Epigenetic Mechanisms of Immunoresistant and Chemoresistant Colon Cancer Stem Cells

Abstract

Advanced-stage colon cancer is nearly always treated with a conventional chemotherapy known as 5-Fluorouracil (5-FU). However, colon cancer patients nearly always develop a resistance to 5-FU therapy. Understanding how colon carcinoma cells can overcome the mechanisms by which 5-FU induces cell death can thus show great promise for improving chemotherapies for patients with advanced stages of the disease. We initially hypothesized that 5-FU treatment selectively targets rapidly-dividing cells; thus, slowly-dividing cells would be able to survive 5-FU therapy and could continue to proliferate after receiving high doses of 5-FU. These cells would therefore be enriched and give rise to 5-FU-resistant tumors in vivo. In this study, we developed human metastatic colon carcinoma cell lines that are resistant to 5-FU treatment. We determined that these 5-FU-resistant cells possess properties commonly associated with cancer stem cells, such as the ability to form tumorispheres in ultra-low attachment conditions and increased expression of certain markers, such as CD133 and ALDH. We also show here that 5-FU-resistant cells may use epigenetic mechanisms to overcome immune-mediated tumor growth control via silencing of the death receptor, Fas. Our experiments show that Fas protein expression is decreased in metastatic colon carcinoma cells, and that loss of Fas function increases spontaneous tumor growth in a mouse model. We show that trimethylation of the H3K9 residue is highly enriched at the FAS promoter region in 5-FU resistant cell lines. Using a selective histone methyltransferase inhibitor known as verticillin A, we were able to restore Fas expression in these colon cancer cells. Our in vivo model shows that treatment of tumor-bearing mice with verticillin A can enhance the efficiency of 5-FU therapy and inhibit tumor growth. We thus conclude that 5-FU treatment enriches a subpopulation of cancer stem cells which are resistant to 5-FU and FasL-mediated cytotoxicity via host immune cells, and that inhibiting H3K9 trimethylation can overcome resistance to 5-FU and restore Fas expression.