Chlorpropamide br Materials and methods br Results br

Materials and methods
Results
Discussion The COMPASS family of histone methyltransferases exhibits HMT activity towards histone 3 lysine 4 residue with KMT2F and KMT2G being the major H3K4 methylases (Shilatifard, 2006). They play an important role in the formation and progression of leukemia and various solid tumors (Muntean & Hess, 2009). Till date, their role in ccRCC is largely undetermined. Therefore, the main goal of this study was to explore the role of COMPASS family of HMTase during the progression of ccRCC. In the present study, MLL family of histone methyltransferases showed differential gene expression profile with tumor stage, grade, and metastasis. Among seven members of the family, only KMT2G gene was found to be significantly over-expressed with higher stages of the tumor. The studies on the role of KMT2G gene in human cancers are limited. Loss of KMT2G gene was reported in colorectal and gastric cancers, due to frameshift mutations in the gene (Choi et al., 2014). The significantly higher expression of KMT2G gene in high stages of ccRCC from the present study indicates its role during the progression of the tumor. Further, we observed a significant increase in KMT2G mRNA levels in metastatic tumors. Similarly, the protein level of KMT2G was also significantly augmented in metastatic tumors as compared to non-metastatic. Recently, it has been shown that tumor-induced myeloid-derived suppressor Chlorpropamide exhibited high levels of KMT2G gene as compared to tumor-free mice. This resulted in the induction of inducible nitric oxide synthase (iNOS) which ultimately inhibited T-cell activation and antitumor immune response (Redd et al., 2017). Previously, Hara et al. reported that iNOS is required for an acquaintance of hypoxic conditions by tumor cells and in the tumor metastasis (Hara et al., 2003). As iNOS was also shown to stimulate Wnt pathway in human cancers (DuQ et al., 2013), therefore, further study was carried out to examine the protein levels of some of the Wnt target genes. From our study, it was concluded that CTNNB1, Sox2, Sox9 and EGFR were over-expressed in metastatic tumors in comparison to non-metastatic. Therefore, the over-expression of Wnt target proteins in the present study might be due to the KMT2G genes through the activation iNOS. This might be one of the mechanisms, which explains the increased levels of KMT2G gene with the progression of the tumor from the present study. Therefore, KMT2G can be used as a potential marker for tumor progression as well as therapeutic target.
Acknowledgments
Histone posttranslational modifications (PTMs), including methylation, acetylation, phosphorylation and many others, play an important role in human gene regulation. Methylation of lysine residues is catalyzed by members of histone lysine methyltransferases (KMTs) that transfer the methyl group from S-adenosylmethionine (SAM) to lysine residues on histone -terminal tails, core histones and non-histone proteins. Recent structural, mutagenesis and molecular modelling studies provided basic mechanistic insight into histone methyltransferase catalysis., , , SET domain-containing proteins G9a and its highly related homologue G9a-like protein (GLP) (also known as EHMT2 and EHMT1, respectively) catalyze mono-, di- and trimethylation of lysine 9 on histone 3 (H3K9me1/2/3, a) and several other proteins. The highest methylation mark (H3K9me3) results in formation of heterochromatin, i.e. the transcriptionally inactive form of chromatin, and has been linked to the development and maintenance of various types of cancer. For instance, recent work has shown that increased expression of G9a in aggressive lung cancer cells is associated with greater mortality in patients. Therefore G9a and GLP have been recognized as validated targets for development of small molecule inhibitors for therapies against a variety of diseases, including cancer. Recent medicinal chemistry studies have demonstrated that inhibition of G9a and GLP (and other KMTs) can be achieved by small molecules that act as histone-competitive or/and SAM-competitive inhibitors., , BIX-01294, the first known selective inhibitor of G9a, was reported in 2007. This small molecule inhibitor, which was identified by high throughput screening, targets the histone binding site. Since its discovery, the structure of BIX-01294 has been used in various structure-activity relationship (SAR) explorations and structure-based design studies, which led to the development of inhibitors with an improved potency and selectivity, and reduced toxicity to cells, such as UNC0638, A-366, E72, and DCG066. Few SAM-competitive inhibitors of G9a and GLP have also been recently reported, including BIX-01338 and BRD4770., However, SAM-competitive inhibitors are often unselective due to high homology in SAM-binding sites between different methyltransferases. Most known inhibitors have similar inhibitory activity against G9a and GLP, and developing selective inhibitors for either one is considered challenging due to their high protein homology and similarity of the histone and SAM binding sites (∼80%). Nonetheless, recent work has shown that a high degree of selective inhibition can be achieved; MS012 and related structures have up to 140-fold selectivity for GLP over G9a.