The continuously changing scienceFrom infection to cancer and back

2012

Ana Maria Barral

What is Science?

“We shall not cease from exploration. And the end of all our exploring will be to arrive where we started

and know the place for the first time.”

T. S. Eliot

This talkA bit of personal history

Cancer and inflammation through the history of Tumor Necrosis Factor alpha (TNFa)

Philosophy of science (guaranteed brief)

“What we’ve got here is a failure to communicate:” cancer biologists and immunologists

Projects: Ca-pterinScience course

The dry facts B.Sc of Biochemistry, University of Havana, Cuba

Thesis: protease inhibitors from sea anemones

National Institute of Oncology and Radiobiology Development and characterization of mAbs against malignant

melanoma

University of Linkoping, Sweden Ph.D. thesis: Melanoma, thioredoxin, and cytokine regulation

La Jolla Institute for Allergy and Immunology, San Diego Postdoc projects: Exosomes, cytokines and chemokines in Type

1 diabetes

Nereus Pharmaceuticals Characterization of proteasome inhibitors with anti-tumor effect

from marine microorganisms

Malignant melanoma

Tumor derived from the pigment producing cells (melanocytes) from the skin

Least abundant but most lethal skin cancer

Presence of tumor antigens and often strong immune response

TNF and receptorsTumor necrosis factor

alpha

26 kD protein cleaved to the 17 kD mature TNF (soluble & intracellular)

2 receptors, TNF-R1 and TNF-R2

Currently a whole superfamily of conserved factors and receptors

TNF superfamily

TNF as a link between cancer and infection

(inflammation)Original observation: bacterial extracts could

provoke tumor necrosis

This was caused by a factor released by the host cells in response to bacterial endotoxin

TNF= cachexin, circulating factor in parasite infected animals

TNF is an important mediator of the inflammatory response, needs to be controlled (septic shock)

Tumor cells can also produce TNF

The devil is in the details…

There are 2 receptors

Human TNF only acts on mTNFR1, mouse TNF on both

Some pathways are the same, but the default is for growth

Response will depend on context

TNF tumor toxicity was mainly observed with concurrent metabolic inhibition

TNFa signaling pathways

Tumor-promoting effect of TNFa

Balkwill, Nat Rev Cancr VOLUME 9 | MAY 2009 | 361

TNF and melanomaPrevious study: TNF+ primary melanomas

present less CD3+ T cell infiltration (Sander & Boeryd, 1996)

Goal : how is TNF expressed in melanoma cells and how does it affect the resistance against cytotoxic attack

Multiple approaches: cell lines and patient samples, detection of intracellular and secreted TNFa, transfection of cell lines with tagged pro-TNF

TNFa expression protects melanoma cells

TNF is present in Golgi but not in melanosomes

TNF and WGA TNF and HMB-45 (melanosome marker)

Immunohistochemical studies of TNF in melanoma patients

TNF staining in primary melanomas

Hazard Rate P Confidence intervalTumorThickness 1.555 0.010* 1.113-2.171Clark level 2.462 0.247 0.536-11.309Mitotic index 1.227 0.039* 1.010-1.490Age 0.996 0.834 0.960-1.034TNFa 0.113 0.046* 0.013-0.966

Patients with TNFa+ tumorshad a significantly bettersurvival than those with TNFa-tumors

TNFa : an independentprognostic factor?

Study of the intracellular dynamics of TNF in melanoma

cells

Pro-TNF Mature TNF GFP

FLAG Pro-TNF Mature TNF GFP

N-

N-

-C

-C

TNF is correctly cleaved in melanoma cell lines by TACE

Uncleaved TNF (yellow), mature TNF (green),Pro-TNF (red)

TNF is transported to the dendrites and transferred to neighboring cells after PMA

stimulation

TNF and TNF-receptors are released in exosomes

Colocalization of TNF and TNFR1 afterPMA stimulation in the dendrites

TNF and TNF-receptors arepresent in exosomes

TNF in exosomes provoke higher levels of ROS in T-

cells

Conclusions TNF-melanoma project

TNF is correctly cleaved in melanoma cell lines

Upon stimulation it is transported to the dendrites and transferred to neighboring cells or

Released via exosomes

Possible local or systemic functional role?

CANCER-INFLAMMATION CONNECTION REDUX

Who was first, the hen or the egg?

What are Exosomes?

Small (60-90 nm) vesicles of endocytic originSecreted by APCs, B-cells, tumor cellsCapable to prime against tumor antigens: immunotherapy“Trojan exosome” hypothesis: HIV uses exosome pathway for budding?

Some cytokines can be released via exosomes

Possible role of exosomes during viral infection

Exosomes and viruses share similar budding pathways in certain cells

Exosomes from antigen-presenting cells present MHC class I-II antigens and costimulatory molecules

Could exosomes be released by virus-infected cells and “amplify” the antiviral response?

NP: nucleoprotein (np396) CD8+

GP: glycoprotein (gp33) CD8+

(gp61) CD4+

Viral infection: viremia peaks day 3 CTL response/viral clearance day 6-8Intracranial infection: mice die because of CNS damage by CTLs

LCMV lymphocytic choriomeningitis virus

RIP-LCMV Mouse Model for Type 1 Diabetes

T CellPool

bGP

GPGP

GP

ToleranceIgnorance

1

Antigen-specificprecursor T-cells

b-Cell Destruction(antigen specific

adaptive)

GP

GPGP

4

Overt Diabetes(Day 10-14)

Virus elimination(antigen specific

adaptive)

3

bGP

GPGP

GP

No DiabetesNo Diabetes

LCMV2

bGP

GPGP

GP

InflammationCellular attraction

(non-specific / innate)

LCMV

No Diabetes

Exosomes were obtained from LCMV-infected cells and mice

EM of serum exosomes from LCMV-infected miceWestern blot of exosomes from splenic DCsof infected mice

Exosomes derived from LCMV-infected DCs and serum express CD11c, some B7.2 and low FasL

Iad+ PKH62exo

Exosomes isolated with CD11b-Dynabeads

Exosomes isolated with Dynabeads coupled to MHC-II (Iad) from PKH26 (red) labeled DCs

Added 2 hrs to splenic GFP-DCs

PKH26 exosomes

Exosomes as vaccines…nope.

Cancer causes inflammation

“The Dialectical Biologist”An organism does not compute itself from its DNA. The organism is the consequence of a historical process that goes on from the moment of conception until the moment of death; at every moment gene, environment, chance, and the organism as a whole are all participating. . . .Natural selection is not a consequence of how well the organism solves a set of fixed problems posed by the environment; on the contrary, the environment and the organism actively codetermine each other. (Levins and Lewontin, 1985)

My philosophy of science Forest and trees

Avoid mechanical reductionist thinking ALTHOUGH scientists HAVE to apply a reductionist approach

Go back as often as you can to the big picture but avoid superorganic holism

Historical approach (lots can be learned from reading the MatMet sections of the old articles)

Do not be afraid to question established paradigms: there is no such as absolute truth

Exploration of our world is a dynamic process

Cross-pollinate

Lost in TranslationTumor biologists focus on the tumor cell,

especially on its genes

Tumor immunologists study the immune mechanisms reacting (or not) to the tumor cell

Few instances of dialogue

Cytokines involved in beta-cell death

IFNg: direct apoptosis (with TNFa) upregulation of MHC class-1TNFa: direct apoptosisIL1: induction of iNOS, NO productionalso mediates dsRNA mediated damage (viral infection)

SOCS: negative regulator of JAK-STAT responsesInhibits signaling of IL1, IFNg IL2, IL3, IL4 and others

Transgenic mice with cytokine signaling defects in beta-cells

Mice used in this study(T. Kay and E. Thomas, Australia)

RIP-GP+/SOCS-1+ (SOCS: suppressor of cytokine signaling)

RIP-GP+/IFNgRtg+

RIP-GP+/IL1Rko

Mice were infected with LCMV, and diabetes incidenceand immunological parameters were followed

Diabetes incidence

Diabetes incidence

0 25 50 75 1000

10

20

30

40

50

60

70

80

90

GP+SOCS+ (n=12)

GP+SOCS- (n=17)

IFNgRtg (n=42)

IL1Rko (n=9)

days

Per

cen

t d

iab

etes

SOCS mice do not present lymphocytic infiltration

SOCS mice do not present upregulation of the chemokine

IP10 (CXCL-10) after viral infection

SOCS - SOCS+

SOCS+ islets are more resistant to killing by

effector cells or cytokines

SOCS- SOCS+ 0

10

20

30

40SOCS- isletSOCS + islet

% C

ytot

oxic

ity

TNF TNF+IL10

10

20

30

40SOCS- isletSOCS + islet

% C

ytot

ocix

ity

SOCS+ mice do not upregulate Fas and MHC-class I expression

after viral infection

Fas MHC class I

B6 RIP-GP ILR1ko IFNgRtg SOCS+0

500

1000

1500

2000

2500

3000

3500

Me

an

Flu

ore

sc

en

t In

ten

sit

y

B6 RIP-GP ILR1ko IFNgRtg SOCS+0

100

200

Mea

n F

luo

resc

ent

Inte

nsi

ty

SOCS+ local effectors are less activated

GP61

0

1

2

*

*spleen SOCS+spleen SOCS-PLN SOCS+PLN SOCS-

% I

FN

+ c

ell

s

A C

B D

GP33

0

2

4

6

8 *

*

spleen SOCS+spleen SOCS-

PLN SOCS+

PLN SOCS-

% IF

N+

cel

lsRIP-GP ILR1ko IFNgRtg SOCS+

0

10

20

30

40

50

60

70 day 7day 5

Mea

n Fl

uore

scen

t In

tens

ity

SOCS project

Other stuff…

To make things more complicated: effect of

microbiota

Gut microbiota modulates the immune system

Presence of microbes help training of immune system in newborns children

C-section vs vaginal birth: colonization by different microbes

Parasites can be helpful

Autoimmune diseases due to “wrong” microbes

Autism?

Supraorganisms (animal plus microbes and everything in-between

Keep your mind open for more paradigm changes!

An interesting substance…

Calcium pterin as immune modulator

SanRx pharmaceuticals (SD startup) has developed DCP: calcium pterin

Pterins as natural heterocyclic compounds involved in many biochemical reactions

Folates are conjugated pterins

Metabolism of amino acids

Aromatic compounds, NO

Common in vertebrates and also bacteria

DCP storyline

Anti-tumoral in vivo effect in several mouse models

This effect seems to be related to modulation of IDO (Indoleamine-pyrrole 2,3-dioxygenase) activity

IDO degrades tryptophan to kyrunenine

Cytokine pattern is also altered- seem to affect Th1 cytokines (IL6 and IFNg especially)

Recent results: enhances intracellular killing of Mycobacteria

Possible mechanisms?

Mechanism of action: direct effect on IDO? Immunomodulatory effect via cytokines?

Direct DNA binding?

DCP as supplement (ongoing)

Indirect effect through the gut microbiota? There are several bacterial enzymes that require pterin (molybdopterin, cyanopterin)

“Back where you started”Anti-cancer drug development

Nereus Pharmaceuticals

Emphasis: proteasome inhibitors (modulate NFkB) and angiogenesis inhibitors

Angiogenesis inhibitors

Thesis projects

Project: DCP (Ca-pterin)

Currently in the process of gain FDA-approval

Regulatory requirements: formulation strategies

Combination of folate and Ca-compounds?

To explore formulation alternatives based on sound biochemical/pharmacological principles (review process)

Analysis of HPLC products of different formulations

Research: the inverted STEM classroom

Learning for life: active and interactive, project & inquiry-based

Minimal lecturing, use class room time for discussions, projects, activities

Use of technology: recorded lectures (podcasts), mobile learning (see Khan academy, iTunes University, Eric Mazur)

NU ideal: high proportion of engaged, focused, professional students who value real-life learning, in their own time

NU strategic plan is online (competition with state universities & for-profits)

Voice-thread for discussions

Project: online science course

In collaboration with School of Education

Development of a fully online, interactive science course (Intro to Bio)

Pedagogy: development of learning objectives and content/assignment/artifacts to fulfill them

Relevance: up to date content, 21st century tools and digital media

Thank you!