Maria Pascale

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.

bag3 gene expression is regulated by heat shock factor 1

BAG3 protein, a member of the BAG co‐chaperones family, sustains cell survival, through its interaction with the heat shock protein (HSP) 70, in a variety of normal and neoplastic cell types. bag3 gene expression is induced by stressful stimuli. Here we report for the first time that two of the three putative heat shock‐responsive elements (HSEs) in bag3 promoter interact with the heat shock factor (HSF) 1 in vitro and in vivo. Furthermore, downmodulation of HSF1 protein levels by specific small interfering (si) RNAs results in reducing BAG3 protein levels, indicating that the transcription factor plays a major role in bag3 gene expression. Because of the anti‐apoptotic role of BAG3 protein, these results disclose a previously unrecognized pathway, through which HSF1 maintains cell survival. J. Cell. Physiol. 215: 575–577, 2008.

IKKγ protein is a target of BAG3 regulatory activity in human tumor growth

BAG3, a member of the BAG family of heat shock protein (HSP) 70 cochaperones, is expressed in response to stressful stimuli in a number of normal cell types and constitutively in a variety of tumors, including pancreas carcinomas, lymphocytic and myeloblastic leukemias, and thyroid carcinomas. Down-regulation of BAG3 results in cell death, but the underlying molecular mechanisms are still elusive. Here, we investigated the molecular mechanism of BAG3-dependent survival in human osteosarcoma (SAOS-2) and melanoma (M14) cells. We show that bag3 overexpression in tumors promotes survival through the NF-κB pathway. Indeed, we demonstrate that BAG3 alters the interaction between HSP70 and IKKγ, increasing availability of IKKγ and protecting it from proteasome-dependent degradation; this, in turn, results in increased NF-κB activity and survival. These results identify bag3 as a potential target for anticancer therapies in those tumors in which this gene is constitutively expressed. As a proof of principle, we show that treatment of a mouse xenograft tumor model with bag3siRNA-adenovirus that down-regulates bag3 results in reduced tumor growth and increased animal survival.

BAG3 protein regulates cell survival in childhood acute lymphoblastic leukemia cells.

No abstract available.

The activity of hsp90|[alpha]| promoter is regulated by NF-|[kappa]|B transcription factors

Heat-shock proteins (HSP) 90 exert a relevant role in the survival and response to therapy of many neoplastic cell types. Here, we show that the promoter of hsp90α gene, that encodes the inducible form of HSP90, is regulated by nuclear factor-κB (NF-κB) activity. Indeed, we found that NF-κB factors bound to one of the two putative consensus sequences present in the hsp90α-flanking region; mutation of such motif hampered the phorbol-myristate-13-acetate-stimulated expression of a luciferase reporter gene under the control of the hsp90α promoter. Furthermore, the downmodulation of NF-κB (p65) levels by a specific small interfering (si) RNA resulted in reducing the levels of endogenous HSP90α protein. These findings disclose a previously unrecognized mechanism that contributes to connect NF-κB factors and HSPs in cell defence machinery.

Functional and pharmacological characterization of a VEGF mimetic peptide on reparative angiogenesis

Vascular endothelial growth factor (VEGF) is the main regulator of physiological and pathological angiogenesis. Low molecular weight molecules able to stimulate angiogenesis have interesting medical application for example in regenerative medicine, but at present none has reached the clinic. We reported that a VEGF mimetic helical peptide, QK, designed on the VEGF helix sequence 17-25, is able to bind and activate the VEGF receptors, producing angiogenesis. In this study we evaluate the pharmacological properties of peptide QK with the aim to propose it as a VEGF-mimetic drug to be employed in reparative angiogenesis. We show that the peptide QK is able to recapitulate all the biological activities of VEGF in vivo and on endothelial cells. In experiments evaluating sprouting from aortic ring and vessel formation in an in vivo angiogenesis model, the peptide QK showed biological effects comparable with VEGF. At endothelial level, the peptide up-regulates VEGF receptor expression, activates intracellular pathways depending on VEGFR2, and consistently it induces endothelial cell proliferation, survival and migration. When added to angiogenic factors (VEGF and/or FGF-2), QK produces an improved biological action, which resulted in reduced apoptosis and accelerated in vitro wound healing. The VEGF-like activity of the short peptide QK, characterized by lower cost of production and easier handling compared to the native glycoprotein, suggests that it is an attractive candidate to be further developed for application in therapeutic angiogenesis.

BAG3 protein regulates caspase-3 activation in HIV-1-infected human primary microglial cells.

BAG3, a member of the BAG co‐chaperones family, is expressed in several cell types subjected to stressful conditions, such as exposure to high temperature, heavy metals, drugs. Furthermore, it is constitutively expressed in some tumors. Among the biological activities of the protein, there is apoptosis downmodulation; this appears to be exerted through BAG3 interaction with the heat shock protein (Hsp) 70, that influences cell apoptosis at several levels. We recently reported that BAG3 protein was detectable in the cytoplasm of reactive astrocytes in HIV‐1‐associated encephalopathy biopsies. Here we report that downmodulation of BAG3 protein levels allows caspase‐3 activation by HIV‐1 infection in human primary microglial cells. This is the first reported evidence of a role for BAG3 in the balance of death versus survival during viral infection. J. Cell. Physiol. 218: 264–267, 2009.

BAG3 promotes pancreatic ductal adenocarcinoma growth by activating stromal macrophages

The incidence and death rate of pancreatic ductal adenocarcinoma (PDAC) have increased in recent years, therefore the identification of novel targets for treatment is extremely important. Interactions between cancer and stromal cells are critically involved in tumour formation and development of metastasis. Here we report that PDAC cells secrete BAG3, which binds and activates macrophages, inducing their activation and the secretion of PDAC supporting factors. We also identify IFITM-2 as a BAG3 receptor and show that it signals through PI3K and the p38 MAPK pathways. Finally, we show that the use of an anti-BAG3 antibody results in reduced tumour growth and prevents metastasis formation in three different mouse models. In conclusion, we identify a paracrine loop involved in PDAC growth and metastatic spreading, and show that an anti-BAG3 antibody has therapeutic potential.

BAG3 controls angiogenesis through regulation of ERK phosphorylation

BAG3 is a co-chaperone of the heat shock protein (Hsp) 70, is expressed in many cell types upon cell stress, however, its expression is constitutive in many tumours. We and others have previously shown that in neoplastic cells BAG3 exerts an anti-apoptotic function thus favoring tumour progression. As a consequence we have proposed BAG3 as a target of antineoplastic therapies. Here we identify a novel role for BAG3 in regulation of neo-angiogenesis and show that its downregulation results in reduced angiogenesis therefore expanding the role of BAG3 as a therapeutical target. In brief we show that BAG3 is expressed in endothelial cells and is essential for the interaction between ERK and its phosphatase DUSP6, as a consequence its removal results in reduced binding of DUSP6 to ERK and sustained ERK phosphorylation that in turn determines increased levels of p21 and p15 and cell-cycle arrest in the G1 phase.

1-Methoxy-canthin-6-one induces c-Jun NH2-terminal kinase-dependent apoptosis and synergizes with tumor necrosis factor-related apoptosis-inducing ligand activity in human neoplastic cells of hematopoietic or endodermal origin.

We investigated the effects of 1-methoxy-canthin-6-one, isolated from the medicinal plant Ailanthus altissima Swingle, on apoptosis in human leukemia (Jurkat), thyroid carcinoma (ARO and NPA), and hepatocellular carcinoma (HuH7) cell lines. Cultures incubated with the compound showed >50% of sub-G1 (hypodiploid) elements in flow cytometry analysis; the apoptosis-inducing activity was evident at <10 micromol/L and half-maximal at about 40 micromol/L 1-methoxy-canthin-6-one. The appearance of hypodiploid elements was preceded by mitochondrial membrane depolarization, mitochondrial release of cytochrome c, and Smac/DIABLO and procaspase-3 cleavage. We subsequently investigated the effect of 1-methoxy-canthin-6-one in combination with human recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in the four cell lines. Suboptimal concentrations (10 micromol/L 1-methoxy-canthin-6-one and 0.25 ng/mL TRAIL, respectively) of the two agents, unable to elicit apoptosis when used alone, induced mitochondrial depolarization, activation of caspase-3, and 45% to 85% of sub-G1 elements when added together to the cells. The synergism seemed to rely partly on the enhanced expression of TRAIL receptor 1 (TRAIL-R1; DR4), analyzed by immunofluorescence, by 1-methoxy-canthin-6-one. Cell incubation with 1-methoxy-canthin-6-one resulted in activating c-Jun NH2-terminal kinase (JNK), as revealed by Western blotting; induction of apoptosis and TRAIL-R1 up-regulation by 1-methoxy-canthin-6-one were >80% prevented by the addition of the JNK inhibitor (JNKI) SP600125JNKI, indicating that both effects were almost completely mediated by JNK activity. On the other hand, synergism with TRAIL was reduced by about 50%, suggesting that besides up-regulating TRAIL-R1, 1-methoxy-canthin-6-one could influence other factor(s) that participated in TRAIL-induced apoptosis. These findings indicate that 1-methoxy-canthin-6-one can represent a candidate for in vivo studies of monotherapies or combined antineoplastic therapies.