The prostate specific membrane antigen, a biomarker of prostate cancer, is endowed with signaling properties that regulates cytokine production and cell survival of prostate cancer cells

Prostate cancer is the most commonly diagnosed neoplasia in man living developed countries. A number of studies have recently demonstrated that a prominent role in tumor survival and progression can be attributed to soluble mediators present in the tumor microenvironment. Among these, Interleukin-6 (IL-6) has a fundamental role in the regulation of proliferation, apoptosis, angiogenesis and differentiation in many cell types and it is also implicated in the development and progression of several forms of tumours including prostate cancer. In fact, the expression of IL-6 and its receptor is consistently demonstrated in human prostate cancer cell lines and in freshly isolated cells from human prostate carcinoma and benign prostate hyperplasia. Clinically, IL-6 levels in serum are significantly elevated in many cases of advanced, hormone-refractory prostate cancer. Recently, the chemokine CCL5 (RANTES) was found to be expressed by human prostate carcinoma cells and it reported to stimulate their proliferation and invasion. Thus, also CCL5 appears to be directly involved in the behaviour of prostate carcinoma cells. The expression levels of the prostate specific membrane antigen (PSMA) have been proposed as a useful indicator of the severity of the disease in prostate cancer. PSMA expression is augmented in high-grade and metastatic tumors. Observations of the clinical behaviour of prostate tumors suggest that increased secretion of IL-6 and CCL5 may correlate to higher expression of PSMA may be correlated. We hypothesized that PSMA is endowed with signalling properties and that its stimulation regulate IL-6 and CCL5 gene expression. We herein demonstrate that cross-linking of cell surface PSMA with specific antibodies activates the small GTPases RAS and RAC1 and the MAPKs p38 and ERK1/2 in prostate carcinoma LNCaP cells. As downstream effects of PSMA- fostered RAS-RAC1-MAPK pathway activation we observed a strong induction of NF-κB activation associated with an increased expression of IL-6 and CCL5 genes. Pharmacological blockade by specific inhibitors revealed that both p38 and ERK1/2 participate in the phenomenon, although a major role exerted by p38 was evident. Finally we demonstrated that IL-6 and CCL5 synergistically enhanced the proliferative potential of LNCaP cells in a dose-dependent manner and that CCL5 functioned by receptor-mediated activation of the STAT5-Cyclin D1 pro-poliferative pathway. The novel PSMA functions described in the present report may have profound influence on the survival and proliferation of prostate tumor cells, accounting for the observation that PSMA overexpression in prostate cancer patients is related to a worse prognosis. Having assessed for the first time that PSMA signal in cancer cells, we next investigated whether a PSMA-beta1 integrin interaction could occur at the plasma membrane. It was reported that in normal endothelial cell like HUVECs, PSMA co-operates with beta1 integrin in the regulation of cytoskeletal dynamics, thanks to the activation of PAK-1 and to the association to Filamin. Confocal microscopy analysis of adherent, non stimulated LNCaP cells confirmed the co-localization of the two molecules, that, in addition, could be co-immune precipitated together with filamin A. Noteworthy, PSMA-cross-linking determined the conformational change of beta1 molecules leading to the exposure of the activation epitopes recognized by the HUTS-21 mAb and the cross-linking of the two molecules at the same time raised the production of IL-6 and CCL5 by LNCaP cells. Although the role of filamin in PSMA-beta1 interactions and the molecular assembly at the plasma membrane still need to be investigated, we think that this newly discovered functions of PSMA and its possible cooperation with beta1 integrin may help in elucidating the biological effects of PSMA, implicating this molecule as an important regulator of prostate tumor cell survival and proliferation. Moreover, in order to determine whether or not the interaction of PSMA with filamin affects its signalling ability, we silenced filamin expression, in LNCaP, by using RNA interfering method. Preliminary results showed a strong decrease of ERK activation of ERK induced by PSMA cross-linking in silenced cells compared to that of the wild type. Collectively, these results are very suggestive of a PSMA-beta1integrin interaction. If so, FLNa could bridge the cytodomains of the two proteins thereby providing PSMA with signalling complex recruited by beta1 integrin. In all, the newly discovered functions of PSMA and its possible cooperation with beta1 integrin may help in elucidating the biological effects of PSMA, implicating this molecule as an important regulator of prostate tumor, cell survival and proliferation.