oa Targeting pro-apoptotic protein BAD inhibits survival and self-renewal of cancer stem cells

Background: Accumulating evidence suggests that the resistance of cancer stem cells (CSC) to many conventional therapies accounts for the inability of these therapies to cure cancers. Current cancer therapies can only shrink tumors as they target and kill differentiated tumor cells that constitute the bulk of the tumor, but are unable to target rare CSC population. Thus, despite a wealth of the information in differentiated cancer cells, the active survival and self-renewal pathways in CSC have not been characterized in detail. An understanding of the molecular mechanisms involved in the survival, self-renewal, maintenance, and resistance of cancer stem cells to current therapeutic regimens is of immense clinical interest. Objectives: BAD is a proapoptotic protein that has been shown to modulate apoptosis in cancer cells. However, the potential role of BAD in CSC biology has not been investigated. The objectives of this study are to address the role of pro-apoptotic protein BAD in survival and self-renewal of cancer stem cells, and test whether BAD expression can be used as a biomarker. Methods: Sphere formation method was used to enrich cancer stem cells from breast, prostate and melanoma cell lines. BAD expression was reduced by lentiviral-mediated delivery of shRNA specific to BAD. Apoptosis was induced in CSC by inhibiting the survival kinases PI3K and MAPK using pharmacological inhibitors LY294002 and PD98059, respectively. The cytoprotective and self-renewal effects of growth factors and neuropeptides were assessed. RT-PCR was used to quantify the expression of BAD in tumors of prostate cancer patients. Results and conclusion: Using sphere-derived CSC, we show that the BAD phosphorylation is essential for the survival of CSC as they are addicted to the expression of phosphorylated BAD. While none of CSCs could survive in the absence of BAD phosphorylation, both drugs and growth factors delivered their respective cytotoxic and protective effects by modulating the BAD phosphorylation in CSC. We also show that the self-renewal ability of cancer stem cells is significantly reduced at least in part by specifically knocking down the expression of BAD. In addition, about 65% tumors display increased BAD expression compared to tumor adjacent normal tissue, suggesting a role of BAD in tumor advancement. Taken together, our findings suggest that BAD plays a critical role in both differentiated cancer cells and CSC and thus targeting BAD might be an attractive strategy for development of novel therapeutics, and BAD expression might be used as a biomarker for tumor progression.