Numbers on the right represent the percentage of cells with AR nuclear localization
Numbers on the right represent the percentage of cells with AR nuclear localization. Among all the tyrosine kinases expressed in LNCaP cells, we previously showed that Src and FAK are most prominently activated by bombesin (3). migration of the GRP-expressing cell lines, and blocks the nuclear transloation of AR, indicating the involvement of SFK in the aberrant activation of AR and demonstrating the potential use of SFK inhibitor Rabbit polyclonal to INPP5K in the treatment of castration resistant CaP. In vivo study showed AZD0530 profoundly inhibits tumor metastasis in severe combined immunodeficient (SCID) mice implanted with GRP-autocrine LNCaP cells. This xenograft model demonstrates autocrine, neuropeptide- and Src kinase-mediated progression of androgen-independent CaP post-castration, and is potentially useful for testing novel therapeutic agents. 0.001), suggesting GRPs involvement. Migration of GRP1-1 and 4C9 towards ctlCM was two-fold greater than that of LNCaP-zeo, and could be further stimulated by GRP CM, and significantly inhibited by 2A11 (0.001). These data showed that LNCaP-GRP cells release GRP, which confers androgen-independent growth and migration through autocrine loop. Open in a separate window Figure 1 The model of an androgen-independent GRP expressing prostate cancer line, with evidence of enhanced proliferation and migration: and and and and and Chromatin immunoprecipitation assay: AR binding to both the enhancer and proximal ARE in the PSA promoter was revealed through PCR analysis using ChIP assay coupled with amplification with primers described in the Materials and Methods. E, P, and I designate Adoprazine (SLV313) for the upstream enhancer region, proximal ARE region, and the intervening region, respectively. GRP modulates activation of AR We further sought to illustrate GRP-mediated AR Adoprazine (SLV313) activation at the molecular level. Transactivation assay was performed with LNCaP-Zeo, GRP4-9 and GRP-Pro cells in CS media using promoter PSA-Luc as the reporter. Expression of PSA-Luc in GRP4-9 and GRP-Pro is 1.8 and 4.5 fold higher than in LNCaP-Zeo cells (Figure 3B). This suggests GRP secreted from GRP cells is driving the expression. Addition of synthetic androgen R1881 induced PSA-Luc expression in LNCaP-Zeo cells more than 6 fold, but much less in GRP4-9 and GRP-Pro cells probably because the GRP-activated AR, through post-translational modification, already adopted an active conformation and may not be further stimulated by R1881. If GRP activates AR in GRP-Pro cells, AR should be recruited to ARE sites in the PSA promoter. We therefore performed the ChIP assay on LNCaP-Zeo, GRP4-9 and GRP-Pro cells in CS or CS+R1881 conditions. AR binding was analyzed by PCR using respective primers against enhancer (E) and proximal (P) ARE regions, and an intervening (I) region void of any ARE sites. Figure 3C shows AR binds to PSA P region in GRP4-9 and GRP-Pro even in the absence of androgen. When treated with R1881, AR binds preferentially to the E site in LNCaP-Zeo; whereas in GRP4-9 and GRP-Pro, Adoprazine (SLV313) AR binding was evenly detected at both P and E sites. Src and FAK tyrosine kinases play important roles in GRP-mediated androgen-independent growth and migration Exogenous bombesin induces AR nuclear translocation, and this Adoprazine (SLV313) induction is inhibited by Src inhibitor PP2 (25). In our LNCaP GRP mouse model, AR is localized to the nuclei as shown in the tumor IHC staining (Figure 2C). We further compared the GRP cells with the mock control by immunofluorescent staining to confirm AR nuclear localization in GRP cells through autocrine GRP-mediated activation (Figure 4). Staining of AR is limited to the cytoplasm in Zeo cells grown in CS media but concentrated to the nuclei of GRP cells (counted 65% nuclei with AR). This localization was inhibited by AZD0530, a selective SFK inhibitor demonstrating significant effects on prostate cancer cells (27). Almost half of GRP cells (35%.