Microenvironmental factors and tumorigenesis Rama Khokha Ontario Cancer Institute 8 th PHM Conference
The mammary tissue microenvironment Mouse mammary gland H&E Human breast tissue section Stromal Compartment p Epithelial Duct Epithelial Duct ECM Fibroblasts Proteolytic Scissors y Immune Cells Immune Cells • Clipping Epithelial Duct Growth Factors TIMPs • Shedding Stromal Cytokines • RIPping Compartment BV and LV
Cell surface shedding of TNF by TACE Injury Injury Macrophage TNF 26 kD TNF 26 kD TACE T TNF 17 kD TIMP3 Aditya Murthy
Metalloproteinase Substrates Thrombospondin Plasminogen Collagen I, II, III Collagen I, IV, V, VII, X Gelatin Collagen IV Fibronectin a9, b1 Integrin Fibronectin Fibronectin I Fibrillin a2 Actinin a5 integrin ECM M L-Selectin Laminin a Decorin Decorin Aggrecan 1 Aggrecan 1 Laminin Laminin Collagen IV, V, XI Nidogen Elastin E-cadherin a9 Integrin Collagen IV Fibrillin Elastin Aggrecan I Aggrecan 1 MBP b4 Integrin Fibronectin MBP a2M syndecans MT1-MMP MMP7 MMP2 MMP9 TACE/ADAM17 ADAM10 ADAM12 ADAMTS4, 5 TIMP3 FasL FasL NGFR NGFR IL8 IL6R bAPP CCL7 Pro a-defensin Lck CCL11 Notch aling CXCL12 pro-TNFa IL1b Delta IGFBP3 CuZnSOD pro-TGFa CCL7 FGF1 CD40 IFGBP5 ICAM1 p55/TNFR1 p55/TNFR1 CXCL12 CXCL12 FGFR1 FGFR1 pro-TNFa HB-EGF TNF HB EGF Sign p75/TNFR2 TGFb1 CD30 bAPP IL1R-II IL6R c MET c-MET GHBP MUC1 IL15RA MxL1 Fractalkine
Tissue Inhibitors of Metalloproteinases (TIMPs) Tissue Inhibitors of Metalloproteinases (TIMPs) Q Questions: sti ns: - 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)
TIMPs regulate clipping and shedding Clipping Shedding Trimolecular complex MT1MMP TIMP2 TNF MMP2 TACE Zn+ TNFR1, TNFR2 Zn+ Cell membrane Timp3 Timp3 + + + + Timp3, Timp2 ECM-degradation sTNF MMPs Increased MT1-MMP activity Cytokines IL-6 Stromal matrix Abnormal TNF signaling • Increased systemic inflammation • Failure of liver regeneration f g Smookler et al, J Imm, 2006 Smookler et al, J Imm, 2006 • Accelerated heart failure Kassiri et al, Circ Res, 2005 English et al, JBC, 2006 Mohammed et al, Nat Genet 2004
Heart disease model – pressure overload/mechanical stress Compensated Start of Decompensation Decompensation Aortic hypertrophy yp p y LV dilation severe LV dilation banding g ↑ ↔ contractility ↓ contractility ↓↓ Contractility LV wall thinning ↑ 3 wk 6 wks 15 wks WT 1 wk 3 wks 6 wks Heart Failure timp3 − / − Kassiri et al, Circ. Res. 2005
TGF β 1 – TNF conflict A RA LA Atria (discarded) 1-2 day RV old LV pups Ventricles A A Digestion Uncoordinated TGF β 1-TNF signaling Mature TGFß1 (25 kDa) Differential Cleaved TGFß1 (12 kDa) • Transcriptional induction of subset of MMPs adhesion • Mutual cytokine induction p-Smad Total Smad (58 kDa) In vitro Neonatal +v -ve In vivo In vivo Co-culture e e Neonatal N t l Neonatal N t l p-Smad 2/3 (58k Da) cardio-myocytes cardio-fibroblasts ß-actin B 2500 3500 +ve 3000 gen III agen I 2000 TNF (26 kDa) 2500 Neonatal 1500 2000 Colla Colla cardio-myocytes 1500 1000 Cleaved TNF (17 kDa) 1000 500 500 0 0 ß-actin C 2500 3500 3000 Control Ang II PE en III 2000 gen I 2500 Collage 1500 1500 Collag 2000 Neonatal 1500 cardio-fibroblasts 1000 B 1000 500 500 Myocytes Fibroblasts Co-culture 0 0 ß1 (pg/mL) 60 D *‡ 2500 3500 *‡ 50 *‡ 3000 *‡ III 2000 n I *‡ ‡ 40 Collagen 2500 2500 Cleaved TGFß Collagen *‡ 1500 30 Neonatal 2000 1500 Co-culture 1000 20 * * 1000 * * * *‡ 500 10 *‡ 500 * * 0 0 0 Con PE Ang II Con PE Ang II Con PE Ang II Con Ang II PE Con Ang II PE Zamaneh Kassiri
Global gene expression profiling and Genomatix analysis: 3 weeks vs sham A B Cell adhesion Ca 2+ Neuropeptide Hypoxia Wnt Wnt FGFR FGFR DNA damage Integrin JAK/STAT TGF β R GPC R MAPK NGFR Insulin NFkB Insulin NO Genes Wnt Toll Ca 2+ /NFAT IGFR Wnt R Wnt R JNK I κ B/NF κ B NO Signaling JAK/STAT Ca 2+ Cell adhesion Fas MAPK ERK1/ERK2 MAPK Ephrin R Ca 2+ /NFAT Myocyte Adrenergic Neuropeptide Insulin R Insulin R Pathway Pathway NO Signaling ~ 400 Genes JNK DNA Damage G-Prot (via IP3) NO Genes ~16 Major pathways Circadian Pathway FGFR Integrin Rho Prot WntR Adrenergic Pathway Fas Hypoxia GPC R G α s Notch 7 TmR via β -Arrestin TGF β R NF κ B G α q Cytokine and Chemokine ~ 1200 Genes ~ 42 Major pathways Virginie Defamie, Mehrdad Hariri
Top 25 categories (MeSH Filter disease) from Genomatix WT vs Timp3-/- (3 week ) Timp3 -/- WT Term ZScore Term ZScore Osteogenesis Imperfecta 29.96 Neoplasms 60.58 Fibrosis 28.65 Neoplasms by Site 45.25 Neoplasm Invasiveness Neoplasm Invasiveness 28.48 28.48 Neoplasms by Histologic Type Neoplasms by Histologic Type 40.61 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 Cell Transformation. Neoplastic 19 23 19.23 Breast Diseases Breast Diseases 23 8 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
TIMPs regulate potent cytokines which alter tumor microenvironment Clipping Shedding Trimolecular complex MT1MMP TIMP2 TNF MMP2 TACE Zn+ TNFR1, TNFR2 Zn+ Cell membrane Timp3 Timp3 Shedding Shedding + + + + Timp3, Timp2 ECM-degradation Timp3 TGFß activation sTNF Smad2/3—P MMPs Increased MT1-MMP activity Cytokines Activated Fibroblasts Collagen synthesis C ll n nth i IL-6 Stromal matrix Abnormal TNF signaling Unscheduled TGF β 1 signaling • Increased systemic inflammation • Transcriptional signature reflective of cancer • Failure of liver regeneration f g • Accelerated heart failure
TGF β in Cancer Joan Massagué, Cell 2008 • Pleiotropy • Coordination • Context-dependence Cancer: an inflammatory link Balkwill and Coussens, Nature, 2004
The context dependent effects of TNF - TIMP3 axis Aged Timp3 ‐ / ‐ Liver • Aged livers • Liver regeneration Smookler et al, J Imm, 2006 • LPS induced sepsis Mohammed et al, Nat Genet 2004 • Fas ‐ mediated hepatocyte death • HCC
TNF sensitization of Fas-mediated cell deaths is reduced in timp3-/- hepatocytes p p y A B
Absence of early JNK phosphorylation due to elevated TNFR1 shedding A B Primary hepatocytes treated with 1ng/ml TNF + 10ng/ml Jo2 Aditya Murthy
Loss of Timp3 delays cell death and hepatotoxicity A B C Aditya Murthy
TACE mediated EGFR transactivation LPA Carl P. Blobel, 2005
Increased ERK phosphorylation in timp3-/- MEFs Increased ERK phosphorylation in timp3 / MEFs Primary MEF cultures to study LPA ‐ induced EGFR activation Aditya Murthy
Elevated EGFR ligand shedding and signaling in Timp3-/- hepatocytes Timp3-/- hepatocytes Primary hepatocytes cultures treated with TNF + Jo2 Aditya Murthy
Parallel effects on TNFR & EGFR signaling protect from cell death TIMP3 TIMP3 TACE/ADAM17 TACE/ADAM17 Receptor shedding Ectodomain Ectodomain inhibits signaling shedding shedding TNFR1 TNF Amphiregulin activates TGFa TGFa EGFR EGFR activates HB-EGF JNK activation NFkB activation ERK1/2 activation Target Gene BcL inactivation Transcription p cytochrome C release t h C l Target Gene Transcription Inflammation Enhanced TNF signaling Apoptosis Proliferation Survival (E.g. Mcl-1) Apoptosis Dampened TNF signaling Accelerated EGFR signaling • Reduced cell death signal • A critical survival signal Aditya Murthy
TIMP3 in Tumorigenesis timp-3 -/- Inflammation Extracellular Proteolysis Proteolysis Cancer
B16F10 melanoma I.V. Colony count y Histomorphometry 14 days Increased lung metastasis of melanoma cells in timp-3 -/- mice I d l t t i f l ll i ti 3 / i WT timp3- /-
Increased metastatic dissemination in timp3-/- mice Secondary site Secondary site y Bone Lung EL-4 B16F10 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
Timps are normally expressed in the mouse mammary gland p y p y g Timp1 Timp2 Timp3 Timp4 Fata et al, Dev Biol 1999
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