Fig 2: NgR2 induces NE markers and RhoA in CRPrCa and NEPrCa cells in vitro. (A) Immunoblotting analysis of the expression levels of the αVβ3 integrin (on a 7.5% PAGE), NgR2 and Aurora Kinase A (AURKA, on a 7.5% PAGE), and neuron-specific enolase (NSE) and RhoA on a 12.5% PAGE in NCI-H660 cells in which NgR2 expression was downregulated using a pool of siRNAs against RTN4RL2 (NgR2). AURKA and NSE protein expression, measured using densitometric analysis, is reduced 11.22 and 8.4 fold, respectively, when compared to the non-silencing siRNA control (n = 4). (B) Left panel, immunoblotting analysis of the expression levels of NgR2 (on a 7.5% PAGE); RhoA and synaptophysin (SYP, on a 10% PAGE) in C4-2B cells that exogenously express αVβ3 in which NgR2 expression was downregulated using a pool of siRNA against RTN4RL2. Right panel, immunoblotting analysis of the expression levels of the αVβ3 integrin, AURKA, and NSE (on a 12.5% PAGE) in C4-2B cells that exogenously express the αVβ3 integrin and in which NgR2 expression was downregulated using a pool of siRNAs against RTN4RL2 (n = 3). AURKA and NSE protein expression, measured using densitometric analysis, is reduced 3.59 and 3.9 fold, respectively, when compared to the non-silencing siRNA control. (A,B) –, Oligofectamine; NS, non-silencing. (C) Immunoblotting analysis of the expression levels of NgR2 (on a 10% PAGE), the αVβ3 integrin, NSE, and RhoA (on a 10% PAGE), and Chromogranin A (CHGA, on a 10% PAGE) in DU145 cells that exogenously express NgR2 or their mock control cells (n = 2). Actin or CANX was used as loading control (A–C). All immunoblotting analyses were performed under reducing conditions.

Fig 3: Schematic representation of the pathway described in this paper. (A) The αVβ3 integrin, together with its co-activator K2, induces increased levels of NgR2 expression in castrate-resistant prostate cancer cells. In turn, NgR2 regulates the levels of NE markers and stimulates anchorage-independent growth of these cells. NgR2 also upregulates the levels of RhoA that causes increased PrCa cell motility. These cells with increased NgR2 expression are likely to be NEPrCa cells. (B) The schematic drawing summarizes our proposed model.

Fig 4: NgR2 expression is increased in NEPrCa TKO mouse tumors. (A, B) Representative immunohistochemical staining of the αVβ3 integrin, NgR2, and SYP of prostate tumors (top row) and lung metastasis (bottom row) from TKO mice (A, n = 5) and of primary tumors from SKO mice (B, n = 5). (C) Representative immunohistochemical staining of NgR2 of prostate samples from wild-type mice (n = 4). IgG was used as negative control (A–C). The bars at the bottom right corner of each panel represent 20 μm.

Fig 5: The αVβ3 integrin increases NgR2 expression and is associated with NgR2. (A) Immunoblotting analysis of the expression levels of the αVβ3 integrin and NgR2 (n = 3, left panels), and of the αVβ3 integrin, the NE markers chromogranin A (CHGA) and neuron-specific enolase (NSE; n = 1, middle panels), in C4-2B cells that exogenously express αVβ3 or their mock control cells. Right panel, immunoblotting analysis of the expression levels of the αVβ3 integrin, and NgR2 in PC3 cells in which the αVβ3 integrin expression was downregulated using three different siRNAs against ITGB3 (the gene responsible for the β3 integrin subunit). Non-silencing siRNA (NS) was used as control. Calnexin was used as loading control. (B) PC3 cell lysates were immunoprecipitated with Abs against the αVβ3 integrin, NgR2, the β1 integrin, the αVβ6 integrin or their isotype controls (left panel) and the αVβ3 integrin, NgR2, and the αVβ6 integrin (right panel). Immunoprecipitates were analyzed for NgR2, the β1 integrin, the αVβ3 integrin, and the αVβ6 integrin by immunoblotting (n = 2). Immunoblotting analyses were performed under non-reducing (left panel) and reducing conditions (right panel). NgR2 was detected using two different Abs (left panel).