KDM7 Demethylases: Regulation, Function and Therapeutic Targeting.

It was more than a decade ago that PHF8, KDM7A/JHDM1D and PHF2 were first proposed to be a histone demethylase family and were named as KDM7 (lysine demethylase) family. Since then, knowledge of their demethylation activities, roles as co-regulators of transcription and roles in development and diseases such as cancer has been steadily growing. The demethylation activities of PHF8 and KDM7A toward various methylated histones including H3K9me2/1, H3K27me2 and H4K20me1 have been identified and proven in various cell types. In contrast, PHF2, due to a mutation of a key residue in an iron-binding domain, demethylates H3K9me2 upon PKA-mediated phosphorylation. Interestingly, it was reported that PHF2 possesses an unusual H4K20me3 demethylation activity, which was not observed for PHF8 and KDM7A. PHF8 has been most extensively studied with respect to its roles in development and oncogenesis, revealing that it contributes to regulation of the cell cycle, cell viability and cell migration. Moreover, accumulating lines of evidence demonstrated that the KDM7 family members are subjected to post-transcriptional and post-translational regulations, leading to a higher horizon for evaluating their actual protein expression and functions in development and cancer. This chapter provides a general view of the current understanding of the regulation and functions of the KDM7 family and discusses their potential as therapeutic targets in cancer as well as perspectives for further studies.

Identification of novel TGF-ß regulated genes with pro-migratory roles.

Transforming growth factor-ß (TGF-ß) signaling plays fundamental roles in the development and homeostasis of somatic cells. Dysregulated TGF-ß signaling contributes to cancer progression and relapse to therapies by inducing epithelial-to-mesenchymal transition (EMT), enriching cancer stem cells, and promoting immunosuppression. Although many TGF-ß-regulated genes have been identified, only a few datasets were obtained by next-generation sequencing. In this study, we performed RNA-sequencing analysis of MCF10A cells and identified 1166 genes that were upregulated and 861 genes that were downregulated by TGF-ß. Gene set enrichment analysis revealed that focal adhesion and metabolic pathways were the top enriched pathways of the up- and downregulated genes, respectively. Genes in these pathways also possess significant predictive value for renal cancers. Moreover, we confirmed that TGF-ß induced expression of MICAL1 and 2, and the histone demethylase, KDM7A, and revealed their regulatory roles on TGF-ß-induced cell migration. We also show a critical effect of KDM7A in regulating the acetylation of H3K27 on TGF-ß-induced genes. In sum, this study identified novel effectors that mediate the pro-migratory role of TGF-ß signaling, paving the way for future studies that investigate the function of MICAL family members in cancer and the novel epigenetic mechanisms downstream TGF-ß signaling.

Histone demethylase PHF8 regulates hypoxia signaling through HIF1α and H3K4me3.

Hypoxia through transcription factor HIF1α plays a critical role in cancer development. In prostate cancer, HIF1α interplays with androgen receptor (AR) to contribute to the progression of this disease to its lethal form-castration-resistant prostate cancer (CRPC). Hypoxia upregulates several epigenetic factors including histone demethylase KDM3A which is a critical co-factor of HIF1α. However, how histone demethylases regulate hypoxia signaling is not fully understood. Here, we report that histone demethylase PHF8 plays an essential role in hypoxia signaling. Knockdown or knockout of PHF8 by RNAi or CRISPR-Cas9 system reduced the activation of HIF1α and the induction of HIF1α target genes including KDM3A. Mechanistically, PHF8 regulates hypoxia inducible genes mainly through sustaining the level of trimethylated histone 3 lysine 4 (H3K4me3), an active mark in transcriptional regulation. The positive role of PHF8 in hypoxia signaling extended to hypoxia-induced neuroendocrine differentiation (NED), wherein PHF8 cooperates with KDM3A to regulate the expression of NED genes. Moreover, we discovered that the role of PHF8 in hypoxia signaling is associated with the presence of full-length AR in CRPC cells. Collectively, our study identified PHF8 as a novel epigenetic factor in hypoxia signaling, and the underlying regulatory mechanisms likely apply to general cancer development involving HIF1α. Therefore, targeting PHF8 can potentially be a novel therapeutic strategy in cancer therapy.

Histone demethylase PHF8 promotes epithelial to mesenchymal transition and breast tumorigenesis.

Histone demethylase PHF8 is upregulated and plays oncogenic roles in various cancers; however, the mechanisms underlying its dysregulation and functions in carcinogenesis remain obscure. Here, we report the novel functions of PHF8 in EMT (epithelial to mesenchymal transition) and breast cancer development. Genome-wide gene expression analysis revealed that PHF8 overexpression induces an EMT-like process, including the upregulation of SNAI1 and ZEB1. PHF8 demethylates H3K9me1, H3K9me2 and sustains H3K4me3 to prime the transcriptional activation of SNAI1 by TGF-ß signaling. We show that PHF8 is upregulated and positively correlated with MYC at protein levels in breast cancer. MYC post-transcriptionally regulates the expression of PHF8 via the repression of microRNAs. Specifically, miR-22 directly targets and inhibits PHF8 expression, and mediates the regulation of PHF8 by MYC and TGF-ß signaling. This novel MYC/microRNAs/PHF8 regulatory axis thus places PHF8 as an important downstream effector of MYC. Indeed, PHF8 contributes to MYC-induced cell proliferation and the expression of EMT-related genes. We also report that PHF8 plays important roles in breast cancer cell migration and tumor growth. These oncogenic functions of PHF8 in breast cancer confer its candidacy as a promising therapeutic target for this disease.

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

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.

Ossifying fibroma tumor stem cells are maintained by epigenetic regulation of a TSP1/TGF-ß/SMAD3 autocrine loop.

Abnormal stem cell function makes a known contribution to many malignant tumors, but the role of stem cells in benign tumors is not well understood. Here, we show that ossifying fibroma (OF) contains a stem cell population that resembles mesenchymal stem cells (OFMSCs) and is capable of generating OF-like tumor xenografts. Mechanistically, OFMSCs show enhanced TGF-ß signaling that induces aberrant proliferation and deficient osteogenesis via Notch and BMP signaling pathways, respectively. The elevated TGF-ß activity is tightly regulated by JHDM1D-mediated epigenetic regulation of thrombospondin-1 (TSP1), forming a JHDM1D/TSP1/TGF-ß/SMAD3 autocrine loop. Inhibition of TGF-ß signaling in OFMSCs can rescue their abnormal osteogenic differentiation and elevated proliferation rate. Furthermore, chronic activation of TGF-ß can convert normal MSCs into OF-like MSCs via establishment of this JHDM1D/TSP1/TGF-ß/SMAD3 autocrine loop. These results reveal that epigenetic regulation of TGF-ß signaling in MSCs governs the benign tumor phenotype in OF and highlight TGF-ß signaling as a candidate therapeutic target.