We have shown that consecutive treatment of the human bronchial epithelial cells with the environmentally relevant concentration of As3+ (0.125 – 0.25M), an environmental metalloid metal, for six months, induces transformation of the human bronchial epithelial cells, some of which possess characteristics of the cancer stem-like cells (CSCs), such as tumor sphere formation in vitro, self- renewal in vivo, increased expression of the stemness genes, including Oct4, Sox2, KLF4, and c-myc. In addition, these cancer stem-like cells exhibited a pronounced increase in the expression of several microRNAs, most notably, the miR-214, miR-199, miR-10b, miR-34b, etc. Furthermore, integrated transcriptomic and metabolomic analyses demonstrated a higher rate of glycolysis and lower levels of mitochondrial metabolism due to mitochondrial DNA (mtDNA) depletion among these As3+-induced CSCs. Lastly, a unique glycolytic feature that is different from naïve embryonic stem cells (ESCs) and cancer cells was found in these As3+-induced CSCs. Both ESCs and cancer cells direct glycolysis for lactate production. In contrast, the As3+-induced CSCs show increased conversion of the glycolytic intermediates into the subsidiary pathways for the generation of N-acetylglucosamine important for O- GlcNAcylation of the stemness genes and the S-adenosyl methionine (SAM) that contributes to DNA and histone methylation. Accordingly, the goal of this application is to determine: (1) is As3+-induced miRNAs, esp. miR214/199, responsible for the depletion of mtDNA and the consequent inhibition of mitochondria; (2) if so, how miRNAs induced by As3+ impairs the integrity and function of mtDNA and mitochondria; and (3) how the impaired function of mitochondria contributes to the generation of the CSCs induced by As3+. We hypothesize that As3+-induced JNK-dependent pSTAT3S727 and miR-214/199 switch mitochondrial OXPHOS to glycolysis for the formation of CSCs. To test this hypothesis, the following three specific aims are proposed: Specific Aim 1: As3+-activated JNK and pSTAT3S727 enforce expression of miR-214 and miR-199 that down-regulate mitochondrial transcription factor A (TFAM) in BEAS-2B and other lung cells for the generation of CSCs. We will focus on the transcriptional regulation of the miR-214/199 cluster with emphases on promoter DNA methylation and transcription factor binding in cellular response to As3+ and its down-stream signaling; Specific aim 2: Understand how As3+-induced JNK, miR-214/199 and mitochondrial dysfunction contribute to the formation of CSCs with an emphasis on metabolic reprogramming from OXPHOS to glycolysis. Specific Aim 3: Defining the causal roles of As3+-induced JNK- and miR-214/199-dependent metabolic reprogramming in the changes of epigenetics related to chromatin structure and accessibility that linked to self-renewal and/or differentiation of the As3+-induced CSCs through high-throughput profiling. We will identify metabolite- dependent epigenetic and chromatin changes in non-transformed cells, As3+-induced transformed cells and CSCs, which will be further verified through overexpressing or CRISPR-Cas9-based knockdown of the key genes in the related metabolic pathways and monitoring the self-renewal and differentiation status of the CSCs. The high-throughput approaches will include ChIP-seq to map H3K9me3, H3K27me3, and H3K4me3, and RNA-seq to profile transcription of the genes, esp. for those contributing to the pluripotency, self-renewal and differentiation of the CSCs. We anticipate that the results from the proposed studies will unravel importance of As3+-induced miR-214/199 on the generation of CSCs and lead to emerging of new concepts of As3+ carcinogenesis by emphasizing the capability of As3+ in CSC induction. Moreover, we believe that the date generated from this project will help us in developing novel therapeutic strategies by targeting JNK, miR-214/199 and CSCs through utilizing our unique mouse orthotopical lung cancer model in NOD/SCID mice in a separate research project.

Project Narrative Environmental exposure to carcinogenic metal (metalloid) arsenic, especially the trivalent form, As3+, has long been a major public health concern. The research proposed in this application is aimed at increasing our understanding of mechanisms by which As3+ induces generation of the cancer stem-like cells (CSCs). Main efforts will be on the role of As3+-induced JNK, phosphorylation of serine 727 of STAT3 (pSTAT3S727) on the expression of miR-214 and miR-199 that inhibit mitochondrial transcription factor A (TFAM) and the depletion of mitochondrial DNA (mtDNA). Results from these studies are anticipated to generate new concepts indicating that induction of CSCs is the central mechanism of As3+-induced human cancer. The proposed preclinical testing of targeting therapy on miR-214, miR-199, O-GlcNAc, and glycolysis in unique orthotopic mouse model of lung cancer mimics relevant molecular and clinical features of the human cancer, which has the potential of identifying new target-based strategies of cancer therapy.