C-MYC Promotes Prostate Cancer Progression Via Activation Of SOX4

Prostate cancer(PCa)is the most prevalent malignant tumor of the male reproductive system.In recent years,the incidence of PCa has steadily increased.PSA screening test has increased the diagnostic detection rate of PCa greatly,but also led to overdiagnosis and overtreatment of indolent PCa.PCa of advanced stage may metastasize to bone and develop into castration resistance prostate cancer(CRPC),which has a very poor prognosis.Multiple genes and mechanisms participate in the tumorigenesis and development of PCa,among which,castration resistance and epithelial-mesenchymal transition(EMT)are two important mechanisms,and C-MYC and SOX4 play vital roles.Proto-oncogene C-MYC is a transcription factor.C-MYC dysregulation plays a key role in the genesis and development of many malignant tumors,including PCa.In PCa,C-MYC may amplify and overexpress.C-MYC amplification can occur in 29%of PCa and lead to a poor prognosis.C-MYC overexpression is prevalent in PCa.However,its correlation with clinicopathological parameters and survival rate of PCa is still controversial.SOX4 is a member of SOX(SRY-related HMG-box)family.As a transcription factor closely related with development,SOX4 is involved in embryonic development,regulation of cell differentiation and stem cells.In the previous studies,we have reported that SOX4 could not only promote proliferation,migration and invasion of PCa cells,but also initiate a transcriptional program that enables the epithelial-mesenchymal transition(EMT)phenotype.Furthermore,SOX4 might serve as a prognostic marker for Chinese PCa patients.The objective of this research is to study the potential mutual regulations between C-MYC and SOX4 in the malignant progession of PCa via clinical investigations,in vitro experiments and bioinformatics analyses.We show that C-MYC/SOX4 axis may play a key role in the development of PCa,which provides a novel idea for the study of PCa.Methods1.Detection of the protein expression and gene amplification in PCa1.1 Public PCa databases were used to analyze the expression characteristics and correlation of C-MYC and SOX4.1.2 A total of 126 PCa cases(Qilu cohort,105 localized PCa and 21 CRPC)diagnosed between 2007 and 2014 were collected and tissue microarrays were constructed.1.3 Immunohistochemical staining was performed to detect the expression of C-MYC,SOX4 and(?)67.1.4 Fluorescence in situ hybridization(FISH)was carried out to detect the abnormalities of C-MYC gene copy.1.5 SPSS 19.0 was used to analyze the clinicopathological significance of C-MYC overexpression,the correlation between C-MYC overexpression and C-MYC amplification/SOX4 overexpression and cancer-related death.2.Activation of SOX4 by C-MYC2.1 Quantitive Real-time PCR(qRT-PCR)and western blot were used to detect the expression of C-MYC and SOX4 in PCa cell lines.2.2 siRNA knockdown was perform to study the effect of SOX4 on the expresssion of C-MYC.2.3 siRNA knockdown and overexpression experiments were carried out to study the effect of C-MYC on the expression of SOX4.2.4 Bioinformatics analysis was performed to predict the binding sites of C-MYC on the promoter region of SOX4.2.5 Chromatin immunoprecipitation assay(ChIP)was performed to detect the binding of C-MYC to the promoter region of SOX4.2.6 Luciferase reporter assay was used to confirm the direct transcriptional regulation of C-MYC to SOX4.3.Interaction of C-MYC and SOX43.1 The online tool Cistrome was used to obtain the putative overlapping target genes of both C-MYC and SOX4.3.2 Bioinformatics analysis was used to reveal the ChIP-seq read intensity of C-MYC and SOX4 at the TSS region of target genes.3.3 Co-imnunoprecipitation(CoIP),siRNA,overexpression and western blot were performed to study the interaction of C-MYC and SOX4 in PCa cell lines.3.4 siRNA,MTT,colony formation assay,wound healing and transwell were used to detect the effects of both C-MYC and SOX4 on the proliferation,migration and invasion functions of PCa cells.3.5 siRNA,overexpression and western blot were performed to detect the effects of both C-MYC and SOX4 on the immunophenotype of EMI of PCa cell lines.4.C-MYC+/SOX4+defines a subset of PCa with a poor prognosis4.1 Bioinformatics analysis was used to analyze the expression of C-MYC and SOX4 in CRPCs from public PCa databases.4.2 Gene set enrichment analysis(GSEA)was used to analyze the differentially expressed genes(DEGs)in both C-MYC+/SOX4+PCa and CRPC.4.3 SPSS 19.0 was used to analyze the correlation of C-MYC+/SOX4+and Ki67 in Qilu cohort.4.4 SPSS 19.0 was used to analyze the effect of C-MYC and SOX4 expression levels on the survival rate of PCa patients in public databases and Qilu cohort.Results1.Clinicopathological significance of C-MYC overexpression in PCa1.1 In Qilu cohort,C-MYC was overexpressed in 23%(29/126)of cases.16.2%(17/105)of localized PCas and 57.1%(12/21)of CRPCs demonstrated C-MYC overexpression.The proportion of cases with C-MYC overexpression was significantly higher in CRPC than that in localized PCa(P<0.001).1.2 C-MYC amplification was identified in 16.8%(16/95)of the cases in Qilu cohort,significantly associated with C-MYC overexpression.According to the TCGA dataset,PCa cases with C-MYC gain or amplification expressed higher C-MYC mRNA level(P<0.05).1.3 C-MYC overexpression was significantly associated with high Gleason scores(P=0.012),high Ki67 index(P=0.002)and clinical tumor stage(P=0.056).1.4 C-MYC overexpression was significantly linked to cancer-related death in our cohort(P=0.018,Kaplan-Meier survival analysis)and night serve as a prognostic marker in Chinese PCa.2.SOX4 is a direct target of C-MYC in PCa2.1 In PCa databases(TCGA,GSE35988,GSE33316,GSE120006,GSE70769,MSKCC),SOX4 co-expression signature was significantly enriched for C-MYC expression.Patients with concurrent C-MYC and SOX4 expression(C-MYC+/SOX4+)significantly increased in highly aggressive PCas(P<0.05).We also found a significant correlation between C-MYC and SOX4 expression levels(P<0.001).2.2 C-MYC overexpression was significantly correlated with SOX4 overexpression in Qilu cohort(P=0.022).2.3 Both C-MYC and SOX4 expressed in LNCaP and VCaP.2.4 After siRNA knockdown of SOX4,both mRNA and protein expressions of C-MYC increased in LNCaP,but decreased in VCaP,which was inconsistent in the two cell lines.2.5 After siRNA knockdown of C-MYC,both mRNA and protein expressions of SOX4 decreased in LNCaP and VCaP.Furthermore,overexpression of C-MYC increased the expression of SOX4.Therefore,the subsequent study accorded to the hypothesis of transcriptionally regulation of C-MYC to SOX4.2.6 A publicly available C-MYC ChIP-seq dataset(GSM1907205)was used to identify the binding of C-MYC at the SOX4 promoter region.An on online tool was used to predict the binding sites and five regions were selected as potential binding sites.2.7 Five putative binding sites were selected to perform ChIP assay,which revealing that the antibody against C-MYC efficiently immunoprecipitated-1420bp?-1226bp upstream from TSS of SOX4 gene in PCa cells.2.8 Luciferase reporter assay confirmed the direct transcriptional regulation of C-MYC to SOX4.3.C-MYC and SOX4 cooperate to promote the progession of PCa3.1 We used Citrome to obtain the putative target genes of both C-MYC and SOX4.Bioinformatics analysis revealed that both C-MYC and SOX4 had the strongest ChIP-seq read intensity at the TSS region.3.2 Co-IP analysis demonstrated C-MYC interacted with SOX4 in PCa cells.siRNA knockdown of SOX4 suggested that SOX4 expression was essential for C-MYC binding to SOX4 at the protein levels.3.3 Functional experiments of MTT,colony formation assay,wound healing and transwell revealed that siRNA knockdown of C-MYC significantly inhibited the proliferation,migration and invasive capacity of VCaP cells.In contrast,SOX4 re-expression was able to rescue these phenotypes significantly after C-MYC knockdown.3.4 C-MYC inhibition significantly increased the expression of the epithelial marker(E-cadherin)but decreased the mesenchymal marker(Vimentin)levels,and SOX4 overexpression partially reversed these changes.4.C-MYC+/SOX4+characterizes a subset of highly aggressive PCa with a poor prognosis4.1 According to analyses of public databases(GSE35988,GSE70769,GSE74367),patients with concurrent C-MYC and SOX4 expression(C-MYC+/SOX4+)significantly increased in CRPC patients.4.2 In public databases(TCGA,GSE35988 and GSE70769),differentially expressed genes(DEGs)in C-MYC+/SOX4+ patients were congruent with CRPC subgroup,and enriched for statistically significant overlapping gene signature involved in cell cycle.4.3 In PCa of Qilu cohort,C-MYC+/SOX4+was correlated with Ki67 significantly(P<0.001).4.4 C-MYC+/SOX4+patients in both Qilu cohort and public databases had worse overall survival rates.Conclusions1.C-MYC may promote PCa progession by activating SOX4 directly.2.C-MYC and SOX4 may cooperate to promote the development of PCa.3.C-MYC/SOX4 axis may promote PCa aggression by regulating EMT.4.C-MYC/SOX4 axis may promote PCa progression by regulationg cell cycle.5.C-MYC+/SOX4+ may define a highly aggressive subset of PCa with poor prognoses.