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Bag-1 protein is a crucial target in cancer to increase the survival and proliferation of cells. The Bag-1 expression is significantly upregulated in primary and metastatic cancer patients compared to normal breast tissue. Overexpression of Bag-1 decreases the efficiency of conventional chemotherapeutic drugs, whereas Bag-1 silencing enhances the apoptotic efficiency of therapeutics, mostly in hormone-positive breast cancer subtypes. In this study, we generated stable Bag-1 knockout (KO) MCF-7 breast cancer cells to monitor stress-mediated cellular alterations in comparison to wild type (wt) and Bag-1 overexpressing (Bag-1 OE) MCF-7 cells. Validation and characterization studies of Bag-1 KO cells showed different cellular morphology with hyperactive Akt signaling, which caused stress-mediated actin reorganization, focal adhesion decrease and led to mesenchymal characteristics in MCF-7 cells. A potent Akt inhibitor, MK-2206, suppressed mesenchymal transition in Bag-1 KO cells. Similar results were obtained following the recovery of Bag-1 isoforms (Bag-1S, M, or L) in Bag-1 KO cells. The findings of this study emphasized that Bag-1 is a mediator of actin-mediated cytoskeleton organization through regulating Akt activation., Competing Interests: The authors have declared that no competing interests exist.

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., (Copyright © 2015 John Wiley & Sons, Ltd.)

FOXM1 is implicated in genotoxic drug resistance but its role and mechanism of action remain unclear. Here, we establish that γH2AX foci, indicative of DNA double-strand breaks (DSBs), accumulate in a time-dependent manner in the drug-sensitive MCF-7 cells but not in the resistant counterparts in response to epirubicin. We find that FOXM1 expression is associated with epirubicin sensitivity and DSB repair. Ectopic expression of FOXM1 can increase cell viability and abrogate DSBs sustained by MCF-7 cells following epirubicin, owing to an enhancement in repair efficiency. Conversely, alkaline comet and γH2AX foci formation assays show that Foxm1-null cells are hypersensitive to DNA damage, epirubicin and γ-irradiation. Furthermore, we find that FOXM1 is required for DNA repair by homologous recombination (HR) but not non-homologous end joining (NHEJ), using HeLa cell lines harbouring an integrated direct repeat green fluorescent protein reporter for DSB repair. We also identify BRIP1 as a direct transcription target of FOXM1 by promoter analysis and chromatin-immunoprecipitation assay. In agreement, depletion of FOXM1 expression by small interfering RNA downregulates BRIP1 expression at the protein and mRNA levels in MCF-7 and the epirubicin-resistant MCF-7 Epi(R) cells. Remarkably, the requirement for FOXM1 for DSB repair can be circumvented by reintroduction of BRIP1, suggesting that BRIP1 is an important target of FOXM1 in DSB repair. Indeed, like FOXM1, BRIP1 is needed for HR. These data suggest that FOXM1 regulates BRIP1 expression to modulate epirubicin-induced DNA damage repair and drug resistance.