Vasoactive Intestinal Peptide Protects Cancer Stem Cells from Apoptosis by Activating Multiple Signaling Pathways

BackgroundVasoactive intestinal peptide (VIP) is a neuropeptide that controls the proliferation, differentiation, and survival of various normal and cancer cells. Elevated expression of VIP receptors has been found in prostate and breast cancer; and antagonists of VIP inhibit the growth of cancer xenografts. We previously reported the cytoprotective mechanisms activated by VIP in differentiated cancer cells that form the bulk of tumor, however, the signaling mechanisms activated in cancer stem cells (CSC) have not been identified.ObjectivesThe objectives of this study are to unravel the anti-apoptotic mechanisms activated by VIP in CSC, and examine if these pathways converge on a pro-apoptotic protein BAD. Identification of mechanisms responsible for survival of CSC will help design new therapeutic strategies that target and eliminate CSC.MethodsSphere formation assay and Fluorescent Activated Cell Sorter (FACS) were used to enrich and purify CSC (CD44+/CD24 −  and/or CD133+) from prostate and breast cancer cells. Apoptosis of CSC was induced by inhibiting the survival kinase PI3K/Akt using pharmacological inhibitor LY294002. Caspase assay, FACS and soft agar colony formation assays were used to measure the cytoprotective effects of VIP. Western blot was used to address the phosphorylation and quantification of various signaling proteins.Results and conclusionCancer stem cells were sorted to a purity of 95% and characterized for their stemness phenotype. While LY induced apoptosis in CSC by dephosphorylating BAD, VIP rescued by restoring the BAD phosphorylation at Ser112. VIP activated two non-redundant antiapoptotic signaling pathways that converge on BAD. One signaling pathway operate via activation of protein kinase A (PKA), whereas the other pathway predominately relies on MEK/MAPK pathway. Both pathways partially control BAD phosphorylation at Ser112. Either of these pathways is sufficient to protect CSC from apoptosis, thus both have to be inhibited to block cytoprotective effects of VIP. Using phosphorylation deficient mutant BAD, we show that the BAD phosphorylation is essential for the VIP-induced survival of CSC. Taken together, our findings suggest the potential usefulness of VIP receptor inhibition, and that BAD plays a critical role in CSC and thus targeting BAD might be an attractive strategy for development of novel therapeutics.