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. 2011 Mar;18(3):452-64.
doi: 10.1038/cdd.2010.116. Epub 2010 Oct 1.

Downregulation of autophagy by Bcl-2 promotes MCF7 breast cancer cell growth independent of its inhibition of apoptosis

S Oh  1 , E Xiaofei, D Ni, S D Pirooz, J-Y Lee, D Lee, Z Zhao, S Lee, H Lee, B Ku, T Kowalik, S E Martin, B-H Oh, J U Jung, C Liang
Affiliations

Downregulation of autophagy by Bcl-2 promotes MCF7 breast cancer cell growth independent of its inhibition of apoptosis

S Oh et al. Cell Death Differ. 2011 Mar.

Abstract

The anti-apoptotic Bcl-2 protein, which confers oncogenic transformation and drug resistance in most human cancers, including breast cancer, has recently been shown to effectively counteract autophagy by directly targeting Beclin1, an essential autophagy mediator and tumor suppressor. However, it remains unknown whether autophagy inhibition contributes to Bcl-2-mediated oncogenesis. Here, by using a loss-of-function mutagenesis study, we show that Bcl-2-mediated antagonism of autophagy has a critical role in enhancing the tumorigenic properties of MCF7 breast cancer cells independent of its anti-apoptosis activity. A Bcl-2 mutant defective in apoptosis inhibition but competent for autophagy suppression promotes MCF7 breast cancer cell growth in vitro and in vivo as efficiently as wild-type Bcl-2. The growth-promoting activity of this Bcl-2 mutant is strongly correlated with its suppression of Beclin1-dependent autophagy, leading to sustained p62 expression and increased DNA damage in xenograft tumors, which may directly contribute to tumorigenesis. Thus, the anti-autophagic property of Bcl-2 is a key feature of Bcl-2-mediated oncogenesis and may in some contexts, serve as an attractive target for breast and other cancer therapies.

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Figures

Figure 1
Figure 1
Anti-apoptotic and anti-autophagic interaction of WT and mutant Bcl-2 proteins. (a) Schematic representation of the wild type (WT) and mutant Bcl-2 constructs interacting with Beclin1 and/or Bax. The α-helical structures of Bcl-2 is indicated at the top numbered according to previous publication. Colored boxes denote the BH 1–4 domains in Bcl-2; TM, transmembrane domain; ‘+' : positive interaction, ‘−': no interaction. Triple arrows denote the alanine substitutions at Trp144-Gly145-Arg146 within the BH1 domain of Bcl-2. (b and c) Co-immunoprecipitation (Co-IP) of WT and mutant Bcl-2 with Bax. 293T cells were transfected with the indicated Bcl-2 constructs, with (in b) or without (in c) Flag-Bax. Whole-cell lysates (WCLs) were used for immunoprecipitation (IP) with an anti-HA antibody, followed by immunoblotting (IB) with an anti-Flag (b) or a Bax (c) antibody. (d) Bcl-2 interaction with Bak. 293T cells were transfected with WT and mutant forms of Bcl-2 as indicated. At 48 h posttransfection, WCLs were mixed either with GST-BakΔTM fusion protein (right panel) or with GST alone (left panel) for an in vitro GST pulldown (GST PD) assays. GST fusion proteins used for the pulldown assay are indicated (bottom panel). 1% WCL was used as the input. Data are representative of at least three experiments yielding similar results. (e and f) Co-IP of WT or mutant Bcl-2 with Beclin1. 293T cells were transfected with the indicated constructs, followed by IP with an anti-HA antibody and IB with an anti-V5 (e) or an anti-Beclin1 (f) antibody. 1% whole-cell lysates (WCLs) was used as the input
Figure 2
Figure 2
Anti-apoptotic activities of the Bcl-2 mutants. NIH3T3 cells stably expressing the WT or mutant forms of Bcl-2 were treated with DMSO (control) or staurosporine for 24 h, then assayed for cell viability by trypan blue exclusion assay (a), for apoptosis (sub-G1 staining) by PI staining (b), or for caspase-3 activation using flow cytometry (c). Data represents mean±S.D. of combined results from three independent experiments. The M1 gate in (b) marks the population of cells with a sub-G1 DNA content. *P<0.01; **P<0.001; ***P<0.0001. PI, propidium iodide
Figure 3
Figure 3
Anti-autophagic activity of the Bcl-2 mutants. (a) NIH3T3 cells expressing the WT or mutant Bcl-2 proteins as indicated were transfected with GFP-LC3 then treated with 2 μM rapamycin for 6 h. GFP-LC3 signal was detected using an inverted fluorescence microscope (top). Autophagy was quantified (percentage of cells with GFP-LC3 puncta) as mean±S.D. of the combined results from three independent experiments (bottom). Scale bar, 5 μm; **P<0.01; ***P<0.001. (b) NIH3T3 cells as described in (a) were cultured in a complete medium or treated with 2 μM rapamycin with or without 100 nM bafilomycin A1 for 2 h. The cell lysates were subject to immunoblotting with a LC3 (top), a p62 (middle), and an actin (bottom) antibodies. Densitometric quantification of the LC3-II/LC3-I ratios under the indicated conditions is shown at the bottom of the LC3 blot. Similar results were obtained from three independent experiments. NT, no treatment; R, rapamycin treatment; RB, rapamycin and bafilomycin A1 treatment. (c) MCF7.control cells and MCF7.beclin 1 cells stably expressing the WT or mutant forms of Bcl-2 were transfected with GFP-LC3. At 18 h posttransfection, cells were treated with 2 μM rapamycin for 6 h. Autophagy was quantified as mean±S.D. of the combined results from three independent experiments. **P<0.001; ***P<0.0001
Figure 4
Figure 4
Effects of expression of the Bcl-2 mutants on the transformation property and tumorigenicity of MCF7.beclin 1 cells. (a) Cellular proliferation of MCF7.control cells (open triangles), MCF7.beclin1 cells stably expressing empty vector (open squares), WT Bcl-2 (closed squares), Bcl-2Δα1 (open circles), and Bcl-2ΔBH2 (closed circles). Representative result from three independent experiments is shown. (b) Clonogenic assay in vitro. MCF7 cells in (a) were plated at low density (2500 cells per 10 cm plate), grown for 7 days and fixed and stained with crystal violet. Data represent mean±S.D. of duplicate plates from three independent experiments. (c) Anchorage-independence. MCF7 cells (1 × 104) in (a) were incubated in soft-agar as described in Materials and Methods. After 3 weeks, colonies were photographed and counted. Colony numbers were quantified as means±S.D. of pooled results from three independent experiments. (d and e) Tumorigenicity. MCF7 cells as indicated were injected into the breast pad of nude mice (5 × 106 cells/injection), and tumor size and tumor incidence were measured at 21 days postinoculation. Numbers on top of bar (e) denote the number of autopsy-confirmed tumors at 21 days per number of mice injected with each cell line. *P<0.05; **P<0.01; ***P<0.001
Figure 5
Figure 5
Effects of expression of the Bcl-2 mutants on the transformation property and tumorigenicity of MDA-MB-231 cells. (a) Cellular proliferation (left panel) of MDA-MB-231 cells stably expressing empty vector (open squares), WT Bcl-2 (closed squares), Bcl-2Δα1 (open circles), Bcl-2ΔBH2 (closed circles), and Bcl-2AAA (open triangles). Representative result from three independent experiments is shown. (b) Clonogenic assay in vitro. MDA-MB-231 cells in (a) were plated at low density (2500 cells per 10 cm plate), grown for 7 days and fixed and stained with crystal violet. Data represent mean±S.D. of duplicate plates from three independent experiments. *P<0.05; NS, not significant. (c) Tumorigenicity. MDA-MB-231 cells as indicated were injected into the breast pad of nude mice (107 cells/injection), and tumor size were measured at 30 days post-inoculation. Data represent mean±S.D. from three independent experiments. **P<0.01; NS, not significant
Figure 6
Figure 6
Bcl-2Δα1 promotes tumor growth and suppresses autophagy in xenograft tumors in vivo. (a) Hematoxylin/eosin (H&E) staining of xenograft tumors generated by the indicated MCF7 cell lines. Pronounced necrosis (N) was detected in the Bcl-2Δα1 tumor independent of the means of Bcl-2-mediated apoptosis inhibition. Original magnification, × 40. (b) Mitotic activity was brisk in xenografts with WT and Bcl-2Δα1 expression. Original magnification, × 400. The number of mitotic figures per 10 high-power fields (HPF) in WT and Bcl-2Δα1 xenografts were significantly higher than in vector and AAA xenografts. Arrows indicate mitotic cells. (c) Immunohistochemical analysis of HA–Bcl-2 (WT and mutants) expression in breast tumor sections developed from implanted MCF7.beclin1 cells as indicated. (d) Electron microscopic analyses of xenograft tumors. Magnified images of the insets highlighted abundant autophagic vacuoles in tumors derived from the empty vector- and the AAA-expressing MCF7.beclin1 cells. The mean number of autophagic vacuoles per cell in tumors was determined (data are mean±S.D., n=25). Asterisks denote autophagic vacuoles (double- or single-membrane vacuolar structures containing recognizable cytoplasmic materials) counted in each sample
Figure 7
Figure 7
Bcl-2Δα1 expression upregulates the p62 and activates DNA damage response in vivo. Immunohistochemical analysis of p62 (a), GRP78 (b), γ-H2AX (c), cleaved caspase-9 (d), and PARP (e) in breast tumor sections developed from implanted MCF7.beclin1 cells as indicated. The insets highlighted the staining of the indicated proteins. Original magnification, × 400. Arrows indicate apoptotic cells with a positive PARP cleavage
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