis.formation.. Tumors,. which. developed. later. metachronous. metastases,.showed. a. significantly. higher. MACC1. expression. compared. to. non-metastasizing.tumors..The.5-year.survival.rate.for.patients.with.a.high.MACC1.expression.was.only.15%.compared.to.80%.with.a.low.expres-sion..MACC1.regulates.the.expression.of.Met.(met.proto-oncogene).and.plays.a.key.role.in.the.HGF.(Hepatocyte.Growth.Factor)/Met-signaling.which. is. often. deregulated. in. cancer. leading. to. metastasis.. Various.single. nucleotide. polymorphisms. (SNPs). of. cancer-related. genes. are.associated.with.e.g..a.higher.risk.for.or.a.faster.progression.of.cancer..In.this.study,.we.analyzed.the.mutation.status.of.the.genes.MACC1.and.Met.in.colon.tumors..We.wanted.to.evaluate.if.mutations.are.associated.with.expression.modulation.of.MACC1.and.Met,.as.well.as.with.clinicopa-thological.data.and.particularly.with.metastasis.formation..We.amplified.the.coding.exons.of.MACC1.and. the.exons.of. the. juxtamembrane.and.the.tyrosine.kinase.domain.of.Met.by.PCR.and.sequenced.the.products..We.found. in. 13%.of.the.tumors.the.MACC1.SNP.L31V,. in.53%.the.SNP.S515L.and.in.78%.the.SNP.R804T..We.detected.in.only.two.tumors.the.Met.variants.T1010I.and.R988C..We.correlated.the.MACC1.SNPs.to.MACC1.and.Met.mRNA.expression.level,.sex,.tumor.grade,.tumor.stage,.lymph.node.metastasis,. formation.of.metachronous.metastases.and.overall/metastasis-free.survival..None.of.the.SNPs.were.significantly.correla-ted.to.one.of.the.parameters..Tumors.lacking.the.SNP.L31V.showed.the.tendency.to.correlate.to.lymph.node.metastasis..Patients.with.the.SNP.R804T.revealed.the.tendency.for.a.reduced.overall.survival..Thus,.these.two.SNPs.will.be.analyzed.in.further.studies.with.a.larger.tumor.panel..In.order.to.evaluate.the.biological.abilities.of.the.SNPs.we.generated.constructs.containing.MACC1.SNPs..Colon.cancer.cells.were.transfected.with.these.constructs..We.found.no.significant.difference.in.the.migra-tory.behaviour..So.far,.MACC1.mutation.analysis,.even.when.combined.with.MACC1.expression.data,.did.neither. improve.negative.or.positive.predictive.values.nor.sensitivity.or.specificity,.compared.to.MACC1.ex-pression.data.alone.

AEK

21

AEKAbstracts Poster Invasion and Metastasis

Notes:

P-207

Becker, Marina;.Mertsch,.Sonja;.Paulus,.Werner;.Senner,.Volker.(Univer-sity.Hospital,.Münster)

Follistatin is a migration and proliferation enhancing factor in glioma cell lines

Follistatin.is.a.key.regulator.in.TGF-ß.signaling.by.binding.and.neutrali-zing.proteins.of.this.family,.e.g..activinA,.BMPs.or.myostatin..Therefore.follistatin.can. influence.various.cellular.mechanisms. like.proliferation.or.differentiation..Here.we.describe.increased.expression.of.follistatin.in. glioblastoma,. the. most. common. and. most. aggressive. brain. tumor..By.immunofluorescence.staining.follistatin.was.detected.in.glial.tumor.cells,.whereas. in.normal.brain. it.was.restricted.to.neurons.. In.human.tumor. tissue. and. glioma. cell. lines. the. expression. of. follistatin. is. up-regulated.compared.to.normal.brain.tissue..For.studying.the.functional.role.of.follistatin.in.gliomas.we.analysed.migration.and.proliferation.ef-fects..By.blocking.the.expression.of.endogenous.follistatin.with.siRNA.in. four. glioma. cell. lines. migration. and. proliferation. was. attenuated,.whereas. after. addition. of. recombinant. protein. migration. and. prolife-ration.was.increased..Furthermore.we.overexpressed.a.loss-of-function.mutant.isoform.of.follistatin.that.fails.to.bind.activinA.and.observed.no.effect.in.proliferation.and.migration.assays..This.let.us.assume.that.the.effect.on.migration.and.proliferation.induced.by.recombinant.follistatin.is.mediated.through.the.activinA.signaling.pathway..Furthermore.by.cul-turing.glioma.cells.under.glucose-deficient.conditions.the.expression.of.follistatin.was.activated..In.summary.we.propose.that.glucose-deficient.conditions.in.the.tumor.core.may.activate.expression.of.follistatin.and.this.may.in.turn.stimulate.the.tumor.cells.to.migrate.into.the.brain..Inhi-bition.of.follistatin.may.represent.a.novel.promising.target.for.therapy.Supported.by.Wilhelm.Sander.Stiftung.(grant.2005.058.2)

P-208

Demir, Resit (Universitätsklinikum, Erlangen); Pohl,.Johann.(Klinikum,.Bamberg);.Klein,.Peter.(Universitätsklinikum,.Erlangen;.Agaimy,.Abbas.(Universitätsklinikum,.Erlangen)

Necrosis and Angioinvasion Predict Adverse Outcome in Pancreatic Neuroendocrine Tumours after Curative (R0) Surgical Resection: Re-sults of a Single-Center Series

Introduction:.The.prediction.of.outcome.in.pancreatic.neuroendocrine.tumours. (P-NETs). still. represents. a. challenge.. Several. clinicopatholo-gical.parameters.have.been.proposed.to.predict.adverse.outcome..The.aim.of. this. study.was. to.evaluate. the. impact.of. tumour.necrosis.and.angioinvasion.on.the.outcome.after.curative.R0.resection.of.P-NETs..Method:.We.reviewed.our.institutional.experience.over.the.last.30.ye-ars..A.total.of.82.patients.with.a.mean.age.of.54.years.(range,. 17-83.years).underwent.a.surgical.resection.of.P-NET.in.the.period.from.1964.to.2006..The.data.were.collected.prospectively..There.were.41.men.and.41.women..Patient´s.outcome.after.R0.surgical.treatment.was.analyzed.in.relation.to.the.presence.or.absence.of.tumour.cell.necrosis.and.angi-oinvasion.judged.by.histological.methods.Results:.The.overall.(n.=.82).5-year.survival.was.51.8%.(±6.1%)..Forty-eight.of.the.patients.received.a.R0.resection,.successfully..Four.pati-ents.of. this.collective.died.within.3.months.due.to.the.postoperative.complications.and.were.excluded.from.survival.calculations..Forty-four.patients.showed.a.5-year.survival.of.63.3%.(±8.0)..Necrosis.status.was.documented.on.43.of.R0.resected.patients.(97.7%)..The.mean.survival.of.patients.with.tumour.cell.necrosis.was.significantly.shorter.than.tho-se.without.necrosis. (53±14.months.vs.. 176±310.months,. respectively,.p=0.019)..The.patient’s.5-year.survival.was.also.significantly.decreased.(33.9%.±17.6%.vs..70.6%.±8.9%),.accordingly..Angioinvasion.status.was. documented. on. 40. of. R0. resected. patients. (90.9%).. The. mean.survival.of.these.patients.was.decreased.from.187.(±346).to.80.(±18).months,.when.angioinvasion.was.observed.in.the.histological.sections.(p=0.075)..The.patient’s.5-year.survival.was.also.decreased.from.74.3%.±10.1%.to.38.7%.±14.8%,.accordingly.Conclusion:.Long.term.survival.of.patients.with.P-NETs.is.influenced.by.different.pathological.factors..In.our.patients,.we.have.not.seen.signi-ficant.difference. in.overall.survival.based.on.diameter.of.the.primary.tumour. or. the. lymph. node. status.. However,. our. results. showed. that.presence.of. tumour.cell.necrosis.and/or.angioinvasion. is.significantly.associated.with.poor.overall.survival,.even.after.curative.(R0).surgical.resection,.representing.an.independent.variable.for.poor.prognosis.

22


Notes:

P-209

Bartkowiak, Kai; Brandt,. Burkhard;. Pantel,. Klaus. (University. Medical.Center,.Hamburg)

Discovery of hypoxia tolerant disseminated cancer cells with a stem-ness phenotype in the bone marrow

Metastases.arise.from.disseminated.tumor.cells.(DTC).that.are.released.from.primary.tumors.and.are.detected.in.the.bone.marrow.of.breast.and.prostate.cancer.patients.even.in.the.absence.of.clinical.signs.of.distant.metastases.. DTC. can. survive. chemotherapy. (e.g.,. anthracyclines). and.the.hypoxic.bone.marrow.microenvironment.for.many.years..However,.only.a.small.DTC.population.is.able.to.start.metastatic.outgrowth..Due.to. the. lack. of. biomarker. proteins. for. stress. tolerant. DTC. the. in. vivo.processes.selecting.colony.founding.DTC.is.poorly.understood..We.per-formed. proteome. analyses. (2-D. DIGE). of. DTC. cell. lines. that. were. di-rectly.generated.from.bone.marrow.DTC.of.cancer.patients..We.found.a.DTC.population.with.a.stemness.phenotype,.which.confers.immortality.and.limitless.replication..This.DTC.phenotype.is.stress.resistant.by.ove-rexpression.of.the.cytoprotective.program.‘unfolded.protein.response’.(UPR)..We.show.that.breast.cancer.cells.acquire.the.CD44+/CD24-.stem.cell.phenotype.under.persistent.hypoxia.together.with.induction.of.the.UPR.. In. particular,. the. UPR. protein. Grp78. is. specifically. induced. via.Y1248.of.ErbB-2.in.highly.migratory.breast.cancer.cells..Since.we.obser-ved.this.phenotype.for.breast.cancer.DTC.in.vivo,.we.assume.the.iden-tification.of.a.previously.undetected.DTC.population.that.is.extremely.resistant. to.environmental.stress..By.combination.of.stress.tolerance.and.stemness.these.cells.may.be.the.target.of.metastatic.cancer.the-rapy,.rather.than.the.bulk.of.unselected.cancer.cells.released.from.the.primary.tumor.

P-210

Wilde, Cornelia (German Cancer Research Center, Heidelberg); Berger,.Stefan.(ZI,.Mannheim);.Berger,.Martin.(German.Cancer.Research.Center,.Heidelberg)

Evaluation of TPX2 as target in breast cancer therapy

The.microtubule-associated.protein.TPX2,.a.key.component.in.the.mo-lecular.process.of.mitotic.cell.division,.is.under.investigation.as.a.po-tential. therapeutic. target. in.different.types.of.human.cancer..Several.studies. implicated.that.TPX2.expression. is.specifically.upregulated. in.tumor.cells,.which. is. suspected. to.be. important. for. the.maintanence.of.the.tumorigenic.state..Our.aim.was.to.evaluate.the.role.of.TPX2.as.a.target.for.new.anti-mitotic.therapies.in.breast.cancer.TPX2. expression. was. examined. in. non-neoplastic. primary. breast. tis-sues,.the.non-tumorigenic.cell.line.MCF-10A.and.in.various.breast.can-cer.cell.lines..The.expression.level.was.determined.at.the.protein.level.by.Western.Blot.and.at.the.RNA.level.by.real-time.PCR..While.there.was.only.a.marginal.TPX2.expression.in.the.non-neoplastic.patients’.samples.and. in. MCF-10A. cells,. the. different. breast. cancer. cell. lines. showed. a.significant. upregulation. of. TPX2. expression. both. at. the. RNA. and. the.protein.level.To.investigate.the.functional.importance.of.TPX2.in.breast.cancer.cells,.two.cell.lines.were.created,.in.which.TPX2.production.can.be.conditio-nally.inhibited.by.a.TPX2-specific.miRNA.under.the.transcriptional.con-trol.of.the.TetOff.system..The.efficiency.of.the.conditional.knockdown.was.determined.by.real-time.PCR.and.Western.Blot..The.effects.of.the.TPX2.knockdown.on.cell.progression.and.division.were.followed.up.by.life.cell.imaging.under.a.light.microscope.and.by.MTT.assay..Within.both.investigated.cell.lines,.an.almost.complete.knockdown.was.achieved.both.at.the.RNA.and.protein.levels..TPX2.expression.was.sup-pressed.by.71%.three.days.after.induction.of.miRNA.expression.and.by.97%.after.four.days.in.MDA-MB-231-TPX-54-1.1.and.by.89.%.after.three.days.and.by.99%.after.four.days.in.MDA-MB-231-TPX-23-25,.respectively..This.potent.inhibition.of.TPX2.production.led.to.an.increased.number.of.apoptotic.cells.in.both.cell.lines.as.identified.by.life.cell.imaging..In.ad-dition,.cell.proliferation.was.shown.to.be.significantly.reduced.by.34%.(MDA-MB-231-TPX-54-1.1). and. 19%. (MDA-MB-231-TPX-23-25). three. days.after.induction.of.the.knockdown..These.results.show.that.knockdown.of.TPX2.causes.apoptosis.in.breast.cancer.cells.and.has.an.anti-proliferative.effect..In.conclusion,.the.re-sults.provide.evidence.that.TPX2.is.a.suitable.target.in.the.therapy.of.breast.cancer..

AEK

23

AEKAbstracts Poster Invasion and Metastasis

Notes:

P-211

Damm, Friederike; Hoffmann,.Michèle.Janine;.Gabbert,.Helmut.Erich;.Engers,.Rainer.(University.Medical.Center,.Düsseldorf)

Functional relevance of Tiam1/Rac3 signaling in prostate cancer

Aims:.Rho-like.GTPases.are.a.subgroup.of.the.Ras.superfamily.and.in-clude.3.different.isoforms.of.Rac..While.Rac1.is.ubiquitously.expressed.and.Rac2.hematopoiesis-specific,.Rac3.is.less.characterized.regarding.its.functional.role..We.found.increased.expression.of.Rac3.and.its.acti-vator.Tiam1.in.most.prostate.carcinomas.(PCas).as.compared.to.benign.prostatic.tissues.and.strong.overexpression.of.Tiam1.or.Rac3.correlated.with.an.aggressive.clinical.course..Here,.we.investigated.the.functional.role.of.Tiam1.and.Rac3.in.PCa..Methods:.The.human.PCa.cell.line.PC3.was.stably.transfected.with.either.empty.vector,.wild-type.or.constitutively.active. forms.of.Tiam1. (FL-Tiam1,.C1199-Tiam1).and.Rac3. (wt-Rac3,.V12-Rac3)..Functional.assays.were.performed.as.described.earlier.(Engers.et.al,.Int.J.Cancer.2000;.J.Biol.Chem.2001;.Endocr.Rel.Cancer.2007)..Results:.Overexpression.of.FL-Tiam1.and.C1199-Tiam1.induced.a.pancake-like. phenotype. and. pronounced. membrane. ruffling,. whereas. wt-Rac3.and.V12-Rac3.had.no.obvious.effect.on.cell.morphology.. In. functional.in. vitro. assays. C1199-Tiam1. and. V12-Rac3. significantly. stimulated. cell.adhesion. to. laminin. (p<0,001).. Moreover,. in. contrast. to. our. previous.investigations. in. renal. cell. carcinoma,. C1199-Tiam1. stimulated. rather.than. inhibited. both. transwell. migration. and. Matrigel. invasion,. indica-ting.a.more.aggressive.behaviour...Similar.effects.were.also.seen.upon.overexpression.of.V12-Rac3.(p<0,001)..The.effects.of.FL-Tiam1.and.wt-Rac3.were.partly.similar.and.partly.less.pronounced.than.those.of.their.constitutively.active.counterparts,.which.is.in.accordance.with.obser-vations. in.other.cell. systems.. .Cell.proliferation.was.not. significantly.affected.neither.by.wild-type.forms.nor.by.constitutively.active.forms.of. Tiam1. and. Rac3.. Conclusions:. Tiam1/rac3. signaling. significantly. af-fects.cell.adhesion,.migration.and.invasion.of.human.PCa.cells.in.vitro.towards.a.more.aggressive.phenotype..Given.the.fact.that.in.vivo.incre-ased.expression.of.either.Tiam1.or.Rac3.in.PCa.is.associated.with.an.ag-gressive.clinical.course.and.a.poor.clinical.outcome,.our.data.suggest.an.important.role.of.Tiam1/Rac3.signaling.in.prostate.cancer.progression.

P-212

Michèle Janine Hoffmann, University Medical Center, Düsseldorf);.Anne.Maaßen,.University.Medical.Center,.Düsseldorf);.John.G..Collard,.University.Medical.Center,.Düsseldorf);.Arif.B..Ekici,.Humangenetisches.Institut,. Erlangen);. Maciej. Rosolowski,. Interdisziplinäres. Zentrum. für.Bioinformatik,.Leipzig);.Helmut.Erich.Gabbert,.University.Medical.Cen-ter,.Düsseldorf);.Rainer.Engers,.University.Medical.Center,.Düsseldorf)

Candidate factors mediating Tiam1/ Rac3 effects in the development of prostate cancer

Objective:.The.small.GTP-binding.Rac.proteins.belong.to.the.Rho-like.GT-Pase.family.which.includes.different.isofoms.of.Rac..While.Rac1.is.ubiqui-tously.expressed.and.Rac2.hematopoiesis-specific,.only.little.is.known.about.the.functional.role.of.Rac3..We.found.Rac3.and.its.activator.Tiam1.significantly.overexpressed.in.prostate.carcinomas.and.pre-neoplastic.high-grade. PIN. lesions. and. both. Tiam1. and. Rac3. overexpression. pre-dicted. an. aggressive. clinical. course.. Investigations. on. the. functional.role.in.carcinogenesis.revealed.that.constitutive.activation.of.Rac3.in-duces.a.more.aggressive.phenotype.in.benign.prostatic.epithelial.cells.(BPH1)..BPH1.cells,.stably.transfected.with.either.constitutively.active.Rac3.(V12Rac3).or.double.transfected.with.wildtype.Rac3.and.its.activa-tor.Tiam1,.mimicking.the.situation.in.vivo,.gained.significant.capability.of.migration.and.invasion..Further,.these.two.cell.lines.showed.colony-forming.capacity,.also.under.anchorage-independent.conditions..In. this. study,. we. performed. a. microarray. analysis. to. identify. down-stream.factors.mediating.Tiam1/Rac3.effects.Methods:.BPH1. cells.were. stably. transfected.with.either.empty.vector,.wildtype.or.constitutively.active.forms.of.Tiam1.and.Rac3..RNA.was.hybri-dized.on.Affymetrix.U133.Plus.2.0.chips.according.to.the.manufacturer.Results:.Statistical.analysis.of. the.microarray.data.was.performed. to.answer.the.following.questions:1...Which. genes. are. differentially. expressed. in. V12Rac3-. and. double.

transfected.cells.as.compared.to.the.vector.or.other.cell.lines?2..Can.we.find.genes.specifically.regulated.by.Tiam1.or.Rac3?3...Can.we.find.differences/.common.genes.between.constitutive.active.

and.wildtype.forms?A.number.of.differentially.expressed.genes.could.be.assigned.to.the.de-fined.groups..Interestingly,.among.these.genes.nine.components.of.the.uPA.system.and.the.associated.SERPINB.cluster.appeared.to.be.influ-enced.by.Tiam1/Rac3.signaling..UPA.activity.assays.revealed.increased.uPA.activity.in.V12Rac3-.and.double.transfected.BPH1.cells..Conclusions:.Constitutive.activation.of.Rac3.induces.a.more.aggressive.phenotype. in. BPH1. cells.. Searching. for. downstream. factors. we. found.members. of. the. uPA. system. to. be. differentially. expressed. in. stably.transfected.cell.lines..The.uPA.system.plays.a.role.in.key.processes.of.tumor.invasion..Shifting.in.balances.of.activators.and.inhibitors.of.the.uPA.system.may.contribute. to. the. increased.capability.of. invasion.of.such.cell.lines.and.needs.further.investigation..

24


Notes:

P-213

Vormbrock, Kirsten (Max-Delbrück Centrum, Berlin); Schreiber,.Caroline. (Max-Delbrück. Centrum,. Berlin);. Loddenkemper,. Christoph.(Charité,.Berlin);.Hoang-Vu,.Cuong.(University.Medical.Center,.Halle./.S.);.Ziebold,.Ulrike.(Max-Delbrück.Centrum,.Berlin)

Elevated CENPA induces chromosomal instability but promotes sur-vival and transformation creating metastasis in mouse and human medullary thyroid carcinomas

Centromere.identity.is.defined.by.the.histone.H3.variant.Cenpa..Incre-ased.expression.of.Cenpa.has.been. linked. to.chromosomal. instability.(CIN).suggesting.that.Cenpa.is.critical.for.accurate.genomic.fidelity..We.show. that. both. the. retinoblastoma. gene. product. (pRb). and. E2F3. are.required.for.proper.regulation.and.positioning.of.mouse.Cenpa..The.ab-sence.of.pRb.and.E2F3.is.associated.with.a.high.incidence.of.metastasis.of.mouse.medullary.thyroid.carcinomas.(MTCs)..This.also.results.in.ele-vated.levels.of.Cenpa.and.abnormally.large.intercentromeric.distances..Consequently,.Cenpa.accurately.predicts. the. incidence.of.metastasis:.prospectively. in. early. stage. tumors. as. well. as. retrospectively. in. ad-vanced. MTCs. in. pRb/E2F3. deficient. mice.. Importantly,. also. in. human.MTC.patients.elevated.CENPA.was.significantly.associated.with.the.oc-currence.of.metastasis..To.recapitulate.the.underlying.mechanism.we.overexpressed. CENPA. in. human. MTC. cells,. which. leads. to. erroneous.mitosis.and.CIN..Remarkably,.excess.CENPA.profoundly.supported.the.cells.to.cope.with.CIN-induced.genomic.stress..Thus,.by.promoting.CIN,.survival,. cellular. transformation. and. anchorage-independent. growth.excess.CENPA.propels.metastasis..Associating.CENPA.with.metastasis.might.make.more.accurate.prognosis.for.MTC.patients.possible.and.it.also.provides.a.new.therapeutic.target.

P-214

Kovacheva, Marineta (German Cancer Research Center, Heidelberg); Reufsteck,. Christina. (German. Cancer. Research. Center,. Heidelberg);.Berger,.Stefan.(ZI,.Mannheim);.Berger,.Martin.(German.Cancer.Research.Center,.Heidelberg)

Inducible RNAi -based repression of target genes associated with skeletal metastasis impairs metastatic properties in breast cancer subclones

Breast.cancer.is.one.of.the.most.commonly.diagnosed.malignancies.and.second. leading.cause.of.cancer-related.death. in.women..Most.breast.cancer.patients.have.osteolytic.bone.metastasis,.which.is.characterized.by.severe.pain,.high.incidence.of.fractures,.spinal.cord.compression.and.bone.marrow.aplasia.requiring.hospitalization..Potential.targets.for.the.treatment.of.metastasis.might.be.osteopontin.(OPN).as.member.of.the.SIBLING.(small.integrin-binding.ligand.N-linked.glycoprotein).family.and.matrix.metalloproteinase.2.(MMP2)..These.proteins.are.reported.to.play.a.role.in.the.processes.of.proliferation.and.migration,.which.are.essenti-al.for.metastasis.formation..We.hypothesized.that.RNAi-mediated.down-regulation.of.these.proteins.will. impair. the. metastatic. properties. of. invasive. breast. cancer. cells..Therefore,.we.generated.MDA-MB231.subclones,. in.which. the. tetracy-cline-dependent.transactivator.tTA.controls.the.expression.of.the.red.fluorescent. protein. mCherry,. firefly. luciferase. and. an. highly. efficient.and.specific.miRNA.for.the.respective.target.gene..The.consequences.of.conditional.target.gene.“knockdown”.on.cell.pro-liferation.was.determined.by.MTT.assay.and.that.on.spontaneous.migra-tion.by.using.a.cell.culture-based.migration.assay.The.“knockdown”.of.OPN.in.the.relevant.clone.resulted.in.18,.16.and.10%.inhibition.of.cell.proliferation.after.24,.48.and.72.hours. respectively..Also.65%.inhibition.of.cell.migration.was.observed.after.48.h.. In.the.case.of.MMP2,. the.proliferation.of. the.respective. inducible.cell.clone.was.not.inhibited,.whereas.the.migration.was.decreased.by.17%.after.48h.In. conclusion,. the. single. knockdown. of. subclones. expressing. miRNAs.against.OPN.or.MMP2.resulted.in.reduced.spontaneous.migration.and.in.a.mild.suppression.of.proliferation.following.OPN.silencing..

AEK

25

AEKP-215

Milde-Langosch, Karin;. Hein,. Sibyll;. Wikman,. Harriet;. Köhler,. Nadine;.Müller,. Volkmar;. Pantel,. Klaus;. Jänicke,. Fritz. Atashrazm,. Farzaneh;..Pazhakh,.Vahid.(Hamburg.University.Medical.School,.Hamburg)

Biologic role of activated leukocyte cell adhesion molecule (ALCAM) overexpression in breast cancer cell lines and clinical tumor tissue

The. Activated. Leukocyte. Cell. Adhesion. Molecule. (ALCAM). is. overex-pressed.in.many.mammary.tumours,.but.controversial.results.about.its.role.and.prognostic.impact.in.breast.cancer.have.been.reported..The-refore,.we.evaluated.the.biological.effects.of.ALCAM.expression.in.two.breast.cancer.cell.lines.and.a.larger.cohort.of.mammary.carcinomas..Methods:.By.stable.transfections,..MCF7.cells.with.ALCAM.overexpressi-on.and.MDA-MB231.cells.with.reduced.ALCAM.levels.were.generated.and.analyzed. in. functional. assays. and. cDNA. microarrays.. Additionally,. an.immunohistochemical.study.including.347.breast.cancer.patients.with.long-term. follow-up. and. analysis. of. disseminated. tumour. cells. (DTC).was.performed.Results:.In.both.cell.lines,.high.ALCAM.expression.was.associated.with.reduced.cell.motility..Additionally,.ALCAM.silencing.in.MDA-MB231.cells.resulted. in. lower. invasive. potential,. whereas. ALCAM. overexpressi-on.was.associated.with. increased.apoptosis. in.both.cell. lines..Among.genes.which.were.differentially.expressed.in.clones.with.altered.ALCAM.expression,.there.was.an.overlap.of.15.genes.between.both.cell. lines,.among. them. cathepsin. D,. keratin. 7,. gelsolin. and. ets2. whose. deregu-lation.was.validated.by.western.blot.analysis.. In.MDA-MB231.cells,.we.observed. a. correlation. with. VEGF. expression. which. was. validated. by.enzyme-linked.immuno.sorbent.assay.(ELISA)..Our. IHC. results. on. primary. breast. carcinomas. showed. that. ALCAM.expression.was.associated.with.an.estrogen.receptor-positive.pheno-type.. Additionally,. strong. ALCAM. immunostaining. correlated. with. no-dal. involvement.and.the.presence.of.tumour.cells.in.bone.marrow..By.Kaplan-Meier.analysis,.strong.ALCAM.expression. in.ductal.carcinomas.correlated.with.shorter.recurrence-free.intervals.(p=0.048).and.overall.survival.(p=0.003)..Conclusion:.Our.results.indicate.that.the.biological.role.of.ALCAM.in.bre-ast.cancer.is.complex..Depending.on.the.cell.type,.ALCAM.overexpres-sion.can.increase.tumour.invasion.and.angiogenesis,.leading.to.a.more.aggressive.tumour.type,.whereas.its.influence.on.cell.motility.and.apo-ptosis.might.lead.to.a.less.malignant.phenotype.and.a.better.prognosis..These. results. might. offer. an. explanation. for. the. partly. controversial.results.concerning.the.role.of.ALCAM.in.clinical.tumour.tissues.

P-216

Rose1 M.;. Sechi2. A.S.,. Veeck1. J.;. Noetzel1. E.;. Bovi3. A.;. Bornemann3. J.;.Knüchel1.R..and.Dahl1.E.

1..Institute.of.Pathology,.Medical.Faculty.of.the.RWTH.Aachen.University,.Aachen,.Germany.

2..Institute.for.Biomedical.Technology.–.Cell.Biology,.Medical.Faculty.of.the.RWTH.Aachen.University,.Aachen,.Germany

3..Electron. Microscopy. Facilities,. Medical. Faculty. of. the. RWTH. Aachen.University,.Aachen,.Germany.

Metastasis in human breast cancer: ITIH5 mediates tumor-suppressive properties by repressing epithelial-to-mesenchymal transition (EMT)

Background:. ITIH5,. a. novel. member. of. the. inter-a-trypsin. inhibitory.heavy. chain. (ITIH). protein. family. and. prognostic. marker. in. human.breast. cancer,. is. lost. during. breast. cancer. progression,. the. process.being.closely.associated.with.clinical.parameters.of.metastasis..Since.these. data. indicate. a. potential. role. of. ITIH5. in. prevention. of. tumor.invasion,.we.hypothesized.that. ITIH5.may.represent.a.putative.tumor.suppressor.gene.whose.functional.loss.marks.a.key.initiating.event.in.breast.cancer.metastasis.Methods:.We.analyzed.the.functional. influence.of.ITIH5.expression.on.tumor.progression.using.a.model. that. reflects.highly. invasive. cancer.cells.. We. therefore. stably. transfected. basal-type. MDA-MB-231. cancer.cells. with. a. full. length. ITIH5. cDNA. pBK-CMV. expression. vector. (MDA-MB-231-ITIH5.clones).or.empty.vector.alone.(MDA-MB-231-Mock.clones).which. were. afterwards. subjected. to. in-depth. functional. and. morpho-logic.analysis.based.on.the.well.defined.hallmarks.of.cancer.Results:.After.ITIH5.transfection.MDA-MB-231-ITIH5.tumor.cells.exhibit-ed.a.significantly.enhanced.cell:matrix-adhesion.(63%;.p<0.01),.a.reduc-tion.in.cell-growth.(48%;.p<0.001).and,.moreover,.a.strong.retardation.of.both.in.vitro.motility.(85%;.p<0.01).and.invasiveness.(91%;.p<0.05).when.compared.to.control.cells.without.ITIH5.expression..Most.interestingly,.we.observed.a.switch.towards.an.epithelial-like.cell.phenotype.in.ITIH5.transfectands:. Formerly. fibroblastic. post-EMT. MDA-MB-231. cells. went.through.a.dramatically.cytoskeleton.re-organization.since.MDA-MB-231-ITIH5. clones. formed. cortical. actin. bundles. and. exhibited. small. focal.adhesions.being.accompanied.by.distinct.in.cell:cell.contacts,.e.g..tight.junctions..This.strong.cell:cell.regimentation.correlated.with.the.restora-tion.of.established.epithelial.markers.such.as.Zonula.occludens-1.(ZO-1).Conclusions:.Forced. ITIH5.expression. leads. to.a. reversion.to.epithelial.characteristics.in.post-EMT.breast.cancer.cells.which.is.associated.with.an.effective.suppression.of. invasive.properties..Thus,.we.here.provide.clear.evidence. for. the. tumor-suppressive.character.of. ITIH5..Owing. to.the.revealed.functional.linkage.to.the.MET.process,.we.suggest.that.ITIH5.may.act.as.crucial.“gatekeeper”.in.human.breast.cancer.progression.by.repressing.the.initiation.of.tumor.cell.invasion..Further.ITIH5.investiga-tion.may.deliver.new.insights.into.EMT.associated.pathways.and.might.also.open.novel.therapeutic.approaches.for.breast.cancer.treatment.

Abstracts Poster Invasion and Metastasis

Notes:

26

AEKAEK AEKAbstracts Short Talks Mouse Models

Notes:

short talk 301

Thomale, Jürgen (University of Duisburg-Essen, Essen); Mendus,.Diana. (University. of. Duisburg-Essen,. Essen);. Manz,. Daniel. (University.of.Duisburg-Essen,.Essen);.Nickel,.Ann-Christin.(University.of.Duisburg-Essen,.Essen);.Oliver,.Trudy.(Koch.Institute.for. Integrative.Cancer.Re-search,.Cambridge)

Mouse lessons on tumor drug resistance and on the mechanisms of side effects

Successful.cancer.chemotherapy.with.DNA-reactive.drugs.is.hampered.by.two.major.obstacles,.the.development.of.non-responsiveness.in.tu-mor.cells.and. the.accumulating. toxicity. in.non-malignant,.physiologi-cal.cells..The.molecular.mechanisms.underlying.both.problems.are.still.poorly.understood,.which.is.partly.due.to.the.lack.of.appropriate.analy-tical.tools.and.the.usage.of.uninformative.cell.culture.systems.We.have.employed.a.panel.of.transgenic.mouse.models.and.DNA.damage-specific.immuno-analytical.methods.to.uncover.such.mechanisms.for.the.clinically.important.group.of.platinum.anticancer.drugs.such.as.cisplatin.Thereby,.we.have.identified.in.the.inner.ear.and.in.the.kidney.(the.two.tissues. suffering. most. from. the. systemic. toxicity. of. cisplatin). a. small.number.of.specific.“target.cells”.which.accumulate.excessive.levels.of.drug-induced.platination.products.in.their.nuclear.DNA..The.causing.me-chanism.was. identified.as.aberrant. import.of. cisplatin. into. these.cells.by.physiological.membrane.transporters..Measurements.of.DNA.damage.kinetics.in.repair-proficient.and.-deficient.mice.clearly.linked.the.levels.of.cisplatin-DNA.intrastrand.cross.inks.to.the.induction.of.autophagic.cell.death.in.the.target.cells.and.to.the.functional.loss.of.the.inner.ear.and.the.kidney..The.in.vivo.screening.for.potential.inhibitors.of.the.transporter-mediated.uptake.of.cisplatin.disclosed.a.compound.which,.when.given.in.combination.with.cisplatin,.prevented.the.excess.formation.of.DNA.dama-ge.in.the.critical.cells.and.averted.the.nephro-.and.ototoxicity.of.the.drug.without.affecting.its.antineoplastic.activity.for.primary.tumors.in.mice..With.respect.to.tumor.drug.resistance.we.have.exploited.a.Ki-rasmut-driven.mouse.model.for.human.non.small.cell.lung.cancer.(NSCLC)..Like.in.the.clinical.setting.tumors.frequently.forfeit.their.initial.responsive-ness.to.cisplatin.after.several.courses.of.drug.treatment..When.analyzi-ng.such.tumors.in.mice.after.a.last.challenging.dose.of.cisplatin.the.level.of.DNA.adducts.was.strongly.reduced.in.most.of.the.cancer.cells.but.was.still.unaltered.high.in.neighbouring.normal.lung.tissue..Data.from.in.vivo.adduct.kinetics.in.such.tumors.insinuate.augmented.cellular.DNA.repair.capacity.to.cause.the.reduced.damage.burden.in.drug-resistant.primary.mouse.tumors..As.this.type.of.“low.adduct”.tumors.was.not.observed.in.therapy-naïve.mice.our.data.suggest.a.model.of.clonal.selection.for.cells.with.an.up-regulated.nucleotide.excision.repair.function.

short talk 302

Pusterla, Tobias (German Cancer Research Center, Heidelberg); Németh,.Julia.(German.Cancer.Research.Center,.Heidelberg);.Stein,.Ilan.(Hebrew. University. Hadassah. Medical. School,. Jerusalem);. Marhenke,.Silke. (Medizinische. Hochschule,. Jhannover);. Wiechert,. Lars. (DKFZ,.Heidelberg);.Longerich,.Thomas. (University,.Heidelberg);.Vogel,.Arndt.(Medizinische. Hochschule,. Hannover);. Bierhaus,. Angelika. (University,.Heidelberg);.Pikarsky,.Eli.(Hebrew.University.Hadassah.Medical.School,.Jerusalem);.Angel,.Peter.(German.Cancer.Research.Center,.Heidelberg);.Hess,.Jochen.(German.Cancer.Research.Center,.Heidelberg)

Impaired liver tumor formation and oval cell activation in the RAGE knockout mouse

The.Receptor.for.Advanced.Glycation-End.products.(RAGE).is.a.multili-gand.receptor.and.member.of.the.immunoglobulin.superfamily.of.sur-face.receptors..RAGE.is.mainly. involved. in.tissue.damage.and.chronic.inflammatory.disorders,.sustaining.the.inflammatory.response.upon.en-gagement.with.damage.associated.molecular.pattern.molecules.such.as.S100.proteins.and.HMGB1..Besides.chronic.inflammatory.diseases,.RAGE.and.its.potential.ligands.have.also.been.shown.to.be.overexpressed.in.several.types.of.tumors,.enhancing.tumor.progression.and.metastasis.by. still. unknown. mechanisms.. Interestingly,. RAGE-deficient. mice. are.protected.in.skin.and.colon.models.of.chemically-induced.tumorigene-sis,.whereas.in.the.Mdr2-/-.model.of.inflammation-associated.hepato-cellular.carcinoma.expression.of.RAGE.ligands.S100A8/A9.was.shown.to.protect.cancer.cells.from.apoptosis.We.analyzed.the.causal.link.between.RAGE.signaling.and.inflammation-driven. liver.carcinogenesis.by.ablating.RAGE.expression. in. the.Mdr2-/-.mouse.model..Rage-/-.Mrd2-/-.(dKO).mice.developed.smaller.and.fe-wer.hepatocellular.carcinomas.compared.to.Mrd2-/-.mice..Surprisingly,.RAGE.ablation.did.neither.affect.hepatitis.development.nor.recruitment.of.inflammatory.cells.to.the.liver..However,.dKO.mice.developed.dimi-nished. liver.damage.and.fibrosis. accompanied.by. impaired.activation.of.oval.cells..In.the.liver,.RAGE.was.strongly.expressed.by.oval.cells.and.treatment.of.an.immortalized.oval.cell.line.with.its.ligand.HMGB1.promo-ted.ERK1/2.phosphorylation.and.cell.proliferation.in.vitro..These.results.provide.experimental.evidence. for.a.crucial. role.of.RAGE.signaling. in.promotion.and.malignant.progression.of.HCC.

AEK

27

AEKAbstracts Poster Mouse Models

Notes:

P-302

Lipp, Martin (Max-Delbrück Centrum, Berlin); Krause,.Claudia. (Max-Delbrück.Centrum,.Berlin);.Mallem,.Nedjouna.(Max-Delbrück.Centrum,.Berlin);. Thirunarayanan,. Nanthakumar. (National. Institutes. of. Health,.Bethesda);. Dittmar,. Gunnar. (Max-Delbrück. Centrum,. Berlin);. Müller,.Gerd.(Max-Delbrück.Centrum,.Berlin)

A novel HHV8-vGPCR-triggered animal model to study the role of (epi)genetic mechanisms in immune escape and tumor progression

The. Human. herpes. virus. 8. (HHV-8)-encoded. G. protein-coupled. che-mokine. receptor. (vGPCR).has.been. implicated. in. the.pathogenesis.of.Kaposi´s.sarcoma.(KS).particularly.because.of.its.constitutive.signaling.activity.. We. have. used. retroviral. transduction. to. generate. vGPCR-ex-pressing. BALB/c-3T3. fibroblasts. that. are. tumorigenic. in. immunocom-promised.nude.mice,.but.as.expected.fail.to.induce.tumors.in.immuno-competent.wild-type.BALB/c.mice..However,.tumor.fragments.obtained.from.nude.mice.grow.progressively.in.immunocompetent.BALB/c.mice..Unexpectedly,.vGPCR-expressing.tumor.cells.established.from.grafted.tumor.fragments.formed.tumors.in.immunocompetent.mice..These.tu-mors.exhibit.a.striking.histological.resemblance.to.KS.including.plump.spindle.cell.morphology.and.a.high.degree.of. vascularization..Knock-down. of. the. vGPCR. by. shRNA. significantly. delayed. tumorigenesis. in.nude.mice,.demonstrating.that.in.our.model.tumor.development.depen-ds. on. the. vGPCR. oncogene.. Acquired. changes. in. the. tumor. cells. are.the.result.of.successive.passage.of.vGPCR-3T3.cells.through.nude.and.immunocompetent.mice..We.have.now.compared.the.gene.expression.profiles. of. vGPCR-induced. tumors. of. immunocompetent. mice. as. well.as.of.cell.lines.established.from.the.tumors.to.the.profiles.of.parental.BALB/c-3T3.and.vGPCR-transformed.BALB/c-3T3.cells..Moreover,.we.are.extending.our.studies.to.analysis.of.the.proteome.(SILAC),.histone.mo-difications.(Chip-on-chip).and.genetic.aberrations.(aCGH)..This.will.lead.to.the.identification.of.critical.genes.regulated.by.either.epigenetic.or.genetic. mechanisms. that. will. be. further. studied. in. vitro. und. in. vivo..Hence,.this.novel.model.could.contribute.to.our.understanding.of.both.epigenetic.and.genetic.changes.in.tumor.progression.and.immune.es-cape.induced.by.the.tumor.microenvironment.

28

AEKAEK AEKAbstracts Short Talks Carcinogenesis, Genome stability, DNA Damage and Repair, Epigenetics

Notes:

short talk 401

Beyer, Ulrike (University of Goettingen, Göttingen); Moll-Rocek,.Julian.(Stony.Brook.University,.New.York);.Moll,.Ute.(Stony.Brook.University,.New.York);.Dobbelstein,.Matthias.(University.of.Goettingen,.Göttingen)

An endogenous retrovirus drives hitherto unknown pro-apoptotic p63 isoforms in the male germline of humans and great apes

TAp63,.but.not.its.homologue.p53,.eliminates.oocytes.that.suffered.DNA.damage.. An. equivalent. gene. for. guarding. the. male. germ. line. is. cur-rently.not.known..Here.we.identify.hitherto.unknown.human.p63.tran-scripts.with.novel.5‘-ends.derived. from. incorporated.exons.upstream.of.the.currently.mapped.TP63.gene..These.unique.p63.transcripts.are.highly.and.specifically.expressed.in.testis..Their.most.upstream.region.corresponds.to.a.long.terminal.repeat.(LTR).of.the.human.endogenous.retrovirus.9.(ERV9)..The.insertion.of.this.LTR.upstream.of.the.TP63.locus.occurred.only.recently.in.evolution.and.is.unique.to.humans.and.great.apes.(Hominidae)..A.corresponding.p63.protein.is.the.sole.p63.species.in. healthy. human. testis,. and. is. strongly. expressed. in. spermatogenic.precursors.but.not.in.mature.spermatozoa..In.response.to.DNA.damage,.this.human.male.germ.cell-encoded.TAp63.protein.(designated.GTAp63).is.activated.by.caspase.cleavage.near.its.carboxyterminal.domain.and.induces.apoptosis..Human.testicular.cancer.tissues.and.cell.lines.largely.lost.p63.expression..However,.pharmacological.inhibition.of.histone.de-acetylases.completely.restores.p63.expression.in.testicular.cancer.cells.(>3000-fold.increase)..Our.data.support.a.model.whereby.testis-specific.GTAp63.protects.the.genomic.integrity.of.the.male.germ.line.and.acts.as.a.tumor.suppressor..In.Hominidae,.this.guardian.function.was.greatly.enhanced.by.integration.of.an.endogenous.retrovirus.upstream.of.the.TP63.locus.that.occurred.15.million.years.ago...By.providing.increased.germ.line.stability,.this.event.may.have.contributed.to.the.evolution.of.hominids.and.enabled.their.long.reproductive.periods.

short talk 402

Leufke, Christine (German Cancer Research Center, Heidelberg); Krunic,. Damir. (German. Cancer. Research. Center,. Heidelberg);. Boehm-Steuer,.Barbara.(University,.Heidelberg);.Jauch,.Anna.(University,.Hei-delberg);.Boukamp,.Petra.(German.Cancer.Research.Center,.Heidelberg)

A ROLE FOR TELOMERE LENGTH AND ORGANISATION IN HUMAN SKIN CARCINOGENESIS

Telomeres. are. thought. to. guarantee. chromosomal. stability. and. inte-grity. and. critically. short. telomeres. to. cause. genomic. instability.. We.recently.demonstrated.that.also.disorganisation.of.the.telomeres,.i.e..formation. of. telomeric. aggregates. (TA),. leads. to. genomic. instability..Since.we. identified.UV. irradiation.as.one.mechanism.of.TA. induction,.we.asked.whether.and.which.of.these.two.telomere-dependent.mecha-nisms.would.be.relevant.for.non-melanoma.skin.cancer..For.this,.we.in-vestigated.telomeres.in.normal.skin,.actinic.keratoses.(AKs).-.precursor.lesions.of.SCC.-.as.well.as.SCCs.and.BCCs.by.in.situ.hybridization.and.deconvolution.microscopy,. computer-added.3D. reconstruction.and. si-gnal.intensity.evaluation.In.healthy.skin,. telomeres.were.evenly.distributed.within.the.nucleus.with. occasional. associations. of. two. or. three. telomeres. (~15%).. In-terestingly,.AKs.showed.a.significant. increase. in.these.small.associa-tions.(~40%),.TAs.were,.admittedly,.not.detected..However,.in.SCCs.and.BCCs.76%.and.90%.of.the.tumors.showed.associations.of.>3.telomeres.and.TAs..This.demonstrates.that. the.tightly.regulated.organisation.of.telomeres.is.progressively.disturbed.during.tumor.progression.up.to.a.stage.of.severe.disorganisation..Actually,.this.seems.to.be.a.more.gene-ral.mechanism.in.tumor.development,.because.TA.formation.is.also.seen.in.malignant.melanomas..Concerning.telomere.length,.we.could.identify.two.groups.in.SCCs.and.BCCs.. Tumors. either. exhibited. rather. short. telomeres. and. telomere.length. was. homogeneous. throughout. the. tumor. or. telomeres. were.generally. longer. and. the. length. differed. strongly. between. different.tumors.and.most.importantly.between.different.tumor.areas,.pointing.to.a.strong.inter-.and.intratumoral.telomere.length.heterogeneity..In-terestingly,. in.SCCs.telomere. length.distribution.did.neither.correlate.with.telomerase.activity.nor.with.the.amount.of.TAs,.suggesting.that.telomere.length.and.organisation.are.two.independent.parameters.how.telomeres.can.contribute.to.tumor.development..However,.genetic.ana-lysis.suggests.for.a.correlation.of.an.increased.number.of.chromosomal.aberrations.in.SCCs.with.long.and.heterogeneous.telomeres.while.SCCs.with.short.and.homogeneous.telomeres.showed.a.more.simple.karyo-type.Taken.together,.our.results.demonstrate.that.understanding.the.regu-lation.of.telomere.length.and.in.particular.telomere.organisation.(TAs.are.so.far.unique.for.malignant.tumors).may.provide.the.basis.for.novel.approaches.in.skin.cancer.therapy.

AEK

29

AEKAbstracts Poster Carcinogenesis, Genome stability, DNA Damage and Repair, Epigenetics

Notes:

P-401

Goering, Wolfgang; Ribarska,. Teodora;. Schulz,. Wolfgang. (University.Medical.Center,.Düsseldorf)

Selective changes of retroelement expression in human prostate cancer

Retroelements. constitute. a. large. part. of. the. human. genome.. These.sequences.are.mostly.silenced. in.normal.cells,.but.genome-wide.DNA.hypomethylation.in.cancers.might.lead.to.their.reexpression..Whether.this. reexpression. really. occurs. in. human. cancers. is. largely. unkown..We.therefore. investigated.expression.and.DNA.methylation.of.several.classes.of.retroelements.in.human.prostate.cancer.tissues.and.cell.lines.by.quantitative.RT-PCR.and.pyrosequencing,.respectively.The.most.striking.finding.was.strong.and.generalized.increased.expres-sion. of. the. HERV. K_22q11.23. provirus. in. cancers,. including. de. novo-expression. of. a. spliced. accessory. Np9. transcript. in. some. tumors.. In.parallel,. DNA. methylation. in. the. LTR. decreased.. Conversely,. HERVK17.expression.was.significantly.diminished.in.cancer.tissues,.but.this.de-crease.was.unrelated.to.LTR.methylation..Expression.of.both.proviruses.was. restricted. to. androgen-responsive. prostate. cancer. cell. lines. and.LTRs.sequences.containing.steroid.hormone.responsive.elements.con-ferred.androgen.responsiveness.to.reporter.constructs..Expression.of.LINE.1.5’.UTR.and.3’.UTR.sequences.in.prostate.cancers.rather.decrea-sed,.despite.significant.hypomethylation.of.the.internal.LINE.1.promo-ter..Increased.expression.of.the.young.AluYa5.and.AluYb8.families.was.restricted.to.individual.tumors.Our. findings. demonstrate. a. surprising. specificity. of. changes. in. ex-pression.and.DNA.methylation.of.retroelements. in.prostate.cancer.. In.particular,.LINE.1.hypomethylation.does.not.lead.to.generalized.overex-pression,.but.specific.HERV-K.proviruses.display.conspicuous.changes.in.their.expression.hinting.at.significant.functions.during.prostate.car-cinogenesis..

P-402

Koch, Annemarie; Knievel,.Judith;.Ribarska,.Teodora;.Niegisch,.Günter;.Schulz,.Wolfgang.(University.Medical.Center,.Düsseldorf)

Loss of Delta-like1 and Maternally Expressed Gene3 expression in urothelial carcinoma cells is associated with distinctive epigenetic changes at chromosome 14q32.2

Objective:.Delta-like.1.(DLK1).and.Maternally.Expressed.Gene.3.(MEG3).are.two.imprinted.genes.controlled.by.differentially.methylated.regions.(DMR).on.chromosome.14q32.2..This.chromosome.segment.is.frequently.altered.in.urothelial.cancer..Methods:. We. studied. DLK1. and. MEG3. expression. by. quantitative. PCR,.DNA.methylation.by.bisulfite.sequencing.and.histone.modifications.by.chromatin. immunoprecipitation. in.urothelial. cancer.cell. lines.and. tis-sues..Results:. DLK1. and. MEG3. expressions. were. significantly. diminished. in.cancerous.urothelial.tissues.and.undetectable. in.urothelial.carcinoma.cell. lines..These.changes.were.accompanied.by.distinctive.epigenetic.alterations. at. the. DLK1. promoter,. the. IG-DMR. (located. between. both.genes).and.the.MEG3-DMR..The.patchy.methylation.at.the.DLK1.promo-ter.region.in.normal.urothelial.tissues.changed.to.a.clustered.pattern.in.urothelial. cancer. cells. and. tissues.. The.normally.differentially.me-thylated. DMRs. assumed. novel. characteristic. “banded”. patterns.. This.latter.change.was.more.generalized. for. the.MEG3-DMR..Furthermore,.in. cancer. cell. lines. all. three. regions. presented. histone. modifications.indicative.of.repression.and.fixed.nucleosomes.Conclusion:.Distinctive.epigenetic.alterations.appear.to.cause.suppres-sion.of.DLK1.and.downregulation.of.MEG3.in.urothelial.cancer.

30

AEKAEK AEKAbstracts Poster Carcinogenesis, Genome stability, DNA Damage and Repair, Epigenetics

Notes:

P-403

Nindl, Ingo (Charité, Berlin); Muschik,.Dorothea. (German.Cancer.Re-search.Center,.Heidelberg);.Braspenning-Wesch,.Ilona.(German.Cancer.Research. Center,. Heidelberg);. Hofmann,. Thomas. (German. Cancer. Re-search.Center,.Heidelberg)

Stabilization of cutaneous human papillomavirus E6 mediated by UV-induced HIPK2

Ultraviolet. irradiation. is. the.major.risk. factor. for.the.development.of.skin.cancer..However,.increasing.evidence.supports.a.role.of.cutaneous.human. papillomaviruses. (HPV). from. the. beta. genus. as. a. co-factor. in.the.development.of.cutaneous.squamous.cell.carcinoma..Recent.studies.demonstrate.the.ability.of.human.beta-papillomavirus.(betaPV).E6.pro-teins.to.inhibit.UV-induced.apoptosis,.and.therefore.to.act.as.a.survival.factor.in.DNA-damaged.cells..The.tumour.suppressor.HIPK2.is.induced.by.UV-induced.DNA.damage..HIPK2.is.an.important.regulator.of.cellular.proliferation.and.apoptosis,.forms.a.complex.with.the.tumour.suppres-sor.p53,.mediates.phosphorylation.at.Ser.46,.stimulates.CBP-mediated.p53.acetylation.and.act.as.a.potential.suppressor.for.skin.tumourigene-sis..In.our.study,.we.demonstrate.that.cutaneous.beta2PV.E6.proteins.are.a.novel.target.of.HIPK2..E6.proteins. interact.and.co-localize.with.HIPK2.. Moreover,. UVB. induced. HIPK2. re-localizes. HPV. E6. proteins. in.the.nucleus.that.were.not.recruited.to.promyelocytic.leukaemia.nuclear.bodies.(PML.bodies)..HIPK2.expression.as.well.as.DNA.damage.induced.either.by.UVB.or.chemotherapeutics.causes.a.p53-independent.stabi-lization.of.HPV.E6,.whereas.the.kinase-deficient.mutant.of.HIPK2.has.no.effect..Thus,.the.stabilization.of.E6.may.result.in.an.increased.anti-apoptotic. effect. in. UV-induced. DNA. damaged. keratinocytes. infected.with.cutaneous.beta2PV.types.

P-404

Yang, Linlin; Chen,.Yuan;.Cui,.Tiantian;.Knoesel,.Thomas;.Petersen,.Iver.(University,.Jena)

Desmoplakin has potential tumor-suppressive activity in human lung cancer

Aims:. Desmoplakin. (DSP). is. one. of. desmosomal. components. involved.in.carcinogenesis..However,.the.role.of.DSP.in.human.lung.cancer.has.not.yet.been.well.understood..The.aims.of.this.study.were:.1).to.analyse.the. DSP. expression. in. lung. cancer;. 2). to. explore. the. mechanism. for.downregulation.of.DSP;.3).to.investigate.the.functional.role.of.DSP.in.lung.cancer.cells.Methods:.Real-time.RT-PCR.and.Western.blot.analysis.were.performed.to.analyse.the.expression.of.DSP.in.human.lung.cancer.cell. lines..The.protein. expression. of. DSP. in. primary. lung. tumors. was. evaluated. by.immunohistochemistry.on.tissue.microarry..To.investigate.methylation.status.of.DSP,.demethylation.test,.bisulfate.sequencing.(BS),.and.me-thylation-specific-PCR.(MSP).were.carried.out.in.lung.cancer.cell.lines.and. in.primary. lung.tumors..To.study.a. functional. role.of.DSP. in. lung.cancer. cells,. a. full-length. cDNA. of. DSP. was. stably. transfected. into. a.lung.cancer.cell.line.H157,.and.functional.assays.including.proliferation.assay,. soft. agar. test,. wound. healing. assay,. and. migration. as. well. as.invasion. assay. were. performed.. Moreover,. H157. parental. cells,. mock.transfectants.and.DSP.transfectants.were.treated.with.anticancer.drug.gemcitabine,.apoptotic.rate.was.analysed.by.Dye-uptake.bioassay,.and.the.cleavage.of.caspase-3.was.confirmed.by.Western.blot.analysis..Results:. DSP. was. downregulated. in. 8. out. of. 11. lung. cancer. cell. lines..More. than.50%.(34.out.of.56).of.primary. lung. tissues.exhibited. low.or. no. expression. of. DSP. protein.. Treatment. with. 5-aza-2-doxycyidine.restored.DSP.expression.in.all.of.8.lung.cancer.cell.lines.with.low.DSP.expression..Bisulphite.sequencing.and.MSP.from.the.six.lung.cancer.cell.lines.revealed.a.hypermethylation.in.the.region.of.intron.1..In.primary.lung. tumors,. hypermethylation. of. DSP. was. identified. in. 27. out. of. 56.samples.by.MSP..Lower.expression.of.DSP.protein.was.significantly.cor-related.to.DSP.DNA.hypermethylation..Stable.transfection.of.DSP.into.H157.lung.cancer.cell.line.led.to.an.increased.expression.of.DSP.protein..DSP-positive. transfectants. exhibited. remarkably. reduced. colony-for-ming.ability.in.soft.agar,.lower.proliferation.rates,.decreased.migration.and.invasion.ability,.and.increased.apoptotic.cell.number.as.well.as.acti-vated.caspase-3.expression.level.after.gemcitabine.treatment.Conclusions:.DSP.is.a.tumour.suppressor.inactivated.by.DNA.methyla-tion.in.human.lung.cancer..DSP.might. increase.the.sensitivity.of.anti-cancer.drug.through.induction.of.apoptosis..

AEK

31


Notes:

P-405

Ontikatze, Teona (University of Duisburg-Essen, Essen);. Handrick,.René.(University.of.Duisburg-Essen,.Essen);.Grimm,.Fabian.(University.of.Duisburg-Essen,.Essen);.Daniel,.Peter.(Charité,.Berlin);.Belka,.Claus.(University,. München);. Jendrossek,. Verena. (University. of. Duisburg-Essen,.Essen)

Dihydroartemisinin is a promising hypoxia-active anticancer drug

Background:. There. is. accumulated. evidence. that. hypoxia. is. a. major.factor.of. therapy.resistance. in.human.solid. tumors..However,. the.mi-croenvironment. of. solid. tumors. is. mostly. characterized. by. acute. or.chronic.hypoxia..Here,.we.propose.a.novel.strategy.to.overcome.the-rapy.resistance.of.hypoxic.tumor.cells.by.using.the.radical-forming.en-doperoxide.Dihydroartemisinin. (DHA)..We. recently.demonstrated. that.DHA.potently. induces.apoptosis. in.Jurkat.T-lymphoma.cells.acting.on.the.mitochondrial.apoptosis.pathway..Aim.of.our.present.study.was.to.evaluate.the.antineoplastic.efficacy.of.DHA.in.human.colon.cancer.cells.under.normoxic.and.hypoxic.conditions.Methods:.The.cytotoxic.efficacy.of.DHA.was.evaluated.in.colon.cancer.cells. in.vitro.(HCT115,.HCT116.and.Colo205)..HCT116.clones.with.or.wi-thout.expression.of.Bax,.Bak,.or.both.were.used.in.order.to.investigate.the. importance. of. apoptosis. in. the. antineoplastic. efficacy. of. DHA. in.solid.tumors..Cell.death.after.treatment.with.DHA.(0-25µM).in.normoxia.(21%.O2).or.hypoxia.(0.2%.O2).was.analyzed.by.fluorescence.microsco-py.(cytochrome.c.release.and.Hoechst/propidium.iodide.staining),.flow.cytometry. (Δψm,. DNA. fragmentation),. and. immunoblotting. (caspase.cleavage.and.PARP.cleavage)..Clonogenic.survival.was.tested.by.colony.formation.assays..Results:.Under.normoxic.conditions,.DHA.induced.concentration-depen-dent.apoptosis.in.colon.cancer.cells..The.cells.were.also.highly.sensitive.to.DHA-induced.apoptosis.under.conditions.of.acute.hypoxia.(0.2%.O2)..DHA-induced.apoptosis.was.significantly.reduced.by.loss.of.either.Bax,.Bak,.or.both.in.normoxia.and.hypoxia..Although.loss.of.Bax,.Bak.or.Bax/Bak.impaired.DHA-induced.eradication.of.clonogenic.tumor.cells.in.nor-moxia,.clonogenic.cell.death.was.almost.not.affected.by.loss.of.those.proapoptotic.effector.proteins.under.hypoxic.conditions..Conclusions:.DHA.induces.apoptosis.in.HCT116.colon.carcinoma.cells.via.a.mitochondrial.apoptosis.pathway.in.both.normoxic.and.hypoxic.condi-tions.involving.the.mitochondrial.death.pathway..The.observation.that.clonogenic.death.is.less.affected.by.loss.of.Bax.and/or.Bak.under.hypo-xic.conditions.suggests.that.the.drug.may.induce.alternative.cell.death.modes.at.low.pO2...Our.findings.suggest.that.DHA.may.be.of.particular.value.for.the.treatment.of.human.tumors.characterized.by.high.levels.of.tissue.hypoxia.and.apoptosis.resistance.

P-406

Henning, Stefan; Chen,. I-Peng;. Faust,. Alexandra;. Greinert,. Rüdiger;.Volkmer,.Beate.(Elbekliniken,.Buxtehude)

UVA induced long-lasting repression of p16INK4a in human keratino-cytes is attributable to epigenetic alterations

The.tumor.suppressor.p16INK4a.prevents.the.hyperphosphorylation.of.retinoblastoma.(RB).protein.by.the.cyclin.D-CDK4.(or.-CDK6).complex.via.competing.binding. to. the.complex.and. thus.elicits.G1-arrest.upon.DNA.damage.aiming.to.safe-guard.the.integrity.of.the.genome..Dysre-gulation.of.p16INK4a.has.often.been.found.(among.others).in.skin.can-cers.such.as.malignant.melanoma.or.squamous.cell.carcinoma..Etiology.studies.have.provided.strong.evidences.linking.skin.cancers.to.exces-sive.UV.radiation..To.access.effects.of.UVA,.which.constitutes.the.major.UV.fraction.of.the.terrestrial.sunlight,.on.p16INK4a.gene.expression.in.human. keratinocytes,. we. have. treated. primary. human. keratinocytes,.HaCaT.and.KH8.2.00.keratinocytic.cell.lines.with.different.UVA-radiation.regimes.(acute,.chronic.or.heavy.dose)..Whereas.the.acute.UVA-treat-ment.caused.only.a.minor.alteration.at.the.p16INK4a.transcription.level.in. primary. keratinocytes,. chronically. UVA-treated. HaCaT. cells. which.were. able. to. induce. tumors. in. nude. mice,. showed. a. long-lasting. and.drastic.depletion.of.p16INK4a.transcripts..A.reduction.of.p16INK4a.has.also.been.seen.in.KH8.2.00.cells.repeatedly.treated.with.a.heavy.dose.of.UVA..Consistently,.the.p16INK4a.reduction.is.accompanied.by.hyper-methylation.and.altered.histone.methylation.patterns.at.the.p16INK4a.promoter.. Our. results. highlight. the. significance. of. the. epigenetic. re-sponses. upon. UVA-irradiation.. The. consequence. of. the. compromised.expression.of.p16INK4a.via.epigenetic.changes.in.UVA-treated.cells.with.respect.to.skin.carcinogenesis.will.be.discussed.

32


Notes:

P-407

Roos, Wynand (University Medical Center, Mainz);.Eich,.Marcus.(Uni-versity.Medical.Center,.Mainz);.Tsaalbi-Shtylik,.Anastasia.(Leiden);.Dig-weed,. Martin. (Charité,. Berlin);. de. Wind,. Niels. (Leiden);. Kaina,. Bernd.(University.Medical.Center,.Mainz)

Rev3L and NBN: Two independent factors that protect against alky-lating anticancer drugs

The. alkylating. agents. temozolomide. and. fotemustine,. representing.methylating.and.chloroethylating.agents.respectively,.are.used.in.the.therapy.of.glioma.and.malignant.melanoma..Since.chemoresistance.of.these.tumors.is.a.common.phenomenon,.identification.of.the.underlying.mechanisms.is.needed..The.mechanism.of.cellular.defense.against.the-se.agents.primarily.involves.O6-methylguanine-DNA.methyltransferase.(MGMT),.base.excision. repair. (BER).and.DNA.mismatch. repair. (MMR)..Here.we.show.that.Rev3L,.the.catalytic.subunit.of.the.translesion.DNA.polymerase ζ,.and.nibrin.(NBN.alias.NBS-1),.a.critical.component.of.the.DNA.double-strand.break.(DSB).recognition.MRN.complex,.mediates.re-sistance.to.alkylating.agents..Both.Rev3L.knockout.and.NBN.mutated.cells. are. hypersensitive. to. alkylating. agents.. Remarkably,. cells. hete-rozygous. for. Rev3L. showed. an. intermediate. sensitivity.. Rev3L. is. not.involved. in. the. tolerance. of. the. toxic. O6-methylguanine. lesion. while.NBN. is..A. role.of.Rev3L. in. the. tolerance.of.O6-chloroethylguanine.or.the.subsequently.formed.N1-guanine-N3-cytosine.interstrand.crosslink.is. shown.. Neither. Rev3L. nor. NBN. had. an. influence. on. BER. of. the. N-alkylation.lesions,.although.Rev3L.is.likely.involved.in.the.tolerance.of.N-alkylations.or.apurinic/apyrimidinic.sites.originating.from.them..We.also. show. that. Rev3L. exerts. its. protective. effect. in. replicating. cells.and.that.loss.of.Rev3L.leads.to.a.significant.increase.in.DNA.DSBs.fol-lowing. alkylating. agent. treatment.. Hypersensitivity. of. NBN. cells. was.due.to.the. induction.of.both.apoptosis.and.necrosis..Down-regulation.of.NBN.in.melanoma.cells.by.siRNA.rendered.them.more.sensitive.to.al-kylating.agents..The.data.show.that.both.Rev3L.and.NBN.contributes.to.alkylating.agent.resistance.independently.from.each.other,.thus.acting.in.concert.with.MGMT,.BER.and.MMR.in.the.defense.against.alkylating.anticancer.drugs.

P-408

Michele, Inga Dorina (University Hospital Essen, Essen); Nickel,.Ann-Christin.(University.of.Duisburg-Essen,.Essen);.Hananberg,.Helmut.(Uni-versity.Hospital,.Düsseldorf);.Thomale,.Jürgen.(University.of.Duisburg-Essen,.Mainz)

Molecular und functional characterisation of a novel DNA repair me-chanism for O6-methylguanine employing components of the nucle-otide excision and the Fanconi anemia pathways

O6-methylguanine.(O6-meG).adducts.in.DNA.are.induced.by.endogenous.and.exogenous.methylating.compounds.including.a.number.of.alkylating.anti-cancer.drugs.. If.not.repaired,.this. lesion. is.highly.mutagenic.and.cytotoxic..Biochemical.analyses. in.our.group.suggested.that.mamma-lian.cells.have.an.alternative.possibility. to.remove.this.critical. lesion.from.their.genome.by.a.dual.incision.mechanism.in.addition.to.the.well-studied.suicidal.repair.protein.O6-meG-DNA.methyltransferase.(MGMT)..The. alternative. mechanism. resembles. the. nucleotide. excision. repair.(NER).and.removes.lesions.by.dual.incisions,.but.in.contrast.to.NER.its.activity.is.restricted.to.the.pre-replicative.phase..Here,.we.have.further.characterised.the.novel.pathway.at.the.cellular.level.and.have.identified.essential. components.using.adduct-specific.antibodies.and. functional.repair.assays.Measurements. of. O6-meG. adduct. kinetics. in. mouse. and. human. cells.exposed. to. the.methylating.compound.N-methyl-N-nitrosourea. (MNU).demonstrated.an.efficient.removal.of.the.lesion.also.in.the.absence.of.functional. MGMT.. As. biochemical. studies. had. indicated. that. the. NER.component.XPC.binds.specifically.to.O6-meG.in.DNA.and.is.indispensa-ble.for.the.excision.step,.we.measured.adduct.kinetics.in.XPC-deficient,.MGMT-depleted.human.fibroblasts.and.found.a.complete.inability.to.re-pair.O6-meG,.which.was.reversed.by.the.reconstitution.with.a.wild.type.XPC.gene..In.addition.to.NER.constituents,.the.Fanconi.anemia.protein.D2. (FancD2). was. identified. to. be. essential. for. the. excision. repair. of.O6-meG,.whereas.other.Fanconi.anemia.proteins.did.not.seem.to.be.in-volved.. These. observations. could. be. further. substantiated. by. adduct.kinetics.measured.in.different.primary.cell.types.of.MNU-treated.XPC-.or.FancD2-deficient.mice..To.investigate.the.substrate.specificity.of.the.alternative.excision.repair.pathway.we.analysed.its.function.in.the.re-moval.of.Pt-(GpG).intrastrand.cross.links,.the.major.type.of.DNA.adduct.induced.by.the.anti-cancer.drug.cisplatin..Human.cell. lines.as.well.as.primary.mouse.hematopoietic.stem.cells. lacking.functional.FancD2.or.XPC.proteins.are.deficient.for.the.excision.repair.of.Pt-(GpG).and.are.hypersensitive.to.the.cytotoxic.potential.of.cisplatin.Further.analyses.with.corresponding.pairs.of.repair-proficient.and.-defi-cient.mouse.strains.will.uncover.the.role.of.this.novel.repair.mechanism.in.preventing.the.mutagenic,.carcinogenic.and.cytotoxic.effects.of.me-thylating.compounds.in.different.cell.types.in.vivo.

AEK

33


Notes:

P-409

Quiros, Steve; Roos,.Wynand;.Kaina,.Bernd.(University.Medical.Center,.Mainz)

Genotoxic anticancer drugs trigger cell death several cell cycles af-ter DNA damage induction

Methylating.agents.such.as. temozolomide,.procarbazine,.dacarbazine.and.streptozotocine.are.potent.genotoxic.drugs..They.induce.a.dozen.DNA.adducts.from.which.O6-methylguanine.(O6MeG).is.a.highly.muta-genic,. genotoxic. and. apoptotic. lesion.. O6MeG. exerts. its. toxicity. in. a.mechanism.dependent.on.the.DNA.mismatch.repair.and.DNA.replication..Nevertheless,.the.precise.mechanism.leading.to.cell.death.has.not.yet.been.fully.elucidated..Making.use.of.a.highly.synchronized.mammalian.cell.system.proficient.and.deficient.for.the.DNA.repair.enzyme.O6-me-thylguanine-DNA.methyltransferase.(MGMT),.we.show.that.DNA.double-strand.breaks.(DSBs).were.formed.at.high.level.in.the.2nd.S/G2-phase.of.the.cell.cycle.following.O6MeG.formation..They.are.accompanied.by.ATR.and.Chk1.phosphorylation..The.appearance.of.cell.death.temporally.coincided.with.the.beginning.of.the.G2/M.block.in.the.second.cell.cycle.following. treatment. and. continued. to. increase. with. time.. Although. a.fraction.of.cells.underwent.apoptosis.out.of.the.2nd.cell.cycle,.the.ma-jority.of.cells.died.after.subsequent.replication.cycles,.very.late.after.DNA.damage.induction..The.data.provide.evidence.that.two.rounds.of.DNA.replication.are.required.for.converting.O6MeG.adducts.into.potent.killing.lesions..Nevertheless,.the.majority.of.cells.recovered.and.under-went.apoptosis.after.passing.through.additional.replication.cycles..We.propose.a.model.where.O6MeG.can.be.passed.on.to.subsequent.gene-rations.and. that.DNA.breakage/genomic. instability.provoked.by. futile.MMR.replication.cycles.is.responsible.for.cell.death.executed.even.se-veral.cell.cycles.after.DNA.damage.induction..The.data.have.important.implications. for. both. the. delayed. onset. of. the. therapeutic. response.after.O6MeG.based.chemotherapy.and.the.need.for.long.lasting.MGMT.inhibition.for.proper.effectiveness.of.MGMT.inactivating.drugs.

P-410

Krunic, Damir (German Cancer Research Center, Heidelberg); Bernig,.Manuel.(German.Cancer.Research.Center,.Heidelberg);.Odunsi,.Yetunde.(German. Cancer. Research. Center,. Heidelberg);. Kuhn,. Anne. (German.Cancer.Research.Center,.Heidelberg);.von.Zglinicki,.Thomas.(Newcastle.University,.Newcastle);.Boukamp,.Petra.(German.Cancer.Research.Cen-ter,.Heidelberg)

UV-dependent telomere erosion an early event in skin cancer deve-lopment?

Telomeres.are.DNA-protein.structures.at.chromosomal.ends. that.pre-vent.DNA.degradation.and.hinder.the.recognition.of.these.ends.as.bro-ken.DNA;.maintaining.chromosomal.integrity..We.used.3D.telomere.Q-FISH/immunofluorescence.to.investigate.telomere.length.in.human.skin.and. found. significant. inter-. and. intra-personal. variations. in. telomere.lengths.within.the.epidermis.bur.we.did.not.see.an.overall.significant.age-dependent.telomere.loss..Instead,.we.identified.regions.in.normal.skin.with.significantly.shorter.telomeres.which.were.characterised.by.p53.and.53BP1.foci.co-localised.with.telomeres..Accordingly,.we.obser-ved.shorter.telomeres.in.skin.from.sun-exposed.versus.sun-protected.sites..Furthermore,.we.showed.that.acute.UV. irradiation.of.volunteer.skin.caused.distinct.telomere.shortening.within.3.days.after.irradiati-on..Similarly.UV-radiation.of.2D.cultured.keratinocytes.and.fibroblasts.show.distinct.telomere.loss.already.after.24.h..Also.in.organotypic.cul-tures.telomere.loss.can.be.induced.upon.UV.radiation.thus.allowing.now.for.mechanistical. studies..Aiming.at.understanding. the.mechanism.of.UV-dependent.telomere.shortening.we.can.show.that.reactive.oxygen.species. (ROS). and. the. poly-ADP-ribosylpolymerase. 1. (PARP1). directly.contribute.to.this.process..Thus,.not.age-dependent.telomere.loss.but.UV.radiation.and.consecutive.oxidative.DNA.damage.are.responsible.for.accelerated.telomere.loss.in.human.skin.and.thereby.likely.contribute.to.skin.cancer.development.

34


Notes:

P-411

Mallem, Nedjouna (Max-Delbrück Centrum, Berlin); Krause,. Claudia.(Max-Delbrück.Centrum,.Berlin);.Thirunarayanan,.Nanthakumar.(Nation-al. Institutes.of.Health,.Bethesda);.Fichtner,. Iduna.(Max-Delbrück.Cen-trum,.Berlin);.Müller,.Gerd.(Max-Delbrück.Centrum,.Berlin);.Lipp,.Martin.(Max-Delbrück.Centrum,.Berlin)

The role of tumor microenvironment and epigenetic changes in im-mune escape and tumor progression triggered by HHV8-encoded chemokine receptor vGPCR

The.human.herpes.virus.8.(HHV-8)-encoded.G.protein-coupled.chemoki-ne.receptor.(vGPCR).has.been.implicated.in.the.pathogenesis.of.Kaposi´s.sarcoma.(KS).particularly.because.of.its.high.constitutive.signaling.ac-tivity..We.have.previously.described.that.vGPCR-expressing.BALB/c-3T3.fibroblasts.are.tumorigenic. in.nude.mice,.but. fail. to. induce.tumors. in.their. immunocompetent. counterparts.. However,. tumor. fragments. ob-tained.from.nude.mice.grow.progressively.in.immunocompetent.BALB/c.mice.. Unexpectedly,. vGPCR-expressing. cells. established. from. grafted.tumor.fragments.gave.rise.to.tumors.in.immunocompetent.mice..These.tumors.exhibit.a.striking.histological.resemblance.to.KS.including.plump.spindle.cell.morphology.and.a.high.degree.of.vascularization..Short.in-terfering.RNA.directed.at.vGPCR.abrogated.or.significantly.delayed.tu-morigenesis. of. tumor-derived. cells. in. nude. mice,. demonstrating. that.the.tumor.development.is.specifically.driven.by.the.vGPCR.oncogene,.but.not.by.other.successive.oncogenic.mutations..Interestingly,.vGPCR-expressing.cells.established.from.the.tumor.in.nude.mice.fail.to.indu-ce.tumors.in.immunocompetent.mice.suggesting.that.critical.changes,.which.make.the.vGPCR-expressing.cells.more.tumorigenic,.occur.duri-ng. the.passage. in.a. immunocompetent.host.. Tumor-derived.cell. lines.treated.with.the.DNA.methylation.inhibitor.5-aza-2´-Deoxycytidine.and.the.histone.deacetylase.inhibitor.Trichostatin.A.revert.to.a.phenotype.similar.to.the.vGPCR.expressing.3T3.cells.and.show.a.significant.delay.in.tumor.formation..Consequently,.we.hypothesize.that.mainly.epigenetic.changes.had.occurred.during. the.passage.of.vGPCR-expressing.fibro-blasts.in.immunocompetent.mice..To. identify. genes. that. are. critically. involved. in. immune. escape. and.tumor.progression,.we.have.compared.the.gene.expression.profiles.of.primary.tumor.tissue.of.immunocompetent.mice.and.tumor.derived.cell.lines.with.data.obtained.from.the.parental.cell.lines..We.are.currently.extending.this.analysis.by.large-scale.DNA.methylation.profiling.using.Reduced-Representation.Bisulfite.Sequencing.(RRBS),.a.high.through-put.sequencing.technology.that.allows.us.to.analyze.and.compare.geno-mic.methylation.patterns.between.our.samples..Hence,.we.will.be.able.to.identify.epigenetic.changes.underlying.tumor.growth.and.progressi-on.as.well.as.potential.target.genes,.which.are.suitable.for.therapeutic.strategies.

P-412

Luque, Raquel Mejias (TU München, Institut für Medizinische Mikro-biologie, Immunologie und Hygiene); de.Bolós,.Carme.(IMIM-Hospital.del.Mar,.Barcelona);.Gerhard,.Markus.(TU.München,.Institut.für.Medizi-nische.Mikrobiologie.Immunologie.und.Hygiene)

Regulation of SOX2 by STAT3 in gastric cancer cells

Gastric.cancer.remains. the. fourth.most.common.cancer.and.the.sec-ond.leading.cause.of.cancer-related.deaths.worldwide..Although.envi-ronmental.factors.as.Helicobacter.pylori.infection.have.been.linked.to.gastric.cancer,.the.molecular.mechanisms.underlying.gastric.carcino-genesis.are.poorly.understood..SOX.transcription.factors.are.essential.for.embryonic.development.and.play.critical.roles.in.cell.fate.determination,.differentiation.and.prolif-eration..Sox2.is.expressed.in.the.normal.stomach.mucosa,.whereas.its.expression. pattern. in. gastric. cancers. differs. between. intestinal-type.tumours,. in. which. it. is. downregulated,. and. diffuse. tumours,. in. which.Sox2.is.overexpressed..STAT.transcription.factors.mediate.cytokine-.and.growth.factor-directed.transcription..In.many.human.cancers,.including.gastric.tumours,.STAT3.is.persistently.activated,.and.its.hyper.activation.in.mice.harbouring.a.knock-in.mutation.in.the.gp130.receptor.induces.spontaneous.develop-ment.of.gastric.tumours..Sox2.has.been.recently.described.to.be.regu-lated.by.STAT3.during.neural.differentiation;.however.no.data.in.gastric.cells.has.been.reported.to.date.The.mRNA.levels.of.Sox2,.the.protein.expression.levels.of.Sox2.and.ac-tivated.STAT3.(p-STAT3).and.the.basal.Sox2.transcriptional.activity.were.analysed.in.a.panel.of.gastric.cancer.cell.lines..In.most.of.them,.Sox2.expression.was.low.or.absent..Only.AZ521.cells.presented.high.levels.of.Sox2.expression.and.transcriptional.activity..The.effect.of. the.pro-inflammatory.cytokine. IL-6,.which.activates. the.gp130/STAT3. signalling. pathway,. on. Sox2. expression. was. assessed. in.different.gastric. cancer.cells.after.24.hour. treatment.. Increased. lev-els.of.Sox2.mRNA.were.detected.and.the.novo.protein.expression.was.observed..To. determine. if. STAT3. can. modulate. the. transcriptional. activity. of.Sox2,. cells. were. transfected. with. three. different. STAT3. constructs:.STAT3. wild. type,. constitutively. active. STAT3. and. the. βSTAT3. mutant,.which. possesses. low. transcriptional. activity.. An. increase. in. the.transcriptional.activity.of.Sox2.was.only.observed.in.cells.transfected.with.constitutively.active.STAT3.Also,. the. expression. of. Sox2. was. analysed. in. tumours. from. gp130.Y757F/Y757F. .mice.. Increased. levels.of.Sox2.were.detected.that.cor-related.with.Ki67,.a.marker.of.cellular.proliferation,.suggesting.an.im-portant.role.for.Sox2.in.tumour.proliferation.Our.results.indicate.that.STAT3.regulates.Sox2.expression.and.transcriptional.activity.in.gastric.cancer.cells.

AEK

35

AEKAbstracts Short Talks Oncogene Addicton

short talk 501

Sers, Christine (Charité, Berlin); Bremo,. Gustavo. (Charité,. Berlin);.Saint-Paul,.Janne.(Charité,.Berlin);.Weichert,.Wilko.(University,.Heidel-berg);.Shila.Mang-Fatehi,.(Charité,.Berlin)

Regulation of EZH2 via MAPK signalling, c-MYC and miRNA 101 in co-lorectal cancer cell lines

The.RAS/MEK/ERK.pathway.is.a.key.oncogenic.pathway.involved.in.hu-man.cancer..Point.mutations.of.KRAS.resulting.in.constitutive.activation.of.the.MAPK.pathway.occur.in.about.40%.of.colorectal.cancers..Recen-tly,.we.unraveled.a.link.between.oncogenic.MAPK.signaling.and.specific.DNA.methylation.impinging.on.genes.responsible.for.immune.function..Due.to.the.intimate.connection.between.DNA.and.histone.modification.in. epigenetic. gene. control,. we. investigated. a. potential. role. for. RAS/MEK/ERK.signal.transduction.on.histone.modification.control.and.iden-tified.an.impact.of.the.signaling.pathway.onto.H3K27me3..Furthermore,.EZH2.(Enhancer.of.Zeste.Homolog.2),.which.serves.as.the.catalytic.sub-unit.of.PRC2.and.has.been.reported.as.the.sole.histone.methyltrans-ferase.methylating.H3K27,.is.regulated.in.a.MAPK-dependent.manner.in.KRAS-mutated.colorectal.cancer.cells..Further.studies.showed.that.the.MAPK.signalling.pathway.controls.both.the.EZH2.promoter.and.protein.synthesis.resulting.in.increased.EZH2.expression..Similar.observations.on.EZH2.were.also.made.in.human.colorectal.cancer.samples,.indicating.a.similar.in.vivo.regulation..To.uncover.the.mechanism.of.this.dysregu-lation,.we.analysed.the. impact.of.the.oncogenic.transcription.factors.c-Myc/N-myc.and.of.mirRNAs.on.EZH2..We.identified.c-Myc/N-myc.and.miRNA101,.but.not.miRNA26a,.as.negative.regulators.of.EZH2.expres-sion. in. colorectal. cancer.. Furthermore,. a. functional. impact. of. MAPK.signalling.on.EZH2.target.genes.such.as.KLF4.and.RYBP. in.colorectal.cancer.cells.was.defined..This.data.suggest.that.oncogenic.mutations.such.as.KRAS.and.BRAF.in.colorectal.cancer,.which.induce.a.dysregulati-on.of.MAPK.signalling,.stimulate.consecutive.dysregulation.of.c-Myc.and.miRNA101,.EZH2.and.finally.EZH2.target.genes.resulting.in.increased.cell.proliferation.

short talk 502

Kohlhof, Hella; Schrepfer,.Sabine;.Kronthaler,.Kerstin;.Krüger,.Jacque-line;.Asche,.Julia;.Leban,.Johann.(4SC.AG,.Martinsried)

Eliminating Cancer Stem Cells by Inhibition of the Wnt Pathway via Small Molecule Kinase Inhibitors

One.hallmark.of.cancer.stem.cells.(CSC).is.their.self.renewal.capacity.driven. by. developmental. pathways. like. Wnt,. Hedgehog. and. Notch.. To.eliminate.CSCs,.we.target.the.Wnt.and.the.Hh.pathway..A.multitude.of.protein.kinases.is.involved.in.positive.and.negative.regulation.of.these.pathways.. We. discovered. several. novel. chemotypes. of. kinase. . inhibi-tors.which.inhibited.the.activity.of.Wnt.signaling.in.a.HEK293.reporter.assay.in.the.micromolar.range..Some.of.these.compounds.have.remar-kable.selectivity.towards.certain.kinases. in.the. low.nanomolar.range..Therefore,.we.think.that.we.can.identify.specific.kinases.for.Wnt.or.Hh.inhibition..Selected.compounds.inhibit.colony.formation.of.HCT116.cells.at.submicromolar.concentrations..Additionally,.the.compounds.have.a.negative.impact.on.spheroid.formation.of.the.embryonic.carcinoma.cell.line.NCCIT..Further. in.vitro.and. in.vivo.experiments. to.determine. the.effects.on.cancer.stem.cells.are.in.progress..We.will.continue.to.validate.kinase.inhibitors.of.the.Wnt.and.Hh.pathway.with.the.ultimate.goal.to.halt.tumor.initiating.and.metastasis.forming.cells.

Notes:

36

AEKAEK AEKAbstracts Posters Oncogene Addicton

P-501

Nitsche, Ulrich (Technical University, Munich); Balmert,. Alexander.(Technical. University,. Munich);. Schuster,. Tibor. (Technical. University,.Munich);.Abal,.Miguel. (Universitario,.Santiago.de.Compostela);. Friess,.Helmut. (Technical. University,. Munich);. Rosenberg,. Robert. (Technical.University,.Munich);.Janssen,.Klaus-Peter.(Technical.University,.Munich)

Individualized recurrence risk assessment for stage II colorectal cancer patients by molecular methods

In.40%.of.patients.with.colorectal.cancer,.the.tumor.is.locally.invasive.but.has.not.spread.at.the.time.of.diagnosis.(UICC.stage.II)..After.resec-tion.of.the.primary.tumor,.these.patients.are.considered.to.be.cured..However,.tumor.recurrence.occurs.in.20-30%.of.the.cases..To.date,.it.is. not. possible. to. reliably. identify. these. high. risk. patients,. who. may.benefit.from.adjuvant.therapy..Thus,.we.investigated.the.feasibility.of.individualized.risk.assessment.by.robust.screening.methods,.based.on.established.as.well.as.novel.molecular.markers..We.have.focused.on.two.crucial.signaling.pathways. involved. in.colorectal.carcinogenesis.(Wnt,.and.KRAS/BRAF),.on.microsatellite.instability,.and.on.the.putative.me-tastatic.and.invasion.marker.genes.ETV5.(Ets.variant.gene.5).and.SASH1.(SAM.and.SH3.domain.containing.1)..Our.own.previous.data.demonstrate.a.prognostic.influence.of.elevated.expression.of.osteopontin.(OPN),.as.a.surrogate.marker.for.Wnt.signaling.activity..Moreover,.reduced.SASH1.expression.and.elevated.ETV5.expression.are.correlated.with.invasion.and.formation.of.metachronous.metastasis.Tumor.samples.of. stage. II. colorectal.cancers.were.analyzed. (n=239)..All. patients. underwent. complete. resection. (R0),. and. median. follow.up.was.85.months..21. patients. (8.8%).developed.disease. recurrence.(metachronous.metastasis),.and.20.(8.4%).died.due.to.tumor-related.causes..We.assessed.the.mutational.status.of.the.genes.KRAS.(exon.2),.and.BRAF.(exon.15).by.high.resolution.melting,.the.microsatellite.insta-bility.status.by.multiplex.PCR,.and.the.expression.levels.of.OPN,.ETV5.and.SASH1.by.qRT-PCR..Mutations.in.the.KRAS.gene.were.detected.in.30%.of.patients.(71/237),.in.the.BRAF.gene.in.16%.(37/237),.and.microsatellite.instability.(MSI-H).in.27%.(62/227)..Overexpression.of.OPN.and.ETV5.above.previously.es-tablished.threshold.levels.was.found.in.34%.(64/189).and.7.1%.(17/185),.respectively..SASH1.expression.was.significantly.reduced.in.tumor.tis-sue.compared.to.normal.mucosa.levels.(p<0.001)..Patients.with.tumors.bearing.a.KRAS.mutation,.as.well.as.microsatellite.stable.tumors,.had.a.significantly.higher.incidence.of.metachronous.metastasis.(p=0.009.and.p=0.018,.respectively)..However,.our.approach.including.six.complementary.DNA.and.RNA.ba-sed.parameters.so.far.did.not.provide.satisfying.stratification.potency.for.this.inhomogeneous.patient.group..Thus,.it.remains.challenging.to.establish.robust.tools.for.risk.stratification.of.stage.II.colorectal.cancer.patients.

P-502

Bug, Monika; Dobbelstein,. Matthias. (University. Medicine. Göttingen,.Göttingen)

Anthracyclines induce the accumulation of mutant p53 through E2F1-dependent and –independent mechanisms

Mutant.p53.frequently.accumulates.in.cancer.cells.and.promotes.tumor.cell.invasion,.as.part.of.its.gain.of.function..Its.accumulation.is.partially.due.to.enhanced.stability,.but.little.is.known.about.how.the.mRNA.levels.of.mutant.p53.can.be.regulated..Likewise,.the.impact.of.cancer.therapy.on.the. levels.of.mutant.p53. is.poorly.understood..We.show.here.that.the. anthracyclines. doxorubicin,. daunorubicin,. and. epirubicin. further.increase.the.amounts.of.mutant.p53.mRNA.and.protein.in.cancer.cells..Moreover,.we.show.for.the.first.time.that.the.transcription.factor.E2F1.associates.with.the.promoter.DNA.of.TP53..Upon.genotoxic.treatment,.E2F1. contributed. to. the. expression. of. mutant. p53,. both. directly. and.through. induction. of. TAp73.. In. contrast,. the. anthracycline. idarubicin.and.also.another.topoisomerase.inhibitor,.etoposide,.failed.to.increase.the.levels.of.p53.mRNA,.despite.their.ability.to.induce.the.synthesis.of.TAp73.mRNA..Instead,.a.natural.antisense.transcript.of.TP53,.WRAP53,.was.strongly.augmented.by.idarubicin.and.etoposide,.but.only.less.so.by.the.other.anthracyclines.under.study..RNA.corresponding.to.the.first.exon.of.WRAP53.was.mainly.found.in.cell.nuclei.and.reduced.the.levels.of. mutant. p53.. Taken. together,. this. suggests. a. reciprocal. activation.pattern.of.TP53.and.WRAP53.by.different.chemotherapeutics..Reducing.the.levels.of.mutant.p53.by.siRNA.increased.chemosensitivity,.and.ida-rubicin.prevented.cell. survival.more.efficiently. than. the.mutant.p53-inducing.doxorubicin..We.conclude.that.even.closely.related.anthracy-clines.induce.the.synthesis.of.different,.opposing.transcripts.from.the.TP53.locus..When.using.these.drugs.for.cancer.therapy,.the.increased.levels.of.mutant.p53.may.augment.its.gain.of.function.and.thus.favor.unwanted.chemoresistance.and.tumor.progression.

Notes:

AEK

37

AEKAbstracts Poster Oncogene Addicton

Notes:

P-503

Cui, Tiantian; Chen,.Yuan;.Yang,.Linlin;.Knoesel,.Thomas;.Petersen,.Iver.(University,.Jena)

Identification of a tumor suppressor gene desmocollin 3 in human lung cancer

Aims:. Desmocollin. 3(DSC3). is. a. member. of. cadherin. family. involved.in.carcinogenesis..However. its.role. in.human. lung.cancer.has.not.yet.been.well.elucidated..The.aims.of.this.study.were.to.analyse.the.DSC3.expression;.to.explore.the.mechanism.for.downregulation.of.DSC3;.to.investigate.the.regulation.of.DSC3.expression.and.to.analyze.the.func-tional. role. of. DSC3. in. lung. cancer.Methods:. Expression. of. DSC3. was.analyzed.by.RT-PCR.and.Western.blotting.in.lung.cancer.cell. lines.and.normal. bronchial. epithelial. cells. (HBEC).. In. primary. lung. tissues,. the.protein.expression.of.DSC3.was.evaluated.by.IHC.on.tissue.microarray..Methylation. status.of.DSC3.was.examined.by.demethylation. test,.BS,.and.MSP..To.investigate.the.effect.of.the.tumor.suppressor.gene.p53.on.DSC3.(a.putative.target.gene.of.p53),.transfection.with.a.wild-type.p53.expression.vector.was.performed.in.lung.cancer.cell.lines..For.the.func-tional.analysis,.an.expression.vector.containing.the.full-length.cDNA.of.DSC3.was.constructed.and.stably.transfected.into.lung.cancer.cell.lines..Results:.In.a.majority.of.lung.cancer.cell.lines,.mRNA.expression.of.DSC3.was.downregulated..In.100.primary.lung.tumors,.83%.samples.exhibi-ted.no.expression.of.DSC3..Higher.expression.of.DSC3.was.significantly.correlated.to.squamous.cell.lung.cancer.(SCC).(P=0.0001)..Expression.of.DSC3.was.restored.in.4.out.9.of.lung.cancer.cell.lines.by.5’-aza-2’-DC..BS.and.MSP.showed.DNA.methylation.of.DSC3.in.the.regions.of.promo-ter.and.exon.1..In.primary.lung.tumors,.methylation.of.DSC3.was.found.in.44.6%.samples.by.MSP,.which.was.associated.with.poor.prognosis..Transfection.with.the.p53-expression.vector.resulted.in.an.increased.ex-pression.of.DSC3.in.H2170.(DSC3.unmethylated).but.not.in.H1299.(DSC3.methylated).. Combination. of. transfection. with. 5-aza-2-DC. treatment.led.to.increased.expression.of.DSC3.in.H1299..After.stable.transfection,.overexpression.of.DSC3.protein.was.detected.in.transfectants..Prolife-ration.assay.showed.that.DSC3.positive.transfectants. inhibited.tumor.growth.in.comparison.to.parental.cells.and.mock.transfectants.(empty.vector)..Wound.healing.assay.revealed.that.DSC3.positive.transfectants.exhibited.reduced.mobility.compared.to.control.cells..Conclusions:.Gene.silencing.of.DSC3.could.be.explained.by.DNA.hypermethylation..DSC3.could.be.a.marker.for.patients.with.SCC,.and.methylation.status.of.DSC3.predicts. poor. survival. in. patients. with. lung. cancer.. DSC3. might. be. a.potential.tumor.suppressor.gene.in.human.lung.cancer.

P-504

Stelniec, Iwona (Charité, Berlin); Legewie,. Stefan. (German. Cancer.Research.Center,.Heidelberg);.Tchernitsa,.Oleg. (Charité,.Berlin);.Sers,.Christine.(Charité,.Berlin);.Herzel,.Hanspeter.(Humboldt.University,.Ber-lin);.Blüthgen,.Nils.(Charité,.Berlin);.Schäfer,.Reinhold.(Charité,.Berlin)

Hierarchical regulation in a KRAS pathway-dependent transcriptional network revealed by a reverse-engineering approach

RAS.mutations.are.highly.relevant.for.progression.and.therapy.response.of.human.tumors,.but.the.genetic.network.that.ultimately.executes.the.oncogenic.effects.is.poorly.understood..Tumor.models,.in.which.the.ma-lignant. transformation.were.experimentally. triggered.and.maintained.through.defined.onkogenes,.give.manifold.possibilities.to.determine.the.complex.mechanisms.of.tumor.progression.Consequently,.we.used.a.model.for.ovarian.cancer.based.on.ROSE.(„Rat.ovarian.surface.epithelium“).cells.to.determined.regulation.principles.of.KRAS-dependent.cytoplasmatic.and.transcriptional.networks..The.KRAS.transformed.derivates.of.the.normal.ROSE.cells.exhibit.typi-cal.characteristics.of.anchorage-independent.and.invasive.tumor.cells..The.transcription.factors.Fosl1,.Hmga2,.Klf6,.JunB,.Otx1,.Gfi1.and.RelA,.which.are.up-regulated.through.KRAS.dependent.signaling,.were.syste-matically.eliminated.by.transient.RNA.interference.in.KRAS.transformed.ROSE.cells..For.network.reconstruction.we.measured.mRNA.and.protein.levels.in.manipulated.cells.by.microarray,.RT-PCR.and.Western.Blot.ana-lysis,.respectively,.and.analysed.them.by.reverse-engineering.approach.(MRA)..The. reconstructed. model. shows. a. complex. KRAS-depending. network.composed.of.activating.and.inhibitory.interactions.among.the.transcrip-tional. and. cytoplasmatic. components.. The. model. predicted. a. strong.involvement. of. each. network. component. in. the. maintaince. of. KRAS.dependent.cell.transformation..This.prediction.was.documented.by.the.partial.reduction.of.epithelial-mesenchymal.transition.(EMT)-properties.after.each.siRNA.treatment..In.the.reconstructed.model.the.transcrip-tion.factors.decomposed.into.two.hierarchically.arranged.groups.with.different.cellular.functions.as.determined.by.growth.parameters.after.siRNA.treatment.of.KRAS.transformed.cells..The.experiments.thus.confirmed.the.predicted.hierarchical.transcripti-on.factor.regulation.and.showed.that.the.hierarchy.manifests.itself.in.downstream.gene.expression.patterns.and.phenotype..Furthermore,.an. interdependence.of. the.transcriptional.networks.and.cytoplasmatic. signaling. was. observed. by. protein. analysis. of. the. mi-togen.dependent.signal.kinases.(MAPK)..This.was.interpreted.as.com-pensatory. regulation,. which. in. spite. of. perturbation,. experimentally.through.siRNA,.assured.the.efficient.survival.of.the.transformed.cells..Potentially,. such.regulatory.networks.constrict. the.efficient.effect.of.target.specific.therapies.in.clinical.situations.

38

AEKAEK AEKAbstracts Short Talks Tumor Metabolism

Notes:

short talk 601

Gdynia, Georg; Kopitz,. Jürgen;. Bergmann,. Marion;. Weber,. Alexander;.Zentgraf,.Hans-Walter;.Schirmacher,.Peter;.Roth,.Wilfried.(DKFZ,.Heidel-berg)

The danger signaling protein HMGB1 induces a novel form of cell de-ath accompanied by formation of giant mitochondria

Cells.dying.by.necrosis.release.the.high.mobility.group.box.1.(HMGB1).protein.which.has.immune.stimulatory.effects..However,.little.is.known.about.the.direct.actions.of.extracellular.HMGB1.protein.on.cancer.cells..Here.we.show.that.recombinant.human.HMGB1.(rhHMGB1).exerts.strong.cytotoxic. effects. on. malignant. tumor. cells.. The. rhHMGB1-induced. cy-totoxicity.depends.on.the.presence.of.mitochondria.and. leads.to.fast.depletion. of. mitochondrial. DNA,. severe. damage. of. the. mitochondrial.proteome. by. toxic. malondialdehyde. adducts,. and. formation. of. giant.mitochondria.. The. formation. of. giant. mitochondria. is. independent. of.direct.nuclear.signaling.events,.since.giant.mitochondria.are.also.ob-served.in.cytoplasts.lacking.nuclei..Further,.the.ROS.scavenger.NAC.as.well.as.JNK.kinase.blockade.inhibited.the.cytotoxic.effect.of.rhHMGB1..Importantly,. various. types.of. cancer. cells. but.not.normal,. non-trans-formed.cells.were.susceptible.to.rhHMGB1-induced.cell.death..Systemic.treatment.with.rhHMGB1.results.in.significant.growth.inhibition.of.xe-nografted.tumors.in.vivo..In.summary,.rhHMGB1.induces.a.distinct.form.of.cell.death.in.cancer.cells.which.differs.from.the.known.forms.of.apo-ptosis,.autophagy,.and.senescence,.possibly.representing.an.important.novel.mechanism.of.specialized.necrosis..Further,.our.findings.suggest.that. rhHMGB1. may. offer. therapeutic. applications. in. the. treatment. of.patients.with.certain.types.of.malignant.tumors,.such.as.colorectal.car-cinomas.or.brain.tumors.

short talk 602

Nazarenko, Irina (Karlsruhe Insitute of Technology, Eggenstein-Leopoldshafen);.Giselbrecht,.Stefan.(Karlsruhe.Insitute.of.Technology,.Eggenstein-Leopoldshafen);. Sleeman,. Jonathan. (University. of. Heidel-berg,.Mannheim)

Horizontal transfer of genetic information by mean of extracellular vesicles: the role in tumorigenesis and potential application in tumor diagnostic

Extracellular.membrane.vesicles.originally.described.as.cargo.for.excessive.proteins,.are.in.the.meantime.recognized.as.a.powerful.mechanism.for.the.horizontal.transfer.of.genetic. information..According.to.the.current.state.of.knowledge,.there.are.three.major.types.of.vesicles.defined:.exosomes,.40-100.nm.diameter.vesicles,.derived.from.the.intraluminal.membranes.of.multivesicular.bodies.(MVB);.microvesicles,.200-1000.nm.diameter.vesicles.which.bud.directly.from.the.cell.membrane.upon.different.stimuli,.and.the.apoptotic.bodies.produced.by.the.cells.undergoing.apoptosis..Although.the.mechanisms.of.functions.of.exosomes.and.microvesicles.are.still.poorly.un-derstood,.their.biological.activity.has.been.demonstrated.in.many.physiolo-gical.and.pathological.processes..Tumor.cells.produce.increased.amounts.of.extracellular.vesicles.as.compa-red.to.their.non-transformed.counterparts..One.being.released,.exosomes.and.microvesicles.either.bind.target.cells.in.their.microenvironment,.media-ting.for.instance.sharing.of.mutated.oncogenes.between.tumor.cells..Alter-natively,.they.can.be.distributed.by.the.circulation.through.the.whole.body.and.bind.different.target.cells.changing.their.properties..We.are.particularly.interested.in.the.function.of.extracellular.vesicles.during.metastatic.tumor.progression,.starting.with.the.formation.of.the.so-called.pre-metastatic.niches,.allowing.tumor.cells.to.home.in.specific.sites.of.fu-ture.metastasis,.in.the.homing.of.tumor.cells.in.these.sites.and.in.the.selec-tive.activation.of.dormant.tumor.cells.allowing.secondary.tumors.to.grow..We.demonstrated.that.donor.tumor.cells.selectively.sort.certain.mRNAs.and.proteins.into.the.exosomes,.which.can.be.transferred.within.the.exosomes.to.different.types.of.the.host.acceptor.cells,.such.as.endothelial.cells.and.fibroblasts..Via.a.specific.regulation.of.signaling.cascades,.tumor.exosomes.activate.these.cells.to.proliferation.and.morphogenesis,.contributing.to.the.formation.of.tumor.stroma.and.tumor.vascularization..Most.importantly,.our.findings.and.work.of.other.groups.suggest.that.tumor.exosomes.target.not.only. differentiated. normal. cells. but. also. adult. stem. and. progenitor. cells.from.the.bone.marrow,.modulating. their. fate..Thus,. tumor.exosomes.and.mircrovesicles.represent.not.only.a.powerful.tool.for.gene.regulation.across.the.long.distances..Additionally,.being.enriched.in.tumor.fluids.and.in.the.circulation,.they.can.serve.for.diagnostic.and.prognostic.purposes..

AEK

39

AEKAbstracts Poster Tumor Metabolism

Notes:

P-601

Braun, Norbert; Rudner,.Justine;.Bleif,.Martin;.Huber,.Stephan.(Univer-sity.Hospital,.Tuebingen)

Upregulation of UCP-3 uncoupling protein confers hypoxia resistance in renal epithelial cells

Tumor. cells. can. adapt. to. a. hostile. environment. with. reduced. oxygen.supply..Chronic.or.repetitive.acute.hypoxia,.in.turn,.may.foster.neoplas-tic.transformation.or.malignant.progression.of.tumor.cells..The.present.study.aimed.to.identify.molecules.that.confer.resistance.to.hypoxia.by.the.use.of.immortalized.proximal.convoluted.tubule.(PT).microdissected.from.newborn.mouse.kidney..To.this.end,.partially.hypoxia-resistant.PT.cells.were.selected.by.exposing.PT.cultures.to.repetitive.cycles.of.hy-poxia.and.reoxygenation..Thereafter,.hypoxic.stress-induced.changes.in.mRNA.(quantitative.RT-PCR).and.protein.expression.(immunoblotting),.inner. mitochondrial. membrane. potential. (DeltaPsi),. reactive. oxygen.species. (ROS). formation,. and. apoptotic. cell. death. (flow. cytometry.with.TMRE,.CM-H2DCFDA.and.propidium.iodide.as.DeltaPsi-,.redox-.and.DNA-specific.fluorescence.dyes,.respectively).were.compared.between.hypoxia-selected.and.control.PT.cultures..As.a.result,.hypoxia-selected.PT. cells. constitutively. up-regulated. mRNAs. associated. with. oxidative.defense,. DNA-repair. and. apoptosis.. In. addition,. hypoxia-selected. PT.cells. (but.not.control.cultures).exhibited,.besides.chronically.upregu-lated. UCP-3. mRNA,. highly. hypoxic-stress. inducible. UCP-3. mRNA. and.protein..UCP-3.reportedly.short-circuits.the.inner.mitochondrial.memb-rane.potential..Accordingly,.hypoxia-selected.PT.cells.developed.a. lo-wer.hypoxic.stress-induced.rise.in.DeltaPsi.than.control.cells..Moreover,.hypoxia-selected. PT. cells. produced. less. ROS. upon. hypoxic. stress. as.compared.to.control.cells..Finally,.hypoxic.stress.triggered.break-down.of.DeltaPsi.and.DNA.degradation.in.a.lower.number.of.hypoxia-selected.than.control.PT.cells..Taken.together,.our.data.suggest.that.repetitive.hypoxia/reoxygenation.cycles.select.cells.with.higher.oxidative.defense.and.lower.hypoxic-stress.stimulated.ROS.formation..The.latter.is.achie-ved.by.upregulating.UCP-3.uncoupling.protein.which.confines.hypoxic.stress-induced.hyperpolarization.of.DeltaPsi.

P-602

Otto, Angela (Technical University, Garching)

Effects of limiting nutrients and extracellular pH on the metabolism and growth of tumor cells

During.the.course.of.tumor.growth,.cells.become.exposed.to.a.micro-environment.of.limited.growth.factor.and.metabolite.supply.as.well.as.altered.physical-chemical.conditions..The.aim.of.this.study.was.to.study.the.effect.of.limiting.glutamine.and.glucose.concentrations.at.varying.extracellular.pH.on.the.metabolic.activity.and.growth.of.cancer.cells..The.breast.cancer.cell.line.MCF-7.was.maintained.in.Dulbecco’s.Modified.Eagle’s.Medium.(DMEM).containing.25.mM.glucose.and.4.mM.glutamine.and.supplemented.with.5%.FCS.and.3.7g/L.NaHCO3.for.maintaining.a.pH.of.7.4.in.a.CO2-gased.incubator..Experiments.were.performed.in.DMEM-base.with.selected.low.glucose.and.glutamine.concentrations.and.sup-plemented.with.1%.FCS.and.50.nM.insulin..The.pH.was.set.to.selected.values.by.varying.the.NaHCO3-concentration..Cell.growth.was.quanti-fied.by.electronic.counting.of.a.nuclei.suspension..The.transformation.of.the.tetrazolium.salts.XTT.and.WST.to.formazans.in.cells.reflects.the.activity.of.mitochondrial.dehydrogenases.(DH);.the.optical.density.mea-sured.was.calculated.per.cell.and.denoted.here.as.specific.DH.activity.At. limiting.concentrations,.glucose.utilization. is.highly.pH-dependent:.1.mM.glucose.(in.the.presence.of.4.mM.glutamine).was.able.to.maxi-mally. enhance. cell. growth. at. pH. 7.0,. while. about. 5. mM. glucose. was.required.at.pH.7.4...Specific.DH.activity.at.low.glucose.levels.was.also.pH-dependent:.it.was.highest.at.pH.7.4.in.the.range.of.1.0.to.2.5.mM.glu-cose,.declining.at.5.mM.glucose.to.a.plateau.level..At.pH.7.0.this.plateau.was.reached.already.at.1.mM.glucose..No.glucose-dependent.changes.were.observed.in.cells.still.viable.at.pH.6.6..In.contrast,.glutamine.de-pendency.for.growth.was.very.similar.at.all.pH.tested,.with.the.high-est.cell.numbers.obtained.at.pH.7.0..Specific.DH.activity,.however,.was.always.higher.at.pH.7.4.than.at.pH.7.0.with.glutamine.concentrations.up.to. 1.mM..When.testing.different.ratios.of. limiting.nutrient.concen-trations,.the.combination.of.0.1.mM.glutamine.with.2.5.mM.glucose.was.remarkable.in.eliciting.DH.activity.up.to.10-fold.higher.than.expected..Growth.kinetics.revealed.superior.cell.survival.under.these.conditions..This.effect.was.most.pronounced.at.pH.7.0..This.indicates.that.there.are.selective.combinations.of.nutrient.levels.and.pH.which.are.particularly.advantageous. for.maintaining. tumor.cell. viability.even.under. limiting.growth.conditions.Acknowledgements:.This.work.was.supported.by.a.grant.from.the.Deut-schen.Forschungsgemeinschaft.

40

AEKAEK AEKAbstracts Poster Tumor Metabolism

Notes:

P-603

Hamid, Syed; Varisli,.Lokman;.Tepedelen,.Burcu;.Debelec,.Bilge;.Kork-maz,.Kemal.(EBILTEM.Hall,.Izmir)

HoxB13 directly interacts with Cyclin D1 to modulate cell cycle in prostate cancer cell line LNCaP

Prostate.cancer.is.among.the.leading.causes.of.cancer.related.deaths.in.men,.especially.in.north.American.and.European.region..Although.an-drogen.ablation. therapy. results. in.early. shrinkage.of. the. tumour.but.ultimately.leads.to.the.development.of.more.aggressive.and.lethal.type.of.cancer.that.is.resistant.to.this.type.of.therapy..HoxB13.is.a.homeobox.protein. that. is. exclusively. expressed. in. adult. prostate. and. colon.. Its.deregulated.expression.has.been.reported.in.a.number.of.malignancies.including. prostate. and. breast. cancer.. In. prostate. cancer,. HoxB13. has.been.reported.as.a.tumour.suppressor.gene.that.causes.G1.cell.cycle.arrest.in.PC-3.and.LNCaP.cells..This.G1.arrest.has.been.linked.to.down-regulation.of.TCF4.trans-activation.which.results.in.decreased.expres-sion.of.Cyclin.D1,.c-myc.and.beta.catenin.thus.halting.the.cell.cycle...We.have.questioned.the.function.of.HoxB13.in.androgen.receptor.positive.prostate.cancer.cell.line.and.have.found.that.the.expression.pattern.of.this.protein.changes.as.cell.cycle.proceeds,.showing.an.increased.ex-pression.at.G0/G1.and.G2/M.while.decreased.levels.at.S.phase..Further,.it.was. found. that.HoxB13.directly. interacts.with.a.major. regulator.of.the.G1-S.transition,.namely.Cyclin.D1..Forced.expression.of.HoxB13.led.to.G1.arrest,. indicating.that. interaction.of.HoxB13.with.Cyclin.D1. is.of.inhibitory.nature.for.the.activity.of.Cyclin.D1..While.silencing.of.HoxB13.led.to.increased.expression.of.Cyclin.D1,.B1.and.pH3S10.which.indicates.increased. mitogenic. potential. of. the. cells.. It. was. also. observed. that.the.expression.of.HoxB13.is.positively.regulated.by.a.epidermal.growth.factor.through.PI3.kinase.pathways.as.inhibition.of.this.pathway.leads.to. down-regulation. of. its. expression.. So. it. seems. that. expression. of.HoxB13.is.positively.regulted.by.growth.signals.but.negatively.regulates.the. cell. cycle. progressiong.. Direct. interaction. of. this. protein. with. a.major.regulator.of.the.cell.cycle.may.have.important.consequences.in.deciding.the.proliferative.fate.of.the.cells.

P-604

Hippe, Andreas (University Medical Center, Düsseldorf); Schorr,.Anne.(University.Medical.Center,.Düsseldorf);.Müller-Homey,.Anja.(University.Medical.Center,.Düsseldorf);.Seeliger,.Stephan.(University.Medical.Cen-ter,.Göttingen);.Jannasch,.Katharina.(University.Medical.Center,.Göttin-gen);.Buhren,.Bettina.Alexandra.(University.Medical.Center,.Düsseldorf);.Sleemann,.Jonathan.(University.Medical.Center,.Karlsruhe);.Stoecklein,.Nikolas.(University.Medical.Center,);.Alves,.Frauke.(University.Medical.Center,. Göttingen);. Hoffmann,. Thomas. K.. (University. Medical. Center,.Düsseldorf);.Homey,.Bernhard.(University.Medical.Center,.Düsseldorf)

Tumor derived CCL20 production critically contributes to angiogene-sis and tumor progression

Mutational.activation.of.the.EGFR/Ras/ERK-signaling.pathway.is.a.cru-cial.step.in.the.malignant.transformation.of.a.wide.variety.of.tumors..Modulation.of.gene.expression.by.EGFR/Ras/ERK.promotes.cell.prolife-ration.and.survival..Here,.we.demonstrate.that.the.activation.of.Ras.re-gulates.chemokine.expression.in.a.dichotomic.manner.with.an.inducible.set.demonstrating.pro-tumor.and.a.repressible.set.showing.anti-tumor.properties..In.particular,.tumors.enhance.angiogenesis.by.upregulating.the.production.of.CCL20.through.the.activation.of.the.EGFR/Ras/ERK-si-gnaling.pathway..In.vivo,.the.chemokine.CCL20.is.over-expressed.in.are-as.of.melanoma,.breast.cancer,.colon.cancer,.and.head.and.neck.squa-mous.cell.carcinoma.(HNSCC).with.increased.ERK.posphorylation..High.CCL20.expression.in.breast.cancer.tumors.reduces.cumulative.survival.of.patients..Moreover,. in.334.cases.of.breast.cancer,.colon.carcinoma.and.HNSCC,.a.higher.pT/pN-category.of.the.observed.tumors.correlated.significantly.to.higher.expression.of.CCL20..Its.specific.corresponding.receptor,. CCR6,. is. abundantly. expressed. on. endothelial. cells. in. vitro.and.in.vivo..Activation.of.CCR6.signaling.in.endothelial.cells.induces.cell.migration,. in. vitro-repair. and. leads. to. enhanced. vessel. formation.. In.vivo,.CCL20.specifically. induced. increased.vascularization.of.Matrigel.plugs. in. wildtype. mice,. which. was. abrogated. in. CCR6-deficient. mice..Furthermore,.CCL20-expressing.B16F10.melanomas.showed.significant-ly.decreased.tumor.growth.and.vascularization.in.CCR6-deficient.com-pared.to.wildtype.mice.using.flat.panel.volume.computertomography..Collectively,.our.findings.identify.a.novel.chemokine-driven.mechanism.of.tumors.to.promote.angiogenesis.and.progression.

AEK

41

AEKAbstracts Short Talks Molecular Diagnostics and Targeted Therapy

Notes:

short talk 701

Maak, Matthias (Technische Universität, München); Nitsche,. Ulrich.(Technische.Universität,.München);.Keller,.Larissa.(Technische.Univer-sität,.München);.Johannes,.Ludger.(CNRS-UMR.144,.Paris);.Klaus-Peter.(Technische.Universität,.Klinik.für.Chirurgie,.München)

Specific targeting of colorectal and pancreatic cancer with Shiga to-xin B-subunit

Tumor.targeting.is.defined.as.the.specific.transport.of.molecules.to.a.tumor.for.diagnostic.or.therapeutic.purposes..Efficient.methods.for.tu-mor.targeting.are.eagerly.awaited.for.gastrointestinal.cancers..A.targe-ting.vector.should.have.molecular.specificity,.fast.uptake.in.tumor.cells,.low.immunogenic.potential.and.the.capacity.to.withstand.degradation.or. inactivation.. These. requirements. are. satisfied. by. the. non-toxic. B-subunit.of.the.bacterial.Shiga.toxin.(STxB)..The.glycosphingolipid.Glo-botriaosylceramide.(Gb3;.CD77),.the.specific.cellular.receptor.of.Shiga.toxin,.is.significantly.increased.on.a.wide.range.of.solid.tumors.in.com-parison.to.healthy.tissue,.as.we.and.others.could.demonstrate.for.pan-creatic.carcinoma,. lymphoma,.primary.colorectal.carcinoma.and. liver.metastases...Up-regulation.of.Gb3-levels.is.observed.in.85%.of.colon.cancer.(n=66),.91%.of.liver.metastases.of.colorectal.cancer.(n=10),.and.78%.of.pancreatic.cancer. (n=27)..We.are.currently.analyzing.cholan-giocellular.carcinomas.(CCC).for.putative.over-expression.of.the.STxB.receptor.. However,. the. GB3. expression. was. not. dependent. on. tumor.stage.or.grading,.and.showed.no.correlation.with.clinical.outcome..STxB.is.taken.up.rapidly.into.many.tumor.cell.types.via.the.retrograde.route,.thus.avoiding. intracellular.degradation.and. inactivation. in. lysosomes..Our. studies. showed. that. STxB. reaches. the. Golgi. apparatus. within. 15.minutes,.and.stays.there.for.up.to.5.days..This.could.be.shown.for.esta-blished.colon.and.pancreas.cancer.cell.lines,.as.well.as.for.short-term.primary.cell.cultures.of.human.colorectal.cancer.and.liver.metastases..In.contrast,.primary.cultures.of.benign.colonic.adenomas.with.low.ex-pression.of. the.Gb3. receptor. showed.only. low. levels.of. STxB.uptake..Based.on.these.findings.we.propose.STxB.be.used.as.an.efficient.tool.for.specific.tumor.targeting,.e.g.,.as.a.carrier.for.chemotherapeutic.or.contrast.agents.We. are. currently. evaluating. therapeutic. use. of. STxB. for. the. specific.delivery.of.covalently.coupled.SN38,.an.active.metabolite.of.the.topoi-somerase.I.inhibitor.Irinotecan..The.cytotoxic.effect.of.the.STxB-SN38.compound.was.tested.on.pancreatic.and.colorectal.cancer.cell.lines,.and.was.found.to.be.increased.more.than.100-fold.as.compared.to.Irinote-can..Moreover,.this.effect.was.effectively.blocked.by.competing.incuba-tion.with.non-labeled.STxB,.demonstrating. the. receptor-specificity.of.the.cytotoxicity..Thus,.STxB.constitutes.a.promising.new.tool.for.speci-fic.tumor.targeting.

short talk 702

Bühler, Helmut (Ruhr-Universität Bochum, Herne); Kochanneck,.Anja.(Ruhr-Universität. Bochum,. Herne);. Priesch,. Bettina. (Ruhr-Universität.Bochum,.Herne);.Polz,.Katja.(Ruhr-Universität.Bochum,.Herne);.Adami-etz,.Irenäus.(Ruhr-Universität.Bochum,.Herne)

Na+K+ATPase activity might be the crucial factor for selective inhi-bition of tumor stem cells

Background:.In.2009.Gupta.et.al..did.show.that.some.ionophores.for.mo-novalent.kations.could.act.as.selective.inhibitors.for.tumor.stem.cells.with. salinomycin. beeing. the. most. active. compound.. As. experimental.system.they.used.an.epithelial.breast.cancer.cell. line.and.a.subclone.that.gained.tumor.stem.cell.like.properties.by.silencing.E-cadherin.Results:.In.a.different.cellular.model.system.we.could.show.a.very.simi-lar.selective.inhibition.of.tumor.stem.cell.proliferation.by.salinomycin..An. isolated.stem.cell. fraction. from.the.human.breast.cancer.cell. line.MDA.231.was.checked.against.an.epithelial.subclone.of.this.cell.line.ge-nerated.by.the.transfection.of.keratin.18.A.first.approach.to.clarify.the.underlying.mechanism.revealed.that.the.Na+K+ATPase.might.play.a.role.in.this.selective.inhibition:.the.dose.de-pendent. inhibition. of. stem. cells. by. salinomycin. was. approximated. in.epithelial.cells. if.additionally.the.Na+K+ATPase.was.partially. inhibited.by.increasing.concentrations.of.hellebrin.Conclusion:.The.selective.inhibition.of.tumor.stem.cells.by.salinomycin.might.be.caused.by.a.weakly.active.Na+K+ATPase.in.these.cells.

42

AEKAEK AEKAbstracts Poster Molecular Diagnostics and Targeted Therapy

Notes:

P-701

Schönfeld, Kurt; Knopp,.Christiane;.Brendel,.Christian;.Köhler,.Sylvia;.Grez,.Manuel;.Wels,.Winfried.(Georg-Speyer-Haus,.Frankfurt/M.)

Retargeted natural killer cells for cancer immunotherapy

Natural.killer.(NK).cells.are.the.body‘s.first.line.of.defense.against.vi-ral. infections.and.malignant.cells..In.addition.to.autologous.or.donor-derived.primary.NK.cells,.also.continuously.growing.cytotoxic.cell.lines.such. as. NK-92. hold. promise. for. cancer. immunotherapy.. In. preclinical.studies.NK-92.showed.high.cytotoxicity.against.malignant.cells.of.he-matologic.origin,.and.safety.of.infusion.of.high.doses.of.NK-92.cells.was.established.in.phase.I.clinical.trials.utilizing.irradiated.cells.to.prevent.permanent. engraftment.. To. further. enhance. their. therapeutic. utility.and.provide.NK-92.cells.with.pre-determined.tumor-cell.specificity,.we.previously.generated.variants.genetically.modified.with.retroviral.vec-tors. to. express. chimeric. antigen. receptors. (CAR). with. specificity. for.tumor-associated. surface. and. differentiation. antigens. such. as. ErbB2.(HER2),.EpCAM,.and.CD20..These.prototypic.CAR.were.composed.of.a.tumor-specific. single. chain. Fv. antibody. fragment. fused. via. a. flexible.hinge.region.to.the.murine.CD3.zeta.chain.as.an.intracellular.signaling.moiety..The.retargeted.NK.cells.displayed.high.cytotoxic.activity,.and.selectively.killed.antigen.positive.and.otherwise.NK-resistant.targets..As.a.prerequisite.for.application.of.ErbB2-specific.NK.cells. in.cancer.patients,.here.we.developed.novel.CAR.constructs.that.were.humanized.to. reduce. potential. immunogenicity,. and. codon. optimized. to. improve.expression. in.NK-92.cells.. In.addition. to.an.optimized.CAR.employing.the.ErbB2-specific.scFv(FRP5).antibody.fragment.for.target-cell.reco-gnition.and.human.CD3.zeta.chain.as.a.signaling.moiety,.we.also.inve-stigated.CAR.with.composite.signaling.domains..CAR.constructs.were.stably.introduced.into.NK-92.by.lentiviral.gene.transfer,.CAR-expressing.single.cell.clones.were.isolated.by.FACS.sorting,.and.functionality.was.characterized..Thereby.CAR.containing.CD3.zeta.(5.z),.or.CD28.and.CD3.zeta.domains.(5.28.z).showed.highest.expression.levels,.with.NK.cells.expressing.the.latter.displaying.most.selective.killing.activity.towards.ErbB2-expressing.breast.carcinoma,.medulloblastoma.and.glioblastoma.cells..Based.on.these.results,.a.clinically.applicable.lentiviral.5.28.z.CAR.vector.was.derived,. and.protocols. for.GMP-compliant. transduction.of.NK-92.cells.were.developed..Ongoing.work.now.focuses.on.molecular.and. functional. characterization. of. the. resulting. NK-92/5.28.z. cells. to.identify.clonal.cell.lines.most.suitable.for.clinical.development.

P-702

Hirschfeld, Marc; Jäger,.Markus;.Neumann,.Vivi;.Gitsch,.Gerold;.Stick-eler,.Elmar.(University.Medical.Center,.Freiburg)

Hyperthermia triggers down-regulation of Estrogen receptor α iso-forms and its co-activators DEAD-box5 and DEAD-box17 in breast cancer cells

Recently,. the. clinical. application. of. hyperthermal. therapy. becomes.more.and.more.reintroduced.and.is.used.concomitant.to.chemothera-py. or. radiotherapy,. that. might. improves. the. effect. of. those. classical.anti-cancer. treatments.. RNA. helicases. p68. (DEAD-box5,. DDX5). and.p72. (DEAD-box17,.DDX17).act.as. transcriptional. co-activators.of. seve-ral.tumor-relevant.genes,.e.g..estrogen.receptor.α.(ERα)..Both.factors.regulate.ERα-activity.in.breast.cancer..We.investigated.potential.regula-tory.effects.of.hyperthermia.on.the.expression.of.these.breast.cancer-related.factors.Various.ERα-positive.breast.cancer.cell.lines.(MCF-7,.ZR-75-1,.T47D,.BT-474).were.cultured.under.hyperthermia.(42°C,.2.hrs).followed.by.main-tenance.under.regular.culture.conditions.(37°C,.4.hrs)..As.a.negative.control.the.same.cell.lines.were.cultivated.under.regular.temperature.conditions. permanently.. mRNA. and. protein. expression. levels. of. ESRα.isoforms,.DDX5.and.DDX17.were.analyzed.by.RT-PCR,.Western.blot.and.immunocytochemistry.technique.The.analyses.revealed.markedly.decreased.mRNA.and.protein.levels.of.ERα.isoforms,.as.well.as.of.DDX5.and.DDX17.in.cells.exposed.to.hyper-thermia.compared.to.cells.cultured.under.regular.conditions.Our.results.clearly.indicate.a.regulatory.effect.of.hyperthermal.treat-ment.on.both,.the.mRNA.and.protein.expression.of.the.breast.cancer-relevant.gene.ERα.and.its.co-activators.DDX5.and.DDX17..According.to.our.findings,.hyperthermal.treatment.seems.to.represent.a.method.that.may. improve. classical. anti-cancer. therapies. by. down-regulating. the.activity.of.important.factors.in.breast.cancer.biology..We.hypothesize.that.hyperthermia. inhibits.the.expression.of.ERα. isoforms.and. its.co-activators,.thus.probably.leading.to.a.suppression.of.tumor.progression..However,.the.molecular.background.of.signalling.pathways.that.undergo.hyperthermia-dependent.alterations.and.its.concomitant.effects.on.tu-mor.biology.still.need.to.be.investigated.in.more.detail.

AEK

43

AEKAbstracts Poster Molecular Diagnostics and Targeted Therapy

Notes:

P-703

Riehle, Ulrike (University Hospital, Freiburg); Mader,. Andreas. (De-partment.of.Microsystems.Engineering,.Freiburg);.Brandstetter,.Thomas.(Department. of. Microsystems. Engineering,. Freiburg);. Rühe,. Jürgen.(Department.of..Microsystems.Engineering,.Freiburg);.zur.Hausen,.Axel.(Maastricht. University. Medical. Center,. Maastricht);. Stickeler,. Elmar.(University.Medical.Center,.Freiburg)

Microarray-based amplification and detection of microRNAs by NAS-BA

MiRNAs.(microRNAs).are.small.(22-24.nt).non-coding.RNAs.that.regulate.gene.expression.and.have.been.shown.to.play.a.crucial.role.in.the.pa-thology.of.breast.cancer..Existing.miRNA.amplification.and.detection.techniques.comprise.a.high-ly.complex.workflow.and.are.therefore.rather.suitable.for.research.pur-poses.than.for.usage.in.clinical.routine..Aim.of.this.study.is.to.establish.a. biochip. technology. platform. that. combines. an. on-chip. nucleic. acid.amplification.of.miRNAs.(by.NASBA,.(nucleic.acid.sequence.based.am-plification)).with.a.microarray.based.detection.and.subsequent.readout..The.simplified.workflow.makes.this.technique.interesting.for.usage.in.clinical.routine.A.conventional.NASBA.assay.was.adapted.to.the.amplification.of.the.rat-her.small.miRNA.molecules..Therefore.a.reverse.transcription.step.using.a.stem-loop.RT.Primer.was.combined.with.the.miRNA.specific.NASBA.assay.For. miroarray-based. detection. miRNA-specific. oligonucleotides. were.immobilized.in.a.hydrogel.mounted.on.a.polymethylmetacrylat.(PMMA).chip.. Total. RNA. was. amplified. utilizing. a. labelling,. multiplex. miRNA-NASBA.assay..Labelled.miRNA-NASBA.products.were.immobilized.to.the.probes.and.fluorescence.data.collected..Northern.blot.analysis.confir-med.specificity.of.the.miRNA-NASBA.reaction..For.the.proof.of.concept.we.chose.seven.different.breast.cancer.related.miRNAs.(hsa-miR-16,.hsa-miR-21,.hsa-miR-10b,.hsa-miR-125a,.hsa-miR-125b,.hsa-miR-206,.hsa-miR-210).and.showed.that.parallel.amplification.of. these.7.miRNAs.by.means.of.multiplex.microarray-based.NASBA. is.possible.. Furthermore. we. showed. that. the. established. miRNA. NASBA.assays.are.sensitive.enough.to.amplify.a.minimum.of.10.to.100.copies.of.a.respective.synthetic.miRNA.template..In.terms.of.clinical.applications,.we. showed. that. the.multiplex. miRNA. NASBA. assay. can.be. applied. to.RNA.extracted.from.various.clinical.samples.such.as.fresh.frozen.and.FFPE.breast.cancer.tissue.Our.study.indicates.that.amplification.and.detection.of.miRNAs.by.NAS-BA.and.subsequent.detection.on.a.microarray.is.possible..This.test.can.be.easily.performed.in.a.single.step.and.also.applied.to.RNA.extracted.from.patient.samples.such.as.FFPE.tissues..

P-704

Oberoi, Pranav; Jabulowsky,.Robert;.Dälken,.Benjamin;.Mahmud,.Hayat;.Wels,.Winfried.(Georg-Speyer-Haus,.Frankfurt/M.)

Targeted induction of tumor-cell death by chimeric granzyme B fusion proteins

Tumor.cells.are.frequently.insensitive.to.apoptotic.stimuli.due.to.dere-gulation.of.apoptotic.cell.death.pathways..Targeted.delivery.of.a.potent.apoptosis. inducer. into.such.cells. that.concurrently.activates.multiple.targets. within. the. apoptotic. cascade. may. effectively. overcome. apo-ptosis. resistance. and. induce. cancer. cell. death.. The. serine. protease.granzyme.B.(GrB).is.naturally.produced.by.cytotoxic.T-lymphocytes.and.natural.killer.(NK).cells,.where.it.resides.together.with.other.granzymes.and.the.pore-forming.protein.perforin.in.cytotoxic.granules..Upon.de-granulation,.GrB.is.released.and.enters.target.cells.along.with.perforin,.where.it.rapidly.induces.programmed.cell.death.via.caspase-dependent.and.caspase-independent.mechanisms..Based.on.its.broad.pro-apoptotic.activity,.we.are.investigating.GrB.as.an.effector.function.in.tumor-specific.chimeric.molecules.with.potenti-al. therapeutic.activity..We.previously.described.prototypic.GrB.fusion.proteins.that.carry.an.ErbB2-specific.scFv.antibody.fragment.or.the.EG-FR-specific.peptide.ligand.TGFalpha.for.cell.targeting..These.molecules.were.selectively.internalized.by.cancer.cells.expressing.the.respective.target.receptors,.and.rapidly.induced.cell.death.in.the.presence.of.an.exogenous.endosomolytic.activity..Ongoing.work.now.aims.at.improving.the.design.of.such.molecules.to.further.enhance.their.selectivity.and.cytotoxicity..As.a.first.step,.we.modified.the.surface.charge.of.GrB.by.site-directed.mutagenesis.to.reduce.remaining.intrinsic.cell.binding.of.the.GrB.domain..In.parallel,.we.are.exploring.alternative.strategies.to.enhance.intracellular.delivery.of.chimeric.GrB.molecules..Perforin.is.be-lieved.to.facilitate.escape.of.wildtype.GrB.from.vesicular.compartments.upon.cellular.uptake,.and.allow.access.to.its.substrates.in.the.cytoplasm.of. target. cells.. To. test. whether. the. pore-forming. activity. of. perforin.can.also.be.employed.for.intracellular.delivery.of.GrB.fusion.proteins,.we.are.investigating.potential.cooperation.of.targeted.GrB.derivatives.with.endogenous.perforin.upon.co-expression. in.human.NK.cells.as.a.model.system.

44


Notes:

P-705

Kloke, Björn-Philipp; Burkhardt,. Ute;. Wels,. Winfried. (Georg-Speyer-Haus,.Frankfurt/M.)

Immunotherapy of ErbB2/HER2-expressing tumors with gene-modi-fied producer cells that secrete APC-targeted fusion vaccines

Presentation. of. tumor-associated. antigens. by. professional. antigen-presenting.cells.(APC).is.critical.for.the.induction.of.tumor-specific.T-cell. responses..Dendritic.cells.are.thereby.considered.to.be.the.most.important. APC. mediating. T-cell. activation.. For. targeted. delivery. of. a.tumor.antigen.to.APC.in.vivo,.we.previously.generated.a.fusion.protein.that.contains.the.extracellular.domain.of.CTLA-4.for.specific.binding.to.costimulatory.B7.molecules.on.APC,.fused.to.an.antigenic.fragment.of.the.human.ErbB2.(HER2).receptor.tyrosine.kinase..Protective.vaccina-tion.of.BALB/c.mice.by.subcutaneous.injection.of.bacterially.expressed.chimeric.vaccine.or. intramuscular. injection.of.a.DNA.construct. for. in.vivo. production. of. the. fusion. protein. induced. ErbB2-specific. immuni-ty.and. improved. tumor-free. survival. upon. subsequent. challenge.with.ErbB2-expressing.Renca.renal.carcinoma.cells.. For. immunotherapy. of. established. tumors,. we. generated. syngeneic.HC11.mammary.epithelial.cells.continuously.secreting.the.CTLA-4-ErbB2.fusion.protein..Therapeutic.vaccination.of.tumor.bearing.BALB/c.mice.by.implantation.of.these.non-tumorigenic.producer.cells.close.to.sub-cutaneously.growing.Renca-lacZ/ErbB2.tumors.resulted.in.tumor.rejec-tion,.accompanied.by.the.induction.of.ErbB2-specific.antibody.and.T-cell.responses..Furthermore,.long-term.protection.was.induced.in.vaccina-ted.animals,.indicated.by.the.rejection.of.a.lethal.dose.of.Renca-lacZ/ErbB2.tumor.cells.two.months.later..While.these.vaccine-producing.cells.of.epithelial.origin.are.locally.restricted.and.do.not.harbor.endogenous.immunomodulatory.activities,.lymphocytes.like.natural.killer.(NK).cells.have.the. intrinsic.potential. to. infiltrate.tissues,.and.to.modulate.APC.activity..Hence,.to. investigate.the.potential.of.NK.cells.as.alternative.producer.cells.for.in.vivo.production.of.APC-targeted.vaccines,.we.ge-nerated.lentiviral.vectors.encoding.secreted.CTLA-4-ErbB2.fusion.pro-teins..These.vectors.were.used.for.transduction.of.human.NK-92.cells..Gene-modified.NK.cells.were.enriched.by.FACS.sorting,.and.their.ability.to.secrete.functional.vaccine.proteins.was.confirmed.in.in.vitro.assays..The.antitumoral.activity.of.vaccine-secreting.NK-92.cells.in.vivo.is.cur-rently.being.analyzed.in.a.murine.tumor.model.

P-706

Niegisch, Günter; Knievel,. Judith;. Koch,. Annemarie;. Albers,. Peter;.Wolfgang.Schulz.(University.Medical.Center,.Düsseldorf)

Expression patterns of histone deacetylases in urothelial cancer and their implications for the action of histone deacetylase inhibitors

Objective:.To.investigate.the.expression.of.classical.histone.deacetylase.(HDAC).1-11.and.the.efficacy.of.HDAC.inhibitors.as.targeted.therapeutics.depending.on.differences.in.expression.patterns.Material.and.methods:.mRNA.expression.of.HDAC.1.-.11.was.determined.by.quantitative.RT-PCR.in.16.urothelial.cancer.cell.lines.(UCC).compared.to.urothelial.primary.cultures..Expression.of.HDACs.2,.3,.6,.8,.and.9.was.additionally. determined. in. urothelial. cancer. tissues. from. cystectomy.specimens. (n=25). compared. to. normal. controls. (n=12).. Up-. or. down-regulation.was.defined.as.a.more.than.2-fold.difference.compared.to.median.expression.of.controls..For.external.validation,.public.available.tissue.microarrays.were.examined..Effects.of.suberoylanilide.hydroxa-mic.acid.(SAHA).treatment.in.UCCs.were.assessed.by.viability/apoptosis.assays,.cell.cycle.analysis.and.via.target.protein.expression.(p21,.thy-midylate.synthase,.EZH2.and.PARP)..Effects.of.specific.HDAC6.inhibition.by.Tubacin.were.determined.by.cell.viability.assay.and.measurements.of.p21.and.EZH2.protein.expression...Results:.Common.changes.in.UCC.were.upregulation.of.HDAC2.(7/16).and.HDAC8.(4/16),.downregulation.of.HDAC6.(12/16).and.a.broad.decrease.in.class.IIa.HDACs..In.our.small.tissue.series.only.HDAC8.(P=0.006).was.significantly.upregulated.overall..However,.upregulation.of.HDAC2.and.downregulation. of. HDAC6,. respectively,. were. detected. in. 9/25. tissue.samples.each..Analysis.of.published.microarray.data.(n=6,.60–266.pa-tient.samples).confirmed.HDAC2.and.HDAC8.upregulation.and.revealed.high.variability.of.HDAC6.expression..EC50. for.UCC.viability.after. treatment.with. the.broad.HDAC. inhibitor.SAHA.ranged.between.2.and.6.µM..SAHA.generally.induced.G2/M.arrest.and.apoptosis,.elevated.p21.and.diminished.thymidylate.synthase.pro-tein..Effects.on.EZH2.and.PARP.cleavage.after.SAHA.treatment.differed.between.the.cell.lines,.with.the.best.correlation.to.the.severity.degree.of. HDAC. class. IIa. downregulation.. Inhibition. of. HDAC6. by. its. specific.inhibitor.Tubacin.resulted.in.decreased.viability.of.5637.cells.retaining.HDAC6.compared.to.639v.cells.with.diminished.HDAC6..Conclusion:. Characteristic. changes. of. HDAC. expression. take. place. in.urothelial. cancer,. albeit. in. a. heterogeneous. manner.. These. changes.affect.the.response.to.different.HDAC.inhibitors..Targeting.of.HDACs.re-lying.on.specific.tumor.expression.profiles.may.therefore.provide.new.options.for.urothelial.cancer.treatment.

AEK

45


P-707

Hülsmann, Helen (German Cancer Research Center, Heidelberg); Bender,.Christian.(German.Cancer.Research.Center,.Heidelberg);.Rolff,.Jana. (Max-Delbrück-Center,. Berlin);. Fichtner,. Iduna. (Max-Delbrück-Center,.Berlin);.Herwig,.Ralf. (Max-Planck-Institut,.Berlin);.Korf,.Ulrike.(DKFZ,.Heidelberg);.Sültmann,.Holger.(DKFZ,.Heidelberg).Kuner,.Rupre-cht.(DKFZ,.Heidelberg)

Identification of Drug-Associated Proteins in NSCLC Xenograft Mo-dels by Reverse-Phase-Protein-Arrays

The. treatment. of. non-small. cell. lung. cancer. (NSCLC). patients. is. cur-rently. limited.by.the.fact.that.most.therapies.are.not.adapted.to.the.individual.response.of.a.patient..The.stratification.of.patients. for.the.most.efficient.response.to.conventional.chemotherapeutics.and.targe-ted. therapies. will. improve. established. therapy. schemes.. Our. project.aims.at.unravelling.the.influence.of.specific.signalling.molecules.on.the.response.to.common.NSCLC.drugs..We.quantified.protein.expression.levels.in.patient.derived.NSCLC.xeno-graft.models..The.tumor.models.are.characterized.by.different.response.rates. upon. treatment. with. established. chemotherapeutics. (e.g.. Gem-citabine,.Paclitaxel,.Carboplatin).and.EGFR-targeted.therapies.(Cetuxi-mab,.Erlotinib).Expression. of. 77. proteins. of. cancer-relevant. pathways. (e.g.. MAPK,.JAK/STAT,.PI3K/AKT).was.analyzed.using.the.reverse.phase.protein.ar-ray. technology. (RPPA).. Protein. lysates. of. 53. xenograft. models. were.spotted.in.dilution.series.and.replicates..Response.data.were.associa-ted.with.protein.expression.levels.in.the.tumor.samples..Furthermore,.protein.expression.changes.after.Cetuximab.treatment.were.analyzed.in.dependence.on.the.response.rate..The.major.goal.in.this.experimental.subset.was.to.find.links.between.the.activities.of.specific.proteins.and.the.response.to.Cetuximab.treatment..Statistical.analysis. indicated.significant.associations.between.the.ex-pression. of. distinct. proteins. and. the. response. rate. to. certain. drugs.(Erlotinib,. Cetuximab,. Paclitaxel,. Carboplatin,. Gemcitabine).. Proteins.of.the.ErbB.signalling.pathway.were.differentially.expressed.in.Carbo-platin.responders.and.non-responders..MEK1.upregulation.was.observed.upon.Cetuximab.treatment. in.responders..We.revealed.an.association.between.higher.P-SRC.expression.and.increased.Gemcitabine.response.rates..This.result.fits.to.observations.that.describe.coherence.between.SRC. inhibition.and.reduced.suppression.of.cell.growth.and.survival. in.the.presence.of.Gemcitabine.in.cancer.cell.lines..Next.we.will.corroborate.the.results.in.various.validation.experiments..The.dependency.between.target.protein.activity,.downstream.signaling.of.these.interactions.and.response.to.distinct.drugs.will.be.further.inve-stigated.in.tumor.cell.lines.in.a.pharmacogenomic.approach.In.summary,.our.study.suggests.predictive.markers.for.the.response.of.NSCLC.tumors.to.certain.drugs,.which.may.be.useful.for.therapy.decisi-ons.towards.a.more.efficient.personalized.treatment.

P-708

Fritsche, Raphaela; Witzel,. Franziska;. Sieber,. Anja;. Schmidt,. Nadine;.Sers,.Christine;.Blüthgen,.Nils.(Charité,.Berlin)

Strong negative feedback from Erk to Raf confers robustness to MAPK signalling

Protein. levels.within.signal.transduction.pathways.vary.strongly.from.cell.to.cell..Here.we.analysed.how.signalling.pathways.can.still.process.information.quantitatively.despite.strong.heterogeneity. in.protein. le-vels..We.systematically.perturbed.the.protein.levels.of.Erk,.the.terminal.kinase. in.the.MAPK.signalling.pathway. in.a.panel.of.human.cell. lines..We. found.that. the.steady-state.phosphorylation.of.Erk. is.very.robust.against.perturbations.of.Erk.protein.level..Although.a.multitude.of.me-chanisms.exist.that.may.provide.robustness.gainst.fluctuating.protein.levels,.we.found.that.one.single.feedback.from.Erk.to.c-Raf.accounts.for.the.observed.robustness..Surprisingly,.robustness.is.provided.through.a.fast.post-translational.mechanism.although.variation.of.Erk.levels.oc-curs.on.a.timescale.of.days.

Notes:

46


Notes:

P-709

Bier, Carolin (University Medical Center, Mainz); Knauer,.Shirley.(Uni-versity.Duisburg-Essen,.Essen);.Scheider,.Gisbert.(ETH,.Zurich);.Krämer,.Oliver. (University,. Jena);. Marschalek,. Rolf. (University,. Frankfurt/M.);.Stauber,.Roland.(University,.Mainz)

Probing the Structure-Function of Taspase1: A protease with unresol-ved (patho)biology and oncological relevance

Background:.Proteases.play.crucial.roles.not.only.in.physiological.but.also.pathophysiological.processes,.such.as.cancer..Besides.the.tumour-promoting.activity.of.MMPs,.there.is.growing.evidence.that.intracellular.proteases.can.also.trigger.oncogenic.mechanisms,.and.hence.represent.promising.therapeutic.targets.Taspase1.is.a.threonine.protease.processing.(patho)biologically.relevant.nuclear.and.cytoplasmic.substrates,.such.as.the.Mixed.Lineage.Leuke-mia.protein.and. leukemia.provoking.MLL-fusions.. In.contrast. to.other.proteases.in.oncology,.the.understanding.of.Taspase1’s.(patho)biologi-cal.relevance.is.still.limited..Neither.Taspase1’s.degradome,.the.spatial.and.exact.biochemical.requirements.controlling.its.proteolytic.activity.nor.chemical.Taspase1.inhibitors.are.known.Results:.Here,.we.present.first.cell-based.high-throughput.biosensor.as-says.to.probe.Taspase1.trans-cleavage.in.vivo,.which.were.used.to.iden-tify.bioactive.compounds..The.assay.was.further.exploited.to. identify.the.sequence.and.spatial.requirements.for.efficient.Taspase1.processing.in.vivo,.allowing.the.first.genome-wide.bioinformatic. identification.of.the. human. Taspase1. degradome. (Bier;. JBC. 2011).. Cleavage. site. reco-gnition.and.proteolytic.processing.of.selected.targets.were.verified.for.selected.targets..To.provide.a.molecular.rationale.how.the.predominantly.nuclear/nucleo-lar.Taspase1.could.also.cleave.its.cytoplasmic.substrates,.we.show.that.Taspase1’s.function.is.regulated.by.the.importin-alpha/nucleophosmin-axis.Conclusions:.We.present.novel.insights.into.Taspase1’s.molecular.regula-tion.and.provide.tools.allowing.a.focussed.analysis.of.the.(patho)biolo-gical.and.potential.therapeutic.relevance.of.this.protease.

P-710

Bormann, Felix (Charité, Berlin); Tierling,. Sascha. (Universität,. Saar-brücken);. Rivera,. Maria. (Max-Delbrück. Centrum,. Berlin);. Hoffmann,.Jens.(Max-Delbrück.Centrum,.Berlin);.Fonfara,.Sabine.(Charité,.Berlin);.Sers,.Christine.(Charité,.Berlin)

Epigenetic regulation of the EGFR ligand Amphiregulin and its role in predicting the outcome of EGFR-targeted therapies in colorectal cancer

The. EGF. tyrosine. kinase. receptor. has. become. one. of. the. most. in-teresting. therapeutic. targets. in. human. colorectal. carcinoma. (CRC)..KRAS.mutations.occurring.in.up.to.40%.of.colon.cancer.patients.ser-ve.as.exclusion.criteria.for.EGFR-directed.therapy..However,.only.30%.of.the.patients.bearing.KRAS-wild-type.genes.show.a.positive.therapy.response.. Therefore,. further. reliable.biomarkers.are.urgently.needed.enabling.improved.patient.stratification.One. candidate,. potentially. predictive. for. a. positive. EGFR. therapy. re-sponse,.is.the.EGFR.ligand.Amphiregulin.(AREG)..Since.it.was.described.to.correlate.negatively.or.positively.with.the.response.towards.EGFR-targeted.therapeutics.in.NSCLC,.HNSCC.and.CRC,.we.were.interested.to.decipher.the.mechanisms.of.AREG.regulation.in.colorectal.carcinomas.To.understand.the.regulation.of.AREG,.several.cell.lines.and.xenograft.tumor.samples.harboring.either.KRAS.or.BRAF.mutations.were.tested.for.AREG.expression.by.Real-Time.PCR.and.ELISA.following.interference.with.oncogenic.pathways.Interestingly,.HCT116.cells.revealed.an.unusual.methylation.dependent.regulation.of.AREG..DKO,.a.DNMT1.and.3b.knockout.cell.line,.expressed.significantly. less.AREG.than. its.parental.cell. line.HCT116..Thus,.deme-thylation.leads.to.a.decrease.of.AREG.expression..When.testing.the.me-thylation.pattern.of.the.AREG.gene,.we.found.no.methylation.at.the.pro-moter.site,.however.there.is.a.CpG-rich.region.covering.exon.2.,.where.differential.methylation.occurs.in.different.cell.lines..This.in-gene-me-thylation.could.be.an.epigenetic.regulatory.element.for.the.AREG.gene..Further.experiments. in.human.xenografts.derived. from.CRC,. indicate.that.there.is.a.connection.between.sensitivity.towards.EGFR.targeted.therapeutics. and. the. expression. of. Amphiregulin.. Whether. in-gene-methylation.of.AREG.is.an.indicator.for.this.correlation.is.currently.in-vestigated..Together,. these.results.suggest,. that.the.outcome.of. that.therapies. in.colorectal. cancer.might.be. influenced.by.epigenetic.me-chanisms.

AEK

47


Notes:

P-711

Kuznia, Christina (Charité, Berlin); Klinger,.Bertram.(Charité,.Berlin);.Keil,. Jana. (Charité,. Berlin);. Seliger,. Barbara. (University,. Halle./.S.);..Sers,.Christine.(Charité,.Berlin)

Mechanisms of apoptosis induction by Sorafenib in human tumor cell lines

The. RAS/MEK/ERK. pathway. is. a. key. oncogenic. pathway. involved. in.human.cancer..Point.mutations. in.RAS.genes. resulting. in.constitutive.activation.of. the.RAS.proteins.occur. in.about.30%.of.all.human.can-cers..RAS.activation.results.in.an.overactivation.of.signaling.pathways,.thereby.inducing.cell.proliferation.and.invasion.but.also.preventing.apo-ptosis..We.investigated.the.Raf.kinase.inhibitor.Sorafenib.and.the.MEK.inhibitor.U0126.in.human.embryonal.kidney.cells.transformed.by.onco-genic.HRAS..Both.substances.target.the.RAF/MEK/ERK.pathway;.howe-ver,. treatment.with.Sorafenib. induces.apoptosis.while.U0126.addition.does.not..A.time-resolved.analysis.of.this.effect.using.protein.and.mRNA.arrays,.revealed.an.involvement.of.Redd1/DDIT4.and.mTOR.signaling.in.the.differential.action.of.Sorafenib.and.U0126..To.investigate.whether.mTOR.signaling.and.Redd1/DDIT4.expression.is.induced.by.Sorafenib.in.human.cancer,.we.tested.six.different.human.renal.carcinoma.cell.lines.MZ1257RC,.MZ1795RC,.MZ1851LN,.MZ1851RC,.MZ1854RC.and.MZ1940RC.and.one.melanoma.cell.line.A375..Cells.were.treated.with.Sorafenib.or.U0126. and. apoptosis. induction. was. examined. under. the. microscope..For. a. quantitative. description. of. apoptosis,. cleaved. caspase-3. was.examined.by.flow.cytometry.. In.addition,.mRNA.expression.of.Redd1/DDIT4.was.analyzed.using.Real-Time-PCR..Cell.lines.1851RC,.1854RC.and.1940RC.were.badly.damaged.after.treatment.with.Sorafenib,.while.this.was.not.observed.after.U0126.addition..The.same.cells.also.showed.a.significant. increase. in.cleaved.caspase-3.and. in.expression.of.Redd1/DDIT4.within. 12.hours.. The. results.of. this. study.encourage. the.hypo-thesis.that.interference.with.the.mTOR.pathway.by.induction.of.Redd1/DDIT4.might.play.an.important.role.during.the.apoptosis. induction.by.Sorafenib.in.human.renal.cancer.

P-712

Seliger, Barbara; Leich,.Franziska;.Giersberg,.Corinna;.Müller,.Anja.(Uni-versity,.Halle./.S.)

The effects of tyrosine kinase inhibitors on growth properties and immunogenicity of human renal cell carcinoma and melanoma cells

The.orally.available.small.molecule.tyrosine.kinase.inhibitors.(TKI).so-rafenib,. sunitinib. and. axitinib. preferentially. inhibit. the. vascular. and.endothelial.growth.factor.receptors,.the.platelet-derived.growth.factor.receptor.and.c-Kit.in.addition.to.other.kinases..It.has.been.shown.that.the.three.TKIs.exert.significant.anti-angiogenic.and.anti-tumor.effects..Furthermore,.the.treatment.of.patients.with.these.agents.showed.a.mo-dulation.of.circulating.angiogenesis.markers.as.well.as.a.reduction.of.the.tumor.metabolism..We.here.compared.the.effects.of.all.three.sub-stances.on.the.tumor.growth,.apoptosis.and.immune.modulatory.mole-cules.of.renal.cell.carcinoma.(RCC).and.melanoma.cell. lines..All.three.TKIs.exert.distinct.dose-dependent.anti-proliferative.and.anti-apoptotic.effects.on.the.human.tumor.cell.lines.analyzed,.which.were.associated.with.a.distinct.protein.expression.pattern..In.addition,.immune.modula-tory.molecules,.including.MHC.class.I.surface.antigens.and.B7-H.mole-cules,.were.modulated.by.these.substances,.suggesting.an.effect.on.the.immunogenicity.of.the.different.tumor.cell.lines..A.better.understanding.of.the.mechanisms.involved.in.the.modulation.of.growth.properties.as.well.as. immunogenicity.will.provide.the.basis. for.combination.strate-gies. and. should. enhance. the. clinical. effect.. 1. Molecular. diagnostics.and.targeted.therapy

48


P-713

Becker, Michael (Experimental Pharmacology & Oncology, Berlin); Hinzmann,. Bernd. (Signature. Diagnostics. AG,. Potsdam);. Pechanska,..Paulina. (Signature. Diagnostics. AG,. Potsdam);. Pauli,. Roland. (Institute.of. Pathology,. Brandenburg);. Mantke,. René. (Institute. of. Pathology,.Brandenburg);. Hertel,. Kay. (Institute. of. Pathology,. Erfurt);. Pertschy,.Jörg. (Dept..of.General-.and.Visceral.Surgery,.Erfurt).Hellwig,.Karsten.(Institute. of. Pathology,. Magdeburg);. Ridwelsji,. Kartsen. (Institute. of.Pathology,. Magdeburg). Radke,. Cornelia. (DRK-Kliniken,. Berlin);. Pross,.Matthias.(DRK-Kliniken);.Adams,.Hans-Peter.(Signature.Diagnostics.AG,.Postdam);.Mayr,.Tobias.(Signature.Diagnostics.AG,.Potsdam);.Rosenthal,..André. (Signature. Diagnostics. AG,. Potsdam);. Hoffmann,. Jens. (Experi-mental. Pharmacology. &. Oncology,. Berlin);. Fichtner,. Iduna. (Max-Del-brück-Center,.Berlin)

Establishment of a large panel of „early“ colon carcinoma xeno-grafts as a preclinical tool for identification of predictive biomarkers

The.development.and.growth.of.tumours.is.based.on.a.complex.process.as.it.results.from.a.combination.of.several.genetic.alterations.and.in-dividual.cellular.and.environmental.factors..In.addition,.there.is.a.high.molecular. variability.even.among. tumours.of. the. same.classification..Each.tumour. is. individual.and.every.patient.will.react.differently.to.a.particular.treatment..Accurate.prediction.of.response.to.therapy.is.the-refore.a.prerequisite.for.individualized.approaches.to.colorectal.cancer.treatment.Human.tumour.xenografts.directly.derived.from.patient.cancer.speci-men.can.provide.a.preclinical.research.alternative.considering.both.he-terogeneity.and. individuality.of.malignomas..Xenografts.allow.to. test.novel.anti-tumour.agents.in.a.fast.and.standardised.manner.and.provide.sufficient.tumour.tissue,.even.post.treatment,.for.the.search.of.corre-sponding.predictive.biomarkers..During.a.two.year.period,.240.primary.colon.carcinoma.tissue.samples.representing.all.four.Dukes.stages.were.collected.by.a.network.of.col-laborating.clinics..Tumour.pieces.were.transplanted.onto.immunodefi-cient.mice.immediately.after.surgery.using.a.standardised.procedure..Therefrom,.a.panel.of. 148.stably.passagable.colon.cancer.xenografts.could.be.established.as.permanent.tumour.models..These.patient-de-rived.colon.cancer.models.feature.a.high.coincidence.with.the.original.tumour.regarding.histology.and.genome-wide.gene.expression.profiling..They.are.subjected.to.an.extensive.molecular.and.pharmacological.cha-racterization..The.pharmacological.testing.performed.so.far.with.75.of.the.xenografts.resulted. in.the.following.therapy.response.rates.for.single.drug:.oxa-liplatin.7%,.cetuximab.25%,.and.bevacizumab.3%..About.59%.of.tu-mour.models.carry.at.least.one.mutation.in.one.of.the.three.hotspots.relevant.for.EGFR-targeted.therapy.(Kras-,.BRAF-.and.PIK3CA.gene).as.conducted. by. allele-specific. RT-PCR.. We. could. confirm. the. frequently.described. finding. that. mutations. in. the. Kras. gene. and. cetuximab. re-sponse.are.strongly.alternative.and.that.Kras.and.BRAF.mutations.ex-clude.each.other..The.corresponding.primary.tumours.showed.the.same.mutation.profile.By.characterizations.performed.so.far.we.could.prove.our.xenografts.to.be.valid.preclinical.models.of.their.originals.

P-714

Armbruster, Holger (German Cancer Research Center, Heidelberg); Starmann,.Julia.(German.Cancer.Research.Center,.Heidelberg);.Bender,.Christian. (German. Cancer. Research. Center,. Heidelberg);. Korf,. Ulrike.(German.Cancer.Research.Center,.Heidelberg);.Sers,.Christine.(Charité,.Berlin);.Sültmann,.Holger.(German.Cancer.Research.Center,.Heidelberg)

Identification and functional analyses of tumor-associated and the-rapy-relevant proteins in colorectal carcinomas

Despite. of. novel. targeted. therapy. approaches. and. new. combination.therapies.of.conventional.chemotherapeutics,.many.colorectal.cancer.patients.can.still.not.be.treated.efficiently..Ineffective.therapies.cause.undesirable.side.effects.and.generate.high.costs..Although.EGFR. tar-geting.drugs,. like.Cetuximab.and.Panitumumab,.are.only.applied.if.no.activating.mutations.in.the.KRAS.and.BRAF.genes.(downstream.of.EGFR).are.detected,.the.response.rates.hardly.reach.30%..This.project.aims.at.an.improved.understanding.of.the.signal.transduction.processes.un-derlying.the.sensitivity.and.resistance.towards.drugs.targeting.EGFR..To.this.end,. four.colorectal.carcinoma.cell. lines.(HCT116,.SW403,.LIM1215.and.HT29).were.treated.with.all.combinations.of.six.small.molecule.in-hibitors.and.seven.activators,.targeting.signaling.proteins.of.the.MAPK-,.PI3K/AKT-,. WNT-. and. JAK/STAT-pathways.. The. effects. of. the. inhibitors.and.activators.on.these.pathways.were.analyzed.using.the.reverse.pha-se.protein.array.(RPPA).technology..With.this.high.throughput.protein.detection.method.we.are.currently.able. to.detect.more. than.80.pro-teins.from.cancer.relevant.signaling.pathways..Furthermore,.the.acti-vation.of.key.proteins.can.be.analyzed.using.phosphorylation.specific.antibodies..In.agreement.with.literature.data,.the.RPPA.data.revealed.a.strong.correlation.between.the.SW403.and.LIM1215.cell.lines.concerning.overall.protein.expression..In.SW403.and.LIM1215.cells,.an.activation.of.the.MAPK.pathway.was.observed.upon.stimulation.with.TGFα,.a.ligand.of.EGFR..This.activation.was.blocked.by.AZD6244.and.U0126,.both.inhi-bitors.of.MEK..In.contrast,.TGFα.could.not.activate.the.MAPK-pathway.in.HCT116.cells.. IGF1,.a. ligand.of. IGFR,.had.a.much.stronger.effect.on.the.PI3K/AKT-pathway.in.HCT116.cells,.compared.to.the.other.cell.lines..The.PI3K/AKT-pathway.activation.could.not.be.blocked.by.the.PI3K.in-hibitor.LY294002..In.summary.we.found.significant.different.effects.on.intracellular. signal. transduction. in. HCT116,. SW403,. LIM1215. and. HT29.colorectal.cancer.cell. lines..These.differences.may.also.contribute. to.resistance.or.sensitivity.towards.drugs.targeting.EGFR.

Notes:

AEK

49


Notes:

P-715

Bednarz, Natalia (University Medical Center, Hamburg); Semjonow,.Axel. (University. Clinic,. Münster);. Eltze,. Elke. (Institute. of. Pathology,.Saarbrücken);. Pantel,. Klaus. (University. Medical. Center,. Hamburg);.Brandt,.Burkhard.(University.Medical.Center,.Hamburg)

Gains of EGFR gene and their clinical relevance in prostate cancer patients

EGFR. plays. a. pivotal. role. in. variable. cellular. processes. including. cell.signalling,. cell. cycle. regulation. and. migration.. Its. amplification. and.overexpression.are.reported.to.be.associated.with.higher.invasiveness.and.poor.prognosis. in.different. types.of. tumors..Some.screening.and.in.vitro.studies.suggest.that.also.in.prostate.cancer.it.might.be.useful.in.diagnosis.of.patients.with.more.advanced.disease.and.serve.as.the-rapeutic.target..Therefore,.the.goal.of.the.current.study.was.to.assess.the.prevalence.of.EGFR.gains.in.prostate.cancer.and.to.determine.their.clinical.relevance.as.well.as.basic.molecular.characteristics.of.tumors.demonstrating.such.genotype.EGFR.gene.dosage.was.assessed.and.compared.to.clinical-pathological.data. and. selected. protein. expression. (proliferation. marker. Ki-67,. CK.5/6,. 14,.8/18.and. 19)..398.tumors.corresponding. to. 199.patients.were.examined.on.tissue.microarray.sections.by.the.usage.of.fluorescent.in.situ.hybridization.and. immunohistochemistry.. .Additionally,. 11.pairs.of.corresponding.primary.tumors.and.lymph.node.metastases.were.analy-sed.for.allelic.imbalances.of.EGFR.with.the.usage.of.PCR.EGFR.gains.were.detected.in.13.(11%).of.121.evaluable.prostate.cancer.patients..They.co-existed.frequently.with.centromer.7.gains..EGFR.gains.correlated.to.higher.Gleason.score,.higher.T.status.and.lymph.node.po-sitivity. (p<0.05)..Allelic. imbalances.of.EGFR.gene.were.observed. in.6.(55%).of.11.pairs.of.primary.tumor.and.corresponding.lymph.node.meta-stasis..When.analysed.in.individual.tumors,.EGFR.gains.were.associated.with.proliferation.marker.Ki-67.(p<0.001)..Tumors.with.such.a.genotype.expressed.also. less. frequently. luminal.CK8/18.(p<0.05).and.more.fre-quently.basal.CK5/6.(p>0.05)..In.conclusion,.EGFR.gains.were.detected.in.a.small.subset.of.prostate.cancer.patients.which.were.characterised.by.a.more.advanced.tumor.stage.and.aggressive.phenotype..Thus,.EGFR.gene.gains.might.be. im-portant.for.prostate.cancer.progression..

P-716

Kahn, Nicolas (German Cancer Research Center, Heidelberg); Meis-ter,. Michael. (University. of. Heidelberg,. Heidelberg);. Eberhardt,. Ralf.(University. of. Heidelberg,. Heidelberg);. Muley,. Thomas. (University. of.Heidelberg,.Heidelberg);.Schnabel,.Philipp.A..(University.of.Heidelberg,.Heidelberg);.Bender,.Christian.(German.Cancer.Research.Center,.Heidel-berg);.Johannes,.Marc. (German.Cancer.Research.Center,.Heidelberg);.Keitel,. Denise. (German. Cancer. Research. Center,. Heidelberg);. Herth,.Felix.J..(University.of.Heidelberg,.Heidelberg);.Sültmann,.Holger.(Ger-man.Cancer.Research.Center,.Heidelberg);.Hoffmann,.Hans.(University.of.Heidelberg,.Heidelberg);.Kuner,.Ruprecht.(German.Cancer.Research.Center,.Heidelberg)

Early detection of lung cancer by molecular markers in endobronchi-al lining fluid

Molecular. biomarkers. in. tissues. and. body. fluids. represent. a. promi-sing. source. to. improve. cancer. diagnosis. and. risk. assessment.. In. the.lung,.early.detection.of.malignancies.by.less.invasive.methods.aims.at.achieving. efficient. intervention. and. subsequently. a. reduction. of. the.high. mortality. rate.. We. investigated. whether. biomarker. analysis. in.endobronchial. epithelial. lining. fluid. (ELF). collected. by. bronchoscopic.microsampling. may. be. useful. for. a. definitive. preoperative. diagnosis..ELF.was.collected.from.subsegmental.bronchi.close.to.the. indetermi-nate.pulmonary.nodule,.which.was.detected.by.CT.scans,.and.from.the.contralateral.lung..Diagnosis.was.confirmed.by.transbronchial.biopsy.or.surgery..The.study.includes.51.non-small.cell.lung.cancer.patients.and.20.benign.cases..Biomarker.candidate.selection.was.based.on.microar-ray.analysis.of.a.subset.of.ELF.samples..Thirteen.potential.biomarkers.have.been.further.analyzed.by.qRT-PCR.in.independent.ELF.samples..No-tably,.one.biomarker.candidate.was.clearly.up-regulated.in.ELF.samples.of.NSCLC.patients.independent.of.the.tumor.subtype..Combined.analysis.of.clinical.parameters.like.nodule.size.and.biomarker.expression.further.improved.the.prediction.of.early.malignancies..In.conclusion,.our.study.indicates.that.specific.marker.genes.in.ELF.collected.by.bronchoscopic.microsampling.may.be.useful.to.distinguish.between.malignant.and.be-nign.pulmonary.nodules.

50


Notes:

P-717

Christmann, Markus;.Nagel,.Georg;.Horn,.Sigrid;.Krahn,.Ulrike;.Wiewrodt,.Dorothee;. Sommer,. Clemens;. Kaina,. Bernd. (University. Medical. Center,.Mainz)

MGMT activity, promoter methylation and immunohistochemistry of pre-treatment and recurrent malignant gliomas

The. DNA. repair. protein. O6-methylguanine-DNA. methyltransferase.(MGMT).removes.methyl.groups.from.the.O6-position.of.guanine..If.not.repaired,.O6-methylguanine.leads.to.the.induction.of.cell.death..Several.anticancer.drug,.such.as.temozolomide,.procarbazine,.ACNU,.CCNU.and.fotemustine.utilize.the.cytotoxic.properties.of.O6-alkylguanine.adducts.to.kill.cancer.cells..The.therapeutic.outcome.of.therapy.with.so-called.O6-alkylating.agents.is.decisively.determined.by.the.amount.of.active.MGMT.molecules.present.in.tumour.cells..Besides.direct.measurement.of.MGMT.activity,.determination.of.the.methylation.status.of.the.MGMT.promoter. and. immunohistochemistry. can. be. used. for. the. evaluation.of.the.MGMT.status..However,.there.is.still.controversy.whether.MGMT.promoter.methylation.correlates.with.MGMT.activity.and.immunohisto-chemistry..It.is.also.unknown.whether.MGMT.promoter.methylation.and/or.MGMT.activity.of.the.pre-treatment.tumor.predict.the.MGMT.status.of.recurrences..To.address.these.questions,.we.determined.in.pre-treat-ment.and.recurrent.glioblastoma.multiforme.(GBM).MGMT.activity,.pro-moter.methylation.and.immunohistochemistry..We.show.that.GBM.that.were.promoter.methylated.display.a.range.of.0-62.fmol/mg.MGMT,.and.tumors.that.were.non-methylated.0-423.fmol/mg.protein..For.astrocy-tomas,.promoter.methylated.samples.displayed.0-28.fmol/mg,.and.non-methylated.samples.23-107.fmol/mg..No.correlation.was.found.between.the.intensity.of.promoter.methylation.and.MGMT.enzyme.activity..Given.a.threshold.level.of.30.fmol/mg.protein.we.found.a.correlation.between.promoter. methylation. (yes/no. decision). and. no/low. MGMT. activity. in.82.4%.of.the.tumors..This.high.correlation.level.is.only.achieved.when.tumors. were. excluded. showing. a. hemi-methylated. promoter. (aprox..20%).. Therefore,. classification. of. hemi-methylated. tumors. as. to. the.MGMT.status.remains.questionable..Further,.we.show.that.39.1%.of.pre-treatment. GBM. and. 5.3%. of. recurrences. were. promoter. methylated,.which.is.in.line.with.increase.of.MGMT.activity.in.recurrences..Although.individual.exceptions.were.found,.the.data.show.an.overall.correlation.between.promoter.methylation.and.lack/low.MGMT.activity.(≤30.fmol/mg.protein).in.GBM.and.astrocytomas..Promoter.methylation.was.supe-rior.over.immunohistochemistry.in.determining.the.MGMT.status.

Work.was.supported.by.German.Cancer.Research.Foundation.and.DFG

P-718

Chen, Yuan; Yang,.Linlin;.Cui,.Tiantian;.Knoesel,.Thomas;.Petersen,.Iver.(University,.Jena)

Identification of biomarkers to distinguish clear cell sarcoma from malignant melanoma

Clear.cell.sarcoma.(CCS).or.malignant.melanoma.of.soft.parts.is.a.rare.malignancy. that. shows. phenotypic. and. immunohistochemical. overlap.with. cutaneous. malignant. melanoma. (MM),. and. sometimes. it. is. diffi-cult. to. distinguish. one. from. another.. Identification. of. biomarkers. to.differentiate.between.the.two.malignancies.is.therefore.needed..In.this.study,.we.analysed.the.mutation.status.of.BRAF.and.NRAS,.EWSR1.gene.rearrangement,.and.the.protein.expression.of.IGF2.as.well.as.IGF-1R.in.formalin-fixed.paraffin-embedded.tissues.(FFPE).of.16.CCS.and.31.MM..By.direct.sequencing.and.high.resolution.melting.analysis.(HRM),.we.found.that.51.6%.(16.out.of.31).and.12.9%.(4.out.of.31).of.MM.showed.BRAF.and.NRAS.mutations. respectively,.while.none.of.CCS.harboured.BRAF.and.NRAS.mutations..Meanwhile,.we.analyzed.the.EWSR1.rearrangement.by.fluorescence.in.situ.hybridization.(FISH)..It.turned.out.that.78.6%.(11.out.of.14.informative.cases).of.CCS.exhibited.the.t(12;22)(q13;q12).trans-location..The.presence.of.type.1,.2,.and.3.EWSR1/ATF1.fusion.gene.tran-scripts.were.confirmed.in.FISH-positive.patients.by.RT-PCR.analysis..In.contrast,.no.fusion.transcripts.could.be.detected.in.MM.samples..Additi-onally,.we.evaluated.the.IGF2.and.IGF-1R.protein.expression.in.these.two.malignancies.by.immunohistochemistry.on.tissue.microarray..We.found,.that.both.IGF2.and.IGF-1R.were.expressed.in.melanoma.and.CCS,.howe-ver,.in.comparison.of.melanoma,.the.expression.of.IGF2.and.IGF-1R.was.higher.in.CCS.(p=0.0001.and.p=0.006,.respectively)..Our.results.suggest.that.combination.of.BRAF.and.NRAS.mutation.analysis.with.EWSR1/ATF1.fusion.gene.detection.contributes.to.diagnosis.of.MM.and.CCS..Since.IGF.system.is.emerging.as.a.new.target.in.cancer.therapy,.high.expression.of.IGF2.and.IGF-1R.in.melanoma.and.CCS.indicates.the.potential.clinical.utility.of.their.inhibitors.in.treatment.of.these.two.malignancies.

AEK

51


Notes:

P-719

Schultz1.S.;.Bartsch2.H.;.Sotlar2.K.;.Petat-Dutter2.K.;.Bonin3.M.;.Poths3.S.;.Walter3.M.;.Riess3.O.;.Wallwiener1.D.;.Fehm1.T.;.Neubauer1.H.

1)..Department.of.Obstetrics.and.Gynecology,.Eberhard-Karls-University,.Tuebingen

2)..Institute.for.Pathology,.Ludwig-Maximilians-University,.Munich3)..Microarray.Facility.Tuebingen,.Eberhard-Karls-University,.Tuebingen

Differential expression of mRNAs and miRNAs during the progression from DCIS to IDC in breast cancer

Background:.The.ductal.carcinoma.in.situ.(DCIS).of.the.breast.is.consi-dered.to.be.the.pre-invasive.form.of.the.invasive.duct.carcinoma.(IDC)..The.analysis.of.molecular.mechanisms.is.an.important.prerequisite.to.understand.the.biology.of.tumor.progression..Aims:.Aims.of.this.project.were.to.(1).identify.and.validate.miRNAs.and.mRNAs.that.are.differentially.expressed.between.DCIS.and.IDC,.and.(2).to.identify.miRNAs.or.mRNAs.that.detect.aggressive.DCIS.with.a.high.potential.invasive.growth.Material/Methods:. In. formalin. fixed. and. in. parafin. embedded. (FFPE).breast. cancer. tissues. were. selected. and. examined:. (a). 15. tissue.samples.with. ‘pure’.DCIS.without.detectable. IDC.component.obtained.from.patients.with.a.recurrence-free.survival.of.at.least.five.years,.and.(b).15.tissue.samples.with.mixed.DCIS/IDC.tumors..Tissue.sections.were.prepared.and.stained.with.hematoxylin-eosin..After.characterization.of.the. tissue. sections. by. a. pathologist,. tumor. cells. were. isolated. using.laser.capture.microdissection.(LCM)..RNA.was.extracted.with.the.High.Pure.miRNA. Isolation.kit. (Roche),.200.ng.of.which.was.hybridized. to.the. ‘Universal.Bead.miRNA.chip’.or.to.the. ‘Whole-Genome.DASL.Gene.Expression.bead.chip’.(both.Illumina)..Bioinformatic.analysis.was.per-formed.with.GeneSpring.GX.(Agilent)..The.expression.of.selected.miRN-As.and.mRNAs.was.validated.by.qRT-PCR.(Roche.LightCycler®.System).Results:.We.identified.32.miRNAs.which.were.differentially.expressed.bet-ween.‘pure’.DCIS.and.the.DCIS.component.of.DCIS/IDC.mixed.tumors.and.16.miRNAs.which.were.differentially.expressed.between.the.two.compon-ents.of.DCIS/IDC.mixed.tumors..Regarding.mRNAs.700.and.691.differenti-ally.expressed.transcripts.were.found,.respectively..Quantitative.RT-PCR.confirmed. increased. expression. of. Mmp11. in. DCIS. with. IDC. component.compared.to.‘pure’.DCIS..Between.DCIS.and.IDC.Thbs2,.Cspg2,.Krt14,.and.Pleckhc1. were. differentially. expressed.. Hsa-miR-214. and. hsa-miR-199a.were.upregulated.in.IDC.compared.to.the.DCIS.areas.of.the.same.tumor.Conclusion:.We.successfully.analyzed.miRNA.and.mRNA.of.FFPE.breast.cancer.tissues.using.LCM.and.microarray.technology..Further.functio-nal.analyses.are.under.way.to.evaluate.the.biologic.importance.of.the.differentially.expressed.miRNAs.and.mRNAs.as.prognostic.markers.for.breast.cancer.progression.

P-720

Hoffmann1.J.;.Walter1..M.;.Staebler2.A.;.Poths1.S.;.Riess1.O.;.Wallwiener3.D.;Fehm3.T.;.Neubauer3.H.;.Bonin3.M.

1)..Microarray.Facility.Tuebingen,.Eberhard-Karls-University,.Tuebingen2)..Institute.for.Pathology,.Eberhard-Karls-University,.Tuebingen3)..Department.of.Obstetrics.and.Gynecology,.Eberhard-Karls-University,.

Tuebingen

Identification of new targets of cisplatin resistance in ovarian cancer patients using combined transcriptome and methylation analyses

Background:. Ovarian. cancer. is. one. of. the. most. malignant. tumors. in.women. and. the. leading. cause. of. death. from. gynecological. cancer. in.the. Western. civilization.. The. standard. therapy. consists. of. aggressive.cytoreductive. surgery. followed. by. platinum-based. chemotherapy.. In.addition.to.the.advanced.stage.of.the.disease.at.the.time.of.diagnosis.in.most.instances,.the.intrinsic.or.acquired.platinum.resistance.in.the.majority.of.patients.leads.to.its.high.mortality.rate.Aims:.To.gain.new.insights.in.resistance.mechanisms.and.modified.pa-thways,. we. performed. combined. whole-genome. expression. and. me-thylation.studies.using.Illumina.BeadArrays..Material.and.Methods:.Eleven.cryopreserved.tissues.samples.from.each.platinum.resistant.and.platinum.sensitive.ovarian.carcinomas.were.se-lected..Tissue.sections.(10µm).were.prepared,.stained.with.hematoxi-lin/eosin.and.characterized.by.a.pathologist..Only.samples.with.serous.ovarian. cancers. containing. at. least. 50%. malignant. tissue. were. pro-cessed.further..From.consecutive.sections.RNA.and.DNA.was.extracted.and.analysed.for.differences. in.gene.expression.and.methylation.pat-terns. by. using. Illumina. BeadArray. platforms.. Results. obtained. were.confirmed.using.qRT-PCR.and.pyrosequencing.Results:. We. assessed. the. DNA. methylation. profile. of. approx.. 27,000. CpG.sites.(associated.with.approx..14000.transcripts).and.found.613.differenti-ally.methylated.promoter.sites.using.M-value.statistics..To.investigate.the.relationship.between.DNA.methylation.status.and.gene.expression,.we.mea-sured.levels.of.transcripts.from.the.same.set.of.genes.in.the.same.samples,.using.an.Illumina.whole.genome.expression.array..After.normalization.and.biostatistical.analysis.of.these.arrays.we.detected.115.significantly.differ-entially.expressed.transcripts.with.a.fold.change.>1.6.and.a.p-value.<0.05..Promoter.DNA.methylation.and.levels.of.the.corresponding.gene’s.transcript.have.been.found.to.be.inversely.correlated.in.some.cases..Overall,.we.were.able.to.detect.14.negativly.correlated.transcripts..Further.characterization.of.these.genes.by.pathway.and.network.analysis.strong.enrichment.of.genes.involved.in.cell.differentiation.and.tumorigenesis.processes.was.obtained.Conclusion:. By. combining. whole-genome. expression. and. methylation.analyses.we.reveal.molecular.changes.in.ovarian.cancers.which.might.be.involved.in.establishing.platinum.resistance..Functional.verification.and.pathway.analysis.is.under.way.

52

AEKAEK AEKAbstracts Short Talks Novel Approaches to Therapy

Notes:

short talk 801

Eke, Iris; Förster,.Claudia;.Cordes,.Nils.(OncoRay,.Dresden)

The radiosensitizing effect of Cetuximab is attenuated by activation of JNK2 signaling

Tumor.cell.response.to.radiotherapy,.cytotoxic.drugs.or.novel.molecular.therapeutics.like.the.inhibitory.EGFR.antibody.Cetuximab.is.modulated.by.cell-matrix.interactions..The.molecular.mechanisms.which.contribute.to.these.effects.are.still.largely.unknown..The.aim.of.this.study.was.to.compare.the.impact.of.Cetuximab.on.the.radiosensitivity.and.the.signal.transduction.of.conventional.two.dimensional.monolayer.squamous.cell.carcinoma.(SCC).cultures.(2D).with.cells.grown.in.a.three.dimensional.extracellular.matrix.(3D)..Colony.formation.assays,.siRNA.transfections,.Phosphoproteom. arrays,. Western. blotting,. immunoprecipitations. and.mass.spectrometry.analysis.were.performed..While.under.2D.conditions.treatment.with.Cetuximab.was.radioprotective,.Cetuximab.significant-ly. radiosensitized.3D.SCC.cultures.dependent.on.EGFR-integrin. linker.proteins.PINCH1.and.Nck2..As.we.observed.enhanced.phosphorylation.of.JNK2.and.the.downstream.molecule.c-Jun.after.Cetuximab.we.de-pleted. or. deactivated. JNK2. with. siRNA. or. a. small. molecule. inhibitor.(SP600125;. 10.µM).and.analyzed.the.efficacy.of.Cetuximab.on.clono-genic.survival..Interestingly,.both.approaches.synergistically.increased.the.cellular.sensitivity.to.X-ray.irradiation.and.radiosensitization.by.Ce-tuximab.. Immunoprecipitations. revealed. a. direct. interaction. between.EGFR.and.the.JNK.activator.JIP-4.which.might.be.responsible.for.the.hy-perphosphorylation.of.JNK2.upon.Cetuximab.treatment..Our.data.show.that. cell-matrix. interactions. impact. on. the. radiosensitizing. potential.of.Cetuximab. in.a.PINCH1.and.Nck2.dependent.manner..By.activation.of.JNK2,. .Cetuximab.clearly.reduced. its.own.efficacy.suggesting.that.a.combined.inhibition.of.EGFR.and.JNK2.will.be.beneficial.for.patients.with.SCC.receiving.radiotherapy.

short talk 802

Fritz, Gerhard; Hamalikic,. Melanie;. Kaina,. Bernd. (University. Medical.Center,.Mainz)

Radiotherapy stimulates pro-metastatic normal tissue responses, which are attenuated by the lipid-lowering drug lovastatin

Extravasation.of.circulating.tumor.cells.is.an.essential.step.in.metasta-sation.. In.order.to.escape.from.the.blood.vessel,. tumor.cells.have.to.overcome. the.endothelial. cell. barrier.. Therefore,. tumor. cell. adhesion.to. the.endothelium,.which. is.mediated.by.endothelial.adhesion.mole-cules.such.as.E-selectin.and.tumor.specific.counterligands,.is.required..The.expression.of.E-selectin.is.induced.by.ionizing.radiation.(IR)..This.brings.concern.about.possible.pro-metastatic.adverse.effects.of.radi-otherapy..Here,.we.analyzed.the. impact.of.pharmacological. inhibition.of.Rho.GTPases.by.lovastatin.on.IR-induced.pro-metastatic.processes.in.vitro.and.in.vivo..We.show.that.IR.exposure.of.primary.human.umbilical.vein.endothelial.cells.(EC).stimulated.the.expression.of.different.pro-adhesive.factors.and.increased.the.adhesion.of.human.colon.carcinoma.(TC).cells.to.EC..Induction.of.cell.adhesion.molecules.and.increase.in.EC-TC.adhesion.were.attenuated.by.pre-treatment.of.the.endothelial.cells.with. the. lipid-lowering. drug. lovastatin.. Pre-treatment. of. tumor. cells.with. lovastatin.was. ineffective.. To. scrutinize. the. in. vivo. relevance.of.these.data,.we.investigated.the.effect.of.total.body.irradiation.(TBI).on.tumor.cell.extravasation.and.metastasation.in.vivo..To.this.end,.tumor.cells.were. injected. into.the. lateral. tail.vein.of. immunodeficient.mice,.followed.by.total.body.irradiation.(TBI)..After.4.weeks,.the.formation.of.lung.metastases.was.monitored..The.results.of.extensive.experiments,.comprising.two.different.mouse.strains.and.tumorigenic.cell.lines,.con-cordantly.show.that.IR.largely.increases.the.number.of.lung.metastases..Notably,.enhanced.metastasation.was.also.observed.after.injection.on.non-irradiated. tumor. cells. into. pre-irradiated. mice.. This. shows. that.normal.tissue.responses.following.irradiation.are.sufficient.to.provoke.tumor.cell.extravasation.and.metastasis..TBI-stimulated.metastasation.was.blocked.by.pre-administration.of. lovastatin..A.model.of. IR-driven.metastasation. is. presented.. Summarizing,. we. provide. evidence. that.ionizing.radiation. increases.EC-TC. interactions. in.vitro.and,.moreover,.stimulates. the. extravasation. of. circulating. tumor. cells. in. vivo.. These.pro-metastatic.IR.effects.are.driven.by.normal.tissue.responses.and.are.mitigated.by.lovastatin..The.data.provide.novel.evidence.that.radiation.exposure.increases.the.metastatic.risc..Co-administration.of.clinically.widely. used. lipid-lowering. drugs. is. suggested. to. counteract. this. ad-verse.radiation.effect.

AEK

53

AEKAbstracts Poster Novel Approaches to Therapy

Notes:

P-801

Müller, Nina (Georg-Speyer-Haus, Frankfurt/M.); Hartmann,. Cord.(Georg-Speyer-Haus,. Frankfurt/M.);. Koch,. Joachim. (Georg-Speyer-Haus,.Frankfurt/M.);.Lissanu-Deribe,.Yonathan.(University.of.Frankfurt,.Frankfurt/M.);.Dikic,. Ivan.(University.of.Frankfurt,.Frankfurt/M.);.Wels,.Winfried.(Georg-Speyer-Haus,.Frankfurt/M.)

Antibody-fusion proteins targeting the intracellular domain of epi-dermal growth factor receptor

The.epidermal.growth.factor.receptor.(EGFR).plays.an.essential.role.in.the.regulation.of.cellular.processes.such.as.proliferation,.survival.and.migration..Aberrant.activation.of.EGFR.has.been.found.in.human.cancers.of.various.origin,.and.has.been.implicated.in.cancer.pathogenesis..We.previously.generated.single-chain.Fv.(scFv).antibody.fragments.specific.for.the.intracellular.domain.(ICD).of.EGFR,.which.were.functional.upon.expression.as.cytoplasmic.intrabodies.in.EGFR-positive.tumor.cells,.and.colocalized.with.EGFR.at.the.cell.membrane..Nevertheless,.direct.scFv-mediated.effects.on.EGFR.signaling.and.cell.growth.were.not.detected.Here.we.further.characterized.the.binding.specificities.of.the.scFv.an-tibodies,.which.recognized.an.identical.linear.peptide.in.peptide.array.analysis.corresponding.to.a.sequence.close.to.the.C-terminus.of.EGFR..To. investigate. the. potential. utility. of. the. scFv. molecules. for. specific.intracellular. targeting. of. heterologous. protein. domains. to. EGFR,. we.generated. a. chimeric. scFv-Cbl. fusion. protein.. The. E3-ubiquitin-ligase.c-Cbl.is.an.important.regulator.of.intracellular.signaling.pathways,.and.is.critically.involved.in.EGFR.downregulation..We.fused.the.RING.domain.of.c-Cbl.that.lacks.the.intrinsic.EGFR.binding.activity.of.the.protein.to.a.selected.EGFR-specific.scFv..The.resulting.scFv-Cbl-RING.fusion.protein.displayed.specific.binding. to.a. recombinant.EGFR. fragment.and.EGFR.from.tumor.cell.lysates.upon.bacterial.expression,.and.interaction.with.endogenous. EGFR. upon. expression. as. a. cytoplasmic. protein. in. mam-malian. cells.. Furthermore,. recombinant. scFv-Cbl-RING. mediated. the.ubiquitination.of.a.GST-EGFR.fusion.protein.in.a.cell-free.assay,.demons-trating.that.scFv-Cbl-RING.is.bifunctional..Ongoing.work.now.aims.at.the.further.functional.characterization.of.the.scFv-Cbl-RING.fusion.protein,.and.the.generation.of.similar.bifunctional.molecules.that.may.interfere.with.EGFR.signaling.

P-802

Zaharieva, Maya M. (German Cancer Research Center, Heidelberg); Kolev,.Andrey.(Medical.University.of.Sofia,.Sofia);.Konstantinov,.Spiro.M..(Medical.University.of.Sofia,.Sofia);.Berger,.Martin.(DKFZ,.Heidelberg)

The anti-apoptotic factor BCL-XL influences the antineoplastic acti-vity of erufosine in human leukemic cell lines

The.ether.lipid.analogue.erufosine.is.a.new.antineoplastic.agent.inter-fering. with. signal. transduction. and. conferring. apoptosis. in. leukemic.cells,.but.its.exact.mode.of.action.is.not.fully.understood.yet..The.aim.of.our.study.was.to.investigate.the.impact.of.the.anti-apoptotic.prote-in.BCL-XL.on.the.antineoplastic.activity.of.erufosine..We.compared.the.influence.of.erufosine.on.cell.proliferation,.apoptosis.and.autophagy.in.the.sensitive.lymphoid.cell. line.BV-173.and.the.resistant.myeloid.cells.K-562..Treatment.of.BV-173.cells.with.erufosine.(5-40.µM,.18.h).led.to.PARP-cleavage,.activation.of.caspases-3.and.-9,.and.inhibition.of.BCL-XL,.p-mTor,.p-Raptor,.p-PRAS40.and.p-p70S6K.as.shown.by.Western.blot..These.effects.were.not.observed.in.K-562.cells..Therefore.we.generated.a.stable.BCL-XL-knockdown.in.this.cell.line.using.a.lentiviral.system.and.studied.the.cytotoxicity.of.erufosine.(12.5-50.µM).at.conditions.of.80%.reduced.BCL-XL.protein.expression..Treatment.with.erufosine.reduced.the.cell.proliferation.(MTT-assay).and.clonogenicity.(CFU-assay).of.the.transformed.cells.by.more.than.50%.as.compared.to.the.wild.type.cells.or. the. NSO-control.. It. also. led. to. an. inhibition. of. the. mTor. signalling.pathway,.induction.of.apoptosis.and.autophagy.as.evidenced.by.Western.blot,.acridine.orange.staining.and.a.luminescence.assay.for.caspases-3.and.-7..In.addition,.we.combined.erufosine.with.As2O3.(0.156-0.625.µM).which.exerts.apoptosis. in.human.leukemic.cells.and.reduces.the.BCL-XL.expression. in.K-562.cells..An.additive.combination.effect.between.erufosine.and.As2O3.was.observed.in.K-562.cells..The.combination.was.more.effective.in.cells.with.BCL-XL-knockdown..In.conclusion,.our.data.indicate.that.erufosine’s.antineoplastic.activity.is.influenced.by.the.ex-pression.levels.of.BCL-XL..The.cytotoxicity.of.erufosine.in.leukemic.cells.with.high.BCL-XL.expression.could.be.increased.after.BCL-XL-knockdown.or.in.combination.with.As2O3.and.this.finding.could.be.of.relevance.for.the.clinical.application.of.erufosine.

54

AEKAEK AEKAbstracts Poster Novel Approaches to Therapy

Notes:

P-803

Kobelt, Dennis (Max Delbrück Center, Berlin); Schmidt,.Manuel.(MOLO-GEN.AG,.Berlin);.Aumann,.Jutta.(ECRC,.Berlin);.Schlag,.Peter.(Charité,.Berlin);.Walher,.Wolfgang.(Max-Delbrück-Center,.Berlin)

Employment of the MIDGE vector system for improved chemosensiti-zation by cancer gene therapy

In. a. clinical. phase. I. study. we. were. able. to. show. that. plasmid. based.vectors. are. safely. applicable. in. patients. using. the. jet-injection. tech-nology..However,.nonviral.vectors.need.further. improvement. in.terms.of.efficiency.and.safety..One.possible.way.to.achieve.this.is.the.vector.size.reduction.using.the.MIDGE.technology..Those.linear.vectors.contain.additionally.to.the.expression.cassette.small.end-sealing.hairpins.and.can.be.smaller.than.2kb..We.have.previously.shown.that.MIDGE-based.vectors. lead. to. higher. reporter. gene. expression. after. lipofection. or.electroporation.compared.to.other.vectors.like.plasmids.Using. TNF. alpha. (TNF). as. a. therapeutic. transgene. the. MIDGE. vector.shows.improved.expression.after.lipofection.and.electroporation.in.dif-ferent.cell.lines.(A375,.MeWo,.SkMel5,.SkMel28,.HCT116.and.SW480).in.vitro..To.determine.the.therapeutic.potential.of.combined.TNF.and.vin-desine.treatment,.the.survival.of.human.cancer.cell.lines.was.analyzed.after.TNF.gene.transfer.in.combination.with.increasing.concentrations.of.vindesine..We.show.that.MIDGE-mediated.TNF.expression.is.sensiti-zing. all. used. cancer. cells. lines. towards. vindesine. treatment.. In. vitro.analysis.of.caspase.3/7.as.apoptosis.marker.after.TNF.gene.transfer.in.combination.with.a.fixed.concentration.of.vindesine.showed,.that.the.increase.of. caspase.3/7.activation. is.accelerated.by. the.combination.treatment.In.vivo.we.were.able.to.show,.that.gene.transfer.of.MIDGE.based.vec-tors. leads. to. safe. and. efficient. transgene. expression. in. a. melanoma.xenograft.mouse.model..The.vector.was.rapidly.cleared.from.the.blood.and. led. to. TNF. levels. known. to. be. sufficient. for. therapeutic. effects..The.expressed.TNF.was.detectable.in.the.tumor.tissue.only..The.com-bination.of.MIDGE-based.TNF.gene.transfer.and.vindesine.showed.a.si-gnificant.decrease.of.tumor.growth..Detailed.analysis.showed.that.the.application.of.the.gene.therapy.vector.and.the.chemotherapeutic.drug.was.well.tolerated..There.were.no.significant.changes.in.body.weight,.temperature.and.blood.parameters..These.data.demonstrate.that.local.and. high-level. expression. of. TNF. sensitizes. tumors. towards. systemic.treatment.with.cytostatic.drugs,.such.as.vindesine.

P-804

Bayer, Helene (German Cancer Research Center, Heidelberg); Voss,.Cristina.(Heidelberg.Pharma,.Ladenburg);.Berger,.Martin.(DKFZ,.Heidel-berg)

Riproximin: comparison of its variants and investigation of the bin-ding specificity

The.lectin.riproximin.(Rpx).is.a.glycoprotein.that.was.initially.isolated.from.Ximenia.americana.African.plant.material.and.later.from.fruit.ker-nels.of.this.plant..It.was.characterized.as.potent.antineoplastic.agent.(Voss.et.al,.2006)..The.aim.of.this.study.was.to.compare.the.available.Rpx.variants. in.their.biological.activity.to.determine.the.most.potent.species.. Furthermore,. the. lectin. binding. specificity. of. Rpx. was. to. be.investigated.in.order.to.characterize.the.unknown.binding.partners.sin-ce.it.is.known.that.malignant.cells.express.aberrant.sugar.structures.For.the.characterisation.of.the.biological.activity.tumorigenic.MCF7.and.non-tumorigenic.MCF10A.cells.were.used,.established.from.human.bre-ast. cancer. and. human. fibrocystic. mammary. tissue,. respectively.. The.cytotoxic.activity.of.Rpx.was.determined.by.MTT-assay..Based.on.gly-cobinding.experiments,.the. influence.of.sialic.acids.on.the.activity.of.Rpx.was.investigated.by.measuring.the.activity.of.Rpx.after.desialyla-tion.of.MDA-MB-231,.HeLa.and.MCF7.cells..To.validate.the.results,.model.proteins.were.used.in.their.natural.and.desialylated.forms.for.binding.analysis.in.dot.blot.and.cell.culture.experiments.Kernel. Rpx. showed. the. same. sensitivity. profile. in. the. two. cell. lines.as. Rpx. from. plant. material:. the. non-tumorigenic. MCF10A. cells. were.1000fold. less. sensitive. (IC50:. 0.1µg/ml). than. the. tumorigenic. MCF7.cells.(IC50:.0.1ng/ml)..In.dot.blot.analyses,.Rpx.showed.higher.binding.to. the.desialylated.than.the.sialylated.natural.model.proteins.. In. line.with.this,.the.Rpx.activity.in.cell.culture.experiments.was.inhibited.by.adding. desialylated. proteins. but. not. their. sialylated. form.. Enzymatic.removal.of.sialic.acids.from.cell.surface.the.led.to.a.higher.Rpx.activity.in.MDA-MB-231.cells.but.had.no.effect.in.MCF7.or.HeLa.cells.exposed.to.Rpx,.thus.indicating.differences.in.the.glycosylation.pattern.of.the.cell.surfaces..In.spite.of.differences.in.the.SDS-PAGE.pattern,.kernel.Rpx.showed.the.same. remarkable. differential. activity. on. tumour. and. non-malignant.cells.as.Rpx.from.plant.material..The.binding.affinity.indicates.a.relation.between.cellular.sialylation.and.the.activity.of.Rpx..In.future.investiga-tions.special.glycostructures.will.be.correlated.with.the.sensitivity.to.Rpx,.thus.supporting.the.identification.and.characterization.of.a.tumor.target.for.Rpx.

AEK

55

AEKAbstracts Poster Novel Approaches to Therapy

Notes:

P-805

Jaeger, Markus; Hirschfeld,.Marc;.Gitsch,.Gerald;.Stickeler,.Elmar.(Uni-versitätsklinikum,.Freiburg)

Hyperthermia triggers suppression of alternatively spliced CD44 isoforms

The. clinical. application. of. hyperthermal. therapy. is. recently. getting.more.attention.and.is.applied.in.addition.to.chemotherapy.or.radiothe-rapy.in.malignant.diseases..This.approach.might.improve.the.effect.of.these.classical.anti-cancer.treatment.strategies.The.CD44.cell-surface.glycoprotein. is. involved. in.various.cellular.bio-logical.processes. like.cell.adhesion.and.signal. transduction.. In.breast.cancer,. variable. alternatively. spliced. isoforms. are. induced. and. their.expression.is.positively.correlated.to.tumor.progression.and.formation.of.distant.metastasis.We. investigated. potential. regulatory. effects. of. hyperthermia. on. the.expression.of.alternative.spliced.CD44.isoforms..Various.breast.cancer.cell.lines.(MCF-7,.T47D,.MDA-MB-231,.Jimt-1).were.cultured.under.hyper-thermia. (42°C,.2.hrs). followed.by.maintenance.under. regular. culture.conditions.(37°C,.4.hrs)..As.a.negative.control.the.same.cell.lines.were.permanently. cultivated. under. regular. temperature. conditions.. Tran-script. and.protein.expression. levels.of. selected.CD44. isoforms,.were.analyzed. by. RT-PCR,. Western. blot. and. immunocytochemistry,. respec-tively..The.analyses.presented.a.decreased.mRNA.and.protein.level.of.alternative.spliced.CD44.isoforms..Our.results.show.a.regulatory.effect.of.hyperthermal.treatment.on.tumor.relevant.alternative.spliced.CD44.isoforms.triggered.by.hyperthermia..We.hypothesize.that.hyperthermia.can.regulate.the.expression.of.tumor.relevant.alternative.spliced.CD44.isoforms,.thus.probably.leading.to.a.suppression.of.tumor.progression..According.to.our.findings,.hyperthermal.treatment.seems.to.represent.a.method.that.may.improve.classical.anti-cancer.therapies.by.adirect.influence.on.the.regulation.of.gene.expression.of.important.factors.in.breast.cancer.biology.

P-806

Salem, Ahmed;. Khader,. Jamal;. Almousa,. Abdelatief;. Hashem,. Sameh.(King.Hussein.Cancer.Center,.Amman)

Bibliometric analysis of cancer nanotechnology research: Global trends over the past 10 years

Objective:. Evaluate. the. overall. scientific. research. output. and. assess.worldwide. geographical. contributions. to. cancer. nanotechnology. re-search.via.biometric.literature.search..Methods:. On. 6. October. 2010,. a. Medline. search. was. conducted. using.the. National. Library. of. Medicine’s. search. service. PubMed. to. retrie-ve. research. papers. in. cancer. nanotechnology.. Retrieval. was. limited.to.papers.published.from.1.January.2000.to.6.October.2010..„Nano“,.„Cancer“.and.the.country.of.interest.were.used.as.search.phrases..Data.pertaining.to.the.number.of.articles,.date.of.publication.and.country.of.origin.were.analyzed...Results:. Five. hundred. and. twenty. three. reports. addressing. cancer.nanotechnology.research.were.published.from.2000.to.2010..The.Uni-ted.States.had.the.greatest.gross.productivity.of.over.the.years.with.196.(37.5%),. followed.by.China.with.60.(11.5%),.South.Korea.with.50.(9.6%),.Japan.with.35.(6.7%),.Germany.with.24.(4.6%),.United.King-dom. with. 15. (2.9%),. Canada. with. 14. (2.7%),. followed. by. India. and.Australia.both.with. 11. published. reports. compromising.2.1%.of.global.publication..Other.world.countries.with.less.than.10.published.reports.include.Sweden,.The.Netherlands,.Spain,.Denmark,.Greece,.Turkey,.Sin-gapore,.Egypt,.South.Africa,.Brazil,.Iran.and.Chile..The.world.output.of.cancer.nanotechnology.research.increased.progressively.over.the.years.from.4.(0.8%).in.2000,.6.(1.1%).in.2001,.8.(1.5%).in.2002,.18.(3.4%).in.2003,.31.(5.9%).in.2004,.47.(9%).in.2005.and.2006,.66.(12.6%).in.2007,.77.(14.7%).in.2008,.109.(20.8%).in.2009.and.110.(21%).in.2010.to.the.time.of.this.study..The.research.outputs.of.China.and.Korea.have.increased.substantially.over.the.past.3.years.with.78%.and.80%.of.the.respective.total.number.of.reports.published.between.2007.and.2010...Conclusions:.These.results.provide.crucial.information.on.the.worldwide.geography.of.nano-related.cancer.research..The.United.States.plays.a.leading.role.in.advancement.of.this.branch.of.biomedical.research.pos-sibly.reflecting.the.huge.resources.involved.in.this.field..China.and.South.Korea.provide.major.and.rapidly.increasing.research.contributions..Lack.of.basic.research.infrastructure,.human.expertise.and.funding.appear.to.be.significant.issues.pertaining.to.the.lack.of.widespread.global.boom.in.cancer.nanotechnology..Our.study.shows.that.cancer.nanotechnology.research.is.growing.indicating.strong.global.commitment;.however,.the.inadequacy. of. funding. appears. to. hinder. more. comprehensive. global.participation.

56

AEKAEK AEKAbstracts Poster Novel Approaches to Therapy

Notes:

P-807

Oden, Felix; Koch,.Mathias;.Kriegel,.Cathleen;.Panjideh,.Hossein;.Wilde,.Frank;.Müller,.Gerd;.Lipp,.Martin.(Max-Delbrück.Centrum,.Berlin)

Bispecific antibodies targeting CXCR5 for the treatment of B cell Non-Hodgkin’s lymphoma and autoimmune diseases

The.homeostatic.chemokine.receptor.CXCR5.appears.to.be.critically.in-volved.in.autoimmune.diseases.and.cancer.. It. is.expressed.on.mature.recirculating.B.cells.and.some.T.cell. subsets.such.as. follicular.B.hel-per.T.cells.(TFH)..Furthermore,.it.is.highly.expressed.on.90.%.of.Non-Hodgkin’s.B.cell.lymphoma.(NHL)..CXCR5.regulates.compartmentalization.of.B.and.T.cells.within.seconda-ry.lymphoid.tissues..In.addition,.CXCR5.expressing.antigen-specific.TFH.cells.promote.the.proliferation.of.CXCR5+.B.cells.during.the.germinal.center.reaction..This.T.cell.subset.therefore.plays.a.central.role.in.the.formation.of.germinal.centers.and.maturation.of.plasma.cells..Hence,.TFH.cells.are.also. involved. in. the. formation.of.autoreactive. lymphoid.structures.(ectopic.follicles).and.the.continuous.synthesis.of.(auto).an-tibodies.in.autoimmune.diseases..In.conclusion,.the.elimination.of.CXCR5.expressing.cells.or.the.functio-nal.inactivation.of.the.chemokine.receptor.might.be.particularly.suited.for.the.therapy.of.NHL.as.well.as.chronic.inflammatory.and.autoimmune.diseases..Receptor.blockade.and.cell.depletion.may.be.achieved.by.ap-plication.of.therapeutic.antibodies.with.novel.bispecific.functionalities..For.this.purpose.a.hybrid.hybridoma.was.generated.by.the.quadroma.technique,.which.produces.a. trifunctional.bispecific.antibody. (bspAb).recognizing.CXCR5.and.CD3..In.addition,.we.have.generated.a.series.of.monospecific.(mspAb).and.bispecific.antibodies.(bspAb).based.on.sin-gle-chain.variable-domain.fragments.(scFv)..The.latter.where.designed.to. show. an. enhanced. efficiency. and. reduced. immunogenicity. which.is.of.high. interest. for.clinical.applications..The. recombinant.antibody.constructs. either. recognize. CXCR5. alone. or. both. CXCR5. and. CD3. or.CXCR5.and.CD5,.respectively..After.purification.of.the.antibody.constructs,.in.vitro.cytotoxicity.assa-ys.have.been.carried.out..Further.investigation.of.therapeutic.effects.in.vivo.will.follow.using.murine.tumor.and.autoimmune.models..Prelimina-ry.results.have.already.shown.that.anti-CXCR5-CD5.recombinant.anti-bodies.and.the.anti-CXCR5xCD3.quadroma.antibody.can.successfully.kill.CXCR5.expressing.NHL.cell.lines.and.primary.B.cells.

P-808

Mack, Laura (Georg-Speyer-Haus, Frankfurt/M.); Weber,. Axel.(Georg-Speyer-Haus,. Frankfurt/M.);. Brill,. Boris. (Georg-Speyer-Haus,.Frankfurt/M.);. Delis,. Natalia. (Georg-Speyer-Haus,. Frankfurt/M.);. Borg-houts,.Corina.(GANYMED.Pharmaceuticals.AG,.Mainz);.Weiss,.Astrid.(In-stitut.für.Experimentelle.Tumorforschung,.Frankfurt/M.);.Groner,.Bernd.(Georg-Speyer-Haus,.Frankfurt/M.)

Interference with protein-interactions: Inhibition of Stat-signaling by peptide ligands in cancer

Members. of. signal. transducers. and. activators. of. transcription. (STAT).family. have. been. found. to. be. constitutively. activated. in. a. wide. ran-ge.of.human.tumors..STAT3.is.present.in.its.activated.form.in.breast-,.prostate-.and.pancreatic.cancer;.STAT5.in.prostate-,.breast.cancer.and.myeloid. leukemia..Since.STAT.proteins.do.not.entail.enzymatic.activi-ties,.they.have.been.considered.as.non-druggable.and.developing.spe-cific.inhibitors.has.been.largely.neglected..We.are.developing.novel.ap-proaches.for.the.derivation.of.STAT.inhibitors.and.devised.peptides.able.to.mask.defined.functional.domains.of.STAT3.or.STAT5..These.peptides.are.able.to.specifically.inhibit.their.function.and.affects.STAT.addicted.tumor.cells..We. isolated.peptide.aptamer.sequences.which.specifically.bind. to.di-stinct. functional.domains.of.STAT3.and.STAT5..The.peptide.sequences.are.inserted.into.a.scaffold.molecule.(human.thioredoxin;.hTRX).which.increases.stability.of.the.peptide.thereby.enhancing.binding.specificity..Recombinant.expression.and.purification.of.the.protein.scaffold.contai-ning.the.Stat.targeting.peptide.and.addition.of.a.protein.transduction.domain.consisting.of.9.arginines.led.to.functional.inhibitors,.which.can.be.efficiently.introduced.into.cultured.cells.A.peptide.aptamer.(rS3-PA).is.able.to.specifically.bind.to.the.SH2-do-main.of.STAT3..It.inhibits.expression.of.STAT3.target.genes.and.blocks.growth.and.migration.of.STAT3.addicted.tumor.cells.at.concentrations.of. 1. μM.. We. also. identified. a. peptide. aptamer. sequence. (S5-DBD-PA).with.specific.binding.affinity.for.the.DNA-binding.domain.of.Stat5..S5-DBD-PA.impairs.the.expression.of.STAT5.target.genes.and.inhibits.the.growth. of. STAT5. dependent. human. and. mouse. leukemia. cell. lines. at.concentrations.of. 1µM..The.mechanisms.with.which.the.peptide.apta-mers.interfere.with.STAT.mediated.transactivation.include.inhibition.of.tyrosine.phosphorylation.and.DNA.binding.as.well.as.targeted.protein.degradation..We.also.derived.a.STAT3.specific.binding.peptide. from.PIAS3. (protein.inhibitor.of.activated.Stat)..This.negative.regulator.of.STAT3.is.downre-gulated.in.tumor.cells.and.tissues.in.which.constitutive.STAT3.activity.has.been.observed..This.peptide.(rPP-C8).is.able.to.mimic.PIAS3.activity.and.block.oncogenic.STAT3.functions..The.STAT3.and.STAT5.specific.peptide.ligands.have.been.tested.for.their.activity.to.inhibit.tumor.cell.growth.in.vitro.and.in.vivo.and.will.serve.as.tools.to.develop.clinically.applicable.STAT.protein.inhibitors.

AEK

57

AEKAbstracts Short Talks Tumor Inflammation and Immunology

Notes:

short talk 901

Seifert, Christin (University Hospital, Essen); Lubojanski,. Silke.(University.Medical.Centre,.Mainz);.Zhao,.Fang.(University.Hospital,.Es-sen);. Seifert,.Ulrike. (University.Medicine.Charite,.Berlin);. Schadendorf,.Dirk.(University.Hospital,.Essen);.Paschen,.Annette.(University.Hospital,.Essen)

Activity of ER Aminopeptidase 1 in Malignant Melanoma Determines the Strength of the Overall CD8+ T Cell Response Against Autologous Tumor Cells

The.knowledge.of.HLA.class.I-restricted.tumor.antigen.epitopes.reco-gnized.by.cytotoxic.CD8+.T.lymphocytes.(CTL).is.currently.exploited.in.immunotherapy.of.malignant.melanoma,.in.vaccination.as.well.as.adop-tive.T.cell. therapy..However,.the.generation.of.CTL.epitopes. in.tumor.cells.has.been.studied.only.marginally.. In.general,. the.multi-catalytic.proteasome.complex.cleaves.a. tumor.antigen. into.peptide. fragments.of.different.length,.thereby.defining.the.C-terminus.for.the.majority.of.peptides.presented.by.HLA.class.I.molecules..While.some.of.the.prote-asome.products.are.of. the.correct.size. for.direct.HLA.class. I.binding.others.are.N-terminally.elongated.peptide.precursors.that.require.fur-ther.trimming.by.aminopeptidases..Recent.data.suggested.that.the.ER.aminopeptidase.1.(ERAP1).might.be.of.specific.relevance.for.the.efficient.generation.of.HLA.class.binding.peptides.in.tumors.as.it.was.demons-trated.that.for.cervical.carcinoma.low.in.situ.expression.of.ERAP1.is.as-sociated.with.reduced.overall.survival..So.far,.the.significance.of.ERAP1.activity.for.the.overall.recognition.of.tumor.cells.by.CD8+.T.cells.has.not.been.determined..To.do.so,.we.employed.the.autologous.tumor-CD8+.T.cell.system.of.patient.Ma-Mel-86..Two.melanoma.cell.lines.(Ma-Mel-86a,.Ma-Mel-86c). were. established. from. different. metastasis. of. this. pati-ent,.in.which.expression.of.ERAP1.was.downregulated.by.shRNA..ERAP1.knockdown,.confirmed.by.quantitative.RT-PCR.and.Western.Blot.analy-sis,.did.not.influence.the.expression.level.of.total.HLA.class.I.molecules.in.comparison.to.control.cells.(mock.transfectants)..To.determine.the.impact.of.ERAP1.knockdown.on.T.cell.stimulation.Ma-Mel-86a.cells.were.used. to. repeatedly. stimulate. autologous. CD8+. T. cells.. Subsequently,.the.T.cells.were.analyzed.for.their.capability.to.recognize.Ma-Mel-86a.(mock. transfectants). in. comparison. to. ERAP1-shRNA. transfected. Ma-Me-86a.cells..Interestingly,.ERAP1.knockdown.impaired.the.recognition.of.melanoma.cells.by.autologous.bulk.CTL,.indicating.that.ERAP1.indeed.impacts.the.overall.epitope.presentation.by.this.tumor.cells..Furthermo-re,.from.bulk.CD8+.T.cells.we.established.T.cell.clones.whose.anti-tumor.response.was. indeed.decreased.upon.ERAP1. knockdown. in.melanoma.cells..Thus,.our.results.suggest.that.ERAP1.plays.an.important.role.in.the.generation.of.HLA.class.I.peptides.from.tumor.antigens,.as.its.activity.influences.the.strength.of.the.antitumor.CTL.response.

short talk 902

Keller, Larissa;.Doll,.Dietrich;.Janssen,.Klaus-Peter.(Technische.Univer-sität,.München)

CXC-CHEMOKINES AS CRUCIAL IMMUNE MEDIATORS IN COLORECTAL CARCINOGENESIS

The.progression.and.aggressiveness.of.solid.tumors.are.strongly.influ-enced.by.the.tumor.microenvironment,.notably.by.components.of.the.immune. system.. In. an. earlier. transcriptome. analysis,. we. have. com-pared. colorectal. cancer. patients. with. either. good. or. bad. prognosis.(n=25. in.each.group),.but.with. identical. tumor.stages. (T3N0M0)..Our.data.indicated.that.an.elevated.expression.of.interferon-regulated.CXC-chemokines.(CXCL9,.CXCL10.and.CXCL11). is.significantly.and.positively.correlated.with.postoperative.survival..These.chemokines.represent.a.group.of.soluble.modulators.of.the.immune.system,.which.lead.to.the.chemotactic.recruitment.of.T-cells,.and.are.furthermore.able.to.block.angiogenesis.. Moreover,. high. intratumoral. numbers. of. T-lymphocytes.have.been.reported.to.predict.good.prognosis.in.colorectal.cancer..The.chemokine.expression.data.were.confirmed.by.qRT-PCR.on.an.indepen-dent.collective.of.97.colon.cancer.patients..Thus,.CXCL9-11.chemokines.emerge.as.an.excellent.independent.prognostic.predictor.for.postope-rative.survival,.as.evidenced.by.microarray.analysis.and.qRT-PCR.assays.on. an. independent. patient. collective.. Next,. intratumoral. immune. cell.populations.and.blood.vessel.density.were.assessed.by. immunohisto-chemistry.on.tissue.sections.of.the.same.patients..The.apparent.protec-tive.effect.of.high.chemokine.expression.was.associated.to.the.attrac-tion. of. cytotoxic. CD8-positive. T-cells,. and. CD4-positive. T-helper. cells.into.the.tumor,.whereas.no.clear.association.with.blood.vessel.density.was. found.. Thus,. interferon-regulated. CXC-chemokines. are. excellent.prognostic.predictors.in.human.colorectal.cancer,.and.high.chemokine.levels.are.correlated.with.increased.intratumoral.T-cell.numbers..We.are.currently.establishing.an.orthotopic.mouse.model,.as.well.as.an.ex.vivo.tissue.culture.model.based.on.primary.human.colon.tumors,.to.find.out.if.CXC-chemokines.indeed.exert.a.causal.impact.during.colorectal.tumor.formation,.and.if.they.are.suitable.targets.for.therapeutic.intervention..The.orthotopic.model.is.based.on.an.isogenic.murine.colon.carcinoma.cell.line,.which.was.engineered.to.express.CXC-chemokines..This.model.will.allow.us.to.investigate.colorectal.tumor.formation.in.immunocom-petent.or. immunodeficient.host.animals. in.dependence.of.chemokine.expression..

58

AEKAEK AEKAbstracts Poster Tumor Inflammation and Immunology

Notes:

P-901

Rohwer, Nadine (Charité, Berlin); Bogdanoff,. Birgit. (Charité,. Berlin);.Glauben,.Rainer.(Charité,.Berlin);.Wiedemann,.Bertram.(Charité,.Berlin);.Loddenkemper,. Christoph. (Technische. Universität,. München); Cramer,.Thorsten.(Charité,.Berlin)

HIF-1α is essential for inflammation-associated colon carcinogenesis

A.substantial.amount.of.cancer.entities.is.driven.by.inflammation,.e.g..liver.and.gastric.cancer..Chronic. inflammation.might.also.represent.a.key.factor.in.the.pathogenesis.of.colorectal.cancer.(CRC),.as.evidenced.by.the.inhibiting.effect.of.anti-inflammatory.drugs.on.CRC.progression..Despite.the.intriguing.interrelation.of.chronic.inflammation.and.cancer,.its.molecular.nature. remains. largely.elusive.. The. transcription. factor.HIF-1α.is.upregulated.in.most.human.cancers.and.controls.central.pro-tumorigenic.pathways.such.as.glycolysis,.angiogenesis.and.therapy.re-sistance..Interestingly,.HIF-1α.constitutes.a.pivotal.regulator.of.myeloid.cell.function.and.is.activated.strongly.by.pro-inflammatory.cytokines..To.achieve.a.comprehensive.characterization.of.HIF-1α,.we.have.analyzed.colitis-associated.cancer.in.mice.lacking.HIF-1α.specifically.in.intestinal.epithelial.or.myeloid.cells,.respectively..Tissue-specific.knockout.(KO).of.HIF-1α.was.achieved.by.the.Cre-loxP-system..Colon.tumors.were.ge-nerated.by.intraperitoneal.injection.of.azoxymethan.followed.by.three.cycles.of.oral.dextrane.sodium.sulphate..Enterocyte-specific.deletion.of.HIF-1α.led.to.decreased.tumor.size,.without.affection.of.tumor.inci-dence,.at.early.(8.weeks),.but.not.at.later.(12.weeks).time.points.after.tumor.initiation..Remarkably,.the.number.of.HIF-1α-positive.tumor.cells.in.Villin-Cre.expressing.mice.increased.over.time.until,.at.12.weeks,.no.more.difference.between.WT.and.enterocyte-specific.KO.tumors.could.be.observed..We.conclude.from.these.data.that.in.enterocyte-specific.HIF-1α-KO. mice. a. few. remaining. cells. escaped. recombination. and. can.apparently.participate.in.colon.tumorigenesis.due.to.accelerated.pro-liferation.. Deletion. of. HIF-1α. in. myeloid. cells. resulted. in. a. significant.decrease.of.both.tumor. incidence.and.tumor.size. independent.of. the.time.point..Furthermore,.reduction.of.tumor.formation.in.both.conditio-nal.KO.models.was.linked.to.decreased.angiogenesis.in.HIF-1α-deficient.tumors.. In. conclusion,. specific. inactivation. of. . HIF-1α. in. both. entero-cytes.and.myeloid.cells.attenuates.the.course.of.colitis-associated.can-cer..The.participation.of.HIF-1α-expressing.(non-recombined).epithelial.cells.to.tumor.formation.in.enterocyte-specific.HIF-1α-KO.mice.empha-sizes.a.pivotal.role.for.HIF-1α.in.colon.carcinogenesis..Whether.HIF-1α-inactivating.drugs.can.be.regarded.as.innovative.and.effective.agents.for. the. therapy. of. inflammation-associated. cancer. is. currently. being.evaluated.in.our.group.

P-902

Goepfert, Katrin; Sieben,.Maike;.Schäfer,.Petra;.Galle,.Peter.R.;.Moehler,Markus.(Universitätsklinikum,.Mainz)

Reactivation of the human immune system by CTLA-4 blocking mono-clonal antibody tremelimumab in different human immune cells

Tumors.have.distinct.mechanism.to.circumvent.the.human.immune.sy-stem..Besides.the.low.immunogeneity.of.tumors,.the.dysregulation.of.the.immune.response.leads.to.a.loss.of.effective.immune.effector.cells.and.thus.to.uncontrolled.tumor.growth..The.deficiency.is.caused.by.an-tigen-presenting.cells,.especially.matured.dendritic.cells.(DC)..They.are.no.longer.able.to.present.sufficient.tumor.associated.antigens..Otherwise,.loss.of.immune.defence.can.also.be.caused.by.expression.of.CTLA-4..Several.mechanisms.of.CTLA-4.have.been.proposed.including.li-gand.competition.with.CD28.and.interference.of.TCR.signaling..A.possible.function.of.CTLA-4.in.regulatory.CD4+CD25+.T-.cells.has.generated.wi-despread.interest.indicating.another.mechanism.by.which.CTLA-4.might.downregulate.immune.response.and.promote.peripheral.tolerance.In.negatively.immune.modulating.and.often.chemoresistent.tumor.types,.we.previously.showed.that.parvovirus.H1.induced.tumor.cell.lysates.are.able.to.activate.tumor.cell.antigen.specific.cytotoxic.T-.cells.(CTLs).by.improvement.of.direct.and.indirect.cross-.presentation.via.DCs.It.has.been.shown,.that.tumors.express.CTLA-4..To.our.knowledge,.the.underlying.immune.escape.mechanisms.of.CTLA-4.expressing.tumors.are.still.unclear.and.CTLA-4.mediated.blockage.of.human.DCs.by.CTLA-4.ex-pressing.tumors.have.not.been.analysed.so.far.With.this.question.in.mind,.we.analyse.if.tremelimumab.induce.a.stron-ger. DC. activation.. The. DC-mediated. tumor. antigen. cross. presentation.may.be.even.stronger.after.phagocytosis.of.CTLA-4.expressing.tumors.in.the.presence.of.tremelimumab..It.is.a.new.antibody.possibly.enhancing.the.CTL.response.by.blocking.the.CTLA-4.mediated.silencing.of.T-.cells.First.we.established.the.expression.of.CTLA-4.profiles.of.different.hu-man.tumor.cells.by.FACS..We.were.able.to.show.a.CTLA-4.expression.in.melanoma.and.colorectal.cells.Secondly,.we.showed.that. tremelimumab.did.not.negatively. influence.cell.viability.of.tumor.cells,.measured.by.MTT.assay..Combination.of.tre-melimumab.with.different.cytotoxic.agents.did.neither.block.nor.enhan-ce.the.induction.of.apoptosis.in.cell.culture.Furthermore,.we.isolated.regulatory.CD4+CD25+.T.cells..They.were.cha-racterized.by.expression.of.FoxP3,.CD127,.CD25.and.CTLA-4..In.coculture.experiments.with.iDCs.or.mDCs;.with.or.without.tremelimumab.at.diffe-rent.time.points,.we.want.to.overcome.the.negative.feedback.of.Tregs.on.mDCs..The.further.analysis.will.be.presented.at.the.meeting.

AEK

59

AEKAbstracts Poster Tumor Inflammation and Immunology

Notes:

P-903

Seibt, Stephanie (Charité, Berlin); Bormann,. Felix. (Charité,. Berlin);.Dahl,.Andreas.(Max-Delbrück.Centrum,.Berlin);.Braun,.Monika.(Univer-isty,.Heidelberg);.Falk,.Christine.(University,.Heidelberg);.Sers,.Christine.(Charité,.Berlin)

DOWNREGULATION OF HLA AND ULBP EXPRESSION BY ONCOGENIC RAS AND EPIGENETIC MECHANISMS IN COLORECTAL CANCER CELLS

RAS. proteins. control. proliferation. and. differentiation. by. transmitting.signals. from. the.cell. surface. to. the.nucleus..RAS.oncogenes.are.mu-tated.and.constitutively.activated.in.40%.of.human.colorectal.cancer..Recently,. a. correlation. between. RAS. oncogene. activation,. decreased.expression.of.HLA.class.I.and.ULBP2.genes.as.well.as.epigenetic.alte-rations. in. colorectal. cancer. was. found.. This. indicates. that. activation.of.the.RAS.oncogene.might.have.a.negative. impact.on.HLA.and.ULBP.expression..A.strong.methylation.pattern.was.detected.for.the.ULBP2.promotor.but.not.at.the.HLA-A.promotor.–.supposing.different.mecha-nisms.of.epigenetic.suppression.of.HLA-A.and.ULBP2.genes.by.RAS.on-cogene.activation..To. further.analyze,.which.genes.within.genomic.regions.encoding. im-mune. modulatory. proteins. are. affected. by. DNA. methylation,. a. more.detailed. investigation. was. performed.. Bisulphite-treated. DNA. from. 8.colorectal.cancer.cell.lines.was.tested.by.ligation-mediated.PCR.com-bined. with. a. TaqMan-based. readout. using. universal. reporter. probes..In.addition.to.HLA-A.and.ULBP2.genes.HLA-B,.β2M,.ULBP3,.TAP1,.TAP2,.TAPBP,.LMP2,.LMP7.and.PA28α.gene.methylation.analyses.were.done..The.results.showed.only.methylation.within.the.HLA-B.promotor.and.a.sporadic.methylation.in.LMP7.and.TAP2..Strong.methylation.was.obser-ved.at.the.ULBP2.and.ULBP3.promotors.To. determine. the. mechanisms. of. RAS-dependent. and. epigenetic. sup-pression.of.HLA.and.ULBP.genes,.an.embryonic.kidney.cell.line.(HEK-RAS.ER),.harboring.an.inducible.HRASG12V,.was.used..The.RAS.pathway.can.be. activated. by. 4-hydroxytamoxifen. via. an. estrogen. receptor–HRAS-G12V.fusion.protein..In.this.cell.line.the.HLA.class.I.and.ULBP2.protein.expression. was. transiently. decreased. 72. hours. after. HRASG12V. acti-vation.and.reached.pre-induction.levels.72.hours.after.removal.of.the.inducing. agent.. The. treatment. with. inhibitors. of. the. MAPK. pathways.leads.to.an.re-expression.of.HLA.and.ULBP.proteins..Treatments.with.the.de-methylating.agent.5-aza-2’-deoxycytidine.or.the.applications.of.the.histone.deacetylase.inhibitor.Valproat.showed.HLA.and.ULBP.re-ex-pression.as.well..These.cells.are.now.further.used.to.analyze.the.effects.of.prolonged.HRASG12V.expression.and.its.signaling.pathway.activation.onto.HLA.and.ULBP.genes.

P-904

Akhmetzyanova, Ilseyar; Zelinskyy,.Gennadiy.(University.Clinics.Essen,.Essen)

The local T cell response in tumor development: biological role of CD8+ T cells

Immune.cells.infiltrate.tumors.and.become.a.significant.component.of.the. multicellular. cancer. microenvironment;. however. the. immune. sy-stem.often.fails.to.prevent.tumor.development..Mechanisms.of.immune.escape. contributing. to. tumor. development. are. largely. unknown.. The.biological.role.of.cytotoxic.T.cells.(CTL).and.regulatory.T.cells.(Tregs).that.counteract.antitumor.immune.cells.is.essential.in.the.local.tumor.immune.response..FBL-3.cells.are.transformed.erythroid.leukemia.cell.lines.from.a.Friend.Virus.infected.C57BL/6.mouse,.which.express.immu-nogenic.epitopes.recognized.by.T.cells..The.local.interaction.of.tumor.cells.and.different.populations.of.immune.cells.was.studied.in.this.mo-del.by.exploiting. the.draining. lymph.nodes.compared. to.non-draining.lymph.nodes..The.level.of.activated.CD8+.T.cells.in.the.draining.lymph.nodes. was. significantly. higher. compared. to. the. non-draining. lymph.nodes...Tumor.specific.CD8.T.cells.have.a.potent.in.vivo.killing.activity.at. the.site.of. tumor.growth.due.to. increased.production.of.cytotoxic.molecule.GzmB..Currently,.we.are.trying.to.characterize.the.functional.features.of.CTL.in.draining.and.non-draining.lymph.nodes.

60

AEKAEK AEKAbstracts Poster Tumor Inflammation and Immunology

Notes:

P-905

Bukur, Juergen (University, Halle / S.); Respa,. Annedore. (University,.Halle./.S.);.Steven,.André.(University,.Halle./.S.);.Handke,.Diana.(Univer-sity,. Halle./.S.);. Ferrone,. Soldano. (Hillman. Cancer. Research. Institute,.Pittsburgh);.Seliger,.Barbara.(University,.Halle./.S.)

Transcription factors and signal transduction pathways involved in MHC class I abnormalities of tumor cells

Abnormalities.of.the.constitutive.and.interferon.(IFN)-γ.inducible.MHC.class. I. surface. expression. in. human. and. murine. tumor. cells. is. often.associated.with.an.impaired.expression.of.components.of.the.antigen.processing.machinery.(APM)..In.order.to.determine.the.underlying.mo-lecular. mechanisms. leading. to. these. deficiences. we. determined. the.transcriptional. regulation. of. APM. components. and. factors. mediating.their.repression.and.the.role.of.the.interferon.signal.transduction.pa-thway.for.basal.and.IFN-γ-induced.APM.component.expression..We.could.demonstrate.a.transcriptional.downregulation.of.the.major.MHC.class.I.APM.components.upon.HER-2/neu.transformation,.which.is.associated.with.low.MHC.class.I.surface.expression..Site-directed.mutagenesis.of.the.p300.and.E2F1.transcription.factor.binding.sites.of.the.APM.promo-ters.demonstrated.a.E2F-directed.control.of.tapasin.expression,.which.was.confirmed.by.chromatin. immunoprecipitation..SiRNA-mediated.si-lencing.of.E2F1.was.associated.with.an. increased. tapasin.expression,.whereas.E2F1.overexpression.was.associated.with.reduced.tapasin.tran-scription.suggesting. that.E2F1. is.an.essential. transcription. factor. for.APM.component.expression..In.addition,.siRNA-mediated.knock.down.of.CREB.was.also.associated.with.an.increase.in.MHC.class.I.expression..Furthermore,.constitutive.expression.of.JAK2,.a.component.of.the.IFN.signal.transduction.pathway,.is.essential.for.basal.MHC.class.I.APM.com-ponent.expression,.whereas.downregulation.or.deletion.of.JAK2.caused.not.only.a.resistance.to.the.anti-proliferative.effect.of.IFN-γ,.but.also.a.decreased.APM.component.expression..In.conclusion,.the.IFN.signal.transduction.and.defined.transcription.factors.appear.to.be.involved.in.the. regulation.of.APM.component.expression. thereby.modulating. the.immunogenicity.of.tumor.cells.

AEK

61

AEKAbstracts Poster Free Topics

Notes:

P-001

Eke, Iris (OncoRay, Dresden); Hehlgans,.Stephanie.(OncoRay,.Dresden);.Shevchenko,.Anna. (Max.Planck. Institute,.Dresden); Baumann,.Michael.(OncoRay,.Dresden);.Zips,.Daniel.(OncoRay,.Dresden); Cordes,.Nils.(On-coRay,.Dresden)

Interactions of focal adhesion protein PINCH1 and Protein Phospha-tase 1 alpha control Akt1 function in tumor cell radio- and chemore-sistance

The.response.of.cancer.cells.treated.with.X-rays.or.cytotoxic.drugs.is.critically.regulated.by.cell-extracellular.matrix.(ECM).interactions..The.adaptor,.LIM-only.protein.PINCH1.is.part.of.the.multiprotein.complexes,.called.focal.adhesions,.which.mediate.these.interactions..To.analyze.the.role.of.PINCH1.for.the.cellular.radio-.and.chemoresistance,.embryonic.PINCH1.wildtype.and.PINCH1.knockout.mouse.fibroblasts.as.well.as.seve-ral.cancer.cell.lines.of.different.origin,.in.which.PINCH1.expression.was.depleted.by.siRNA,.were.examined..Colony.formation.assays,.immuno-precipitations,. Western. blotting,. mass. spectrometry. analysis,. protein.phosphatase.assays.and.in.vivo.tumor.control.probability.assays.were.performed..We.found.that.knockout.or.knockdown.of.PINCH1.resulted.in.a.highly.significantly.enhanced.radiosensitivity.associated.with.dephos-phorylation.and. reduced.kinase.activity.of.Akt1.and.Akt1.downstream.targets. in. vitro. and. in. vivo.. Immunoprecipitation. and. mass. spectro-metry.on.PINCH1.and.Akt1.identified..the.known.Akt1.regulator.Protein.Phosphatase.1.(PP1).as.interactor.of.both.PINCH1.and.Akt1..Mutation.of.the.putative.PP1.binding.motif,.i.e..KFVEF,.within.the.5th.LIM.domain.of.PINCH1.resulted.in.a.loss.of.PP1.binding.to.PINCH1..Mechanistically,.loss.of. .PP1.binding. to.PINCH1. increased.PP1.phosphatase.activity.causing.Akt1.dephosphorylation..The.data. identified.PINCH1.as.novel.determi-nant.of.Akt1.kinase.activity.and.evidently.show.how.PINCH1.controls.ra-diation.and.chemoresistance..Further.studies.have.already.commenced.to.show.whether.targeting.PINCH1.may.provide.new.potent.strategies.to.reduce.tumor.cell.resistance.and.to.improve.cancer.therapy.

P-002

Rudner, Justine (University Hospital, Tuebingen); Elsaesser,. Simon.(University.Hospital,.Tuebingen);.Trivigno,.Donatella.(University.Hospi-tal,.Tuebingen);.Jendrossek,.Verena.(University.of.Duisburg-Essen,.Es-sen);.Huber,.Stephan.(University.of.Duisburg-Essen,.Essen)

The anti-apoptotic Bcl-2 and Bcl-xL use different mechanisms to protect from apoptosis induction

Members.of. the.Bcl-2. family. regulate. intrinsic.apoptosis. induction.at.the.mitochondria.and.endoplasmic.reticulum..According.to.their.func-tion.the.proteins.are.divided.in.anti-apoptotic.and.pro-apoptotic..During.apoptosis. induction,. anti-apoptotic. members. maintain. mitochondrial.and.ER.integrity.preventing.the.release.of.pro-apoptotic.factors.into.the.cytosol.and.subsequent.caspase.activation..The.protective.Bcl-2.mem-bers.Bcl-xL,.Bcl-2,.and.Mcl-1.are.often.up-regulated.during.tumorigene-sis.and.contribute.to.resistance.against.common.anti-neoplastic.thera-pies..Especially.Bcl-xL.and.Bcl-2.display.high.structural.and.functional.homology.and.might. substitute.each.other..However,. their. regulation.differs.in.many.aspects..Amongst.others,.Bcl-xL,.Bcl-2,.and.Mcl-1.bind.a.distinct.set.of.pro-apoptotic.Bcl-2-related.proteins,.co-called.BH3-only.proteins,.which.neutralize.their.function.Here.we.examined.the.protective.role.of.the.anti-apoptotic.Mcl-1,.Bcl-2,.and.Bcl-xL.in.response.to.ionizing.radiation.(IR).and.the.anti-inflamm-atoric,.pro-apoptotic.drug.Celecoxib..We.were.able.to.show.that.Mcl-1.was.down-regulated.during.apoptosis. induction. in.response.to. IR.and.Celecoxib.in.Jurkat.cells..The.depletion.of.Mcl-1.by.siRNA.was.sufficient.for. apoptosis. induction.. The. overexpression. of. either. Bcl-2. or. Bcl-xL.prevented. apoptosis. induction. in. response. to. IR.. Interestingly,. when.over-expressed,.only.Bcl-xL.but.not.Bcl-2.was.able.to.inhibit.Celecoxib-induced.apoptosis..Immunoprecipitation.studies.showed.that.only.Mcl-1.and.Bcl-xL,.but.not.Bcl-2,.sequestered.pro-apoptotic.multi-domain.pro-tein.Bak.in.healthy.cells.to.prevent.its.activation..Thus,.when.abundantly.expressed,. Bcl-xL. can. substitute. for. the. loss. of. Mcl-1. in. response. to.Celecoxib.whereas.Bcl-2.could.not..We.further.conclude.that.radiopro-tection.by.Bcl-2. is.mediated.by.a.mechanism.which.does.not. rely.on.sequestration.of.Bak.

62

AEKAEK AEKAbstracts Poster Free Topics

Notes:

P-003

Braeuning, Albert (University, Tuebingen); Schmidt,.Andreas.(Univer-sity,.Tuebingen);.Ruck,.Peter.(Leonberg);.Fuchs,.Jörg.(University,.Tue-bingen);. Warmann,. Steven. (Tuebingen);. Schwarz,. Michael. (Tuebingen).

Differential expression of Glutamine synthetase and Cytochrome P450 isoforms in human hepatoblastoma

Carcinogenesis. is. often. linked. to. aberrant. activation. of. Wnt/beta-Catenin. signalling,. in.many.cases.caused.by.activating.CTNNB1.muta-tions.(encoding.beta-Catenin)..Recently,.beta-catenin.was.established.as. a. decisive. regulator. of. hepatic. glutamine. synthetase. (GS). and. cy-tochrome.P450.(CYP).expression.in.mouse.hepatocarcinogenesis..This.study.was.aimed.to.analyse.the.connection.of.beta-Catenin.signalling.and. GS/CYP. expression. in. human. paediatric. tumours.. Samples. were.analysed.for.activating.mutations.in.CTNNB1..Protein.expression.of.the.model.beta-Catenin.target.GS.and.of.various.CYP.isoforms.was.analy-sed.and.correlated.with.CTNNB1.mutational.status.and.histological.fin-dings..Activating.CTNNB1.mutations.were. frequent. in.hepatoblastoma.(80%). and. nephroblastoma. (31%),. but. absent. from. neuroblastoma,.rhabdomyosarcoma,.and.other.soft.tissue.sarcoma..In.CTNNB1-mutated.hepatoblastoma,. expression. of. GS. was. only. detected. in. tumour. are-as. with. epithelial,. not. with. mesenchymal. differentiation.. Particularly.high.expression.of.glutamine.synthetase.was.found.in.hepatoblastoma.cells.directly.neighbouring.a.mesenchymal-type.tumour.area.or.stroma.cells,. associated.with.above-average.cell. proliferation..GS.expression.was.not.observed.in.CTNNB1-mutated.nephroblastoma..Hepatoblastoma.with.activated.beta-Catenin.expressed.different.CYPs.relevant.for.the.metabolism.of.cytostatic.drugs,.but.with.high. interindividual.variance.and. heterogeneity. within. a. single. tumour.. GS. and. different. CYPs. are.co-expressed. in. hepatoblastoma. with. activated. beta-Catenin.. More-over,. other. factors. like. histological. subtype. of. tumour. cells. and. cell-cell-interactions.at.the.borders.between.different.areas.of.the.tumours.affect.expression.of.these.beta-Catenin.target.genes..Analysis.of.CYP.expression.in.resected.tumour.tissue.might.be.useful.for.the.selection.of.appropriate.cytostatics.for.post-operative.chemotherapy.

P-004

Monz, Miriam; Moll,.Roland;.Morokina,.Viktoria;.Schmidt,.Ansgar.(Uni-versity,.Institute.of.Pathology,.Marburg)

Desmosomes and Glycolysis: A novel connection of different cellular functions?

The. adhesion. of. cells. is. not. only. of. importance. for. the. development.and.maintenance.of.tissues.and.organs,.but.is.also.implicated.into.the.formation,. invasion. and. metastasis. of. tumors.. Plakophilins. (PKPs). as.essential. cytoplasmic. components. of. desmosomal. cell. adhesion. have.been.associated.with. some.of. these.events.and.have.been.moreover.identified.as.part.of.additional.cellular.procedures.such.as.post-tran-scriptional.gene.regulation..Glycolysis.on.the.other.hand.is.a.fundamen-tal.process.that.is.essential.for.all.living.cells..Here.we.describe.for.the.first.time.a.physical.interaction.of.componen-ts.of.these.different.cellular.functions..We.identified.Triosephosphate-Isomerase.1.(TPI.1).as.a.supposed.interacting.partner.of.Plakophilin.3.using.a.two-hybrid-assay..Co-immunoprecipitation.from.soluble.protein.extracts.of.epithelial.cells.of.line.A-431.with.specific.antibodies.against.the.proteins.revealed.that.both.proteins.also.bind.to.each.other.in.euka-ryotic.cells..This.interaction.seems.to.be.fairly.robust.since.incubation.of. the. complexes. with. buffers. containing. non-physiological. high. salt.concentration.did.not.break.up. the. interactions..Separating. the.com-plexes.of.PKP.3.and.TPI.1.using.sucrose-density-gradient-centrifugation.revealed. complexes. of. up. to. 8. S. to. where. both. proteins. co-migrate..Immunoprecipitation. of. the. proteins. from. respective. fractions. of. the.gradient.confirmed.their.interaction.in.these.fractions..The.analysis.of.the.binding.of.PKP.3.and.TPI. 1.on.cultured.cells.using. immunofluore-scence-microscopy. revealed. a. co-localization. of. the. both. proteins. in.part.in.the.cytoplasm..Surprisingly.we.found.a.particulate.co-alignment.of.both.proteins.at.the.cell-boundary.in.a.desmosome-like.fashion.that.was.especially.apparent.in.simple.epithelia-derived.cells.of.colon.carci-noma.line.CaCo-2..This.co-alignment.was.not.disturbed.when.inhibitory.chemical. compounds. of. glycolysis. such. as. 3-bromopyruvic. acid. were.applied.to.cells.before.immunofluorescence.analysis,.whereas.reagents.that.induce.environmental.stress.such.as.sodium.arsenite.seem.to.inhi-bit.the.co-alignment.to.some.degree...Since. the. functional. relevance. of. the. interaction. is. enigmatic. at. the.moment,.pursuing.experiments.have.to.clarify.a.possible.impact.of.the.interaction.of.the.desmosomal.plaque-protein.PKP.3.with.the.glycoly-tic.TPI.1.for.normal.cellular.functions.or.possibly.dysregulated.cellular.functions.that.lead.to.tumors.

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AEKList of Speakers and Poster Authors

A

Akhmetzyanova, Ilseyar (Essen) University.Clinics.Essen.Institut.of.Virology

Armbruster, Holger (Heidelberg) German.Cancer.Research.Center.NCT.National.Center.for.Tumor.Diseases

B

Bartkowiak, Kai (Hamburg) University.Medical.Center.Institute.for.Tumor.Biology

Bastians, Holger (Göttingen) Universitätsklinikum.Abteilung.für.molekulare.Onkologie

Bayer, Helene (Heidelberg) German.Cancer.Research.Center.

Becker, Marina (Münster) University.Hospital..Institute.of.Neuropathology

Becker, Michael (Berlin) Experimental.Pharmacology.&.Oncology.Berlin-Buch.GmbH.

Bednarz, Natalia (Hamburg) University.Medical.Center.Institute.of.Tumor.Biology

Beyer, Ulrike (Göttingen) University.of.Goettingen.Goettingen.Center.of.Molecular.Biosciences.

Bier, Carolin (Mainz) University.Medical.Center.Molecular.and.Cellular.Oncology

Bormann, Felix (Berlin) Charité,.Institute.of.Pathology

Brabletz, Thomas (Freiburg) University.of.Freiburg.Dept..of.Visceral.Surgery.and.Comprehensive.Cancer.Center

Braeuning, Albert (Tuebingen) University,.Institute.of.Experimental.and.Clinical.Pharmacology.and.Toxicology

Braun, Norbert (Tuebingen) University,.Department.of.Radiation.Oncology

Bug, Monika (Göttingen) University.Medicine.Göttingen.Ernst.Caspari.Haus

Bühler, Helmut (Herne) Ruhr-Universität.Bochum,.Marienhospital

Bukur, Juergen (Halle / S.) University,.Institute.of.Medical.Immunology

Bünger, Stefanie (Lübeck) University.of.Lübeck,.Department.of.Surgery

C

Chen, Yuan (Jena) University,.Institute.of.Pathology

Christmann, Markus (Mainz) University.Medical.Center.Department.of.Toxicology

Croce, Carlo (Columbus)

Cui, Tiantian (Jena) University,.Institute.of.Pathology

D

Damm, Friederike (Düsseldorf) University.Medical.Center.Institute.of.Pathology

Demir, Resit (Erlangen) Universitätsklinikum..Klinik.für.Chirurgie.

Der, Channing (Chapel Hill)

Dikic, Ivan (Frankfurt) Klinikum.der.Goethe-Universität.Institut.für.Biochemie.II

Domschke, Christoph (Heidelberg) University.of.Heidelberg.Department.of.Obstetrics.and.Gynecology

E

Eke, Iris (Dresden) OncoRay.National.Center.for.Radiation.Research.in.Oncology

Engers, Rainer (Düsseldorf/Neuss) Universitätsklinikum.Institut.für.Pathologie

F

Faethe, Christina (Mainz) University.Medical.Center.Institute.of.Physiology.and.Pathophysiology

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AEKAEK AEKFalkowska-Hansen, Berit (Heidelberg) German.Cancer.Research.Center.Genetics.of.Skin.Carcinogenesis

Felsher, Dean (Stanford) Stanford.University.Division.of.Oncology

Fichtner, Iduna (Berlin) Max-Delbrück.Centrum..for.Molecular.Medicine

Friedl, Peter (Nijmegen) Radboud.University.Nijmegen.Medical.Centre.Department.of.Cell.Biology

Fritsche, Raphaela (Berlin) Charité,.Institute.of.Pathology

Fritz, Gerhard (Mainz) University.Medical.Center,.Toxicology

Fröhling, Stefan (Ulm) Universitätsklinikum.Klinik.für.Innere.Medizin.III

G

Galon, Jérôme (Paris) Integrative.Cancer.Immunology.Laboratory

Gdynia, Georg (Heidelberg) German.Cancer.Research.Center.Institute.of.Pathology

Georges, Rania (Heidelberg) German.Cancer.Research.Center

Goepfert, Katrin (Mainz) Universitätsklinikum.1..Medizinische.Klinik

Goering, Wolfgang (Düsseldorf) University.Medical.Center.Department.of.Urology.

Gomis, Roger (Barcelona) Institute.for.Research.in.Biomedicine

Greten, Florian (München) Klinikum.rechts.der.Isar.Institut.für.Molekulare.Immunologie

H

Hamid, Syed (Izmir) EBILTEM.Hall,.Department.of.Bioengineering.

Hanenberg, Helmut (Indianapolis) Indiana.University.School.of.Medicine.Herman.B.Wells.Center.for.Pediatric.Research

Henning, Stefan (Buxtehude) Elbekliniken.Buxtehude.Dermatologisches.Zentrum

Hippe, Andreas (Düsseldorf) University.Medical.Center.Department.of.Dermatology

Hirschfeld, Marc (Freiburg) University.Medical.Center.Gynecological.Hospital

Hoffmann, Jessica (Tübingen) Universitätsfrauenklinik

Hoffmann, Michèle Janine (Düsseldorf) University.Medical.Center.Institute.of.Pathology

Hülsmann, Helen (Heidelberg) German.Cancer.Research.Center.NCT.National.Center.for.Tumor.Diseases

J

Jaeger, Markus (Freiburg) Universitätsklinikum,.Frauenklinik.

Jonkers, Jos (Amsterdam) The.Netherlands.Cancer.Institute

Jung, Andreas (München) Ludwigs-Maximilians.Universität.Pathologisches.Institut

K

Kahn, Nicolas (Heidelberg) German.Cancer.Research.Center.Unit.Cancer.Genome.Research

Keller, Larissa (München) Technische.Universität.Klinische.Forschergruppe.„Molekulare.Tumorbiologie“

Kloke, Björn-Philipp (Frankfurt/M.) Georg-Speyer-Haus.Chemotherapeutisches.Forschungsinstitut.

Kobelt, Dennis (Berlin) Max.Delbrück.Centrum.for.Molecular.Medicine

Koch, Annemarie (Düsseldorf) University.Medical.Center.Department.of.Urology

Kohlhof, Hella (Martinsried) 4SC.AG

List of Speakers and Poster Authors

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AEKKollar, Jasmin (Heidelberg) German.Cancer.Research.Center.Genetics.of.skin.carcinogenesis

Kovacheva, Marineta (Heidelberg) German.Cancer.Research.Center

Krause, Claudia (Berlin) Max-Delbrück.Centrum.Molecular.Tumor.Genetics.and.Immunogenetics

Kroemer, Guido (VILLEJUIF CEDEX) Institut.Gustave.Roussy.Pavillon.de.Recherche

Krunic, Damir (Heidelberg) German.Cancer.Research.Center

Kuznia, Christina (Berlin) Charité,.Institute.of.Pathology

L

Leufke, Christine (Heidelberg) German.Cancer.Research.Center.Genetics.of.Skin.Carcinogenesis

M

Maak, Matthias (München) Technische.Universität.Klinik.für.Chirurgie

Mack, Laura (Frankfurt/M.) Georg-Speyer-Haus

Malek, Nisar (Hannover) Medizinische.Hochschule.Hannover.Klinik.für.Gastroenterologie,.Hepatologie.und.Endokrinologie

Mallem, Nedjouna (Berlin) Max-Delbrück.Centrum.for.Molecular.Medicine

Mejias Luque, Raquel (München) Technische.Universität

Michele, Inga Dorina (Essen) University.Hospital.Essen.Institute.of.Cell.Biology

Milde-Langosch, Karin (Hamburg) Universitätsklinikum

Monz, Miriam (Marburg) University,.Institute.of.Pathology

Müller, Annett (Mainz) University.Chemotherapeutisches.Forschungsinstitut

Müller, Eva Margarethe (Düsseldorf) University.Medical.Center.Institute.of.Pathology

Müller, Nina (Frankfurt/M.) Georg-Speyer-Haus.First.Department.of.Internal.Medicine

N

Nazarenko, Irina (Karlsruhe) Karlsruhe.Insitute.of.Technology.Institute.of.Toxicology.and.Genetics

Neri, Dario (Zurich) Swiss.Federal.Institute.of.Technology.(ETH).Institute.of.Pharmaceutical.Sciences

Niegisch, Günter (Düsseldorf) University.Medical.Center.Department.of.Urology

Nindl, Ingo (Berlin) Charité,.Viral.Skin.Carcinogenesis

Nitsche, Ulrich (Munich) Technical.University.Clinic.of.surgery

O

Oberoi, Pranav (Frankfurt/M.) Georg-Speyer-Haus.Chemotherapeutisches.Forschungsinstitut

Oden, Felix (Berlin) Max-Delbrück.Centrum.for.Molecular.Medicine

Olszewski, Ulrike (Vienna) Ludwig.Boltzmann.Cluster.Translational.Oncology

Ontikatze, Teona (Essen) University.of.Duisburg-Essen.Institute.of.Cell.Biology

Otto, Angela (Garching) Technical.University.Institute.of.Medical.Engineering

P

Pantel, Klaus (Hamburg) University.Medical.Center.Hamburg-Eppendorf.Institute.of.Tumor.Biology

Pusterla, Tobias (Heidelberg) German.Cancer.Research.Center.Signal.trasnduction.and.growth.control


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AEKAEK AEKQ

Quiros, Steve (Mainz) University.Medical.Center.Institute.of.Toxicology

R

Riehle, Ulrike (Freiburg) University.Hospital.Department.of.Obstetrics.and.Gynecology

Rohwer, Nadine (Berlin) Charité

Roos, Wynand (Mainz) University.Medical.Center.Institute.of.Toxicology

Rose, Michael (Aachen) Universitätsklinikum,.Institut.für.Pathologie

Rudner, Justine (Tuebingen) University.Hospital.Department.of.Radiation.Oncology

S

Salem, Ahmed (Amman) King.Hussein.Cancer.Center

Schmid, Felicitas (Berlin) Max-Delbrück.Centrum.Molekulare.Medizin

Schönfeld, Kurt (Frankfurt/M.) Georg-Speyer-Haus

Schultz, Silke (Tübingen) Universitätsfrauenklinik

Seibt, Stephanie (Berlin) Charité,.Institute.of.Pathology

Seifert, Christin (Essen) University.Hospital.Department.of.Dermatology

Seliger, Barbara (Halle/S.) University.Institute.of.Medical.Immunology

Sers, Christine (Berlin) Charité,.Institute.of.Pathology

Smalley, Matthew (London) Breakthrough.Breast.Cancer.Centre.Institute.of.Cancer.Research

Stelniec, Iwona (Berlin) Charité,.Institute.of.Pathology

T

Thomale, Jürgen (Essen) University.of.Duisburg-Essen.Institute.for.Cell.Biology

V

Vafaizadeh, Vida (Frankfurt/M.) Georg-Speyer-Haus

Vormbrock, Kirsten (Berlin) Max-Delbrück.Centrum.for.Molecular.Medicine

W

Welte, Yvette (Berlin) Charité,.Institute.of.Pathology

Wilde, Cornelia (Heidelberg) German.Cancer.Research.Center

Wolf, Jürgen (Köln) Universitätsklinikum.Klinik.I.für.Innere.Medizin

Wu, Hui (Heidelberg) German.Cancer.Research.Center.Divison.of.Molecular.Genetics

Y

Yang, Linlin (Jena) University,.Institute.of.Pathology

Z

Zaharieva, Maya M. (Heidelberg)

German.Cancer.Research.Center.Toxicology.and.Chemotherapy.Unit

Ziebold, Ulrike (Berlin) Max-Delbrück.Centrum.for.Molecular.Medicine

Zimmerer, Rüdiger (Hannover) Medizinische.Hochschule.Klinik.für.Mund-,.Kiefer-.und.Gesichtschirurgie


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AEK AEK16th International AEK Cancer CongressAbteilung Experimentelle Krebsforschung


March 16th – 18th, 2011 | Heinrich-Heine-University Düsseldorf

Congress Program and Abstracts

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AEK th International AEK Cancer Congress · e-mail:. [email protected] URL:.. ... D-99407 Weimar or via...

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Transcript of AEK th International AEK Cancer Congress · e-mail:. [email protected] URL:.. ... D-99407 Weimar or via...