The possible reason could be that USP9x has diverse oncogenic substrate proteins and these proteins also promote PCa cell survival and proliferation. activity, we screened for PBX1-specific deubiquitinases (Dubs) and found that ubiquitin-specific peptidase 9 X-linked (USP9x) interacted with and stabilized the PBX1 protein by attenuating its Lys-48Clinked polyubiquitination. Moreover, the USP9x inhibitor WP1130 markedly induced PBX1 degradation and promoted PCa cell apoptosis. The results in this study indicate that PBX1 confers EPZ-6438 (Tazemetostat) to PCa chemoresistance and identify USP9x as a Dub of PBX1. We concluded that targeting the USP9x/PBX1 axis could be a potential therapeutic strategy for managing advanced prostate cancer. representative immunohistochemical analyses of PBX1 in BPH, PIN, and PCa tissues. the rate of PBX1 expression in BPH, PIN, and PCa specimens. PC3 and DU145 cells were treated with DOX or CDDP at the indicated concentrations for 24 h, whole cell lysates were subjected to IB with specific antibodies against PARP or cleaved caspase-3. DU145 cells were treated with DOX or CDDP EPZ-6438 (Tazemetostat) with the indicated concentrations and incubation times. Whole cell lysates were applied for IB assay. PBX1-expressing PC3, PC-3M, and 22RV1 cells were treated with DOX or CDDP at increasing concentrations for 24 h before being harvested for IB assays against PBX1, PARP, and caspase-3. DU145 and PC3 cells were treated with DOX and CDDP at increasing concentrations for 24 h, followed by Annexin V-FITC and PI staining and flow cytometric assays. To determine the therapeutic implications of PBX1 in PCa, we next evaluated the significance of PBX1 in anti-cancer treatment. PC3 that expresses high PBX1 and DU145 that lacks PBX1 were treated with cisplatin (CDDP) or doxorubicin (DOX), two typical cytotoxic anti-cancer drugs that are also used for advanced PCa, followed by measurement of the cleavage of PARP and caspase-3, two hallmarks of apoptosis, by immunoblotting assay. As shown in Fig. 1and and and and an HA-PBX1a plasmid was transfected into DU145 cells Bnip3 for 48 h before cell lysates were prepared for IB assays (and DU145 cells were EPZ-6438 (Tazemetostat) transfected with Myc-PBX1b plasmids for 48 h. Cells were harvested for IB assays (and PBX1 was knocked down in PC3 cells by specific siPBX1, followed by IB assays ( 0.05; **, 0.01; ***, 0.001. Next, we wondered whether PBX1 directly contributed to PCa chemoresistance. To this end, PBX1 was overexpressed in drug-sensitive DU145 or knocked down from drug-resistant PC3 cells followed by drug treatment and analyses on cell viability and apoptosis. As shown in Fig. 3, and and and and and siRNAs of PBX1 and negative control (and siPBX1 was transfected into PC3 cells for 24 h, followed by DOX treatment for another 24 h. Cell lysates were subjected to IB assays with PARP and PBX1 antibodies (and plasmids of Myc-PBX1a and Myc-PBX1b were transfected into DU145 cells for 36 h followed by DOX treatment for another 24 h. PBX1 was measured by IB assays (and the PBX1a ( 0.05; **, 0.01; ***, 0.001. PBX1 protein stability is modulated by the ubiquitin-proteasome pathway The above results have clearly demonstrated that PBX1 is a critical factor in PCa chemoresistance, targeting at PBX1 degradation will be a potential therapeutic strategy for PCa treatment. Because most transcription factors (such as c-Maf, p53, and NF-B) are processed via the UPP pathway (13,C15), we wondered whether PBX1 stability could be modulated by UPP. To this end, we first measured PBX1 in HEK293T cells, followed by treatment with MG132, one of the typical proteasomal inhibitors, or bafilomycin A1 (BMA1), one of the typical inhibitors of lysosomes. The IB assays showed that PBX1 was accumulated by MG132 but not by BMA1 (Fig. 4HEK293T cells were transfected with HA-PBX1a plasmids for 24 h, followed by DMSO, MG132, and BMA treatment for 12 h. The cell lysates were subjected to IB assays. HEK293T cells were transfected with an HA-PBX1a plasmid for 24 h, followed by MG132 treatment for 6 h before being assayed by IB anti-HA antibody. PC3 cells were treated with BMA, chloroquine (HEK293T cells were transfected with HA-PBX1a plasmids for 24 h, followed by treatment of DOX and BZ for.
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