Biliary tract cancers BTC are categorized into intrahepatic

Biliary tract cancers (BTC) are categorized into intrahepatic, perihilar, extrahepatic BTC (or cholangiocarcinoma) as well as gallbladder cancer (GBC). Although the incidence of BTC is low in developed countries (3% of gastrointestinal cancers), BTC is the second most common primary liver tumor after HCC [11]. Current therapies include surgery (applicable in only 30%), chemotherapy (cisplatin, gemcitabine), radiation, and photodynamic therapy (PDT) [11], [12], [13]. However, due to the heterogeneous and aggressive nature as well as high therapeutic resistance, prognosis remains poor with a 5-year survival rate of only 5% to 10% [11]. A better understanding of the molecular oncogenesis of BTC and identification of new therapeutic targets is therefore of utmost importance.
Materials and methods
Results
Discussion Here, we provide first information on the relevance of histone methyltransferase G9a in BTC carcinogenesis. In accordance with previous publications regarding other cancer entities, we observed significant Cy3-dUTP of G9a in human BTC samples [3], [5], [7], [8], [9], [27]. G9a clearly correlated with unfavorable clinicopathological characteristics, specifically with tumor size and grading. This first description of such correlations for BTC is in line with a previous study in esophageal squamous cell carcinoma where G9a expression was associated with histological grade and tumor stage [10]. Likewise, in HCC, G9a correlated with aggressive tumor characteristics such as TNM stage [9]. Of note, this study describes a step-wise increase of G9a from normal liver (lowest expression), over chronic hepatitis, cirrhotic liver and, finally, to early and advanced HCC, suggesting a role of G9a in HCC development and progression [9]. Interestingly, we observed a highly significant increase in G9a expression from G2- and G3-graded BTC cases. Sub-analysis of G3 tumor with a detectable G2 area showed that this G9a expression pattern is also mirrored within a given BTC case. For future studies it will be interesting to see whether an (stepwise) increase of G9a expression is observable in progression of BTC on a histological level. Importantly, G9a was a predictor of poor overall survival and an independent prognostic factor in our BTC cohort—in line with studies describing a connection between G9a expression and poor survival in lung [3], ovarian [8] and esophageal squamous cell carcinoma [10]. EMT is a complex process involved in cancer invasion and metastasis, in which E-Cadherin serves as a negative and Vimentin as a positive effector [14], [28]. Other studies reported a significant correlation of G9a expression and formation of local and distant metastases [5], [9], [10] and that G9a directly represses E-Cadherin as a marker of (benign) epithelial cell phenotype [2], [7], [29]. Although a highly significant negative correlation between G9a and E-Cadherin exists in BTC, in vitro G9a inhibition using BIX01294 failed to consistently restore E-Cadherin protein levels in our study. This discrepancy may be explained by generally high cytotoxic effects of the BIX01294 -IC90 concentration as well as by the fact that in (artificial) 2D cell culture systems, mechanisms regulating cell attachment and extracellular matrix association might be different from the physiological situation. Additionally, several publications describe different interaction partners (DNMT1 [3], [30]) and non-histone targets (p53 [31]) of G9a, which might explain cell line– or cancer-specific regulatory mechanisms of G9a. We recently published that another SET-domain histone methyltransferase called EZH2 is involved in BTC [18]. EZH2 is the enzymatic part of the polycomb repressive complex 2 (PRC2) that specifically tri-methylates lysine 27 at histone 3 (H3K27me3), a repressive epigenetic mark [32]. There is evidence that G9a directly coordinates PRC2 and H3K27me3, because G9a was necessary for mono-methylation of H3K27 (a prerequisite for PRC2-dependent H3K27me3 [33]) and because G9a directly interacts with PRC2 [34]. Of note, this study also describes several genes directly regulated (at promoter level) by coordinated G9a and PRC2 recruitment [34]. Dual G9a and PRC2 inhibition might therefore be an interesting therapeutic strategy which also addresses the commonly observed problem that removal of H3K27me3 (PRC2 inhibition) is not sufficient to reactivate PRC2-silenced genes [35]. In fact, dual inhibition of G9a and PRC2 using small-molecule inhibitors (but not single treatments) massively enhanced Spink1 target mRNA levels [35]. Therefore, subsequent studies should evaluate dual G9a and PRC2 inhibition in BTC cells and tumors.