| Objective:PLCε(Phospholipase C epsilon)is an oncogene with high expression in hormone-sensitive prostate cancer.The Hedgehog/Gli signaling pathway is over-activated in prostate cancer(PCa).Previous studies suggest that both PLCεand Hedgehog signaling pathways are involved in androgen receptor(AR)regulation in PCa.However,the role of PLCεand Hedgehog/Gli in castration-resistant prostate cancer(CRPC)remains unclear.The purpose of this study was to investigate the interaction and mechanism of PLCε,Hedgehog/Gli and AR in CRPC,and whether knocking down PLCεin CRPC cells affects its drug sensitivity to Enzluramide by regulating AR activity.Methods:1)A total of 30 benign prostatic hyperplasia(BPH)tissue samples,64PCa tissue samples and 27 CRPC tissue samples were obtained from the Department of Urology of the First Affiliated Hospital of Chongqing Medical University between April 2010 and September 2015.The expressions of PLCε,Gli-1 and Gli-2 protein was investigated by using Immunohistochemistry(IHC).The correlation between PLCεand Gli-1,Gli-2 expression in BPH,PCa and CRPC tissues was analyzed by using Pearson’s correlation.And the correlation between PLCεexpression and clinical pathological data in CRPC tissues was analyzed.The overall survival(OS)and progression-free survival(PFS)of CRPC patients were further analyzed.2)The expression of PLCεprotein and mRNA level was detected in LNCaP,22RV1 and EN-R(22RV1 and EN-R represent CRPC cells)cell lines by Western blot and RT-qPCR.The effect of PLCεon cell proliferation and invasion was assessed in CRPC cell lines by using CCK-8,Clone formation and Transwell assay,and the sensitivity of EN-R and 22RV1cells to enzalutamide following the downregulation of PLCεexpression was determined using lentivirus-mediated shPLCε.3)The expression of PLCε,Hedgehog signaling pathway-related molecules,androgen receptor(AR)proteins and mRNA levels were detected by Western blot and RT-qPCR in 22RV1 and EN-R cell lines followingthedownregulationofPLCεexpressionbyusing lentivirus-mediated shPLCεand/or treated with Hedgehog signaling pathway inhibitor GANT61.4)En-R cells were treated by knocking down PLCεand/or overexpressing Gli-1/Gli-2.The expression of AR protein in nucleus and plasma was detected by immunofluorescence assay,and the expression levels of PLCε,Gli-1,Gli-2,AR,STAT3 and phosphorylated STAT3 in En-R cell lines were detected by Western blot and RT-q PCR respectively.5)Four-week-old nude mice were subcutaneously inoculated with2x10~6 EN-R(cont group)cells or LV-shPLCε-EN-R(LV-shPLCεgroup)cells.Then the mice in each group were divided into two groups and fed with 10mg/kg PBS or enzalutamide respectively.The mice were euthanized after 32 days,the volume of the tumors were calculated and the growth curve of the tumors was plotted.Results:1)A high expression of PLCεwas identified in most PCa and CRPC tissue samples.By contrast,none of the BPH tissues stained positive for PLCε,and the expression of PLCεin the CRPC tissues was significantly higher than that in the PCa tissues(P=0.0447).It was also observed that the expression of Gli-1 and Gli-2 in the tumor(PCa and CRPC)tissues was significantly higher than that in the benign(BPH)tissues,the expression of Gli-1,but not Gli-2,in the CRPC tissues was significantly upregulated,as compared to that in the PCa tissues(P=0.152).Pearson’s linear correlation results showed that PLCεexpression was positively correlated with Gli-1(r=0.581,P=0.01)and Gli-2 expression(r=0.409,P=0.034).Kaplan-Meier survival analysis results showed that the median progression-free survival(PFS)was 24 months in the PLCε-positive CRPC patients,while the median PFS was 28 months in the PLCε-negative patients,PLCεpositivity in the CRPC tissues was associated with a shorter PFS in the CRPC patients(P=0.025).Similarly,the overall survival(OS)of PCa patients with a negative PLCεexpression was significantly longer than that of PCa patients with a positive PLCεexpression(P=0.027).2)PLCεwas highly expressed in LNCaP,22RV1 and EN-R cells.Compared with LNCaP cells,the expression of PLCεin 22RV1 and EN-R cells was significantly higher.Gli-1/Gli-2 was highly expressed in 22RV1and EN-R cells,but slightly expressed in the LNCaP cells.CCK-8 and colony formation assays showed that PLCεknockdown and/or GANT61treatment suppressed the proliferation of the 22RV1 and EN-R cells.Transwell assay showed that PLCεknockdown and/or GANT61 treatment inhibited the invasion of the 22RV1 and EN-R cells.CCK-8 assay also showed that PLCεknockdown and/or Gli-1/Gli-2 inhibition could sensitize CRPC cells to enzalutamide in vitro.3)PLCεknockdown inhibited the mRNA and protein expression levels of Gli-1/Gli-2 and AR,while their inhibition did not affect the expression of PLCεin 22RV1 and EN-R cells,suggesting that PLCεis the upstream regulator of Gli-1/Gli-2.However,following PLCεknockdown,Smo(upstream regulatory gene of Gli in the classical Hh signaling pathway)showed no significant mRNA and protein expression change.These data indicated that PLCεknockdown inhibited the expression of Gli-1/Gli-2through the non-canonical Hh signaling pathway.4)Treatment of EN-R cells by knocking down PLCεand/or overexpressing Gli-1/Gli-2.Immunofluorescence assay showed that PLCεknockdown suppressed AR nuclear translocation via the Gli-2/AR signaling pathway.Western blot and RT-qPCR data showed that knockdown of PLCεinhibited the AR target gene PSA expression via the p-STAT3 signaling pathway.5)Animal xenograft model data suggested that PLCεknockdown inhibited the tumor growth of the EN-R cell xenografts and contributed to the sensitization of CRPC cells to enzalutamide in vivo.Conclusion:1.High expression of PLCεcontributes to unfavorable disease phenotype and poor outcomes in patients with PCa.2.PLCεknockdown suppressed cell proliferation and invasion in CRPC cells.3.PLCεsensitizes castration-resistant prostate cancer cells to enzalutamide by suppressing the androgen receptor through the non-canonical Hh/Gli signaling pathway and the p-STAT3 signaling pathway. |
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