Apoptosis inhibition in cancer cells: a novel molecular pathway that involves BAG3 protein

Stress-induced apoptosis regulates neoplasia pathogenesis and response to therapy. Indeed, cell transformation induces a stress response, that is overcome, in neoplastic cells, by alterations in apoptosis modulators; on the other hand, antineoplastic therapies largely trigger the apoptosis stress pathway, whose impairment results in resistance. Therefore, the study of the roles of apoptosis-modulating molecules in neoplasia development and response to therapy is of key relevance for our understanding of these processes. Among molecules that regulate apoptosis, a role is emerging for BAG3, a member of the BAG co-chaperone protein family. Proteins that share the BAG domain are characterized by their interaction with a variety of partners (heat shock proteins, steroid hormone receptors, Raf-1 and others), involved in regulating a number of cellular processes, including proliferation and apoptosis. BAG3, also known as CAIR-1 or Bis, forms a complex with the heat shock protein (Hsp) 70. This assists polypeptide folding, can mediate protein delivery to proteasome and is able to modulate apoptosis by interfering with cytochrome c release, apoptosome assembly and other events in the death process. It has been recently shown that, in human primary lymphoid and myeloblastic leukemias and other neoplastic cell types, BAG3 expression sustains cell survival and underlies resistance to therapy, through downmodulation of apoptosis. This review summarizes findings that assign an apoptotic role to BAG3 in some neoplastic cell types and identify the protein as a candidate target of therapy.     

BAG3 gene silencing sensitizes leukemic cells to Bortezomib-induced apoptosis

Proteasome inhibition has emerged as a powerful option for the treatment of a number of malignancies including leukemias. However, Bortezomib showed limited single-agent activity for patients with leukemia. Here, we report for the first time that Bortezomib up-regulated a novel antiapoptotic protein, BAG3, in human leukemic cells. BAG3 gene knockdown with shRNA greatly potentiated the generation of apoptosis by Bortezomib in leukemia cells. Furthermore, BAG3 silencing enhanced the antitumor activity of Bortezomib dramatically in a nude mouse model. Our results indicate that knocking down BAG3 gene is a promising new approach to enhance the therapeutic potency of Bortezomib in leukemia.     

BAG1: the guardian of anti-apoptotic proteins in acute myeloid leukemia

BCL2 associated Athano-Gene 1 (BAG1) is a multifunctional protein that has been described to be involved in different cell processes linked to cell survival. It has been reported as deregulated in diverse cancer types. Here, BAG1 protein was found highly expressed in children with acute myeloid leukemia at diagnosis, and in a cohort of leukemic cell lines. A silencing approach was used for determining BAG1's role in AML, finding that its down-regulation decreased expression of BCL2, BCL-XL, MCL1, and phospho-ERK1/2, all proteins able to sustain leukemia, without affecting the pro-apoptotic protein BAX. BAG1 down-regulation was also found to increase expression of BAG3, whose similar activity was able to compensate the loss of function of BAG1. BAG1/BAG3 co-silencing caused an enhanced cell predisposition to death in cell lines and also in primary AML cultures, affecting the same proteins. Cell death was CASPASE-3 dependent, was accompanied by PARP cleavage and documented by an increased release of pro-apoptotic molecules Smac/DIABLO and Cytochrome c. BAG1 was found to directly maintain BCL2 and to protect MCL1 from proteasomal degradation by controlling USP9X expression, which appeared to be its novel target. Finally, BAG1 was found able to affect leukemia cell fate by influencing the expression of anti-apoptotic proteins crucial for AML maintenance.     

HIV-1 Tat protein induces glial cell autophagy through enhancement of BAG3 protein levels

BAG3 protein has been described as an anti-apoptotic and pro-autophagic factor in several neoplastic and normal cells. We previously demonstrated that BAG3 expression is elevated upon HIV-1 infection of glial and T lymphocyte cells. Among HIV-1 proteins, Tat is highly involved in regulating host cell response to viral infection. Therefore, we investigated the possible role of Tat protein in modulating BAG3 protein levels and the autophagic process itself. In this report, we show that transfection with Tat raises BAG3 levels in glioblastoma cells. Moreover, BAG3 silencing results in highly reducing Tat- induced levels of LC3-II and increasing the appearance of sub G0/G1 apoptotic cells, in keeping with the reported role of BAG3 in modulating the autophagy/apoptosis balance. These results demonstrate for the first time that Tat protein is able to stimulate autophagy through increasing BAG3 levels in human glial cells.     

BAG1L enhances trans-activation function of the vitamin D receptor

The vitamin D receptor (VDR) is a member of the steroid/retinoid receptor superfamily of nuclear receptors that has potential tumor-suppressive functions. We show here that VDR interacts with and is regulated by BAG1L, a nuclear protein that binds heat shock 70-kDa (Hsp70) family molecular chaperones. Endogenous BAG1L can be co-immunoprecipitated with VDR from prostate cancer cells (ALVA31; LNCaP) in a ligand-dependent manner. BAG1L, but not shorter non-nuclear isoforms of this protein (BAG1; BAG1M/Rap46), markedly enhanced, in a ligand-dependent manner, the ability of VDR to trans-activate reporter gene plasmids containing a vitamin D response element in transient transfection assays. Mutant BAG1L lacking the C-terminal Hsc70-binding domain suppressed (in a concentration-dependent fashion) VDR-mediated trans-activation of vitamin D response element-containing reporter gene plasmids, without altering levels of VDR or endogenous BAG1L protein, suggesting that it operates as a trans-dominant inhibitor of BAG1L. Gene transfer-mediated elevations in BAG1L protein levels in a prostate cancer cell line (PC3), which is moderately responsive to VDR ligands, increased the ability of natural (1alpha,25(OH)(2) vitamin D(3)) and synthetic (1alpha, 25-dihydroxy-19-nor-22(E)-vitamin D(3)) VDR ligands to induce expression of the VDR target gene, p21(Waf1), and suppress DNA synthesis. Thus, BAG1L is a direct regulator of VDR, which enhances its trans-activation function and improves tumor cell responses to growth-suppressive VDR ligands.     

BAG3: a multifaceted protein that regulates major cell pathways

Bcl2-associated athanogene 3 (BAG3) protein is a member of BAG family of co-chaperones that interacts with the ATPase domain of the heat shock protein (Hsp) 70 through BAG domain (110–124 amino acids). BAG3 is the only member of the family to be induced by stressful stimuli, mainly through the activity of heat shock factor 1 on bag3 gene promoter. In addition to the BAG domain, BAG3 contains also a WW domain and a proline-rich (PXXP) repeat, that mediate binding to partners different from Hsp70. These multifaceted interactions underlie BAG3 ability to modulate major biological processes, that is, apoptosis, development, cytoskeleton organization and autophagy, thereby mediating cell adaptive responses to stressful stimuli. In normal cells, BAG3 is constitutively present in a very few cell types, including cardiomyocytes and skeletal muscle cells, in which the protein appears to contribute to cell resistance to mechanical stress. A growing body of evidence indicate that BAG3 is instead expressed in several tumor types. In different tumor contexts, BAG3 protein was reported to sustain cell survival, resistance to therapy, and/or motility and metastatization. In some tumor types, down-modulation of BAG3 levels was shown, as a proof-of-principle, to inhibit neoplastic cell growth in animal models. This review attempts to outline the emerging mechanisms that can underlie some of the biological activities of the protein, focusing on implications in tumor progression.     

Exposure to 50 Hz electromagnetic field raises the levels of the anti-apoptotic protein BAG3 in melanoma cells

The expression of the anti‐apoptotic protein BAG3 is induced in several cell types by exposure to high temperature, oxidants, and other stressful agents. We investigated whether exposure to 50 Hz electromagnetic fields raised BAG3 levels in the human melanoma cell line M14, in vitro and in orthotopic xenografts. Exposure of cultured cells or xenografts for 6 h or 4 weeks, respectively, produced a significant (P < 0.01) increase in BAG3 protein amounts. Interestingly, at the same times, we could not detect any significant variation in the levels of HSP70/72 protein or cell apoptosis. These results confirm the stressful effect of exposure to ELF in human cells, by identifying BAG3 protein as a marker of ELF‐induced stress. Furthermore, they suggest that BAG3 induction by ELF may contribute to melanoma cell survival and/or resistance to therapy. J. Cell. Physiol. 226: 2901–2907, 2011. © 2011 Wiley‐Liss, Inc.     

BAG3: a new therapeutic target of human cancers?

Bcl-2-associated athanogene (BAG) family proteins share the BAG domain, which is characterized by their interaction with a variety of partners (heat shock proteins, steroid hormone receptors, Raf-1 and others) and is involved in regulating a number of cellular processes. BAG3, also known as CAIR-1 or Bis, mediates protein delivery to proteasome and modulates apoptosis by interfering with cytochrome c release, apoptosome assembly and other events in the cellular death program. Moreover, it takes part in the processes of cell adhesion and migration. It has been shown that, in human cancer cells, including lymphocytic and myeloblastic leukemic cells, BAG3 sustains cell survival and underlies resistance to chemotherapy, through down-modulation of apoptosis. BAG3 knocking down could enhance the effectiveness of chemotherapy. This review summarizes the physiological and pathological roles of BAG3 in cancer cells and its potential as a therapeutic target of human malignancies.     

BAG3 induces the sequestration of proteasomal clients into cytoplasmic puncta: Implications for a proteasome-to-autophagy switch

Eukaryotic cells use autophagy and the ubiquitin–proteasome system as their major protein degradation pathways. Upon proteasomal impairment, cells switch to autophagy to ensure proper clearance of clients (the proteasome-to-autophagy switch). The HSPA8 and HSPA1A cochaperone BAG3 has been suggested to be involved in this switch. However, at present it is still unknown whether and to what extent BAG3 can indeed reroute proteasomal clients to the autophagosomal pathway. Here, we show that BAG3 induces the sequestration of ubiquitinated clients into cytoplasmic puncta colabeled with canonical autophagy linkers and markers. Following proteasome inhibition, BAG3 upregulation significantly contributes to the compensatory activation of autophagy and to the degradation of the (poly)ubiquitinated proteins. BAG3 binding to the ubiquitinated clients occurs through the BAG domain, in competition with BAG1, another BAG family member, that normally directs ubiquitinated clients to the proteasome. Therefore, we propose that following proteasome impairment, increasing the BAG3/BAG1 ratio ensures the “BAG-instructed proteasomal to autophagosomal switch and sorting” (BIPASS).     

BAG3 sensitizes cancer cells exposed to DNA damaging agents via direct interaction with GRP78

Bcl-2 associated athanogene 3 (BAG3) has a modular structure that contains a BAG domain, a WW domain, a proline-rich (PxxP) domain to mediate potential interactions with chaperons and other proteins that participate in more than one signal transduction. In search for novel interacting partners, the current study identified that 78 kDa glucose-regulated protein (GRP78) was a novel partner interacting with BAG3. Interaction between GRP78 and BAG3 was confirmed by coimmunoprecipitation and glutathione S-transferase (GST) pulldown. We also identified that the ATPase domain of GRP78 and BAG domain of BAG3 mediated their interaction. Counterintuitive for a prosurvival protein, BAG3 was found to promote the cytotoxicity of breast cancer MCF7, thyroid cancer FRO and glioma U87 cells subjected to genotoxic stress. In addition, the current study demonstrated that BAG3 interfered with the formation of the antiapoptotic GRP78-procaspase-7 complex, which resulted in an increased genotoxic stress-induced cytotoxicity in cancer cells. Furthermore, overexpression of GRP78 significantly blocked the enhancing effects of BAG3 on activation of caspase-7 and induction of apoptosis by genotoxic stress. Overall, these results suggested that through direct interaction BAG3 could prevent the antiapoptotic effect of GRP78 upon genotoxic stress.     

BAG1 down-regulation increases chemo-sensitivity of acute lymphoblastic leukaemia cells

BCL2-associated athanogene-1 (BAG1) is a multi-functional protein that is found deregulated in several solid cancers and in paediatric acute myeloid leukaemia. The investigation of BAG1 isoforms expression and intracellular localization in B-cell acute lymphoblastic leukaemia (B-ALL) patient-derived specimens revealed that BAG1 levels decrease during disease remission, compared to diagnosis, but drastically increase at relapse. In particular, at diagnosis both BAG1-L and BAG1-M isoforms are mainly nuclear, while during remission the localization pattern changes, having BAG1-M almost exclusively in the cytosol indicating its potential cytoprotective role in B-ALL. In addition, knockdown of BAG1/BAG3 induces cell apoptosis and G1-phase cell cycle arrest and, more intriguingly, shapes cell response to chemotherapy. BAG1-depleted cells show an increased sensitivity to the common chemotherapeutic agents, dexamethasone or daunorubicin, and to the BCL2 inhibitor ABT-737. Moreover, the BAG1 inhibitor Thio-2 induces a cytotoxic effect on RS4;11 cells both in vitro and in a zebrafish xenograft model and strongly synergizes with pan-BCL inhibitors. Collectively, these data sustain BAG1 deregulation as a critical event in assuring survival advantage to B-ALL cells.     

Implication of Bcl-2-associated athanogene 3 in fibroblast growth factor-2-mediated epithelial–mesenchymal transition in renal epithelial cells

The epithelial–mesenchymal transition (EMT) of tubular epithelial cells to myofibroblast-like cells plays a substantial role in renal tubulointerstitial fibrosis, which is a common pathological character of end-stage renal disease (ESRD). Fibroblast growth factor-2 (FGF-2) triggers EMT in tubular epithelial cells and increases Bcl-2-associated athanogene 3 (BAG3) expression in neural progenitor and neuroblastoma cells. In addition, a novel role of regulation of EMT has been ascribed to BAG3 recently. These previous reports urged us to study the potential involvement of BAG3 in EMT triggered by FGF-2 in renal tubular epithelial cells. The current study found that FGF-2 induced EMT, simultaneously increased BAG3 expression in human kidney 2 (HK2) cells. Although FGF-2 induced EMT in nontransfected or scramble short hairpin RNA (shRNA) transfected HK2 cells, it was ineffective in BAG3-silenced cells, indicating a favorable role of BAG3 in EMT of tubular cells induced by FGF-2. Knockdown of BAG3 also significantly suppressed motion and invasion of HK2 cells mediated by FGF-2. Furthermore, we confirmed that BAG3 was upregulated in kidney of unilateral ureteral obstruction (UUO) rats, a well-established renal fibrosis model, in which EMT is supposed to exert a substantial influence on renal fibrosis. Importantly, upregulation of BAG3 was limited to tubular epithelial cells. Results of the current study identify BAG3 as a potential player in EMT of tubular epithelial cells, as well as renal fibrosis.     

Evidence for the impact of BAG3 on electrophysiological activity of primary culture of neonatal cardiomyocytes

Homeostasis of proteins involved in contractility of individual cardiomyocytes and those coupling adjacent cells is of critical importance as any abnormalities in cardiac electrical conduction may result in cardiac irregular activity and heart failure. Bcl2-associated athanogene 3 (BAG3) is a stress-induced protein whose role in stabilizing myofibril proteins as well as protein quality control pathways, especially in the cardiac tissue, has captured much attention. Mutations of BAG3 have been implicated in the pathogenesis of cardiac complications such as dilated cardiomyopathy. In this study, we have used an in vitro model of neonatal rat ventricular cardiomyocytes to investigate potential impacts of BAG3 on electrophysiological activity by employing the microelectrode array (MEA) technology. Our MEA data showed that BAG3 plays an important role in the cardiac signal generation as reduced levels of BAG3 led to lower signal frequency and amplitude. Our analysis also revealed that BAG3 is essential to the signal propagation throughout the myocardium, as the MEA data-based conduction velocity, connectivity degree, activation time, and synchrony were adversely affected by BAG3 knockdown. Moreover, BAG3 deficiency was demonstrated to be connected with the emergence of independently beating clusters of cardiomyocytes. On the other hand, BAG3 overexpression improved the activity of cardiomyocytes in terms of electrical signal amplitude and connectivity degree. Overall, by providing more in-depth analyses and characterization of electrophysiological parameters, this study reveals that BAG3 is of critical importance for electrical activity of neonatal cardiomyocytes.     

BAG3 蛋白Ser187位点磷酸化诱导人甲状腺癌FRO 细胞上皮间质转化

Bcl-2相关抗凋亡蛋白3（Bcl-2 associated athanogene 3,BAG3）是BAG家族的重要成员,调节肿瘤细胞的粘附、迁移和侵袭,促进恶性肿瘤的复发和转移.本室前期工作证明,PKCδ可催化BAG3的Ser187位点磷酸化.本文研究BAG3蛋白磷酸化修饰对甲状腺癌FRO 细胞EMT表型转化的影响.稳定转染野生型WT-BAG3、模拟磷酸化型S187D-BAG3、阻碍磷酸化型S187A-BAG3 FRO细胞后,观察细胞形态的变化.结果显示,稳定转染模拟磷酸化型S187D-BAG3引起甲状腺癌FRO细胞呈现明显的间质细胞形态.实时PCR 和Western印迹,结果显示,稳定表达S187D-BAG3显著上调间质细胞标记物N-cadherin和波形蛋白mRNA与蛋白质在FRO细胞的表达,但下调上皮细胞标记物E-cadherin的mRNA和蛋白质的表达.同时,免疫荧光结果显示,稳定过表达S187D-BAG3的FRO细胞,E-cadherin和β-catenin出现向核周的内化.本文结果提示,BAG3蛋白磷酸化修饰可诱导甲状腺癌FRO细胞上皮间质转化.     

BAG3 regulates total MAP1LC3B protein levels through a translational but not transcriptional mechanism

Autophagy is mainly regulated by post-translational and lipid modifications of ATG proteins. In some scenarios, the induction of autophagy is accompanied by increased levels of certain ATG mRNAs such as MAP1LC3B/LC3B, ATG5 or ATG12. However, little is known about the regulation of ATG protein synthesis at the translational level. The cochaperone of the HSP70 system BAG3 (BCL2-associated athanogene 3) has been associated to LC3B lipidation through an unknown mechanism. In the present work, we studied how BAG3 controls autophagy in HeLa and HEK293 cells. Our results showed that BAG3 regulates the basal amount of total cellular LC3B protein by controlling its mRNA translation. This effect was apparently specific to LC3B because other ATG protein levels were not affected. BAG3 knockdown did not affect LC3B lipidation induced by nutrient deprivation or proteasome inhibition. We concluded that BAG3 maintains the basal amount of LC3B protein by controlling the translation of its mRNA in HeLa and HEK293 cells.