Epigenetic regulation of breast cancer metastasis.

Breast cancer is the most frequently diagnosed malignancy and the second leading cause of cancer-related mortality among women worldwide. Recurrent metastasis is associated with poor patient outcomes and poses a significant challenge in breast cancer therapies. Cancer cells adapting to a new tissue microenvironment is the key event in distant metastasis development, where the disseminating tumor cells are likely to acquire genetic and epigenetic alterations during the process of metastatic colonization. Despite several decades of research in this field, the exact mechanisms governing metastasis are not fully understood. However, emerging body of evidence indicates that in addition to genetic changes, epigenetic reprogramming of cancer cells and the metastatic niche are paramount toward successful metastasis. Here, we review and discuss the latest knowledge about the salient attributes of metastasis and epigenetic regulation in breast cancer and crucial research domains that need further investigation.

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.

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).

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.

Acute exposure to abuse-like concentrations of toluene induces inflammation in mouse lungs and brain.

Toluene is an aromatic hydrocarbon commonly abused by young adolescents for its central nervous system depressant effects. Although toluene's pharmacological effects at high concentrations are relatively well-known, few studies have assessed toluene's effects on lung and brain tissues. The present study characterized the pathological effects of acute inhaled toluene exposure in the lungs and brains of male Swiss-Webster mice (N = 68). Using a static vapor exposure chamber, mice (PND 28) received a single 30-min toluene administration (0, 1000, 2000, or 4000 ppm). Lung and brain tissues were extracted 24-h post-exposure. Histology results revealed significant changes in the morphology of lung tissue (e.g., irregular cellular architecture) with the 2000- and 4000-ppm exposures expressing greater signs of pathology than control 0-ppm exposure. Markers of immune system activity (F4/80 and Ly-6G) and cellular proliferation (Ki-67) in the lung revealed no significant differences. Additionally, brain tissues were analyzed for changes of astrogliosis (glial fibrillary acidic protein [GFAP]) and oxidative stress (glutathione peroxidase [GPx]). GFAP showed increased astrogliosis in the striatum with 2000-ppm toluene showing significantly higher expression than control (p < 0.05) and a marginal effect in the hippocampus. No other markers showed significant changes. The increased signs of inflammation and cellular damage suggest that exposure to a single high concentration of toluene, typical of abuse, is capable of producing pathology in both lung and brain tissue.

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.

Connections between metabolism and epigenetics in cancers.

In the past half century, our version on cancer, from tumor initiation, growth, to metastasis, is dominated by genetic mutation. The importance of metabolism and epigenetics was not recognized until most recently. Extensive cell proliferation is one of the hallmarks of cancers. To support the energetic and anabolic demands of enhanced proliferation, tumors reprogram the pathways of nutrient procurement and metabolism. In this context, a new link between metabolic alterations and cancer progression has been unraveled over the last decade by the studies conducted in the area of cancer cell metabolism. Cancer cells are known to alter their metabolic profile during the course of tumorigenesis and metastasis thereby exhibiting a tightly regulated program of metabolic plasticity. Noteworthy, certain metabolic alteration are known to occur at the epigenetic level, thus making epigenetics and metabolism highly interwoven in a reciprocal manner. Metabolites that are generated during metabolic pathways, such as in glycolytic cycle and oxidative phosphorylation, serve as cofactors or substrates for the enzymatic reactions that catalyze the epigenetic modifications and transcriptional regulation. Several studies also indicate that the epigenome is sensitive to cellular metabolism. Since many of the metabolic alterations and consequently aberrated epigenetic regulation are common to a wide range of cancer types, they serve as promising targets for anti-cancer therapies. Here we discuss the latest findings in cancer cell metabolism, elucidating the major anabolic, catabolic and energetic demands required for sustaining cancer growth, and the influence of altered metabolism on epigenetics and vice versa. A comprehensive research pertaining to metabolomic profiling and epigenome interactors/mediators in malignant neoplasias is imperative in deciphering the potential targets that can be exploited for the development of robust anti-cancer therapies.

New discoveries of mdig in the epigenetic regulation of cancers.

Mineral dust-induced gene (mdig) encodes a member of the evolutionarily conserved JmjC family proteins that play fundamental roles in regulating chromatin-based processes as well as transcription of the genes in eukaryotic cells. This gene is also named as myc-induced nuclear antigen 53 (MINA), nucleolar protein 52 (NO52) and ribosomal oxygenase 2 (RIOX2). Increased expression of mdig had been noted in a number of human cancers, esp. lung cancer. Emerging evidence suggests that the oncogenic activity of mdig is most likely achieved through its regulation on the demethylation of histone proteins, despite it lacks the structural identities of the demethylases. Here, we discuss the latest discoveries on the characteristics of the mdig protein and its roles in a wide variety of normal and carcinogenic processes. We will also provide perspectives on how mdig is involved in the maintenance and differentiation of the embryonic stem cells, somatic stem cells and cancer stem cells.

Metabolic and epigenetic reprogramming in the arsenic-induced cancer stem cells.

At present, the belief that genetic mutations control every aspect of tumorigenesis is still very popular. Even for the highly debated "bad luck" theory of cancers, it ascertained that random mutation of genes during the self-renewal of somatic stem cells is responsible for cancer initiation. Logically, most of the new therapeutic strategies so far, from molecular targeting to precision medicine or personalized medicine, are genome-obsessed and focused on identifying and targeting these mutated genes. Accordingly, a rather simplified therapeutic regimen was formulated: cancers with the same mutations, e.g., lung cancer, pancreatic cancer, breast cancer, ovarian cancer, etc, were managed with the same chemo or targeting medicine, whereas for a particular cancer, such as breast cancer or lung cancer, with different mutational spectrums was treated with different, so-called personalized medicine. The outcomes of this strategy, however, are mixed with encouraging and disappointing findings. In this review article, we will address the importance of non-genetic factors, the metabolic and epigenetic reprogramming, during the induction of cancer stem cells in response to arsenic, a major environmental human carcinogen. The information provided may not only advance our understanding of carcinogenic mechanism to a new level but also help in designing new strategies through targeting the metabolic and epigenetic signaling pathways for cancer therapy.

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.

Reactive oxygen species contribute to arsenic-induced EZH2 phosphorylation in human bronchial epithelial cells and lung cancer cells.

Our previous studies suggested that arsenic is able to induce serine 21 phosphorylation of the EZH2 protein through activation of JNK, STAT3, and Akt signaling pathways in the bronchial epithelial cell line, BEAS-2B. In the present report, we further demonstrated that reactive oxygen species (ROS) were involved in the arsenic-induced protein kinase activation that leads to EZH2 phosphorylation. Several lines of evidence supported this notion. First, the pretreatment of the cells with N-acetyl-l-cysteine (NAC), a potent antioxidant, abolishes arsenic-induced EZH2 phosphorylation along with the inhibition of JNK, STAT3, and Akt. Second, H2O2, the most important form of ROS in the cells in response to extracellular stress signals, can induce phosphorylation of the EZH2 protein and the activation of JNK, STAT3, and Akt. By ectopic expression of the myc-tagged EZH2, we additionally identified direct interaction and phosphorylation of the EZH2 protein by Akt in response to arsenic and H2O2. Furthermore, both arsenic and H2O2 were able to induce the translocation of ectopically expressed or endogenous EZH2 from nucleus to cytoplasm. In summary, the data presented in this report indicate that oxidative stress due to ROS generation plays an important role in the arsenic-induced EZH2 phosphorylation.

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.

Association of progesterone receptor gene polymorphism with male infertility and clinical outcome of ICSI.

PURPOSE: To investigate the association of Progesterone Receptor (PR) gene variations and male infertility METHODS: DNA extraction, PCR and sequencing of PR gene, PROGINS insertion by PCR. Association of the variations with seminal parameters and outcomes of ICSI. RESULTS: Four known SNPs in the PR gene were identified in the study of which three (rs3740753, rs1042838, rs104283) were co-inherited and in complete linkage disequilibrium with the PROGINS Alu insertion. There were no differences in their frequencies between fertile and infertile males. The rs2020880 was found at a very low frequency only in the controls but not in the infertile subjects. The sperm counts, fertilization rate, embryo quality or pregnancy rates were not different in individuals with or without PROGINS allele. CONCLUSION: PR gene alterations are not associated with male infertility or ICSI outcome.

Disruption of the tumor suppressor-like activity of aryl hydrocarbon receptor by arsenic in epithelial cells and human lung cancer.

As the most classic and extensively studied transcription factor in response to environmental toxic chemicals, the human aryl hydrocarbon receptor (AHR) has been implicated in mediating some oncogenic responses also. Limited information is available, however, on whether arsenic, a widely presented environmental carcinogen, can regulate AHR to exert its carcinogenic activity. Through chromatin immunoprecipitation and sequencing (ChIP-seq), CRISPR-Cas9 gene editing, RNA-seq, and immunohistochemistry (IHC), in this report we provided evidence showing that arsenic enforces TGFß and other oncogenic signaling pathways in bronchial epithelial cells through disrupting the tumor suppressor-like activity of AHR. AHR is normally enriched on a number of oncogenic genes in addition to the known phase I/II enzymes, such as genes in TGFß and Nrf2 signaling pathways and several known oncogenes. Arsenic treatment substantially reduced the binding of AHR on these genes followed by an increased expression of these genes. CRISPR-Cas9-based knockout of AHR followed by RNA-seq further demonstrated increased expression of the TGFß signaling and some oncogenic signaling pathway genes in the AHR knockout cells. IHC studies on human tissue samples revealed that normal human lung tissues expressed high level of AHR. In contrast, the AHR expression was diminished in the lung cancer tissues. Accordingly, the data from this study suggest that AHR has tumor suppressor-like activity for human lung cancer, and one of the carcinogenic mechanisms of arsenic is likely mediated by the inhibition of arsenic on the tumor suppressor-like activity of AHR.

Combinatorial treatment of mammospheres with trastuzumab and salinomycin efficiently targets HER2-positive cancer cells and cancer stem cells.

A major obstacle in the successful treatment of cancer is the occurrence of chemoresistance. Cancer cells surviving chemotherapy and giving rise to a recurrence of the tumor are termed cancer stem cells and can be identified by elevated levels of certain stem cell markers. Eradication of this cell population is a priority objective in cancer therapy. Here, we report elevated levels of stem cell markers in MCF-7 mammospheres. Likewise, an upregulation of HER2 and its differential expression within individual cells of mammospheres was observed. Sorting for HER2(high) and HER2(low) cells revealed an upregulation of stem cell markers NANOG, OCT4 and SOX2 in the HER2(low) cell fraction. Accordingly, HER2(low) cells also showed reduced proliferation, ductal-like outgrowths and an increased number of colonies in matrigel. Xenografts from subcutaneously injected HER2(low) sorted cells exihibited earlier onset but slower growth of tumors and an increase in stem cell markers compared to tumors developed from the HER2(high) fraction. Treatment of mammospheres with salinomycin reduced the expression of SOX2 indicating a selective targeting of cancer stem cells. Trastuzumab however, did not reduce the expression of SOX2 in mammospheres. Furthermore, a combinatorial treatment of mammospheres with trastuzumab and salinomycin was superior to single treatment with each drug. Thus, targeting HER2 expressing tumors with anti-HER2 therapies will not necessarily eliminate cancer stem cells and may lead to a more aggressive cancer cell phenotype. Our study demonstrates efficient killing of both HER2 positive cells and cancer stem cells, hence opening a possibility for a new combinatorial treatment strategy.

Epigenetics and environment in breast cancer: New paradigms for anti-cancer therapies.

Breast cancer remains the most frequently diagnosed cancer in women worldwide. Delayed presentation of the disease, late stage at diagnosis, limited therapeutic options, metastasis, and relapse are the major factors contributing to breast cancer mortality. The development and progression of breast cancer is a complex and multi-step process that incorporates an accumulation of several genetic and epigenetic alterations. External environmental factors and internal cellular microenvironmental cues influence the occurrence of these alterations that drives tumorigenesis. Here, we discuss state-of-the-art information on the epigenetics of breast cancer and how environmental risk factors orchestrate major epigenetic events, emphasizing the necessity for a multidisciplinary approach toward a better understanding of the gene-environment interactions implicated in breast cancer. Since epigenetic modifications are reversible and are susceptible to extrinsic and intrinsic stimuli, they offer potential avenues that can be targeted for designing robust breast cancer therapies.

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.

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.

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.

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.