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1 Matrixnedbrydning ved cancerinvasion -Biokemiske aspekter Ekstracellulære Proteaser og endocytosereceptorer Niels Behrendt The Finsen Laboratory Rigshospitalet Copenhagen

2 Nødvendige begivenheder i metastasering Primary tumor Primary tumor growth Angiogenesis Invasion

3 Nødvendige begivenheder i metastasering Primary tumor Clinically evident metastasis Primary tumor growth Angiogenesis Survival in circulation Invasion Intravasation Arrest in new organ Extravasation Local migration Initiation of growth Sustained growth From Chambers and Matrisian 1997

4 Matrixnedbrydning og cancer ce invasion Collagen Fibroblasts Myoepithelial cell Basement membrane: Collagen IV, Tenascin, laminin, entactin, PGs Cancer cells

5 Matrix degradation Extracellular/pericellular, intracellular (following internalization) Serine proteases (PA system), MMPs, Endocytic receptors Localization mechanisms Activity rescue and regulation Redundancy and tumor-stroma interplay

6 The proteolytic cascade principle Enzyme 1 Proenzyme 2 Enzyme 2 Proenzyme 3 Enzyme 3 Additional features etc. Amplification Inhibitors Binding components (localization) Feed-back systems Enhancement mechanisms Inactivation mechanisms

7 The Plasminogen Activation System Cascade system of serine proteases i i fib i l i i d li Active in fibrinolysis, tissue remodeling and cancer invasion

8 Lokalisation af kritiske komponenter ved den invasive i front Ekspression af upar i primær colorectal cancer og afledte metastaser Primary tumor Liver metastasis Liver metastasis Expression of upar in liver metastasis does require the presence of desmoplastic stroma formation

9 The plasminogen activation system: A proteolytic cascade PLASMINOGEN/PLASMIN BINDING SITES FIBRIN u-pa RECEPTOR? PRO-u-PA (SC) u-pa (TC) PAI 1 PAI 2 PLASMINOGEN PAI 1 PAI 2 t-pa (SC) t-pa (TC)? t-pa RECEPTOR? SC TC 2 ANTI PLASMIN, SEVERAL PROTEASE INHIBITORS CATALYSIS INHIBITION SURFACE LOCALIZATION SINGLE CHAIN TWO-CHAIN GROWTH FACTOR PRECURSORS ACTIVE GROWTH FACTORS FIBRIN DEGRADATION PRODUCTS PLASMIN PLASMINOGEN/PLASMIN BINDING SITES EXTRACELLULAR MATRIX PROTEINS DEGRADATION PRODUCTS PRO-MMP's ACTIVE MMP's

10 Effector protease: Plasmin Fairly broad spectrum protease with Lys/Arg specificity Lysine binding sites in Kringles (fibrin, cell surface, α 2 -apli, etc.) K K K K K SP Highly efficient fibrinolytic enzyme Additional ECM substrates Activates pro-upa and certain pro-mmps Activates/releases certain growth factors

11 Serine proteases - Substrate cleavage Serine protease Ser OH NH CO COOH Protein substrate (Exposed peptide bond) NH 2 NH 2 Ser O CO NH 2 COOH Acyl enzyme e intermediate ed (Ester bond) H O 2 NH 2 Serine protease released Ser OH COOH Cleaved protein (Dissociates or remains non-covalently associated) COOH NH 2

12 Pro-enzyme: Plasminogen Pro-enzyme of M r ~ 92,000 K K High concentration in plasma (1.5 2 μm) COOH-terminal serine protease domain K K K SP Kringles containing lysine binding sites NH 2 -terminal domain modulating rate of NH 2 terminal domain modulating rate of activation

13 Plasminogen activation K K K K Plasminogen K SP upa tpa Activation cleavage at K K Arg 561 Val 562 Plasmin K K K SP

14 Trigger protease: upa upar GFD K SP Plg High specificity COOH-terminal serine protease domain One kringle NH 2 -terminal growth factor domain (upar binding)

15 Pro-uPA / upa upar GFD K SP M r ~ 50,000 Trace component (~ 20 pm in plasma) Activation cleavage at Lys158 Ile159 Plasmin, other proteases upar GFD K SP Plg

16 Reciprocal proenzyme activation pro-upa + Plasminogen? upa + Plasmin

17 Pro-uPA and plasminogen: Time course of plasminogen activation Plasmin activity delta(a )/min x Cascade initiation in vivo? - Intrinsic pro-upa activity - Plasmin (from tpa) Other serine proteases (or even minutes other classes)

18 PAI-1 (Dominant) PA system inhibitors PAI-2 (Intra-/extracellular) PAI-3 (Protein C inhibitor) (Serpin p type inhibitors against PAs) ) α 2 -antiplasmin (Serpin type plasmin inhibitor) α 2 -macroglobulin (Non-serpin type inhibitor of plasmin (Non serpin type inhibitor of plasmin and several other serine proteases)

19 Serpins Irreversible inhibitors of serine proteases Form covalent complexes with their target proteases

20 upa - PAI-1 complex: Ester linkage Ser 356 Ser OH NH upa PAI-1 COOH CO NH 2 upa Ser 356 O CO NH 2 COOH- terminal PAI-1 f (non-covalently bound) PAI-1 COOH NH 2 Ester linked complex

21 The active proteases exist in an environment with a general excess of protease inhibitors How can activity be maintained and how does cascade amplification proceed?

22 Activation / Inhibition Balance pro-upa Plasminogen upa Plasmin a2-antiplasmin PAI-1

23 Steady-state activity in solution upa PAI-1 Plg Plg PAI-1 2aPli Pli upa Plg Pro-uPA Pro-uPA Plg upa PAI-1 Pro-uPA 2aPli Plg 2aPli Pli Plg Pro-uPA Pli Integrated system allows time window of activity

24 0.06 Activity ty rescue with pro-upa and plasminogen Solution Plasmin activity Delta(A4 405) / min Reciprocal activation survives in the presence of inhibitors min Pro-uPA Pro-uPA PA+PAI PAI-1 tc-upa tc-upa + PAI-1 Behrendt, N. et al. (2003) Biochem. J. 371:

25 The PA system and cell surfaces Localization of activity Stimulation of activation reactions Protection against inhibition

26 upar Cell surface pro-upa Plasminogen upa Plasmin a2-anti-anti plasmin PAI-1

27 Cell bound plasmin activity is protected against α 2 -antiplasmin 100 activity, % Plasmin minutes Addition of α 2 -antiplasmin to cells with surface-bound plasmin (upper) or with simultaneous release of the bound plasmin (lower)

28 Plasminogen activation system on the cell surface A B C D 2 PAI-1 Pro-uPA Plg Pro-uPA Pli upa Plg upa Pli upar upar upar upar

29 Biochemistry of the PA system Some features to (try to) remember Enzyme cascade Effector protease (Plasmin) Matrix degradation Reciprocal pro-enzyme activation Steady-state activity model Trigger components (PAs) Fibrinolysis Downstream effects Irreversible inhibitors Surface bound reactions Localization of proteolysis Steric favorization of activation Inhibitor protection ti

30 Matrix metalloproteases (MMPs) Zinc-dependent proteases Extracellular l or membrane-associated Some degrade collagen Additional matrix substrates Specific inhibitors (TIMPs) Important roles in tissue remodeling and probably in cancer

31 De forskellige MMP inddeles efter domæne sammensætning s Egeblad et al, 2002

32 MMP 14 (MT1 MMP) MMP 15 (MT2 MMP) MMP 16 (MT3 MMP) MMP 1 (Collagenase 1) MMP 13 (Collagenase 3) MMP 2 (Gelatinase A) Cathepsin K

33 Fibroblast collagenolytic activity abolished by MT1-MMP gene e knock-out out cell number G.3:G2 Fibroblasts on reconstituted collagen-i matrix MT1-MMP-/- MT1-MMP+/+

34 MMP ekspression i cancer Egeblad et al. Nature Reviews, 2002

35 A collagenolytic MMP in matrix degradation MMP-13 in microinvasion Myoepithelium MMP-13 Nielsen, B.S. et al. (2001) Cancer Res. 61:

36 Part 2 Samspil mellem ekstracellulær proteolyse og endocytose i collagennedbrydning

37 Collagen Main protein component in extracellular matrix Characteristic triple helix Forms insouble fibres and sheets Only few proteases can cleave native collagen

38 uparap/endo180 Mannose receptor 1 1 1/ 1 integrin 2 1 2/ 1 integrin

39 uparap An endocytic receptor for collagen NH 2 Cys-rich FN-II Type-1 membrane protein CRDs Member of MMR family uparap = Endo180 COOH TM Cyto

40 uparap/endo180 in invasive ductal carcinoma Expression of uparap in tumor-associated fibroblast-like cells Tumor cells are uparapnegative B.S.Nielsen & N. Behrendt group

41 uparap knock-out mice Phenotype largely normal But: Cells from uparap-/- mice are unable to internalize collagen Internalized protein (fg/cell ) Col V * Genotype: -/- +/+ Col V * -/- -/- Col I * /- +/+ Col IV Transferrin * -/- +/ /- +/+ 0.2 MMP-13 Fibronectin /- +/+ -/- +/+ Transfection: Neo o 180 PARAP/End up Engelholm, L.H. et al., J. Cell Biol. 2003

42 Internalization of fluorescencelabeled collagen IV uparap+/+ uparap-/- Kjøller, j, L. et al. Exp p Cell Res. 2004

43 Complementary routes of collagen breakdown? Combined deficiency of uparap and MT1-MMP

44 U+/M+, N=46 U-/M+, N= p= U+/M-, N= p= U-/M-, N= Age (days) The combined deficiency leads to early postnatal death Wagenaar-Miller, R.A. et al. (2007) Mol Cell Biol. 27:

45 The importance of uparap for PymT tumor growth 95 day-old mice 105 day-old mice 25 t-test P < Wilcoxon P < t-test P < Wilcoxon P < Tum mor burde en (g) Tum mor burde en (g) Control uparap-/- (N=19) (N=18) Control uparap-/- (N=48) (N=41) Curino, A.C. et al. (2005) J. Cell Biol. 169:

46 Accumulation of type-i collagen in uparap-/- tumors uparap + Treshol darea (% of im age) Collagen I P< uparap -/- 0 uparap+ uparap-/- N=7 N=7

47 Uptake of collagen exclusively in uparap+ fibroblasts (explants from uparap + and -/- tumors) uparap+ uparap-/- Collagen Lysosomes Nucleus

48 Collagenolysis summary MMP-mediated and/or endocytic Redundant pathways Integrated pathways Involved in bone growth and cancer invasion in vivo

49

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