Targeting BCL-xL improves the efficacy of bromodomain and extra-terminal protein inhibitors in triple-negative breast cancer by eliciting the death of senescent cells

Abstract

Inhibitors of bromodomain and extra-terminal proteins (BETi) suppress oncogenic gene expression and have been shown to be efficacious in many in vitro and murine models of cancer, including triple-negative breast cancer (TNBC), a highly aggressive disease. However, in most cancer models, responses to BETi can be highly variable. We previously reported that TNBC cells either undergo senescence or apoptosis in response to BETi, but the specific mechanisms dictating these two cell fates remain unknown. Using six human TNBC cell lines, we show that the terminal response of TNBC cells to BETi is dictated by the intrinsic expression levels of the anti-apoptotic protein B-cell lymphoma-extra large (BCL-xL). BCL-xL levels were higher in cell lines that senesce in response to BETi compared with lines that primarily die in response to these drugs. Moreover, BCL-xL expression was further reduced in cells that undergo BETi-mediated apoptosis. Forced BCL-xL overexpression in cells that normally undergo apoptosis following BETi treatment shifted them to senescence without affecting the reported mechanism of action of BETi in TNBC, that is, mitotic catastrophe. Most importantly, pharmacological or genetic inhibition of BCL-xL induced apoptosis in response to BETi, and inhibiting BCL-xL, even after BETi-induced senescence had already occurred, still induced cell death. These results indicate that BCL-xL provides a senescent cell death-inducing or senolytic target that may be exploited to improve therapeutic outcomes of TNBC in response to BETi. They also suggest that the basal levels of BCL-xL should be predictive of tumor responses to BETi in current clinical trials.

Keywords: B-cell lymphoma 2 (Bcl-2) family; B-cell lymphoma-extra large; BCL-xL; BCL2L1; BET inhibitor; apoptosis; breast cancer; bromodomain-containing protein 4 (BRD4); senescence; senolytic agent.

Figure 1.

TNBC cells have varied apoptotic/senescent responses to sustained BET inhibition. A, TNBC cell line panel: MDA-MB-231, HCC1143, MDA-MB-468, BT549, HCC38, and HCC70 cells were exposed to vehicle (VEH) or 500 nm JQ1 for 96 h and the number of viable cells counted using trypan blue exclusion. B, TNBC cell line panel was treated with VEH or 500 nm JQ1 and apoptosis measured by annexin V–FITC plus PI staining and FACS analysis. Bars show the number of cells that were apoptotic. C, representative images (10×) of MDA-MB-231 and HCC1143 cells that were treated with vehicle or 1 μm JQ1 for 8 days and then stained for senescence-associated β-gal activity. For graphs, data are expressed as mean ± S.D. (* = p < 0.05 compared with vehicle), n = 3 independent experiments.

Figure 2.

Cell lines that senesce and survive following BETi exposure express higher basal levels of BCL-xL. A, RT-qPCR of basal level mRNA expression of BCL2 family members in TNBC panel: MDA-MB-231, HCC1143, MDA-MB-468, BT549, HCC38, and HCC70. Data are presented as mean ± S.D., n = 3 independent experiments. B, Western blot showing protein expression of various BCL-2 family members or β-actin loading control in a panel of TNBC cell lines. C, MDA-MB-231, HCC1143, MDA-MB-468, and HCC70 cells were treated with vehicle (V) or 500 nm JQ1 (J) for 72 h and cell lysates evaluated by Western blotting for BCL-xL and β-actin. D, MDA-MB-231, HCC1143, MDA-MB-468, and HCC70 cells were treated with vehicle (V) or 500 nm JQ1 (J) for 72 h, and cell lysates were evaluated by Western blotting for BCL2, MCL1, BAX, and BIM. Each protein was normalized to total protein.

Figure 3.

BCL-xL inhibition shifts cell fate in response to BETi from senescence to apoptosis. A, MDA-MB-231 cells were treated with DMSO (VEH), 50 nm JQ1, 5 nm Obatoclax, or the combination (COMBO), and HCC1143 cells were treated with DMSO (VEH), 200 nm JQ1, 40 nm Obatoclax, or the combination (COMBO). Viable cells were counted using trypan blue exclusion. Error bars are S.D. *** = p < 0.05. B, MDA-MB-231 and HCC1143 cells were treated with DMSO (VEH), 125 nm JQ1, 40 nm Obatoclax, or the combination (COMBO). Apoptotic cells were quantified using annexin V–FITC plus PI staining and FACS analysis. Error bars are S.D. + = p < 0.05 compared with VEH and COMBO, # = p < 0.05 compared with VEH and COMBO, and * = p < 0.05 compared with VEH, JQ1, and OBAT. C, MDA-MB-231 and HCC1143 cells were transfected with either siRNA targeting BCL2L1 (BCL-xL) or NS siRNA. Reduced BCL-xL expression was confirmed by Western blotting with β-actin serving as a loading control. Cell viability was quantified using the trypan blue exclusion assay after a 72-h treatment of JQ1 (250 nm). D, left, MDA-MB-231 cells were treated with either DMSO (VEH) or 100, 250, or 500 nm Navitoclax for 72 h. Viable cells were counted using the trypan blue exclusion assay. Right, senescence was induced in MDA-MB-231 cells by pretreatment with 1 μm JQ1 for 8 days. Cells were then treated with Navitoclax (250 nm) for 72 h. Error bars are S.D. * = p < 0.05, *** = p < 0.001.

Figure 4.

BCL-xL overexpression suppresses BETi-induced apoptosis. A and B, 468EV and 468BCLxL cells were treated with vehicle or 500 nm JQ1 for 72 h. A, apoptotic cells were stained with Hoechst, and pyknotic nuclei (cells undergoing apoptosis) were counted. Inset, Western blot confirming overexpression of BCL-xL in MDA-MB-468 cells (468BCLxL) compared with MDA-MB-468 cells expressing the empty vector (468EV). B, representative images of Hoechst staining. White arrows indicate pyknotic nuclei. C, 468EV and 468BCLxL cells were treated with increasing concentrations of JQ1, and viable cells were counted after 72 h. For graphs, data are presented as mean ± S.D., n = 3 independent experiments. * = p < 0.05 compared with vehicle for 468EV cell line. # = p < 0.05 compared with vehicle for 468BCLxL cell line.

Figure 5.

BCL-xL modulation does not affect BETi-induced multi-nucleation. A, left, representative images (20×) of 468EV and 468BCLxL cells treated with vehicle (VEH) or 500 nm JQ1 for 4 days and stained with DAPI (blue, nucleus) and phalloidin (red, F-actin). White arrows indicate multi-nucleated cells; scale bars are 120 μm. Right, quantitation of multi-nucleated cells. Data are mean ± S.D., * = p < 0.05 compared with vehicle. B, left, representative images (20×) of MDA-231-shLuc and MDA-231-shBCLxL cells treated with vehicle (VEH) or 500 nm JQ1 for 8 days and stained with DAPI (blue, nucleus) and phalloidin (red, F-actin). White arrows indicate multi-nucleated cells. Right, quantitation of multi-nucleated cells. Data are mean ± S.D., * = p < 0.05 compared with vehicle.

Figure 6.

BCL-xL regulation of apoptosis occurs downstream of BETi-induced mitotic catastrophe. A, bee swarm plot of the length of time required by individual 468EV and 468BCLxL cells to complete mitosis starting 6 h after the addition of vehicle or 1000 nm JQ1. Red lines are mean ± S.E. p < 0.05 between 1) 468EV+VEH and 468EV+JQ1 and 2) 468BCLxL+VEH and 468BCLxL+JQ1 according to Tukey's multiple comparison test. B, quantitation of the percent of single daughter cells produced following mitosis in 468EV and 468BCLxL cells. * = p < 0.05 compared with vehicle. C and D, pie charts showing the percentage of vehicle- and JQ1-treated 468EV (C) and 468BCLxL (D) cells that underwent different mitosis-associated cell fates: divide (black), die in mitosis (red), exit and die (gray), or prolonged interphase (white).