Career Development Award: Center for Appalachian Research in Environmental Sciences: Determining the Role of the Long Non-Coding RNA ABHD11-AS1 in Cr(VI)-Induced Cell Transformation and Cancer Stem Cell-Like Property

Hexavalent chromium [Cr(VI)] is one of the most common environmental and occupational pollutants and a well-recognized carcinogen causing lung and other cancer in humans. However, the mechanism of Cr(VI) causing cancer has not been elucidated. The goal of this study is to generate critical preliminary data to support my NIEHS R01 grant application that investigates the epigenetic mechanism of Cr(VI) carcinogenesis focusing on the role of long non-coding RNAs. Epigenetics refers to heritable alterations in the pattern of gene expression that are not caused by changes in DNA sequence, but are mediated by DNA methylation, histone posttranslational modifications, and non-coding RNAs (miRNAs and long non-coding RNAs, etc.). It is now well established that epigenetics is often dysregulated in cancer and that epigenetics dysregulation plays important roles in carcinogenesis. Cancer stem cells (CSCs) are cancer cells possessing characteristics of normal stem cells and CSCs or CSC-like cells are considered as cancer initiating and maintaining cells. While the mechanisms of generating CSCs or CSC-like cells are not elucidated, studies showed that dysregulation of epigenetics play crucial roles in producing CSCs or CSC-like cells. Non-coding RNAs refer to a variety of RNA molecules that are not translated into proteins. The long non-coding RNAs (lncRNAs) are RNA molecules that are longer than 200 bp in length and are not translated into proteins. With the completion of human genome sequencing projects, a large number of lncRNAs have been identified. Studies showed that lncRNAs are key players in regulation of gene transcription through the recruitment of chromatin epigenetic modifying enzymes and other mechanisms. Although emerging evidence suggests that lncRNAs are implicated in important physiology and pathology processes such as development and carcinogenesis, the biological functions of the majority of lncRNAs remain largely unknown. Previous studies showed that that Cr(VI) exposure causes genotoxic effects, which is thought to play important roles in Cr(VI) carcinogenicity. However, studies also showed that Cr(VI) exposure causes epigenetic changes. For example, studies on lung tumors from workers exposed to chromate revealed increased DNA methylation in the promoter regions of several tumor suppressor genes. In addition, studies also revealed that Cr(VI) exposure changes histone posttranslational modifications in cultured cells. However, it remains largely unknown whether Cr(VI) exposure dysregulates non-coding RNAs, particularly long non-coding RNAs, an important epigenetic mechanism regulating gene expression. Moreover, it is not clear whether long non-coding RNAs play an important role in Cr(VI)-induced cell transformation, cancer stem cell-like property and tumorigenesis. Our preliminary studies found: (i) Chronic low dose Cr(VI) exposure induces cancer stem cell-like property and malignant transformation of immortalized human bronchial epithelial cells; (ii) the expression level of the long non-coding RNA ABHD11-AS1 is significantly higher in Cr(VI)-transformed two human bronchial epithelial cells (BEAS-2B and 16HBE) than that in the passage-matched control cells; and (iii) siRNA knockdown of the expression of ABHD11-AS1 in Cr(VI)-transformed cells significantly reduces their transformed phenotypes such as anchorage-independent growth and cancer stem cell-like property. Moreover, ours and other recent studies showed that the expression level of ABHD11-AS1 is significantly increased in bladder, gastric, ovarian and lung cancer tissues, suggesting that up-regulation of the long non-coding RNA ABHD11-AS1 may play an important role in cancer development and progression. Based on recent literature reports and our preliminary studies, we hypothesize that down-regulation of ABHD11-AS1 significantly reduces chronic low dose Cr(VI) exposure-induced cell transformation, cancer stem cell-like property and tumorigenesis. This proposed study will demonstrate a critical role of the long non-coding RNA ABHD11-AS1 in Cr(VI)-induced cell transformation and cancer stem cell-like property, generating key preliminary data for my NIEHS R01 application. Two aims are proposed: Specific Aim 1: Determine the role of ABHD11-AS1 in chronic low dose Cr(VI) exposure-induced cell transformation. We will use lentiviral-mediated potent gene knockdown technology to generate ABHD11-AS1 stable knockdown cell lines for studying the role of ABHD11-AS1 in chronic low dose Cr(VI) exposure-induced cell malignant transformation and tumorigenesis. Specific Aim 2: Determine the role of ABHD11-AS1 in Cr(VI)-induced cancer stem cell (CSC)-like property. We will use a series of in vitro cell culture and in vivo mouse tumorigenesis models to determine the role of ABHD11-AS1 in Cr(VI)-induced cancer stem cell (CSC)-like property