The E2F4 transcriptional repressor is a key mechanistic regulator of colon cancer resistance to irinotecan (CPT-11)

Background. Colorectal carcinomas (CRCs) are seldom eradicated by cytotoxic chemotherapy. Cancer cells with stem-like functional properties, often referred to as “cancer stem cells” (CSCs), display preferential resistance to several anti-tumor agents used in cancer chemotherapy, but the molecular mechanisms underpinning their selective survival remain only partially understood. Methods. In this study, we used Transcription Factor Target Genes (TFTG) enrichment analysis to identify transcriptional regulators (activators or repressors) that undergo preferential activation by chemotherapy in CRC cells with a “bottom-of-the-crypt” phenotype (EPCAM ⁺ /CD44 ⁺ /CD166 ⁺ ; CSC-enriched) as compared to CRC cells with a “top-of-the-crypt” phenotype (EPCAM ⁺ /CD44 ^neg /CD166 ^neg ; CSC-depleted). The two cell populations were purified in parallel by fluorescence-activated cell sorting (FACS) from a patient-derived xenograft (PDX) line representative of a moderately differentiated human CRC, following in vivo chemotherapy with irinotecan (CPT-11). The transcriptional regulators identified as differentially activated were tested for differential expression in normal vs. cancer tissues, and in cell populations enriched in stem/progenitor cell-types as compared to differentiated lineages (goblet cells, enterocytes) in the mouse colon epithelium. Finally, the top candidate was tested for mechanistic contribution to drug-resistance by selective down-regulation using short-hairpin RNAs (shRNAs). Results. Our analysis identified E2F4 and TFDP1, two core components of the DREAM transcriptional repression complex, as transcriptional modulators preferentially activated by irinotecan in EPCAM ⁺ /CD44 ⁺ /CD166 ⁺ as compared to EPCAM ⁺ /CD44 ^neg /CD166 ^neg cancer cells. The expression levels of both genes ( E2F4 , TFDP1 ) were found up-regulated in CRCs as compared to human normal colon tissues, and in a sub-population of mouse colon epithelial cells enriched in stem/progenitor elements (Epcam ⁺ /Cd44 ⁺ /Cd66a ^low /Kit ^neg ) as compared to other sub-populations enriched in either goblet cells (Epcam ⁺ /Cd44 ⁺ /Cd66a ^low /Kit ⁺ ) or enterocytes (Epcam ⁺ /Cd44 ^neg /Cd66a ^high ). Most importantly, E2F4 down-regulation using shRNAs dramatically enhanced the sensitivity of human CRCs to in vivo treatment with irinotecan , across three independent PDX models. Conclusions. Our data identified E2F4 and the DREAM repressor complex as critical regulators of human CRC resistance to irinotecan , and as candidate targets for the development of chemo-sensitizing agents.