G-protein-coupled receptors, as a large group of evolutionarily related proteins, are well known to mediate a variety of physiological and pathologic processes [12, 27, 28]. By contrast, only limited data have suggested the involvement of LPHNs, a group of G-protein-coupled receptors, in neoplastic diseases [12, 29,30,31,32]. In the present study, we have investigated the functional role of LPHN3 in the progression of urothelial cancer, which is a distinct step or event from urothelial tumorigenesis (i.e. tumor initiation, neoplastic transformation, tumor development), in relation to AR signaling, primarily via their activation (e.g. ligand treatment) and inactivation (e.g. knockdown) in cell line models for bladder cancer.

As aforementioned, we had originally identified LPHN3 as a potential AR target from our DNA microarray analysis in a control AR-positive UMUC3 cell line versus its AR-knockdown subline [20]. In not only UMUC3 cells but also other human bladder cancer sublines stably expressing a wild-type AR, we confirmed up-regulation of the expression of ADGRL3 gene and LPHN3 protein by androgen treatment, the latter of which was blocked by AR antagonists. Using ChIP assay in bladder cancer lines, we further demonstrated the interactions of AR with ADGRL3 at its promoter region, indicating the direct regulation of LPHN3 expression by AR. These results indicate that LPHN3 represents a downstream target of AR signaling in bladder cancer cells.

Again, it is known that LTX, a neurotoxin naturally found in the venom of widow spiders, binds and activates all 3 LPHNs [10,11,12]. In addition, it has been documented that FLRT3, a member of the fibronectin leucine rich transmembrane protein family, is an endogenous ligand for LPHN3 (and LPHN1) [12, 14, 33]. In UMUC3 cells, we demonstrated that α-LTX and FLRT3 induced the expression of ADGRL3.

The involvement of ADGRL3/LPHN3 in the progression, as well as therapeutic resistance, has been suggested in several types of malignancy. The observations in these studies include: (1) an association of reduced ADGRL3 expression in tumor tissues with shorter overall survival in patients with ependymoma [32]; and (2) elevated ADGRL3 expression in acute myeloid leukemia cell lines possessing P-glycoprotein variants that have been implicated in chemoresistance [30]. Recently, we have additionally demonstrated that LPHN3 promotes: (1) the chemical carcinogen-mediated neoplastic transformation of urothelial cells [22]; and (2) the cell proliferation and migration of both AR-positive and AR-negative prostate cancer lines [23]. Nevertheless, loss-of-function mutations within the ADGRL3 gene have been identified in a few malignancies, including bladder cancer [12, 34], implying its role as a tumor suppressor. We herein found that α-LTX and FLRT3 could induce the proliferation of bladder cancer cells derived primarily from invasive urothelial carcinoma. Correspondingly, LPHN3 knockdown resulted in the reduction of their growth. These findings suggest that ADGRL3/LPHN3 functions as a promoter of bladder cancer progression. However, signal transduction pathways downstream of LPHN3 in tumor cells, as well as the roles of other LPHNs in urothelial cancer, need to be further elucidated. Moreover, it is interesting to determine if LPHN3 also induces the growth of low-grade and/or non-invasive urothelial cancer cells.

The expression status of ADGRL genes and LPHN proteins in the bladder or urothelial tumor remained uncertain. We immunohistochemically examined LPHN3 expression in transurethral resection specimens and its prognostic significance in patients with muscle-invasive disease. There were no significant associations of LPHN3 expression with the histopathology including tumor grade and stage. Nonetheless, LPHN3 positivity in muscle-invasive bladder tumors, as an independent prognosticator, was associated with a significantly higher risk of postoperative disease progression and resultant disease-specific mortality. Meanwhile, positivity of LPHN3 vs. AR in bladder tumors was strongly correlated. All these immunohistochemical findings in surgical specimens strongly support our in vitro data indicating that LPHN3, presumably as a consequence of AR activation, induces the progression of urothelial tumor. In addition, it would be possible that, as documented in not only AR itself [35] but also some effectors as its direct downstream targets, such as FOXO1 [36], GABBR2 [24], and GULP1 [37], LPHN3 might contribute to reducing cisplatin sensitivity, and LPHN3 overexpression was thereby associated with a poorer prognosis particularly in patients with muscle-invasive bladder cancer who received cisplatin-based chemotherapy.

There have been sex-related differences in the incidence and prognosis of bladder cancer [1,2,3], while sex hormone receptors, including AR and estrogen receptors (ERs), have been implicated in the development and progression of urothelial cancer [7, 25, 38]. Although the expression of AR was found to be positively correlated with that of LPHN3, as a direct downstream, in bladder cancer cells, there was no significant difference in LPHN3 expression between male vs. female tumors in our cohort. Remarkably, the majority of the previous studies, where the expression levels of AR, ERα, and/or ERβ were compared between male and female bladder tissues, failed to show significant differences [7, 25, 38, 39], although their activities in cells might vary depending on the levels of sex hormones. As aforementioned, LPHN3 could similarly induce the growth of AR-positive and AR-negative prostate cancer cells [23]. LPHN3 might thus simply represent a direct target of AR. Otherwise, as demonstrated in other AR targets including FOXO1 [40] and GULP1 [41], ERβ may also possibly regulate the expression of LPHN3 in bladder cancer cells, particularly leading to the promotion of the proliferation of female tumor cells. In any case, although it remains to be determined, LPHN3 may not mediate distinct further downstream pathways to promote the growth of bladder cancer cells in males vs. females.

In conclusion, LPHN3 was found to be a downstream effector of AR in bladder cancer cells and promote their growth. Accordingly, although no specific inhibitors are currently available, LPHN3 represents a potential therapeutic target for advanced bladder cancer. Moreover, LPHN3 overexpression may serve as a useful prognosticator especially in patients with muscle-invasive bladder cancer. Meanwhile, further studies are warranted to not only validate our results but also elucidate the molecular mechanisms underlying LPHN-mediated urothelial cancer progression.