Histone Demethylase PHF8 Drives Neuroendocrine Prostate Cancer Progression By Epigenetically Upregulating FOXA2

Background:Prostate cancer is the most prevalent malignant tumor in male,and the second leading cause of cancer-related death in developed countries.In China,as to the development of economy and diagnosis technologies,prostate cancer more and more becomes one of the most common diseases affecting the aging male survival and living qualities.For metastatic prostate cancer,androgen deprivation therapy is the standard therapeutic strategy.However,the majorities of patients benefit in the early stage and suffer from resistance to androgen deprivation therapy,which were referred to as castration resistance prostate cancer(CRPC).Adminstration of the second generation of anti-androgen therapy,abiraterone and enzalutamide,prolonged the survival of CRPC patients,but the benefiting period is limited.Proportion of CRPC developed independently of androgen receptor,which express no androgen receptor but neuroendocrine markers,referred to as neuroendocrine prostate cancer(NEPC).NEPC is aggressive and lethal,and lack of therapy strategy because of the confusion of mechanism in initiation and development.The incident rate of primary NEPC is nearly 2%,but that of NEPC after androgen deprivation therapy(ADT)is 20%-30%,which suggests the stress of ADT plays crucial role in the development of NEPC.Previous study demonstrated that lineage plasticity of prostate cancer regulated by epigenetic factors is the main event during the development of NEPC.Illustration the mechanism of the link between ADT and lineage plasticity toward NEPC would benefit the patients.Clarifying the mechanism of how ADT inducing lineage plasticity will provide new insight into the development of NEPC,and basics for development of new strategies for diagnosis and treatment.Materials and Methods:1.Model in vitro.Several cell lines including PC-3,LNCaP,NE1.3 were used to study the effect of PHF8 in proliferation,migration and invasion of neuroendocrine prostate cancer cell.2.Model in vivo.a.Patient derived tumor xenograft(PDX)classified as castration-resistance-prostate-cancer and neuroendocrine prostate cancer were applied to study the difference of specific markers.b.Model with spontaneous prostate cancer,transgenic adenocarcinoma of mouse prostate(TRAMP)were taken advantage of to observe the effect of PHF8 in development of prostate cancer.c.Xenograft in nude mice of PC-3 cell line was applied to study the effect of PHF8 in proliferation and migration.3.Samples from patients.Prostate cancer tissues from different patients with divergent pathology diagnosis were applied to produce tissue microarrays,with which immunohistochemistry staining was applied to study the differenc e in expression of several markers.Sample from patients whose tumor were classified as adenocarcinoma before androgen deprivation therapy and neuroendocrine prostate cancer after androgen deprivation therapy were applied to study the change of several rel ated markers.Public data were analyzed to study the difference of related markers.4.Experiment approaches.Divergent approaches including wound scratch assay,clonogenic assay,invasion and migration assay,immunohistochemistry staining,immunofluorescence,co-immunoprecipitation,Western blotting,chromatin immunoprecipitation,luciferase assay,RNA-seq and so on were applied to demonstrate the effect of PHF8 in prostate cancer,Results:1.Knockingout of PHF8 inhibited NEPC development but did not influence the development of adenocarcinoma in Tramp mouse.Comparing PHF8 knock out TRAMP with wild type TRAMP at 12 weeks,25 weeks and 37 weeks,no difference was observed in occurrence of prostatic intraepithelial neoplasia and adenocarcinoma,while significant reduction of neuroendocrine prostate cancer was noticed,which indicated significant effect of PHF8 in neuroendocrine prostate cancer occurrence.2.Knocking down of PHF8 inhibited the proliferation,migration and invasion of NEPC cells.Applying wild type and PHF8 knock down PC-3 and NE1.3 cell lines,obvious downregulation in proliferation,invasion and migration were identified.By establishing wild type and knock down and overexpression of PHF8 LNCaP cell lines,we confirmed that knocking down of PHF8 inhibited the proliferation,migration and invasion of LNCaP cells,and overexpression of PHF8 upregalated the proliferation and resistance to bicalutamide and enzalutamide.3.PHF8 promoted NEPC by transcriptionally upregulate FOXA2.By RNA-seq,we identified impaired signature of neuroendocrine prostate cancer in PHF8 knock out TRAMP comparing with wild type TRAMP,and significant downregulation of FOXA2 was identified,which is well-known driver gene of neuroendocrine prostate cancer.Using model in vivo and vitro,we demonstrated that knock down of PHF8 significant downregulated FOXA2 and specific markers of NEPC,while overexpression of PHF8 could rescue the expression of FOXA2 and specific markers of NEPC.According to the results of luciferase assay and chromatin immunoprecipitation,PHF8 was identified to promote NEPC development by combining to the promoter of FOXA2 and regulating FOXA2 by epigenetic approach.4.PHF8 and FOXA2 were upregulated in NEPC patients.Expression of PHF8 and FOXA2 were studied in public database and samples of patients and PDX.We identified that PHF8 and FOXA2 were significantly upregulated in neuroendocrine prostate cancer,which means PHF8 and FOXA2 could be biomarker of neuroendocrine prostate cancer.Conclusion:Neuroendocrine prostate cancer is a highly aggressive and lethal subtype of prostate cancer with poor prognosis,and a challenge of treatment of prostate cancer.Strategies for early diagnosis and treatment of NEPC are insufficient nowadays for the confussion of mechanism in development of NEPC.In our research,to our knowledge,we for the first time applied PHF8 knockout Trampto study the role of PHF8 in NEPC.We discussed a previously unknown axis involving PHF8 and FOXA2 in development of neuroendocrine prostate cancer for the first time.It was revealed in present study that PHF8 transcriptionally regulate FOXA2 by the fuction of demethylase domain and promote NEPC development.Considering the research we established before,that PHF8 could be upregulated by ADT,the present study provided the mechanism of the link between ADT and the epigenetic factors,FOXA2,in prostate cancer lineage plasticity in NEPC development,which is PHF8 upregulated by ADT,transcriptionally upregulate FOXA2,and then promote prostate cancer neuroendocrine transdifferentiation.It is suggested that combination of ADT and inhibitor of PHF8 could delay or inhibit the development of NEPC.These findings provided new insight into mechanism of development of neuroendocrine prostate cancer,suggesting new target for early diagnosis and strategies for treatment of neuroendocrine prostate cancer.