c-MYC drives histone demethylase PHF8 during neuroendocrine differentiation and in castration-resistant prostate cancer

Abstract

Epigenetic factors play critical roles in prostate cancer (PCa) development. However, how they contribute to neuroendocrine differentiation (NED) and castration-resistant PCa (CRPC) is not fully understood. Using bioinformatics and biochemical approaches to analyze cell-based models of NED and CRPC, we found a cluster of epigenetic factors whose expression is downregulated during NED and upregulated in CRPC (i.e. follow a Down-Up pattern). Two histone demethylases within this cluster, PHF8 and KDM3A, are post-transcriptionally regulated by c-MYC through miR-22, which targets both PHF8 and KDM3A. We also found that the c-MYC/miR-22/PHF8 axis is downstream of androgen receptor (AR) signaling in CRPC cells. The co-expression of PHF8 with AR in clinical CRPC samples, normal mouse prostate, and adenocarcinomas of the prostate during PCa progression in a transgenic (TRAMP) mouse model supports the connection between PHF8 and AR. Knockdown of PHF8 impedes cell cycle progression in CRPC cells and has more profound effects on their growth than on the parental LNCaP cell line. Furthermore, PHF8 knockdown sensitizes LNCaP-Abl cells to the AR antagonist enzalutamide. Our data reveal novel mechanisms that underlie the regulation of PHF8 and KDM3A during NED and in CRPC, and support the candidacy of PHF8 as a therapeutic target in CRPC.

Histone demethylase PHF8 regulates hypoxia signaling through HIF1α and H3K4me3

“…They were subcloned into the XhoI and NotI sites of pOZ-N-Flag-HA vector. Control and PHF8 shRNAs and siRNAs were described previously [17]. The sequence to shPHF8_2 was GGCCTAGAAATGCCAACTTCA.…”

“…This latter PHF8 shRNA and the two shRNA (1 and 2) sequences for KDM3A [20] were cloned into the AgeI and EcoRI sites of a TetON-pLKO-puro vector, which was a gift from Dmitri Wiederschain (Addgene plasmid # 21915) [21]. Retrovirus and lentivirus packaging of the pOZ and TetON-pLKO-puro vectors, infection, and stable selection were performed as described previously [17]. Doxycycline at 1 μg/ml and 0.5 μg/ml was applied to induce the expression of target shRNAs for experiments lasting < 72 h and 6 days, respectively.…”

“…Transfection of siRNA duplexes targeting PHF8 and western blotting were conducted as previously described [17]. The antibodies against KDM3A, PHF8, Chromogranin A/CgA, γTubulin, HIF1α, β-Actin, ENO2 and HA have been described previously [17].…”

“…The antibodies against KDM3A, PHF8, Chromogranin A/CgA, γTubulin, HIF1α, β-Actin, ENO2 and HA have been described previously [17]. In addition, antibodies against the following proteins were utilized: H3K27me2 (39245) from Active Motif; H3K9me2 (#1220) and H3 (#1791) from Abcam; H3K4me3 (#07-473), and WDR5 (#07-706) from Millipore; and WDR5 (#A302-429A) from Bethyl Labs.…”

“…We recently reported that PHF8 (PHD finger protein 8) is dynamically regulated during the neuroendocrine differentiation (NED) that occurs in prostate cancer, and that the c-MYC-miR-22 axis contributes to the regulation of PHF8 in the context of androgen depletion and IL-6 treatment [17]. However, the c-MYC-miR-22-PHF8 axis is decoupled under hypoxia (1% O 2 , 6 day treatment), with c-MYC downregulated but PHF8 post-transcriptionally upregulated.…”