Epigenetic Reprogramming of the Glucose Metabolic Pathways by the Chromatin Effectors During Cancer.

Glucose metabolism plays a vital role in regulating cellular homeostasis as it acts as the central axis for energy metabolism, alteration in which may lead to serious consequences like metabolic disorders to life-threatening diseases like cancer. Malignant cells, on the other hand, help in tumor progression through abrupt cell proliferation by adapting to the changed metabolic milieu. Metabolic intermediates also vary from normal cells to cancerous ones to help the tumor manifestation. However, metabolic reprogramming is an important phenomenon of cells through which they try to maintain the balance between normal and carcinogenic outcomes. In this process, transcription factors and chromatin modifiers play an essential role to modify the chromatin landscape of important genes related directly or indirectly to metabolism. Our chapter surmises the importance of glucose metabolism and the role of metabolic intermediates in the cell. Also, we summarize the influence of histone effectors in reprogramming the cancer cell metabolism. An interesting aspect of this chapter includes the detailed methods to detect the aberrant metabolic flux, which can be instrumental for the therapeutic regimen of cancer.

Epigenetic regulation of Fructose-1,6-bisphosphatase 1 by host transcription factor Speckled 110 kDa during hepatitis B virus infection.

Hepatitis B virus (HBV) is the leading cause of liver disease ranging from acute and chronic hepatitis to liver cirrhosis and hepatocellular carcinoma (HCC). Studies have revealed that HBV infection broadly reprogrammes the host cellular metabolic processes for viral pathogenesis. Previous reports have shown that glycolysis and gluconeogenesis are among the most deregulated pathways during HBV infection. We noted that despite being one of the rate-limiting enzymes of gluconeogenesis, the role and regulation of Fructose-1,6-bisphosphatase 1 (FBP1) during HBV infection is not much explored. In this study, we report FBP1 upregulation upon HBV infection and unravel a novel mechanism of epigenetic reprogramming of FBP1 by HBV via utilizing host factor Speckled 110 kDa (Sp110). Here, we identified acetylated lysine 18 of histone H3 (H3K18Ac) as a selective interactor of Sp110 Bromodomain. Furthermore, we found that Sp110 gets recruited on H3K18Ac-enriched FBP1 promoter, and facilitates recruitment of deacetylase Sirtuin 2 (SIRT2) on that site in the presence of HBV. SIRT2 in turn brings its interactor and transcriptional activator Hepatocyte nuclear factor 4-alpha to the promoter, which ultimately leads to a loss of DNA methylation near the cognate site. Interestingly, this Sp110 driven FBP1 regulation during infection was found to promote viral-borne HCC progression. Moreover, Sp110 can be used as a prognostic marker for the hepatitis-mediated HCC patients, where high Sp110 expression significantly lowered their survival. Thus, the epigenetic reader protein Sp110 has potential to be a therapeutic target to challenge HBV-induced HCCs.

Suppression of poised oncogenes by ZMYND8 promotes chemo-sensitization.

The major challenge in chemotherapy lies in the gain of therapeutic resistance properties of cancer cells. The relatively small fraction of chemo-resistant cancer cells outgrows and are responsible for tumor relapse, with acquired invasiveness and stemness. We demonstrate that zinc-finger MYND type-8 (ZMYND8), a putative chromatin reader, suppresses stemness, drug resistance, and tumor-promoting genes, which are hallmarks of cancer. Reinstating ZMYND8 suppresses chemotherapeutic drug doxorubicin-induced tumorigenic potential (at a sublethal dose) and drug resistance, thereby resetting the transcriptional program of cells to the epithelial state. The ability of ZMYND8 to chemo-sensitize doxorubicin-treated metastatic breast cancer cells by downregulating tumor-associated genes was further confirmed by transcriptome analysis. Interestingly, we observed that ZMYND8 overexpression in doxorubicin-treated cells stimulated those involved in a good prognosis in breast cancer. Consistently, sensitizing the cancer cells with ZMYND8 followed by doxorubicin treatment led to tumor regression in vivo and revert back the phenotypes associated with drug resistance and stemness. Intriguingly, ZMYND8 modulates the bivalent or poised oncogenes through its association with KDM5C and EZH2, thereby chemo-sensitizing the cells to chemotherapy for better disease-free survival. Collectively, our findings indicate that poised chromatin is instrumental for the acquisition of chemo-resistance by cancer cells and propose ZMYND8 as a suitable epigenetic tool that can re-sensitize the chemo-refractory breast carcinoma.

A novel role of tumor suppressor ZMYND8 in inducing differentiation of breast cancer cells through its dual-histone binding function.

Accumulating evidences indicate the involvement of epigenetic deregulations in cancer. While some epigenetic regulators with aberrant functions in cancer are targeted for improving therapeutic outcome in patients, reinstating the functions of tumor-suppressor-like epigenetic regulators might further potentiate anti-cancer therapies. Epigenetic reader zinc-finger MYND-type-containing 8 (ZMYND8) has been found to be endowed with multiple anti-cancer functions like inhibition of tumor cell migration and proliferation. Here, we report another novel tumor suppressor role of ZMYND8 as an inducer of differentiation in breast cancer cells, by upregulating differentiation genes. Interestingly, we also demonstrated that ZMYND8 mediates all its antitumor roles through a common dual-histone mark binding to H4K16Ac and H3K36Me2. We validated these findings by both biochemical and biophysical analyses. Furthermore, we also confirmed the differentiationinducing potential of ZMYND8 in vivo, using 4T1 murine breast cancer model in Balb/c mice. Differentiation therapy holds great promise in cancer therapy, since it is non-toxic and makes the cancer cells therapysensitive. In this scenario, we propose epigenetic reader ZMYND8 as a potential therapeutic candidate for differentiation therapy in breast cancer.

Human sirtuin 3 (SIRT3) deacetylates histone H3 lysine 56 to promote nonhomologous end joining repair.

Human sirtuin 3 (SIRT3) is a conserved NAD+ dependent deacetylase, which functions in important cellular processes including transcription, metabolism, oxidative stress response. It is a robust mitochondrial deacetylase; however, few studies have indicated its nuclear functions. Here we report interaction of SIRT3 with core histones and identified acetylated histone H3 lysine 56 (H3K56ac) as its novel substrate, in addition to known substrates acetylated H4K16 and H3K9. Further, we showed in response to DNA damage SIRT3 localizes to the repair foci colocalizing with γH2AX and nonhomologous end joining (NHEJ) marker p53-binding protein 1 (53BP1). However, it does not colocalize with homologous repair (HR) marker BRCA1. By ChIP break assay, we demonstrated the recruitment of SIRT3 at the double strand-break site in response to DNA damage. Additionally, the relocalization of SIRT3 to the nucleus on MMS treatment led to concurrent decrease in H3K56ac, which is an important step in NHEJ. Depletion of SIRT3 by si-RNA mediated knock down affected recruitment of 53BP1, resulting in compromised NHEJ efficiency, and survival defect as seen by colony formation assay. Altogether, our results demonstrated that SIRT3 recruits 53BP1 to the site of damage thereby plays a significant role in NHEJ pathway.

A novel SIRT1 inhibitor, 4bb induces apoptosis in HCT116 human colon carcinoma cells partially by activating p53.

The NAD+-dependent protein deacetylase SIRT1 has emerged as an important target for epigenetic therapeutics of colon cancer as its increased expression is associated with cancer progression. Additionally, SIRT1 represses p53 function via deacetylation, promoting tumor growth. Therefore, inhibition of SIRT1 is of great therapeutic interest for the treatment of colon cancer. Here, we report discovery of a novel quinoxaline based small molecule inhibitor of human SIRT1, 4bb, investigated its effect on viability of colon cancer cells and molecular mechanism of action. In vitro, 4bb is a significantly more potent SIRT1 inhibitor, compared to ß-naphthols such as sirtinol, cambinol. Increasing concentration of 4bb decrease viability of colon cancer cells but, does not affect the viability of normal dermal fibroblasts depicting cancer cell specificity. Further, 4bb treatment increased p53 acetylation, Bax expression and induced caspase 3 cleavage suggesting that the death of HCT116 colon cancer cells occur through intrinsic pathway of apoptosis. Overall, our results presents 4bb as a new class of human SIRT1 inhibitor and suggest that inhibition of SIRT1 by 4bb induces apoptosis of colon cancer cells at least in part via activating p53 by preventing p53 deacetylation, increasing Bax expression and inducing caspases. Therefore, this molecule provide an opportunity for lead optimization and may help in development of novel, non-toxic epigenetic therapeutics for colon cancer.

p300-mediated acetylation of histone H3 lysine 56 functions in DNA damage response in mammals.

The packaging of newly replicated and repaired DNA into chromatin is crucial for the maintenance of genomic integrity. Acetylation of histone H3 core domain lysine 56 (H3K56ac) has been shown to play a crucial role in compaction of DNA into chromatin following replication and repair in Saccharomyces cerevisiae. However, the occurrence and function of such acetylation has not been reported in mammals. Here we show that H3K56 is acetylated and that this modification is regulated in a cell cycle-dependent manner in mammalian cells. We also demonstrate that the histone acetyltransferase p300 acetylates H3K56 in vitro and in vivo, whereas hSIRT2 and hSIRT3 deacetylate H3K56ac in vivo. Further we show that following DNA damage H3K56 acetylation levels increased, and acetylated H3K56, which is localized at the sites of DNA repair. It also colocalized with other proteins involved in DNA damage signaling pathways such as phospho-ATM, CHK2, and p53. Interestingly, analysis of occurrence of H3K56 acetylation using ChIP-on-chip revealed its genome-wide spread, affecting genes involved in several pathways that are implicated in tumorigenesis such as cell cycle, DNA damage response, DNA repair, and apoptosis.