Colemon et al. Genes and Environment (2020) 42:24 https://doi.org/10.1186/s41021-020-00162-2 RESEARCH Open Access DNA hypomethylation drives changes in MAGE-A gene expression resulting in alteration of proliferative status of cells Ashley Colemon1, Taylor M. Harris2 and Saumya Ramanathan2,3* Abstract Melanoma Antigen Genes (MAGEs) are a family of genes that have piqued the interest of scientists for their unique expression pattern. A subset of MAGEs (Type I) are expressed in spermatogonial cells and in no other somatic tissue, and then re-expressed in many cancers. Type I MAGEs are often referred to as cancer-testis antigens due to this expression pattern, while Type II MAGEs are more ubiquitous in expression. This study determines the cause and consequence of the aberrant expression of the MAGE-A subfamily of cancer-testis antigens. We have discovered that MAGE-A genes are regulated by DNA methylation, as revealed by treatment with 5-azacytidine, an inhibitor of DNA methyltransferases. Furthermore, bioinformatics analysis of existing methylome sequencing data also corroborates our findings. The consequence of expressing certain MAGE-A genes is an increase in cell proliferation and colony formation and resistance to chemo-therapeutic agent 5-fluorouracil and DNA damaging agent sodium arsenite. Taken together, these data indicate that DNA methylation plays a crucial role in regulating the expression of MAGE-A genes which then act as drivers of cell proliferation, anchorage-independent growth and chemo-resistance that is critical for cancer-cell survival. Keywords: Cancer, Cancer-testis antigens, Melanoma antigen genes, Anchorage-independent growth, Epigenetics, DNA methylation, Gene expression, Cell proliferation, Chemo-resistance Introduction antigens, and located on the X-chromosome, whereas Type Melanoma Antigen Genes (MAGEs) were first discovered II MAGEs are ubiquitous in expression and some members because a patient with melanoma and a few melanoma cell such as MAGEL2 are located on autosomes [3]. All MAGE lines presented an antigen that was recognized by cytotoxic proteins share a MAGE homology domain (MHD) and some T-cells. Subsequent autologous typing led to the discovery of members of this enigmatic family bind to E3-ubiquitin ligases MAGE-1, now known as MAGEA1, as the tumor associated and enhance their activity, by as yet unknown mechanisms antigen [1]. Since then, based on sequence similarity, more [4]. This adaptor function of the MAGE proteins results in gene members have been added to the family in humans, regulation of many biological processes. For example, with a total of about 60 genes including pseudogenes [2]. MAGEA3/6, a Type I MAGE regulates degradation of MAGEs can be divided into Type I and Type II based on AMPK, a master metabolic regulator and tumor-suppressor their expression pattern and their chromosomal location [5], and activation of cancer-specific MAGEA11-HUWE1 (Fig. 1a). Type I MAGEs are all reported to be cancer-testis ligase complex leads to alternate polyadenylation of core oncogenic and tumor suppressor transcripts [6], whereas * Correspondence: [email protected] MAGEL2,whichisatypeIIMAGE, regulates protein traf- 2Department of Life and Physical Sciences, Fisk University, Nashville, TN, USA ficking by ubiquitination of WASH, a known mediator of the 3Department of Pharmacology, Vanderbilt University, Nashville, TN, USA retromer complex [7, 8]. 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The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Colemon et al. Genes and Environment (2020) 42:24 Page 2 of 13 Fig. 1 Introduction of MAGE-A subfamily of genes and their protein products. a Schematic illustrating the focus of this paper. Melanoma Antigen Genes are divided into Type I and Type II based on their expression pattern. MAGE-A genes are considered bona fide cancer-testis antigens and are located on the X-chromosome. Type II MAGEs are ubiquitously expressed, and all members are not located on the X chromosome. b Clustal W sequence alignment shows the different percentages of sequence identity among the MAGE-A proteins. c Alignment of individual protein sequences shows that MAGE-A proteins share a MAGE homology domain (pink region) and an invariant dileucine motif (indicated by **) Colemon et al. Genes and Environment (2020) 42:24 Page 3 of 13 Type I MAGEs have garnered a lot of interest because A gene family are regulated by DNA methylation. We of their unique expression pattern. As cancer therapy is present evidence that each MAGE-A gene might have a becoming more personalized, being able to target cancer different role to play in the process of cellular cells specifically, is attractive. Therefore, genes and their transformation. protein products that are exclusively expressed in cancer cells such as the MAGE proteins have good therapeutic Results potential. However, there is a significant gap in the MAGE-A genes are enriched in the bone marrow, cancer knowledge of how the expression of each of these genes and testis is regulated and their individual contribution towards In order to determine whether all members of the the process of either initiation or maintenance of cancer MAGE-A gene family strictly adhered to the expression phenotypes. Furthermore, if we target one, do we need pattern of a cancer-testis antigen, we used UCSC Xena to target them all? functional genomics browser to analyze mRNA expres- Many germline genes are regulated by epigenetic mech- sion of MAGE-A genes from RNA-Sequencing datasets anisms, such as promoter methylation, histone methyla- from the TCGA (PAN-CANCER) and GTEx dataset for tion, and other post-translational modifications of bone marrow, colon, heart, liver and, testis samples [20]. histones that affects chromatin state [9–11]. In fact, the We used these tissues specifically because, the heart is a epigenetic landscape of the spermatozoon is thought to highly differentiated tissue [21], while colon and liver are contribute transgenerational epigenetic inheritance [12]. differentiated but still proliferative [22, 23] and the bone In addition, there is evidence that MAGEA1 and marrow, which is enriched in stem cells [24]. As a nega- MAGEA11 are both regulated by CpG methylation [13– tive control, we also determined the expression pattern 15]. However, what is not clear is whether expression of of MAGEL2, a more ubiquitously expressed Type II all MAGE-A genes is regulated by methylation, as would MAGE gene [3, 7]. Not surprisingly, MAGEL2 was be expected since many MAGE-A genes are co-expressed expressed in all tissues, except the bone marrow (Fig. 2a). in cancers, and whether aberrant expression of each of With the exception of MAGEA8, we found that all the these genes contributes to the process of cellular trans- MAGE-A genes, were exclusively expressed in the bone formation in any way. A recent study has shown that in marrow, testis and in cancer tissue (Fig. 2a). Moreover, mice, MAGE-A genes protect spermatogonial cells from the level of expression of the MAGE-A genes was highly genotoxic stress [16]. The role that these genes, either col- variable within the PAN-CANCER samples. To confirm lectively as a gene family, or individually, play in cancer- if this expression profile translates into cell culture associated phenotypes is a little less clear. models, we next performed RT-qPCR analysis on normal This study is focused on MAGE-A sub-family of genes and cancer cell lines. We determined the relative mRNA and its protein products and the role they play in cancer. levels of MAGE-A genes in normal non-transformed cell This sub-family has garnered interest because MAGEA3 lines from three different tissue origins, breast (Human peptides were used in a clinical trial for non-small cell Mammary Epithelial Cells, HMEC), lung (Human Bron- lung cancer, as an immunostimulant and more recently, chial Epithelial Cells, HBEC), and colon (Human Colonic a study established MAGE-A proteins as predictors of Epithelial Cells, HCEC). We detected no expression of resistance to anti-CTLA4 therapy in patients with meta- MAGE-A genes in these non-transformed cell lines (Fig. static melanoma [17–19]. Sequence alignment of the 2b). HEK 293 cells have either undetectable or low basal full-length MAGE-A proteins reveals varying levels of expression of a few MAGE-A genes, however patient de- sequence identity. For example, MAGEA3 and MAGEA6 rived cancer cell lines such as SKBR3 and MCF7 (breast are nearly identical (96% identity) and more similar to cancer), HCT116 and HT29 (colon cancer), A549 and MAGEA2 and MAGEA12 (84 and 85% identity), while H209 (lung cancer) cells lines expressed high levels of MAGEA5 is an outlier with a truncated MHD. In most of the MAGE-A genes (Fig. 2b). Within cell lines of addition, MAGEA10 and MAGEA11 at most share only each type of cancer, SKBR3, HCT116 and H209 dis- 58 and 63% identity with other MAGEA proteins, re- played higher expression of MAGE-A genes.