Prostate-specific membrane antigen (PSMA)-mediated laminin proteolysis generates a pro-angiogenic peptide

Abstract

Prostate-specific membrane antigen (PSMA) is a membrane-bound glutamate carboxypeptidase expressed in a number of tissues. PSMA participates in various biological functions depending on the substrate available in the particular tissue; in the brain, PSMA cleaves the abundant neuropeptide N-acetyl-aspartyl-glutamate to regulate release of key neurotransmitters, while intestinal PSMA cleaves polyglutamated peptides to supply dietary folate. PSMA expression is also progressively upregulated in prostate cancer where it correlates with tumor progression as well as in tumor vasculature, where it regulates angiogenesis. The previous research determined that PSMA cleavage of small peptides generated via matrix metalloprotease-mediated proteolysis of the extracellular matrix protein laminin potently activated endothelial cells, integrin signaling and angiogenesis, although the specific peptide substrates were not identified. Herein, using enzymatic analyses and LC/MS, we unequivocally demonstrate that several laminin-derived peptides containing carboxy-terminal glutamate moieties (LQE, IEE, LNE) are bona fide substrates for PSMA. Subsequently, the peptide products were tested for their effects on angiogenesis in various models. We report that LQ, the dipeptide product of PSMA cleavage of LQE, efficiently activates endothelial cells in vitro and enhances angiogenesis in vivo. Importantly, LQE is not cleaved by an inactive PSMA enzyme containing an active site mutation (E424S). Endothelial cell activation by LQ was dependent on integrin beta-1-induced activation of focal adhesion kinase. These results characterize a novel PSMA substrate, provide a functional rationale for the upregulation of PSMA in cancer cells and tumor vasculature and suggest that inhibition of PSMA could lead to the development of new angiogenic therapies.

PSMA redirects cell survival signaling from the MAPK to the PI3K-AKT pathways to promote the progression of prostate cancer

“…We found that tumors in wild-type animals were larger and of higher grade with a higher microvessel density as compared to tumors in the PSMA knockout animals, which is consistent with our previous results implicating PSMA as an angiogenic regulator (36, 37, 42). In addition, PSMA-positive tumor cells were viable at greater distances from the vasculature than their PSMA knockout counterparts, suggesting that cell-intrinsic survival components also contribute to tumor growth.…”

“…We have previously shown that PSMA regulates angiogenesis by modulating integrin signal transduction and β 1 integrin–dependent FAK-Tyr 925 phosphorylation in endothelial cells (36, 37, 42) and show here that its expression alters FAK phosphorylation and IGF-1R signaling pathways in PCa cells (Fig. 6).…”

A Perspective on the Evolving Story of PSMA Biology, PSMA-Based Imaging, and Endoradiotherapeutic Strategies

“…In a collaboration with Shapiro's research group on our knockout mouse model, we used an in vivo assay to evaluate activation of endothelial cell ingrowth (neovascularization) into an extracellular matrix plug (Matrigel; Corning). The PSMAknockout animals responded with dramatically attenuated vascular growth and reduced hemoglobin content, compared with the wild-type animals (39). Similarly, the PSMA inhibitor 2phosphonomethyl pentanedioic acid also blocked neovascularization.…”

Consensus guidelines for the use and interpretation of angiogenesis assays

“…These assays have been instrumental in the study of vascular biology in growth and development [6–8] but also play a key role in the design, development, and evaluation of drugs that positively or negatively modulate vessel function for the treatment of many diseases [9–11]. Some examples of where the use of such bioassays has been imperative are: (1) the development of angiostatic drugs for the treatment of cancer, ocular diseases, and other pathologic conditions where angiogenesis is implicated and also angiogenic treatment strategies in ischemic cardiovascular disease [12, 13], (2) screening of natural anti-angiogenic compounds [14], (3) the efforts to design combination therapies including angiogenesis inhibitors [15–20], (4) the unraveling of mechanisms regulating lymphangiogenesis [21, 22], (5) the interrelationship of angiogenesis and immunity [23–25], (6) the development of imaging as diagnostic strategy [26], (7) the study of drug resistance mechanisms [27–29], (8) development of compounds and strategies for the revascularization of ischemic injuries [30, 31], and (9) to improve the vascular fitness in aging vessels [32, 33]. The current paper describes a large collection of assays and techniques for the evaluation of angiogenesis and aims at explaining their respective advantages and limitations.…”