Cooperation between NRF2-mediated transcription and MDIG-dependent epigenetic modifications in arsenic-induced carcinogenesis and cancer stem cells

Environmental exposure to arsenic, a well-established carcinogen linked to a number of human cancers, is a public health concern in many areas of the world. Despite extensive studies on the molecular mechanisms of arsenic-induced carcinogenesis, how initial cellular responses, such as activation of stress kinases and the generation of reactive oxygen species, converge to affect the transcriptional and/or epigenetic reprogramming required for the malignant transformation of normal cells or normal stem cells remains to be elucidated. In this review, we discuss some recent discoveries showing how the transcription factor NRF2 and an epigenetic regulator, MDIG, contribute to the arsenic-induced generation of cancer stem-like cells (CSCs) as determined by applying CRISPR-Cas9 gene editing and chromosome immunoprecipitation followed by DNA sequencing (ChIP-seq)

New Discoveries and Ambiguities of Nrf2 and ATF3 Signaling in Environmental Arsenic-Induced Carcinogenesis

Environment exposure to arsenic had been linked to increased incidents of human cancers. In cellular and animal experimental systems, arsenic has been shown to be highly capable of activating several signaling pathways that play critical roles in cell growth regulation, malignant transformation and the stemness of cancer stem-like cells. Emerging evidence indicates certain oncogenic properties of the Nrf2 transcription factor that can be activated by arsenic and many other environmental hazards. In human bronchial epithelial cells, our most recent data suggested that arsenic-activated Nrf2 signaling fosters metabolic reprogramming of the cells through shifting mitochondrial TCA cycle to cytosolic glycolysis, and some of the metabolites in glycolysis shunt the hexosamine biosynthesis and serine-glycine pathways important for the energy metabolism of the cancer cells. In the current report, we further demonstrated direct regulation of oncogenic signals by arsenic-activated Nrf2 and connection of Nrf2 with ATF3 stress transcription factor. Meanwhile, we also highlighted some unanswered questions on the molecular characteristics of the Nrf2 protein, which warrants further collaborative efforts among scientists for understanding the important role of Nrf2 in human cancers either associated or not to environmental arsenic exposure

Regulation of PKM2 and Nrf2-ARE pathway during benzoquinone induced oxidative stress in yolk sac hematopoietic stem cells.

Benzene is an occupational toxicant and an environmental pollutant that is able to induce the production of reactive oxygen species (ROS), causing oxidative stress and damages of the macromolecules in target cells, such as the hematopoietic stem cells. We had previously found that embryonic yolk sac hematopoietic stem cells (YS-HSCs) are more sensitive to benzene toxicity than the adult bone marrow hematopoietic stem cells, and that nuclear factor-erythroid-2-related factor 2 (Nrf2) is the major regulator of cytoprotective responses to oxidative stress. In the present report, we investigated the effect of PKM2 and Nrf2-ARE pathway on the cellular antioxidant response to oxidative stress induced by benzene metabolite benzoquinone (BQ) in YS-HSC isolated from embryonic yolk sac and enriched by magnetic-activated cell sorting (MACS). Treatment of the YS-HSC with various concentrations of BQ for 6 hours induces ROS generation in a dose-dependent manner. Additional tests showed that BQ is also capable of inducing expression of NADPH oxidase1 (NOX1), and several other antioxidant enzymes or drug-metabolizing enzymes, including heme oxygenase 1 (HMOX1), superoxide dismutase (SOD), catalase and NAD(P)H dehydrogenase quinone 1 (NQO1). Concomitantly, only the expression of PKM2 protein was decreased by the treatment of BQ but not the PKM2 mRNA, which suggested that BQ may induce PKM2 degradation. Pretreatment of the cells with antioxidant N-acetylcysteine (NAC) decreased ROS generation and prevented BQ-induced PKM2 degradation, suggesting involvement of ROS in the PKM2 protein degradation in cellular response to BQ. These findings suggest that BQ is a potent inducer of ROS generation and the subsequent antioxidant responses of the YS-HSC. The accumulated ROS may attenuate the expression of PKM2, a key regulator of the pyruvate metabolism and glycolysis

Arsenic Activates the ER Stress-Associated Unfolded Protein Response via the Activating Transcription Factor 6 in Human Bronchial Epithelial Cells

Arsenic is a well-known human carcinogen associated with a number of cancers, including lung cancers. We have previously shown that long-term exposure to an environmentally relevant concentration of inorganic arsenic (As3+) leads to the malignant transformation of the BEAS2B cells, and some of the transformed cells show cancer stem-like features (CSCs) with a significant upregulation of glycolysis and downregulation of mitochondrial oxidative phosphorylation. In the present report, we investigate the short-term effect of As3+ on the endoplasmic reticulum (ER) stress response—the “unfolded protein response (UPR)” and metabolism in human bronchial epithelial cell line BEAS-2B cells. Treatment of the cells with inorganic As3+ upregulated both glycolysis and mitochondrial respiration. Analysis of ER UPR signaling pathway using a real-time human UPR array revealed that As3+ induced a significant up-regulation of some UPR genes, including ATF6, CEBPB, MAPK10, Hsp70, and UBE2G2. Additional tests confirmed that the induction of ATF6, ATF6B and UBE2G2 mRNAs and/or proteins by As3+ is dose dependent. Chromosome immunoprecipitation and global sequencing indicated a critical role of Nrf2 in mediating As3+-induced expression of these UPR genes. In summary, our data suggest that As3+ is able to regulate the ER stress response, possibly through activating the ATF6 signaling

The ROS-generating NADPH oxidase NOX1 was induced by BQ.

<p>Immunoblotting data were shown in the upper panel. GAPDH was measured as loading control. Densitometry of immunoblotting results was shown in the lower panel. Values are presented as means ± SDs (n = 3−5). *, <i>P</i><0.05; **, <i>P</i><0.01; #, <i>P</i><0.001 compared with controls.</p

Increased expression of Nrf2-ARE pathway proteins were observed in the BQ-treated YS-HSCs.

<p>Induction of catalase, SOD1, SOD2, HMOX1 and NQO1 protein levels by BQ. Immunoblotting data were shown in the upper panel. GAPDH was measured as loading control. Densitometry of immunoblot results was shown in the lower panel. Values are presented as means ± SDs (n = 3−5). *, <i>P</i><0.05; **, <i>P</i><0.01; #, <i>P</i><0.001 compared with controls.</p

Expression of hematopoietic surface markers in E8-10 (embryonic day 8–10) murine yolk sacs.

<p>Representative flow cytometric data from more than three independent analyses were shown.</p

Expression of hematopoietic surface makers in E8-10d yolk sac cells.

<p>Expression of hematopoietic surface makers in E8-10d yolk sac cells.</p

BQ induced ROS and two possible pathways involved in the ROS detoxification.

<p>Exposure to BQ causes NOX1 up-regulation, resulting in ROS generation in YS-HSCs. Intracellular ROS elevation promotes two possible pathways: (a) Nrf2 nuclear accumulation and activation of Nrf2-ARE pathways, including detoxification enzymes and antioxidant enzymes; (b) PKM2 degradation and promoting glucose flux into the pentose phosphate pathway (PPP), leading to generate NADPH responsible for the maintain of reducing glutathione, which plays a critical role in detoxification of ROS.</p