MGMT Down-Regulation in the Carcinogenicity of Hexavalent Chromium

Hexavalent chromium [Cr(VI)] exposure causes multiple toxic effects in humans. The main concern of Cr(VI) toxicity is its carcinogenicity as Cr(VI) is one of the most well-recognized environmental and occupational carcinogens causing lung and other cancer in humans. The mechanism of Cr(VI) carcinogenicity remains elusive. Previous studies showed that Cr(VI) exposure causes genotoxic effects, which are thought to play important roles in Cr(VI) carcinogenicity. However, ours and other studies showed that Cr(VI) exposure also causes epigenetic dysregulations such as increased DNA methylation and abnormal histone posttranslational modifications. While these findings open new directions for studying Cr(VI) carcinogenicity, it remains to be determined how dysregulated epigenetics contributes to Cr(VI) carcinogenicity and whether Cr(VI)-caused epigenetic changes play a role in its genotoxic effect. The goal of this study is to investigate the mechanism of Cr(VI) carcinogenicity focusing on the role of histone H3 methyltransferases-mediated epigenetic downregulation of an important DNA repair gene O6-methylguanine-DNA methyltransferase (MGMT). MGMT is a unique and critical DNA repair protein playing a crucial role in inhibiting carcinogenesis through maintaining genome stability by repairing highly mutagenic and carcinogenic DNA lesion O6-methylguanine (O6-MeG). The O6-MeG is produced either by exposure to exogenous alkylating carcinogens or formed endogenously during cellular metabolism and inflammation process, and is repaired only by MGMT. Unrepaired O6-MeG causes G:C to A:T transition mutations, sister chromatid exchanges, chromosomal aberrations thus causing genome instability and increasing the risk of cancer. The ability of a cell to repair the O6-MeG lesion is directly related to the MGMT expression level; loss of MGMT expression generates a new mutator phenotype facilitating cancer development. Indeed, studies showed that MGMT deficient mice are hypersensitive to alkylating agents-induced carcinogenesis. In contrast, MGMT overexpression not only significantly protects mice from carcinogens-induced carcinogenesis, but also greatly reduces the frequency of spontaneous liver tumor formation in mice, indicating that endogenous O6-MeG also plays an important role in tumorigenesis. The importance of MGMT in cancer is further evidenced by the fact that loss of MGMT expression is observed in many kinds of human cancers including lung cancer. Moreover, studies also showed that cells exposed to some metal carcinogens such as cadmium or nickel exhibit reduced MGMT levels. However, whether other metal carcinogen such as Cr(VI) exposure also decreases MGMT expression level and whether MGMT plays a role in metal carcinogenesis are unknown. Our preliminary studies found: (i) Chronic Cr(VI) exposure increases the levels of histone H3 repressive methylation marks and their related histone methyltransferases (HMTases), which contribute causally to Cr(VI)-induced cancer stem cell-like property and cell transformation; (ii) Cr(VI) exposure down-regulates the expression of MGMT, which plays a causal role in Cr(VI)-induced cell transformation; (iii) Knocking down the expression of HMTases prevents the down-regulation of MGMT by Cr(VI) exposure; (iv) Cr(VI) exposure significantly increases the level of O6-MeG in cultured cells, mouse and human lung tissues. In contrast, stably expressing MGMT drastically reduces Cr(VI)-induced O6-MeG level; (v) Cr(VI)-transformed cells display impaired DNA repair capacity but stably expressing MGMT significantly reduces Cr(VI) exposure-caused DNA repair deficiency; (vi) Treatment with dihydromethysticin (DHM), a natural compound, is capable of increasing MGMT expression and drastically reducing O6-MeG level in Cr(VI)-transformed cells with no significant cytotoxicity. Thus, our central hypothesis is that up-regulation of HMTases by chronic Cr(VI) exposure down-regulates the expression of MGMT leading to increased level of highly mutagenic DNA lesion O6-MeG and promoting Cr(VI) carcinogenesis. The results to be generated from this study will provide novel mechanistic insights for understanding Cr(VI) carcinogenicity by well integrating its epigenetic and genetic effects. Three Aims are proposed: Specific Aim 1: To determine the mechanism by which chronic Cr(VI) exposure causes MGMT down-regulation. We hypothesize that Cr(VI) exposure reduces MGMT expression by up-regulating HMTases (G9a, SUV39H1 or EZH2), which increases histone H3 repressive methylation modifications and recruits DNMTs (DNA methyltransferases) to MGMT promoter region silencing its expression. The presence of H3 repressive methylation marks and DNMTs at MGMT promoter region and the underlying mechanism will be examined. Specific Aim 2: To demonstrate that enforced MGMT expression in mouse lungs using a conditional and lung specific MGMT transgenic animal model impairs Cr(VI) lung tumorigenesis. We hypothesize that enforced MGMT expression in mouse lungs impairs Cr(VI) lung tumorigenesis. A conditional and lung specific MGMT transgenic mouse model has been generated and will be used to test the hypothesis. Specific Aim 3: To investigate the protective effect of DHM treatment against Cr(VI)-induced cell transformation and tumorigenesis. We hypothesize that DHM treatment that is capable of increasing MGMT expression and reducing O6-MeG level will inhibit Cr(VI)-induced cell transformation and tumorigenesis in mice