New molecular interactions of c‐Myc in cholangiocarcinoma may open new therapeutic opportunities
Authors: Carmen Berasain, Maite G. Fernández‐Barrena, Matías A. Avila
Journal: Hepatology (2016)

Potential conflict of interest: Nothing to report. See Article on Page 439 Cholangiocarcinoma (CCA) is the second‐most frequent liver cancer after hepatocellular carcinoma (HCC). Currently, surgical resection is the only curative intervention, but unfortunately most patients present with advanced unresectable tumors. Diagnostic methods using serum markers are limited, and no reliable diagnosis of CCA is currently available.1 Risk factors for CCA involve biliary inflammation and cholestasis, including primary sclerosing cholangitis, choledocholithiasis, and parasitic infections.1 The current standard chemotherapy for patients with unresectable diseases consists of gemcitabine and cisplatin, although it confers only modest survival rates.1 Targeted drugs are currently being tested, but none has proved effective so far.2 It is important to understand the pathogenesis of CCA and its underlying mechanisms for the development of new diagnostic and therapeutic methods.2 The application of next‐generation sequencing technologies is essential to unveil the genomic landscape of biliary tumors and identify driver genes.2 Biochemical analyses, however, are required to understand protein functions and interactions for identification of potential targets for cancer therapy, including CCA. In this issue of Hepatology, Yang et al. identify novel cross‐talks of the c‐MYC (myelocytomatosis oncogene) oncoprotein contributing to cholestasis‐associated cholangiocarcinogenesis.3 Previously, these investigators demonstrated that c‐MYC induction promoted cholestatic liver injury and CCA progression.4 Along with c‐MYC, they also showed that two MAF (avian musculoaponeurotic fibrosarcoma oncogene homolog) proteins, MAFG and c‐MAF, involved in gene regulation and antioxidant defense, were also up‐regulated in cholestasis, contributing to liver damage through impairment of glutathione levels.5 Importantly, chronic liver disease, including cholestasis, leads to reduced S‐adenosylmethionine (SAMe) levels. SAMe, the main methyl group donor and a hepatic glutathione precursor, is the product of methionine adenosyltransferase (MAT).6 MAT is encoded by two different genes, MAT1A, mainly expressed in adult hepatocytes, and MAT2A, widely expressed across different cell types.6 The mechanisms leading to c‐MYC up‐regulation in cholestatic liver injury are not well understood. In this regard, these investigators previously described that SAMe could down‐regulate c‐MYC expression in hepatocytes.6 These previous findings prompted them to investigate whether chronic cholestasis could affect the molecular interactions of MATα1 protein, the product of MAT1A. The investigators analyzed the interactome of MATα1 in control and cholestatic mouse livers through proteomic analysis of anti‐MATα1 antibody immunoprecipitates. This unbiassed assay revealed that in control liver, MATα1 interacted with c‐MYC, MNT (MAX network transcriptional repressor), MAX (MYC‐associated factor X), MAFG, and c‐MAF proteins. MAX is an essential binding partner for c‐MYC target promoter activation (i.e., promoters containing E‐boxes).7 On the other hand, MNT‐MAX heterodimers may repress gene expression in concert with histone deacetylases.7 Importantly, these novel interactions of MATα1 were markedly altered in cholestasis. The investigators found attenuated interactions of MATα1 with MNT and enhanced interactions with c‐MYC, MAFG, and c‐MAF, whereas interactions with MAX were found unchanged. Similar changes in protein‐protein interactions were observed in murine CCA. One important finding relevant to the interpretation of the above results was the alterations in expression of all these proteins in chronic cholestasis and CCA. c‐MYC, MAFG, and c‐MAF up‐regulation in cholangiocytes during cholestasis had been already reported by these investigators, although induction of these proteins in human and murine CCA is a novel finding. Notwithstanding the significance of these changes, one most intriguing observation was the detection of high MATα1 expression in normal cholangiocytes, and its down‐regulation in cholestatis and CCA. MATα1 expression, like that of other relevant genes in methionine/SAMe metabolism, such as glycine‐N‐methyltransferase (GNMT), was thought to be mainly confined to hepatocytes, being compromised in chronic liver injury and HCC.6 The present report demonstrates that MAT1A is expressed in cholangiocytes, being reduced in cholestasis and CCA. Interestingly, a previous study also found high GNMT levels in biliary epithelial cells, which were lost in a significant proportion of CCAs.8 Together, these observations suggest that SAMe metabolism might be similar in hepatocytes and cholangiocytes, being also similarly affected upon chronic liver disease and tumorigenesis. Next, Yang et al. went on to demonstrate the functional significance of MATα1 protein interactions. One important aspect to consider was the presence of E‐boxes in c‐MYC, MAFG, c‐MAF, and MAT1A promoters. By chromatin immunoprecipitation assays, it was found that these E‐boxes in all four promoters were occupied by MATα1 and MNT, and that this pattern switched to an increased presence of c‐MYC, MAFG, and c‐MAF not only in cholestatic livers, but also in CCA cells (Fig. 1). These findings prompted the investigators to examine the possibility of a reciprocal regulation of expression of these genes. In CCA cell lines, it was found that MATα1 overexpression increased promoter activity for MAT1A, but reduced for c‐MYC, MAFG, and c‐MAF. In contrast, c‐MYC overexpression attenuated promoter activity for MAT1A and enhanced for MAFG and c‐MAF. These cross‐regulatory effects were confirmed on the endogenous genes (i.e., their messenger RNA [mRNA] levels) upon knockdown and overexpression of each gene. As a result, the investigators have demonstrated that MATα1 and c‐MYC/MAF proteins transcriptionally regulated each other in a reciprocal fashion (Fig. 1). These interactions took place at the E‐boxes present in the promoters of all these genes, where MATα1 was able to bind in the presence of c‐MYC and MAX. These are indeed very inspiring findings to understand the regulation of these important genes during cholestasis and cholangiocarcinogenesis. Particularly, induction of c‐MYC and MAFG/c‐MAF may contribute to explain the progressive down‐regulation of MAT1A expression not only in CCA, but also in HCC (Fig. 1). Moreover, c‐MYC‐mediated gene repression has also been linked to DNA methylation,7 and hypermethyation of MAT1A promoter, which occurs in HCC,9 is now demonstrated to happen also in CCA (Fig. 1). Nevertheless, the differential effects of MATα1‐MNT‐c‐MYC‐MAX complexes on gene expression in healthy liver, namely, repression of c‐MYC, c‐MAF, and MAFG transcription versus MAT1A gene activation, warrant further studies. Perhaps alternative recruitment of additional factor(s) (denoted as "X" in Fig. 1) could underlie the promoter‐specific activity of these complexes, as occurs for c‐MYC‐mediated gene activation and repression.7 Similarly, in the context of cholestasis and CCA, the same MATα1‐c‐MAF‐c‐MYC‐MAFG‐MAX complexes appear to promote c‐MYC, c‐MAF, and MAFG up‐regulation while inhibiting MAT1A expression. Involvement of additional factor(s) ("Y" in Fig. 1) may be speculated.Figure 1: Expression and functional interplay between c‐MYC, MNT, MATα1, and the MAF proteins, c‐MAF and MAFG, in cholestasis and CCA. In healthy liver and cholagiocytes, c‐Myc directly interacts with MATα1, Max, and Mnt. This complex binds to E‐boxes present in the promoters of c‐MYC, c‐MAF, MAFG, and MAT1A genes, leading to their activation or repression as indicated. Upon chronic cholestasis and in CCA cells, expression of MATα1 is reduced, whereas that of c‐MYC, c‐MAF, and MAFG is increased. Under these conditions, MATα1 interactions change, and complexes encompassing MATα1, c‐MYC, and MAX now include the MAF proteins, MAFG and c‐MAF. These complexes are shown to up‐regulate expression of c‐MYC, c‐MAF, and MAFG genes through binding and activation of E‐boxes in their respective promoters, while repressing expression of MATα1. MAT1A promoter is also found to be hypermethylated in CCA. MAT1A knockdown, or MafG or c‐Maf overexpression, enhance CCA growth and invasion. X and Y denote additional factors that could modulate the differential activity (i.e., activation vs. repression) of the same protein complexes when recruited to different gene promotors.This work provides strong evidence on the biological significance of MAT1A, MAFG, and c‐MAF dysregulation in cholangiocarcinogenesis. MAT1A overexpression, or c‐MYC, MAFG, or c‐MAF inhibition in CCA cells markedly inhibited their in vitro and in vivo growth. In contrast, overexpression of c‐MYC, MAFG, and c‐MAF or attenuation of MATα1 expression by knocking down resulted in larger tumors. Although it has already been reported that c‐MYC is associated with cholangiocarcinogenesis, the investigators have demonstrated, for the first time in this study, the oncogenic functions of c‐MAF and MAFG, and the antitumoral role of MATα1 in CCA. These findings suggest that it may be important to investigate relative expression of these proteins under different liver conditions using larger cohorts to evaluate heterogeneity of CCA and underlying mechanisms. MATα1 catalyzes the synthesis of SAMe, the most important biological methyl donor.6 Therefore, implication of epigenetic mechanisms in MATα1‐mediated gene expression regulation through changes in SAMe levels could be possible. Interestingly, a recent report also described the nuclear presence of MATα2, the product of MAT2A, in complex with MAFK, another MAF protein, and a number of chromatin remodeling factors and histone methyltransferases.10 In this study, MATα2 contributed to gene repression through in situ production of SAMe, which was utilized for histone methylation. Yang et al. tested whether the corepressor activity of MATα1 depended on methylation reactions by using DNA and histone methylation inhibitors. However, these compounds did not revert the inhibitory effect of MAT1A overexpression on c‐MYC, MAFG, and c‐MAF mRNA levels, suggesting the lack of involvement of methylation reactions. Nevertheless, a catalytically inactive MATα1 mutant showed little or no repressive activity, which could indeed support an epigenetic mechanism. Although the loss of oligomerization ability may be responsible for the lack of corepressor activity in the mutant as the investigators suggested, further structural studies are required for this issue. In summary, this study demonstrates novel molecular interactions of the c‐MYC oncogene contributing to cholangiocarcinogenesis. MATα1 is identified as a novel coregulator of gene expression and a tumor suppressor for CCA. Interventions aimed at the preservation of MAT1A expression may be explored for CCA treatment and/or prevention. Author names in bold designate shared co‐first authorship.