Matrixnedbrydning ved cancerinvasion -Biokemiske aspekter Ekstracellulære Proteaser og endocytosereceptorer Niels Behrendt The Finsen Laboratory Rigshospitalet Copenhagen
Nødvendige begivenheder i metastasering Primary tumor Primary tumor growth Angiogenesis Invasion
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
Matrixnedbrydning og cancer ce invasion Collagen Fibroblasts Myoepithelial cell Basement membrane: Collagen IV, Tenascin, laminin, entactin, PGs Cancer cells
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
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
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
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
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
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
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
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
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
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)
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
Reciprocal proenzyme activation pro-upa + Plasminogen? upa + Plasmin
Pro-uPA and plasminogen: Time course of plasminogen activation 300 250 200 Plasmin activity delta(a )/min x 10 3 150 405 Cascade initiation in vivo? - Intrinsic pro-upa activity - Plasmin (from tpa) 100 50 0 0 5 10 15 20 25 30 35 - Other serine proteases (or even minutes other classes)
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)
Serpins Irreversible inhibitors of serine proteases Form covalent complexes with their target proteases
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
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?
Activation / Inhibition Balance pro-upa Plasminogen upa Plasmin a2-antiplasmin PAI-1
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
0.06 Activity ty rescue with pro-upa and plasminogen Solution 005 0.05 Plasmin activity Delta(A4 405) / min 0.04 003 0.03 0.02 Reciprocal activation survives in the presence of 001 0.01 inhibitors 0.00 0 20 40 60 80 100 min Pro-uPA Pro-uPA PA+PAI PAI-1 tc-upa tc-upa + PAI-1 Behrendt, N. et al. (2003) Biochem. J. 371: 277-87
The PA system and cell surfaces Localization of activity Stimulation of activation reactions Protection against inhibition
upar Cell surface pro-upa Plasminogen upa Plasmin a2-anti-anti plasmin PAI-1
Cell bound plasmin activity is protected against α 2 -antiplasmin 100 activity, % Plasmin 10 1 0 5 10 15 20 minutes Addition of α 2 -antiplasmin to cells with surface-bound plasmin (upper) or with simultaneous release of the bound plasmin (lower)
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
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
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
De forskellige MMP inddeles efter domæne sammensætning s Egeblad et al, 2002
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
Fibroblast collagenolytic activity abolished by MT1-MMP gene e knock-out out cell number 2500 7500 22500 2500 7500 22500 +3.G.3:G2 Fibroblasts on reconstituted collagen-i matrix MT1-MMP-/- MT1-MMP+/+
MMP ekspression i cancer Egeblad et al. Nature Reviews, 2002
A collagenolytic MMP in matrix degradation MMP-13 in microinvasion Myoepithelium MMP-13 Nielsen, B.S. et al. (2001) Cancer Res. 61: 7091-100
Part 2 Samspil mellem ekstracellulær proteolyse og endocytose i collagennedbrydning
Collagen Main protein component in extracellular matrix Characteristic triple helix Forms insouble fibres and sheets Only few proteases can cleave native collagen
uparap/endo180 Mannose receptor 1 1 1/ 1 integrin 2 1 2/ 1 integrin
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
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
uparap knock-out mice Phenotype largely normal But: Cells from uparap-/- mice are unable to internalize collagen Internalized protein (fg/cell ) 20 15 10 5 Col V * 6 4 2 0 0 Genotype: -/- +/+ Col V * -/- -/- Col I 30 10 25 20 5 15 10 * 5 0 0 -/- +/+ Col IV Transferrin * -/- +/+ 6 4 2 0 -/- +/+ 0.2 MMP-13 Fibronectin 0.2 0.1 0.1 0.0 0.0 -/- +/+ -/- +/+ Transfection: Neo o 180 PARAP/End up Engelholm, L.H. et al., J. Cell Biol. 2003
Internalization of fluorescencelabeled collagen IV uparap+/+ uparap-/- Kjøller, j, L. et al. Exp p Cell Res. 2004
Complementary routes of collagen breakdown? Combined deficiency of uparap and MT1-MMP
100 90 U+/M+, N=46 U-/M+, N=46 80 70 p=.0009 60 50 U+/M-, N=17 40 30 20 p=.0063 10 0 U-/M-, N=29 0 10 20 30 40 50 60 70 Age (days) The combined deficiency leads to early postnatal death Wagenaar-Miller, R.A. et al. (2007) Mol Cell Biol. 27: 6309-22
The importance of uparap for PymT tumor growth 95 day-old mice 105 day-old mice 25 t-test P < 0.006 Wilcoxon P < 0.009 25 t-test P < 0.0002 Wilcoxon P < 0.0006 Tum mor burde en (g) 20 15 10 Tum mor burde en (g) 20 15 10 5 5 0 0 Control uparap-/- (N=19) (N=18) Control uparap-/- (N=48) (N=41) Curino, A.C. et al. (2005) J. Cell Biol. 169: 977-985
Accumulation of type-i collagen in uparap-/- tumors uparap + Treshol darea (% of im age) 25 20 15 10 5 Collagen I P<0.000001 000001 uparap -/- 0 uparap+ uparap-/- N=7 N=7
Uptake of collagen exclusively in uparap+ fibroblasts (explants from uparap + and -/- tumors) uparap+ uparap-/- Collagen Lysosomes Nucleus
Collagenolysis summary MMP-mediated and/or endocytic Redundant pathways Integrated pathways Involved in bone growth and cancer invasion in vivo