| Background: Androgen-deprivation therapy?ADT?remains the mainstream treatment for both locally advanced and metastatic prostate cancers.Unfortunately,although the majority of patients are initially responsive to ADT,most tumors eventually progress from hormone-dependent prostate cancer?HDPC?to castration-resistant prostate cancer?CRPC?.Importantly,recent studies indicate that the androgen receptor?AR?still plays a pivotal role even in CRPC.Castration-induced hypoxia promotes AR transactivation and the development of CRPC.Plant Homeo Domain Finger protein 8?PHF8?,a member of the histone demethylase family,is overexpressed in kinds of cancers including prostate cancer.The present study is designed to investigate the critical roles of PHF8 in castration-induced hypoxia and AR transactivation.Methods: Firstly,immunohistochemistry?IHC?analysis was performed on tissue samples from 97 patients including 16 benign prostatic hyperplasia?BPH?,16 prostate intraepithelial neoplasia?PIN?and 65 prostate cancers from tissue array.Based on PHF8 immunostaining scores,the 65 prostate cancer patients could be categorized into strong?33 patients?and weak?32 patients?PHF8 groups,respectively.The relationship between the levels of PHF8 and tumor-related variables was analyzed by a Pearson ?2 test.We conducted Kaplan-Meier survival analyses on the follow-up data obtained from these 65 patients.Secondly,color Doppler imaging of transrectal ultrasound was used to qualitatively analyze blood flow to the pre-and post-castration prostate cancer tissues.We compared the levels of PHF8,HIF1? and HIF2? in pre-and post-castration prostate cancer tissues from 14 patients with advanced prostate cancer by IHC and western blot analysis.Thirdly,we cultured three prostate cancer cell lines,LNCa P,22RV1 and DU145 under hypoxic conditions for 0,12 and 24 h and then examined the levels of PHF8 protein by western blot analysis and m RNA by RT-PCR.To examine if hypoxia-induced PHF8 expression is dependent on HIF family transcription factors,we knocked down HIF1a or HIF2a in LNCa P cells with their specific sh RNAs and then subjected the cells to normoxic or hypoxic conditions for 12 h to observe PHF8 expression.We investigate if HIF transcription factors can directly regulate PHF8 expression through luciferase reporters assays.Fourthly,we first compared the global levels of H3K9me1,H3K9me2 and H4K20me1 which are known substrates for PHF8,by western blot analysis in hypoxia.We also analyzed the effect of PHF8 knockdown on global histone methylation in LNCa P and PC-3 cells.Toward this end,we transiently transfected LNCa P and PC-3 cells with plasmids coding for PHF8 and determined the ability of PHF8 overexpression to demethylate histones under both normoxic?21% O2?and hypoxic?1% O2?conditions by immunofluorescent staining.Fifthly,we tested the effect of PHF8 knockdown on DHT-induced transcriptional activation of PSA and NKX3.1 by quantitative RT-PCR in LNCa P and VCa P cells and by western blot analysis in normoxic and hypoxic condition in LNCa P cells.Chromatin immunoprecipitation?Ch IP?was used to analyze AR transactivation by PHF8 in the absence or presence of DHT and PHF8 recruitment to PSA or NKX3.1 promoter in hypoxia.Sixthly,we co-expressed a GFP-tagged AR and Flag-tagged PHF8 wild type or mutant in 293 FT cells and treated the transfected cells with or without DHT for 24 hours.Co-immunoprecipitation?Co-IP?was then conducted with antibodies against Flag or GFP.Endogenous Co-IP between PHF8 and AR was conduced in LNCa P cell.Toward this end,we transfected a 4x UAS-TK-luc reporter,a plasmid expressing a fusion protein of Gal4 DNA-binding domain and AR?Gal-AR?,and various concentrations?as indicated?of plasmids encoding the wild-type or H247 A mutant PHF8 into Hela cells,followed by a luciferase reporter assay to examine AR transactivation by PHF8.Results:1.Elevated levels of PHF8 in prostate cancer correlate with higher Gleason grades and poor prognoses.PHF8 is progressively upregulated from BPH to PIN to higher Gleason score prostate cancer.Compared to the weak-PHF8 group,the strong-PHF8 group contained more poorly differentiated patients with Gleason scores 8-10.It appears that more cytoplasmic staining in tumors with lower Gleason grades and stronger stain of PHF8 in nuclear compartment of the cells with higher Gleason grades.The levels of PHF8 in BPH,castration-sensitive and castration-resistant specimens are highly correlated with differentiation grade and the highest level of PHF8 was observed in castration-resistant cancers.2.Elevated levels of PHF8 in prostate cancer correlate with castration-induced hypoxiaCastration treatment reduced blood flow to the prostate cancer tissues.HIF1?,HIF2? and PHF8 were found in both the cytoplasm and nuclei and the levels of these factors are relatively higher in the samples from the post-castration treatment.3.Hypoxia induces PHF8 expression in prostate cancer cellsHypoxia?1% O2?resulted in elevated levels of PHF8 protein and m RNA in three prostate cancer cell lines,LNCa P,22RV1 and DU145 under hypoxic conditions for 0,12 and 24 h.Knockdown of either HIF1a or HIF2a impaired hypoxia-induced PHF8 expression.Both HIF1a and HIF2a strongly induced the transcription of the luciferase reporter with the-1281 to +1 region of PHF8 promoter.HIF transcription factors could bind to PHF8 proximal promoter and activate PHF8 transcription under hypoxic conditions.4.Hypoxia-induced PHF8 expression is functional and alters global histone methylationHypoxia induced PHF8 expression and resulted in a concomitant global reduction of H3K9me1,H3K9me2 and H4K20me1,but had no effect on the level of H3K9me3 in LNCa P and 293 FT cells,which is not a substrate for PHF8.Knockdown of PHF8 resulted in elevated levels of H3K9me1/2 and H4K20me1 but did not increase H3K9me3 in LNCa P and PC-3 cells.PHF8 overexpression in LNCa P and PC-3 cell lines correlated with reduced levels of H3K9me1,H3K9me2 and H4K20me1,but not H3K9me3 under normoxic?21% O2?and hypoxic?1% O2?conditions.5.PHF8 is recruited to and required for optimal transcriptional activation of AR target genesKnockdown of PHF8 significantly impaired DHT-induced transcriptional activation of PSA and NKX3.1 genes.Knockdown of PHF8 also reduced the DHT-induced transcriptional activation of AR,although to a lesser extent in comparison to PSA and NKX3.1 in LNCa P and VCa P cells,which is also shown in 22RV1 cells in the absence of DHT.PHF8 is recruited to AR target genes PSA and NKX3.1 upon DHT treatment.6.PHF8 interacts with and transactivates the AR in a demethylase activity-dependent mannerExogenous and endogenous PHF8 and AR interact with each other in 293 FT and LNCa P cells respectively.Co-expression of PHF8 enhanced AR transcriptional activation in a dose-dependent manner.This coactivator activity is dependent on its demethylase activity as it was not observed in the PHF8?H247A?mutant.Conclusion: Altogether,our data suggest the existence of a HIF/PHF8/AR axis that promotes prostate cancer progression.We propose that prostate cancer subjected to castration treatment generates a hypoxic microenvironment which activates HIFs.The activation of HIFs in turn stimulates PHF8 expression.The elevated levels of PHF8 augment AR transcriptional activity and are likely to promote prostate cancer progression at least in part through its ability to promote the AR signaling pathway.Our study suggests that the HIFs/PHF8/AR axis is likely a driving force for prostate cancer progression and may also be a target for therapy.Given the elevated levels of hypoxia and increased PHF8 expression in CRPCs,targeting PHF8 and/or the HIF signaling pathway could be an attractive therapeutic approach in CRPC therapy. |
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