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. Ó2018 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 Ó2018 American Association for Cancer Research. approval of the rapamycin family of allosteric drugs (rapalogs) www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst April 24, 2018; DOI: 10.1158/1078-0432.CCR-18-0177 Veiga et al. 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