Supplementary Material for: Enhancer of Zeste Homolog 2 in Colorectal Cancer Development and Progression

Background: Colorectal cancer (CRC) is the leading gastrointestinal malignancy. The development from premalignant intraepithelial lesions leading to invasive cancer is paradigmatic for the stepwise carcinogenesis of epithelial cancers, but the knowledge of the underlying mechanism of carcinogenesis and progression of CRC is still incomplete. The understanding of epigenetic mechanisms of carcinogenesis has led to new therapeutic approaches during the last years. Enhancer of zeste homolog 2 (EZH2) is one central epigenetic silencer of the polycomb repressor complex 2 (PRC2) that is already in clinical use as a novel drug target and is associated with poorer prognosis in several cancer entities. Patients and Methods: The protein expression of EZH2 and other members of the PRC2 as well as resulting posttranslational modifications were investigated by immunohistochemistry in 187 patients with CRC and in 94 patients with premalignant colorectal lesions and correlated with their clinical outcome. Furthermore, the corresponding mRNA expression levels were analyzed in 217 patients with rectal cancer that were enrolled in a prospective clinical trial. Results: We found a weak expression of EZH2 in normal colon mucosa that increased in low grade, peaked in high grade intraepithelial neoplasia, and decreased again in invasive CRC. The posttranslational modification caused by EZH2 as a measure of EZH2 activity showed the same behavior. Strong protein and mRNA expression of EZH2 were significantly correlated with favorable prognosis in both investigated cohorts. Conclusion: The expression and activity of EZH2 are associated with colorectal carcinogenesis and most expressed in intraepithelial high-grade lesions. Strong expression of EZH2 is associated with a significantly favorable prognosis in patients suffering from CRC.背景：结直肠癌（Colorectal cancer, CRC）是最常见的消化道恶性肿瘤。从癌前上皮内病变进展为浸润性癌的过程，是上皮源性肿瘤逐步癌变的经典范式，但目前对于结直肠癌癌变及进展的潜在分子机制仍未完全阐明。近年来，对癌变表观遗传机制的解析推动了新型治疗策略的开发。zeste增强子同源物2（Enhancer of zeste homolog 2, EZH2）是多梳抑制复合体2（Polycomb Repressive Complex 2, PRC2）的核心表观遗传沉默因子之一，现已作为新型药物靶点进入临床应用阶段，且在多种肿瘤实体中均与不良预后相关。 患者与方法：本研究通过免疫组化方法，检测了187例结直肠癌患者、94例结直肠癌前病变患者的EZH2及PRC2其他成员的蛋白表达水平，以及相关的翻译后修饰情况，并分析其与患者临床结局的相关性。此外，本研究还对一项前瞻性临床试验中纳入的217例直肠癌患者的对应mRNA表达水平进行了分析。 结果：本研究发现，正常结肠黏膜中EZH2呈弱表达，在低级别上皮内病变中表达升高，在高级别上皮内瘤变中达到峰值，随后在浸润性结直肠癌中表达再次下降。以EZH2介导的翻译后修饰作为EZH2活性的检测指标，也呈现出一致的表达趋势。在两个研究队列中，EZH2的高蛋白及mRNA表达水平均与良好的临床预后显著相关。 结论：EZH2的表达与活性与结直肠癌发生发展密切相关，且在高级别上皮内病变中表达水平最高。结直肠癌患者的EZH2高表达与显著更优的临床预后密切相关。