Apoptosis (2015) 20:1373–1387 DOI 10.1007/s10495-015-1158-5
ORIGINAL PAPER
Metformin synergizes 5-fluorouracil, epirubicin, and cyclophosphamide (FEC) combination therapy through impairing intracellular ATP production and DNA repair in breast cancer stem cells Jaslyn Sian-Siu Soo1,2,3 • Char-Hong Ng2,3 • Si Hoey Tan4 • Rozita Abdul Malik6 Yew-Ching Teh2,3 • Boon-Shing Tan4,5 • Gwo-Fuang Ho6 • Mee-Hoong See2,3 • Nur Aishah Mohd Taib2,3 • Cheng-Har Yip2,3 • Felicia Fei-Lei Chung4 • Ling-Wei Hii4 • Soo-Hwang Teo1,2,3 • Chee-Onn Leong4
•
Published online: 15 August 2015 Ó Springer Science+Business Media New York 2015
Abstract Metformin, an AMPK activator, has been reported to improve pathological response to chemotherapy in diabetic breast cancer patients. To date, its mechanism of action in cancer, especially in cancer stem cells (CSCs) have not been fully elucidated. In this study, we demonstrated that metformin, but not other AMPK activators (e.g. AICAR and A-769662), synergizes 5-fluouracil, epirubicin, and cyclophosphamide (FEC) combination chemotherapy in non-stem breast cancer cells and breast cancer stem cells. We show that this occurs through an AMPK-dependent mechanism in parental breast cancer cell lines. In contrast, the synergistic effects of metformin and FEC occurred in an AMPK-independent mechanism in breast CSCs. Further analyses revealed that metformin accelerated glucose consumption and lactate production more
severely in the breast CSCs but the production of intracellular ATP was severely hampered, leading to a severe energy crisis and impairs the ability of CSCs to repair FECinduced DNA damage. Indeed, addition of extracellular ATP completely abrogated the synergistic effects of metformin on FEC sensitivity in breast CSCs. In conclusion, our results suggest that metformin synergizes FEC sensitivity through distinct mechanism in parental breast cancer cell lines and CSCs, thus providing further evidence for the clinical relevance of metformin for the treatment of cancers.
Jaslyn Sian-Siu Soo and Char-Hong Ng are joint first authors and shared equal contribution.
Introduction
Electronic supplementary material The online version of this article (doi:10.1007/s10495-015-1158-5) contains supplementary material, which is available to authorized users. & Soo-Hwang Teo
[email protected] & Chee-Onn Leong
[email protected] 1
Cancer Research Initiatives Foundation, Sime Darby Medical Centre, 1 Jalan SS12/1A, 47500 Subang Jaya, Selangor, Malaysia
2
Department of Surgery, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia
3
Breast Cancer Research Unit, Faculty of Medicine, University Malaya Medical Centre, University Malaya Cancer Research Institute, University of Malaya, 50603 Kuala Lumpur, Malaysia
Keywords Metformin Breast cancer stem cell Drug combination AMPK
Breast cancer is a major cause of cancer-related deaths among women, largely because the control of recurrent and metastatic disease remains a major challenge [1, 2]. One 4
Center for Cancer and Stem Cell Research, International Medical University, 126 Jalan 19/155B, 57000 Bukit Jalil, Kuala Lumpur, Malaysia
5
Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan
6
Department of Clinical Oncology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia
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possible explanation is that while many cancer therapies eliminate the bulk of tumor cells, they may ultimately fail because they do not eliminate cancer stem cells (CSCs) [2]. CSCs are a source of tumor cells that are more resistant to therapies and may lead to cancer progression, recurrence and metastasis [3, 4]. Indeed, the CD44?/CD24-/low breast CSCs are enriched in residual breast cancers after chemotherapy and endocrine therapy [5, 6]. These observations suggest that to be effective, cancer therapies should include drugs that target CSCs and non-CSCs to prevent the regrowth of neoplastic cell populations. Metformin, an AMP-activated protein kinase (AMPK) activator, is a relatively safe drug that is widely used for the treatment of Type II diabetes, obesity and polycystic ovarian syndrome (PCOS) [7]. Pre-clinical studies have shown that metformin have anti-cancer and CSCs-targeting properties in cancer cell lines, such as breast, endometrial, prostate and pancreatic cancer [8]. In breast cancer, metformin selectively targeted CSCs in cell lines and blocked tumor growth and prolong remission in mouse xenografts when combined with chemotherapy [8–10]. These findings are consistent with epidemiological studies indicating a reduction of cancer incidence and mortality across different cancer types in diabetic patients treated with metformin compared to non-users [11–14]. Despite the promising findings from numerous studies, it is currently unclear whether other AMPK activators might have similar CSCs-targeting properties. In this study, we investigated the anti-tumor effects of three AMPK activators (metformin, AICAR and A-769662) in combination with chemotherapeutic drugs commonly used in the treatment of breast cancer [doxorubicin, paclitaxel, and 5-flurouracil/epirubicin/cyclophosphamide (FEC)] in a panel of parental breast cancer cell lines and CD44?/CD24-/low CSCs. Our results indicate that metformin, but not AICAR or A-769662, synergizes with FEC to induce cytotoxicity through an AMPK-dependent mechanism in the parental cells but in an AMPK-independent manner in CSCs. Further analyses revealed that metformin accelerated glucose consumption and lactate production (indicative of increased in glycolysis) in breast CSCs, but the production of intracellular ATP was severely hampered, leading to a severe energy crisis and lack of DNA repair, resulting in a striking synergistic elimination of these cells in vitro with FEC chemotherapeutic drug. Taken together, our results suggest that metformin synergizes FEC sensitivity through distinct mechanism in parental breast cancer cell lines and CSCs, thus providing support for future clinical studies using metformin in combination with conventional chemotherapy for the treatment of refractory breast cancers.
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Materials and methods Chemicals and reagents Metformin hydrochloride, 5-fluorouracil, epirubicin hydrochloride and cyclophosphamide monohydrate were purchased from Sigma Aldrich, St. Louis, MO, USA. Doxorubicin, paclitaxel, AICAR, A-769662 and Compound C were purchased from Selleckchem, Houston, USA. FEC was prepared with a combination of 5-fluorouracil, epirubicin, and cyclophosphamide at a molar ratio of 22:1:11, similar to the clinical dosage. Any reference on FEC utilized in this study would be based on the concentration of 5-fluorouracil. All drugs were dissolved in DMSO except for metformin which was dissolved in water, and stored at -20 °C. Compound C was stored at 4 °C. Cell lines and cell culture MDA-MB-468, MDA-MB-231, HCC1937, MCF7, SKBR3, and T47D breast cancer cell lines, and MRC-5 human embryonic lung fibroblast were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA) and were maintained in RPMI 1640 containing 10 % fetal bovine serum (FBS), 100 IU/ml penicillin, and 100 lg/ml streptomycin (Sigma-Aldrich, St. Louis, MO, USA). MCF-10A cells were grown in DMEM-F12 (SigmaAldrich, USA) supplemented with 5 % horse serum, 20 ng/ ml EGF, 0.5 g/ml hydrocortisone, 100 ng/ml cholera toxin, 10 lg/ml insulin, 100 IU/ml penicillin, and 100 lg/ml streptomycin. All cells were maintained at 37 °C in an environment containing 5 % CO2. Mammosphere culture Mammosphere cultures were performed as described previously [8, 9, 15]. Briefly, single-cell suspensions were cultured in six-well ultra-low attachment plates (Corning Inc., New York, USA). Cells were grown in MammoCult medium supplemented with 4 lg/ml heparin (Stem Cell Technologies, Vancouver, BC, Canada) and 0.48 lg/ml hydrocortisone (Sigma-Aldrich, St. Louis, MO, USA). Mammospheres were collected by gentle centrifugation (1500 rpm) after 5 days and dissociated with trypsinEDTA (Sigma-Aldrich, St. Louis, MO, USA) to single cells. Analysis of cancer stem cells Analysis of breast CSCs populations were performed on single-cell suspensions using flow cytometry. Briefly, cells were stained with CD44-APC and CD24-PE (BD
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Biosciences, San Jose, CA, USA) for 30 min, washed, and re-suspended in PBS supplemented with 1 % FBS. CSCs populations in breast cancer cell lines or mammospheres were identified as CD44?/CD24-/low. All cells were analyzed using a FACSCalibur flow cytometer and the CellQuest Pro software (version 5.1.1; BD Biosciences, USA) for acquisition and FlowJo VX (Version 10.0.6; TreeStar Inc., Ashland, OR, USA) for data analysis. Cell proliferation and apoptosis assay Cell growth assay was performed using Cell Titer 96 AQueous One Solution cell proliferation assay (Promega, Madison, WI, USA). Attached cells and mammospheres were seeded in 96-well plates overnight and treated with different concentrations of agents for 72 h. Cell Titer 96 AQueous One Solution reagent was added to each well, and the plate was incubated for 4 h, after which the absorbance was recorded at 490 nm with a TECAN Infinite F200 96-well plate reader. Quantitation of apoptosis by Annexin V-PE/7-AAD staining was performed as described previously [16, 17]. Drug combination analysis Effects of drug combinations were evaluated using Bliss independence model [18, 19] and Excess Over Highest Single Agent (EOHSA) statistical analysis. The Bliss independence model is defined by the equation Exy = Ex ? Ey - (ExEy), where (Exy) is the additive effect of drugs x and y as predicted by their individual effects (Ex and Ey). If the experimentally measured fractional inhibition is more than Exy, the combination is said to be synergistic. If the experimentally measured fractional inhibition is less than Exy, the combination is said to be antagonistic [20]. In the single-concentration experiment, hits were identified using: (1) a cut off of 10 % excess over Bliss additivity; and (2) a statistically significant improvement in the combination compared with the highest single agent alone (P \ 0.01, Student’s t test). For dose–response curves, the Bliss additivity value was calculated for varying concentrations of drug 1 when combined with varying concentrations of drug 2 at a fixed concentration ratio with MacSynergy II program version 1.0 [20]. A synergy volume was calculated by adding all of the synergy values (positive values) for each drug combination. Likewise, all of the antagonistic values (negative values) were added to give an antagonistic volume. These synergy and antagonism volumes were then statistically evaluated at 95 % confidence level and are expressed in lM2%. Values between -25 and ?25 lM2% were considered additive, values below -25 lM2% represented
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antagonism, and values above 25 lM2% represented synergism. Protein isolation and western blot analysis Protein lysates from cells were extracted in ice-cold lysis buffer (1 % NP-40, 1 mM DTT, phosphatase inhibitor cocktail and protease inhibitor cocktail in PBS) [21, 22]. Total protein (25 lg) was subjected to SDS-PAGE followed by immunoblotting. All primary antibodies were obtained from Cell Signaling Technology Inc., Danvers, MA, USA except for antibody against actin and SREBP1 were obtained from Santa Cruz Biotechnology, USA. Transfection of shRNA and RagB GTPase Lentiviral shRNA construct targeting AMPKa were purchased from Sigma-Aldrich (St. Louis, MO, USA). The constitutively active Ras-related GTP binding B (RagB GTPase Q99L) plasmid was obtained from Addgene (Cambridge, MA, USA; plasmid 19030) [23]. Transient transfections were performed using X-tremeGENE HP DNA transfection reagent (Roche Diagnostics, IN, USA) as described previously [21, 24, 25]. The shRNA target sequence for AMPKa was 5´-CCTGGAAGTCACACAA TAGAA-3´ (AMPKsi). Glucose and lactate assay Cells were cultured in complete medium in exponential growth phase. Media from the samples were collected at various time intervals and analysed for glucose and lactate concentration using a glucose colorimetric kit (Biovision, Mountain View, CA, USA) and a lactate assay kit (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer’s instruction. The absorbance was measured at 570 nm for glucose assay and fluorescence were measured by excitation at 530 nm and emission at 585 nm in the lactate assay, using a SpectraMax M3 plate reader (Molecular Devices, Sunnyvale, CA, USA). The concentration of glucose and lactate in the media was calculated by comparison with a standard curve. Determination of complex 1 activity and ATP concentration Complex 1 activity was determined with the Complex I Enzyme Activity Microplate Assay Kit (Abcam, Cambridge, MA, USA). Cellular ATP levels were determined using the ATP-based CellTiter-Glo luminescent kit (Promega, Madison, WI, USA) as described previously [26]. ATP generation was normalized by the cell number.
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Single-cell gel electrophoresis assay Single-cell gel electrophoresis for detection of DNA damage was performed as described previously [27]. The percentage of tail DNA was analyzed using the CASPLab software program (version 1.2.3) [28].
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t test) (Fig. 1c). Similar patterns were also observed in T47D cells (Supplemental Table 1). No significant toxicity was observed in MCF-10A non-transformed myoepithelial cells and MRC-5 human embryonic lung fibroblast (Supplemental Fig. 1b). These results show that metformin, but not the other AMPK activators, has additional anti-proliferative effects on CD44?/CD24-/low-enriched mammospheres.
Results CD441/CDC242/low breast cancer stem cells are resistant to chemotherapeutic drugs, but are sensitive to metformin To study the effect of drug treatments on CSCs, we utilized mammosphere culture, which has previously been verified to represent an in vitro model of breast CSCs [8, 9, 15, 29]. Mammospheres were obtained by culturing breast cancer cell lines in a serum-free suspension culture for 5 days and dissociating the cells prior to analysis or drug treatments (Fig. 1a and Supplemental Fig. 1a). Flow cytometry analysis showed that the basal-like breast cancer cells, HCC1937 and MDA-MB-231, had a higher percentage of CD44?/CD24-/ low cells (23 and 35 %, respectively) compared with the MCF-7 and T47D luminal cell lines (3 and 2 %, respectively) and the HER2-positive SKBR3 cells (4 %) (Fig. 1b). Importantly, the population of CD44?/CD24-/low cells was significantly enriched (P \ 0.01, Student’s t test) in mammospheres compared with the respective parental cell lines, indicating at least 50–85 % of the cells in the mammospheres were indeed CD44?/CD24-/low breast CSCs (Fig. 1b). Given that breast CSCs are resistant to conventional chemotherapy [5, 6] but sensitive to metformin [4, 30], we investigated the effect of other AMPK activators on CD44?/CD24-/low-enriched mammospheres. Parental and mammosphere-derived MDA-MB-468 cells were treated with chemotherapeutic drugs (doxorubicin, paclitaxel, and FEC), and AMPK activators (metformin, AICAR, and A-769662) for 72 h and the cell viability was assessed. MDA-MB-468 CD44?/CD24-/low-enriched mammospheres were 10-fold more resistant than parental cells to all chemotherapy drugs tested, particularly to FEC, a clinically used combination therapy for first-line treatment in breast cancer patients (IC50 of 2.66 ± 0.29 lM in parental cells compared to [20 lM in mammospheres) (Fig. 1c and Supplemental Table 1). Similarly, MDA-MB-468 CD44?/CD24-/low-enriched mammospheres were more resistant to AICAR and A-769662 compared to the parental MDA-MB-468 cells. In contrast, we found that metformin had a higher potency ([5fold) toward CD44?/CD24-/low-enriched mammospheres compared to the parental cells with an IC50 of 2.32 ± 0.29 and 12.00 ± 2.54 mM, respectively (P \ 0.01, Student’s
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Metformin synergizes FEC sensitivity in parental breast cancer cells and breast cancer stem cells through induction of apoptosis Next, we sought to evaluate the effect of combining chemotherapeutic drugs with the AMPK activators. The parental and CD44?/CD24-/low-enriched mammospheres from MDA-MB-468 cell line were treated with an equally potent concentration (IC50) of drugs as a single treatment and in combination treatment. As shown in Fig. 2a, the AMPK activators (metformin, AICAR, and A-769662) significantly potentiated the anti-proliferative effects of FEC in the parental cell lines (P \ 0.01, Student’s t-test), while metformin and AICAR were found to enhance the anti-proliferative effects of chemotherapeutic drugs in CD44?/CD24-/low-enriched mammospheres. To further analyze the effects of the combination of AMPK activators and chemotherapeutic drugs, the Bliss independence model was used to define the combined effect of two drugs. The model assumes that the two drugs act by independent mechanisms [19]. Using the Bliss independence analysis, the combination of metformin/FEC exhibited significant synergistic effects in both parental and CD44?/CD24-/low-enriched mammospheres, while the combination of metformin/doxorubicin had a synergistic effect in CD44?/CD24-/low-enriched mammospheres only (Fig. 2b). Given that there are different subtypes of breast cancer that have varying dependence on the AMPK pathway, we determined whether the synergistic effects were present in other breast cancer cell lines, such as the basal-like breast cancer cell lines (MDA-MB-231and HCC1937), luminallike breast cancer cell lines (MCF-7 and T47D) and HER2positive cell line (SKBR3). As shown in Fig. 3a, we found that the synergistic effects of metformin and FEC were observed in the MDA-MB-468, MDA-MB-231 and SKBR3 parental cell lines (25.42 , 26.80 and 82.23 lM%, respectively). In addition, similar synergistic effects of metformin and FEC were also observed in MDA-MB-468, MCF-7 and SKBR3 CD44?/CD24-/low-enriched mammospheres (38.93, 50.85 and 70.67 lM%, respectively)(Fig. 3b) while additive effects were observed in the rest of the cell lines being tested. These results suggest that metformin enhances FEC sensitivity in selected breast
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a MDA-MB-231
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Fig. 1 Cancer stem cells are resistant to chemotherapy agents but sensitive to metformin. a Cell morphology of mammospheres derived from breast cancer cell lines. Breast cancer cells were grown in MammoCult medium supplemented with hydrocortisone and heparin in ultra-low attachment plates for 5 days. Cell morphology was examined using inverted phase-contrast microscopy (2009). b CD44?/CD24-/low breast cancer stem cells are enriched in mammosphere culture. Cells were grown as described in (a). The expression of CD44 and CD24 in parental cells and mammospheres (CSCs) were determined by flow cytometry. Note that at least
50–85 % of the cells in the mammospheres were enriched with CD44?/CD24-/low breast cancer stem cells. c Metformin selectively targets CD44?/CD24-/low breast cancer stem cells derived from MDA-MB-468 basal-like breast cancer cells. Parental cells and mammospheres derived from the MDA-MB-468 cell line were treated with different concentrations of chemotherapeutic drugs and AMPK activators for 72 h followed by determination of cell viability using the Cell Titer 96 Aqueous One assay. CSCs, CD44?/CD24-/lowenriched mammospheres
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Fig. 2 Metformin synergizes FEC sensitivity in parental cancer cells and CD44?/CD24-/low-enriched mammospheres. a Percent inhibition of MDA-MB-468 proliferation as measured by using the Cell Titer 96 Aqueous One assay. Cells were treated with IC50 concentrations of chemotherapeutic drugs and/or AMPK activators in triplicate. The color of the squares indicates the level of cell growth inhibition (blue, no inhibition; red, complete inhibition). Asterisk indicates statistically significant improvement with the combination compared with the highest single agent alone (P \ 0.01, Student’s t test). Data represents
mean of at least three independent experiments. b The calculated excess inhibition over the predicted Bliss additivity model. The predicted Bliss additive effect (see Materials and methods) was subtracted from the experimentally observed inhibition at each pair of concentrations. A cutoff of 10 % excess or deficiency over Bliss additivity was considered as synergism or antagonism, respectively. Red color represents high synergism. Green color represents antagonism. Data represents mean of at least three independent experiments. CSCs, CD44?/CD24-/low-enriched mammospheres
cancer cell lines and CD44?/CD24-/low-enriched mammospheres, independent of ER, PR, and HER2 expression. To further investigate the cellular mechanisms explaining the anti-proliferative action of metformin and FEC combination, we investigated the effect of the combinations on the cell cycle and apoptosis. In parental cells, metformin alone induced significant G1/S arrest and a concomitant decrease in proportion of cells in the S and G2/M phase of the cell cycle (P \ 0.01, Student’s t test) (Supplemental Fig. 2a). In contrast, FEC induced profoundly sub-G1, S and G2/M accumulation (P \ 0.01, Student’s t test). Similarly, FEC also induced significant S and G2/M accumulation in CD44?/CD24-/low-enriched mammospheres while
significant sub-G1 population of cells were observed following metformin treatment (Supplemental Fig. 2a). Interestingly, the combination of metformin and FEC induced profound sub-G1 accumulation in both parental cells and CD44?/CD24-/low-enriched mammospheres as compared to cells receiving either agent alone (P \ 0.01, Student’s t test). Annexin V/7-AAD staining also revealed a significant amount of apoptotic cells in parental cells and CD44?/ CD24-/low-enriched mammospheres following treatment with metformin/FEC combination (Supplemental Fig. 2b). Of note, no significant changes in Beclin-1 expression were observed suggesting that autophagy was not affected by the combination treatment (data not shown). Together, these
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Metformin (mM) Fig. 3 Metformin enhances the anti-proliferative activity of FEC in parental breast cancer cells and CD44?/CD24-/low-enriched mammospheres, independent of ER, PR, or HER2 expression. a Effect of metformin and FEC on the proliferation of parental and b breast cancer stem cells. All cells were treated with metformin and/or FEC at a fixed concentration ratio of 176000:22:1:11 (metformin:5FU:epirubicin:cyclophosphamide) for 72 h, followed by determination of cell viability using the Cell Titer 96 Aqueous One
assay. The Bliss additivity value and synergistic/antagonistic volume were calculated as described in the Materials and Methods section. Values between -25 and ?25 lM2% were considered additive, values below -25 lM2% represented antagonism, and values above 25 lM2% represented synergy. All measurements were statistically evaluated at the 95 % confidence level. Points represent mean ± S.D. of at least three independent experiments. CSCs, CD44?/CD24-/lowenriched mammospheres
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results suggest that the synergistic effects of metformin/FEC combination were mainly mediated through enhanced induction of apoptosis in both parental cells and CD44?/ CD24-/low-enriched mammospheres. The synergistic effects of metformin and FEC is AMPK-dependent in parental breast cancer cells but AMPK-independent in breast CD441/CD242/low cancer stem cells The molecular mechanisms involved in the anti-proliferative effects of metformin are most likely very diverse, involving both AMPK-dependent [31, 32] and/or AMPKindependent [12, 33, 34] pathways. To test the dependence
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Fig. 4 The synergistic effect of metformin/FEC is AMPKdependent in parental breast cancer cells but AMPKindependent in CD44?/CD24-/ low -enriched cancer stem cells. a Inhibition of AMPK phosphorylation by Compound C. Cells were treated with metformin/FEC in the presence or absence of Compound C for 72 h and protein expression was analyzed by immunoblotting. b Inhibition of AMPK signaling by Compound C abrogated the synergistic effects of metformin/FEC in parental cells but not in CD44?/CD24-/lowenriched cancer stem cells. Parental cells and CD44?/ CD24-/low-enriched mammospheres were treated as in (a). Cell viability was determined by the Cell Titer 96 Aqueous One assay. Note that no significant effect was observed in control cells treated with 1 lM compound C. Bars represent mean ± S.D. of at least three independent experiments
on the AMPK pathway, we treated MDA-MB-468 cells with an IC50 concentration of metformin and/or FEC in the presence or absence of the AMPK inhibitor, Compound C. On its own, Compound C had no significant effect on the viability of the tested cell lines (data not shown). As shown in Fig. 4a and Supplemental Fig. 3, treatment with Compound C (1 lM) significantly inhibited the phosphorylation of AMPK and its downstream mTOR signaling pathways following metformin treatment. Inhibition of AMPK activation by Compound C abrogated the synergistic effects of metformin/FEC in MDAMB-468 and SKBR3 parental cells (Fig. 4b). In contrast, inhibition of AMPK had no effect on the metformin/FEC synergism in the CD44?/CD24-/low-enriched
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Fig. 5 Depletion of endogenous AMPK abrogated the synergistic effects of metformin/FEC in parental cells but not in CD44?/CD24-/lowenriched mammospheres. a MDA-MB-468 and SKBR3 cells were reverse-transfected with AMPK shRNA followed by treatment of cells with 10 mM metformin and/or FEC for 72 h and protein expression was analyzed by immunoblotting. b Cell viability was determined by the Cell Titer 96 Aqueous One assay. NS represents control cells transfected with a non-targeting shRNA. Asterisk indicates statistical significance compared with untreated control cells (P \ 0.05, Student’s t test). CSCs, CD44?/CD24-/lowenriched mammospheres
mammospheres in both cell lines. Similar results were observed in cells depleted for AMPK using shRNA (Fig. 5a, b). Together, these results suggest that metformin has synergistic effect with FEC in parental breast cancer cells through an AMPK-dependent mechanism while the synergistic effects in breast CD44?/CD24-/low-enriched CSCs are AMPK-independent. Metformin severely hampers intracellular ATP production in parental cells and CD441/CD242/lowenriched mammospheres Numerous studies have suggested that metformin is capable of inhibiting tumor cell proliferation through
inhibition of RagB GTPase [35] or induction of REDD1 expression [36]. However, ectopic expression of the constitutively active RagB GTPase Q99L mutant did not affect the synergistic effects of metformin/FEC in parental cells and CD44?/CD24-/low-enriched mammospheres, suggesting that the synergistic effect is independent of RagB GTPase activity (Supplemental Fig. 4). Similarly, we did not detect any endogenous nor induced REDD1 expression in MDA-MB-468 and SKBR3 parental cells and CD44?/ CD24-/low-enriched mammospheres following metformin treatment suggesting that REDD1 is dispensable in mediating the synergistic effects of metformin (data not shown). Recent studies have suggested that metformin might inhibit tumor cell proliferation through inhibiting complex
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I of the respiratory chain in mitochondria and production of ATP [34, 37–39]. To determine the mechanism by which metformin might affect metabolism, we analyzed its effects on mitochondrial complex 1 activity, glucose consumption, lactate production and intracellular ATP generation in
SKBR3
MDA-MB-468
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b
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d
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Fig. 6 Metformin severely hampers intracellular ATP production in parental cells and CD44 ?/CD24-/low-enriched mammospheres. a, b and c Metformin stimulates glycolysis but severely hampers intracellular ATP production in CD44?/CD24-/low-enriched mammospheres. Metformin suppresses mitochondrial complex I activity but stimulates glucose consumption and lactate production in breast cancer cells. MDA-MB-468 (top) and SKBR3 (bottom) parental cells and CD44?/ CD24-/low-enriched mammospheres were treated with 10 mM metformin for 72 h. The mitochondrial complex I activity, glucose consumption and lactate production were measured as described in the Materials and Methods section. d Metformin reduces intracellular ATP in breast CSCs. Points and bars represent mean ± standard deviation (s.d.) of at least three independent experiments. Asterisk indicates statistical significance compared with untreated control cells (P \ 0.05, Student’s t test)
MDA-MB-468 and SKBR3 parental cells and CD44?/ CD24-/low-enriched mammospheres. Metformin decreased complex 1 activity by more than 70 % in both parental cell lines and up to 90 % in the CD44?/CD24-/low-enriched mammospheres (Fig. 6a). In addition, metformin
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accelerated glucose consumption from medium more severely in the CD44?/CD24-/low-enriched mammospheres than in the parental cells and augmented production of lactate, indicative of increased in glycolysis (Fig. 6b, c). Interestingly, despite the increased in glycolysis, the production of intracellular ATP was severely hampered, particularly in the CD44?/CD24-/low-enriched mammospheres (Fig. 6d). Metformin impairs the ability of CSCs to repair FEC-induced DNA damage FEC have been shown to cause DNA damage by formation of DNA adducts and interstrand cross-links, resulting in DNA double-strand breaks (DSBs) [40]. DSBs can be repaired by non-homologous end-joining and homologous recombination repair through induction of histone H2A.X phosphorylation and formation of cH2A.X foci [40, 41]. Because ATP is required for H2A.X phosphorylation and the subsequent complicated DNA repair process [42–44], we speculated that the depletion of ATP in CD44?/CD24-/ low -enriched mammospheres by metformin might compromise the DNA repair process. Using a comet assay, we showed that treatment of parental cells and CD44?/CD24-/low-enriched mammospheres with FEC alone or in combination with metformin for 24 h caused severe DNA damage, resulting in the increased of broken DNA as the comet tails after single-cell gel electrophoresis (Fig. 7a and Supplemental Fig. 5a). In parental cells treated with FEC alone or in combination with metformin, the DNA damage could be repaired, as evidenced by the reduction of the DNA tails at 72 h after treatment (Fig. 7a and Supplemental Fig. 5a). However, the CD44?/ CD24-/low-enriched mammospheres were unable to repair the DNA damage when treated with a combination of metformin and FEC. In fact, the percentage of DNA in the tails increased from 14 to 42 % in MDA-MB-468 CD44?/ CD24-/low-enriched mammospheres following treatment with metformin/FEC combination. Immediately after formation of DSBs, the MRN (MRE11, RAD50, and NBS1) complex binds to broken DNA ends and recruits ATM and/or ATR serine/threonine kinase, resulting in phosphorylation of H2A.X (cH2A.X) and initiation of the DNA-repair process [40, 41]. Dephosphorylation and removal of cH2AX in the cancer cells turns off the DNA damage response. In our study, we observed that parental cells and CD44?/CD24-/low-enriched mammospheres treated with FEC alone or in combination with metformin for 24 h induced substantial accumulation of cH2A.X (Fig. 7b and Supplemental Fig. 5b). When the CD44?/CD24-/low-enriched mammospheres were treated with FEC alone for 72 h, significant amount of phospho-ATM and cH2A.X were present,
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indicating DNA damage and active repair were ongoing (Fig. 7b, Supplemental Fig. 5b and Supplemental Fig. 6). However, in CD44?/CD24-/low-enriched mammospheres treated with metformin/FEC combination for 72 h, cH2A.X decreased substantially, despite the persistence of high phospho-ATM expression, suggesting a plausible dephosphorylation of H2A.X due to a lack of ATP to support the DNA repair process. Indeed, supplementation of 0.1 mM of ATP completely abrogated the de-phosphorylation of H2A.X, reduced DNA damage and rescue CD44?/CD24-/low-enriched mammospheres cell death induced by metformin/FEC combination (Fig. 7 and Supplemental Fig. 5). Together, these results indicate that the striking synergistic elimination of CSCs following metformin/FEC treatment is due to the impaired ATP production induced by metformin to support the repair of DNA damage induced by FEC.
Discussion In the present study, we demonstrated that metformin synergizes FEC sensitivity in parental breast cancer cells and CD44?/CD24-/low-enriched mammospheres in vitro, independent of ER, PR, and HER2 status. Our data is consistent with in vitro and in vivo data showing that metformin inhibits cancer cell growth in various cancers including leukemia, lymphoma, prostate, gastric, colon, lung, pancreatic, endometrial, ovarian, thyroid cancers [45–49]. Our data also showed that metformin had synergistic activity with doxorubicin in CD44?/CD24-/low-enriched MDA-MB-468 cells, but not in the parental cells. This is consistent with other studies showing that metformin inhibits the growth of breast cancer cell lines [50– 52], suppresses breast epithelial cell transformation [8, 9], selectively inhibits the growth of CSCs in a distinct subtype of breast cancer cell lines [8], and increases the rate of tumor regression and prolongs tumor remission in mouse xenografts involving multiple cancer cell types in combination with doxorubicin [8, 10]. Interestingly, we observed no synergistic effect in cells treated with a combination of other AMPK activators (e.g., AICAR and A-769662), suggesting that metformin possesses distinct anti-tumor properties in addition to activation of AMPK. Several potential mechanisms have been suggested for the ability of metformin to suppress cancer growth: (1) activation of the AMPK pathway; (2) induction of cell cycle arrest and/or apoptosis; (3) inhibition of protein synthesis; (4) reduction in circulating insulin levels; (5) inhibition of the unfolded protein response (UPR); (6) suppression of HER2; (7) activation of the immune system; and (8) inhibition of the cross talk between receptors and decrease in local estrogen production by breast adipose tissue by
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Fig. 7 Metformin impairs the ability of CSCs to repair FEC-induced DNA damage. a Effect of metformin on repair of DNA damage induced by FEC in MDA-MB-468 parental cells and CD44?/CD24-/ low -enriched mammospheres. Cells were treated with the metformin and/or FEC for 24 and 72 h. DNA damage was evaluated by Comet assay. At least 50 cells in each sample were quantitatively analyzed for percentage of DNA tail that eluted from the cellular nuclei. Bars represent mean ± S.D. of at least three independent experiments. Asterisk indicates statistical significance increased in DNA damage compared with cells analyzed at 24 h post-treatment. Hash indicates statistical significance reduction in DNA damage compared with cells
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analyzed at 24 h post-treatment (P \ 0.01, Student’s t test). b Immunoblot analysis of cH2A.X and total H2A.X proteins in parental cells and CD44?/CD24-/low-enriched mammospheres. c Supplementation of ATP rescues CD44?/CD24-/low-enriched mammospheres cell death induced by metformin/FEC indicating that the synergistic effects of metformin in CSCs is ATP-dependent. Bars represent mean ± S.D. of at least three independent experiments. Asterisk indicates statistical significance compared with control cells in the absence of ATP supplementation (P \ 0.01, Student’s t test). CSCs, CD44?/CD24-/low-enriched mammospheres
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inhibiting aromatase expression [53–55]. Although some of the proposed mechanisms of metformin would explain the effect of the agent in vivo, its in vitro effects cannot be explained by the anti-insulin and immune-activating action. Despite the various proposed mechanisms of action of metformin, the precise mechanisms of the metformin-induced effects in non-CSCs and CSCs are not fully understood. In particular, controversy remains about whether metformin mediates any of its anti-tumor effects through AMPK, and whether AMPK is required for metformin to inhibit cancer cells (non-CSCs and CSCs) growth synergistically with chemotherapeutic drugs. To test these hypotheses, the AMPK activity in breast parental breast cancer cell lines and CD44?/CD24-/low-enriched mammospheres were inhibited using Compound C, a potent AMPK inhibitor, or shRNA targeting endogenous AMPK. We demonstrated that the growth inhibitory and synergistic effects of metformin in parental cells were highly dependent on AMPK activities, as inhibition of AMPK completely abrogated the antitumor effects of metformin and its synergistic effects in combination with FEC. In stark contrast, the inhibition of AMPK had no effect on metformin sensitivity in the CD44?/CD24-/low-enriched mammospheres, suggesting that AMPK is dispensable for the antitumor effects of metformin in CSCs. These apparently diverse effects imply that metformin might regulate other signaling pathways in addition to AMPK in CSCs. Energy metabolism has emerged as a potential candidate target for the treatment of cancers. Multiple studies have confirmed that nutrient metabolism in cancer cells may be different from that in normal cells. As first proposed by Warburg, cancer cells might rely more on glycolysis for metabolism and survival, due in part to mitochondrial dysfunction [56]. The glycolytic shift is even more pronounced in CSCs, as a number of new studies have demonstrated that CSCs show decreased mitochondrial mass and membrane potential, decreased mitochondrial respiratory activity, consume far less oxygen per cell, have higher levels of the hypoxic response protein HIF-1a (even under normoxic conditions), and produce markedly reduced levels of reactive oxygen species (ROS) [56, 57]. Interestingly, recent studies have suggested that metformin might stimulate AMPK indirectly, through a decreased in ATP levels as a result of inhibiting complex I of the respiratory chain in mitochondria [34, 37–39]. Consistently, we also observed significant reduction in mitochondrial complex I activity following treatment of metformin in breast CSCs and non-CSCs. Although glucose consumption and lactate production was significantly increased (indicative of increased in glycolysis) following metformin treatment, the production of intracellular ATP was severely hampered, particularly in the CSCs. Further analyses revealed that the ATP depletion induced by
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metformin could lead to a severe energy crisis, which causes severe compromise of CD44?/CD24-/low-enriched mammospheres to repair the DNA damage induced by FEC, resulting in a striking synergistic elimination of these cells in vitro. These new findings imply that metformin might have a more profound effect in changing the energy state or other related pathways at the cellular level other than the activation of AMPK in CD44?/CD24-/low-enriched mammospheres. Given the synergistic effects of metformin and FEC on breast non-CSCs and CSCs in the preclinical studies and that epidemiologic studies have shown a significant risk reduction in cancer incidence and mortality in patients with diabetes receiving metformin relative to other anti-diabetic drugs [11, 58], it was hypothesized that neoadjuvant treatment with metformin and FEC would reduce CSCs proportion and improve response in breast cancer patients. Notably, a recent study has demonstrated that metformin increases survival in hormone receptor-positive, HER2positive breast cancer patients with diabetes [59]. Also, a Phase III randomized trial of metformin in early-stage breast cancer conducted by the National Cancer Institutes is currently ongoing (ClinicalTrials.gov Identifier: NCT01101438) and will be able to directly address the potential of combination therapy with metformin and chemotherapeutic agents. Whether the reduction of CSCs in tumor may eventually contribute to better survival and lower recurrence rate will need to be further validated. In conclusion, we show that metformin, unlike other AMPK activators, synergizes with FEC to induce cytotoxicity in breast non-CSCs and breast CSCs independent of ER, PR, and HER2 status. Furthermore, the synergistic effects of metformin in breast CSCs and non-CSCs is mediated through distinct mechanisms, highlighting the importance of metformin in inducing metabolic synthetic lethality in a cell-type dependent manner. Overall, our findings suggest that combination therapy with metformin and FEC may be a useful approach for treating patients with breast cancers. Acknowledgments This work was supported by the funds raised through the Yayasan Sime Darby LPGA Tournament, donors of Cancer Research Initiatives Foundation and the Ministry of Higher Education Grant [UM.C/HIR/MOHE/06 and ERGS/1/2013/SKK01/ IMU/02/1]. Compliance with ethical standards Conflict of interest The authors report no conflicts of interest pertaining to this study. Research involving human participants and/or animals No human participants and/or animals was involved in this study. Informed consent
No informed consent was required for this study.
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