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. 2013 Mar 8;288(10):6980-90.
doi: 10.1074/jbc.M112.414177. Epub 2013 Jan 22.

The stress protein BAG3 stabilizes Mcl-1 protein and promotes survival of cancer cells and resistance to antagonist ABT-737

Mariana Boiani  1 , Cristina Daniel, Xueyuan Liu, Michael D Hogarty, Lawrence J Marnett
Affiliations

The stress protein BAG3 stabilizes Mcl-1 protein and promotes survival of cancer cells and resistance to antagonist ABT-737

Mariana Boiani et al. J Biol Chem. .

Abstract

Members of the Bcl-2 family of proteins are important inhibitors of apoptosis in human cancer and are targets for novel anticancer agents such as the Bcl-2 antagonists, ABT-263 (Navitoclax), and its analog ABT-737. Unlike Bcl-2, Mcl-1 is not antagonized by ABT-263 or ABT-737 and is considered to be a major factor in resistance. Also, Mcl-1 exhibits differential regulation when compared with other Bcl-2 family members and is a target for anticancer drug discovery. Here, we demonstrate that BAG3, an Hsp70 co-chaperone, protects Mcl-1 from proteasomal degradation, thereby promoting its antiapoptotic activity. Using neuroblastoma cell lines, with a defined Bcl-2 family dependence, we found that BAG3 expression correlated with Mcl-1 dependence and ABT-737 resistance. RNA silencing of BAG3 led to a marked reduction in Mcl-1 protein levels and overcame ABT-737 resistance in Mcl-1-dependent cells. In ABT-737-resistant cells, Mcl-1 co-immunoprecipitated with BAG3, and loss of Mcl-1 after BAG3 silencing was prevented by proteasome inhibition. BAG3 and Mcl-1 were co-expressed in a panel of diverse cancer cell lines resistant to ABT-737. Silencing BAG3 reduced Mcl-1 protein levels and overcame ABT-737 resistance in several of the cell lines, including triple-negative breast cancer (MDA-MB231) and androgen receptor-negative prostate cancer (PC3) cells. These studies identify BAG3-mediated Mcl-1 stabilization as a potential target for cancer drug discovery.

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Figures

FIGURE 1.
FIGURE 1.
BAG3 expression is related to ABT-737 resistance in neuroblastoma. A, classification of neuroblastoma cell lines according to BH3 profile and sensitivity to the Bcl-2 antagonist, ABT-737. B, protein immunoblot showing the levels of BAG proteins in neuroblastoma cell lines; BAG5 was not detected. Actin was included as a loading control.
FIGURE 2.
FIGURE 2.
BAG3 binds to Mcl-1 in ABT-737-resistant cell lines. Endogenous proteins were immunoprecipitated using specific antibodies for BAG3 (A) or Mcl-1 (B and C). Co-immunoprecipitated proteins were detected by immunoblot. Normal sera IgG were used as control. CL, cell lysate; IP, immunoprecipitate.
FIGURE 3.
FIGURE 3.
BAG3 silencing reduces Mcl-1 protein levels. A, neuroblastoma cells were transfected with nontargeting siRNA (NEG) or BAG3 siRNA (BAG3), and after 48 h, the levels of Mcl-1 and Bcl-2 were determined by immunoblot. Actin was included as a loading control. B, IMR5 cells were transfected with nontargeting siRNA (NEG) or BAG3 siRNA (BAG3), and the expression of BAG3 and Mcl-1 was determined by real time PCR (RT-PCR) 24 h after transfection. Data are expressed relative to nontargeting siRNA as mean ± S.E. (n = 3). C, IMR5 cells were treated with 10 μg/ml cycloheximide (CHX) 24 h after transfection with nontargeting siRNA (NEG) or BAG3 siRNA (BAG3), and the level of Mcl-1 was determined by immunoblot. Actin was included as a loading control. D, the Mcl-1 level in the cycloheximide chase experiment was quantified by densitometry, and data are expressed as mean ± S.D. (n = 3). Mcl-1 half-life was calculated in each experiment, and comparison between groups was done using an unpaired Student's t test (nontargeting siRNA versus BAG3, p value < 0.05).
FIGURE 4.
FIGURE 4.
BAG3 prevents Mcl-1 degradation by the proteasome. A, IMR5 cells were transfected with nontargeting siRNA (NEG) or BAG3 siRNA (BAG3). After 48 h, cells were treated for 4 h with MG-132 (10 μm) or DMSO, and protein levels were determined by immunoblot. B, IMR5 cells were transfected with nontargeting siRNA (NEG) or BAG3 siRNA (BAG3) with or without PSMD14 siRNA. After 48 h, proteins levels were assessed by immunoblot. C, IMR5 cells were transfected with nontargeting siRNA (NEG) or BAG3 siRNA (BAG3). After 24 h, cells were treated with MG-132 (10 μm, 4 h) and then collected, and Mcl-1 was immunoprecipitated (IP). Ubiquitin conjugates were detected by immunoblot (IB). A band possibly corresponding to mono-ubiquitinated Mcl-1 is indicated with an arrow. D, HEK293T cells were transfected with nontargeting siRNA (NEG) or BAG3 siRNA (BAG3) alone or along with the corresponding FLAG-tagged BAG3 constructs: FLAG-BAG3 (WT), FLAG-R480A (BAG3R480A), and FLAG-P209L (BAG3P209L). Proteins levels were determined by immunoblot. Actin was included as a loading control.
FIGURE 5.
FIGURE 5.
Silencing BAG3 increases apoptosis in Mcl-1-dependent cells. A, neuroblastoma cells were transfected with BAG3 siRNA (BAG3), Mcl-1 siRNA (Mcl-1), or nontargeting siRNA (NEG). Relative viability was determined using WST-1 72 h after transfection. Data are expressed relative to the corresponding negative control as mean ± S.D. (n ≥ 8). Statistically significant values relative to the negative control are marked with asterisks (**, p value < 0.01, *, p value < 0.05). B, immunoblot of IMR5 cells showing the levels of PARP and cleaved PARP 24–48 h after transfection with BAG3 siRNA (BAG3) or nontargeting siRNA (NEG). C, IMR5 cells were pretreated with 10 μm Z-VAD-FMK (zVAD) or DMSO 1 h before transfection with nontargeting siRNA (NEG) or BAG3 siRNA (BAG3), and the pan-caspase inhibitor was maintained in the medium until cell collection (48 h). Protein levels were determined by immunoblot. Actin was included as loading control.
FIGURE 6.
FIGURE 6.
Silencing BAG3 increases ABT-737 sensitivity in Mcl-1-dependent cell lines. A, representative concentration-dependent response curves for ABT-737 in neuroblastoma cell lines. B–D, cells were transfected with nontargeting siRNA (NEG), BAG3 siRNA (BAG3), or Mcl-1 siRNA (Mcl-1). After 24 h, ABT-737 was added, and cells were treated for an additional 48 h. Relative viability was determined by WST-1. Data are expressed relative to the corresponding DMSO-treated cells as mean ± S.E. (n = 8).
FIGURE 7.
FIGURE 7.
BAG3 regulates Mcl-1 levels and is related to ABT-737 resistance in cancer cells. A, protein immunoblot showing the levels of BAG3 and Mcl-1 in cancer cell lines that are resistant to ABT-737. Actin was included as a loading control. B, selected cancer cell lines were transfected with nontargeting siRNA (NEG) or BAG3 siRNA (BAG3), and 48 h later, the level of Mcl-1 was determined by immunoblot. Actin was included as a loading control. C, cells were transfected with nontargeting siRNA (NEG) or BAG3 siRNA (BAG3). After 24 h, ABT-737 was added, and cells were treated for an additional 48 h. Relative viability was determined by WST-1. Data are expressed relative to the corresponding DMSO-treated cells as mean ± S.E. (n = 8).
FIGURE 8.
FIGURE 8.
BAG3 supports Mcl-1 antiapoptotic function in cancer cells. Mcl-1 is a short-lived protein that is actively degraded by the proteasome after binding to Hsp70. In Mcl-1-dependent cells, BAG3 expression prevents Mcl-1 degradation in the proteasome and supports Mcl-1 antiapoptotic activity. Neutralization of BAG3 (dashed arrows) increases Mcl-1 degradation by the proteasome, rendering the cell sensitive to the Bcl-2 antagonist ABT-737.
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