Mechanism of Hexavalent Chromium Carcinogenesis - Role of Long Non-coding RNA Dysregulation

Hexavalent chromium [Cr(VI)] is one of the most common and well-recognized environmental and occupational carcinogens causing lung and other cancers, however, the mechanism of Cr(VI) carcinogenesis remains elusive. Previous studies on the mechanism of Cr(VI) carcinogenesis mostly focus on its genotoxic effects mainly due to the fact that Cr(VI) undergoes a series of metabolic reductions inside cells to generate reactive Cr metabolites and reactive oxygen species, which produce various genotoxic effects playing important roles in Cr(VI) carcinogenesis. On the other hand, studies showed that Cr(VI) exposure also causes non-genotoxic effects such as epigenetic changes as evidenced by dysregulations in DNA methylation and histone posttranslational modifications. While these observations open new avenues for studying the mechanism of Cr(VI) carcinogenesis, it remains to be determined how Cr(VI) exposure-caused epigenetic dysregulations contribute to Cr(VI) carcinogenesis. Moreover, very little is known about the role of non-coding RNAs (ncRNAs) especially the long non-coding RNAs (lncRNAs), another important epigenetic mechanism regulating gene expression, in Cr(VI) carcinogenesis. The goal of this study is to investigate the mechanism of Cr(VI) carcinogenesis focusing on the role of lncRNA dysregulation. The lncRNAs are RNA molecules that are longer than 200 bp in length and are not translated into proteins. Accumulating evidence shows that lncRNAs are emerging key regulators of gene expression. Recent studies show that lncRNAs play crucial roles in cancer stem cell (CSC) differentiation and maintenance and lncRNAs are critically involved in carcinogenesis. CSCs or CSC-like cells are considered as cancer initiating and maintaining cells and are thought to be responsible for cancer initiation, metastasis, relapse and treatment resistance. Excitingly, ours and other recent study showed that chronic Cr(VI) exposure generates CSC-like cells, which may play a critical role in Cr(VI) carcinogenesis. However, the mechanism of how Cr(VI) exposure produces CSC-like cells is unclear. Moreover, whether chronic Cr(VI) exposure dysregulates lncRNA expression and whether lncRNA dysregulation plays a role in Cr(VI)-induced cell malignant transformation, CSC-like property and tumorigenesis are unknown. Our preliminary studies found: (i) Chronic Cr(VI) exposure up-regulates the expression of the lncRNA ABHD11-AS1, which contributes causally to Cr(VI)-induced cell transformation, CSC-like property and tumorigenesis; (ii) Chronic Cr(VI) exposure down-regulates the expression of miR-182-5p; but knockdown of ABHD11-AS1 increases the level of miR-182-5p. Moreover, overexpressing miR-182-5p in Cr(VI)-transformed cells significantly reduces their transformed phenotypes, phenocopying the effect of ABHD11-AS1 knockdown; (iii) The Rho GTPase Rac1 and Erk1/2 MAPK are highly activated in Cr(VI)-transformed cells and inhibition of Rac1 or Erk1/2 significantly reduce their transformed phenotypes; (iv) The expression level of the oncogenic Rac-GEF (guanine nucleotide exchange factor) Tiam1 is significantly up-regulated in Cr(VI)-transformed cells; but overexpressing miR-182-5p or knockdown of ABHD11-AS1 greatly decrease Tiam1 level; (v) The expression level of poly (ADP-ribose) polymerase-1 (PARP-1), a critical DNA repair gene and a key regulator of gene expression, is significantly up-regulated in Cr(VI)-transformed cells; (vi) The level of protein PARylation is drastically increased in Cr(VI)-transformed cells; inhibition of PARP-1 activity or knockdown of PARP-1 expression greatly decrease the levels of protein PARylation and ABHD11-AS1 and the transformed phenotype of Cr(VI)-transformed cells; (vii) The nuclear interaction between PARP-1 and histone lysine demethylase KDM5B is significantly increased in Cr(VI)-transformed cells. Based on literature review and our novel preliminary data, our central hypothesis is: “Chronic Cr(VI) exposure-upregulated lncRNA ABHD11-AS1 sponges miR-182-5p and causes its down-regulation, which leads to increased level of the oncogenic Rac-GEF Tiam1 promoting Cr(VI) carcinogenesis”. This study focusing on lncRNA dysregulation not only provides novel mechanistic insights for understanding Cr(VI) carcinogenicity, our lncRNA studies may also be applicable to other metal carcinogenesis studies and may have a broad impact on metal carcinogenesis field and move the field forward. Three aims are proposed: Specific Aim 1: To determine the mechanism of how chronic Cr(VI) exposure up-regulates the expression of lncRNA ABHD11-AS1 focusing on the role of PARP-1-regulated key chromatin modifiers. We will analyze ABHD11-AS1 promoter recruitment of H3K4me3, RNA polymerase II, PARP-1, histone lysine demethylase KDM5B and its PARylation, the underlying mechanisms and their role in ABHD11-AS1 expression. Specific Aim 2: To demonstrate that ABHD11-AS1 sponges miR-182-5p causing its down-regulation and promoting chronic Cr(VI)-exposure-induced CSC-like property, cell transformation and tumorigenesis. We will demonstrate a direct interaction between ABHD11-AS1 and miR-182-5p by 3’UTR luciferase reporter-like assay and MS2-TRAP (MS2-tagged RNA affinity purification) assay; and determine the effect of miR-182-5p overexpression on chronic Cr(VI) exposure-induced CSC-like property, cell transformation and tumorigenesis. Specific Aim 3: To demonstrate that down-regulation of miR-182-5p increases level of the oncogenic Rac-GEF Tiam1 promoting chronic Cr(VI)-exposure-induced CSC-like property, cell transformation and lung tumorigenesis in mice. We will use luciferase reporter assay, Tiam1 stable knockdown cells and Tiam1 knockout mice to demonstrate that Tiam1 is a direct target of miR-182-5p, playing an important role in chronic Cr(VI) exposure-induced cell transformation, CSC-like property and lung tumorigenesis in mice