Lysine Demethylase 6B Regulates Prostate Cancer Cell Proliferation by Controlling c-MYC Expression.

Elevated expression of lysine demethylase 6A (KDM6A) and lysine demethylase 6B (KDM6B) has been reported in prostate cancer (PCa). However, the mechanism underlying the specific role of KDM6A/B in PCa is still fragmentary. Here, we report novel KDM6A/B downstream targets involved in controlling PCa cell proliferation. KDM6A and KDM6B mRNAs were higher in prostate adenocarcinoma, lymph node metastatic site (LNCaP) but not in prostate adenocarcinoma, bone metastatic site (PC3) and prostate adenocarcinoma, brain metastatic site (DU145) cells. Higher KDM6A mRNA was confirmed at the protein level. A metastasis associated gene focused oligonucleotide array was performed to identify KDM6A/B dependent genes in LNCaP cells treated with a KDM6 family selective inhibitor, ethyl-3-(6-(4,5-dihydro-1H-benzo[d]azepin-3(2H)-yl)-2-(pyridin-2-yl)pyrimidin-4-ylamino)propanoate (GSK-J4). This identified five genes [V-myc myelocytomatosis viral oncogene homolog (avian) (c-MYC), neurofibromin 2 (merlin) (NF2), C-terminal binding protein 1 (CTBP1), EPH receptor B2 (EPHB2), and plasminogen activator urokinase receptor (PLAUR)] that were decreased more than 50% by GSK-J4, and c-MYC was the most downregulated gene. Array data were validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR), which detected a reduction in c-MYC steady state mRNA and prespliced mRNA, indicative of transcriptional repression of c-MYC gene expression. Furthermore, c-MYC protein was also decreased by GSK-J4. Importantly, GSK-J4 reduced mRNA and protein levels of c-MYC target gene, cyclinD1 (CCND1). Silencing of KDM6A/B with small interfering RNA (siRNA) confirmed that expression of both c-MYC and CCND1 are dependent on KDM6B. Phosphorylated retinoblastoma (pRb), a marker of G1 to S-phase transition, was decreased by GSK-J4 and KDM6B silencing. GSK-J4 treatment resulted in a decrease in cell proliferation and cell number, detected by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay and conventional cell counting, respectively. Consequently, we conclude that KDM6B controlling c-MYC, CCND1, and pRb contribute regulation of PCa cell proliferation, which represents KDM6B as a promising epigenetic target for the treatment of advanced PCa. SIGNIFICANCE STATEMENT: Lysine demethylase 6A (KDM6A) and 6B (KDM6B) were upregulated in prostate cancer (PCa). We reported novel KDM6A/B downstream targets controlling proliferation. Amongst 84 metastasis associated genes, V-myc myelocytomatosis viral oncogene homolog (avian) (c-MYC) was the most inhibited gene by KDM6 inhibitor, ethyl-3-(6-(4,5-dihydro-1H-benzo[d]azepin-3(2H)-yl)-2-(pyridin-2-yl)pyrimidin-4-ylamino)propanoate (GSK-J4). This was accompanied by decreased c-MYC targets, cyclinD1 (CCND1) and phosphorylated retinoblastoma (pRb), which were KDM6B dependent. GSK-J4 decreased proliferation and cell counting. We conclude that KDM6B controlling c-MYC, CCND1, and pRb contribute regulation of PCa proliferation.

Regulation of Epigenetic Modifiers, Including KDM6B, by Interferon-γ and Interleukin-4 in Human Macrophages.

BACKGROUND: Interferon-γ (IFN-γ) or interleukin-4 (IL-4) drives widely different transcriptional programs in macrophages. However, how IFN-γ and IL-4 alter expression of histone-modifying enzymes involved in epigenetic regulation and how this affects the resulting phenotypic polarization is incompletely understood. METHODS AND RESULTS: We investigated steady-state messenger RNA levels of 84 histone-modifying enzymes and related regulators in colony-stimulating factor-1 differentiated primary human macrophages using quantitative polymerase chain reaction. IFN-γ or IL-4 treatment for 6-48 h changed 11 mRNAs significantly. IFN-γ increased CIITA, KDM6B, and NCOA1, and IL-4 also increased KDM6B by 6 h. However, either cytokine decreased AURKB, ESCO2, SETD6, SUV39H1, and WHSC1, whereas IFN-γ alone decreased KAT2A, PRMT7, and SMYD3 mRNAs only after 18 h, which coincided with decreased cell proliferation. Rendering macrophages quiescent by growth factor starvation or adenovirus-mediated overexpression of p27kip1 inhibited expression of AURKB, ESCO2, SUV39H1, and WHSC1, and mRNA levels were restored by overexpressing the S-phase transcription factor E2F1, implying their expression, at least partly, depended on proliferation. However, CIITA, KDM6B, NCOA1, KAT2A, PRMT7, SETD6, and SMYD3 were regulated independently of effects on proliferation. Silencing KDM6B, the only transcriptional activator upregulated by both IFN-γ and IL-4, pharmacologically or with short hairpin RNA, blunted a subset of responses to each cytokine. CONCLUSION: These findings demonstrate that IFN-γ or IL-4 can regulate the expression of histone acetyl transferases and histone methyl transferases independently of effects on proliferation and that upregulation of the histone demethylase, KDM6B, assists phenotypic polarization by both cytokines.