Matrilysin Matrix Metalloproteinase-7 Research Articles

Matrilysin/MMP-7 Cleavage of Perlecan/HSPG2 Complexed with Semaphorin 3A Supports FAK-Mediated Stromal Invasion by Prostate Cancer Cells

Interrupting the interplay between cancer cells and extracellular matrix (ECM) is a strategy to halt tumor progression and stromal invasion. Perlecan/heparan sulfate proteoglycan 2 (HSPG2) is an extracellular proteoglycan that orchestrates tumor angiogenesis, proliferation, differentiation and invasion. Metastatic prostate cancer (PCa) cells degrade perlecan-rich tissue borders to reach bone, including the basement membrane, vasculature, reactive stromal matrix and bone marrow. Domain IV-3, perlecan’s last 7 immunoglobulin repeats, mimics native proteoglycan by promoting tumoroid formation. This is reversed by matrilysin/matrix metalloproteinase-7 (MMP-7) cleavage to favor cell dispersion and tumoroid dyscohesion. Both perlecan and Domain IV-3 induced a strong focal adhesion kinase (FAK) dephosphorylation/deactivation. MMP-7 cleavage of perlecan reversed this, with FAK in dispersed tumoroids becoming phosphorylated/activated with metastatic phenotype. We demonstrated Domain IV-3 interacts with the axon guidance protein semaphorin 3A (Sema3A) on PCa cells to deactivate pro-metastatic FAK. Sema3A antibody mimicked the Domain IV-3 clustering activity. Direct binding experiments showed Domain IV-3 binds Sema3A. Knockdown of Sema3A prevented Domain IV-3-induced tumoroid formation and Sema3A was sensitive to MMP-7 proteolysis. The perlecan-Sema3A complex abrogates FAK activity and stabilizes PCa cell interactions. MMP-7 expressing cells destroy the complex to initiate metastasis, destroy perlecan-rich borders, and favor invasion and progression to lethal bone disease.

Abstract 1563: Interplay between perlecan/HSPG2 and matrilysin/MMP-7 in the prostate cancer tumor microenvironment directs metastatic programming through focal adhesion kinase and FoxM1

Abstract Prostate cancer (PCa) cells interact with the stroma and extracellular matrix (ECM) during tumor expansion and invasion during metastasis. Dissecting the complex matrix-cancer interplay is essential to halting tumor progression. A vital component of the ECM is perlecan/heparan sulfate proteoglycan 2 (HSPG2). As a large extracellular proteoglycan, perlecan helps orchestrate tumor angiogenesis, proliferation, differentiation and invasion. Actively metastasizing cancer cells must proteolytically degrade several tissue borders which perlecan patrols, including the basement membrane, vasculature, reactive stromal matrix and bone marrow. We have demonstrated that perlecan, even fully decorated with glycosaminoglycan chains and in the context of the basement membrane, is cleaved efficiently by the protease matrilysin/matrix metalloproteinase 7 (MMP-7). MMP-7 levels increase, especially in relation to its endogenous inhibitor, during metastatic programming in several cancers including PCa. Recent analysis of a tissue microarray of 157 prostatectomy patients showed perlecan and MMP-7 are increased in tandem, with perlecan disappearing at the sites where MMP-7 is high, indicative of a protease-substrate relationship in tissue. This idea is supported by the finding that perlecan fragments are present in serum of PCa patients, with a majority from domain IV. Additionally, higher perlecan staining in cancer related stroma positively correlated with higher grade cancer. Investigating the effects of perlecan on metastatic PCa cells showed full length perlecan and specifically domain IV of perlecan triggers clustering of PCa cells. Perlecan proteolysis by MMP-7 reverses this, favoring cell dispersion and tumor dyscohesion. To discover the molecular signaling events associated with perlecan binding to PCa cells, a reverse phase protein array was analyzed for protein interactions that were plotted in Cytoscape. Network analysis revealed, and western blotting confirmed, perlecan and MMP-7 mediated proteolysis altered focal adhesion kinase (FAK) signaling, with intact perlecan and domain IV suppressing FAK activity during clustering, and MMP-7 cleaved perlecan activating FAK, consistent with the induction of dispersion. Further work showed that cleavage of perlecan increased the pro-cancer transcription factor forkhead box protein M1 (FOXM1). This work reveals that degradation of perlecan by MMP-7 in the tumor microenvironment can unlock stable epithelial contacts and reprogram PCa cells for invasion and metastasis through processes involving FAK and FoxM1. Citation Format: Brian J. Grindel, Jerahme Martinez, Hamim Zafar, Luay K. Nakhleh, Leland W.K. Chung, Mary C. Farach-Carson. Interplay between perlecan/HSPG2 and matrilysin/MMP-7 in the prostate cancer tumor microenvironment directs metastatic programming through focal adhesion kinase and FoxM1. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1563.

Matrilysin/matrix metalloproteinase-7(MMP7) cleavage of perlecan/HSPG2 creates a molecular switch to alter prostate cancer cell behavior

Perlecan/HSPG2, a large heparan sulfate (HS) proteoglycan, normally is expressed in the basement membrane (BM) underlying epithelial and endothelial cells. During prostate cancer (PCa) cell invasion, a variety of proteolytic enzymes are expressed that digest BM components including perlecan. An enzyme upregulated in invasive PCa cells, matrilysin/matrix metalloproteinase-7 (MMP-7), was examined as a candidate for perlecan proteolysis both in silico and in vitro. Purified perlecan showed high sensitivity to MMP-7 digestion even when fully decorated with HS or when presented in native context connected with other BM proteins. In both conditions, MMP-7 produced discrete perlecan fragments corresponding to an origin in immunoglobulin (Ig) repeat region domain IV. While not predicted by in silico analysis, MMP-7 cleaved every subpart of recombinantly generated perlecan domain IV. Other enzymes relevant to PCa that were tested had limited ability to cleave perlecan including prostate specific antigen, hepsin, or fibroblast activation protein α. A long C-terminal portion of perlecan domain IV, Dm IV-3, induced a strong clustering phenotype in the metastatic PCa cell lines, PC-3 and C4-2. MMP-7 digestion of Dm IV-3 reverses the clustering effect into one favoring cell dispersion. In a C4-2 Transwell® invasion assay, perlecan-rich human BM extract that was pre-digested with MMP-7 showed loss of barrier function and permitted a greater level of cell penetration than untreated BM extract. We conclude that enzymatic processing of perlecan in the BM or territorial matrix by MMP-7 as occurs in the invasive tumor microenvironment acts as a molecular switch to alter PCa cell behavior and favor cell dispersion and invasiveness.

Abstract 5063: Matrilysin cleavage of perlecan produces bioactive fragments acting as a molecular switch to alter prostate cancer cell behavior.

Abstract Perlecan/HSPG2[PLN], a large heparan sulfate (HS) proteoglycan, is expressed in the basement membrane (BM) underlying epithelial and endothelial cells. During prostate cancer (PCa) cell invasion, a variety of proteolytic enzymes are expressed that digest BM components including PLN. After proteolysis, fragments of PLN can be released into circulation. A sandwich ELISA with antibodies generated across PLN detected fragments of PLN from IG-repeat domain IV in PCa patient serum. An enzyme upregulated in invasive PCa cells, matrilysin/matrix metalloproteinase-7 (MMP-7), was examined as a candidate for PLN proteolysis both in silico and in vitro. Purified PLN showed high sensitivity to MMP-7 digestion even when fully decorated with HS or when embedded with other BM proteins. MMP-7 fragments of PLN were detected as limit peptides by western blot. While not predicted in silico, MMP-7 cleaved every subpart of recombinantly generated PLN domain IV. The resultant MMP-7 generated PLN Dm IV fragments were identified by mass spectrometry (LC-MS/MS and MALDI) and Edman degradation N-terminal sequencing. Other PCa relevant enzymes had limited ability to cleave PLN including prostate specific antigen, hepsin and fibroblast activation protein. To determine if PLN digestion affects metastatic PCa cells, C4-2B cells were seeded on plates coated with different forms of PLN. Cells on intact PLN formed large clusters of poorly adherent cells, while those on PLN digested with MMP-7 or heparitinase to remove the HS chains attached and spread within 30 minutes. Immunohistochemistry of PCa patient tissue indicates MMP-7 and PLN are in direct contact at interfaces in the stroma. We conclude that enzymatic processing of PLN in the BM and stroma as occurs in the tumor microenvironment can alter PCa cell behavior and favor cell dispersion and invasiveness. Citation Format: Brian J. Grindel. Matrilysin cleavage of perlecan produces bioactive fragments acting as a molecular switch to alter prostate cancer cell behavior. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5063. doi:10.1158/1538-7445.AM2013-5063

Abstract: 586 MODIFIABLE CVD RISK FACTORS IN COSTA RICA: COMPLEXITIES OF AFFLUENCE AND POVERTY IN MIDDLE INCOME COUNTRIES

Perlecan/HSPG2, a large heparan sulfate (HS) proteoglycan, normally is expressed in the basement membrane (BM) underlying epithelial and endothelial cells. During prostate cancer (PCa) cell invasion, a variety of proteolytic enzymes are expressed that digest BM components including perlecan. An enzyme upregulated in invasive PCa cells, matrilysin/matrix metalloproteinase-7 (MMP-7), was examined as a candidate for perlecan proteolysis both in silico and in vitro. Purified perlecan showed high sensitivity to MMP-7 digestion even when fully decorated with HS or when presented in native context connected with other BM proteins. In both conditions, MMP-7 produced discrete perlecan fragments corresponding to an origin in immunoglobulin (Ig) repeat region domain IV. While not predicted by in silico analysis, MMP-7 cleaved every subpart of recombinantly generated perlecan domain IV. Other enzymes relevant to PCa that were tested had limited ability to cleave perlecan including prostate specific antigen, hepsin, or fibroblast activation protein α. A long C-terminal portion of perlecan domain IV, Dm IV-3, induced a strong clustering phenotype in the metastatic PCa cell lines, PC-3 and C4-2. MMP-7 digestion of Dm IV-3 reverses the clustering effect into one favoring cell dispersion. In a C4-2 Transwell® invasion assay, perlecan-rich human BM extract that was pre-digested with MMP-7 showed loss of barrier function and permitted a greater level of cell penetration than untreated BM extract. We conclude that enzymatic processing of perlecan in the BM or territorial matrix by MMP-7 as occurs in the invasive tumor microenvironment acts as a molecular switch to alter PCa cell behavior and favor cell dispersion and invasiveness.