Microenvironmental factors and tumorigenesis Rama Khokha Ontario - - PowerPoint PPT Presentation

SLIDE 1

Microenvironmental factors and tumorigenesis Rama Khokha

Ontario Cancer Institute 8th PHM Conference

SLIDE 2

The mammary tissue microenvironment Human breast tissue section

Epithelial Duct

Mouse mammary gland H&E

Stromal Compartment Epithelial Duct Proteolytic Scissors ECM Fibroblasts Immune Cells p Stromal Compartment y

• Clipping
• Shedding
• RIPping

Immune Cells Growth Factors Cytokines BV and LV TIMPs Epithelial Duct

SLIDE 3

Cell surface shedding of TNF by TACE

Injury Injury

Macrophage

TNF 26 kD

TIMP3

TNF 26 kD

TACE

TNF 17 kD

T

Aditya Murthy

SLIDE 4

Metalloproteinase Substrates

Collagen I, II, III Gelatin Fibronectin a5 integrin Laminin Collagen IV Fibronectin I Laminin Thrombospondin Plasminogen Collagen I, IV, V, VII, X Fibronectin Fibrillin Decorin L-Selectin a9, b1 Integrin a2 Actinin Aggrecan 1

M

Laminin a Nidogen E-cadherin Elastin b4 Integrin syndecans Decorin Collagen IV, V, XI Elastin Fibrillin MBP a2M Collagen IV Aggrecan 1 MBP Aggrecan 1 a9 Integrin Aggrecan I Fibronectin

ECM TIMP3

MT1-MMP MMP7 MMP2 MMP9 TACE/ADAM17 ADAM10 ADAM12 ADAMTS4, 5

FasL NGFR CCL7 CXCL12 FasL IL6R Pro a-defensin IL1b FGF1 FGFR1 IL8 CCL7 CCL11 CXCL12 CuZnSOD ICAM1 pro-TNFa pro-TGFa p55/TNFR1 NGFR bAPP Lck Notch Delta CD40 TNF IGFBP3 IFGBP5 HB EGF

aling

CXCL12 FGFR1 TGFb1 p55/TNFR1 p75/TNFR2 CD30 bAPP IL1R-II IL6R c MET pro-TNFa HB-EGF

Sign

c-MET GHBP MUC1 IL15RA MxL1 Fractalkine

SLIDE 5

Tissue Inhibitors of Metalloproteinases (TIMPs)

Q sti ns:

Tissue Inhibitors of Metalloproteinases (TIMPs)

Questions:

• Which substrates are linked to Timp activity in biological systems
• Which signaling pathways are influenced by Timp proteins

Approach:

• Disease Models (perturbed tissue homeostasis)
• Cancer Models (perturbed stroma/epithelium)

SLIDE 6

TIMPs regulate clipping and shedding

Shedding

Trimolecular complex MT1MMP

Clipping

Timp3 TNF TACE TNFR1, TNFR2 Zn+

+ +

TIMP2 MMP2 Zn+

Cell membrane

Timp3 MMPs ECM-degradation sTNF

+ +

Timp3, Timp2 Cytokines Increased MT1-MMP activity

Stromal matrix Smookler et al, J Imm, 2006

IL-6 Abnormal TNF signaling

• Increased systemic inflammation
• Failure of liver regeneration

Smookler et al, J Imm, 2006 Kassiri et al, Circ Res, 2005 English et al, JBC, 2006 Mohammed et al, Nat Genet 2004

f g

• Accelerated heart failure

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Heart disease model – pressure overload/mechanical stress

Aortic banding Compensated hypertrophy Start of Decompensation LV dilation Decompensation ↑ severe LV dilation g yp p y ↔ contractility ↓ contractility ↓↓ Contractility ↑ LV wall thinning

6 wks 15 wks 3 wk

WT

Heart Failure

1 wk 6 wks 3 wks

timp3−/− Kassiri et al, Circ. Res. 2005

SLIDE 8

TGFβ1 – TNF conflict

1-2 day

• ld

pups Atria (discarded) Ventricles

LV RV LA RA

A A

Digestion Differential adhesion

N t l N t l Neonatal Co-culture

A

Cleaved TGFß1 (12 kDa) Total Smad (58 kDa) Mature TGFß1 (25 kDa) +v e

• ve

p-Smad

Uncoordinated TGFβ1-TNF signaling

• Transcriptional induction of subset of MMPs
• Mutual cytokine induction

In vitro In vivo

B

Neonatal

agen I

1500 2000 2500

gen III

2000 2500 3000 3500

Neonatal cardio-myocytes Neonatal cardio-fibroblasts

p-Smad 2/3 (58k Da) ß-actin e TNF (26 kDa) +ve

In vivo

C

cardio-myocytes

Colla

500 1000

Colla

500 1000 1500

gen I

1500 2000 2500

en III

2500 3000 3500

Control Ang II PE Cleaved TNF (17 kDa) ß-actin

Neonatal cardio-fibroblasts

Collag

500 1000 1500

Collage

500 1000 1500 2000

*‡ *‡

2000 2500

n I *‡

3000 3500 2500

III

D B

ß1 (pg/mL)

40 50 60

*‡ *‡

Co-culture Myocytes Fibroblasts Neonatal Co-culture

* ‡ *

500 1000 1500

Collagen *‡ *

500 1000 1500 2000 2500

Collagen PE Con Ang II PE Con Ang II Con PE Ang II Cleaved TGFß Con PE Ang II *‡ *‡ * *

10 20 30

* * Con PE Ang II Zamaneh Kassiri

SLIDE 9

Global gene expression profiling and Genomatix analysis: 3 weeks vs sham

Ca2+ Neuropeptide Wnt Hypoxia FGFR Cell adhesion

A B

Integrin Insulin TGFβR Wnt R Wnt NO Genes DNA damage NFkB GPC R MAPK FGFR Wnt IGFR NGFR Insulin Toll Ca2+/NFAT JAK/STAT NO Signaling Wnt R JNK JAK/STAT Fas Ca2+/NFAT IκB/NFκB Ca2+ Insulin R Myocyte Adrenergic Pathway ERK1/ERK2 MAPK MAPK Ephrin R Neuropeptide Cell adhesion

~ 400 Genes ~16 Major pathways

JNK FGFR NO Genes Integrin NO Signaling Circadian Pathway WntR Rho Prot Adrenergic Pathway DNA Damage Insulin R Pathway G-Prot (via IP3) NFκB Fas Notch G α s 7 TmR via β-Arrestin TGFβR Cytokine and Chemokine G α q GPC R Hypoxia

~ 1200 Genes ~ 42 Major pathways

Virginie Defamie, Mehrdad Hariri

SLIDE 10

Top 25 categories (MeSH Filter disease) from Genomatix WT vs Timp3-/- (3 week) WT Timp3-/-

Term ZScore Term ZScore Osteogenesis Imperfecta 29.96 Neoplasms 60.58 Fibrosis 28.65 Neoplasms by Site 45.25 Neoplasm Invasiveness 28.48 Neoplasms by Histologic Type 40.61 Neoplasm Invasiveness 28.48 Neoplasms by Histologic Type 40.61 Neoplastic Processes 25.52 Neoplastic Processes 30.58 Cardiomegaly 24.98

• Neoplasms. Glandular & Epithelial

29.97 Heart Diseases 23.8 Cell Transformation. Neoplastic 28.57 Ventricular Dysfunction 23.55 Prostatic Neoplasms 27.14 Ventricular Dysfunction. Left 22.73 Urogenital Neoplasms 26.92 Hypertrophy 22.46 Prostatic Diseases 26.16 Heart Failure. Congestive 20.71 Genital Neoplasms. Male 26.05 Cardiovascular Diseases 20.4 Digestive System Neoplasms 25.05 Collagen Diseases 19.69 Breast Neoplasms 24.43 Marfan Syndrome 19.31 Carcinoma 24.26 Cell Transformation Neoplastic 19 23 Breast Diseases 23 8 Cell Transformation. Neoplastic 19.23 Breast Diseases 23.8 Aneurysm 17.84

• Leukemia. Myeloid

23.13 Aortic Aneurysm 16.31 Neoplasm Invasiveness 22.12 Patholog Conditions, anatomical 15.11

• Neoplasms. Neuroepithelial

21.77 Aortic Diseases 14.85 Leukemia 21.13 Heart Valve Diseases 13.53 Osteogenesis Imperfecta 20.6 Cutis Laxa 13.08 Cardiomegaly 20.39 Aortic Valve Stenosis 12.01 Genital Diseases. Male 20.08

SLIDE 11

TIMPs regulate potent cytokines which alter tumor microenvironment

Shedding

Trimolecular complex MT1MMP

Clipping

Timp3 TNF TACE TNFR1, TNFR2 Zn+

+ +

TIMP2 MMP2 Zn+

Cell membrane

Shedding

Timp3 MMPs ECM-degradation sTNF

+ +

Timp3, Timp2 Smad2/3—P TGFß activation

Shedding

Timp3 Cytokines Increased MT1-MMP activity Activated Fibroblasts C ll n nth i

Stromal matrix

IL-6 Abnormal TNF signaling

• Increased systemic inflammation
• Failure of liver regeneration

Collagen synthesis Unscheduled TGFβ1 signaling

• Transcriptional signature reflective of cancer

f g

• Accelerated heart failure

SLIDE 12

TGFβ in Cancer

Joan Massagué, Cell 2008

• Pleiotropy
• Coordination
• Context-dependence

Balkwill and Coussens, Nature, 2004

Cancer: an inflammatory link

SLIDE 13

The context dependent effects of TNF - TIMP3 axis

Aged Timp3‐/‐ Liver

• Aged livers
• Liver regeneration
• LPS induced sepsis
• Fas‐mediated hepatocyte death
• HCC

Smookler et al, J Imm, 2006 Mohammed et al, Nat Genet 2004

SLIDE 14

TNF sensitization of Fas-mediated cell deaths is reduced in timp3-/- hepatocytes p p y

A B

SLIDE 15

Absence of early JNK phosphorylation due to elevated TNFR1 shedding

A B

Primary hepatocytes treated with 1ng/ml TNF + 10ng/ml Jo2

Aditya Murthy

SLIDE 16

Loss of Timp3 delays cell death and hepatotoxicity

A B C

Aditya Murthy

SLIDE 17

TACE mediated EGFR transactivation LPA

Carl P. Blobel, 2005

SLIDE 18

Increased ERK phosphorylation in timp3-/- MEFs Increased ERK phosphorylation in timp3 / MEFs

Primary MEF cultures to study LPA‐induced EGFR activation

Aditya Murthy

SLIDE 19

Elevated EGFR ligand shedding and signaling in Timp3-/- hepatocytes Timp3-/- hepatocytes

Primary hepatocytes cultures treated with TNF + Jo2

Aditya Murthy

SLIDE 20

Parallel effects on TNFR & EGFR signaling protect from cell death

TIMP3 TIMP3 TACE/ADAM17 Amphiregulin TGFa EGFR Ectodomain shedding TACE/ADAM17 TNF TNFR1 Ectodomain shedding Receptor shedding inhibits signaling activates TGFa HB-EGF EGFR ERK1/2 activation activates JNK activation NFkB activation Target Gene Transcription BcL inactivation t h C l Target Gene Transcription Proliferation p Inflammation Enhanced TNF signaling

Apoptosis

cytochrome C release Survival (E.g. Mcl-1)

Apoptosis

Dampened TNF signaling

• Reduced cell death signal

Accelerated EGFR signaling

• A critical survival signal

Aditya Murthy

SLIDE 21

TIMP3 in Tumorigenesis

timp-3-/-

Extracellular Proteolysis Inflammation Proteolysis Cancer

SLIDE 22

B16F10 melanoma I.V.

Colony count

I d l t t i f l ll i ti 3 / i

14 days

y Histomorphometry

Increased lung metastasis of melanoma cells in timp-3-/- mice

WT timp3- /-

SLIDE 23

Increased metastatic dissemination in timp3-/- mice

Secondary site Secondary site

Bone

y

B16F10 Lung EL-4 Kidney Liver

• Increased colonization of kidney, lung, liver and bone
• Angiogenic, proliferative and inflammatory responses not altered
• Increased extravasation, increased pro-MMP-2 activation

Cruz et al, Oncogene 2004 Cruz et al, Oncogene 2006

SLIDE 24

Timps are normally expressed in the mouse mammary gland p y p y g

Timp1 Timp2 Timp3 Timp4

Fata et al, Dev Biol 1999

SLIDE 25

BC Models: TIMP3 deficiency inhibits mammary tumors

MMTV-PyMT MMTV-Neu Timp1-/-

No effect No effect

Timp3-/-

Tumor Suppression Tumor Suppression

Timp3-/- WT

80 100

• ur-Free

7.5 10.0

1500

n=17 n=17 T umour Number Burden (mm2) Timp3-/-

20 40 60

Percent Tumo

0.0 2.5 5.0

t3+/ + t3-/ -

n=17n=17

*

500 1000

t3+/ + t3-/ -

*

WT

Carlo Hojilla

100 200 300 400 500 600 700

Days

Py-positive Py-positive

SLIDE 26

Haploinsfficiency of Timp3 and mammary tumor suppression MMTV-PyMT MMTV-Neu

dy Weight

• 0. 3

* * 80 100

• ur-Free

mmary Gland:Bo

• 0. 1
• 0. 2

n= 5 n= 5 n= 5 n= 17 n= 16 n= 17 20 40 60

Percent Tumo

Mam

• 0. 0

t3+ /+ t3+ /- t3-/- Py-negative t3+ /+ t3+ /- t3-/- Py-positive 100 200 300 400 500 600 700 800

Days

Carlo Hojilla

SLIDE 27

SUMMARY: Timp3 and Tissue Microenvironment

• Timp3 is a negative regulator of inflammation
• It couples ECM and cytokine homeostasis
• TIMP3 co-regulates TNFR, TGFβR, EGFR signaling
• Its loss offsets the remodeling pathways towards

non-heart centric responses; affects cell death pathways

• Complex effects on metastasis and tumorigenesis

SLIDE 28

TACE and/or MT1-MMP TIMP3

Shedding Shedding Clipping

Cell membrane

Shedding

MT1MMP MMP2 Activity TNF signaling Death receptor signaling TGFβ1 signaling EGFR signaling

Shedding

ECM State Wnt signaling Wnt signaling

SLIDE 29

Invasion/Metastasis vs Primary Tumor Context-dependent effects Heterogeneity/Redundancy Hi hl ifi i hibit f MMP ADAM Invasion/Metastasis vs Primary Tumor Highly specific inhibitors for MMPs vs ADAMs

SLIDE 30

Funding: CI HR, CBCRA, NCI C, CAN, CPBN, H&S Foundation Komen Fdn, US Army, HFSP