Abstract 3773: Intestinal glutathione peroxidase (GPx2) promotes differentiation of colorectal cancer stem cells by modulating the rate of protein synthesis.
Authors: Emmink Benjamin , Laoukili Jamila , Kipp Anna , Govaert Klaas , Fatrai Szabolcs , Verheem Andre , Brigelius-Flohe Regina , Jimenez Connie , Borel Rinkes Inne , Kranenburg Onno 
Published: August 2014

Abstract
Primary colorectal tumors and liver metastases can be established in vitro as colonosphere cultures. These 3D cultures are enriched in cancer stem cells (CSCs) and create phenocopies of the original patient tumor upon transplantation into mice. We have performed proteomics analysis of a series of colonosphere cultures and show high expression of the Wnt-target gene intestinal glutathione peroxidase (GPx2). GPx2 is a member of the GPx family of ROS scavenging enzymes but its function in colorectal cancer is not known. Excess oxidative damage in normal and cancer stem cell (SC) populations can lead to (C)SC exhaustion. Therefore, we tested whether GPx2 may play a role in colorectal CSC maintenance. We found that GPx2 is predominantly expressed by differentiated tumor cells in human colorectal tumors. A construct in which the GPx2-promoter drives GFP expression revealed that GPx2high cells express differentiation markers and proliferate rapidly, while GPx2low cells express stem cell markers (Oct4, Nanog, Sox2, OLFM4) and proliferate slowly. To study the function of GPX2 in CSC maintenance we generated GPx2 knockdown (kd) cultures. Depletion of GPX2 greatly increased the fraction of immature CSCs and inhibited cellular differentiation. GPx2-kd cells formed slowly growing tumors with high CSC content, while GPx2 overexpression resulted in the formation of rapidly proliferating well-differentiated tumors with low CSC content. Gene Set Enrichment Analysis (GSEA) using gene expression profiles of two independent series of colorectal tumors showed that GPx2 expression was inversely correlated with published gene signatures for immature cells (Nanog, Oct4 and Sox2 target genes) and for cell proliferation. Low expression of GPx2 also correlated with poor patient survival. To explore how GPX2 affects CSC maintenance we performed gene ontology analyses and found that GPx2 expression is most strongly correlated with genes governing protein synthesis. GPx2 knockdown resulted in strongly reduced ribosomal gene expression and reduced protein synthesis. Strikingly, chronic cycloheximide-mediated suppression of protein synthesis in colonosphere cultures also increased the pool of slow-cycling CSCs, similar to GPx2 knockdown. Taken together, our results identify the rate of protein synthesis as a critical new determinant of the CSC phenotype. By stimulating protein synthesis GPx2 drives the differentiation, proliferation and exhaustion of colorectal CSCs. Complete elucidation of this pathway may identify targets for differentiation-stimulating anti-cancer therapy.
Citation Format: Benjamin Emmink, Jamila Laoukili, Anna Kipp, Klaas Govaert, Szabolcs Fatrai, Andre Verheem, Regina Brigelius-Flohe, Connie Jimenez, Inne Borel Rinkes, Onno Kranenburg. Intestinal glutathione peroxidase (GPx2) promotes differentiation of colorectal cancer stem cells by modulating the rate of protein synthesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3773. doi:10.1158/1538-7445.AM2013-3773