The Role And Mechanism Of Fork-Head Domain Mutaions In Prostate Cancer Progression

According to the 2020 Global Cancer Statistics Report released by CA: A Cancer Journal for Clinicians in 2021,prostate cancer is the most common malignant tumor in112 countries: in 2020,there are 1.4 million new cases and 375,000 deaths worldwide.Moreover,it is the second most deadly cancer in male tumors after lung cancer.In recent years,due to the aging of population,the popularity of prostate specific antigen(PSA)screening,widespread use of prostate biopsy,and the improvement of imaging examinations such as ultrasound,CT,MRI,etc.,the incidence rate of prostate cancer in a national wide has been increasing rapidly.Therefore,how to find accurate diagnose and treatment for prostate cancer has remained a major medical problem.Prostate cancer generally follows the pattern of ‘high grade prostate intraepithelial neoplasia-hormone dependent localized cancer-non-hormone-dependent metastatic cancer’ model and its outcome eventually leads to death.In this process,the activation of multiple oncogenes and the inactivation of tumor suppressor genes are involved.More researches have suggested that gene mutation and single nucleotide polymorphisms are associated with the risk of prostate cancer incidence and progression,while comprehensive molecular analyses of localized and metastatic prostate cancer have recently identified FOXA1,a fork-head family transcription factor(an oncogene in prostate cancer),as one of the most frequent mutated genes.The total mutation frequency of this gene is 35% in the United States and 41% in China,which highly suggests that FOXA1 mutations may play an important role in the occurrence and development of prostate cancer.Previous studies have suggested that FOXA1 plays an essential role in the regulation of androgen receptor(AR)transcriptional activity and the occurrence and development of Prostate cancer.As one of the epigenetic transcription factors of Prostate cancer,the main role of FOXA1 protein is to recruit prostate cancer genes such as androgen receptor to regulate transcription as a trans-acting factor,including androgen receptor pathway.However,the mechanism that how FOXA1 mutations promote localized prostate cancer,metastasic prostate cancer and CRPC development and progression remains unknown.Based on TCGA and Chinese Prostate cancer epigenemic and gene atlas(CPEGA),we conclude that the FOXA1 mutation mostly occur on fork-head domain(FKHD)followed by high risks of metastasis.Therefore,we carried out a series of experiments to explain the mechanism that how FOXA1 mutation regulates Prostate cancer progression.Methods:FOXA1 mutation sites and clinical data were downloaded from TCGA and C-Bio Portal and statistic methods were applied to analyze clinical outcomes of FOXA1 mutants.By overexpressing FOXA1 mutation lentivirus and knocking down endogenous FOXA1 in prostate cancer cell lines,we conducted FOXA1 mutants model.FOXA1-mediated downstream genes affected by FKHD mutants were screened by micro-array and q-PCR.Ch IP,Ch IP-sequence and EMSA were used to evaluate FOXA1 chromatin accessibility and nuclear localization.Ch IP,Ch IP-sequence,Co-IP and immunofluorescence were implemented to evaluate AR nuclear localization mediated by FOXA1 mutants.Besides,GSEA analysis was conducted to deeply analyze the enriched pathway by FKHD mutation and western blotting was used to evaluate downstream protein expression.Transwell assay was conducted to evaluate migration ability of prostate cancer cells by FKHD mutantions.Results:By searching TCGA&C-bioportal,we conclude that FOXA1 mutation site mainly located on FKHD.FKHD mutants occurance predicted worse clinical outcomes in FOXA1 mutanted patients.FOXA1 mutants expression model in vitro could be conducted by LNCa P cell lines and FKHD mutants failed to rescue FOXA1-mediated gene expression.FOXA1-up or down regulated mediated genes expression can be changed by FKHD mutants.EMT biomarker TGFB3 expression was increased by FKHD mutants.FKHD mutant proteins gained less chromatin-bound and nuclear localization compared with its wild-type protein.FKHD mutant protein could affect its co-transfactor(AR)nuclear localization and binding.EMT biomarker TGFB3 expression was increased by FKHD mutants.FKHD mutant proteins gained less chromatin-bound and nuclear localization compared with its wild-type protein.FKHD mutant protein could affect its co-transfactor(AR)nuclear localization and binding.FKHD mutants could retain AR expression in CRPC cells.FKHD mutants may significantly activate EMT pathway and mediate related biomarker’s expression.Compared with WT under androgen deprived condition,FKHD mutant could promote prostate cancer cell metastasis.Conclusion:Occurrence of mutation in FKHD of FOXA1 gene predicts poor clinical outcomes in prostate cancer.Vitro experiments have shown that mutations in FKHD affect FOXA1 transcriptional activity,which may be related to the FKHD mutations weakening its ability to bind to chromatin and affecting the nuclear localization and transcriptional activity of AR.Consequently,it ultimately promotes metastatic ability and androgen independent progression of prostate cancer.