The Role And Mechanism Of SOX2 In The Progression Of Neuroendocrine Prostate Cancer

BackgroudProstate cancer is one of the common malignant tumors in male urinary system and is the sixth leading cause of cancer-related deaths in males worldwide.In recent years,the incidence of prostate cancer in China has increased significantly.The onset of prostate cancer is hidden,asymptomatic early,and patients with advanced metastatic prostate cancer account for a larger proportion.Progression from primary prostate cancer to advanced metastatic disease is heavily dependent on the androgen receptor,therefore,androgen deprivation therapy(ADT)is the main treatment for advanced metastatic prostate cancer.ADT are used to deplete circulating androgens to abrogate AR signalling for prostate cancer cell survival and prevent disease progression,most patients are effective in the early stage,however,almost all patients will eventually progress to castration resistant prostate cancer(CRPC)after undergoing ADT treatment with median time of 18-36 months.Despite low levels of serum androgens in men with CRPC,reactivation of the AR occurs;thus it remains central to tumor cell proliferation and metastatic spread.Thus,targeting the AR is a cornerstone therapeutic for CRPC patients.Enzalutamide(ENZ)is an effective AR pathway inhibitors which becomes the mainstay in the CRPC treatment,however,the treatment benefits of ENZ are short lived in CRPC patients and resistance rapidly occurs.Neuroendocrine prostate cancer(NEPC)is a subtype of CRPC with extremely poor prognosis.Histologically,NEPC is characterized by the presence of small round cells with a prominent nucleus.NEPC cells stain positive for neuroendocrine markers such as chromogranin A(CHGA)and synaptophysin(SYP)but negative for PCa markers like androgen receptor(AR)and prostate-specific antigen(PSA).Primary NEPC is not common.It is mostly derived from prostate adenocarcinoma(AdPC)through neuroendocrine differentiation(NED)under ADT selection pressure.The median survival time of patients is less than one year.Currently,there are limited clinical treatment programs and no specific drugs.Exploring the drivers of NED formation is of great significance for revealing the pathogenesis of NEPC,finding accurate and sensitive early diagnostic indicators and clinical therapeutic targets.The transition from AdPC to NEPC is molecularly characterized by the acquisition of NE phenotypes and a decrease/loss in the AD luminal phenotype.At present,the molecular mechanism of NED is still in the exploratory stage.As molecularly targeted cancer therapy improves,lineage plasticity is increasingly appreciated as a potential mechanism under-lying therapeutic resistance.Under the pressure of castration,prostate adenocarcinoma(AdPC)can be re lineated into neuroendocrine like cells independent of drug target growth through cell plasticity,which is gradually considered as a potential pathogenesis of NEPC by researchers.SOX2 is a SOX family member that belongs to the SOXB1 group and is an important transcriptional regulator in pluripotent stem cells,which plays an important role in maintaining the pluripotency of pluripotent stem and neural differentiation.Previous studies have shown that SOX2 levels are significantly higher in CRPC tumors with neuroendocrine like histology than in adenocarcinomas.Besides,SOX2 and the NED marker chromogranin-A are primarily coexpressed in both prostate cancer and lymph node metastases.Recent study shows that SOX2 promotes lineage plasticity and antiandrogen resistance in TP53 and RBI deficient prostate cancer.So,will SOX2 directly mediate the formation of NEPC or what role will it play in the conversion process from AdPC to NEPC?This study will focus on this issue.ObjectiveBased on the analysis of public database sequencing data,this study aims to explore the potential drivers of malignant progression of prostate cancer,and further explore the role of SOX2 in the progression of NEPC and related molecular mechanisms,providing theoretical basis for clinical search for new early diagnosis indicators and therapeutic targets of NEPC.Part 1 The role of SOX2 in the progression of prostate cancerMethods1.Download the RNA-Seq data of 34 CRPC clinical samples and 15 NEPC clinical samples in the TCGA database and a series of PDXs model RNA-Seq data in the literature to simulate the dynamic progress of NEPC,analyze the gene expression changes and the transcriptional regulation of differentially up-regulated and down-regulated gene sets using CHEA2016 dataset in the Enrichr gene enrichment analysis tool.2.Taking prostate cancer androgen-sensitive cell lines LNCaP and CWR22RV1 as research objects,antibiotic-labeled lentivirus was used to transfect cells to establish prostate cancer androgen-sensitive cell lines LNCaP-SOX2 and C WR22RV1-SOX2 with overexpression of SOX2,respectively.The overexpression efficiency was detected by real-time quantitative PCR(RT-qPCR)and Western blot.3.RT-qPCR and Western blot were used to detect the effect of SOX2 on AdPC lineage specific gene and protein expression in LNCaP cells and CWR22RV1 cells under androgen deprivation and non-androgen deprivation conditions.RNA interference technology was used to restore SOX2 gene expression in LNCaP-SOX2 cells.Western blot was used to detect the effect on AdPC lineage specific protein expression4.RT-qPCR and Western blot were used to detect the effect of SOX2 on the expression of NE marker gene and protein in LNCaP cells and CWR22RV1 cells.RNA interference technology was used to inhibit REST expression in LNCaP-SOX2 cells.RT-qPCR and Western blot were used to further detect the effect on the expression of NE marker gene and protein.5.Edu labeling method was used to detect the effect of SOX2 on proliferation of LNCaP cells.Results1.The expression level of SOX2 gene in NEPC samples was significantly higher than that in CRPC samples.With the prolongation of castration in the AdPC PDXs model,the expressions of AR,NKX3-1 and KLK3 decreased or even disappeared,while the expression of SOX2 began to express after 12 weeks and persisted to NEPC formation.Transcriptional regulation analysis showa that SOX2 was highly correlated with the transcriptional regulation of differentially expressed sets of down-regulated genes in NEPC samples.2.SOX2 gene mRNA and protein expression levels were significantly higher in LNCaP cells and CWR22RV1 cells transfected with SOX2 gene-encapsulated cells than in the negative control group,and the cells grew well under antibiotic screening conditions.3.Overexpression of SOX2 can significantly inhibit the mRNA expression of AR,NKX3-1,PSA and ADAM7 genes and the expression of NKX3-1 and PSA proteins in LNCaP cells.shSOX2 can promote the expression of AR,NKX3-1,PSA protein after interfering with the expression of SOX2 in LNCaP-SOX2 cells.Meanwhile,overexpression of SOX2 in CWR22RV1 cells can significantly inhibit the expression of AR and FOXA1 proteins.4.Overexpression of SOX2 alone in LNCaP cells can only promote the expression of EN02 and SYP gene mRNA and the expression of ASCL1 protein,while combined REST deletion can significantly promote the expression of SCG3 gene mRNA and SYP protein in LNCaP cells.Overexpression of SOX2 in CWR22RV1 cells can also promote the expression of some NE markers(e.g.SYP and NSE),but overexpression of SOX2 cannot improve the proliferation ability of LNCaP cells.Part 2 Molecular mechanism of SOX2 inhibiting AdPC lineage-specific genesMethods1.Transcriptome sequencing was carried out on LNCaP-Lev cells and LNCaP-SOX2 cells respectively.DEseq was used to obtain the differentially expressed genes related to SOX2 and the accuracy of sequencing data was verified by RT-qPCR.In addition,Meatascape was used for functional annotation of differentially expressed genes,and the CHEA2016 data set in Enrichr gene enrichment analysis tool was used for transcriptional regulation analysis of differentially expressed gene sets.2.Chromatin immunoprecipitation assay(ChIP)was used to obtain the DNA target sequence directly bound to SOX2 and perform sequencing analysis.Peaks were extracted by the MACS software,and the distribution of peaks in the whole genome was annotated by the ChIPseeker software package.3.Western blot was used to detect the effect of SOX2 on histone methylation level in LNCaP cells and CWR22RV1 cells.4.Western blot was used to detect the effect of SOX2 on the expression of LSD1 total protein in LNCaP cells and CWR22RV1 cells,immunofluorescence technique,IF)was used to detect the expression of LSD 1 nucleoprotein,and colorimetry was used to detect the effect of SOX2 on the activity of LSD1 demethylase.5.3mM pargyline was applied to SOX2+ and SOX-prostate cancer cells,respectively.Colorimetric method was used to detect the effect on LSD1 enzyme activity,and Western blot was used to detect the effect of LSD 1 inhibitor on AdPC lineage specific protein expression.Results1.A total of 1295 differentially expressed annotation genes(difference multiple>1.5 and FDR?0.05)were obtained from the expression profile analysis of LNCaP-SOX2 cells and LNCaP-Lev cells,including 889 up-regulated genes and 406 down-regulated genes(LNCaP-SOX2 vs LNCaP-Lev).Cluster analysis of the selected differentially expressed genes showed that overexpression SOX2 in LNCaP cells could significantly inhibit the expression of AR,NKX3-1,KLK3,FKBP5,TMEFF2 and ADAM7 genes,and could also promote the expression of pluripotent genes CD24 and KLF4 and some NE marker(e.g.,EN02 and CENPE).Transcriptional regulation analysis of the 406 differentially genes revealed that AR and FOXA1 are both transcription factors with strong correlation with the transcriptional regulation of this down-regulated gene set.The results of gene function annotation show that the functions related to AR signal pathway and epigenetic regulation were significantly enriched.2.A total of 2115 significantly enriched SOX2 protein-DNA interaction sites were identified by ChIP-seq(P<0.001).Annotation of the genome wide distribution of peaks detected in samples revealed that there was no significant enrichment of peaks in the transcriptional regulatory regions of AdPC lineage specific genes of interest,and the vast majority of Peaks fell in the intergenic regions.3.Western blot results showed that overexpression of SOX2 in LNCaP cells and CWR22RV1 cells could significantly reduce the levels of monomethylation and demethylation of H3K4 and H3K9.4.Overexpression of SOX2 could promote the expression of LSD1 protein in LNCaP cells and CWR22RV1 cells.IF results showed that SOX2 can promote the expression of LSD 1 in LNCaP cells.In addition,overexpression of SOX2 could also promote the activity of LSD1 demethylase in LNCaP cells and CWR22RV1 cells.5.3mM pargyline could significantly inhibit LSD1 demethylase activity in LNCaP cells and CWR22RV1 cells,but it cannot reverse the inhibitory effect of SOX2 on AdPC lineage-specific genes,but instead enhanced the inhibitory effect.6.Pargyline can inhibit the demethylase activity of LSD1 in LNCaP cells and CWR22RV1 cells,and increase the expression of H3K4 and H3K9 methylated proteinsConclusions1.Analysis of sequencing data from public databases indicates that SOX2 may be a potential driver of NEPC progress.we have successfully constructed SOX2 overexpressed cell models LNCaP-SOX2 cells and CWR22RV1-SOX2 cells in vitro,and further proved that SOX2 can inhibit the expression of AdPC lineage specific genes and weakly promote the formation of NE phenotype.2.Mechanistically,the inhibitory effect of SOX2 on AdPC lineage-specific genes is not through direct binding but through global hypomethylation mediated by LSD1.LSD1 is expected to be a new target for clinical treatment of NEPC patients in the future.