Abstract 5108: Silencing of Bcl-2 by liposomal siRNA enhances Doxorubicin-induced autophagy and inhibits in vivo tumor growth of ER(−) and ER(−) breast cancers

Abstract

Abstract Apoptosis (type I) and autophagic death (type II) are types of programmed cell death and play important roles in development, homeostasis, and elimination of unwanted cells. Autophagy is an evolutionary conserved process of lysosomal digestion of cytoplasm, misfolded proteins and organelles and may lead to autophagic cell death depending on the duration and severity of the process. In contrast to apoptosis, autophagicdeath is caspase independent and does not involve classical DNA laddering. The Bcl-2 proto-oncogene is overexpressed in 50-70% of breast cancers, potentially leading to resistance to chemotherapy, radiation and hormone therapy induced apoptosis. We recently reported that silencing of Bcl-2 by siRNA leads to predominantly autophagic cell death that is associated with induction of ATG5 expression in estrogen receptor ER(+) MCF-7 breast cancer cells that express high levels of Bcl-2 but lack caspase 3, which is also not expressed in 70% breast cancer patients. Bcl-2 siRNA also enhanced doxorubicin-induced autophagy and growth inhibition in MCF-7 cells. Here we investigated whether silencing of Bcl-2 by systemically administered liposomal siRNA alone or in combination with doxorubicin induce autophagic cell death in vivo breast cancers models of MCF-7 and highly aggressive and metastatic ER(−) and low Bcl-2 expressing MDA-MB-231 tumors in mice. In vitro inhibition of Bcl-2 by siRNA in MDA-MB-231 cells inhibited colony formation and significantly enhanced (3-fold) autophagy as indicated by LC3-II protein expression (Western blot), a marker of autophagy, following doxorubicin treatment. Mice bearing orthotopic MCF-7 and luciferase expressing MDA-MB231 breast tumors received liposomal Bcl-2 siRNA (150ug/kg/mouse or 5ug/mouse twice a week i.v.) or control siRNA. Bcl-2 siRNA group had significantly smaller tumors in both models detected by tumor weight and luciferease expression (by IVIS imaging) compared to control siRNA group (p<0.05). Inclusion of doxorubicin (3 mg/kg/mouse, i.p, once a week) further inhibited tumor growth of MDA-MB-231/luciferase and MCF-7 in mice (p<0.05). Induction of ATG5 and caspase 9 cleavage was observed in tumors from mice treated with liposomal Bcl-2 siRNA but not with liposomal control siRNA by Western blot analysis. Reduced expression of cleaved caspase 9 and increased expression of ATG5 protein in Bcl-2 siRNA +Doxorubicin treated mice were found compared with mice that received control siRNA + Doxorubicin treatment in MCF-7, suggesting that inhibition of Bcl-2 shifts doxorubicin-induced apoptotic response to autophagy in vivo. In conclusion, our results demonstrate that targeting of Bcl-2 in vivo tumors induces autophagy and apoptosis and that induction of autophagic cell death alone or in combination with chemotherapy may be used as a therapeutic strategy in breast cancers expressing Bcl-2 cells. 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 5108.