BackgroundBag-1 (Bcl-2-associated athanogene) is a multifunctional anti-apoptotic protein frequently overexpressed in cancer. Bag-1 interacts with a variety of cellular targets including Hsp70/Hsc70 chaperones, Bcl-2, nuclear hormone receptors, Akt and Raf kinases. In this study, we investigated in detail the effects of Bag-1 on major cell survival pathways associated with breast cancer.MethodsUsing immunoblot analysis, we examined Bag-1 expression profiles in tumor and normal tissues of breast cancer patients with different receptor status. We investigated the effects of Bag-1 on cell proliferation, apoptosis, Akt and Raf kinase pathways, and Bad phosphorylation by implementing ectopic expression or knockdown of Bag-1 in MCF-7, BT-474, MDA-MB-231 and MCF-10A breast cell lines. We also tested these in tumor and normal tissues from breast cancer patients. We investigated the interactions between Bag-1, Akt and Raf kinases in cell lines and tumor tissues by co-immunoprecipitation, and their subcellular localization by immunocytochemistry and immunohistochemistry.ResultsWe observed that Bag-1 is overexpressed in breast tumors in all molecular subtypes, i.e., regardless of their ER, PR and Her2 expression profile. Ectopic expression of Bag-1 in breast cancer cell lines results in the activation of B-Raf, C-Raf and Akt kinases, which are also upregulated in breast tumors. Bag-1 forms complexes with B-Raf, C-Raf and Akt in breast cancer cells, enhancing their phosphorylation and activation, and ultimately leading to phosphorylation of the pro-apoptotic Bad protein at Ser112 and Ser136. This causes Bad’s re-localization to the nucleus, and inhibits apoptosis in favor of cell survival.ConclusionsOverall, Bad inhibition by Bag-1 through activation of Raf and Akt kinases is an effective survival and growth strategy exploited by breast cancer cells. Therefore, targeting the molecular interactions between Bag-1 and these kinases might prove an effective anticancer therapy.

Bag-1, Bcl-2 associated athanogene-1, is a multifunctional protein that can regulate a wide variety of cellular processes: proliferation, cell survival, transcription, apoptosis and motility. Bag-1 interacts with various targets in the modulation of these pathways; yet molecular details of Bag-1's involvement in each cellular event are still unclear. We first showed that forced Bag-1 expression promotes cell survival and prevents drug-induced apoptosis in MCF-7 breast cancer cells. Increased mRNA expressions of c-myc protooncogene and ornithine decarboxylase (ODC), biosynthetic enzyme of polyamines, were detected in Bag-1L+ cells, and western blots against the protein product of c-Myc and ODC confirmed these findings. Once ODC, a c-Myc target, gets activated, polyamine biosynthesis increases. We observed enhanced polyamine content in the Bag-1L+ cells. On the contrary, when polyamine catabolic mechanisms were investigated, Bag-1 silencing suppressed biosynthesis of polyamines because of the downregulation of ODC and upregulation of PAO. Exposure of cells to apoptotic inducers enhances the cell death mechanism by producing toxic products such as H2 O2 and aldehydes. Bag-1L+ cells prevented drug-induced PAO activation leading to a decrease in H2 O2 production following cisplatin or paclitaxel treatment. In this line, our results suggested that Bag-1 indirectly affects cell survival through c-Myc activated signalling that causes elevation of ODC levels, leading to an increase of the polyamine content.

Bag-1 is a multifunctional protein that regulates Hsp70 chaperone activity, apoptosis, and proliferation. The three major Bag-1 isoforms have different subcellular localizations and partly non-overlapping functions. To identify the detailed interaction network of each isoform, we utilized mass spectrometry-based proteomics and found that interactomes of Bag-1 isoforms contained many common proteins, with variations in their abundances. Bag-1 interactomes were enriched with proteins involved in protein processing and degradation pathways. Novel interaction partners included VCP/p97; a transitional ER ATPase, Rad23B; a shuttling factor for ubiquitinated proteins, proteasome components, and ER-resident proteins, suggesting a role for Bag-1 also in ER-associated protein degradation (ERAD). Bag-1 pull-down from cells and tissues from breast cancer patients validated these interactions and showed cancer-related prominence. Using in silico predictions we detected hotspot residues of Bag-1. Mutations of these residues caused loss of binding to protein quality control elements and impaired proteasomal activity in MCF-7 cells. Following CD147 glycosylation pattern, we showed that Bag-1 downregulated VCP/p97-dependent ERAD. Overall, our data extends the interaction map of Bag-1, and broadens its role in protein homeostasis. Targeting the interaction surfaces revealed in this study might be an effective strategy in the treatment of cancer.

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