Abstract 1646: Molecular pathways associated with Reolysin and gemcitabine synergy in ras-mutated human HCT116 cells

Abstract

Abstract Reolysin, a type 3 reovirus, is a ubiquitous double-stranded RNA virus that exhibits selective oncolytic activity in ras-activated tumor cells and is non-pathogenic in humans. Our laboratory has previously demonstrated that Reolysin (reo) is synergistic when used in combination with gemcitabine (gem) and other commonly used cytotoxic agents against human colon carcinoma cells in vitro and in vivo. To determine the molecular pathways associated with gem/reo synergy, we exposed HCT116 cells, a human colon carcinoma cell line, to gem, reo, or the combination for 24, 48, 72 and 96 hrs in triplicate. The drug concentrations used were those found to be synergistic in previous in vitro studies (24 pfu/cell reo and/or 4nM gem). We harvested the cells, extracted RNA and performed microarray analysis using the HU133 2.0 plus array (Affymetrix). Gene expression data analysis was performed using GeneSpring GX 7.3.1 (Agilent Technologies). Raw intensity data were imported and preprocessed using the RMA algorithm. Data was then transformed, chips were normalized to the 50th percentile and raw data were filtered with a 2 fold minimum gene expression cutoff. Each experiment was run in triplicate and gene expression levels were averaged among replicates. We performed a 2-way ANOVA to test for time and treatment effects. The genes presenting the most significant treatment effects (160 genes, p<0.01) were uploaded to Ingenuity IPA 6.5 (Ingenuity Systems) for further pathway analysis. The top three canonical pathways significantly affected by the combination treatment were interferon signaling, antigen presentation and the protein ubiquitination pathways. These data suggest that the combination of gem and reo stimulates the immune system to increase surveillance/recognition of cancer cells. Further analyses were performed comparing the combination of gem/reo to gem alone. Approximately 300 genes were significantly (2 fold change, p<0.01) up or down-regulated in the combination therapy compared to gem alone. The combination of gem/reo downregulated PI3Kinase signaling while upregulating IKB signaling. The downstream effects of IKB upregulation are antiviral and stress responses. Single agent gemcitabine has proven to be inactive in colon cancer, yet HCT116 cells treated with a combination of gem and reo proved to be an effective pre-clinical therapy in these experiments. This enhanced preclinical efficacy is potentially due to an enhancement of tumor surveillance by the immune system. Clinical trials with Reolysin in combination with other chemotherapeutic drugs are ongoing. Understanding the mechanisms associated with cytotoxic synergy will allow us to better select drug combinations for specific tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1646.