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.
