Promotive Effect Of PLC? On Androgen Receptor Antagonist Resistance Via The BMP-6/SMAD Axis In Castration-resistant Prostate Cancer

ObjectivePrimary therapy for patients with advanced prostate cancer?PCa?consists of androgen deprivation therapy targeting the androgen receptor?AR?axis.However,most tumors progress to castration-resistant prostate cancer?CRPC?within 18-24 months.The purpose of the present study was to investigate the mechanisms through which PCa acquires drug resistance after long-term treatment with AR antagonists.Phospholipase C??PLC??,a member of the phospholipase C family,had been shown to be upregulated in primary prostate cancer?PPC?.The BMP-6 signaling pathway was significantly over-activated in metastatic prostate cancer.This study was to investigate the changes of PLC?and BMP-6 signaling pathways when PCa cells resistance to AR antagonists,the regulation pathway of PLC?on BMP-6,and its regulation mechanism on drug resistance,proliferation and invasion.MethodsOnline database analysis and Bioinformatics analysis were performed to identify signaling activated during anti-androgen treatment.MTT assay was performed to detect cell viability.RT-qPCR was performed to examine the mRNA expression of the indicated genes.Colony formation assay was used to observe cell proliferation.Transwell assay was conducted to demonstrate invasive ability.Protein levels were determined by western blot analysis and immunofluorescence assays.An online database search and Bioinformatics analysis indicated that bone morphogenetic protein?BMP?-6/SMAD signaling was activated in enzalutamide-resistant LNCaP cells.Furthermore,this signaling interaction was experimentally verified in bicalutamide-and enzalutamide-resistant LNCaP cells,which may be regulated by phospholipase C?PLC??and induced cell proliferation and invasion.Of note,a positive correlation was observed between PLC?and BMP-6 in CRPC tissue samples,which may promote bone metastasis and suggested a poor prognosis.The differentially expressed genes?DEGs?between the prostate cancer cell line LNCaP and the enzalutamide-resistant LNCaP cell line?LNCaP-Bica^R?was screened using online tool GEO2R by analyzing the GSE78201 dataset in the Gene Expression Omnibus?GEO?database.Gene Ontology?GO?enrichment analysis and signaling pathway analysis were then performed using WebGestalt to identify signaling activated during anti-androgen treatment.LNCaP cells were continuously cultured with 10?M bicalutamide or enzalutamide.After 6 months of culture,cells resistant to bicalutamide or enzalutamide?referred to as LNCaP-Bica^R and LNCaP-Enza^R cells,respectively?were obtained.An MTT assay was performed to determine the half maximal inhibitory concentration(IC₅₀)of LNCaP,LNCaP-Bica^R,and LNCaP-Enza^R.The RNA and protein of the above three cells were extracted,PLC?,BMP-6 and ID2 mRNA was detected by Q-PCR,and PLC?,BMP-6,p-SMAD1/5/9,SMAD1/5/9 and ID2 proteins were assessed using western blot analysis.Lentivirus?LV?expressing small hairpin?sh?RNA targeting human PLC??LV-shPLC??and negative control?LV-NC?were transfected into LNCaP-BicaR and LNCaP-EnzaR cells.Q-PCR and WB were performed to detect the expression of PLC?and BMP-6.10 nM phorbol 12-myristate13-acetate?PMA?was added to the drug-resistant cells with PLC?knockdown.And the expression of BMP-6 was tested by Q-PCR and WB.Two drug-resistant cell lines were treated with LV-shPLC?/LV-NC and/or 10 ng/ml human recombinant?rBMP-6?.The cell viability and sensitivity of drug-resistant cells to corresponding drugs were evaluated by using CCK-8 assay.The protein levels of p-SMAD1/5/9,SMAD1/5/9,ID2,AR and EMT-associated molecules E-cadherin,N-cadherin and MMP9were detected by using Western blot assay.Colony formation assay was performed to observe cell proliferation.Transwell assay was conducted to demonstrate invasive ability.We collected 48 primary prostate cancer?PPC?tissures and 33 CRPC tissues.The expressions of PLC?protain and BMP-6 protein were detected by using immunohistochemistry?IHC?.The correlation between PLC?and BMP-6 expression in CRPC tissues was analyzed by Spearman's correlation analysis.The correlation between these two proteins expression and time to progression was retrospectively analyzed.The correlation between protein expression?PLC?and BMP-6?and clinical data in CRPC and PCC tissues was analyzed.ResultsThe dataset GSE78201 was retrieved from the GEO database and analyzed.By using GEO2R,a total of 206 DEGs were identified between LNCaP and LNCaP-Enza^R cells.Pathway enrichment analysis was performed to identify pathways activated during anti-androgen treatment.And BMP-6 signaling was identified as the focus of subsequent in vitro research.The IC₅₀0 of LNCaP,LNCaP-Bica^R and LNCaP-Enza^R was detected by MTT assay,suggesting that drug-resistant cells?LNCaP-Bica^R and LNCaP-Enza^R?were successfully generated.Q-PCR and western blot indicated that compared with the parental LNCaP cells,the expression of PLC?was upregulated and the BMP-6/SMAD signaling pathway was activated in LNCaP-Bica^R and LNCaP-Enza^R.Compared with those in the negative controls?Blank and LV-NC?,RT-qPCR and western blot analysis indicated that the mRNA and protein levels of PLC?and BMP-6 were downregulated while PLC?knockdown?LV-shPLC??in LNCaP-Bica^R and LNCaP-Enza^R cells.To further elucidate the mechanisms through which PLC?mediates BMP-6 expression,PKC activator?PMA?we added to the PLC?knockdown group.The result indicated that PMA treatment increased BMP-6 at the mRNA and protein level,suggesting that PLC?may increase BMP-6 expression in a PKC-dependent manner.An MTT assay indicated that the viability of cells was markedly inhibited in a time-dependent manner after transfection with LV-shPLC?.The protein levels of p-SMAD1/5/9,ID2 and AR were significantly downregulated following knockdown of PLC?.PLC?knockdown also resulted in an increased protein expression of E-cadherin and had a suppressive effect on N-cadherin and MMP9 expression.Consistently with this,a Transwell assay demonstrated that the invasive ability was significantly decreased by PLC?knockdown.Furthermore,a colony formation assay suggested that PLC?inhibited the proliferation of cells.At the same time,rBMP-6 replenishment in the LV-shPLC?group partly recovered the suppressive effect produced by PLC?knockdown.Taken together,these results suggest that PLC?knockdown partially restored the sensitivity of in CRPC cells to AR antagonist and simultaneously inhibited cell proliferation and invasion via BMP-6/SMAD signaling.The results of IHC demonstrated that PLC?and BMP-6 were upregulated in CRPC compared with PCC tissue samples?P=0.0221?.Furthermore,Spearman's correlation analysis revealed that PLC?expression was positively associated with BMP-6 expression?r=0.5063,P<0.01?.Of note,Kaplan-Meier survival analysis demonstrated that PLC?-positive patients had a shorter TTP than PLC?-negative patients?19months vs.29 months;P=0.012?.A similar result was obtained for the comparison between BMP-6-positive and-negative patients?19 months vs.27 months;P=0.022?.Regarding the clinical characteristics,PLC?and BMP-6 expression were associated with bone metastasis?P<0.05?.In summary,the expression of PLC?and BMP-6 predicted a poor prognosis for patients with PCa.ConclusionBMP-6/SMAD pathway was activated by PLC?in drug-resistant cells,which could induced cell proliferation,invasion and drug resistance,and PLC?knockdown increases the sensitivity of drug-resistant cells to AR antagonist.The present results suggest that targeting of PLC?/BMP-6/SMAD signaling may increase the sensitivity of CRPC to AR antagonists and inhibit tumor progression.