Phenformin-Induced Mitochondrial Dysfunction Sensitizes Hepatocellular Carcinoma for Dual Inhibition of Mtor Sonia� R

Published OnlineFirst April 24, 2018; DOI: 10.1158/1078-0432.CCR-18-0177

Cancer Therapy: Preclinical Clinical Cancer Research Phenformin-Induced Mitochondrial Dysfunction Sensitizes Hepatocellular Carcinoma for Dual Inhibition of mTOR Sonia R. Veiga1, Xuemei Ge1, Carol A. Mercer2, María I. Hernandez- Alvarez 3, Hala Elnakat Thomas2, Javier Hernandez-Losa4, Santiago Ramon y Cajal4, Antonio Zorzano3,5,6, George Thomas1,2,7, and Sara C. Kozma1,2

Abstract

Purpose: Hepatocellular carcinoma (HCC) ranks second in Results: We found phenformin caused mitochondrial dys- cancer mortality and has limited therapeutic options. We recently function and fragmentation, inducing a compensatory shift to described the synergistic effect of allosteric and ATP-site compet- glycolysis. In contrast, dual inhibition of mTOR impaired cell itive inhibitors against the mTOR for the treatment of HCC. growth and glycolysis, while increasing mitochondrial fusion However, such inhibitors induce hyperglycemia and increase and efficiency. In a mouse model of human HCC, dual inhi- mitochondrial efficiency. Here we determined whether the mito- bition of mTOR, together with phenformin, was highly effica- chondrial complex I inhibitor phenformin could reverse both side cious in controlling tumor burden. However, more strikingly, effects, impose an energetic stress on cancer cells, and suppress the pretreatment with phenformin sensitized tumors to dual inhi- growth of HCC. bition of mTOR, leading to a dramatic improvement in Experimental Design: Human HCC cell lines were used in vitro survival. to access the signaling and energetic impact of mTOR inhibitors Conclusions: Treatment of HCC cells in vitro with the bigua- and phenformin, either alone or in combination. Next, the nide phenformin causes a metabolic shift to glycolysis, mitochon- therapeutic utility of these drugs alone or in combination was drial dysfunction and fragmentation, and dramatically sensitizes investigated preclinically in human orthotopic tumors implanted orthotopic liver tumors to dual inhibition of mTOR. We therefore in mice, by analyzing their impact on the tumor burden and propose this therapeutic approach should be tested clinically in overall survival. HCC. Clin Cancer Res; 1–14. 2018 AACR.

Introduction insults leading to HCC are hepatitis B or C virus (HBV or HCV) and alcohol (1); however, in Western societies, the abrupt increase Worldwide, liver cancer is the second cause of cancer-related in HCC has been linked to nonalcoholic steatohepatitis (NASH) death, second in most years of lost life, and the most rapidly and the recent obesity epidemic (2, 3). Despite being a very expanding malignant tumor in Western countries. Accounting for heterogeneous tumor type without clear genetic drivers, the most >70% of the total liver cancer burden, hepatocellular carcinoma aggressive forms of HCC are associated with the TGFb1, PI3K/Akt/ (HCC) is its major histologic subtype. The most common liver mTOR, Wnt/b-catenin, and NOTCH proliferative pathways (4, 5). The Cancer Genome Atlas (TCGA) consortium has recently per- 1Laboratory of Cancer Metabolism, Oncobell Program, Bellvitge Biomedical formed the first large-scale multi-platform analysis of HCC, which 2 Research Institute (IDIBELL), Barcelona, Spain. Division of Hematology/Oncol- showed a prevalence of alterations in WNT and p53 signaling as ogy, Department of Internal Medicine, University of Cincinnati, Cincinnati, Ohio. well as TERT upregulation (6). Clinically, surgical resection leads 3Complex Metabolic Diseases and Mitochondria Group, Institute for Research in Biomedicine (IRB), Barcelona, Spain. 4Department of Anatomy/Pathology, Vall to approximately 70% disease-free survival over 5 years in HCC d'Hebron University Hospital, Universitat Autonoma de Barcelona, Barcelona, patients (7, 8), although it is only an option for a small portion of Spain. 5Department of Biochemistry and Molecular Biomedicine, Faculty of early-stage patients with solitary tumors and normal liver func- Biology, University of Barcelona, Barcelona, Spain. 6Centro de Investigacion tion. For advanced stage HCC, the standard of care is sorafenib Biomedica en Red de Diabetes y Enfermedades Metabolicas Asociadas (CIBER- (9), followed upon progression by the recently approved regor- 7 DEM), Instituto de Salud Carlos III (ISCIII), Madrid, Spain. Physiological Sciences afenib, both multi-protein kinase inhibitors, which render mod- Department, Faculty of Medicine and Health Science, University of Barcelona, est life-extending benefits (10, 11). Recently, striking effects have Barcelona, Spain been reported with nivolumab, an immunotherapeutic agent Note: Supplementary data for this article are available at Clinical Cancer (12). Of note, such treatments may benefit from combination Research Online (http://clincancerres.aacrjournals.org/). therapy with targeting agents (13). Corresponding Author: Sara C. Kozma, IDIBELL, Gran Via de l'Hospitalet, mTOR emerged several years ago as an attractive target for þ L'Hospitalet de Llobregat, Barcelona 199 08908, Spain. Phone: ( 34) 932 HCC, as it is hyperactivated in 50%–60% of HCC and is associ- 607 138; Fax: (þ34) 932 607 776; E-mail: [email protected] ated with high-grade tumors and worst case prognosis (14–17). doi: 10.1158/1078-0432.CCR-18-0177 Blocking tumor growth by inhibiting mTOR has led to the 2018 American Association for Cancer Research. approval of the rapamycin family of allosteric drugs (rapalogs)

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not require a transporter and is readily taken up by all tissues (34). Translational Relevance Phenformin was initially withdrawn from clinical use due to Hepatocellular carcinoma is the second cause of cancer deleterious lactic acidosis in a subset of renal patients (35). mortality worldwide, with available therapeutic options hav- However, arguments that phenformin could be considered as an ing poor outcomes. Allosteric mTOR inhibitors were predicted anticancer agent, given distinct dosing and usage (36), have aided to be effective in HCC, but failed in showing efficacy in a phase in the recent approval of a phase I clinical trial for its use in III clinical trial. However, subsequent trial sample analyses melanoma (37). suggested that genotypic stratification would have benefited a Here we set out to determine the effects of RAD001/BEZ235 large percentage of patients, particularly those with loss of and phenformin treatment in human HCC cell lines, tumoroids, TSC2. Here we examined the use of the combined allosteric and mouse orthoxenografts. In cells, we found that both treat- and ATP-binding site mTOR complex 1 inhibitors in HCC, ments inhibited mTOR signaling, but that mTOR inhibition was together with the mitochondrial complex1 inhibitor phenfor- not augmented by the triple combination. In parallel, phenformin min, as recent data suggested that the mTOR inhibitors may induced mitochondrial dysfunction and fragmentation, consis- promote cancer cell survival by maintaining mitochondrial tent with the activation of AMP-activated protein kinase (AMPK; oxidation and/or autophagy. We show that treatment of HCC ref. 38), and led to a metabolic shift toward glycolysis, increased cells in vitro with phenformin causes a metabolic shift to ROS production, and cell death. Unexpectedly, a number of the glycolysis, mitochondrial dysfunction and fragmentation, phenformin responses appeared to be mitigated by the energy- with phenformin pretreatment sensitizing orthotopic liver sparing effects of RAD001/BEZ235 treatment. The growth inhi- tumors to dual inhibition of mTOR, leading to a striking bition of orthotopically implanted tumors was more dramatic increase in overall survival. when using mTOR inhibitors, as compared with phenformin alone, but more penetrant with the triple drug combination, an effect also observed in overall survival. Given that the mTOR inhibitors suppressed the effects of phenformin on mitochondria homeostasis in vivo, HCC-bearing mice were first treated with the in the treatment of a number of cancers (18, 19). Nevertheless, the biguanide, followed by the mTOR inhibitors. Such a treatment rapalog everolimus (RAD001) did not improve overall survival in schedule dramatically sensitized tumors to mTOR inhibitors, the phase III EVOLVE-1 trial for advanced HCC after sorafenib profoundly increasing overall survival. Given the recent approval failure (20). However, a recent retrospective analysis of the of phenformin IND in melanoma (37), this regimen should be EVOLVE-1 data showed that patients exhibiting mutations in the explored clinically in HCC. Tuberous Sclerosis 2 (TSC2) protein, a tumor suppressor, which results in constitutive activation of mTOR complex 1 (mTORC1), Materials and Methods tended to benefit from second-line treatment with RAD001 (21). Cell culture and proliferation assay Importantly, the same authors reported that loss of TSC2 is Huh7 cells, purchased from the JCRB Cell Bank, were main- observed in approximately 20% of the Asian HBV(þ) HCC tained in DMEM (Gibco) containing 10% heat-inactivated FBS patients (21). These findings have further underscored the neces- (Sigma-Aldrich) and 1% penicillin/streptomycin (10,000 U/mL sity of stratifying patients as a function of subpopulations in liver and 10 mg/mL, respectively; Sigma-Aldrich). Cell number was cancer clinical trial designs (22). measured by crystal violet staining (Sigma-Aldrich). The failure of rapalogs in a number of clinical trials may also be attributed in part to their incomplete inhibition of mTORC1 and Animal studies nu mTORC2 (23), as well as the loss of the negative feedback loop Four-week-old male Athymic Nude-Foxn1 mice were pur- from ribosomal protein S6K1 kinase to Akt (24, 25). We recently chased from Harlan Laboratories (Envigo). Six- to 8-week-old showed that these two shortcomings could be overcome by mice were injected intrahepatically with 1–2 106 Huh7 cells in combining an allosteric (RAD001) and an ATP-site competitive an ice-cold PBS (Gibco)/Matrigel growth factor–reduced phenol- (BEZ235) mTOR inhibitor (26). These observations have led to free (Corning, BD Biosciences) solution. After approximately four the proposal that the rapalogs should be reconsidered in the weeks, tumors were collected and equal size tumor pieces treatment of HCC. However, in the clinic mTOR inhibitors hinder (0.05 g) were implanted surgically in the liver of experimental glycolysis, inducing hyperglycemia (27), which may indepen- athymic mice cohorts. Two weeks after tumor implantation, dently drive tumor growth (28, 29). Moreover, it was recently mice were treated orally daily for 15 or 21 days with RAD001 demonstrated that mTOR inhibitors decrease mitochondrial bio- (2.5 mg/kg) and/or BEZ235 (6 mg/kg) and/or INK128 genesis and activity, but counterintuitively increase mitochondri- (0.3 mg/kg), and intraperitoneally with the specified doses of al efficiency (30). This overall increase in mitochondrial efficiency phenformin (70 or 100 mg/kg). The mice were starved for 2 hours may provide cancer cells the potential to survive the catabolic state before sacrifice and received their last dose of drug 1 hour before induced by mTOR inhibitors, particularly a small population of sacrifice. Liver and tumor tissues were snap-frozen in liquid stem-like cells. In the clinic, hyperglycemia induced by rapalogs is nitrogen for protein extraction or fixed in 10% formalin for IHC treated with the biguanide metformin (31, 32). Given that bigua- analysis. For survival experiments, mice were weaned from drug nides inhibit complex I (CI) and mTOR inhibitors suppress and monitored every other day, or daily when signs of stress glycolysis, we reasoned that their combination or their sequential emerged (weight-shedding >10% and blood glucose <90 mg/dL). use might place a further energetic stress on HCCs. However, Glucose levels <60 mg/dL, visible ascites, weight shedding >15%, metformin requires the OCT1 transporter to enter tumor cells, and loss of body temperature were endpoint criteria. All in vivo which is largely downregulated in HCC (33), making the hydro- studies were approved by the IDIBELL's Animal Care and Use phobic analogue phenformin an attractive alternative, as it does Committee and the Generalitat de Catalunya.

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Statistical analyses Effect of combined mTOR inhibitors on the tumorigenic Data are presented as mean SD. All the statistical analyses capacity of Huh7 tumors were performed using GraphPad Prism 6. For one-group vari- Others have reported that mTOR inhibition by rapalogs ables, statistical significance was determined by unpaired two- protects a small population of stem-like cells (41, 42), poten- tailed t test; whenever variances were different, we applied the tially due to the maintenance of high mitochondrial oxidation unpaired two-tailed t test with Welch correction. For two- capacity and/or the induction of autophagy (42, 43). To grouping variables, two-way ANOVA was performed. Kaplan– determine whether RAD001/BEZ235 would impact cell's Meier survival curve differences were analyzed with the log-rank tumorigenic capacity, we first generated tumoroids from Huh7 (Mantel–Cox) test. All statistical analyses were made in a 95% orthotopic tumors treated with or without RAD001/BEZ235 confidence interval, with P values of <0.05, <0.01, <0.001 in vivo. The results showed that cells derived from RAD001/ considered to be significant and represented with , ,and, BEZ235–treated tumors were impaired in their ability to grow respectively. as tumoroids in 3D culture, as compared with controls (Fig. For details on tumoroid cultures, tumorigenic capacity (limit- 1D). To further validate this finding, we conducted tumor ing dilution) assay, immunoblot analysis, GST-BHMT assay, initiation assays in vivo under conditions of limiting dilution oxygen consumption rate (OCR), and extracellular acidification with fixed numbers of cells. Huh7 tumor-bearing mice were rate (ECAR), mitochondrial probes for FACS analyzes, high- treated for 15 days, tumor cells were isolated as for tumoroids, performance liquid chromatography (HPLC) analysis of nucleo- but instead were subcutaneously implanted in nude mice and tides, newly synthesized RNA and protein synthesis rate, oxygen monitored for tumor growth (Fig. 1E). In contrast to the effects consumption of tumor mitochondria, refer to Supplementary reported for the rapalogs as single agents (41), the time of Materials and Methods. tumor appearance is reduced by the dual inhibition of mTOR as compared with the vehicle (Fig. 1E). Together, our results demonstrate that combined mTOR inhibition in vivo Results diminishes, rather than enhances, the tumorigenic potential Effect of combined mTOR inhibitors on HCC orthoxenograft of Huh7 cells. and tumoroid growth We initially set out to determine whether mTOR inhibition was Impact of phenformin on glycemia, proliferation, and signaling effective in blocking the proliferation of a panel of six HCC cell Clinically, persistent hyperglycemia is most commonly trea- lines both sensitive and resistant to sorafenib, the current first-line ted with the biguanide metformin (44), including those patients treatment of HCC (39). The RAD001/BEZ235 combination was whose hyperglycemia is induced by rapalogs (31, 32). This more effective than either agent alone in all HCC cell types raised the possibility of adding a biguanide together, or poten- (Supplementary Fig. S1A), including the two we have previously tially sequentially, with the RAD001/BEZ235 combination, analyzed (26). To test the sensitivity of HCC cells to mTOR which would effectively inhibit both glycolysis and mitochon- inhibitors in vivo, we established orthotopic models from Huh7 drial oxidative phosphorylation (OXPHOS), directing tumors and Hep3B cell lines. After 14 days of tumor engraftment, cohorts toward energetic stress. As previously reported for mTOR inhi- of tumor-bearing mice were treated with mTOR inhibitors alone bition (27), treatment of Huh7 tumor–bearing animals with the or in combination. In all cases, suppression of tumor growth was RAD001/BEZ235 combination (RB) leads to hyperglycemia due observed within 15 days, with the strongest effect elicited by the to lower peripheral glucose uptake, an effect blunted by the RAD001/BEZ235 combination (Fig. 1A; Supplementary Fig. addition of phenformin, which is known to decrease gluconeo- S1B), consistent with our earlier findings in DEN-induced tumors genesis (Fig. 2A). To assess the mechanism of phenformin in C57BL/6 mice (26). Likewise, the combined mTOR inhibitor action, we examined a number of proliferative and signaling treatment had a small impact on the weight of tumor-bearing parameters in Huh7 cells in vitro. We found that cell prolifer- mice (Supplementary Fig. S1C) and induced the dephosphory- ation is inhibited by either phenformin or RAD001/BEZ235 lation of mTORC1 kinase target RPS6 (Fig. 1B). Strikingly the on- alone and that the inhibitory response is further enhanced when target effects were more pronounced in tumor tissue (Fig. 1B), all three drugs are used in combination (Fig. 2B). Nevertheless, possibly due to their higher vascularization. Moreover, in com- the kinetics of inhibition of mTORC1 signaling were much bination with RAD001, the effects of BEZ235 in Huh7 tumors slower with phenformin than RAD001/BEZ235, as measured were recapitulated by a second-generation ATP-site specific mTOR by S6K1, RPS6, and 4EBP1 phosphorylation (Fig. 2C). More- inhibitor INK128 (Supplementary Fig S1D), with similar effects over, phenformin in combination with RAD001/BEZ235 on body weight (Supplementary Fig. S1E). In addition, as we appeared to have no further effect on mTORC1 signaling (Fig. previously showed for RAD001/BEZ235 (26), we found the 2C), suggesting that the effects of phenformin on cell prolifer- RAD001/INK128 combination more effective than either agent ation are mediated through mTORC1-dependent and -indepen- alone in inhibiting mTORC1 signaling and the proliferation of dent pathways. Consistent with this observation, in contrast to Huh7 cells (Supplementary Figs. S1F and S1G). To further test this treatment with RAD001/BEZ235, phenformin strongly induced response, we generated three-dimensional tumoroids (40) from AMPK activation, as measured by increased AMPK T172 phos- Huh7 orthotopic tumors, and treated them in vitro for 6 days with phorylation, with concomitant increases in the phosphorylation the RAD001/BEZ235 combination. The combined mTOR inhib- of AMPK substrate ACC S79 (Fig. 2C). Unexpectedly, these itor treatment in vitro was as effective in inhibiting the growth of responses are blunted in the presence of RAD001/BEZ235, even tumoroids (Fig. 1C), as it was inhibiting orthotopic tumors (Fig. at later times (Fig. 2C). Despite the latter findings, the combined 1A). These studies suggest that the combination of an mTOR effect of the three drugs in impeding proliferation may be allosteric and an ATP-site competitive inhibitor is efficacious in explained by the inhibition of mTORC1 signaling and the treating tumors derived from human HCC cell lines. activation of AMPK.

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A Tumor Graft Treatment B Veh RB implantation 14 days 15 days pRPS6S236/S236 t0 sac

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Figure 1. RAD001 and BEZ235 suppress HCC orthoxenograft growth without promoting tumorigenic capacity. Huh7 orthotopically implanted tumor pieces were

allowed to graft for 15 days (t0) in athymic mice. Mouse cohorts were treated with either vehicle, RAD001 (2.5 mg/kg), or BEZ235 (6 mg/kg), or the latter two in combination. A, Tumor weight before (t0) and after 15 days of treatment (n ¼ 5–6). B, Amount of phosphorylated and total proteins in tumor lysates; b-actin was used as loading control. C, Tumoroid cultures established from Huh7 tumors (scale bar, 200 mmol/L) treated for 6 days with RAD001 (5 nmol/L) and BEZ23 (20 nmol/L; n ¼ 4). D, Tumoroid cultures established from Huh7 tumors (n ¼ 2–3) pretreated in vivo for 7 days with RAD001 (2.5 mg/kg) and BEZ235 (6 mg/kg). E, Tumor incidence over time following a limiting-dilution assay performed with the indicated numbers of cells obtained from tumors pretreated in vivo for 15 days as in D (representative experiment, n ¼ 3). Veh (vehicle); RAD (RAD001); BEZ (BEZ235); RB (RAD001/BEZ235).

Impact of phenformin on glycolysis they should be sensitive to external glucose levels. In agreement The induction of AMPK activation and ACC phosphorylation with this hypothesis, lowering glucose levels in the culture by phenformin (Fig. 2C) supports a change in cellular energy media from 25 mmol/L to either 12.5 mmol/L or 6.25 usage. As phenformin inhibits OXPHOS, we hypothesized that mmol/L for 72 hours led to the death of all the Huh7 cells it would induce a metabolic shift toward glycolysis. To test this subjected to 1 mmol/L phenformin alone, but had no impact on possibility, we followed ECAR, a surrogate for lactate produc- the survival of RAD001/BEZ235–treated cells (compare both tion and glucose utilization. The results show that glucose panelsinFig.2E).Theeffectofphenforminoncellgrowthis addition to culture media in the presence of phenformin led blunted by the RAD001/BEZ235 combination at the higher to sharp increase in ECAR, whereas the combined mTOR inhi- concentrations of glucose, an effect that is almost completely bitors had a small inhibitory effect alone and blunted the effect lost at the lowest concentration of glucose (compare both panels of phenformin (Fig. 2D; Supplementary Fig. S2A). We predicted in Fig. 2E). Phenformin-induced cell death appears to occur that, if phenformin-treated cells rely on glycolysis for survival, through apoptosis, as measured by the induction of caspase 3/7

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AB C RB Phf RBP 16 h * 100 0’ 15’ 45’ 60’ 120’ 0’ 15’ 45’ 60’ 120’ 0’ 15’ 45’ 60’ 120’ Ctrl RB Phf RBP 75 S79 300 ** pACC 25 ** *** pAMPKT172 20 200 * T389 15 pS6K1 Cell number 100 10 ppRPS6S236/S236 Glucose (mg/dL)

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Figure 2. Phenformin activates AMPK, induces a shift toward glycolysis, and increases susceptibility to cell death by decreased glucose levels. A, Circulating glucose levels in mice fed ad libitum and treated for 14 days with RAD001 (2.5 mg/kg) and BEZ235 (6 mg/kg) together, or in combination with phenformin (70 mg/kg; n ¼ 5). B, Relative number of Huh7 cells after 72 hours of exposure to RAD001 (5 nmol/L) and BEZ235 (20 nmol/L) together, or in combination with phenformin (1 mmol/L; n ¼ 3). C, Extent of phosphorylation of indicated proteins in Huh7 cell lysates after treatment for the stated times with RAD001 (5 nmol/L), BEZ235 (20 nmol/L) together, or in combination with phenformin (1 mmol/L); b-actin was used as loading control. D, ECAR after addition of 25 mmol/L glucose (n ¼ 3) to Huh7 cells pretreated with RAD001 (5 nmol/L) and BEZ235 (20 nmol/L) together, or in combination with phenformin (1 mmol/L) for 16 hours. E, Relative cell number of Huh7 after 72 hours of exposure to RAD001 (5 nmol/L) and BEZ235 (20 nmol/L) together, or in combination with phenformin (1 mmol/L), in culture medium containing the stated glucose concentrations (n ¼ 3). F, Relative cell number of Huh7 after 24 hours of exposure to phenformin (0.5 and 1 mmol/L) in culture medium containing 5 mmol/L glucose, with or without pan-caspase inhibitor Q-VDOPH (20 mmol/L; n ¼ 3). G, Tumoroid cultures established from Huh7 tumors treated for 6 days with RAD001 (5 nmol/L) and BEZ235 (20 nmol/L) alone, or in combination with phenformin (30 mmol/L), at the stated glucose concentrations (scale bar, 200 mmol/L; representative experiment, n ¼ 4). Ctrl, (Control); RB, RAD001/BEZ235; Phf, phenformin; RBP, RAD001/BEZ235/phenformin. activation (Supplementary Fig. S2B) and the inhibition of cell (Fig. 2G). The results demonstrate that in the presence of death by pan-caspase inhibitors (Fig. 2F). More strikingly, in phenformin, cells become more sensitive to the glycolytic input. tumoroids derived from Huh7 tumors, we found that when we lowered glucose levels in culture medium from 17.5 mmol/L to Mitochondrial activity and energy status physiologic levels of 5 and 1 mmol/L, we achieved inhibition of Recent studies have shown that mTORC1 inhibition also m proliferation at phenformin concentrations as low as 30 mol/L induces a reduction in the mitochondrial O2 consumption rate

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AB 125 125

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Figure 3. Phenformin, but not mTOR inhibition, reduces the energy state of Huh7 cells. Huh7 cells were treated with RAD001 (5 nmol/L) and BEZ235 (20 nmol/L) together, or in combination with phenformin (1 mmol/L). A, Routine oxygen consumption rate (OCR) of Huh7 cells treated for the stated times, relative to control cells (n ¼ 3–5); , Student t test versus control. B, Mitochondrial mass measured by ﬂow cytometry analyses of Huh7 cells stained with MitoTracker Green FM, after 16 hours of the indicated treatments (n ¼ 6). ATP levels analyzed by HPLC after 16 hours of indicated treatment relative to control (C) and AMP/ATP ratio from the same samples (n ¼ 3; D). E, Newly synthesized RNA, after 3H-Uridine pulse for 30 minutes or 3.5 hours, following 16 hours of the indicated treatment (representative experiment, n ¼ 2). Top, loading controls in agarose gel stained with ethidium bromide; bottom, analysis of nascent rRNA synthesis by pulse labeling with 3H-Uridine. F, Rate of global protein synthesis as determined by pulse-labeling cells with 3H-Leucine, following 16 hours of the indicated treatments; cycloheximide for 50 minutes (100 mg/mL) as a positive control (representative experiment, n ¼ 2).

(OCR) and mass, leading to an approximately 30% decrease in mentary Fig. S3A–S3C), an effect that is established within min- cellular ATP levels, responses attributed to inhibition of transla- utes of treatment, consistent with phenformin directly inhibiting tion of nucleus-encoded mitochondrial gene transcripts (30). CI (Supplementary Fig. S3B). When combined with mTOR inhi- Given this observation, it was important to distinguish the impact bitors, phenformin induced a further decrease in OCR (Fig. 3A; of RAD001/BEZ235 versus phenformin on mitochondrial activ- Supplementary Fig. S3C). Furthermore, phenformin caused a ity. We found that both phenformin and RAD001/BEZ235 dramatic reduction in mitochondrial mass measured by Mito- reduced mitochondrial OCR (Fig. 3A; Supplementary Fig. S3A; Tracker Green FM probe (Fig. 3B) and although ATP levels do not Supplementary Table S1). By 3 hours of treatment, the combined drop (Fig. 3C), the AMP/ATP and ADP/ATP ratios were markedly mTOR inhibitors caused a 25% reduction in OCR, while phen- increased (Fig. 3D; Supplementary Fig. S3D–S3E), consistent with formin led to a striking 75% reduction in OCR (Fig. 3A; Supple- phenformin inducing AMPK activation (Fig. 2C). Taken together,

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these results suggest that the phenformin-induced shift to glycol- mitophagy and ROS production, leading to apoptosis (48). ysis is not sufficient to relieve the energetic stress, with cells Unexpectedly, we found that phenformin treatment alone did striving to maintain ATP levels. not induce autophagy, and even blocked autophagy initiated by In agreement with recent findings (30) we observe a small effect RAD001/BEZ235 treatment, as measured by the cargo-based of RAD001/BEZ235 treatment on mitochondrial mass (Fig. 3B), endpoint GST-BHMT assay (Fig. 4D; ref. 49). This would argue but, in contrast, we observe no effect on ATP levels (Fig. 3C), that in the presence of phenformin, damaged mitochondria are consistent with no changes in the AMP/ATP or ADP/ATP ratios not being cleared by autophagic mechanisms. In parallel, phen- (Fig. 3D; Supplementary Figs. S3D–S3E). The latter findings agree formin induced mitochondrial ROS as early as 3 hours posttreat- with the lack of AMPK activation by RAD001/BEZ235 treatment ment, at levels significantly higher than that of RAD001/BEZ235 (Fig. 2C). Given that RAD001/BEZ235 treatment reduces OCR, treatment, with no further effect of the triple combination (Fig. 4E). but does not alter AMP/ATP or ADP/ATP ratios, we asked whether Critically, by applying the mitochondrial ROS scavenger Mito- this could result from lower energy needs. Inhibition of mTOR Tempo together with pan-caspase inhibitors, we rescued phenfor- negatively affects ribosome biogenesis and protein synthesis, two min-induced cell death at drug doses as high as 1 mmol/L (Fig. 4F). of the most energy consuming catabolic cellular processes (45). Thus phenformin, either alone or in combination with RAD001/ We first analyzed steady-state levels of 18S and 28S rRNA by BEZ235, inhibits mitochondrial efficiency, evoking mitochondrial ethidium bromide (EB) staining, and nascent rRNA levels by pulse network fragmentation, ROS production, and apoptosis. labeling with 3H-uridine. The EB-stained agarose gel shows that that there is no selective impact on the ratio of mature 18S to 28S Tumor growth suppression by RAD001/BEZ235 and rRNA (Fig. 3E). However, 3H-Uridine labeling of 47S precursor phenformin rRNA and the newly synthesized 18S and 28S mature rRNA To determine the tumor growth–suppressing efficacy of phen- species were strongly impaired by the RAD001/BEZ235 combi- formin alone, or in combination with RAD001/BEZ235, we used nation, with no apparent damage to processing, but an overall the orthotopic Huh7 tumor–bearing mouse model (Fig. 1A). reduction in rRNA biogenesis (Fig. 3E). These effects are paralleled Establishing the highest tolerated dose of phenformin in combi- by a strong reduction of 3H-Leucine incorporation into nascent nation with RAD001/BEZ235 required lowering its dosage as a protein by RAD001/BEZ235 treatment (Fig. 3F). In contrast, the single agent in tumor-bearing nude mice from 100 mg/kg/day to effects of phenformin on either response are not as profound 70 mg/kg/day. Although not as effective as the RAD001/BEZ235 (Fig. 3E and F). Taken together, the results suggest that mTOR combination, phenformin alone at 70 mg/kg had a significant inhibitors sustain a healthy cellular energetic state where energy inhibitory effect on tumor weight (Fig. 5A) and minimal effect on production is balanced by controlling the rate of major energy- body weight (Supplementary Fig. S4A). Phenformin treatment in consuming biochemical processes, and such effects suppress combination with RAD001/BEZ235, appeared to improve tumor AMPK activation by phenformin. suppression beyond that of the RAD001/BEZ235 combination (Fig. 5A). In addition, phenfomin combined with either mTOR Mitochondrial dysfunction and fragmentation inhibitor alone, did not reach the same extent of tumor suppres- The inability of cells to maintain the AMP/ATP ratio in the sion as the triple combination (Supplementary Fig. S4B). In four presence of phenformin raised the possibility that, beyond independent experiments, the tumor suppressor effect exerted by lowering mitochondrial activity, the biguanide may also cause RBP led to more homogeneously small tumors when compared mitochondrial dysfunction and fragmentation, similar to other with the RAD001/BEZ235 combination alone. Using the Bartlett mitochondrial electron transport chain inhibitors and AMPK test of homogeneity of variances, we determined that the variance activators (38). Although treatment with RAD001/BEZ235 within the RBP group versus the RAD001/BEZ235 combination decreases mitochondrial activity (Fig. 3A; Supplementary Fig. had a p value 0.76 0.033, statistically different in a 90% S3A), it did not perturb mitochondrial efficiency, as measured confidence interval. Analyses of treated tumor tissues revealed by O2 consumption in the presence of oligomycin, a CV ATPase strong inhibition of phosphorylation of mTORC1 targets RPS6 inhibitor (Fig. 4A). In contrast, the dramatic drop in mitochon- and 4E-BP1 by RAD001/BEZ235 alone, or in combination with drial activity induced by phenformin (Fig. 3A) is accompanied by phenformin, with a less pronounced effect of phenformin alone a decrease in mitochondrial O2 consumption coupled to ATP (Supplementary Fig. S4C). In parallel, RAD001/BEZ235, in the production (Fig. 4A), indicative of mitochondrial dysfunction absence or presence of phenformin, had little effect on mTORC2 (46). Although RAD001/BEZ235 does not negatively affect mito- signaling, as measured by Akt phosphorylation (Supplementary chondrial efficiency, it slightly augments the effects of phenfor- Fig. S4C). This was most likely due to the low concentration of min (Fig. 4A). Consistent with the biguanide inducing mitochon- RAD001/BEZ235 administered to nude mice versus the higher drial damage, by confocal imaging of a MitoTracker probe, we doses, which we employed earlier in C57BL/6 mice (26). Con- could observe that the fusion/fission equilibrium of the mito- sistent with the inhibition of mTORC1 signaling, RAD001/ chondrial network was progressing towards fragmentation in the BEZ235, alone or in combination with phenformin, causes a presence of phenformin (Fig. 4B), consistent with the activation sharp drop in cyclin D1 levels, indicative of G1 cell-cycle arrest of AMPK (38). This was not the case for RAD001/BEZ235 alone, (Supplementary Fig. S4D). As described above, RAD001/ where mitochondria appeared to be more fused (Fig. 4B), as BEZ235–treated mice showed a significant increase in glycemia recently reported for mTOR inhibitors (47). In agreement with (Fig. 2A), whereas phenformin treatment suppressed the glycemic these findings, phenformin, but not RAD001/BEZ235, induced effect of the mTOR inhibitors (Fig. 5B). Although, RAD001/ cleavage of optic atrophy 1 (OPA1; Fig. 4C), an inner mitochon- BEZ235–treated mice did not display a significant inhibition of drial membrane GTPase required for fusion, whose cleavage is lactate production, the combined mTOR inhibitors blunted associated with extensive mitochondrial damage (38). Phenfor- the strong proglycolitic effect of phenformin on lactate produc- min-induced mitochondrial damage has been shown to stimulate tion (Fig. 5C). These results show that RAD001/BEZ235 and

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A B 100 Ctrl ##

75 * ** ** **

(% relative to control) Figure 4. Coupled mitochondrial OCR 0 Phenfomin, but not mTOR inhibitors, Ctrl RB Phf RBP Ctrl RB Phf RBP induces mitochondrial fragmentation 3 h 16 h and high levels of ROS. Huh7 cells were treated with RAD001 (5 C Ctrl RB Phf RBP nmol/L), BEZ235 (20 nmol/L), and RB phenformin (1 mmol/L). A, ATP- OPA1 coupled mitochondrial oxygen consumption of Huh7 cells after 3 and 16 hours of treatment (n ¼ 3–5). B, TIM44 Representative confocal microscopy images of the mitochondria network of 16-hour–treated Huh7 cells D Ctrl RB Phf RBP visualized by Mitotracker Red CMXROS probe (scale bar, 10 mm). GST-BHMT frag C, OPA1 protein isoforms in total extracts from 16-hour–treated Huh7 GFPmyc cells; TIM44 as mitochondrial loading control. D, Autophagy cargo BHMT- assay in Huh7 cells treated for 6 hours; Phf GFP-myc as a transfection control. E 250 E, Mitochondrial superoxide analyzed *** *** by ﬂow cytometry of Huh7 with 200 MitoSox Red probe, after 3 hours of *** *** treatment; Antimycin A (AA; 50 m 150 mol/L) and Rotenone (Rot; 25 *** mmol/L) for 45 minutes as positive control (n ¼ 4). F, Relative cell 100 number of Huh7 after 24 hours of exposure to phenformin (0.5 and 1 50 MitoSox red intensity (% relative to control) mmol/L) in culture medium containing 5 mmol/L glucose, with 0 Ctrl RB PhfRBP AA/Rot or without pan-caspase inhibitor Q-VD-OPH (20 mmol/L) and mitochondrial ROS scavenger F *** RBP 120 MitoTempo (MT; 20mmol/L; n ¼ 3). *** Ctrl (Control); RB (RAD001/BEZ235); 100 Phf (phenformin); RBP (RAD001/ BEZ235/phenformin). 80

Cell number 40

(% relative to control) 20

0 Phf (mmol/L): 0 0.51.0 0 0.5 1.0 Q-VD-OPH & MT

phenformin treatment is highly efficacious in suppressing tumor BEZ235 translated into a greater overall survival benefit after drug growth (Fig. 5A; Supplementary Fig. S4B). Moreover, phenformin exposure for three weeks. RAD001/BEZ235 treatment alone sig- decreases circulating glucose and increases lactic acid production, nificantly increased median survival by 26% over vehicle-treated suggesting that, as in cells, it is acting to impede mitochondrial control as shown by Kaplan–Meier analysis (Fig. 5D), with little activity and favoring glycolysis. effect on body weight (Supplementary Fig. S5A). In combination with RAD001/BEZ235, phenformin treatment further increased Survival benefit of phenformin treatment followed by RAD001/ the median overall survival by 33% (Fig. 5D), with similar tumor BEZ235 weights at sacrifice (Supplementary Fig. S5B), suggesting that Given the findings above, we asked whether the extent of tumor mice eventually perish due to primary tumor burden. Likewise, suppression induced by phenformin when added to RAD001/ in a second experiment, the addition of phenformin to RAD001/

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A * B C 6 * 400 * * 20 *** 300 4 15 ** 200 *** *** 10 2 * 100 Glucose (mg/dL) Tumor weight (g) 5 Plasma lactate (nmol/L/ m L) 0 0 0 t0 Veh RB Phf RBP Veh RB Phf RBP Veh RB Phf RBP

D E F * * Treatment end 400 * 1,000 100 Veh 320 RB 750 RBP 75 240 500 50 160 2 SCR (Succinate) 250 O flux per mass

(ratio pmol/(s*mg)) 80 Survival (%) 25 0 0 *** Veh RB Phf RBP Veh RB Phf RBP 0 *** GM : SCR: 20 30 40 50 60 P Glutamate + Malate + ADP Substrate control ratio Days (after treatment start) (CI substrates) (GMP vs. GMS P )

G Treatment end H 100 10 Veh P100 8 75 P100 + RB

6 50 4

Survival (%) 25 Tumor weight (g) 2 *** 0 0 20 30 40 50 60 70 Veh P100 P100 +RB Days (after treatment start)

Figure 5. Combining RAD001/BEZ235 and phenformin in vivo efﬁciently halts Huh7-tumor growth, and the sequential administration of phenformin followed by

RAD/BEZ235 dramatically improves Huh7-tumor-bearing mouse survival. A, Huh7-tumor weight before (t0) and after 15 days of treatment with RAD001 (2.5 mg/kg) and BEZ235 (6 mg/kg) together, or in combination with phenformin (70 mg/kg; n ¼ 8–9). Circulating levels of glucose (B) and lactate in mice treated for 15 days, while fed ad libitum (n ¼ 6-8; C). D, Kaplan–Meier survival curve of Huh7-tumor–bearing mice after 22 days of treatment with RAD001 (2.5 mg/kg) and BEZ235 (6 mg/kg) together, or in combination with phenformin (70 mg/kg; n ¼ 6–9). E, Oxygen consumption of mitochondria from 7-day–treated tumors in the presence of glutamate, malate, and ADP (GMp; n ¼ 3). F, Succinate substrate control ratio: calculated as the ratio between oxygen consumption in the presence of glutamate, malate, and ADP with (GMSp) or without succinate (GMp; n ¼ 3). Kaplan–Meier survival curve of Huh7 tumor-bearing mice after 8 days of treatment with phenformin (100 mg/kg), followed by 15 days treatment with RAD/BEZ235 (2.5 mg/kg and 6 mg/kg) or phenformin (100 mg/kg) once daily

(G) and tumor weight at ethical time of sacriﬁce (n ¼ 8–10; H). Veh, vehicle; RB, RAD001/BEZ235; Phf, phenformin; RBP, RAD001/BEZ235/phenformin; P100, phenformin 100 mg/kg; P100þRB, phenformin 100 mg/kg followed by RAD001 plus BEZ235.

BEZ235 increased median survival from 26% to 37%, with the extent to which the treatments affected tumor mitochondrial animals dying with similar tumor weights (Supplementary Figs. function in vivo, we isolated mitochondria from treated tumors S5C and S5D, respectively). In vivo, lactic acid production indi- and measured their ability to consume O2 employing a succession cated a systemic inhibition of mitochondrial oxidation and a shift of OXPHOS substrates. In brief, the addition of glutamate, malate, toward glycolysis under phenformin administration. To ascertain and ADP (GMP) shows that RAD001/BEZ235 treatment improves

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the usage of CI substrates, as compared with phenformin treat- RAD001 treatment (21). Interestingly, conditional depletion of ment alone or in the triple combination (Fig. 5E). However, the TSC1 in the liver leads to HCC in the mouse (52). Such mice were addition of succinate led to higher substrate control ratio after cleared of disease if treated with rapamycin before the onset of treatment with phenformin alone (Fig. 5F; Supplementary Fig. tumorigenesis (52). In contrast, others have found that chronic S5E), suggesting that, unlike under the RAD001/BEZ235 combi- treatment with rapamycin accelerates HCC in the DEN-induced nation, such mitochondria are relying more on CII versus CI to mouse model, potentially due to IL6 production (53), or the maintain mitochondrial oxidation. When combined with phen- protection of a specific subpopulation of HCC cells (42, 43). In formin, mTOR inhibition appears to decrease CII usage forced by apparent agreement with these findings, using carcinogen- phenformin inhibition of CI. These mitochondrial alterations did induced (26) or orthotopically implanted HCC human cell line not appear due to altered OXPHOS complex expression (Sup- mouse models, we found little protection against tumor progres- plementary Fig. S5F). The data support the notion that, in tumors sion with RAD001 alone. However, in combination with the treated with phenformin, CI function is hindered, evoking a mTOR ATP-site inhibitor BEZ235, we observed strong tumor compensatory mechanism by which mitochondrial electron suppression with no apparent overall enhancement of tumori- transport is channeled through CII. genic capacity. Despite the fact that the development of BEZ235 in Although we observed benefit from adding phenformin to the cancer has been discontinued, the successor of INK128, TAK228, RAD001/BEZ combination, the results above suggested that the has had encouraging results in phase I/II clinical trials (54); and, in phenformin concentrations we were achieving in vivo were not our preliminary studies, when combined with RAD001, is as sufficient to attain a more dramatic effect on tumor mitochondrial effective as BEZ235 in repressing mTORC1 signaling and tumor activity. Moreover, we find that mTOR inhibitors appear to growth. In support of this approach, others have developed a dual increase mitochondria efficiency and fusion, in agreement with inhibitor which can bind to both the allosteric- and ATP- binding others (30, 47), events that could potentially counteract phen- sites of mTOR, which is as effective as targeting the two sites in formin-induced mitochondrial dysfunction and fragmentation. combination (55, 56). The approach of combining allosteric and Therefore, we reasoned that if we first treated tumors with phen- catalytic site inhibitors to increase efficacy is not confined to formin alone, we could potentially drive mitochondrial dysfunc- mTOR, as such an approach has been recently reported for tion, such that they would be more sensitive to the subsequent Abl/Bcr–positive chronic myeloid leukemia (57). On the basis addition of mTOR inhibitors. In a sequential design, we treated of the synergistic activity of the new allosteric inhibitor ABL001 tumor-bearing mice with phenformin 100 mg/kg for 8 days, and with nilotinib, imatinib, or dasatinib, this combination is being then either maintained them on phenformin or switched them to tested in clinical trials (58). the RAD001/BEZ235 combination for 14 days. After 8 days The utility of targeting mitochondrial metabolism in cancer has treatment, phenformin at 100 mg/kg, had to be administered been recognized for some time (59). In addition, clinically, every other day (QOD), to be tolerated. Treatment with phen- biguanides have been used to resolve hyperglycemic states asso- formin alone showed no enhancement in survival, whereas ciated with mTOR inhibitors (31, 32). We chose phenformin over sequential addition of RAD001/BEZ235 had a dramatic effect metformin because the hydrophobic biguanide does not require (Fig. 5G). The combination of phenformin followed by mTOR cell surface organic cation transporter 1 (OCT1; ref. 60), and inhibitors increased median overall survival by 70% from 28 days because OCT1 appears downregulated in advanced HCC (33). to 48 days (Fig. 5G), without effecting body weight (Supplemen- Both in vitro and in vivo studies show that phenformin acts mainly tary Fig. S5G), effectively doubling the response observed by through inhibition of the mitochondrial OXPHOS CI (34, 48), adding all three agents together (Fig. 5D). Critically, at the time and we find that phenformin recapitulates the effects of other of sacrifice, the tumor size was similar in each treatment group, mitochondrial electron transport chain inhibitors by inducing suggesting that the mice succumb to primary tumor burden (Fig. acute activation of AMPK (38), as a consequence of the increased 5H). Although we cannot discount a systemic effect, the results are AMP/ATP ratio. Moreover, even though phenformin is a potent consistent with the recent findings in cell culture suggesting that inhibitor of mTORC1 through AMPK-dependent and -indepen- mTOR inhibitors are hindered by their capacity to promote dent mechanisms (61, 62), it seems to have little additional effect mitochondrial fusion and increase mitochondrial efficiency on this pathway in presence of RAD001 and BEZ235. Therefore, it (30, 47). Here we propose that in vivo pretreatment of HCC- appears to contribute to tumor suppression by distinct mechan- bearing mice with phenformin compromises mTOR inhibitors isms. This may be through the activation of AMPK, whose role as undesirable effects, by inducing mitochondrial dysfunction and tumor suppressor is complex, but which is largely thought to stem fragmentation while generating high ROS levels, such that sub- from its function as a suppressor of major anabolic process and sequent mTOR inhibitor treatment results in a striking increase in the activation of catabolic responses (63, 64). It is clear from our overall mouse survival (see model in Fig. 6). study that activation of AMPK by phenformin suppresses both ribosome biogenesis and protein synthesis, two of the most major anabolic processes in the cell. However, we find that the activation Discussion of AMPK by phenformin is strongly suppressed in the presence of Sorafenib has shown an unprecedented clinical activity in RAD001/BEZ235, potentially due to the energy-sparing effects of advanced HCC and is presently the standard of care (50). How- mTOR inhibition, underscoring the antagonist effects that such ever, its effects are not durable, with patients eventually succumb- therapies may exert. ing to disease (51). Given these observations and that the mTOR The rationale for pursuing phenformin in combination with pathway is hyperactivated in 50%–60% of HCC patients, the RAD001/BEZ235 was that together they would significantly mTOR inhibitor Everolimus phase III trial EVOLVE was initiated. impact cell metabolism by attacking both mitochondrial and Although no benefits were observed (20), retrospective stratifica- glycolytic capacity, eventually leading to energetic catastrophe. tion of patients suggested those with loss of TSC2 benefited from Cellular low energy-state and mitochondrial dysfunction are

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HCC Cell

AMPK mTOR Glycolysis Mitochondria

Mitophagy OXPHOS Homeostasis

Phenformin AMPK mTOR Glycolysis

? Mitochondria CI OXPHOS Dysfunction Mitophagy ROS

Glycolysis mTOR RAD001/BEZ235

Mitochondria

OXPHOS Mitophagy Efficiency

RAD001/BEZ23 mTOR Glycolysis 5 Overall survival Mitochondria

OXPHOS Mitophagy Dysfunction ROS

Figure 6. Model of RAD001/BEZ235 and phenformin effects on mitochondrial dynamics, OXPHOS, and glycolysis; and their impact on overall survival of tumor-bearing mice. known to further stimulate the generation of mitochondrial ROS induce OPA1 cleavage, a fragmentation-related event that is only (65), consistent with our findings that the phenformin-induced triggered after more extensive mitochondrial damage (38, 68), apoptotic effects appear to be ROS-mediated. Moreover, the such as we observe here with phenfomin. This more extensive effects on ROS may be further exacerbated by a lack of autophagic damage may also be a consequence of phenformin's inhibitory clearance of damaged-mitochondria in the presence of phenfor- effect on autophagy. From a bioenergetics point of view, one min. The mitochondrial impact of phenformin is highlighted by would have predicted that the initiation of autophagy and mito- the apparent effort of treated cells to use alternative ATP-gener- chondrial biosynthetic pathways would be activated to reestablish ating processes to survive, including glycolysis and potentially an efficient mitochondrial network and decrease ROS levels (69, fatty acid oxidation (66). Nevertheless, the observed glycolitic 70). However, it has been reported that autophagy initiates at shift does not seem to resolve the energetic stress imposed by mitochondria-associated ER membranes (MAM; ref. 71), such phenformin in vitro, because the AMP/ATP ratios are not normal- that phenformin-induced fragmentation may structurally com- ized, and cells become strikingly reliant on glucose levels. promise this contact sites, blocking the initiation of autophagy. Although energetic catastrophe, leading to apoptosis, appears Consistent with our data, mTOR inhibitors were previously to be ultimately dictated by ROS levels, the managing of energetic found to decrease cell respiration and mitochondrial mass, while stress seems to be largely mediated by the regulation of mito- maintaining coupled ATP production, although we did not chondrial OXPHOS capacity, efficiency, and dynamics (67). Here observe a drop in ATP levels, as reported by Morita and colleagues we show that phenformin induces a low energy-state character- (30). This finding was also consistent with RAD001/BEZ235 ized by an increase in AMPK activation, mitochondrial dysfunc- treatment having no effects on the AMP/ATP and ADP/ATP ratios tion, and mitochondrial fragmentation, all potentially contrib- or on AMPK activation. Instead, the maintenance of ATP levels uting to phenformin's tumor-suppressive effects. Recent studies appears to be the result of increased mitochondrial efficiency and have shown that the electron transport inhibitors rotenone and lower cellular ATP demand. In agreement, we observed an antimycin A induce mitochondrial fragmentation by abrogating increased mitochondrial network fusion phenotype following the mitochondrial localization of the fission protein DRP1, in an RAD001/BEZ235 treatment, which was recently reported for AMPK-dependent manner (38). However, these agents do not mTOR inhibitors (47), and that may be responsible for the

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increase in OXPHOS efficiency (67). The data suggest that Disclosure of Potential Conflicts of Interest mTOR control of mitochondrial dynamics may partially be No potential conflicts of interest were disclosed. responsible for the cytostatic effects of mTOR inhibitors; inducing a hyperfusion state that protects mitochondria against Authors' Contributions apoptosis, reminiscent of nutrient deprivation (72). Indeed, Conception and design: S.R. Veiga, C.A. Mercer, H.E. Thomas, G. Thomas, Morita and colleagues have demonstrated that inducing mito- S.C. Kozma chondrial fragmentation could render mTOR inhibitors more Development of methodology: S.R. Veiga, X. Ge, C.A. Mercer, J. Hernandez- cytotoxic (47). However, in vivo, the potential impact of phen- Losa, G. Thomas, S.C. Kozma Acquisition of data (provided animals, acquired and managed patients, formin on mitochondrial inhibition of CI is apparently much in vitro provided facilities, etc.): S.R. Veiga, C.A. Mercer, M.I. Hernandez-Alvarez, less than that achieved , suggesting that it might be J. Hernandez-Losa, S.R. y Cajal, A. Zorzano fi insuf cient to overcome the protective effects of the mTOR Analysis and interpretation of data (e.g., statistical analysis, biostatistics, inhibitors when added in combination. computational analysis): S.R. Veiga, C.A. Mercer, M.I. Hernandez-Alvarez, Given the findings above, our goal was to maximize each drugs S.R. y Cajal, G. Thomas, S.C. Kozma effect in suppressing tumor growth, leading to the hypothesis that Writing, review, and/or revision of the manuscript: C.A. Mercer, H.E. Thomas, their sequential use may be more efficacious. This reasoning J. Hernandez-Losa, G. Thomas, S.C. Kozma suggested that an initial treatment with phenformin followed by Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): G. Thomas, S.C. Kozma mTOR inhibitors would favor tumor growth suppression by Study supervision: G. Thomas, S.C. Kozma limiting the capacity of cells to respond to mTOR inhibition by fi hyperfusion of mitochondria and increased mitochondrial ef - Acknowledgments ciency. Concomitantly, phenformin-induced mitochondrial dys- These studies were supported by grants from the Spanish Ministry of function forces tumor cells to use glycolysis, which might render Economy and Competitiveness (BFU2012-38867; to S.C. Kozma), the Instituto them more prone to the mTOR inhibitors, which impair this de Salud Carlos III (II12/00002 and PI16/01762; to C.A. Mercer) the NIH/NCI response. Such a strategy of metabolic synthetic lethality was National Cancer Institute grant R21-CA158768, and the Spanish Ministry of recently reported in a breast cancer model using a tyrosine kinase Economy and Competitiveness grant (SAF2014-52162-P; to to G. Thomas), the receptor inhibitor and either phenformin or ME344, a CI inhib- CIG European Commission (PCIG10-GA-2011-304160), the NIH/NCI Nation- al Cancer Institute (R01-CA191814), the Asociacion Espanola~ Contra el Cancer itor in clinical trials (73). The administration of phenformin prior (GCB-142035THOM), the ISCIII-RTICC (RD12/0036/0049), the AGAUR the use of mTOR inhibitors dramatically increased the overall (2014SGR-870), and by shared funding from the IDIBELL and the Vall d'Heb- survival of tumor-bearing mice, supporting the notion that there ron Institute of Oncology (VHIO). In addition, S.C. Kozma and G. Thomas are is a metabolic synthetic lethality that could be exploited when funded by a grant from ISCIII (PIE13/00022; to M. Esteller). The Spanish combining these agents. The paradigm of combining phenformin Ministry grants to G. Thomas and S.C. Kozma are cofunded by FEDER – awayto ~ in vivo with the mTOR inhibitors needs to be further developed, in terms build Europe. We are grateful to F. Riano for assisting with the models and A. Gentilella for discussions in planning and executing the nascent rRNA and of dose, frequency, and time of drug exposure. In this context, protein experiments. We also thank E. Battle for advice on the culturing of FDG-PET could be used to ascertain the effects of phenformin on tumoroids, M. Sanchez-Feutrie and D. Sebastian for providing help in the the induction of glucose uptake when cells shift to glycolysis, mitochondrial respirometry experiments and G. Doerman with the preparation which should recede when mTOR inhibitors are administered, as of figures. In addition we thank A. Tauler for his insightful discussions and I. recently proposed by Navarro and colleagues in a similar context Fabregat for providing HCC cell lines and critical consultations. Finally, we are (73). This will be of interest given the recent preclinical studies indebted to Novartis Oncology for supplying RAD001 and NVP-BEZ235. showing the benefit of combining phenformin with BRAF inhi- The costs of publication of this article were defrayed in part by the payment of bitors in melanoma (74, 75), which have led to the initiation of a page charges. This article must therefore be hereby marked advertisement in phase I clinical trial with phenformin in melanoma (37). It will be accordance with 18 U.S.C. Section 1734 solely to indicate this fact. of interest to determine its efficacy in such a setting and whether phenformin with the dual mTOR inhibitors can be expanded to Received January 20, 2018; revised April 5, 2018; accepted April 19, 2018; HCC and other cancers. published first April 24, 2018.

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OF14 Clin Cancer Res; 2018 Clinical Cancer Research

Phenformin-Induced Mitochondrial Dysfunction Sensitizes Hepatocellular Carcinoma for Dual Inhibition of mTOR

Sónia R. Veiga, Xuemei Ge, Carol A. Mercer, et al.

Clin Cancer Res Published OnlineFirst April 24, 2018.

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