Ikkγ Protein Is a Target of BAG3 Regulatory Activity in Human Tumor Growth
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IKKγ protein is a target of BAG3 regulatory activity in human tumor growth Massimo Ammirantea,b,1, Alessandra Rosatib,c,1, Claudio Arrac,d, Anna Basileb,c, Antonia Falcob,c, Michela Festab,c, Maria Pascaleb,c, Morena d’Aveniab,c, Liberato Marzullob,c, Maria Antonietta Belisariob, Margot De Marcob,c, Antonio Barbierid, Aldo Giudiced, Gennaro Chiappettae, Emilia Vuttarielloe, Mario Monacoe, Patrizia Bonellid, Gaetano Salvatoref, Maria Di Benedettof, Satish L. Deshmaneg, Kamel Khalilig, Maria Caterina Turcob,c,2, and Arturo Leoneb aLaboratory of Gene Regulation and Signal Transduction, Department of Pharmacology and Cancer Center, School of Medicine, University of California, San Diego, La Jolla, CA 92093-0723; bDepartment of Pharmaceutical Sciences (DiFarma), University of Salerno, 84084 Fisciano, Italy; cBioUniverSA S.R.L., 84084 Fisciano, Italy; dAnimal Facility and eFunctional Genomic Unit National Cancer Institute, “Tumor Institute Fond. Pascale,” 80131 Naples, Italy; fPathological Anatomy, Federico II University, 80131 Naples, Italy; and gCenter For Neurovirology, Department of Neuroscience, Temple University, Philadelphia PA 19122 Edited* by Michael Karin, School of Medicine, University of California, La Jolla, CA, and approved March 17, 2010 (received for review July 10, 2009) BAG3, a member of the BAG family of heat shock protein (HSP) 70 leukemia results in a dramatic increase of basal as well as drug- cochaperones, is expressed in response to stressful stimuli in a number induced apoptosis (17, 18). Furthermore, BAG3 is overexpressed in of normal cell types and constitutively in a variety of tumors, including thyroid carcinomas, where higher levels of expression are reached in pancreas carcinomas, lymphocytic and myeloblastic leukemias, and anaplastic tumors compared with well-differentiated forms. In these thyroid carcinomas. Down-regulation of BAG3 results in cell death, but tumors, down-modulation of BAG3 results in sensitization to TNF- the underlying molecular mechanisms are still elusive. Here, we related apoptosis-inducing ligand-dependent apoptosis (20). investigated the molecular mechanism of BAG3-dependent survival The influence of BAG3 on cell survival is potentially mediated, at in human osteosarcoma (SAOS-2) and melanoma (M14) cells. We show least in part, by regulation of HSC70/HSP70 function (11). Some of that bag3 overexpression in tumors promotes survival through the NF- the apoptosis-regulating proteins that are known to interact with κB pathway. Indeed, we demonstrate that BAG3 alters the interaction HSP70 are therefore potential targets of BAG3 regulatory activity. between HSP70 and IKKγ, increasing availability of IKKγ and protect- Among these regulating proteins, the IKKγ subunit of the IKK ing it from proteasome-dependent degradation; this, in turn, results in complex is of particular interest. Indeed, HSP70 has been shown to MEDICAL SCIENCES increased NF-κB activity and survival. These results identify bag3 as a interact with IKKγ (29, 30) and to suppress NF-κB activity. Inter- potential target for anticancer therapies in those tumors in which ference with the HSP70-IKKγ interaction through a dominant this gene is constitutively expressed. As a proof of principle, we show negative mutant restores NF-κB activity (30). In this paper, we show that treatment of a mouse xenograft tumor model with bag3siRNA- that BAG3 affects NF-κB activity by interfering with the binding adenovirus that down-regulates bag3 results in reduced tumor between HSP70 and IKKγ, thus favoring IKK complex formation growth and increased animal survival. and preventing the proteosomal degradation of IKKγ. Moreover, we show that down-regulation of bag3 in vivo can be a potential BAG3 | IKK-gamma | apoptosis anticancer therapeutical strategy. he bag3 gene belongs to a highly conserved gene family iden- Results Ttified in yeast (Saccharomyces cerevisiae and Schizosaccharomyces Apoptosis Modulation by BAG3 Is Dependent on NF-κB Activity. We pombe), invertebrates (Caenorhabditis elegans, Ciona intestinalis,and investigated the effects of bag3 silencing in SAOS-2 human osteo- Drosophila), amphibians (Xenopus laevis), mammals (Homo sapiens sarcoma cells, which express high constitutive levels of BAG3 (Fig. fi and Mus musculus), and plants (Oryza sativa and Arabidopsis thaliana) 1A). Our results show that down-regulation of BAG3 by a speci c (1–9). All BAG proteins are characterized by a conserved domain of siRNA in these cells results in increased death (detected as sub-G1 110–124 amino acids (BAG domain) (3, 10) through which they bind peak of propidium iodide-stained cells) in response to etoposide to the HSC70/HSP70 ATPase domain regulating the heat shock (Fig. 1B) or serum deprivation (Fig. 1 C and D). Consistent over- expression ofbag3 in thesame cell lineprotects cells from etoposide- protein (HSP) polypeptide folding activity (2, 11). In addition, BAG3 fi contains a WW domain and a proline-rich repeat (PXXP) through induced death (Fig. 1 E and F), thus also con rming the anti- which it interacts with other partners, such as phospholipase C apoptotic property of BAG3 in this tumor. Because apoptosis is also γ known to be regulated by NF-κB factors in human osteosarcoma (PLC)- (12) and Akt kinase (13). κ BAG3 is a 74-kDa cytoplasmic protein mainly localized in the (31, 32) and, as mentioned before, NF- B is a potential target of rough endoplasmic reticulum; a slightly different molecular weight BAG3 action, we investigated if BAG3 modulation affected the NF- κB pathway. By ChIP experiments (Fig. 2A), we were able to show or a doublet form can be observed in some cell types and/or fol- that down-regulation of bag3 by siRNA results in dramatically lowing cell exposition to stressors (11, 14, 15). In humans, bag3 is reduced binding of NF-κB to two well-known responsive elements constitutively expressed in myocytes and a few other normal cell on the IL-8 and IκB-α promoters. These results suggested that bag3 types and in several tumors (leukemia and lymphoma, myeloma, and pancreas and thyroid carcinomas) (11, 16–20). In addition, its expression is induced in different normal cell types (leukocytes and Author contributions: M.C.T. and A.L. designed research; M.A., A.R., C.A., M.F., A. Basile., epithelial and glial cells) in response to cell stressors, such as oxi- A.F., M.P., M.dA., L.M., M.A.B., M.D.M., A. Barbieri, A.G., G.C., E.V., M.M., P.B., G.S., and dants, high temperature, heavy metals, and HIV-1 infection (15, 21– M.D.B. performed research; S.L.D. and K.K. contributed new reagents/analytic tools; M.A., 23). Interestingly, induction of bag3 in response to stress is under the A.R., M.P., M.C.T., and A.L. analyzed data; and M.C.T. wrote the paper. control of HSF1 (24), a member of the heat shock factor family of The authors declare no conflict of interest. transcription factors (25, 26) involved in tumor initiation and *This Direct Submission article had a prearranged editor. maintenance (27, 28). Actually, several lines of evidence suggest 1M.A. and A.R. contributed equally to this work. that overexpression of BAG3 in tumors plays a role in survival of 2To whom correspondence should be addressed. E-mail: [email protected]. these cells. Indeed down-modulation of BAG3 in primary samples This article contains supporting information online at www.pnas.org/cgi/content/full/ of B-cell chronic lymphocytic leukemia and acute lymphoblastic 0907696107/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.0907696107 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 Fig. 2. BAG3 protein levels influence NF-κB activity. (A) SAOS-2 cells at 30% confluency were transfected with bag3 or control scrambled siRNA. Chroma- tin was immunoprecipitated after 72 h with p65 antibody or control IgGs, and purified DNA was subjected to PCR assay to amplify a segment spanning the κB-responsive elements on the IL-8 and IκB-α promoters. β-actin promoter Fig. 1. BAG3 protein levels influence starvation or etoposide-induced primers were used as a negative control. The graph depicts band densitometry fi apoptosis in SAOS-2 cells. (A) SAOS-2 cells (30% confluency) were transfected values from three separate experiments. ns, not signi cant. (B) Lysates from with bag3siRNA or control scrambled RNA. After 72 h, whole-cell extracts SAOS-2 cells, WT or transfected (12 weeks of stable transfectants), with either β β were analyzed by immunoblotting with anti-BAG3 TOS-2 polyclonal anti- a construct expressing a constitutively active form of IKK [IKK EE over- body or control anti-α-tubulin antibody. (B) Cells were transfected as in A. expressing (o.e.)] or the void vector were analyzed in immunoblotting with α β After 72 h, cells were washed and incubated with medium alone or in the anti- -tubulin antibody. (C) WT, void vector-transfected, and IKK EE-trans- fl presence of etoposide (20 μM). Twenty-four hours later, apoptosis was fected cells were plated at 30% con uency and transfected with scrambled or evaluated as the percentage of sub-G cells by cell permeabilization and bag3 siRNA. After 72 h, cells were washed and incubated either with medium 1 μ propidium iodide staining in flow cytometry. (C) Cells were transfected as in alone or in presence of etoposide (20 M). Twenty-four hours later, apoptosis fl A. After 72 h, cells were washed and incubated with complete medium or in was analyzed by ow cytometry. starvation medium (absence of FCS). Twenty hours later, the sub-G1 cell percentage was analyzed. (D) Transfected cells, treated as described in C, were assessed for caspase-3 activity after 8 h of treatment with starvation modulated by NF-κB (34–36). As shown in Fig. 3B, coimmuno- medium. (E) SAOS-2 cells, WT or transfected (12 weeks with stable trans- precipitation experiments performed in M14 cells confirm the fectants), with a bag3 cDNA construct in either the pcDNA3.1 vector [bag3 interaction between BAG3, HSP70, and IKKγ.