Microenvironmental factors and tumorigenesis. Rama Khokha Ontario Cancer Institute. 8 th PHM Conference

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1 Microenvironmental factors and tumorigenesis Rama Khokha Ontario Cancer Institute 8 th PHM Conference

2 The mammary tissue microenvironment Mouse Human mammary breast tissue gland section H&E Stromal Compartment Epithelial Duct Epithelial Duct Stromal Compartment ECM Fibroblasts Immune Cells Growth Factors Cytokines BV and LV Proteolytic Scissors Clipping Shedding TIMPs RIPping

3 Cell surface shedding of TNF by TACE Injury Macrophage TACE T TIMP3 TNF 26 kd TNF 17 kd Aditya Murthy

4 Metalloproteinase Substrates Collagen I, II, III Gelatin Fibronectin Collagen IV a5 integrin Fibronectin I Fibrillin Laminin Laminin Decorin Nidogen E-cadherin Elastin b4 Integrin syndecans Thrombospondin Plasminogen Collagen I, IV, V, VII, X Fibronectin L-Selectin Collagen IV, V, XI Elastin Fibrillin MBP a2m Collagen IV Aggrecan 1 MBP a9, b1 Integrin a2 Actinin Aggrecan 1 a9 Integrin Aggrecan I Fibronectin ECM TIMP3 MT1-MMP MMP7 MMP2 MMP9 TACE/ADAM17 ADAM10 ADAM12 ADAMTS4, 5 CCL7 CXCL12 FasL IL6R Pro a-defensin IL1b FGF1 FGFR1 TGFb1 IL8 CCL7 CCL11 CXCL12 CuZnSOD ICAM1 pro-tnfa pro-tgfa p55/tnfr1 p75/tnfr2 CD30 bapp IL1R-II IL6R c-met GHBP MUC1 IL15RA MxL1 Fractalkine NGFR bapp Lck Notch Delta CD40 pro-tnfa IGFBP3 IFGBP5 HB-EGF aling Sign

5 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)

signaling Stromal matrix Increased systemic inflammation Failure of liver regeneration Smookler et al, J Imm, 2006

6 TIMPs regulate clipping and shedding TACE Zn+ TNF Shedding TNFR1, TNFR2 Cell membrane Trimolecular complex MT1MMP TIMP2 MMP2 Clipping Zn+ + Timp3 + Timp3, Timp2 ECM-degradation stnf MMPs Cytokines Increased MT1-MMP activity IL-6 Abnormal TNF signaling Stromal matrix Increased systemic inflammation Failure of liver regeneration Smookler et al, J Imm, 2006 Accelerated heart failure Smookler et al, J Imm, 2006 Kassiri et al, Circ Res, 2005 English et al, JBC, 2006 Mohammed et al, Nat Genet 2004

7 Heart disease model pressure overload/mechanical stress Aortic Compensated Start of Decompensation Decompensation banding hypertrophy p y LV dilation severe LV dilation contractility contractility Contractility LV wall thinning WT 3 wk 6 wks 15 wks timp3 / 1 wk 3 wks 6 wks Heart Failure Kassiri et al, Circ. Res. 2005

8 TGFβ1 TNF conflict A 1-2 day old pups RA LA RV LV Ventricles Atria (discarded) Digestion Uncoordinated TGFβ1-TNF signaling Differential Transcriptional adhesion induction of subset of MMPs Mutual cytokine induction In vitro Neonatal Co-culture Neonatal In Neonatal vivo cardio-myocytes cardio-fibroblasts A p-smad +v -ve e Mature TGFß1 (25 kda) Cleaved TGFß1 (12 kda) Total Smad (58 kda) p-smad 2/3 (58k Da) B Neonatal cardio-myocytes C Neonatal cardio-fibroblasts D Neonatal Co-culture Colla agen I Collag gen I Collagen I * * gen III Colla Collage en III III Collagen * * B Cleaved TGFß ß1 (pg/ml) ve Control Ang II PE Myocytes Fibroblasts * * * * * * * 0 0 Con Ang II PE Con Ang II PE Con PE Ang II Con PE Ang II 30 ß-actin TNF (26 kda) Cleaved TNF (17 kda) ß-actin Co-culture * * * * Con PE Ang II Zamaneh Kassiri

2+ Neuropeptide TGFβR JNK JAK/STAT ~ 400

damage NO Genes Insulin R GPC R ERK1/ERK2

NGFR IκB/NFκB Myocyte Adrenergic Pathway NO

Ephrin R Wnt Ca 2+ DNA Damage Adrenergic

TmR via β-arrestin Fas NFκB Cytokine and

9 Global gene expression profiling and Genomatix analysis: 3 weeks vs sham A B Ca 2+ Neuropeptide TGFβR JNK JAK/STAT ~ 400 Genes ~16 Major pathways Hypoxia Insulin Wnt R Ca 2+ /NFAT FGFR Integrin Cell adhesion Wnt MAPK NFkB NO Signaling Fas DNA damage NO Genes Insulin R GPC R ERK1/ERK2 MAPK JNK FGFR Integrin Insulin Ca 2+ /NFAT NGFR IκB/NFκB Myocyte Adrenergic Pathway NO Signaling NO Genes JAK/STAT Toll IGFR Ephrin R Wnt Ca 2+ DNA Damage Adrenergic Pathway MAPK Rho Prot WntR Cell adhesion Neuropeptide G-Prot (via IP3) Circadian Pathway GPC R TGFβR Hypoxia G α s Notch 7 TmR via β-arrestin Fas NFκB Cytokine and Chemokine G α q ~ 1200 Genes ~ 42 Major pathways Virginie Defamie, Mehrdad Hariri

10 Top 25 categories (MeSH Filter disease) from Genomatix WT vs Timp3-/- (3 week) WT Timp3 -/- Term ZScore Term ZScore Osteogenesis Imperfecta Neoplasms Fibrosis Neoplasms by Site Neoplasm Invasiveness Neoplasms by Histologic Type Neoplastic Processes Neoplastic Processes Cardiomegaly Neoplasms. Glandular & Epithelial Heart Diseases 23.8 Cell Transformation. Neoplastic Ventricular Dysfunction Prostatic Neoplasms Ventricular Dysfunction. Left Urogenital Neoplasms Hypertrophy Prostatic Diseases Heart Failure. Congestive Genital Neoplasms. Male Cardiovascular Diseases 20.4 Digestive System Neoplasms Collagen Diseases Breast Neoplasms Marfan Syndrome Carcinoma Cell Transformation. Neoplastic Breast Diseases 23.8 Aneurysm Leukemia. Myeloid Aortic Aneurysm Neoplasm Invasiveness Patholog Conditions, anatomical Neoplasms. Neuroepithelial Aortic Diseases Leukemia Heart Valve Diseases Osteogenesis Imperfecta 20.6 Cutis Laxa Cardiomegaly Aortic Valve Stenosis Genital Diseases. Male 20.08

11 TIMPs regulate potent cytokines which alter tumor microenvironment TACE Zn+ TNF Shedding TNFR1, TNFR2 Cell membrane Trimolecular complex MT1MMP TIMP2 MMP2 Clipping Zn+ + Shedding Timp3 + Timp3, Timp2 ECM-degradation Timp3 TGFß activation stnf MMPs Smad2/3 P Cytokines Increased MT1-MMP activity Activated Fibroblasts IL-6 Abnormal TNF signaling Increased systemic inflammation Failure of liver regeneration Accelerated heart failure Collagen synthesis Unscheduled TGFβ1 signaling Stromal matrix Transcriptional signature reflective of cancer

12 TGFβ in Cancer Joan Massagué, Cell 2008 Pleiotropy Coordination Context-dependence Cancer: an inflammatory link Balkwill and Coussens, Nature, 2004

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

14 TNF sensitization of Fas-mediated cell deaths is reduced in timp3-/- hepatocytes A B

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

16 Loss of Timp3 delays cell death and hepatotoxicity A B C Aditya Murthy

17 TACE mediated EGFR transactivation LPA Carl P. Blobel, 2005

18 Increased ERK phosphorylation in timp3-/- MEFs Primary MEF cultures to study LPA induced EGFR activation Aditya Murthy

19 Elevated EGFR ligand shedding and signaling in Timp3-/- hepatocytes Primary hepatocytes cultures treated with TNF + Jo2 Aditya Murthy

20 Parallel effects on TNFR & EGFR signaling protect from cell death TIMP3 TIMP3 Ectodomain shedding TACE/ADAM17 Receptor shedding inhibits signaling Ectodomain shedding TACE/ADAM17 TNF TNFR1 activates JNK activation NFkB activation Amphiregulin TGFa HB-EGF EGFR activates BcL inactivation cytochrome C release Apoptosis Target Gene Transcription Inflammation Enhanced TNF signaling ERK1/2 activation Target Gene Transcription Proliferation Survival (E.g. Mcl-1) Apoptosis Dampened TNF signaling Accelerated EGFR signaling Reduced cell death signal A critical survival signal Aditya Murthy

21 TIMP3 in Tumorigenesis timp-3 -/- Extracellular Proteolysis Inflammation Cancer

22 B16F10 melanoma I.V. 14 days Colony count Histomorphometry Increased lung metastasis t of melanoma cells in timp-3 -/- mice WT timp3- /-

23 Increased metastatic dissemination in timp3-/- mice Secondary site Bone B16F10 EL-4 Lung 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

24 Timps are normally expressed in the mouse mammary gland Timp1 Timp2 Timp3 Timp4 Fata et al, Dev Biol 1999

BC Models: TIMP3 deficiency inhibits mammary

effect Timp3 -/- Tumor Suppression Tumor

Py-positive 1500 n=17 n=17 1000 t3+/+ t3-/-

25 BC Models: TIMP3 deficiency inhibits mammary tumors MMTV-PyMT MMTV-Neu Timp1 -/- No effect No effect Timp3 -/- Tumor Suppression Tumor Suppression WT Tumour Number Burden (mm 2 ) * 500 n=17n=17 0 t3+/+ Timp3-/- t3-/- Py-positive 1500 n=17 n= t3+/+ t3-/- Percent Tumo our-free * WT Py-positive Timp3-/ Days Carlo Hojilla

26 Haploinsfficiency of Timp3 and mammary tumor suppression MMTV-PyMT MMTV-Neu dy Weight Mam mmary Gland:Bo * * n=5 n=5 n=5 n=17 n=16 n=17 t3+/+ t3+/- t3-/- t3+/+ t3+/- t3-/- Py-negative Py-positive Percent Tumo our-free Days Carlo Hojilla

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

28 TIMP3 TACE and/or MT1-MMP Shedding Shedding Clipping Cell membrane MT1MMP MMP2 Activity TNF signaling Death receptor signaling EGFR signaling Shedding TGFβ1 signaling ECM State Wnt signaling

29 Context-dependent effects Invasion/Metastasis vs Primary Tumor Heterogeneity/Redundancy Highly specific inhibitors for MMPs vs ADAMs

30 Funding: CIHR, CBCRA, NCIC, CAN, CPBN, H&S Foundation Komen Fdn, US Army, HFSP

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