Pivotal role for S-nitrosylation of DNA methyltransferase 3B in epigenetic regulation of tumorigenesis.

DNA methyltransferases (DNMTs) catalyze methylation at the C5 position of cytosine with S-adenosyl-L-methionine. Methylation regulates gene expression, serving a variety of physiological and pathophysiological roles. The chemical mechanisms regulating DNMT enzymatic activity, however, are not fully elucidated. Here, we show that protein S-nitrosylation of a cysteine residue in DNMT3B attenuates DNMT3B enzymatic activity and consequent aberrant upregulation of gene expression. These genes include Cyclin D2 (Ccnd2), which is required for neoplastic cell proliferation in some tumor types. In cell-based and in vivo cancer models, only DNMT3B enzymatic activity, and not DNMT1 or DNMT3A, affects Ccnd2 expression. Using structure-based virtual screening, we discovered chemical compounds that specifically inhibit S-nitrosylation without directly affecting DNMT3B enzymatic activity. The lead compound, designated DBIC, inhibits S-nitrosylation of DNMT3B at low concentrations (IC50 ≤ 100 nM). Treatment with DBIC prevents nitric oxide (NO)-induced conversion of human colonic adenoma to adenocarcinoma in vitro. Additionally, in vivo treatment with DBIC strongly attenuates tumor development in a mouse model of carcinogenesis triggered by inflammation-induced generation of NO. Our results demonstrate that de novo DNA methylation mediated by DNMT3B is regulated by NO, and DBIC protects against tumor formation by preventing aberrant S-nitrosylation of DNMT3B.

Potent anti-tumor effects of receptor-retargeted syncytial oncolytic herpes simplex virus.

Most oncolytic virotherapy has thus far employed viruses deficient in genes essential for replication in normal cells but not in cancer cells. Intra-tumoral injection of such viruses has resulted in clinically significant anti-tumor effects on the lesions in the vicinity of the injection sites but not on distant visceral metastases. To overcome this limitation, we have developed a receptor-retargeted oncolytic herpes simplex virus employing a single-chain antibody for targeting tumor-associated antigens (RR-oHSV) and its modified version with additional mutations conferring syncytium formation (RRsyn-oHSV). We previously showed that RRsyn-oHSV exhibits preserved antigen specificity and an ∼20-fold higher tumoricidal potency in vitro relative to RR-oHSV. Here, we investigated the in vivo anti-tumor effects of RRsyn-oHSV using human cancer xenografts in immunodeficient mice. With only a single intra-tumoral injection of RRsyn-oHSV at very low doses, all treated tumors regressed completely. Furthermore, intra-venous administration of RRsyn-oHSV resulted in robust anti-tumor effects even against large tumors. We found that these potent anti-tumor effects of RRsyn-oHSV may be associated with the formation of long-lasting tumor cell syncytia not containing non-cancerous cells that appear to trigger death of the syncytia. These results strongly suggest that cancer patients with distant metastases could be effectively treated with our RRsyn-oHSV.

Antibody Screening System Using a Herpes Simplex Virus (HSV)-Based Probe To Identify a Novel Target for Receptor-Retargeted Oncolytic HSVs.

Herpes simplex virus (HSV) is a promising tool for developing oncolytic virotherapy. We recently reported a platform for receptor-retargeted oncolytic HSVs that incorporates single-chain antibodies (scFvs) into envelope glycoprotein D (gD) to mediate virus entry via tumor-associated antigens. Therefore, it would be useful to develop an efficient system that can screen antibodies that might mediate HSV entry when they are incorporated as scFvs into gD. We created an HSV-based screening probe by the genetic fusion of a gD mutant with ablated binding capability to the authentic HSV entry receptors and the antibody-binding C domain of streptococcal protein G. This engineered virus failed to enter cells through authentic receptors. In contrast, when this virus was conjugated with an antibody specific to an antigen on the cell membrane, it specifically entered cells expressing the cognate antigen. This virus was used as a probe to identify antibodies that mediate virus entry via recognition of certain molecules on the cell membrane other than authentic receptors. Using this method, we identified an antibody specific to epiregulin (EREG), which has been investigated mainly as a secreted growth factor and not necessarily for its precursor that is expressed in a transmembrane form. We constructed an scFv from the anti-EREG antibody for insertion into the retargeted HSV platform and found that the recombinant virus entered cells specifically through EREG expressed by the cells. This novel antibody-screening system may contribute to the discovery of unique and unexpected molecules that might be used for the entry of receptor-retargeted oncolytic HSVs.IMPORTANCE The tropism of the cellular entry of HSV is dependent on the binding of the envelope gD to one of its authentic receptors. This can be fully retargeted to other receptors by inserting scFvs into gD with appropriate modifications. In theory, upon binding to the engineered gD, receptors other than authentic receptors should induce a conformational change in the gD, which activates downstream mechanisms required for viral entry. However, prerequisite factors for receptors to be used as targets of a retargeted virus remain poorly understood, and it is difficult to predict which molecules might be suitable for our retargeted HSV construct. Our HSV-based probe will allow unbiased screening of antibody-antigen pairs that mediate virus entry and might be a useful tool to identify suitable pairs for our construct and to enhance our understanding of virus-cell interactions during infection by HSV and possibly other viruses.

Modulation of Unfolded Protein Response by Methylmercury.

Methylmercury (MeHg) results in cell death through endoplasmic reticulum (ER) stress. Previously, we reported that MeHg induces S-mercuration at cysteine 383 or 386 in protein disulfide isomerase (PDI), and this modification induces the loss of enzymatic activity. Because PDI is a key enzyme for the maturation of nascent protein harboring a disulfide bond, the disruption in PDI function by MeHg results in ER stress via the accumulation of misfolded proteins. However, the effects of MeHg on unfolded protein response (UPR) sensors and their signaling remain unclear. In the present study, we show that UPR is regulated by MeHg. We found that MeHg specifically attenuated inositol-requiring enzyme 1α (IRE1α)-x-box binding protein 1 (XBP1) branch, but not the protein kinase RNA-like endoplasmic reticulum kinase (PERK) and activating transcriptional factor 6 (ATF6) branches. Treatment with GSK2606414, a specific PERK inhibitor, significantly inhibited MeHg-induced cell death. These findings suggest that MeHg exquisitely regulates UPR signaling involved in cell death.

Regulation of Histone Deacetylase 6 Activity via S-Nitrosylation.

Nitric oxide (NO) is a gaseous regulatory factor produced by NO synthases (NOS) and it plays several critical roles via S-nitrosylation of protein cysteine residues. Histone deacetylase (HDAC) functions in the maintenance/balance of chromatin acetylation and contributes to transcriptional supression. It has been reported that S-nitrosylation of HDAC2 is involved in the regulation of deacetylase activity. However, it remains unknown whether other subtypes of the HDAC family are S-nitrosylated. In the present study, we found that HDAC6 is a target of NO. A biotin-switch assay revealed that endogenous HDAC6 is S-nitrosylated by both NO donors and NO derived from the inducible type of NOS in cells treated with cytokines. NO led to suppressed deacetylase activity in vitro and increased acetylated α-tubulin, a major substrate for HDAC6, in A549 cells. These findings suggest that S-nitrosylation of HDAC6 plays a pivotal role in the regulation of protein acetylation.

Endogenous S-sulfhydration of PTEN helps protect against modification by nitric oxide.

Hydrogen sulfide (H2S) is a gaseous regulatory factor produced by several enzymes, and plays a pivotal role in processes such as proliferation or vasodilation. Recent reports demonstrated the physiological and pathophysiological functions of H2S in neurons. PTEN is a target of nitric oxide (NO) or hydrogen peroxide, and the oxidative modification of cysteine (Cys) residue(s) attenuates its enzymatic activity. In the present study, we assessed the effect of H2S on the direct modification of PTEN and the resulting downstream signaling. A modified biotin switch assay in SH-SY5Y human neuroblastoma cells revealed that PTEN is S-sulfhydrated endogenously. Subsequently, site-directed mutagenesis demonstrated that both Cys71 and Cys124 in PTEN are targets for S-sulfhydration. Further, the knockdown of cystathionine ß-synthetase (CBS) using siRNA decreased this modification in a manner that was correlated to amount of H2S. PTEN was more sensitive to NO under these conditions. These results suggest that the endogenous S-sulfhydration of PTEN via CBS/H2S plays a role in preventing the S-nitrosylation that would inhibition its enzymatic activity under physiological conditions.

Correlation between attenuation of protein disulfide isomerase activity through S-mercuration and neurotoxicity induced by methylmercury.

Methylmercury (MeHg), an environmental pollutant, causes neuronal death via endoplasmic reticulum (ER) stress; however, the precise mechanism is not fully understood. The aim of this study was to elucidate the possible mechanism of MeHg-induced neurotoxicity. Treatment with MeHg resulted in a loss of cell viability in a concentration-dependent manner accompanying the expression of ER stress marker genes in human neuroblastoma SH-SY5Y cells. We next attempted to identify a target protein for MeHg in the ER. MeHg covalently modified protein disulfide isomerase (PDI), which is important for disulfide bond formation in nascent proteins in the ER lumen. S-Nitrosylation of the catalytic domains of PDI by nitric oxide was attenuated up to 50 % by a MeHg challenge in cells. The MeHg-modified C-terminal catalytic domain in PDI was detected by MALDI-TOF/MS. Furthermore, treatment with MeHg significantly attenuated the enzymatic activity of PDI. Taken together, these observations suggest that MeHg results in ER stress and following the unfolded protein response pathway via ER dysfunction due to S-mercuration of the C-terminus of PDI.