Introduction: We and others have indicated that androgen receptor (AR) signaling plays a critical role in urothelial cancer outgrowth. Meanwhile, our DNA microarray data suggest that latrophilin-3 (LPHN3), one of latrophilins (LPHNs) that are a group of the G-protein-coupled receptor where a spider venom latrotoxin (LTX) is known to bind, represents a downstream target of AR in bladder cancer cells. The present study aimed to determine the functional role of LPHNs, including LPHN1, LPHN2, and LPHN3, in the development of urothelial cancer.
Methods: We compared the levels of LPHN expression in 83 bladder tumors vs. paired normal bladder tissues (by immunohistochemistry) as well as in human normal urothelial SVHUC sublines stably expressing wild-type AR vs. vector only (by western blot). We then assessed the effects of ligand (e.g. LTX, FLRT3) treatment, as well as silencing of each LPHN via shRNA virus infection, on the neoplastic transformation of SVHUC-derived cells induced by a chemical carcinogen 3-methylcholanthrene (MCA). Bladder tumor development was also compared in C57BL/6 mice treated with a carcinogen BBN and LTX/FLRT3.
Results: The rates of immunoreactivity for LPHN1 (87% vs. 69%, P=0.012), LPHN2 (55% vs. 31%, P=0.002), and LPHN3 (51% vs. 37%, P=0.087) were significantly higher in bladder tumors than in non-neoplastic urothelial tissues. Moreover, LPHN3 positivity in non-muscle-invasive tumors was associated with the risk of disease recurrence after transurethral surgery (P=0.051). The expression levels of each LPHN were also higher in SVHUC-AR cells than in AR-negative SVHUC-vector cells, and androgen treatment in SVHUC-AR further increased them. Chromatin immunoprecipitation assay then revealed the binding of AR to the promoter region of each LPHN in SVHUC-derived cells. Meanwhile, induction in LPHN expression by LTX or FLRT3 treatment was seen in SVHUC cells. The in vitro transformation assay showed that LTX and FLRT3 induced the MCA-mediated oncogenic activity particularly in SVHUC-vector cells, while knockdown of LPHN1, LPHN2, or LPHN3 resulted in similar inhibition in that of MCA-SVHUC-AR cells. LTX (P=0.002) and FLRT3 (P=0.001) also accelerated the development of bladder tumors in BBN-treated female mice.
Conclusions: These findings suggest that LPHNs, which are activated in bladder tumors as key downstream effectors of the AR, promote urothelial tumorigenesis. Accordingly, LPHN inhibition, along with AR inactivation, has the potential of being an effective chemopreventive approach for urothelial cancer.
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