Cellular Basis of Cancer 3 Proteases in Cancer

1. Cellular Basis of Cancer 3 Proteases in Cancer Dr Rosemary Bass rosemary.bass@northumbria.ac.uk

2. Outline • Protease = proteinase • Why proteases are important • Introduction to proteases: • Classes MMP (Matrix Metalloprotease) • ADAM/ADAMTS (A Disintegrin and a • Metalloprotease) • Serine proteases • Inhibitors (TIMPs, Serpins) • Mechanisms of action • How enzymes are regulated – zymogens, spatial, temporal • Why proteases thought to promote cancer - Roles of extracellular proteases in tumour invasion & metastasis • Problems with broad-spectrum MMP inhibitors as anti-cancer therapy • Anti-cancer activities of proteases

3. Proteases – molecular scissors - cleave proteins in vivo - impact on many basic cell functions: • cell adhesion • cell division/ proliferation/multiplication • cell migration – invasion/metastasis • cell survival - apoptosis Because alters cellular environment

4. Cell are in contact with – other cells - extracellular matrix (ECM) • Proteases alter (tumour) cell environment • Cleavage of ECM

5. Extracellular Matrix = ECM • Outside cells • Secreted by cells • Consists of polymeric proteins eg. collagen in skin & bone • Collagens form 25% of total body protein • Glycoproteins eg. Fibronectin • Glycosoaminoglycans/proteoglycans eg. Aggrecan & hyaluronan in cartilage

6. Fibroblast surrounded by collagen fibres Collagen fibres in cross-section Collagen fibres in longitudinal section

7. How do most often hear about collagen?

8. Initial interest in mechanical strength provided by ECM Interaction with ECM important for all major cell biological processes: • cell adhesion • cell proliferation • cell migration • cell apoptosis

9. Proteases Cleave the ECM

10. Integrin • Heterodimer • Cell surface • adhesion • receptors

11. M+ M+ M+ M+ M+ M+ S S S S b a b a Active Inactive Activation state can be regulated to control cell-ECM interactions • Connect cell to ECM

12. Integrin Activation a5b1 Predicted Active ectodomain Inactive allbb3

13. Tissue Remodelling Normal Pathological Tumour growth Metastasis Arthritides Atherosclerosis Reproduction Organogenesis Wound repair • Proteases participate in normal & pathological tissue remodelling • In tissue remodelling proteases  • Normal tissue - tightly controlled, temporally regulated

14. There are 5 Catalytic Classes of Proteases (Neutral pH, cell surface/extracellular) (Intracellular, Important for cancer, caspases in apoptosis, proteasome targeting therapies) 1-Metalloproteinases: Matrix metalloproteinases (MMPs), disintegrin metalloproteinases (ADAMs and ADAMTSs) 2-Serine: plasmin, uPA, tPA, PMN elastase, proteinase 3, cathepsin G, kallikrein, tryptase, chymase, granzymes 3-Cysteine: Cathepsin B, L, S, K, calpains, caspases 4-Aspartate: Cathepsin D 5-Threonine: Proteasomal subunits

15. MMP Number DOMAIN STRUCTURE (23 in human, 24 in mouse) N-terminal domain (catalytic) Propeptide Matrilysins MMP-7, -26 Zn C C-terminal domain (hemopexin-like repeats) Collagenases MMP-1,-8,-13 Metalloelastase MMP-12 Stromelysins MMP-3,-10,-11 Others MMP-19,-20,-27,-28 MMP-23 A/B C Zn C C Cys array Ig-like Zn C Gelatin binding domain (fibronectin-like repeats) II Gelatinases MMP-2, -9 Zn II C C C II MT-MMPs MMP-14,-15,-16, -17,-24,-25 Zn C C C Membrane domain • Classed according to domain structure • Catalytic domain & propeptide • All have Zn2+ binding site – target for inhibitors • C-term region involved in substrate binding & TIMP interactions • Membrane bound by transmembrane domain or GPI anchor

16. MMP in normal development Resorption of tail during tadpole→frog metamorphosis – first identification of collagenase by Jerry Gross in 1965 ECM invasion by cancer cell Wound healing Epithelial migration Angiogenesis MMP = MatrixMetalloprotease

17. REGULATION OF METALLOPROTEINASES: • Gene transcription • Post-transcription • Post-translation • -pro forms require activation (plasmin, MMPs, furin/ • Pro-Protein Convertases) • -intracellular trafficking of membrane bound MPs • -cell and ECM sequestration • -inhibition by endogenous TIMPs

18. Tissue Inhibitors of Metalloproteases (TIMPs) TIMP-1 TIMP-2 TIMP-3 TIMP-4 Soluble Soluble ECM Associated Soluble Leco et al., (1994) J Biol Chem Leco et al., (1997) FEBS Let Baker, Edwards & Murphy (2002) J Cell Sc

19. Characteristic TIMP-1 TIMP-2 TIMP-3 TIMP-4 MW 28kDa 21kDa 24kDa 22kDa Localization soluble soluble ECM soluble Tissue specificity bone, ovary, lung, heart, kidney, heart, heart, (mouse) muscle, brain, brain, lung, muscle, skin, vessels, ovary, brain, testis, ovary placenta ovary Growth promotion yes yes no yes Apoptosis no no yes yes Inhibition of tumour + + + + invasion Angiogenesis inhibition yes yes yes yes Ability to inhibit no yes yes yes MT1-MMP

20. MMP-TIMP Balance Normal ECM Homeostasis MMP TIMP Disrupted Balance Favours ECM Destruction TIMP MMP

21. ADAMs – A Second Class of Metalloproteinase • ADisintegrin and Metalloproteinase • Or Adamalysin • Involved primarily in “ectodomain shedding” eg. TACE = ADAM17 • Cytoplasmic tail of some ADAMs may be involved in inside–out control of proteinase activity or outside–in control of cell signalling • Can be inhibited specifically by TIMP-3 Seals & Courtneidge (2003) Genes and Dev

22. The ADAM Family ADisintegrin and Metalloprotease Metallo Disintegrin Snake venom Metalloproteinases Cys-rich EGF Cytoplasmic P P

23. ADAM = A Disintegrin and A Metalloprotease • 29 Mammalian ADAMs • Pro-domain – intramolecular chaperone inhibiting protease domain • Metalloprotease domain • Disintegrin domain = cell-cell adhesion • Cys-rich domain = cell-cell & cell-ECM domain • EGF repeats • Transmembrane domain • Cytoplasmic tail • Spermatogenesis/fertilisation • De-regulation implicated in development of cancer

24. ADAMs and Cell Adhesion A) Trans Syndecans Dis Cys MP EGF a b Integrin B) Cis • Involved in cell adhesion as well as proteolysis • Signalling through multiple pathways Dis ab

25. Shedding Proteolytic cleavage of membrane-bound proteins (growth factors, cytokines, receptors) Post-translational modulation Activate, inactivate or change properties of processed proteins C Tape, CRI, Cambs

26. TNF convertase, TACE, ADAM-17 Proteins that are shed: Cytokines, growth factors TGF, EGF, HB-EGF, TNF, KL-1, CSF-1, FasL, Delta Receptors TNFRI, TNFRII, p75 NGFR IL6R, TSHR Adhesion proteins, others L-selectin, syndecans, PTP, LAR , ACE, APP pro MP Disintegrin Cys-rich EGF-like TM Cyto TNF Schlondorff J and Blobel C, (1999) J. Cell Sci. 112: 3603-3617 TACE – EGF signalling ADAM 10 – notch, Ephrins

27. MDA-MB-231 ADAM15a BT-549 MCF-7 T47D C B A D ADAM15a MX7 MX8 MX6 DC gr1 DC gr2 DC gr3 LC gr2 C B A D U937 cells PMA timecourse ADAM15a PMA - 1 3 7 24 48 72 96 B A Differentially spliced isoforms of ADAM15cytoplasmic tail in breast cancer Cell lines Signal sequence Prodomain Metalloprotease Disintegrin Cysteine-rich EGF-like Transmembrane Cytoplasmic tail ADAM-15 Isoform Breast tissues and cancers

28. Levels of splice variants differs in breast cancer Association with cell behaviour & agressiveness of cancer Zhong et al 2008 Mol Cancer Res 6:383

29. ADAMTS = A Disintegrin and A Metalloprotease with Thrombospondin like motifs • 19 secreted proteases • ADAMTS1 – discovered 1997 • ECM degradation/assemby • Haemostasis • Organogensis • Angiogenesis • De-regulation associated with development arthritis & cancer

30. Domain Key Signal peptide ADAM SVMP ADAMTS MTMMP MMP Pro domain Metalloproteinase Disintigrin Cysteine-rich EGF like Hemopexin Spacer region TS repeat Transmembrane Region Hinge x Cytoplasmic tail

31. Serine Proteases • plasmin, uPA, tPA, PMN elastase, proteinase 3, cathepsin G, kallikrein, tryptase, chymase, granzymes • Catalytic triad

32. NTP Kr Kr Kr Kr Kr Ser Pr Plasminogen • synthesized in liver, most abundant protease zymogen • very broad substratespecificity • lysine-binding kringle modules uPA, urokinase plasminogen activator • expressed by wide variety of cells • virtually inactive zymogen • binds to uPAR Kr Ser Pr EGF tPA, tissue plasminogen activator • very limited expression • “active zymogen” • activity stimulated by fibrin EGF Fn3 Kr Kr Ser Pr

33. Cell a5b1 uPAR “tPAR” Plasminogen Binding Sites CD82 tPA Pro- uPA a2-anti plasmin uPA PAI-1 Plasmin Plasminogen Activation pro-MMPs & Latent Growth Factors PAI-1 Adhesion Migration Survival Extracellular Matrix (ECM)

34. BM Normal cell Transformed cell Blood vessel ECM Proteases Facilitate Tumour Invasion & Metastasis “Classic” idea that proteases are only acting like molecular scissors to promote invasion & metastasis

35. The main steps in metastasis Since >80% of human tumours derive from epithelial cells (giving rise to carcinomas when malignant) we will mainly consider metastasis of these tumours, but essentially similar steps are involved in the spread of connective tissue tumours (sarcomas) Epithelial cells sit on a basement membrane (basal lamina), with stroma = connective tissue underlying. Stroma includes fibroblasts, blood vessels, immune effector cells. Tumour-stromal interactions are of critical importance in metastasis

36. 1990’s view of MMPs in Cancer Biology • Multiple protease involvement • From different sources - cancer cells/macrophage/stromal cells • Still perceived that proteases all contributing to cancer progression • Therefore good targets for therapy •  protease expression in cancer vs normal tissue

37. Overlapping substrate preferences of MMPs MMP Target Fibroblast collagenase Gelatinase-A Gelatinase-B Matrilysin Stromelysin- 1 Collagen VII Fibronectin Laminin Therefore it was thought to be a good idea to develop broad-spectrum MMP inhibitors that would block many/all family members

38. However broad-spectrum Matrix Metalloprotease Inhibitors (MMPI) have proved disappointing in the clinic Metalloprotease domain BB94 (Batimastat) MMP1

39. MPICANCERRESULT Marimastat x3 Pancreatic II-IV No benefit BB-2516 x1 Gastric (advanced ) Benefit after other treatment x1 Glioblastoma (unres) No benefit x1,x1Non-small/small cell lung No benefit x1 Ovarian (advanced) No benefit Prinomastat x2 Non-small cell lung No benefit AG 3340 x1 Prostate (metastatic) No difference Tanomastat x1 Small cell lung Poorer survival BAY 12-9566 x1 Pancreatic (metastatic) Ditto BMS-275291 x1 Non small cell lung (IIIB,IV) Recruiting Neovastat x1 Renal cell carcinoma Recruiting →Simple notion that all proteases are bad not correct Coussens et al Science 2002

40. The broad-spectrum MMPI batimastat increases metastasis in a murine L-CI.5s lymphoma model Increasing specificity for MMP9 Arlt M. et al., Cancer Research, 2002.

41. PROTEASES IN CANCER THEN: NOW: Coussens et al Science 2002

42. Tumours have elevated levels of many proteases, many of which are expressed by host stromal cells. Tumour-stromal interaction is important for activating the protease cascade leading to MMP activation and ECM destruction Proteases on the cell surface focus proteolysis on the cell surface

43. Proteases also regulated by location – where the activities are

44. Active MMP-2 Pro-MMP-2 TIMP-2 MT1-MMP Good example of this is the requirement for TIMP-2 in the activation of MMP2: Membrane-type-1 MMP activates pro-MMP-2 (Gelatinase-A) on the cell surface

45. How do metastatic cells arise? Fidler’s experiment with B16 melanoma cells suggested that there was a small metastatic sub-population of cells in a primary tumour that could be selected for Fidler IJ 1973 Nature New Biology 242:148-149. Selection of successive tumour lines for metastasis B16F0 = poorly metastatic B16F1 = low metastatic ability B16F2,3,4,5,6,………….. B16F10 = highly metastatic

46. Experimental Metastasis Assay showing metastasis suppression effects of TIMP-1 Tumours B16F10 cells injected in control mice via tail vein Metastasis assay showing lungs from mice injected with B16F10 tumour cells (highly metastatic melanoma cells) B16F10 cells injected along with recombinant TIMP-1 (and treated for 6.5days) Schultz et al., Cancer Res. 1988

47. Other key facts…… Clinical correlation of high expression of certain MMPs with poor outcome – 100s of studies Eg. Colorectal cancer – poor survival with MMP9 expression T N T=tumour, N=normal colon MMP-9 equates with poor survival in colorectal cancer Kaplan-Myer survival curve

48. So why did synthetic MMP inhibitors fail as anti-cancer agents? Trial design – MMPIs (in general) don’t work on very advanced cancers Inhibit not just MMPs but other metalloproteinases eg ADAMs whose existence was unknown MMPs can have unexpected anti-cancer roles

49. So why did synthetic MMP inhibitors fail as anti-cancer agents? Trial design – MMPIs (in general) don’t work on very advanced cancers Inhibit not just MMPs but other metalloproteinases eg ADAMs whose existence was unknown MMPs can have unexpected anti-cancer roles

50. So why did synthetic MMP inhibitors fail as anti-cancer agents? Trial design – MMPIs (in general) don’t work on very advanced cancers If animal model studies had been done properly would have found that MMPI don’t work on late stage cancers before clinical trails