Abstract 3311: Loss of EZH2 at tumor invasive front is correlated with higher aggressiveness

Abstract

Abstract Background: Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths, mainly due to its high metastatic rate, the acquisition of drug resistance and associated recurrences. Today, it is well known that not only genetic alterations but also epigenetic modifications play a major role in these complex processes. The enhancer of zeste homolog 2 (EZH2) represents the catalytic subunit of polycomb repressive complex 2 that preferentially methylates lysine 27 on histone 3 and is involved in epigenetic gene silencing. In addition to its function as a cell cycle regulator, EZH2 has also been associated with the epithelial-to-mesenchymal transition as well as angiogenic and metastatic processes. While an increase in EZH2 expression is well known for CRC and has been associated with both cancer stem-like cell properties and highly aggressive tumors, its prognostic value is still controversially discussed in literature. Methods: Here we investigated the expression of EZH2 in a cohort of colon cancer patients, focusing on the role of EZH2 at the invasion front of CRC tumors. We established a semiautomated software workflow for a time-efficient and objective analysis of EZH2-stained patient samples. In addition, we analyzed HCT116 cells treated with the EZH2 inhibitor DZNep in vitro by crystal violet assay, Western blot and cell cycle analysis and in vivo by the chorioallantoic membrane (CAM) assay in comparison to untreated controls in order to experimentally simulate the clinical situation. Results: While EZH2 expression in the tumor center did not have a prognostic value, EZH2 loss at the invasion front was significantly correlated with poor clinical outcome. Analysis by the trained semiautomated Definiens' Tissue Studio Software (TSS) and manual scoring showed a pronounced interobserver correlation when analyzing tumor centers of TMA punches or whole slides. We were able to mimic the observed EZH2 loss at the invasion front in human CAM xenografts. We found that DZNep-treated HCT116 cells strongly invade into the CAM and their tumors display a higher grade of vascularization, which demonstrates a more aggressive phenotype when EZH2 is lost. Conclusion: From our clinical and experimental findings we propose a potential explanation for the highly contradictory literature data concerning EZH2 expression levels and prognosis in CRC. By establishing the TSS-based analysis for EZH2-stained specimens, we were able to generate a reliable tool to evaluate immunostainings of certain biomarkers to be applied in the daily routine. Citation Format: Julian Boehm, Julienne K. Muenzner, Aylin Caliskan, Benardina Ndreshkjana, Katharina Erlenbach-Wünsch, Susanne Merkel, Roland Croner, Tilman T. Rau, Carol I. Geppert, Arndt Hartmann, Adriana Vial-Roehe, Regine Schneider-Stock. Loss of EZH2 at tumor invasive front is correlated with higher aggressiveness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3311.