Upregulated KDM4B promotes prostate cancer cell proliferation by activating autophagy

Castration-resistant prostate cancer (CRPC) causes most of the deaths in patients with prostate cancer (PCa). The androgen receptor (AR) axis plays an important role in castration resistance. Emerging studies showed that the lysine demethylase KDM4B is a key molecule in AR signaling and turnover, and autophagy plays an important role in CRPC. However, little is known about whether KDM4B promotes CRPC progression by regulating autophagy. Here we used an androgen-independent LNCaP (LNCaP-AI) cell line to assay aberrant KDM4B expression using qPCR and western blot analysis and investigated the function of KDM4B in regulating cell proliferation. We found that KDM4B was markedly increased in LNCaP-AI cells compared with LNCaP cells. KDM4B level was significantly correlated with the Gleason score in PCa tissues. In vitro, KDM4B overexpression in CRPC cells promoted cell proliferation, whereas knockdown of KDM4B significantly inhibited cell proliferation. Upregulated KDM4B contributed to activate Wnt/β-catenin signaling and autophagy. Moreover, KDM4B activated autophagy by regulating the Wnt/β-catenin signaling. Finally, we demonstrated that autophagy inhibition attenuated KDM4B-induced CRPC cell proliferation. Our results provided novel insights into the function of KDM4B-driven CRPC development and indicated that KDM4B may be served as a potential target for CRPC therapy.     

KDM2B promotes cell viability by enhancing DNA damage response in canine hemangiosarcoma

Epigenetic regulators have been implicated in tumorigenesis of many types of cancer; however, their roles in endothelial cell cancers such as canine hemangiosarcoma(HSA) have not been studied. In this study, we find that lysine-specific demethylase 2 b(KDM2 B) is highly expressed in HSA cell lines compared with normal canine endothelial cells. Silencing of KDM2 B in HSA cells results in increased cell death in vitro compared with the scramble control by inducing apoptosis through the inactivation of the DNA repair pathways and accumulation of DNA damage. Similarly, doxycycline-induced KDM2 B silencing in tumor xenografts results in decreased tumor sizes compared with the control. Furthermore, KDM2 B is also highly expressed in clinical cases of HSA. We hypothesize that pharmacological KDM2 B inhibition can also induce HSA cell death and can be used as an alternative treatment for HSA. We treat HSA cells with GSK-J4, a histone demethylase inhibitor, and find that GSK-J4 treatment also induces apoptosis and cell death. In addition, GSK-J4 treatment decreases tumor size. Therefore, we demonstrate that KDM2 B acts as an oncogene in HSA by enhancing the DNA damage response. Moreover, we show that histone demethylase inhibitor GSK-J4 can be used as a therapeutic alternative to doxorubicin for HSA treatment.     

Inhibition of Plk1 represses androgen signaling pathway in castration-resistant prostate cancer

Prostate cancer (PCa) is the second leading cause of cancer-related death in males in the United States. Majority of prostate cancers are originally androgen-dependent and sensitive to androgen-deprivation therapy (ADT), however, most of them eventually relapse and progress into incurable castration-resistant prostate cancer (CRPC). Of note, the activity of androgen receptor (AR) is still required in CRPC stage. The mitotic kinase polo-like kinase 1 (Plk1) is significantly elevated in PCa and its expression correlates with tumor grade. In this study, we assess the effects of Plk1 on AR signaling in both androgen-dependent and androgen-independent PCa cells. We demonstrate that the expression level of Plk1 correlated with tumorigenicity and that inhibition of Plk1 caused reduction of AR expression and AR activity. Furthermore, Plk1 inhibitor BI2536 down-regulated SREBP-dependent expression of enzymes involved in androgen biosynthesis. Of interest, Plk1 level was also reduced when AR activity was inhibited by the antagonist MDV3100. Finally, we show that BI2536 treatment significantly inhibited tumor growth in LNCaP CRPC xenografts. Overall, our data support the concept that Plk1 inhibitor such as BI2536 prevents AR signaling pathway and might have therapeutic potential for CRPC patients.     

Anti-androgen receptor ASC-J9 versus anti-androgens MDV3100 (Enzalutamide) or Casodex (Bicalutamide) leads to opposite effects on prostate cancer metastasis via differential modulation of macrophage infiltration and STAT3-CCL2 signaling

Despite androgen deprivation therapy (ADT) suppression of prostate cancer (PCa) growth, its overall effects on PCa metastasis remain unclear. Using human (C4-2B/THP1) and mouse (TRAMP-C1/RAW264.7) PCa cells–macrophages co-culture systems, we found currently used anti-androgens, MDV3100 (enzalutamide) or Casodex (bicalutamide), promoted macrophage migration to PCa cells that consequently led to enhanced PCa cell invasion. In contrast, the AR degradation enhancer, ASC-J9, suppressed both macrophage migration and subsequent PCa cell invasion. Mechanism dissection showed that Casodex/MDV3100 reduced the AR-mediated PIAS3 expression and enhanced the pSTAT3-CCL2 pathway. Addition of CCR2 antagonist reversed the Casodex/MDV3100-induced macrophage migration and PCa cell invasion. In contrast, ASC-J9 could regulate pSTAT3-CCL2 signaling using two pathways: an AR-dependent pathway via inhibiting PIAS3 expression and an AR-independent pathway via direct inhibition of the STAT3 phosphorylation/activation. These findings were confirmed in the in vivo mouse model with orthotopically injected TRAMP-C1 cells. Together, these results may raise the potential concern about the currently used ADT with anti-androgens that promotes PCa metastasis and may provide some new and better therapeutic strategies using ASC-J9 alone or a combinational therapy that simultaneously targets androgens/AR signaling and PIAS3-pSTAT3-CCL2 signaling to better battle PCa growth and metastasis at castration-resistant stage.     

KDM6B regulates M2 polarization of macrophages by modulating the stability of nuclear β-catenin

Accumulating evidences suggest that the epigenetic regulation plays a pivotal role in establishing phenotype and function of tumor associated macrophages (TAMs). KDM6B is an epigenetic enzyme responsible for the H3K27me3 and reported to influence macrophage polarization. However, the underlying mechanism remains to be determined. Here, we demonstrated that inhibition of KDM6B in TAMs increased M2 polarization induced by coculture of breast cancer cells. Furthermore, we identified that KDM6B downregulation activated β-catenin/c-Myc signaling, and thus promoted the M2-like phenotype. KDM6B accelerated the intranuclear ubiquitination degradation of β-catenin, which depended on its demethylase activity. Therapeutically, our data showed that activated vitamin D analog paricalcitol upregulated the expression of KDM6B and decreased the M2 polarization, consequently protected against tumor progress in the xenograft mouse model of breast cancer. Taken together, our data reveal that epigenetic regulator KDM6B prevents M2 polarization via promoting the intranuclear degradation of β-catenin. Active vitamin D analog induces KDM6B and suppresses tumor progress, suggesting a novel therapeutic potential of epigenetic modulation for the tumor treatment.     

RNA-dependent chromatin localization of KDM4D lysine demethylase promotes H3K9me3 demethylation

The JmjC-containing lysine demethylase, KDM4D, demethylates di-and tri-methylation of histone H3 on lysine 9 (H3K9me3). How KDM4D is recruited to chromatin and recognizes its histone substrates remains unknown. Here, we show that KDM4D binds RNA independently of its demethylase activity. We mapped two non-canonical RNA binding domains: the first is within the N-terminal spanning amino acids 115 to 236, and the second is within the C-terminal spanning amino acids 348 to 523 of KDM4D. We also demonstrate that RNA interactions with KDM4D N-terminal region are critical for its association with chromatin and subsequently for demethylating H3K9me3 in cells. This study implicates, for the first time, RNA molecules in regulating the levels of H3K9 methylation by affecting KDM4D association with chromatin.     

Blocking GRP/GRP-R signaling decreases expression of androgen receptor splice variants and inhibits tumor growth in castration-resistant prostate cancer

Clinical management of castration-resistant prostate cancer (CRPC) resulting from androgen deprivation therapy (ADT) remains challenging. Many studies indicate that androgen receptor splice variants (ARVs) play a critical role in the development of CRPC, including resistance to the new generation of inhibitors of androgen receptor (AR) action. ARVs are constitutively active and lack the ligand-binding domain (LBD), thereby allowing prostate cancer (PC) to maintain AR activity despite therapies that target the AR (full-length AR; AR-FL). Previously, we have reported that long-term ADT increases the neuroendocrine (NE) hormone – Gastrin Releasing Peptide (GRP) and its receptor (GRP-R) expression in PC cells. Further, we demonstrated that activation of GRP/GRP-R signaling increases ARVs expression by activating NF-κB signaling, thereby promoting cancer progression to CRPC. Most importantly, as a cell surface protein, GRP-R is easily targeted by drugs to block GRP/GRP-R signaling. In this study, we tested if blocking GRP/GRP-R signaling by targeting GRP-R using GRP-R antagonist is sufficient to control CRPC progression. Our studies show that blocking GRP/GRP-R signaling by targeting GRP-R using RC-3095, a selective GRP-R antagonist, efficiently inhibits NF-κB activity and ARVs (AR-V7) expression in CRPC and therapy-induced NEPC (tNEPC) cells. In addition, blocking of GRP/GRP-R signaling by targeting GRP-R can sensitize CRPC cells to anti-androgen treatment (such as MDV3100). Further, preclinical animal studies indicate combination of GRP-R antagonist (targeting ARVs) with anti-androgen (targeting AR-FL) is sufficient to inhibit CRPC and tNEPC tumor growth.     

KDM5B promotes self-renewal of hepatocellular carcinoma cells through the microRNA-448–mediated YTHDF3/ITGA6 axis

Histone methylation plays important roles in mediating the onset and progression of various cancers, and lysine-specific demethylase 5B (KDM5B), as a histone demethylase, is reported to be an oncogene in hepatocellular carcinoma (HCC). However, the mechanism underlying its tumorigenesis remains undefined. Hence, we explored the regulatory role of KDM5B in HCC cells, aiming to identify novel therapeutic targets for HCC. Gene Expression Omnibus database and StarBase were used to predict important regulatory pathways related to HCC. Then, the expression of KDM5B and microRNA-448 (miR-448) in HCC tissues was detected by RT-qPCR and Western blot analysis. The correlation between KDM5B and miR-448 expression was analysed by Pearson's correlation coefficient and ChIP experiments, and the targeting of YTH N6-methyladenosine RNA binding protein 3 (YTHDF3) by miR-448 was examined by luciferase assay. Additionally, the effect of KDM5B on the proliferation, migration, invasion and apoptosis as well as tumorigenicity of transfected cells was assessed using ectopic expression and depletion experiments. KDM5B was highly expressed in HCC cells and was inversely related to miR-448 expression. KDM5B demethylated H3K4me3 on the miR-448 promoter and thereby inhibited the expression of miR-448, which in turn targeted YTHDF3 and integrin subunit alpha 6 (ITGA6) to promote the malignant phenotype of HCC. Moreover, KDM5B accelerated HCC progression in nude mice via the miR-448/YTHDF3/ITGA6 axis. Our study uncovered that KDM5B regulates the YTHDF3/ITGA6 axis by inhibiting the expression of miR-448 to promote the occurrence of HCC.     

Studies of H3K4me3 demethylation by KDM5B/Jarid1B/PLU1 reveals strong substrate recognition in vitro and identifies 2,4-pyridine-dicarboxylic acid as an in vitro and in cell inhibitor

Dynamic methylations and demethylations of histone lysine residues are important for gene regulation and are facilitated by histone methyltransferases and histone demethylases (HDMs). KDM5B/Jarid1B/PLU1 is an H3K4me3/me2-specific lysine demethylase belonging to the JmjC domain-containing family of histone demethylases (JHDMs). Several studies have linked KDM5B to breast, prostate and skin cancer, highlighting its potential as a drug target. However, most inhibitor studies have focused on other JHDMs, and inhibitors for KDM5B remain to be explored. Here, we report the expression, purification and characterization of the catalytic core of recombinant KDM5B (ccKDM5B, residues 1-769). We show that ccKDM5B, recombinantly expressed in insect cells, demethylates H3K4me3 and H3K4me2 in vitro. The kinetic characterization showed that ccKDM5B has an apparent Michaelis constant (K(m) (app) ) value of 0.5 μm for its trimethylated substrate H3(1-15)K4me3, a considerably increased apparent substrate affinity than reported for related HDMs. Despite the presence of a PHD domain, the catalytic activity was not affected by additional methylation at the H3K9 position, suggesting that in vitro chromatin cross-talk between H3K4 and H3K9 does not occur for ccKDM5B. Inhibition studies of ccKDM5B showed both in vitro and in cell inhibition of ccKDM5B by 2,4-pyridinedicarboxylic acid (2,4-PDCA) with a potency similar to that reported for the HDM KDM4C. Structure-guided sequence alignment indicated that the binding mode of 2,4-PDCA is conserved between KDM4A/C and KDM5B.     

Androgen Suppresses the Proliferation of Androgen Receptor-Positive Castration-Resistant Prostate Cancer Cells via Inhibition of Cdk2, CyclinA,and Skp2

The majority of prostate cancer (PCa) patient receiving androgen ablation therapy eventually develop castration-resistant prostate cancer (CRPC). We previously reported that androgen treatment suppresses Skp2 and c-Myc through androgen receptor (AR) and induced G1 cell cycle arrest in androgen-independent LNCaP 104-R2 cells, a late stage CRPC cell line model. However, the mechanism of androgenic regulation of Skp2 in CRPC cells was not fully understood. In this study, we investigated the androgenic regulation of Skp2 in two AR-positive CRPC cell line models, the LNCaP 104-R1 and PC-3^AR Cells. The former one is an early stage androgen-independent LNCaP cells, while the later one is PC-3 cells re-expressing either wild type AR or mutant LNCaP AR. Proliferation of LNCaP 104-R1 and PC-3^AR cells is not dependent on but is suppressed by androgen. We observed in this study that androgen treatment reduced protein expression of Cdk2, Cdk7, Cyclin A, cyclin H, Skp2, c-Myc, and E2F-1; lessened phosphorylation of Thr14, Tyr15, and Thr160 on Cdk2; decreased activity of Cdk2; induced protein level of p27^Kip1; and caused G1 cell cycle arrest in LNCaP 104-R1 cells and PC-3^AR cells. Overexpression of Skp2 protein in LNCaP 104-R1 or PC-3^AR cells partially blocked accumulation of p27^Kip1 and increased Cdk2 activity under androgen treatment, which partially blocked the androgenic suppressive effects on proliferation and cell cycle. Analyzing on-line gene array data of 214 normal and PCa samples indicated that gene expression of Skp2, Cdk2, and cyclin A positively correlates to each other, while Cdk7 negatively correlates to these genes. These observations suggested that androgen suppresses the proliferation of CRPC cells partially through inhibition of Cyclin A, Cdk2, and Skp2.     

Depletion of histone demethylase KDM2A inhibited cell proliferation of stem cells from apical papilla by de-repression of p15INK4B and p27Kip1

Mesenchymal stem cells (MSCs) are a reliable resource for tissue regeneration; although, the molecular mechanisms of their differentiation and proliferation are not clearly understood, which restricts the applications of MSCs. The histone demethylase, lysine (K)-specific demethylase 2A (KDM2A), and the mammalian paralog, lysine (K)-specific demethylase 2B (KDM2B), are evolutionarily conserved and ubiquitously expressed members of the JmjC-domain-containing histone demethylase family. A previous study determined that KDM2A and KDM2B can regulate the differentiation of MSCs, and KDM2B has been implicated in cell cycle regulation by de-repressing p15^INK4B (cyclin-dependent kinase inhibitor 2B). It is not known whether KDM2A is involved in the cell proliferation of MSCs. Here, we show that depletion of KDM2A by short hairpin RNAs can inhibit cell proliferation and arrest cell cycle progression at the G1/S-phase in human stem cells from apical papilla (SCAPs). The effect of KDM2A on cell proliferation was found to be mediated through de-repression of the cyclin-dependent kinase inhibitors, p15^INK4B and p27^Kip1 (cyclin-dependent kinase inhibitor 1B), in KDM2A knock-down SCAPs. Furthermore, chromatin immunoprecipitation assays demonstrated that silencing of KDM2A increased histone H3 Lysine 4 (H3K4) trimethylation at the p15^INK4B and p27^Kip1 loci and regulated its expression. Together, our results indicate that KDM2A is a H3K4 demethylase that regulates cell proliferation through p15^INK4B and p27^Kip1 in SCAPs.     

Steroidogenic enzymes and stem cell markers are upregulated during androgen deprivation in prostate cancer

Considerable levels of testosterone and dihydrotestosterone (DHT) are found in prostate cancer (PCa) tissue after androgen deprivation therapy. Treatment of surviving cancer-initiating cells and the ability to metabolize steroids from precursors may be the keystones for the appearance of recurrent tumors. To study this hypothesis, we assessed the expression of several steroidogenic enzymes and stem cell markers in clinical PCa samples and cell cultures during androgen depletion. Gene expression profiles were determined by microarray or qRT-PCR. In addition, we measured cell viability and analyzed stem cell marker expression in DuCaP cells by immunocytochemistry. Seventy patient samples from different stages of PCa, and the PCa cell line DuCaP were included in this study. The androgen receptor (AR) and enzymes (AKR1C3, HSD17B2, HSD17B3, UGT2B15 and UGT2B17 ) that are involved in the metabolism of adrenal steroids were upregulated in castration resistant prostate cancer (CRPC). In vitro, some DuCaP cells survived androgen depletion, and eventually gave rise to a culture adapted to these conditions. During and after this transition, most of the steroidogenic enzymes were upregulated. These cells also are enriched with stem/progenitor cell markers cytokeratin 5 (CK5) and ATP-binding cassette sub-family G member 2 (ABCG2). Similarly, putative stem/progenitor cell markers CK5, c-Kit, nestin, CD44, c-met, ALDH1A1, α2-integrin, CD133, ABCG2, CXCR4 and POU5F1 were upregulated in clinical CRPC. The upregulation of steroidogenic enzymes and stem cell markers in recurrent tumors suggests that cancer initiating cells can expand by adaptation to their T/DHT deprived environment. Therapies targeting the metabolism of adrenal steroids by the tumor may prove effective in preventing tumor regrowth.     

HP1a targets the Drosophila KDM4A demethylase to a subset of heterochromatic genes to regulate H3K36me3 levels

The KDM4 subfamily of JmjC domain-containing demethylases mediates demethylation of histone H3K36me3/me2 and H3K9me3/me2. Several studies have shown that human and yeast KDM4 proteins bind to specific gene promoters and regulate gene expression. However, the genome-wide distribution of KDM4 proteins and the mechanism of genomic-targeting remain elusive. We have previously identified Drosophila KDM4A (dKDM4A) as a histone H3K36me3 demethylase that directly interacts with HP1a. Here, we performed H3K36me3 ChIP-chip analysis in wild type and dkdm4a mutant embryos to identify genes regulated by dKDM4A demethylase activity in vivo. A subset of heterochromatic genes that show increased H3K36me3 levels in dkdm4a mutant embryos overlap with HP1a target genes. More importantly, binding to HP1a is required for dKDM4A-mediated H3K36me3 demethylation at a subset of heterochromatic genes. Collectively, these results show that HP1a functions to target the H3K36 demethylase dKDM4A to heterochromatic genes in Drosophila.     

KDM4B Overexpression Promotes the Growth,Migration, and Invasion of Rheumatoid Arthritis Fibroblast-Like Synoviocytes by Activating STAT3 Pathway

In rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) present a unique aggressive phenotype and have a passive response to the inflammatory microenvironment, which are critical for the disease’s progression. KDM4B, as a histone demethylase, functions as an oncogenic factor in many cancers and is implicated in osteoclastogenesis as well as pro-inflammatory cytokine release in inflammatory diseases. However, the effects of KDM4B on RA FLS have not been reported. To investigate this issue, our study determined the expression of KDM4B in RA FLS using RT-qPCR and western blot. The effects of KDM4B on RA FLS viability, apoptosis, migration, and invasion were detected by MTT, flow cytometry, transwell migration, and invasion assays. Furthermore, the interaction of KDM4B with STAT3 signaling was studied by western blot, MTT, flow cytometry, transwell migration, and invasion assays. The experimental results showed that KDM4B expression was upregulated in RA synovial tissues and FLS as compared to healthy control tissues and normal FLS. Knockdown of KDM4B obviously suppressed RA FLS viability, migration and invasion, and induced apoptosis. In addition, knockdown of KDM4B in RA FLS decreased the expression of p-STAT3 and MMP-9 but increased cleaved caspase-3 expression compared with the control group. Moreover, KDM4B overexpression could promote cell growth, migration and invasion, and suppress apoptosis in RA FLS by activating STAT3 signaling. Therefore, these findings provide new insight for understanding the pathogenesis of RA and indicate that KDM4B may have a potential to be an effective therapeutic target for RA.