The Function And Mechanism Of The Transcription Factor PBX1 In The Chemoresistance Of Prostate Cancer

ObjectivesPBX1(pre-B-cell leukemia homeobox 1)is a TALE class of homeodomain transcriptional factor.It was initially identified as a proto-oncogene in human pre-B acute lymphocytic leukemia(pre-B ALL).Although it has been shown to contribute to many solid tumor progression,the function and mechanism of PBX1 in prostate cancer remain unclear.The present study was aimed to find out the role of PBX1 in prostate cancer progression and chemoresistance and to find its regulators.This study may provide a theoretical basis for the target treatment of prostate cancer.Methods1.Prostate cancer cells DU145 and PC3 were harvested to detect the protein level of PBX1.Cells were treated with anti-cancer drugs(Doxorubicin and Cisplatin),and MTT assay was applied to measure cell viability.2.Over-expression of PBX1 in DU145 cells was transfected with PBX1 plasmids.Knockdown of PBX1 in PC3 cells was performed using si PBX1(PBX1 interfering RNA).3.Cell viability was measured with the MTT assay;Cell apoptosis was evaluated by WB to measure PARP cleavage.4.PC3 and DU145 cells were transfected with PBX1 plasmids,and the cell cycle related proteins Cyclin D2 and Phospho-Rb were detected by WB.5.To determine the degradation pathway of PBX1,cells were transfected with PBX1 plasmids,and then cells were subjected to proteasome or lysosome inhibitors.6.Co-IP experiment was applied to identify the interaction between PBX1 and USP9 X,and examine whether USP9 X could mediate the deubiquitination of PBX1.7.After over-expressing PBX1 and USP9 X in HEK293 T cells,the Western Blot was applied to measure the time-and dose-effect of USP9 X on PBX1.CHX chase experiment was subjected to analyze the half-life of PBX1 in the presence and absence of USP9 X or not.8.PBX1 is the transcriptional factor of RNF6.To detect whether USP9 X affects the transcriptional activity of PBX1,RNF6-Luci plasmids,USP9 X and PBX1 were co-transfected into HEK293 T cells.Luciferase activity was detected using Bright-Glo substrate system.9.WP1130 was used to treat PC3 or HEK293 T cells which were transfected with USP9 X and PBX1 plasmids.The protein level of PBX1 and its target genes were detected.At the same time,the PARP protein level in PC3 cells was measured by Western Blot.Results1.PBX1 was expressed in PC3 but not in DU145 cells.PC3 but not DU145 cells were resistant to the treatment of anti-cancer drugs.2.Over-expressing PBX1 in DU145 promoted its proliferation,and the proliferation of PC3 cells was inhibited when PBX1 was knocked down.3.DU145 became resistant to anti-prostate cancer drugs when PBX1 was over-expressed.The drug sensitivity of PC3 was enhanced when PBX1 was knocked down.4.Cyclin D2 and Phospho-Rb were up-regulated in a concentration dependent manner when PBX1 was over-expressed in PC3 and DU145 cells.Knocking down PBX1 in PC3 cells resulted in reducing indicated proteins and cell cycle arrest.5.Through comparing the effect of lysosome and proteasome inhibitors on PBX1 protein level,we found that the protein level of PBX1 was effectively increased by MG132.It confirmed that PBX1 was degraded through the ubiquitin proteasome pathway.6.Co-IP assay indicated that USP9 X could interact with PBX1 and US9 X could remove the poly-ubiquitin chain of PBX1.These results suggested that USP9 X was the potential deubiquitinase of PBX1.7.USP9 X increased the PBX1 protein level in a time-and dose-dependent manner.CHX chase showed that USP9 X could prolong the half-life of PBX1.8.USP9 X markedly increased the luciferase activity driven by PBX1 recognition element.It indicated that USP9 X could promote the transcriptional activity of PBX1.WP1130,the small molecular inhibitor of USP9 X,decreased the protein level and transcriptional activity of PBX1.9.The experiment both in vitro and in vivo found that WP1130 inhibited the function of USP9 X.Thus the protein level of PBX1 and down-stream genes were decreased and prostate cancer cell was induced to apoptosis.ConclusionsIn the present study,we found that over-expression of PBX1 promotes the proliferation and drug resistance of prostate cancer cells by modulating cell cycle through regulating cell cycle related proteins.USP9 X may be the potential deubiquitinase of PBX1,and it stabilizes the protein level of PBX1 via deubiquitination.The small molecule inhibitor of USP9 X down-regulates PBX1 protein level and inhibits PBX1 transcriptional activity.Thus,targeting at the USP9X/PBX1 could induce apoptosis of prostate cancer cells.