Triple Combinations of Histone Lysine Demethylase Inhibitors with PARP1 Inhibitor-Olaparib and Cisplatin Lead to Enhanced Cytotoxic Effects in Head and Neck Cancer Cells.

PARP inhibitors are used to treat cancers with a deficient homologous recombination (HR) DNA repair pathway. Interestingly, recent studies revealed that HR repair could be pharmacologically impaired by the inhibition of histone lysine demethylases (KDM). Thus, we investigated whether KDM inhibitors could sensitize head and neck cancer cells, which are usually HR proficient, to PARP inhibition or cisplatin. Therefore, we explored the effects of double combinations of KDM4-6 inhibitors (ML324, CPI-455, GSK-J4, and JIB-04) with olaparib or cisplatin, or their triple combinations with both drugs, on the level of DNA damage and apoptosis. FaDu and SCC-040 cells were treated with individual compounds and their combinations, and cell viability, apoptosis, DNA damage, and gene expression were assessed using the resazurin assay, Annexin V staining, H2A.X activation, and qPCR, respectively. Combinations of KDM inhibitors with cisplatin enhanced cytotoxic effects, unlike combinations with olaparib. Triple combinations of KDM inhibitors with cisplatin and olaparib exhibited the best cytotoxic activity, which was associated with DNA damage accumulation and altered expression of genes associated with apoptosis induction and cell cycle arrest. In conclusion, triple combinations of KDM inhibitors (especially GSK-J4 and JIB-04) with cisplatin and olaparib represent a promising strategy for head and neck cancer treatment.

Histone demethylase KDM4A mediating macrophage polarization: A potential mechanism of trichloroethylene induced liver injury.

Trichloroethylene (TCE) is a commonly used organic solvent in industry. Our previous studies have found that TCE can cause liver injury accompanied by macrophage polarization, but the specific mechanism is unclear. The epigenetic regulation of macrophage polarization is mainly focused on histone modification. Histone lysine demethylase 4A (KDM4A) is involved in the activation of macrophages. In this study, we used a mouse model we investigated the role of KDM4A in the livers of TCE-drinking mice and found that the expression of KDM4A, M1-type polarization indicators, and related inflammatory factors in the livers of TCE-drinking mice. In the study, BALB/c mice were randomly divided into four groups: 2.5 mg/mL TCE dose group and 5.0 mg/mL TCE dose group, the vehicle control group, and the blank control group. We found that TCE triggered M1 polarization of mouse macrophages, characterized by the expression of CD11c and robust production of inflammatory cytokines. Notably, exposure to TCE resulted in markedly increased expression of KDM4A in macrophages. Functionally, the increased expression of KDM4A significantly impaired the expression of H3K9me3 and H3K9me2 and increased the expression of H3K9me1. In addition, KDM4A potentially represents a novel epigenetic modulator, with its upregulation connected to ß-catenin activation, a signal critical for the pro-inflammatory activation of macrophages. Furthermore, KDM4A inhibitor JIB-04 treatment resulted in a decrease in ß-catenin expression and prevented TCE-induced M1 polarization and the expression of the pro-inflammatory cytokines TNF-α and IL-1ß. These results suggest that the association of KDM4A and Wnt/ß-catenin cooperatively establishes the activation and polarization of macrophages and global changes in H3K9me3/me2/me1. Our findings identify KDM4A as an essential regulator of the polarization of macrophages and the expression of inflammatory cytokines, which might serve as a potential target for preventing and treating liver injury caused by TCE.

Targeting of H19/cell adhesion molecules circuitry by GSK-J4 epidrug inhibits metastatic progression in prostate cancer.

BACKGROUND: About 30% of Prostate cancer (PCa) patients progress to metastatic PCa that remains largely incurable. This evidence underlines the need for the development of innovative therapies. In this direction, the potential research focus might be on long non-coding RNAs (lncRNAs) like H19, which serve critical biological functions and show significant dysregulation in cancer. Previously, we showed a transcriptional down-regulation of H19 under combined pro-tumoral estrogen and hypoxia treatment in PCa cells that, in turn, induced both E-cadherin and ß4 integrin expression. H19, indeed, acts as transcriptional repressor of cell adhesion molecules affecting the PCa metastatic properties. Here, we investigated the role of H19/cell adhesion molecules circuitry on in vivo PCa experimental tumor growth and metastatic dissemination models. METHODS: H19 was silenced in luciferase-positive PC-3 and 22Rv1 cells and in vitro effect was evaluated by gene expression, proliferation and invasion assays before and after treatment with the histone lysine demethylase inhibitor, GSK-J4. In vivo tumor growth and metastasis dissemination, in the presence or absence of GSK-J4, were analyzed in two models of human tumor in immunodeficient mice by in vivo bioluminescent imaging and immunohistochemistry (IHC) on explanted tissues. Organotypic Slice Cultures (OSCs) from fresh PCa-explant were used as ex vivo model to test GSK-J4 effects. RESULTS: H19 silencing in both PC-3 and 22Rv1 cells increased: i) E-cadherin and ß4 integrin expression as well as proliferation and invasion, ii) in vivo tumor growth, and iii) metastasis formation at bone, lung, and liver. Of note, treatment with GSK-J4 reduced lesions. In parallel, GSK-J4 efficiently induced cell death in PCa-derived OSCs. CONCLUSIONS: Our findings underscore the potential of the H19/cell adhesion molecules circuitry as a targeted approach in PCa treatment. Modulating this interaction has proven effective in inhibiting tumor growth and metastasis, presenting a logical foundation for targeted therapy.

Lysine demethylase 5C inhibits transcription of prefoldin subunit 5 to activate c-Myc signal transduction and colorectal cancer progression.

BACKGROUND: Lysine demethylase 5C (KDM5C) has been implicated in the development of several human cancers. This study aims to investigate the role of KDM5C in the progression of colorectal cancer (CRC) and explore the associated molecular mechanism. METHODS: Bioinformatics tools were employed to predict the target genes of KDM5C in CRC. The expression levels of KDM5C and prefoldin subunit 5 (PFDN5) in CRC cells were determined by RT-qPCR and western blot assays. The interaction between KDM5C, H3K4me3, and PFDN5 was validated by chromatin immunoprecipitation. Expression and prognostic values of KDM5C and PFDN5 in CRC were analyzed in a cohort of 72 patients. The function of KDM5C/PFDN5 in c-Myc signal transduction was analyzed by luciferase assay. Silencing of KDM5C and PFDN5 was induced in CRC cell lines to analyze the cell malignant phenotype in vitro and tumorigenic activity in nude mice. RESULTS: KDM5C exhibited high expression, while PFDN5 displayed low expression in CRC cells and clinical CRC samples. High KDM5C levels correlated with poor survival and unfavorable clinical presentation, whereas elevated PFDN5 correlated with improved patient outcomes. KDM5C mediated demethylation of H3K4me3 on the PFDN5 promoter, suppressing its transcription and thereby enhancing the transcriptional activity of c-Myc. KDM5C knockdown in CRC cells suppressed cell proliferation, migration and invasion, epithelial-mesenchymal transition, and tumorigenic activity while increasing autophagy and apoptosis rates. However, the malignant behavior of cells was restored by the further silencing of PFDN5. CONCLUSION: This study demonstrates that KDM5C inhibits PFDN5 transcription, thereby activating c-Myc signal transduction and promoting CRC progression.

Knockout of KDM3A in MDA-MB-231 breast cancer cells inhibits tumor malignancy and promotes apoptosis.

The histone lysine demethylase 3 A (KDM3A) is vital for the regulation of cancer physiology and pathophysiology. The purpose of this study was to investigate the effect of KDM3A expression with triple-negative breast cancer (TNBC) invasion and metastasis. In our results, knockout of KDM3A in TNBC MDA-MB-231 cells promoted apoptosis and inhibited the proliferation, invasion and metastasis of MDA-MB-231 cells. In addition, we found that in vivo experiments indicated that the growth, invasion and metastasis of metastatic neoplasms were significantly inhibited by knockout of KDM3A in a TNBC metastasis model. These findings suggest that KDM3A may be a potential therapeutic target for the treatment and prevention of TNBC, providing a critical theoretical basis for the effective prevention or treatment of breast cancer disease.

Bisphenol S induces brown adipose tissue whitening and aggravates diet-induced obesity in an estrogen-dependent manner.

Bisphenol S (BPS) exposure has been implied epidemiologically to increase obesity risk, but the underlying mechanism is unclear. Here, we propose that BPS exposure at an environmentally relevant dose aggravates diet-induced obesity in female mice by inducing brown adipose tissue (BAT) whitening. We explored the underlying mechanism by which KDM5A-associated demethylation of the trimethylation of lysine 4 on histone H3 (H3K4me3) in thermogenic genes is overactivated in BAT upon BPS exposure, leading to the reduced expression of thermogenic genes. Further studies have suggested that BPS activates KDM5A transcription in BAT by binding to glucocorticoid receptor (GR) in an estrogen-dependent manner. Estrogen-estrogen receptors facilitate the accessibility of the KDM5A gene promoter to BPS-activated GR by recruiting the activator protein 1 (AP-1) complex. These results indicate that BAT is another important target of BPS and that targeting KDM5A-related signals may serve as an approach to counteract the BPS-induced susceptivity to obesity.

Lysine demethylase 3A in hypoxic macrophages promotes ovarian cancer development through regulation of the vascular endothelial growth factor A/Akt signaling.

BACKGROUND: Hypoxia is a vital feature of the tumor microenvironment of OC. Previous evidence exposes that tumor-associated macrophages (TAMs) are connected with the development of ovarian cancer (OC), whereas the accurate regulatory mechanism of hypoxic macrophages regulating tumor advancement remains unclear. Herein, we examined whether the lysine demethylase 3 A (KDM3A) in hypoxic macrophages expedited the development of OC cells. METHODS: The contents of hypoxia inducible factor-1α (HIF-1α), CD163, CD80, KDM3A, and p-Akt/Akt were detected by western blot. Genomic Spatial Event 4630, Molecular Signatures Database, and Comparative Toxicogenomics Database were utilized for correlated gene prediction. The OC cells viability was scrutinized by cell counting kit-8 assay. The cell proliferation was inspected by 5-Ethynyl-2'-deoxyuridine assay. The vascular endothelial growth factor A (VEGF) level was detected by Enzyme-linked immunosorbent assay. RESULTS: M2 polarization of TAMs was associated with poor prognosis in sufferers with OC. The OC sufferers with high level of CD163 or low level of CD80 were linked with poor overall survival and disease specific survival. Hypoxia induced THP-1-derived macrophages M2 polarization. KDM3A was high-expressed in hypoxia induced macrophages. Upregulated KDM3A in hypoxic macrophages facilitated OC cell proliferation. KDM3A upregulation in hypoxic macrophages stimulated Akt signaling activation in OC cells. KDM3A in hypoxic macrophages promoted VEGF secretion to activate Akt signaling in OC cells. VEGF inhibition or Akt signaling inactivation reversed the effects of KDM3A in hypoxic macrophages on OC cells viability and proliferation. CONCLUSION: The KDM3A content and M2 polarization were enhanced in hypoxic macrophages, and KDM3A in hypoxic macrophages promoted OC development through regulation of the VEGF/Akt signaling pathway.

A novel STAT3/ NFκB p50 axis regulates stromal-KDM2A to promote M2 macrophage-mediated chemoresistance in breast cancer.

BACKGROUND: Lysine Demethylase 2A (KDM2A) plays a crucial role in cancer cell growth, differentiation, metastasis, and the maintenance of cancer stemness. Our previous study found that cancer-secreted IL-6 can upregulate the expression of KDM2A to promote further the transition of cells into cancer-associated fibroblasts (CAFs). However, the molecular mechanism by which breast cancer-secreted IL-6 regulates the expression of KDM2A remains unclear. Therefore, this study aimed to elucidate the underlying molecular mechanism of IL-6 in regulating KDM2A expression in CAFs and KDM2A-mediated paclitaxel resistance in breast cancer. METHODS: The ectopic vector expression and biochemical inhibitor were used to analyze the KDM2A expression regulated by HS-578 T conditioned medium or IL-6 in mammary fibroblasts. Immunoprecipitation and chromatin immunoprecipitation assays were conducted to examine the interaction between STAT3 and NFκB p50. M2 macrophage polarization was assessed by analyzing M2 macrophage-specific markers using flow cytometry and RT-PCR. ESTIMATE algorithm was used to analyze the tumor microenvironment-dominant breast cancer samples from the TCGA database. The correlation between stromal KDM2A and CD163 + M2 macrophages was analyzed using the Pearson correlation coefficient. Cell viability was determined using trypan blue exclusion assay. RESULTS: IL-6 regulates gene expression via activation and dimerization of STAT3 or collaboration of STAT3 and NFκB. However, STAT3, a downstream transcription factor of the IL-6 signaling pathway, was directly complexed with NFκB p50, not NFκB p65, to upregulate the expression of KDM2A in CAFs. Enrichment analysis of immune cells/stromal cells using TCGA-breast cancer RNA-seq data unveiled a positive correlation between stromal KDM2A and the abundance of M2 macrophages. CXCR2-associated chemokines secreted by KDM2A-expressing CAFs stimulated M2 macrophage polarization, which in turn secreted CCL2 to increase paclitaxel resistance in breast cancer cells by activating CCR2 signaling. CONCLUSION: This study revealed the non-canonical molecular mechanism of IL-6 secreted by breast cancer upregulated KDM2A expression in CAFs via a novel STAT3/NFκB p50 axis, which STAT3 complexed with NFκB p50 in NFκB p50 binding motif of KDM2A promoter. KDM2A-expressing CAFs dominantly secreted the CXCR2-associated chemokines to promote M2 macrophage polarization and enhance paclitaxel resistance in breast cancer. These findings underscore the therapeutic potential of targeting the CXCR2 or CCR2 pathway as a novel strategy for paclitaxel-resistant breast cancer.

Lysine Demethylation in Pathogenesis.

Epigenetics has major impact on normal development and pathogenesis. Regulation of histone methylation on lysine and arginine residues is a major epigenetic mechanism and affects various processes including transcription and DNA repair. Histone lysine methylation is reversible and is added by histone lysine methyltransferases and removed by histone lysine demethylases. As these enzymes are also capable of writing or erasing lysine modifications on non-histone substrates, they were renamed to lysine demethylases (KDMs) in 2007. Since the discovery of the first lysine demethylase LSD1/KDM1A in 2004, eight more subfamilies of lysine demethylases have been identified and further characterized. The joint efforts by academia and industry have led to the development of potent and specific small molecule inhibitors of KDMs for treatment of cancer and several other diseases. Some of these inhibitors have already entered clinical trials since 2013, less than 10 years after the discovery of the first KDM. In this chapter, we briefly summarize the major roles of histone demethylases in normal development and human diseases and the efforts to target these enzymes to treat various diseases.

Histone lysine demethylase 3B regulates autophagy via transcriptional regulation of GABARAPL1 in acute myeloid leukemia cells.

Macroautophagy (hereafter referred to as autophagy) is a highly conserved self­digestion process that is critical for maintaining homeostasis in response to various stresses. The autophagy­related protein family, including the GABA type A receptor­associated protein (GABARAP) and microtubule­associated protein 1 light chain 3 subfamilies, is crucial for autophagosome biogenesis. Although the regulatory machinery of autophagy in the cytoplasm has been widely studied, its transcriptional and epigenetic regulatory mechanisms still require more targeted investigations. The present study identified histone lysine demethylase 3B (KDM3B) as a crucial component of autophagy on a panel of leukemia cell lines, including K562, THP1 and U937, resulting in transcriptional activation of the autophagy­related gene GABA type A receptor­associated protein like 1 (GABARAPL1). KDM3B expression promoted autophagosome formation and affected the autophagic flux in leukemia cells under the induction of external stimuli. Notably, RNA­sequencing and reverse transcription­quantitative PCR analysis showed that KDM3B knockout inhibited the expression of GABARAPL1. Chromatin immunoprecipitation­quantitative PCR and luciferase assay showed that KDM3B was associated with the GABARAPL1 gene promoter under stimulation and enhanced its transcription. The present findings demonstrated that KDM3B was critical for regulating the GABARAPL1 gene and influencing the process of autophagy in leukemia cells. These results provide a new insight for exploring the association between autophagy and KDM3B epigenetic regulation in leukemia.

Development of JmjC-domain-containing histone demethylase (KDM2-7) inhibitors for cancer therapy.

Histone methylation is the most common histone modification and a highly dynamic regulator of gene transcription. Methylation of lysine residues can alter the structure of chromatin, helping to regulate DNA-based nuclear activities. Lysine demethylases control and maintain epigenetic factors that affect chromatin structure and cell characteristics. A variety of diseases, including malignant tumors, are connected to their dysregulation. Advances in biochemistry and pathogenesis have prompted the discovery of small molecule inhibitors and tool compounds that disrupt lysine demethylation. In this review, we focus on JmjC-domain-containing histone lysine demethylases (KDM2-7), discussing their structures and biological roles, representative inhibitors, and therapeutic potential in cancer therapy, and aiming to provide unique insights into the development of JmjC-KDM inhibitors.

Bone Marrow Mesenchymal Stem Cells Reversed Ovarian Aging-related m6A RNA Methylation Modification Profile in Aged Granulosa Cells.

BACKGROUND: Ovarian ageing causes endocrine disturbances and the degeneration of systemic tissue and organ functions to seriously affect women's physical and mental health, and effective treatment methods are urgently needed. Based on our previous studies using juvenile rhesus monkey bone marrow mesenchymal stem cells (BMMSCs) to treat ovarian ageing in rhesus monkey, we found that BMMSCs improved ovarian structure and function. This study continues to explore the mechanism by which BMMSCs reversed granulosa cell (GC) ageing. METHODS: A GC ageing model and coculture system of BMMSCs were established, changes in the level of the N6-methyladenosine (m6A) methylation modification were detected, m6A-modified RNA immunoprecipitation sequencing (MeRIP-seq) were performed, correlations between m6A peaks and mRNA expression were determined, and the expression of hub genes was identified using Q-PCR, immunofluorescence staining, and western blot. RESULTS: Our results showed that H2O2 successfully induced GC ageing and that BMMSCs reversed measures of GC ageing. BMMSCs increased the expression of the FTO protein and reduced the overall level of m6A. We identified 797 m6A peaks (348 hypomethylated and 449 hypermethylated peaks) and 817 differentially expressed genes (DEGs) (412 upregulated and 405 downregulated) after aged GCs were cocultured with BMMSCs, which significantly associated with ovarian function and epigenetic modification. The epigenetic repressive mark and important cell cycle regulator lysine demethylase 8 (KDM8) was downregulated at both the mRNA and protein levels, histone H3 was upregulated in aged GCs after BMMSC coculture, and KDM8 was upregulated after FTO was inhibited through FB23. CONCLUSIONS: Our study revealed an essential role for m6A in BMMSCs in reversing GC ageing, and FTO regulated KDM8 mediates histone H3 changes may as a novel regulatory mechanism in BMMSCs to reverse GC ageing.

The catalytic domains of all human KDM5 JmjC demethylases catalyse N-methyl arginine demethylation.

The demethylation of Nε -methyllysine residues on histones by Jumonji-C lysine demethylases (JmjC-KDMs) has been established. A subset of JmjC-KDMs has also been reported to have Nω -methylarginine residue demethylase (RDM) activity. Here, we describe biochemical screening studies, showing that the catalytic domains of all human KDM5s (KDM5A-KDM5D), KDM4E and, to a lesser extent, KDM4A/D, have both KDM and RDM activities with histone peptides. Ras GTPase-activating protein-binding protein 1 peptides were shown to be RDM substrates for KDM5C/D. No RDM activity was observed with KDM1A and the other JmjC-KDMs tested. The results highlight the potential of JmjC-KDMs to catalyse reactions other than Nε -methyllysine demethylation. Although our study is limited to peptide fragments, the results should help guide biological studies investigating JmjC functions.

Lysine Demethylase 1B Promotes Tear Secretion Disorder in Sjogren's Syndrome by Regulating the PAX6/CLU Axis.

The impacts of lysine demethylase 1B (KDM1B) have been probed in multiple diseases, but the effects of KDM1B on SS remained obscure. The study aimed to unravel the efficiency of KDM1B on SS progression via the paired box 6 (PAX6)/clusterin (CLU) axis. NODB10. H2b mice were selected to establish the SS model. KDM1B, Pax6, and CLU expression in SS mice was assessed. Adeno-associated viruses carrying KDM1B, Pax6, and CLU were injected into the SS mice to detect tear secretion, epithelium corneal fluorescein staining scores, and levels of specific markers of lacrimal gland epithelial cells, neurotransmitter receptors that induce secretion from the lacrimal gland, and genes encoding normal tear components. The relation among KDM1B, Pax6, and CLU was examined. The rescue experiments were conducted for verifying the interaction among KDM1B, Pax6, and CLU. KDM1B expression was elevated, while Pax6 and CLU levels were decreased in the lacrimal gland tissues of SS mouse models. KDM1B decrement and Pax6 augmentation improved tear secretion, reduced corneal fluorescein staining score, decreased levels of specific markers of lacrimal gland epithelial cells, and increased levels of neurotransmitter receptors that induce secretion from the lacrimal gland and genes encoding normal tear components. KDM1D suppressed Pax6 expression by mediating H3K4me2 demethylation. Pax6 promoted the expression of CLU at the transcriptional level by binding to the CLU promoter. Silencing of Pax6 or CLU could reverse the effects of KDM1B reduction on improving the tear secretion disorder of SS mice. Silencing KDM1B mitigates the tear secretion disorder of SS mice via modulating the Pax6/CLU axis.

Therapeutic targeting of histone lysine demethylase KDM4B blocks the growth of castration-resistant prostate cancer.

Epigenetics is an emerging mechanism for tumorigenesis. Treatment that targets epigenetic regulators is becoming an attractive strategy for cancer therapy. The role of epigenetic therapy in prostate cancer (PCa) remains elusive. Previously we demonstrated that upregulation of histone lysine demethylase KDM4B correlated with the appearance of castration resistant prostate cancer (CRPC) and identified a small molecular inhibitor of KDM4B, B3. In this study, we further investigated the role of KDM4B in promoting PCa progression and tested the efficacy of B3 using clinically relevant PCa models including PCa cell line LNCaP and 22Rv1 and xenografts derived from these cell lines. In loss and gain-functional studies of KDM4B in PCa cells, we found that overexpression of KDM4B in LNCaP cells enhanced its tumorigenicity whereas knockdown of KDM4B in 22Rv1 cells reduced tumor growth in castrated mice. B3 suppressed the growth of 22Rv1 xenografts and sensitized tumor to anti-androgen receptor (AR) antagonist enzalutamide inhibition. B3 also inhibited 22Rv1 tumor growth synergistically with rapamycin, leading to cell apoptosis. Comparative transcriptomic analysis performed on KDM4B knockdown and B3-treated 22Rv1 cells revealed that B3 inhibited both H3K9me3 and H3K27me3 demethylase activities. Our studies establish KDM4B as a target for CRPC and B3 as a potential therapeutic agent. B3 as monotherapy or in combination with other anti-PCa therapeutics offers proof of principle for the clinical translation of epigenetic therapy targeting KDMs for CRPC patients.

MicroRNA-137-mediated lysine demethylase 4A regulates the recovery of spinal cord injury via the SFRP4-Wnt/ß-Catenin axis.

OBJECTIVE: Spinal cord injury (SCI) causes great harm to the normal life of patients. Histone demethylase is involved in many biological processes, including SCI. Hence, this study explored the role and mechanism of histone lysine demethylase 4A (KDM4A) in SCI. METHODS: The acute SCI (ASCI) rat model was established after spinal compression and the SCI neuronal model was induced via treating PC12 cells with lipopolysaccharide (LPS). KDM4A expression during SCI was detected. The microRNA (miRNA) targeting KDM4A was predicted and verified. The miRNA and KDM4A expression patterns were intervened in LPS-stimulated PC12 cells to evaluate their combined effects on neuronal cells in SCI. The downstream pathways of KDM4A were predicted, and SFRP4 and H3K9me3 expressions were determined. After the intervention of SFRP4 in LPS-treated cells, ß-Catenin expression and the effect of SFRP4 on neuronal cells in SCI were detected. Finally, the effectiveness of the miR-137/KDM4A/SFRP4/Wnt/ß-Catenin axis was verified in vivo. RESULTS: KDM4A was abnormally elevated in SCI. miR-137 targeted KDM4A. miR-137 effectively inhibited the apoptosis of LPS-challenged PC12 cells, which could be reversed after overexpressing KDM4A. KDM4A promoted SFRP4 expression through demethylation of H3K9me3. Overexpression of SFRP4 blocked the Wnt/ß-Catenin pathway and promoted apoptosis of LPS-stimulated cells. In vivo, miR-137 overexpression remarkably improved SCI symptoms, accompanied by obviously increased ß-Catenin expression and notably decreased KDM4A and SFRP4 expressions, while overexpressed KDM4A treatment showed the opposite trend in the presence of miR-137. CONCLUSION: We demonstrated that miR-137 targeted KDM4A and then downregulated SFRP4 to ameliorate SCI in a Wnt/ß-Catenin-dependent manner.

Methylation of the JMJD2B epigenetic regulator differentially affects its ability to coactivate the ETV1 and JUN transcription factors.

OBJECTIVES: Jumonji C domain-containing (JMJD) 2B (JMJD2B) is a transcriptional cofactor and histone demethylase that is involved in prostate cancer formation. However, how its function is regulated by posttranslational modification has remained elusive. Hence, we examined if JMJD2B would be regulated by lysine methylation. METHODS: Through in vitro methylation assays and Western blotting with methyl-lysine specific antibodies, we analyzed lysine methylation within JMJD2B. Identified methylated lysine residues were mutated to arginine residues and the respective impact on JMJD2B transcriptional activity measured with a reporter gene assay in human LNCaP prostate cancer cells. RESULTS: We discovered that JMJD2B is methylated on up to six different lysine residues. Further, we identified the suppressor of variegation 3-9/enhancer of zeste/trithorax (SET) domain-containing protein 7/9 (SET7/9) as the methyltransferase being responsible for this posttranslational modification. Mutating the methylation sites in JMJD2B to arginine residues led to diminished coactivation of the Ju-nana (JUN) transcription factor, which is a known oncogenic protein in prostate tumors. In contrast, methylation of JMJD2B had no impact on its ability to coactivate another transcription factor associated with prostate cancer, the DNA-binding protein E26 transformation-specific (ETS) variant 1 (ETV1). Consistent with a potential joint action of JMJD2B, SET7/9 and JUN in prostate cancer, the expression of JMJD2B in human prostate tumors was positively correlated with both SET7/9 and JUN levels. CONCLUSIONS: The identified SET7/9-mediated methylation of JMJD2B appears to impact its cooperation with selected interacting transcription factors in prostate cancer cells. Given the implicated roles of JMJD2B beyond prostate tumorigenesis, SET7/9-mediated methylation of JMJD2B possibly also influences the development of other cancers, while its impairment might have relevance for obesity or a global developmental delay that can be elicited by reduced JMJD2B activity.

Characterization of KDM5 lysine demethylase family substrate preference and identification of novel substrates.

The KDM5/JARID1 sub-family are 2-oxoglutarate and Fe(II)-dependent lysine-specific histone demethylases that are characterized by their Jumonji catalytic domains. The KDM5 family is known to remove tri-/di-methyl modifications from lysine-4 of histone H3 (i.e. H3-K4me2/3), a mark associated with active gene expression. As a result, studies to date have revolved around the influence of KDM5 on disease through their ability to regulate H3-K4me2/3. Recent evidence demonstrates that KDM5 may influence disease beyond H3-K4 demethylation, making it critical to further investigate KDM5-mediated demethylation of non-histone proteins. To help identify potential non-histone substrates for the KDM5 family, we developed a library of 180 permutated peptide substrates, with sequences that are systematically altered from the wild-type H3-K4me3 substrate. From this library, we characterized recombinant KDM5A/B/C/D substrate preference and developed recognition motifs for each KDM5 demethylase. The recognition motifs developed were used to predict potential substrates for KDM5A/B/C/D and profiled to generate a list of high-ranking and medium/low-ranking substrates for further in vitro validation. Through this approach, we identified 66 high-ranking substrates in which KDM5 demethylases displayed significant in vitro activity towards.

Targeting Histone Epigenetic Modifications and DNA Damage Responses in Synthetic Lethality Strategies in Cancer?

Synthetic lethality strategies are likely to be integrated in effective and specific cancer treatments. These strategies combine different specific targets, either in similar or cooperating pathways. Chromatin remodeling underlies, directly or indirectly, all processes of tumor biology. In this context, the combined targeting of proteins associated with different aspects of chromatin remodeling can be exploited to find new alternative targets or to improve treatment for specific individual tumors or patients. There are two major types of proteins, epigenetic modifiers of histones and nuclear or chromatin kinases, all of which are druggable targets. Among epigenetic enzymes, there are four major families: histones acetylases, deacetylases, methylases and demethylases. All these enzymes are druggable. Among chromatin kinases are those associated with DNA damage responses, such as Aurora A/B, Haspin, ATM, ATR, DNA-PK and VRK1-a nucleosomal histone kinase. All these proteins converge on the dynamic regulation chromatin organization, and its functions condition the tumor cell viability. Therefore, the combined targeting of these epigenetic enzymes, in synthetic lethality strategies, can sensitize tumor cells to toxic DNA-damage-based treatments, reducing their toxicity and the selective pressure for tumor resistance and increasing their immunogenicity, which will lead to an improvement in disease-free survival and quality of life.

Therapeutic potential of inhibiting histone 3 lysine 27 demethylases: a review of the literature.

Histone 3 lysine 27 (H3K27) demethylation constitutes an important epigenetic mechanism of gene activation. It is mediated by the Jumonji C domain-containing lysine demethylases KDM6A and KDM6B, both of which have been implicated in a wide myriad of diseases, including blood and solid tumours, autoimmune and inflammatory disorders, and infectious diseases. Here, we review and summarise the pre-clinical evidence, both in vitro and in vivo, in support of the therapeutic potential of inhibiting H3K27-targeting demethylases, with a focus on the small-molecule inhibitor GSK-J4. In malignancies, KDM6A/B inhibition possesses the ability to inhibit proliferation, induce apoptosis, promote differentiation, and heighten sensitivity to currently employed chemotherapeutics. KDM6A/B inhibition also comprises a potent anti-inflammatory approach in inflammatory and autoimmune disorders associated with inappropriately exuberant inflammatory and autoimmune responses, restoring immunological homeostasis to inflamed tissues. With respect to infectious diseases, KDM6A/B inhibition can suppress the growth of infectious pathogens and attenuate the immunopathology precipitated by these pathogens. The pre-clinical in vitro and in vivo data, summarised in this review, suggest that inhibiting H3K27 demethylases holds immense therapeutic potential in many diseases.