Genomic and epigenetic characterization of the arsenic-induced oncogenic microRNA-21.

As one of the first identified oncogenic microRNAs, the precise details concerning the transcriptional regulation and function of microRNA-21 (miR-21) are still not completely established. The miR-21 gene is situated on chromosome 17q23.2, positioned at the 3'-UTR of the gene that encodes vacuole membrane protein-1 (VMP1). In this current study, we presented evidence indicating that miR-21 possesses its own gene promoter, which can be found in the intron 10 of the VMP1 gene. Chromatin immunoprecipitation followed by global DNA sequencing (ChIP-seq) revealed the presence of a broad H3K4me3 peak spanning the entire gene body of the primary miR-21 and the existence of super-enhancer clusters in the close proximity to both the miR-21 gene promoter and the transcription termination site in arsenic (As3+)-induced cancer stem-like cells (CSCs) and human induced pluripotent stem cells (hiPSCs). In non-transformed human bronchial epithelial cells (BEAS-2B), As3+ treatment enhanced Nrf2 binding to both the host gene VMP1 of miR-21 and the miR-21 gene. Knockout of Nrf2 inhibited both the basal and As3+-induced expressions of miR-21. Furthermore, the As3+-enhanced Nrf2 peaks in ChIP-seq fully overlap with these super-enhancers enriched with H3K4me1 and H3K27ac in the miR-21 gene, suggesting that Nrf2 may coordinate with other transcription factors through the super-enhancers to regulate the expression of miR-21 in cellular response to As3+. These findings demonstrate the unique genetic and epigenetic characteristics of miR-21 and may provide insights into understanding the novel mechanisms linking environmental As3+ exposure and human cancers.

Tumor suppressive activity of AHR in environmental arsenic-induced carcinogenesis.

The aryl hydrocarbon receptor (AHR) is a highly conserved pleiotropic transcription factor that senses environmental pollutants, microbial products, and endogenous ligands. The transcriptional targets of AHR include phase I and phase II detoxification enzymes, as well as numerous signaling molecules that affect a wide spectrum of biological and biochemical processes in a manner of cellular context-dependent. In this review, we systematically assess the latest discoveries of AHR in carcinogenesis with an emphasis on its tumor suppressor-like property that represses the expression of genes in oncogenic signaling pathways. Additionally, we outline recent progress in our studies on the interaction among AHR, TGFb and NRF2 in cellular responses to arsenic and malignant transformation. Our findings indicate that AHR antagonized TGFb and NRF2, suggesting that AHR could serve as a potential tumor suppressor in arsenic-induced carcinogenesis. Notably, while AHR can exhibit both oncogenic and tumor-suppressive properties in cancer development and the generation of the cancer stem-like cells (CSCs), the tumor suppressor-like effect of AHR warrants further extensive exploration for the prevention and clinical treatment of cancers.

Deletion of mdig enhances H3K36me3 and metastatic potential of the triple negative breast cancer cells.

In this report, we provide evidence showing diminished expression of the mineral dust-induced gene (mdig), a previously identified oncogenic gene, in human triple negative breast cancer (TNBC). Using a mouse model of orthotopic xenograft of the TNBC MDA-MB-231 cells, we demonstrate that mdig promotes the growth of primary tumors but inhibits metastasis of these cells in vivo. Knockout of mdig resulted in an enhancement of H3K36me3 in the genome and upregulation of some X chromosome-linked genes for cell motility, invasion, and metastasis. Silencing MAGED2, one of the most upregulated and H3K36me3-enriched genes resulted from mdig depletion, can partially reverse the invasive migration of the mdig knockout cells. The anti-metastatic and inhibitory role of mdig on H3K36me3 was cross-validated in another cell line, A549 lung cancer cells. Together, our data suggest that mdig is antagonist against H3K36me3 that enforces expression of genes, such as MAGED2, for cell invasion and metastasis.

Depletion of Mdig Changes Proteomic Profiling in Triple Negative Breast Cancer Cells.

Triple-negative breast cancers are highly aggressive with an overall poor prognosis and limited therapeutic options. We had previously investigated the role of mdig, an oncogenic gene induced by some environmental risk factors, on the pathogenesis of breast cancer. However, a comprehensive analysis of the proteomic profile affected by mdig in triple-negative breast cancer has not been determined yet. Using label-free bottom-up quantitative proteomics, we compared wildtype control and mdig knockout MDA-MB-231 cells and identified the proteins and pathways that are significantly altered with mdig deletion. A total of 904 differentially expressed (p < 0.005) proteins were identified in the KO cells. Approximately 30 pathways and networks linked to the pathogenicity of breast cancer were either up- or downregulated, such as EIF2 signaling, the unfolded protein response, and isoleucine degradation I. Ingenuity Pathway Analysis established that the differentially expressed proteins have relevant biological actions in cell growth, motility, and malignancy. These data provide the first insight into protein expression patterns in breast cancer associated with a complete disruption of the mdig gene and yielded substantial information on the key proteins, biological processes, and pathways modulated by mdig that contribute to breast cancer tumorigenicity and invasiveness.

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.

Arsenic activates STAT3 signaling during the transformation of the human bronchial epithelial cells.

Arsenic (As3+), a metalloid abundant in environment, is classified as a group I carcinogen associated with several common human cancers, including cancers in lung, skin, bladder, liver, and prostate (Wei et al., 2019). The mechanisms of As3+-induced carcinogenesis had been extensively studied, and different mechanisms might be involved in different types of cancer (Wei et al., 2019). Recent studies showed that exposure to a high dose of arsenic is able to induce lung cancer. Meanwhile, prolonged exposure to a low concentration of arsenic can increase the risk of lung cancer also (Liao et al., 2009; Fernández et al., 2012). Emerging evidence indicated that prolonged exposure to arsenic promotes malignant transformation and some of the transformed cells have cancer-stem-like properties (Ngalame et al., 2014). In the present report, we revealed that exposure to As3+ for short time period inhibited tyrosine-705 phosphorylation of signal transducer and activator of transcription 3 (pSTAT3Y705) and induced Src homology region 2 domain-containing phosphatase-1 (SHP-1) in bronchial epithelial cell line, BEAS-2B. In addition, we found that long term exposure of the cells to As3+ activates phosphorylation of STAT3 at serine 727 (pSTAT3S727) as well as pSTAT3Y705. Moreover, As3+ is able to induce the expression of miRNA-21 (miR-21) and decrease the expression of PDCD4. Taken together, our data suggest that activation of STAT3 and induction of miR-21 are important contributing factors to the reduced expression of PDCD4, which may play significant role in As3+-induced transformation of BEAS-2B cells.

Metabolomic dynamics of the arsenic-transformed bronchial epithelial cells and the derived cancer stem-like cells.

Accumulating evidence indicates a carcinogenic role of environmental arsenic exposure, but mechanisms on how arsenic fosters malignant transformation of the normal cells are not fully established. By applying untargeted global metabolomics approach, we now show that arsenic is highly capable of perturbing the intracellular metabolic programs of the human bronchial epithelial cells, some of which are prominent hallmarks of cancer cell metabolism. To understand the spatiotemporal patterns of arsenic regulation on multiple metabolic pathways, we treated the cells with environmentally relevant concentration of arsenic, 0.25 µM, consecutively for 6 weeks to 24 weeks, and found that arsenic prompted heme metabolism, glycolysis, sphingolipid metabolism, phospholipid catabolism, protein degradation, and cholesterol breakdown constitutively, but inhibited metabolism of uracil-containing pyrimidine, carnitine, serotonin, polyamines, and fatty acid ß-oxidation. A strong inhibition of all metabolites in mitochondrial tricarboxylic acid (TCA) cycle was noted in the cells treated with As3+ for 6 to 13 weeks. However, the metabolites in the earlier, but not the later steps of TCA cycle, including citrate, aconitate and isocitrate, were induced at 16 weeks through 24 weeks of arsenic treatment. This comprehensive metabolomics analysis provides new insights into metabolic perturbation by arsenic and may lead to more precise indications of arsenic in molecular carcinogenesis.

Environmentally-induced mdig contributes to the severity of COVID-19 through fostering expression of SARS-CoV-2 receptor NRPs and glycan metabolism.

The novel ß-coronavirus, SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19), has infected more than 177 million people and resulted in 3.84 million death worldwide. Recent epidemiological studies suggested that some environmental factors, such as air pollution, might be the important contributors to the mortality of COVID-19. However, how environmental exposure enhances the severity of COVID-19 remains to be fully understood. In the present report, we provided evidence showing that mdig, a previously reported environmentally-induced oncogene that antagonizes repressive trimethylation of histone proteins, is an important regulator for SARS-CoV-2 receptors neuropilin-1 (NRP1) and NRP2, cathepsins, glycan metabolism and inflammation, key determinants for viral infection and cytokine storm of the patients. Depletion of mdig in bronchial epithelial cells by CRISPR-Cas-9 gene editing resulted in a decreased expression of NRP1, NRP2, cathepsins, and genes involved in protein glycosylation and inflammation, largely due to a substantial enrichment of lysine 9 and/or lysine 27 trimethylation of histone H3 (H3K9me3/H3K27me3) on these genes as determined by ChIP-seq. Meanwhile, we also validated that environmental factor arsenic is able to induce mdig, NRP1 and NRP2, and genetic disruption of mdig lowered expression of NRP1 and NRP2. Furthermore, mdig may coordinate with the Neanderthal variants linked to an elevated mortality of COVID-19. These data, thus, suggest that mdig is a key mediator for the severity of COVID-19 in response to environmental exposure and targeting mdig may be the one of the effective strategies in ameliorating the symptom and reducing the mortality of COVID-19.

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).

Pathological and Prognostic Indications of the mdig Gene in Human Lung Cancer.

BACKGROUND/AIMS: The mineral-dust-induced gene mdig is a lung-cancer-associated oncogene. The focus of this study is to evaluate the expression status of mdig in lung cancer and to assess its influence in predicting the patient's overall survival. METHODS: Using high-density tissue microarrays and clinical samples of synchronous multiple primary lung cancer (SMPLC), we investigated the expression of mdig through immunohistochemistry and utilized the open-access lung cancer patient databases containing genomic and transcriptomic data from the UCSC Xena and TCGA web platforms to determine the prognostic values of mdig expression status among different subtypes of lung cancer. RESULTS: mdig is upregulated in smokers and in lung squamous cell carcinoma. High mdig expression predicted poor overall survival in lung squamous cell carcinoma and female smokers. Among tumor tissues from SMPLC patients, we not only unraveled the highest positive rate of mdig expression, but also revealed a unique cytoplasmic, rather than nuclear localization of mdig protein. Furthermore, by inspecting some pathological but not cancerous lung tissues, we believe that mdig is required for the transformation of non-cancerous lung cells to the fully-fledged cancer cells. CONCLUSION: These data suggested that mdig is involved in various stages of lung carcinogenesis, possibly through the epigenetic regulation on some critical cancer-associated genes, and increased mdig expression is an important prognostic factor for some types of lung cancer.

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.

CRISPR-Cas9 gene editing causes alternative splicing of the targeting mRNA.

The technique of CRISPR-Cas9 gene editing has been widely used to specifically delete the selected target genes through generating double strand breaks (DSBs) and inducing insertion and/or deletion (indel) of the genomic DNAs in the cells. We recently applied this technique to disrupt mineral dust-induced gene (mdig), a potential oncogene as previously reported, by single guide RNA (sgRNA) targeting the third exon of mdig gene in several cell types, including human bronchial epithelial cell line BEAS-2B, lung cancer cell line A549, and human triple negative breast cancer cell line MDA-MB-231 cells. In addition to the successful knockout of mdig gene in these cells, we unexpectedly noted generation of several alternatively spliced mdig mRNAs. Amplification of the mdig mRNAs during the screening of knockout clones by reverse transcription-polymerase chain reaction (RT-PCR) and the subsequent sanger sequencing of DNA revealed deletion and alternative splicing of mdig mRNAs induced by CRISPR-Cas9 gene editing. The most common deletions include nine and twenty-four nucleotides deletion around the DSBs. In addition, interestingly, some mdig mRNAs showed skipping of the entire exon 3, or alternative splicing between exon 2 and exon 8 using the new donor and accept splicing sites, leading to deletion of exons 3, 4, 5, 6, and 7. Accordingly, cautions should be taken when using CRISPR-Cas9 strategy to edit human genes due to the unintended alterative splicing of the target mRNAs. It is very likely that new proteins, some of which may be highly oncogenic, may be generated from CRISPR-Cas9 gene editing.

Nrf2 and HIF1α converge to arsenic-induced metabolic reprogramming and the formation of the cancer stem-like cells.

In this report, we demonstrated that inorganic arsenic (iAs) induces generation of the cancer stem-like cells (CSCs) through Nrf2-dependent HIF1α activation, and the subsequent metabolic reprogramming from mitochondrial oxidative phosphorylation to glycolysis in epithelial cells. Methods: Genome-wide ChIP-seq analysis was performed to investigate the global binding of Nrf2 and/or HIF1α on the genome in the cells treated with iAs. Both untargeted metabolomics and UDP-13C-glucose flux were applied to determine metabolic reprogramming in the iAs-induced CSCs. The role of Nrf2 on iAs-induced HIF1α and other stemness gene expression was validated by lentiviral transfection of Nrf2 inhibitor Keap1 and CRISPR-Cas9-mediated Nrf2 gene knockout, respectively. Results: The CSCs induced by iAs exhibit a diminished mitochondrial oxidative phosphorylation and an enhanced glycolysis that is actively shunted to the hexosamine biosynthetic pathway (HBP) and serine/glycine pathway. ChIP-seq data revealed that treatment of the cells with iAs amplified Nrf2 enrichment peaks in intergenic region, promoter and gene body. In contrast, a shift of the HIF1α peaks from distal intergenic region to gene promoter and the first exon was noted. Both Nrf2 and HIF1α are responsible for the iAs-induced expression of the glycolytic genes and the genes important for the stemness of the CSCs. Intriguingly, we also discovered a mutual transcriptional regulation between Nrf2 and HIF1α. Inhibition of Nrf2 by lentiviral infection of Keap1, or knockout of Nrf2 by CRISPR-Cas9 gene editing, not only blocked iAs-induced HIF1α activation, but reduced the expression of the key stemness genes for the formation of CSCs also. Conclusion: We demonstrated that Nrf2 activation is an initiating signal for iAs-induced HIF1α activation, and Nrf2 and HIF1α played a concerted role on inducing metabolic reprogramming and the CSCs.

Characterization of Arsenic-Induced Cancer Stem-Like Cells.

Arsenic is a well-known human carcinogen. However, the mechanisms underlying arsenic-induced carcinogenesis remain elusive. Here we show that chronic and low level of arsenic stress induces transformation of the human bronchial epithelial cells, BEAS-2B, and that some of the transformed cells show characteristics of cancer stem-like cells (CSCs). Meanwhile, we demonstrate that arsenic stress dedifferentiates CD61+ BEAS-2B cells into CSC-like CD61- cells featured with noncanonical epithelial-mesenchymal transition (EMT), enhanced chemoresistance, and metastasis. Finally, we show that oncogene c-Myc expression is associated with arsenic-induced tumor initiation and progression. Altogether, our findings highlight a unique mechanism of arsenic-induced transformation of human bronchial epithelial cells and provide a novel therapeutic target for arsenic-initiated lung cancer.