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. Author manuscript; available in PMC: 2019 May 31.
Published in final edited form as: Breast Cancer Res Treat. 2017 Mar 21;163(3):435–447. doi: 10.1007/s10549-017-4201-0

Metformin Sensitizes Triple-Negative Breast Cancer to Proapoptotic TRAIL Receptor Agonists by Suppressing XIAP Expression

Elena Strekalova 1, Dmitry Malin 1, Harisha Rajanala 1, Vincent L Cryns 1
PMCID: PMC6544153  NIHMSID: NIHMS1027818  PMID: 28324269

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: metformin, breast cancer, TRAIL, metastasis, apoptosis, therapeutics

Introduction

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) and agonistic mAbs targeting its proapoptotic receptors (TRAIL-R1/DR4 and TRAIL-R2/DR5) selectively activate the caspase-8/10-mediated extrinsic apoptotic pathway in transformed cells and exhibit robust antitumor effects in diverse murine models of cancer [1, 2]. Despite promising preclinical results, TRAIL receptor agonists have failed to demonstrate significant efficacy, either alone or in combination with chemotherapy, in multiple clinical trials in advanced malignancies [3–8]. These disappointing results in clinical trials have been attributed to a number of factors, including de novo and/or acquired resistance to TRAIL receptor agonists, inadequate receptor oligomerization on ligand binding, limiting procaspase-8/10 activation, and lack of biomarkers to predict treatment response [1, 2].

In an effort to enhance the activity of TRAIL receptor agonists, these proapoptotic agents have been combined with TRAIL-sensitizing agents (e.g., histone deacetylase inhibitors, PPARγ agonists, aspirin, mTOR and other kinase inhibitors) to augment its antitumor effects (9–13). More recently, we have identified a novel nutritional intervention that selectively sensitizes breast cancer cells to TRAIL-R2 agonists [14]. Specifically, we demonstrated that depletion of the essential amino acid methionine metabolically primes breast cancer cells to respond to the agonistic TRAIL-R2 mAb lexatumumab by increasing cell surface expression of TRAIL-R2. Moreover, dietary methionine restriction enhanced the antitumor activity of lexatumumab against mammary tumors and lung metastases in an orthotopic model of metastatic breast cancer [14]. Hence, methionine restriction metabolically primes breast cancer cells to targeted agents that activate cell death by exposing a targetable vulnerability, namely, enhanced cell surface expression of TRAIL-R2.

Intriguingly, the diabetes medication metformin mimics many of the effects of methionine restriction, including disruption of methionine metabolism via inhibition of the functionally linked folate cycle in the one-carbon metabolic pathway, inhibition of the mechanistic target of rapamycin (mTOR), broad antitumor activity, improved insulin sensitivity, and prolonged lifespan [15–19]. Metformin use has been associated with reduced incidence of a broad range of tumor-types and reduced cancer mortality in many epidemiologic studies [19–22]. However, the antitumor mechanisms of metformin are not well understood. Both direct tumor effects (activation of AMPK with resultant inactivation of mTORC1, inhibition of mitochondrial complex I, and suppression of nuclear translocation of NFκB and Stat3 phosphorylation) and indirect systemic effects (reduction in insulin and IGF-1 levels) have been reported and implicated in its antitumor effects [18, 19]. Consistent with preclinical findings, metformin treatment of newly diagnosed breast cancer patients prior to surgery improves metabolic indices, increases tumor apoptosis and decreases tumor proliferation [23].

Given the similarities noted between methionine restriction and metformin, we postulated that metformin may also metabolically prime breast cancer cells to respond to proapoptotic TRAIL receptor agonists. Here we report that metformin sensitizes TNBC cells to TRAIL receptor agonists in vitro and in vivo. Metformin selectively enhances 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 are accompanied by a robust reduction in the expression level of the antiapoptotic protein XIAP. Metformin also enhances the antitumor effects of TRAIL in a murine model of TNBC. Collectively, our findings indicate that metformin enhances the clinical activity of TRAIL receptor agonists and suggests that additional translational studies combining these agents are warranted.

Methods and materials

Cell culture and reagents

Human MDA-MB-231 and GILM2 TNBC cells stably expressing mCherry and parental GILM2 TNBC cells were maintained as described [24]. MDA-MB-468 TNBC cells were cultured in DMEM media supplemented with 10% FBS and 100 IU/mL penicillin/streptomycin (ThermoFisher Scientific/Gibco). Human MCF-10A breast epithelial cells stably expressing oncogenic H-RasV12 or vector were maintained as described [25]. Mapatumumab and lexatumumab were kindly provided by Dr. Robin Humphreys (Human Genome Sciences). Recombinant TRAIL peptide (amino acids 95–281) was purified as described [11].

Crystal violet cell survival assay

A crystal violet cell survival assay was performed as described (10). Cells were seeded on 6-well plates (3 × 105 cells/well) overnight. Cells were then 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 μ.g/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) before staining with crystal violet. The percentage cell confluence of crystal violet-positive cells was determined in 3 fields per treatment condition using NIH ImageJ software. Cells in each field were colored using “color threshold”. Percentage confluence of colored cells was quantified using “analyze particles” which reports the percent area in each field occupied by colored cells.

Immunoblotting

Whole-cell lysates were immunoblotted as described [25] using primary Abs against XIAP, β-actin (Sigma-Aldrich), PARP (BD Biosciences) and procaspase-3 (Cell Signaling Technology).

Caspase-3/7 activity assay

The Caspase-Glo 3/7 Assay System (Promega) was used to measure caspase-3/7 activity in cell lysates according to the manufacturer’s instructions. Briefly, cells were seeded in 96-well plates (2.5 × 103 cells/well) overnight. The next day, cells were 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, GILM2, MCF-10A and MCF-10A-RAS cells or 4 μg/ml for MDA-MB-468 cells) for an additional 24 hours. Caspase-3/7 activity was normalized to cell number and expressed as fold activity compared to vehicle-treated cells.

TRAIL receptor cell surface expression

Cell surface expression of TRAIL receptors (TRAIL-R1 and TRAIL-R2) was determined by flow cytometry using TRAIL-R1, TRAIL-R2 or control IgG1 mAb conjugated with allophycocyanin (BioLegend) as described previously [14].

XIAP siRNA experiments

siRNAs targeting the sequences GAAGCUAGAUUAAAGUCCU (sil-XIAP) or CAGUGAAGACCCUUGGGAA (si2-XIAP) in human XIAP and non-silencing control siRNA were purchased from Sigma-Aldrich. Cells were transfected with siRNAs using Lipofectamine RNAiMAX Reagent (ThermoFisher Scientific) according to the manufacturer’s protocol.

Real-time PCR

Real-time PCR for TRAIL-R1, TRAIL-R2 and GAPDH was performed as described previously [14]. Primers for XIAP (forward 5-AGTGCCACGCAGTCTACAAA, reverse 5-GCATGTGTCTCAGATGGCCT) were purchased from Integrated DNA Technologies and real-time PCR was performed using the same methods. A comparative Ct method was used to normalize RNA expression in samples to the controls in each experiment.

Orthotopic Model of Metastatic TNBC

GILM2-mCherry TNBC cells (2 × 106) were resuspended in Matrigel (BD Biosciences) and injected bilaterally into the 4th mammary gland ducts of 4-to 5-week-old female NOD scid IL2 receptor γ chain knockout (NSG) mice (Jackson Laboratory). Mice were randomized into four treatment groups (10 mice per group) three weeks after tumor inoculation: 1) PBS vehicle i.p. daily; 2) metformin 2 mg/ml in the drinking water; 3) TRAIL (10 mg/kg i.p. daily), or (4) metformin (2 mg/ml in the drinking water) plus TRAIL (10 mg/kg i.p. daily). Mammary tumor volume was calculated as described [26]. Lung metastases were visualized by fluorescence microscopy in isolated whole lungs and scored using NIH ImageJ analysis as described [26]. All animal experiments were carried out as part of an IACUC-approved protocol at the University of Wisconsin-Madison.

Tumor apoptosis assay

Formalin-fixed, paraffin-embedded tumor tissue sections were analyzed for active caspase-3 expression by immunohistochemistry using an Ab against cleaved caspase-3 (Cell Signaling) as described [26].

Statistics

The statistical significance of differences between groups was determined by ANOVA with Bonferroni posttests using GraphPad Prism 4 software.

Results

Metformin sensitizes TNBC cells to TRAIL receptor agonists

To determine whether metformin sensitizes TNBC to TRAIL receptor agonists, three human TNBC cell lines (MDA-MB-231-mCherry, GILM2 and MDA-MB-468) were preincubated with vehicle or metformin and then treated with TRAIL, mapatumumab or lexatumumab. Metformin sensitized all three TNBC cell lines to TRAIL receptor agonists, with the most robust effects observed in MDA-MB-231-mCherry and GILM2 cells. (Fig. 1). A dose-response experiment in MDA-MB-468 cells identified 0.5 mM metformin as the minimal concentration needed to sensitize these TNBC cells to TRAIL receptor agonists (Fig. S1). In addition, preincubation with metformin sensitized human HT29 colon adenocarcinoma and DU145 prostate cancer cell lines to TRAIL receptor agonists (Fig. S2). Collectively, these results demonstrate that metformin augments the cytotoxicity of TRAIL receptor agonists against a broad range of tumor cell types, including TNBC cells.

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

Figure 1.

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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.

Metformin sensitizes transformed breast epithelial cells to TRAIL receptor agonists

To determine whether metformin enhanced the cytotoxicity of TRAIL receptor agonists preferentially in transformed cells, we utilized human MCF-10A breast epithelial cells transformed by oncogenic H-RasV12 and untransformed MCF-10A cells stably expressing empty vector. Strikingly, metformin robustly sensitized MCF-10A-Ras cells to TRAIL and lexatumumab, but had a minimal effect on the sensitivity of untransformed MCF-10A-Vector cells to these agents (Fig. 2). In contrast, metformin had only a modest effect on the cytotoxicity of mapatumumab against MCF-10A-Ras cells. Collectively, these results indicate that metformin preferentially sensitizes transformed cells to TRAIL receptor agonists and support the potential tumor-selectivity of this therapeutic combination.

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

Figure 2.

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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.

Metformin enhances TRAIL-induced caspase activation in TNBC cells

To investigate whether metformin promotes TRAIL-induced caspase activation, TNBC cells were preincubated with metformin, treated with vehicle or TRAIL and then analyzed by immunoblotting. Metformin promoted TRAIL-induced proteolysis of the caspase substrate PARP as detected by diminished full-length PARP and/or increased cleavage product compared to vehicle-treated cells (Fig. 3A). In addition, metformin enhanced TRAIL-induced procaspase-3 proteolytic processing as detected by decreased procaspase-3 levels and/or increased cleaved subunit compared to vehicle-treated cells. Similarly, pretreatment of HT29 and DU145 carcinoma cells with metformin augmented TRAIL-induced caspase activation (Fig. S3). To quantitate more precisely the effects of metformin and TRAIL on caspase activation, we utilized a caspase-3/−7 activity assay. Metformin robustly enhanced TRAIL-induced caspase-3/−7 activity in all three TNBC cells compared to cells treated with either agent alone (Fig. 3B). Metformin also enhanced TRAIL-induced caspase activation in MCF-10A-Ras cells, while untransformed MCF-10-Vector cells were not sensitive to this combination (Fig. 3C and 3D). Consistent with its reported mechanism of action [18, 19], metformin increased p-AMPK levels and selectively inhibited mTORC1 as determined by reduced phosphorylation of the mTORC1 substrate p-S6 and no effect on the mTORC2 substrate p-Akt (Fig. S4). Taken together, these findings indicate that metformin potently augments caspase activation and apoptosis by TRAIL in a broad range of tumor cell types and provide additional evidence for the tumor-selectivity of this combination.

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

Figure 3.

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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.

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

To determine whether metformin sensitizes cancer cells to TRAIL by increasing the expression of its proapoptotic receptors (TRAIL-R1 and TRAIL-R2), TNBC cells were treated with metformin and TRAIL receptor mRNA levels were measured by real-time PCR. Metformin treatment resulted in a modest increase in TRAIL-R2 mRNA levels in MDA-MB-468 TNBC cells, but had little effect on TRAIL-R2 levels in the other TNBC cell lines or on TRAIL-R1 mRNA levels in any of the TNBC cell lines (Fig. 4A). Furthermore, metformin did not significantly affect cell surface expression of either proapoptotic TRAIL-R1 or TRAIL-R2 receptors as determined by flow cytometry (Fig. 4B). Metformin also did not alter protein levels of MAGED2 (Fig. S5), which we previously demonstrated to be downregulated by methionine restriction [14]. These findings indicate that the TRAIL-sensitizing effects of metformin are not due to enhanced TRAIL receptor expression or cell surface localization in TNBC cells.

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

Figure 4.

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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.

Metformin reduces XIAP expression in TNBC cells and silencing XIAP sensitizes TNBC cells to TRAIL

The antiapototic X-linked inhibitor of apoptosis protein (XIAP) has been demonstrated to confer resistance to TRAIL-induced apoptosis by suppressing caspase activation [27, 28]. Therefore, we postulated that metformin might sensitize cancer cells to TRAIL by downregulating XIAP. Consistent with this hypothesis, treatment of MDA-MB-231-mCherry, GILM2 and MDA-MB-468 TNBC cells with metformin resulted in a reduction in XIAP protein levels (Fig. 5A, left panel). In contrast, treatment with metformin did not affect XIAP mRNA levels (Fig. 5A, right panel), indicating that metformin regulates XIAP via a posttranscriptional mechanism. To examine the functional role of XIAP downregulation in the TRAIL-sensitizing effects of metformin, MDA-MB-231 cells were transfected with a scrambled siRNA (si-Control) or one of two different siRNAs targeting XIAP (si-1 XIAP and si-2 XIAP). Both siRNAs targeting XIAP reduced XIAP protein levels compared to the scrambled control siRNA (Fig. 5B). Notably, silencing XIAP in MDA-MB-231-mCherry cells had a modest effect on cell viability and robustly sensitized these cells to TRAIL treatment compared to a scrambled control siRNA (Fig. 5C). To examine whether silencing XIAP enhanced TRAIL-induced caspase activation, MDA-MB-231-mCherry cells were transfected with siRNAs targeting XIAP or a scrambled control and then treated overnight with vehicle or TRAIL. Silencing XIAP enhanced TRAIL-induced cleavage of the caspase substrate PARP (reduction of full-length PARP and/or increased cleavage product), while levels of procaspase-3 proteolysis (reduction of procaspase-3 levels) were comparable in TRAIL-treated cells transfected with scrambled siRNAs or siRNAs targeting XIAP (Fig. 5D). Collectively, these results indicate that metformin sensitizes TNBC cells to TRAIL-induced apoptosis by downregulating expression of XIAP.

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

Figure 5.

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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.

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

To determine whether metformin augments the antitumor effects of TRAIL in an orthotopic model of metastatic TNBC, we treated female NSG mice bearing established GILM2-mCherry mammary tumors with vehicle, metformin alone, TRAIL alone or the combination of metformin and TRAIL. Under the conditions tested, metformin had no significant effect on mammary tumor growth or lung metastases. In contrast, TRAIL inhibited mammary tumor growth, but the combination of TRAIL and metformin was more effective than TRAIL alone (Fig. 6A). Both therapies, TRAIL alone or in combination with metformin inhibited lung metastases to a comparable degree (Fig. 6B). Moreover, both TRAIL alone or in combination with metformin induced apoptosis in mammary tumors and lung metastatic lesions as determined by active, cleaved caspase-3 immunostaining (Fig. 6C). Consistent with our in vitro findings, the combination of TRAIL and metformin resulted in more apoptosis induction in mammary tumors and lung metastases than either TRAIL or metformin alone. Importantly, metformin treatment reduced XIAP protein levels in mammary tumors (Fig. 6D), consistent with our in vitro findings. None of the treatments affected the body weight of the mice at the end of the study compared to control vehicle-treated mice (Fig. 6E). Collectively, these findings indicate that metformin enhances the antitumor activity of TRAIL against mammary tumors and lung metastases in vivo and provide preclinical evidence supporting additional translational studies investigating the combination of metformin and TRAIL receptor agonists in metastatic TNBC.

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

Figure 6.

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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.

Discussion

Based largely on epidemiologic data and promising preclinical studies, the oral diabetes medication metformin has been incorporated into a multitude of clinical trials in diverse early-stage and advanced malignancies, either as monotherapy or in combination with cytotoxic agents or radiation [19]. We have demonstrated that metformin sensitizes TNBC cells to proapoptotic TRAIL receptor agonists, while untransformed breast epithelial cells are largely resistant to this combination. Although other groups have recently demonstrated that metformin enhances TRAIL-induced apoptosis in cultured cancer cells [29–32], we have provided the first in vivo evidence for the therapeutic utility of this combination in an orthotopic model of metastatic TNBC that recapitulates many features of the human disease [24]. Specifically, we showed that metformin augments the antitumor effects of TRAIL against mammary tumors and increases apoptosis in mammary tumors and lung metastases compared to treatment with either agent alone. Importantly, these antitumor effects in vivo were observed at metformin doses that were well tolerated by the mice, a critical point because the TRAIL-sensitizing effects of metformin in vitro typically require millimolar concentrations of metformin in standard cell culture media due to their supraphysiologic glucose concentration [33, 34]. Given the lack of efficacy of TRAIL receptor agonists in clinical trials in advanced solid tumors [3–8], our findings are particularly significant from a translational perspective because they suggest that metformin may enhance the efficacy of TRAIL receptor agonists in clinical trials. As such, our results provide the first murine model evidence for the clinical utility of the combination of metformin and TRAIL receptor agonists, thereby providing critical preclinical evidence to support additional translational studies in poor-prognosis TNBC, which currently lack targeted therapies [35].

Indeed, metformin is a particularly attractive TRAIL-sensitizing agent based on its well documented safety in both diabetic and non-diabetic patients, its low cost and its beneficial impact on metabolic health by improving insulin sensitivity [18, 19]. In addition, our results indicate that the combination of metformin and TRAIL receptor agonists activate apoptosis in p53 mutant TNBC cells and preferentially induce apoptosis in transformed breast epithelial cells with little effect on untransformed breast epithelial cells. Although the mechanism of this tumor-selectivity remains poorly understood, our findings suggest that the toxicity of the combination therapy may be modest. Moreover, our studies may provide insights into the reduced cancer incidence in individuals treated with metformin reported in many epidemiologic studies [19–22]: metformin may enhance the sensitivity of nascent tumors to TRAIL-dependent immune surveillance pathways.

Mechanistically, we have demonstrated that metformin sensitizes TNBC cells to TRAIL receptor agonists by downregulating the antiapoptotic protein XIAP. XIAP is a direct inhibitor of caspases that confers resistance to TRAIL-induced caspase-3 activation and apoptosis, while induction of TRAIL-induced apoptosis requires XIAP to be displaced from caspases by mitochondrial release of Smac/DIABLO [27, 28]. We observed that metformin reduces protein expression of XIAP but does not alter XIAP mRNA levels, thereby pointing to a post-translational mechanism. Intriguingly, rapamycin was previously demonstrated to negatively regulate protein translation of XIAP [36], suggesting that metformin may reduce XIAP protein levels via inhibition of mTORC1. Moreover, metformin treatment reduced XIAP protein levels in mammary tumors in our murine model of TNBC, indicating that the dosing used in our study was sufficient to target this pathway in vivo. To determine the functional relevance of the downregulation of XIAP protein by metformin, we used RNAi and observed that XIAP silencing robustly sensitizes TNBC cells to TRAIL-induced caspase activation and cell death, underscoring the importance of this molecular event in sensitizing TNBC cells to TRAIL. Our results are also consistent with a prior report demonstrating that a small molecule inhibitor of XIAP (embelin) sensitizes inflammatory breast cancer cells lines to TRAIL [37].

Notably, we did not observe any significant effects of metformin on the cell surface expression of TRAIL receptors in contrast to other reports that metformin increases TRAIL-R2 protein levels, although the cellular localization of the receptor was not delineated in these prior studies [29, 31]. Additionally, metformin did not affect protein levels of MAGED2, indicating that metformin sensitizes TNBC cells to TRAIL receptor agonists by a different mechanism than methionine restriction, which increases cell surface expression of TRAIL-R2 by downregulating expression of MAGED2, an inhibitor of TRAIL receptor expression [14, 38]. Furthermore, other groups have implicated downregulation of the antiapoptotic protein FLIP as an important molecular event in the TRAIL-sensitizing effects of metformin [12, 39]. Although our results do not exclude the potential contribution of FLIP or other molecular targets, our studies point to the downregulation of XIAP by metformin as a functionally relevant event for its TRAIL-sensitizing effects.

In conclusion, we have demonstrated that metformin downregulates XIAP and sensitizes clinically aggressive TNBC cells to TRAIL receptor agonists. We have also shown that the combination of metformin and TRAIL has robust antitumor activity in a murine model of metastatic TNBC which recapitulates features of the human disease. Given the lack of targeted therapies for TNBC and the largely disappointing activity of TRAIL receptor agonists in clinical trials to date, our results point to the combination of metformin and TRAIL receptor agonists as a promising approach to enhance activity of these proapoptotic agents. Finally, our studies suggest that inhibition of XIAP expression by metformin may be a useful biomarker to predict response to this combined therapy.

Supplementary Material

Suppl. data

Acknowledgements

We are indebted to Dr. Robin Humphreys for providing mapatumumab and lexatumumab.

Funding: This study was funded by grants from the Breast Cancer Research Foundation (VLC), the Department of Defense PC150221P1 (VLC), and P30CA14520 University of Wisconsin Comprehensive Cancer Center core facility support.

Footnotes

Compliance with Ethical Standards

Conflict of interest: The authors declare that they have no conflict of interest.

Ethical approval: All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Ethical approval: This article does not contain any studies with human participants performed by any of the authors.

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