Metformin sensitizes triple-negative breast cancer to proapoptotic TRAIL receptor agonists by suppressing XIAP expression

Abstract

Purpose: Despite robust antitumor activity in diverse preclinical models, TNF-related apoptosis-inducing ligand (TRAIL) receptor agonists have not demonstrated efficacy in clinical trials, underscoring the need to identify agents that enhance their activity. We postulated that the metabolic stress induced by the diabetes drug metformin would sensitize breast cancer cells to TRAIL receptor agonists.

Methods: Human triple (estrogen receptor, progesterone receptor, and HER2)-negative breast cancer (TNBC) cell lines were treated with TRAIL receptor agonists (monoclonal antibodies or TRAIL peptide), metformin, or the combination. The effects on cell survival, caspase activation, and expression of TRAIL receptors and the antiapoptotic protein XIAP were determined. In addition, XIAP was silenced by RNAi in TNBC cells and the effects on sensitivity to TRAIL were determined. The antitumor effects of metformin, TRAIL, or the combination were evaluated in an orthotopic model of metastatic TNBC.

Results: Metformin sensitized diverse TNBC cells to TRAIL receptor agonists. Metformin selectively enhanced the sensitivity of transformed breast epithelial cells to TRAIL receptor agonist-induced caspase activation and apoptosis with little effect on untransformed breast epithelial cells. These effects of metformin were accompanied by robust reductions in the protein levels of XIAP, a negative regulator of TRAIL-induced apoptosis. Silencing XIAP in TNBC cells mimicked the TRAIL-sensitizing effects of metformin. Metformin also enhanced the antitumor effects of TRAIL in a metastatic murine TNBC model.

Conclusions: Our findings indicate that metformin enhances the activity of TRAIL receptor agonists, thereby supporting the rationale for additional translational studies combining these agents.

Keywords: Apoptosis; Breast cancer; Metastasis; Metformin; TRAIL; Therapeutics.

Figure 1.. Metformin sensitizes TNBC cells to TRAIL receptor agonists.

Crystal violet cell survival assay of TNBC cells preincubated with vehicle or metformin (1 mM for MDA-MB-468 cells or 5 mM for MDA-MB-231-mCherry and GILM2 cells) for 48 hours, and treated with vehicle or TRAIL, mapatumumab or lexatumumab (each 1.5 μg/ml for MDA-MB-231-mCherry and GILM2 cells or 4 μg/ml for MDA-MB-468 cells) for an additional 24 hours (MDA-MB-231-mCherry and GILM2 cells) or 48 hours (MDA-MB-468 cells). Left panels: representative images. Right panels: the percentage confluence of crystal violet-positive cells was scored (mean ± SEM, n = 3). In all panels, *, P < 0.05, **, P < 0.01, ***, P < 0.001.

Figure 2.. Metformin sensitizes transformed cells breast epithelial cells to TRAIL receptor agonists.

Crystal violet cell survival assay of MCF-10A-Vector or MCF-10A-Ras cells preincubated with vehicle or metformin (5 mM) for 48 hours, and then treated with vehicle or TRAIL, mapatumumab or lexatumumab (each 1.5 μg/ml) for an additional 24 hours. Left panels: representative images. Right panels: the percentage confluence of crystal violet-positive cells was scored (mean ± SEM, n = 3). In all panels, *, P < 0.05, **, P < 0.01, ***, P < 0.001.

Figure 3.. Metformin enhances TRAIL-induced caspase activation in TNBC cells.

A, Immunoblots of TNBC cells preincubated with vehicle or metformin (5 mM) for 48 hours and then treated with vehicle or TRAIL (1.5 μg/ml for MDA-MB-231-mCherry and GILM2 cells or 4 μg/ml for MDA-MB-468 cells) for 16 hours. B, TNBC cells were preincubated with vehicle or metformin (5 mM) for 48 hours, treated with vehicle or TRAIL (1.5 μg/ml for MDA-MB-231-mCherry and GILM2 cells or 4 μg/ml for MDA-MB-468 cells) for 24 hours, and caspase-3/7 activity was measured (mean ± SEM, n=3). C, Immunoblots of MCF-10A breast epithelial cells stably expressing vector or H-RasV12 treated preincubated with vehicle or metformin (5 mM) for 48 hours and then treated with vehicle or TRAIL (1.5 μg/ml) for 16 hours. D, MCF-10A-Vector or MCF-10A-RAS cells were preincubated with vehicle or metformin (5 mM) for 48 hours, treated with vehicle or TRAIL (1.5 μg/ml) for 24 hours, and caspase-3/7 activity was measured (mean ± SEM, n=3). In (B) and (D), **, P < 0.01 and ***, P < 0.001.

Figure 4.. Metformin does not alter cell surface expression of TRAIL receptors in TNBC cells.

A, TNBC cells were treated with vehicle or metformin (5 mM) for 72 hours. TRAIL-R2 (left panel) and TRAIL-R1 (right panel) mRNA levels were determined by real-time PCR and were normalized to expression in vehicle-treated TNBC cells. B, TNBC cells were treated with vehicle or MF (5 mM) for 72 hours and then incubated with IgG, TRAIL-R1 or TRAIL-R2 Ab. Cell surface expression of TRAIL receptors was determined by flow cytometry. Grey bar: TNBC cells incubated with IgG. Blue line: TNBC cells treated with vehicle and incubated with TRAIL-R1 (bottom panels) or TRAIL-R2 (top panels) Ab. Red line: TNBC cells treated with metformin and incubated with TRAIL-R1 (bottom panels) or TRAIL-R2 (top panels) Ab.

Figure 5.. Metformin reduces XIAP protein expression in TNBC cells and silencing XIAP sensitizes TNBC cells to TRAIL.

A, Immunoblot of XIAP protein levels (left panel) and real-time PCR analysis of XIAP mRNA levels (right panel) in TNBC cells treated with vehicle or metformin (5 mM) for 72 hours (immunoblots) or 48 hours (real-time PCR). B, MDA-MB-231-mCherry cells were transfected with a non-silencing control siRNA (si-Control) or one of two different siRNAs targeting XIAP (sil-XIAP or si2-XIAP). Immunoblot of XIAP expression 48 hours after transfection. C, Crystal violet cell survival assay of MDA-MB-231-mCherry cells transfected with control or XIAP siRNAs, and 48 hours later treated with vehicle or TRAIL (0.3 μg/ml) for 24 hours. Left panel: representative images. Right panel: quantification of cell confluence in 3 fields for each treatment (mean ± SEM, n = 3) *, P < 0.05, **, P < 0.01, ***, P < 0.001. D, Silencing XIAP enhances TRAIL-induced caspase activation in TNBC cells. MDA-MB-231-mCherry cells were transfected with control or XIAP siRNAs, and 48 hours later were treated with vehicle or TRAIL (0.3 μg/ml) for 16 hours. PARP (full length and caspase-cleaved) and procaspase-3 levels were determined by immunoblotting.

Figure 6.. Metformin enhances the antitumor effects of TRAIL in an orthotopic model of metastatic TNBC.

Three weeks after intraductal injection of tumor cells, female NSG mice with GILM2-mCherry mammary tumors were randomized into 4 groups (10 mice per group): control group (PBS i.p. daily for three weeks), metformin (2 mg per ml in drinking water for three weeks), TRAIL (10 mg/kg i.p., daily for three weeks) or metformin plus TRAIL (same dosing as in single agent arms). A, the percentage of the original mammary tumor volume (at 3 weeks) in each group (mean ± SEM, n = 10 mice per group). B, the percentage of the surface area occupied by lung metastases (mean ± SEM, n = 10 mice per group). C, the percentage active caspase-3-positive tumor cells in mammary tumors (left panel) or metastatic lung tumors (right panel) after treatment (mean ± SEM, n = 3 tumors per group). D, immunoblot of XIAP expression in mammary tumors from control vehicle-treated or metformin-treated mice (n=3 mammary tumors per group). XIAP expression normalized to β-actin was measured by Image J analysis. E, body weight of the mice in each treatment group at the end of the study (mean ± SEM, n = 10 mice per group). In all panels, **, P < 0.01, ***, P < 0.001 versus vehicle-treated mice or the indicated comparison.