Abstract 4902: Targeting APC mutant colon cancer cells by chloroquine

Abstract

Abstract The adenomatous polyposis coli (APC) tumor suppressor is mutated in approximately 80% of sporadic colorectal cancers (CRC) where it is recognized as the critical initiating event. Genetic studies revealed that c-Myc is the crucial mediator of the early stages of tumor development following APC loss. One of the key functions of c-Myc is to repress p21 and thus switches to p53-mediated apoptosis in cancer cells. We therefore hypothesize that p21 might be an attractive target for killing APC-deficient human colon cancer cells preferentially. Here we first utilized p21-deficient human colon cancer cell line (p21−/−HCT116) to elucidate the role of p21 in colon cancer cells for apoptosis. Chloroquine (CQ), a small molecule inhibitor of autophagy, was identified to preferentially initiate p53-PUMA-dependent apoptosis in p21−/−HCT116 cells, but not in isogenic p21+/+HCT116 cells. Although CQ inhibits the late stages of autophagy by targeting lysosmoal degradation, however, the molecular basis of CQ-mediated cancer cell death remains unclear. We found that blockade of the early stage of autophagy by individual knockdown of genes involved in autophagy (Atg5, Atg7, Beclin1 and ULK1) attenuated CQ-induced accumulation of autophagosomes and apoptosis in p21−/−HCT116 cells. In addition, we showed that reactive oxygen species (ROS)-initiated autophagy was essential for CQ-mediated cancer cell death. These results suggest that inhibition of autophagy at a late stage by CQ can be detrimental to cancer cell survival when autophagy is initiated at early stage. To determine the specificity of p21 in mediating CQ-induced cancer cell death, we demonstrated that knockdown of p21 in p21+/+HCT116 cells sensitized, while ectopic expression of p21 in p21−/−HCT116 cells inhibited, CQ-induced apoptosis. Furthermore, administration of CQ in vivo significantly suppressed tumor growth in xenograft mouse model bearing the p21−/−HCT116 cells, but not the p21+/+HCT116 cells. Our data strongly support that inactivation of p21-mediated anti-apoptotic event is required for CQ to kill cancer cells and inhibit tumor growth in vivo. To examine whether what was observed in p21−/−HCT116 cells was also true for killing of APC mutant colon cancer cells due to suppression of p21 through Myc activation upon APC loss, we took shRNA-mediated knockdown approaches to effectively deplete APC in p21+/+HCT116 cells. Treatment of cancer cells with CQ specifically caused apoptosis and blocked p21 induction in APC depleted cancer cells, which correlated with a selective inhibition of tumor growth in xenograft model. Given that CQ has been safely used in patients for many years as anti-malarial and anti-rheumatoid therapies, our findings provide compelling rationale to consider clinic translational trials in colon cancer patients carrying APC mutations using lysosomotropic agents such as CQ. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4902. doi:1538-7445.AM2012-4902