Investigation of Epigenetic Regulation of Tumor Suppressor Genes by Inhibition of Histone Deacetylases and Lipid Raft Signaling in Human Cancers

Tumor development and cancer progression are principally guided by predominant role of oncogenes over tumor suppressor genes. Rejuvenation of tumor suppressor genes (TSGs) could be one of the therapeutic strategies against cancer. This thesis attempts to illuminate the regulatory mechanism behind TSG silencing by targeting histone deacetylases (HDACs) and lipid raft signaling. In view of dynamic and reversible nature of histone acetylation and overexpression of HDAC enzymes in cancer, HDACs were targeted by inhibitors that owe the ability to restore the expression of TSGs. The present thesis elucidates a novel property of Thymoquinone (TQ), an active principle of Nigella sativa, as an efficient inhibitor against histone deacetylases (HDACs). It reveals the molecular interaction of TQ with HDACs, especially HDAC1 and HDAC2. TQ could efficiently reactivate the expression of p21, maspin, insulin like growth factor binding protein7 (IGFBP7) and microtubule-associated tumor suppressor gene (MTUS1). The epigenetic regulation of IGFBP7 in breast cancer has neither been studied adequately nor has its connection with lipid raft been investigated previously. This study reveals that downregulation of IGFBP7 is mostly due to histone modifications rather than DNA methylation in breast cancer. Perturbation of signaling pathways is one of the quintessential factors responsible for aberrant expression of TSGs in cancer. Therefore, modulation of IGFBP7 by lipid raft signaling is examined in breast cancer. It is apparent that lipid raft/RAS-MAPK signaling is involved in epigenetic regulation of IGFBP7 gene. The role and regulation of MTUS1 in lung cancer is still in its infancy. The study delineates that the regulation of MTUS1 is independent of lipid raft inhibition. However, epigenetic mechanism plays a crucial role in its regulation. Human Males absent on the first (hMOF) and DNA methyltransferase 1 (DNMT1) are frequently over expressed in lung cancer. It is apparent from the results in the thesis that both DNMT1 and hMOF may be correlated with MTUS1 silencing in lung cancer. Further experiments through inhibition of DNMT1 and hMOF demonstrated the expression of MTUS1, which in turn attenuate proliferation and migration of lung cancer cells. Taken together this thesis emphasized TQ as an inhibitor of HDACs that helps expression of TSGs, like p21, Maspin and Bax and describes the importance of lipid raft signaling and epigenetic modulation of IGFBP7 and MTUS1

DNA methylation regulates Microtubule-associated tumor suppressor 1 in human non-small cell lung carcinoma

Microtubule associated tumor suppressor 1 (MTUS1) has been recognized as a tumor suppressor gene in multiple cancers. However, the molecular mechanisms underlying the regulation of MTUS1 are yet to be investigated. This study aimed to clarify the significance of DNA methylation in silencing MTUS1 expression. We report that MTUS1 acts as tumor suppressor in non-small cell lung carcinoma (NSCLC). Analysis of in silica database and subsequent knockdown of DNMT1 suggested an inverse correlation between DNMT1 and mars]. function. Interestingly, increased methylation at MTUS1 promoter is associated with low expression of MTUS1. Treatment with DNA methyltransferases (DNMTs) inhibitor, 5-aza-2'-deoxycytidine (AZA) leads to both reduced promoter methylation accompanied with enrichment of H3K9Ac and enhanced MTUS1 expression. Remarkably, knockdown of MTUS1 showed increased proliferation and migration of NSCLC cells in contrast to diminished proliferation and migration, upon treatment with AZA. We concluded that low expression of MTUS1 correlates to DNA methylation and histone deacetylation in human NSCLC

RGG-motif self-association regulates eIF4G-binding translation repressor protein Scd6

Regulation of mRNA translation plays a key role in the control of gene expression. Scd6, a conserved RGG-motif containing protein represses translation by binding to translation initiation factor eIF4G1. Here we report that Scd6 binds itself in RGG-motif dependent manner and self-association regulates its repression activity. Scd6 self-interaction competes with eIF4G1 binding and methylation of Scd6 RGG-motif by Hmt1 negatively affects self-association. Results pertaining to Sbp1 indicate that self-association could be a general feature of RGG-motif containing translation repressor proteins. Taken together, our study reveals a mechanism of regulation of eIF4G-binding RGG-motif translation repressors

Arginine methylation augments Sbp1 function in translation repression and decapping

The fate of messenger RNA in cytoplasm plays a crucial role in various cellular processes. However, the mechanisms that decide whether mRNA will be translated, degraded or stored remain unclear. Single stranded nucleic acid binding protein (Sbp1), an Arginine-Glycine-Glycine (RGG-motif) protein, is known to promote transition of mRNA into a repressed state by binding eukaryotic translation initiation factor 4G1 (eIF4G1) and to promote mRNA decapping, perhaps by modulation of Dcp1/2 activity. Sbp1 is known to be methylated on arginine residues in RGG-motif; however, the functional relevance of this modification in vivo remains unknown. Here, we report that Sbp1 is arginine-methylated in an hnRNP methyl transferase (Hmt1)-dependent manner and that methylation is enhanced upon glucose deprivation. Characterization of an arginine-methylation-defective (AMD) mutant provided evidence that methylation affects Sbp1 function in vivo. The AMD mutant is compromised in causing growth defect upon overexpression, and the mutant is defective in both localizing to and inducing granule formation. Importantly, the Sbp1-eIF4G1 interaction is compromised both for the AMD mutant and in the absence of Hmt1. Upon overexpression, wild-type Sbp1 increases localization of another RGG motif containing protein, Scd6 (suppressor of clathrin deficiency) to granules; however, this property of Sbp1 is compromised in the AMD mutant and in the absence of Hmt1, indicating that Sbp1 repression activity could involve other RGG-motif translation repressors. Additionally, the AMD mutant fails to increase localization of the decapping activator DEAD box helicase homolog to foci and fails to rescue the decapping defect of a dcp1-2 Delta ski8 strain, highlighting the role of Sbp1 methylation in decapping. Taken together, these results suggest that arginine methylation modulates Sbp1 role in mRNA fate determination

Epigenetic silencing of genes enhanced by collective role of reactive oxygen species and MAPK signaling downstream ERK/Snail axis: Ectopic application of hydrogen peroxide repress CDH1 gene by enhanced DNA methyltransferase activity in human breast cancer

Loss of E-cadherin and epithelial to mesenchymal transition (EMT) are key steps in cancer progression. Reactive oxygen species (ROS) play significant roles in cellular physiology and homeostasis. Roles of E-cadherin (CDH1), EMT and ROS are intriguingly illustrated in many cancers without focusing their collective concert during cancer progression. We report that hydrogen peroxide (H2O2) treatment modulate CDH1 gene expression by epigenetic modification(s). Sublethal dosage of H2O2 treatment decrease E-cadherin, increase DNMT1, HDAC1, Snail, Slug and enrich H3K9me3 and H3K27me3 in the CDH1 promoter. The effect of H2O2 was attenuated by ROS scavengers; NAC, lupeol and beta-sitosterol. DNMT inhibitor, AZA prevented the H2O2 induced promoter-CpG-island methylation of CDH1. Treatment of cells with U0126 (inhibitor of ERK) reduced the expression of DNMT1, Snail and Slug, increased CDHL This implicates that CDH1 is synergistically repressed by histone methylation, DNA methylation and histone deacetylation mediated chromatin remodelling and activation of Snail and Slug through ERK pathway. Increased ROS leads to activation of epigenetic machineries and EMT activators Snail/Slug which in their course of action inactivates CDH1 gene and lack of E-cadherin protein promotes EMT in breast cancer cells. ROS and ERK signaling facilitate epigenetic silencing and support the fact that subtle increase of ROS above basal level act as key cell signaling molecules. Free radical scavengers, lupeol and beta-sitosterol may be tested for therapeutic intervention of breast cancer. This work broadens the amplitude of epigenome and open avenues for investigations on conjoint effects of canonical and intrinsic metabolite signaling and epigenetic modulations in cancer