Lez tumori3b retroSHH
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04/05/16 1 HEDGEHOG PATHWAY Hedgehog porcospino Genes in development and cancer
Transcript of Lez tumori3b retroSHH
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HEDGEHOG PATHWAY
Hedgehog
porcospino
Genes in development and cancer
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WNT Colorectal cancer 85% mutated in sporadic cases, Familiar Adenomatosys P >90% in familial Desmoid (Aggressive Fibromatosis) tumor ( 74% sporadic), FAP 10% familial Hepatoblastoma (liver cancer in children) 67% spoardic, FAP 1% familial Germline loss of funcQon of a single copy of a tumor suppressor Hedgehog Basal cell carcinoma 50% sporadic, Basal cell nevus syndrome 100% familial Medulloblastoma (Tumore del cervelleTo, da stem neuroectoderma) 25% sporadic, BCNS 1%familial
Stem Transit amplifying Terminally differenQated
Stem cell connecQon La maggior parte dei tumori nasce da pelle,intesQno e sangue, dove sono cellule staminali
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Sonic Hedgehog Signaling in BASAL CELL CARCINOMA , BCC Tumorigenesis and MEDULLOBLASTOMA (brain) The hedgehog (Hh) pathway has a fundamental role in embryonic development, being responsible for paTerning many developing organs and Qssues. DeregulaQon of Hh signaling causes development of BCC and medulloblastoma and other tumor types There are three Hh proteins known in mammals :Sonic Hh [Shh], Indian Hh, and Desert Hh. Shh is the best characterized homolog, and the only vertebrate Hh expressed in skin
Although the majority of BCCs (Basal Cell Carcinoma) arise sporadically, many cases are aTributable to basal cell nevus syndrome (BCNS), an autosomal dominantly inherited disorder characterized by the occurrence of mulQple BCCs and by extracutaneous tumors. GeneQc studies on paQents with BCNS have led to the idenQficaQon of inacKvaKng mutaKons in the human homolog of the Drosophila gene patched (Ptc), a negaQve regulator of the Hh pathway, as the geneQc defect underlying this syndrome. The Ptch1 gene is also inacQvated in sporadic BCCs.
BASAL CELL NEVUS SYNDROME, BCNS
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BCNS/Gorlin syndrome (AD): Basal Cell Nevus Syndrome: germ line mutaQons in Patched
The basal cell nevus syndrome is associated with odontogenic cysts of the jaws, pitted depressions of the hands and feet (tiny basal cells) and osseous anomalies of the skeleton. Care is given by removal of the carcinomata on a regular basis and genetic counseling, since this is an autosomal dominatly inherited disorder. •Broad nose•Heavy, protruding brow•Jaw that sticks out (in some cases)•Wide-set eyes
Loss Of Heterozygosis in Patched gene (LOH in PTch1)
Cancer of the skin (90% of all skin cancer; UV exposure is a risk factor)
Ci: GLI family zinc finger 1, P08151 GLIoma-‐associated oncogene, transcripQon factor SUFU Suppressor of FUsed
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SonicHedgeHog regola organogenesi e organizzazione del cervello
Christiane Nüsslein-Volhard
Eric F. Wieschaus
Premio Nobel 1995
In Drosophila, Ptc encodes a transmembrane glycoprotein involved in segment polarity that acts by prevenQng Smo from reaching the cell surface. Cubitus interruptus (Ci) forms a complex with a number of interacQng proteins (i.e., Cos2, Fu and Sufu), which promotes Ci processing into an inacQve form
Patched
Smoothened
Cubitus interruptus
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Non-‐moKle primary cilia
The effectors of Hh signaling in vertebrates are the Gli proteins (i.e., Gli1, Gli2 and Gli3), whose acQon is inhibited by Sufu and Iguana or DZIP1 (in zebrafish or humans, respecQvely), and the Drosophila Cos2 primary human homolog Kif7.
COS
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Medulloblastoma : tumore pediatrico del cervelleTo
Posterior Fossa EmbryonalTumor INFRATENTORIAL/PRIMITIVE NEUROECTODERMAL TUMOR
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Corn lily,
Veratrum
californicum
Da questa pianta si estrae la ciclopamina (steroidal alkaloid) teratogeno
Hellebore: was natural treatment VomiQng in pregnancy
ciclope
FeQ umani affei da oloprosencefalia (ciclopi) sono causaQ da mutazioni Nei geni PTC (patched) o SHH (sonic hedgehog) In Idaho (USA) 25% di nuovi naQ fra gli animali allevaQ nei ranch erano ciclopici (1950)
CyclopQc lamb
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LOSS OF Sonic hedghog signal transducQon Cyclopia associated
Ciclopamina blocca SMO
23 mutazioni in SHH sono associate a ciclopia quindi…
Durante lo sviluppo questo Pathway è essenziale
Increase of SH signalling is tumor associated With loss of PTCH GOF of SMO, SHH
,
ReceTori: Patched, 12 transmembrane, suppressor Smoothened: 7 transmembrane, protoncogene
Carcinomi endoteliali
GACCACCCA
Cell cycle genes, cyclin D1, segmentaQon genes
Controllo ciclo cellulare in staminali adulte
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Da ciclopamina a SARIDEGIB, natural product derivaKve ( not syntheQc)
Unlike other Smoothened (Smo) inhibitors, the drug resistance was neither mutaKon-‐dependent nor Gli2 amplificaKon-‐dependent, and saridegib was found to be acQve in cells with the D473H point mutaQon that rendered them resistant to another Smo inhibitor, GDC-‐0449. The fivefold increase in lifespan in mice treated with saridegib as a single agent compares favorably with both targeted and cytotoxic therapies. The absence of geneQc mutaQons that confer resistance disQnguishes saridegib from other Smo inhibitors.
CLINICAL TRIAL PHASES
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Phase 1 Purpose: How much? How? Is it dangerous? To find a safe dose To decide how the new treatment should be given (by mouth, in a vein, etc.) To see how the new treatment affects the human body Number of people taking part: 15–30 Phase 2 Purpose: Is effecQve? Is dangerous? To determine if the new treatment has an effect on a certain cancer To see how the new treatment affects the body Number of people taking part: Less than 100 Phase 3 Purpose: Is it beTer? RandomizaKon: To compare the new treatment (or new use of a treatment)in a InvesKgaKon Group with the current standard treatment (Control Group) Number of people taking part: From 100 to several thousand A computer is usually used to assign paQents to groups. Single blinded: PaQent does not know if is taking the drug or a placebo Double blinded: Not even the doctors know in which group the paQent is
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Controllo dell’integrità del genoma
The care taker genes
XP non riparano danni da UV HNPCC non riparano basi non appaiate
Scopo principale della risposta di danno al DNA: prevenire la replicazione del DNA in presenza di un danno del DNA e la conseguente instabilità genomica
1) Aivazione dei checkpoint cellulari per arrestare il ciclo cellulare
2) Aivazione dei meccanismi di riparo del DNA
3) Riparo del danno e ripresa del ciclo cellulare… oppure, se il danno non è riparabile, si aiva la risposta apoptoQca
Riparo del DNA Apoptosi
Danno al DNA
Arresto del ciclo cellulare
Sopravvivenza
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From Y. Shiloh, 2003, Nat Cancer Rev
Diversi Qpi di danno al DNA e diversi meccanismi di riparo
From R. Hakem, 2008, EMBO J
MMR Mis Match Repair; NER NucleoQde Excision Repair; Base Excision Repair; Homologous RecombinaQon; Non Homologous End Joining NER -‐> Xeroderma Pigmentosus MMR à Human Non Polyposis Colon Cancer
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HR pathway: Homologous RecombinaQon, viene uQlizzata in procarioQ ed eucarioQ per risolvere roTure a doppio filamento (DSB). Viene uQlizzata in circa il 90% dei casi di DSBs nei baTeri ed in lievito, e solo nel 10% dei casi di DSBs in cellule di mammifero, in cui si preferisce l’aivazione della NHEJ. Richiede l’aivazione di una serie di complessi proteici e di una cascata di evenQ di trasduzione del segnale. E’ molto accurata.
NHEJ: Non Homologous End Joining, viene uQlizzata in procarioQ ed eucarioQ per risolvere roTure a doppio filamento (DSB). Viene uQlizzata solo in circa il 10% dei casi di DSBs nei baTeri ed in lievito, e nel 90% dei casi di DSBs in cellule di mammifero. Richeiede l’aivazione di una serie di complessi proteici e di una cascata di evenQ di trasduzione del segnale. Non e’ molto accurata bensi error prone.
Danno al DNA su entrambi i filamenQ: Double Strand Breaks (DSBs)
D. Barilà
Direct reversal: a differenza delle altre risposte non richiede L’aivazione di complessi mulQproteici. Un esempio: O6-‐alkylguanina. L’alchilazione impedisce l’appaiamento con la citosina. Viene rimossa grazie all’azione di un singolo enzima, Ada in E. coli corrispondente a O6-‐methyltransferase, MGMT in cellule di mammifero.
MMR pathway: Mismatch Rapair, presente in eucarioQ e procarioQ viene uQlizzata per il riparo di mismatches, ossia piccole inserzioni o delezioni.
NER pathway: NucleoKde Excision Repair,presente in eucarioK e procarioK viene uKlizzata per il riparo di diverse lesioni tra cui lesioni causate da UV, mutageni chimici e chemioterapici che inducano la formazione di dimeri di pirimidina e addod sproporzionaK. 3 proteine (UvrA, UvrB, UvrC) in procarioK, più di 30 in mammifero. Malade del NER in Xeroderma Pigmentosus: forte predisposizione a tumori della cute
BER pathway: Base Excision Repair, viene uQlizzata in procarioQ ed eucarioQ per rimuovere basi danneggiate.
Daniela Barilà
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Xeroderma Pigmentosus the disease is autosomal recessive
XP Trichothiodystrophy
Difei in NucleoKde Excision Repair ( NER)
Xeroderma Pigmentosus genodermatosi Aut Rec
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Edward Alfred Cockaney 1880-‐1956 descrisse una sindrome di Sensibilità alla luce (UV). I sintomi sono progressivi:statura bassa, Invecchiamento precoce (progeroid), non predispone a tumori, demielinizazzione causa disfunzioni neurologiche Geni CSA/ERCC8( chr.5) e CSB/ERCC6 (chr.10) per RIPARO TRASCRIZIONALE (Aut. Rec) Xeroderma Pigmentosus, autosomico recessivo anormalità della pelle, mulKpli tumori epiteliali (incidenza 2000X), morte neuronale ed anormalità neurologiche 8 gruppi di complementazione implicaK: XP-‐A_XP-‐G e variante. Mutazioni colpiscono il NER NucleoKde Excision Repair TTD TricoKodistrofia, capelli fragili privi di zolfo, proteine con cisteine sono ridoTe, lineamenQ anomali, ritardo mentale, ichQosis , non predispone a tumori Gene assegnato al gruppo di complementazione XP-‐D( chr.10) EREDITA AUTOSOMICA RECESSIVA
ERCC Excision Repair Cross-‐ComplemenQng
Xeroderma Pigmentosus gruppo D:XPD/ERCC2 19q13; ERCC6 10q11
XP-D 80kD è una subunità del fattore di Trascrizione TFIIH
TFIIH 9 subunità: XPB,p62,p52,p44,p34 sono “core” cioè associate strettamente.XP-D è una HELICASE che svolge il DNA 5’-3’ è essenziale in (NER) riparo in trascrizione
OMIM 609413
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CSB/ERCC6 (Excision Repair Cross Complementation group6), 1493aa, 10q11, a member of a subfamily of putative helicases, is involved in Cockayne's syndrome and preferential repair of active genes.Troelstra C et al. Cell 1992 Dec;71(6):939-953PMID: 1339317
The ERCC6 gene is part of the nucleotide Excision Repair (NER) pathway, a complex system that eliminates a broad spectrum of structural DNA lesions, including ultraviolet (UV)-induced, cyclobutane pyrimidine dimers, bulky chemical adducts, and DNA cross-links. One of the NER pathways preferentially repairs lesions on the transcribed strand of active genes; this process occurs more rapidly than repairs on nontranscribed strands that are part of overall genome repair Troelstra et al., 1992
ATP-‐dependent 5'-‐3' DNA helicase, component of the core-‐TFIIH basal transcripQon factor. Involved in nucleoKde excision repair (NER) of DNA by opening DNA around the damage, and in RNA transcripKon by RNA polymerase II by anchoring the CDK-‐acQvaQng kinase (CAK) complex, composed of CDK7, cyclin H and MAT1, to the core-‐TFIIH complex. Involved in the regulaQon of vitamin-‐D receptor acQvity. As part of the mitoQc spindle-‐associated MMXD complex it plays a role in chromosome segregaQon. Might have a role in aging process and could play a causaKve role in the generaKon of skin cancers
P18074
CytogeneQc locaQon: 19q13.32
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Distribution of mutations in XP-D patients with different disorders. P18074, chr 19q13.3
Alan R. Lehmann Genes Dev. 2001;15:15-23
Cold Spring Harbor Laboratory Press
The Xeroderma Pigmentosum group D (XPD) gene: one gene, two funcQons (TranscripQon and Repair), three diseases (Cockayne Syndrome, Trichothiodystrophy).Alan R. Lehmann
The hypothesis that Xeroderma Pigmentosus and Cockayne Syndrome are repair syndrome and TTD a transcripQon syndrome (Bootsma and Hoeijmakers 1993).Autosomal recessive
XPD Arg683
TTD R112 CS
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"SelecKve regulaKon of vitamin D receptor-‐responsive genes by TFIIH." Drane P., Compe E., Catez P., Chymkowitch P., Egly J.-‐M. Mol. Cell 16:187-‐197(2004) Indeed, certain symptoms found in paQents (XP, CS and TTD carrying a mutaQon in XPD could be due to a defect in the vitamin D (vitD) pathway, a hormone known for the maintenance of mineral homeostasis and skeleton architecture (Jones et al., 1998). We therefore invesQgated how VDR transacQvates in XPD-‐deficient cells, and we found that the vitD-‐mediated response can be selecQvely impaired (vitD Response and vitD accumulaQon).
TFHII (CDK7) phosphorylates Ets1 VitaminD-‐Receptor+ phETS1 -‐-‐-‐-‐à transcripQon of OSTEOPONTIN , cyp24 (degradaKon of 1,25OH D3), p21/WAF1
Non solo repair……………….anche mancanza di capacità degrada6ve della vit.D…….
TranscripQon-‐Coupled Global Genome
Cockayne syndrome WD repeat protein A (CSA) and CSB Xeroderma pigmentosum
group C-‐complemenQng protein (XPC)–RAD23B and DNA damage-‐binding protein (DDB);
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HNPCC malada del riparo Human Non Polyposis Colon Cancer Familiare Sporadica INSTABILITA’MICROSATELLITI CROMOSOMI NORMALI (EUPLOIDIA) MMR pathway: Mismatch Rapair, presente in eucarioQ e procarioQ viene uQlizzata per il riparo di mismatches, ossia piccole inserzioni o delezioni.
HNPCC, aut dom Hereditary non-‐polyposis colon cancer
CaraTerizzato da MICROSATELLITE INSTABILITY ( MSI)
è autosomico dominante
MSH2 2p 50-‐60% dei HNPCC (human mutS homolog2)
MSH6 2p
MLH1 3p 30-‐40% HNPCC(h-‐mutL homolog)
PMS1 2q (h-‐post-‐meioQc-‐segregaQon)
PMS2 7p
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Micro Satellite Instability Pathway
• In humans, seven DNA Mis-‐Match Repair (MMR) proteins : MLH1, MLH3, MSH2, MSH3, MSH6, PMS1 and PMS2
Queste proteine riparano ripeQzioni erronee faTe dalla polimerasi nei microsatelliQ, cioè regioni con ripeQzioni monotone della stessa sequenza
Mutazioni Secondarie in geni con microsatelliQ ( TGFbetaRII; BAX) se questo sistema di riparo è perso
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MSI Cancers Microsatellite Instability: 90% dei HNPCC Hereditary Non Poliposis Colon Cancer e 15% dei tumori sporadici al colon. Sono sempre diploidi (euploidia) DNA mismatch repair genes : hMLH1 3p, hMSH2 2p, hPMS1, hPMS2, GTBP/hMSH6. -‐ Germline mutaKons + LOH in familiar HNPCC ( autosomal dominantly inherited disorder of cancer suscepKbility with high penetrance (80–85%). -‐ EpigeneKc change ( HypermetylaKon) in sporadic colon cancer. Prevalent LOF in genes : hMSH2 (human MutS homologue) on chromosome 2p, hMLH1(human MutL homologue) on chromosome 3p
Geni uQlizzaQ per il riparo di appaiamenQ Non correi (indel)
1-‐ DNA mismatch repair genes : hMLH1 3p. 2-‐ Germline mutaKons + LOH in HNPCC ( autosomal dominantly inherited disorder of cancer suscepKbility with high penetrance (80–85%). 3-‐ EpigeneKc change ( HypermetylaKon) in sporadic colon cancer.
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is defined as a relaQvely frequent change of any length of these loci
CIMP pathway : CpG Island Methylated Pathway e mutazioni GOF in Raf. 15% degli sporadici Vengono meKlaK e spenK I promotori di molK geni, fra cui tumor suppressor genes, fra cui spesso hMLH1. Prevalente tumore in donne anziane. Non presenta polipi adenomatosi, bensì polipi sessili formaQ da colonociQ che si affastellano per aività incontrollata di RAS-‐RAF-‐MEK e blocco di apoptosi
GOF RAS + LOF MMR
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CpG -‐-‐-‐à CmetpG la meKlazione della citosina che precede una guanina
CpG promotore-‐-‐-‐-‐-‐-‐à GENE TRASCRITTO-‐-‐-‐ (esempio hMLH1 per il riparo di danni al DNA) CmetG promotore-‐-‐-‐-‐-‐-‐| gene non espresso La ipermeQlazione causa blocco dell’espressione genica
C-‐-‐-‐+ Bisulfito-‐à U Cmet-‐-‐-‐-‐+ Bisulfito—rimane Cmet
Nat Biotec 27 (2009)
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hTp://meeQnglibrary.asco.org/content/43444?media=vm by Sanford D. Markowitz
A biomarker to enrich for COLON STEM CELLS: the AcQvated Leukocyte Cell Adhesion Molecule ALCAM/CD166 as a marker in immature colon epithelial cells expressed at the boTom of colon crypts A marker for CANCER STEM CELL : ALCAM/CD166 was capable of idenQfying colon cancer stem cells, which led the researchers to look for genes that were not expressed in ALCAM/CD166-‐posiQve tumors but that were expressed in ALCAM/CD166-‐negaQve tumors. (2300genes were screened) CDX2 is a master transcripKon factor controlling the expression of many differenQaQon genes in colon epithelial cells. They found significant differences between the paQents with CDX2-‐negaQve tumors (n = 32) and those with CDX2-‐posiQve tumors (n = 434): the 5-‐year DFS was 41% vs 74% (P < .001).
Medscape Medical News > Oncology CDX2 -‐-‐ New PrognosQc Biomarker in Stage II Colorectal Cancer Alexander M. Castellino, PhD; January 20, 2016
