Histone demethylases are involved in

Histone demethylases are involved in the transcriptional output of the AR and hypoxia signaling pathways, and thus contribute to prostate cancer development [12]. For example, the histone demethylase KDM3A serves as a transcriptional coactivator of HIF1α and AR in regulation of their target genes [13]. KDM3A is a JmjC domain-containing histone demethylase. Since the members of this family require oxygen (O2) as a co-factor [14], their O2 thresholds dictate their demethylation activities, and hence their functions under hypoxia [15]. For example, KDM3A and Jumonji Domain Containing 2B (JMJD2B) are active at 1% O2 in HeLa Q-VD Oph [16], and KDM3A is active under 0.5% O2 in LNCaP cells whereas Jumonji Domain-Containing Protein 2A (JMJD2A) and Jumonji/ARID Domain-Containing Protein 1B (JARID1B) lose their demethylation activities under these conditions [13]. The enhancement of the activities of particular histone demethylases by hypoxia results in a feed-forward loop that ensures that they contribute to transcriptional regulation under hypoxia [15]. This regulatory mechanism compensates for the partial loss of their demethylation activities, and those of other histone demethylases under hypoxia. However, how histone demethylases regulate hypoxia signaling is not fully understood. 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% O2, 6day treatment), with c-MYC downregulated but PHF8 post-transcriptionally upregulated. Recently, it was reported that in the context of hypoxia HIF1α and HIF2α transcriptionally upregulate PHF8, which interacts with AR and enhances its transcriptional activity [18]. In the current study, we report PHF8 plays a critical role in hypoxia signaling as it: positively regulates KDM3A which is a critical coactivator of HIF1α; indirectly sustains H3K4me3 levels on select hypoxia-inducible genes; and is required for full activation of HIF1α through various mechanisms. In the context of prostate cancer, PHF8 appears to execute its regulatory function during hypoxia signaling in AR-positive prostate cancer cells.
Materials and methods
Results
Discussion Hypoxia has numerous effects on tumors and their responsiveness to therapy [1]. Thus, understanding the molecular mechanisms, including any epigenetic factors involved in hypoxia signaling is critical for the development of novel cancer therapies. Histone demethylases represent a major class of epigenetic factors, and play important roles in transcriptional regulation [14]. These enzymes require oxygen as a co-factor; therefore, their demethylation activity can be inhibited under hypoxia. However, a compensatory mechanism that upregulates their expression under hypoxia can maintain, even enhance their functions [15]. The histone demethylase PHF8 contributes to oncogenesis by promoting epithelial to mesenchymal transition (EMT) [23] and cell-cycle progression [17], [23], [38], and inhibiting apoptosis [26], [39]. This study identifies a novel regulatory role for PHF8 during hypoxia, showing that it acts through HIF1α and H3K4me3, shedding light on novel functions of PHF8 in cancer biology. Using various loss-of-function systems such as shRNA & siRNA-mediated knockdown of PHF8 in LNCaP, LNCaP-Abl cells, and CRISPR-Cas9 system-mediated knockout of PHF8 in 293T cell, we demonstrate that PHF8 transcriptionally regulates HIF1A. These findings concur with a previous independent study which showed that HIF1A levels are reduced following siRNA-mediated knockdown of PHF8 in LNCaP cells [26]. However, the reduction in levels of HIF1A mRNA expression (20–40%) in these cell lines does not fully explain the reduction or absence of HIF1α protein. Proteasome inhibitors did not restore HIF1α protein levels in the cells with PHF8 loss-of-function. Thus, the regulatory mechanisms whereby PHF8 regulates HIF1α still remain to be studied. PHF8 might indirectly regulate HIF1α through its positive regulators. PHF8 knockdown arrests the cell cycle at G0/G1 [17], and could thereby reduce the level of phosphorylated AKT, which is known to be critical for HIF1α stability [40], [41]. This potential link may also explain the mechanism underlying the association of PHF8-mediated regulation of HIF1α with AR status in prostate cancer cells, in part because, activated AR signaling can activate HIF1α through autocrine loop involving EGF/PI3K/PKB [7] or directly through transcriptional regulation [42]. Additional supporting evidence is the recently identified HIF/PHF8/AR axis, in which PHF8 regulates AR target genes under hypoxia [18], thus, it is also possible that PHF8 indirectly regulates HIF1A through AR signaling. A systematic approach is still needed to tackle these possible mechanisms. Furthermore, PHF8 may regulate HIF1α through protein synthesis as PHF8 is known to positively regulate ribosome RNAs [31].