Phenformin Treatment Research Articles

Phenformin activates ER stress to promote autophagic cell death via NIBAN1 and DDIT4 in oral squamous cell carcinoma independent of AMPK

The efficient clinical treatment of oral squamous cell carcinoma (OSCC) is still a challenge that demands the development of effective new drugs. Phenformin has been shown to produce more potent anti-tumor activities than metformin on different tumors, however, not much is known about the influence of phenformin on OSCC cells. We found that phenformin suppresses OSCC cell proliferation, and promotes OSCC cell autophagy and apoptosis to significantly inhibit OSCC cell growth both in vivo and in vitro. RNA-seq analysis revealed that autophagy pathways were the main targets of phenformin and identified two new targets DDIT4 (DNA damage inducible transcript 4) and NIBAN1 (niban apoptosis regulator 1). We found that phenformin significantly induces the expression of both DDIT4 and NIBAN1 to promote OSCC autophagy. Further, the enhanced expression of DDIT4 and NIBAN1 elicited by phenformin was not blocked by the knockdown of AMPK but was suppressed by the knockdown of transcription factor ATF4 (activation transcription factor 4), which was induced by phenformin treatment in OSCC cells. Mechanistically, these results revealed that phenformin triggers endoplasmic reticulum (ER) stress to activate PERK (protein kinase R-like ER kinase), which phosphorylates the transitional initial factor eIF2, and the increased phosphorylation of eIF2 leads to the increased translation of ATF4. In summary, we discovered that phenformin induces its new targets DDIT4 and especially NIBAN1 to promote autophagic and apoptotic cell death to suppress OSCC cell growth. Our study supports the potential clinical utility of phenformin for OSCC treatment in the future.

Abstract 7170: Pharmacokinetic profiling of Re-Phenformin in vivo: A novel approach in pancreatic cancer therapy

Abstract Phenformin, a biguanide medication, has long been utilized in the treatment of diabetes. Recent research indicates that phenformin may also serve as an effective anticancer agent. This study introduces the synthesis and evaluation of Re tricarbonyl phenformin complex (Re-(CO)3-Phenformin, Re-Phen), as a derivative of Phenformin and novel anticancer agent for pancreatic cancer treatment. Re-Phen was synthesized, purified, and characterized through preparative HPLC, NMR, mass spectrometry, and FT-IR. Subsequent in vitro studies, utilizing the MTS assay, assessed its cytotoxicity against pancreatic cancer cell lines, including Kras* murine PDAC and MiaPaca-2 human PDAC. Notably, Re-Phen exhibited significantly higher cytotoxic effects in MiaPaca-2 cells (IC50= 0.755 mM) and Kras* cells (IC50= 0.410 mM) compared to Phenformin treatment (IC50= 4.02 mM and 2.36 mM, respectively). These results highlight Re-Phen's enhanced anticancer activity, outperforming Phenformin in potency. Further investigation into the pharmacokinetics of Re-Phen involved an in vivo study with rats intravenously administered 2 mg/kg of Re-Phenformin. Blood samples were collected at intervals (10 min to 24 h post-dose) and were analyzed by validated LC-MS/MS. The pharmacokinetic profile of Re-Phen was best described by a 2-compartment model, using Phoenix® WinNonlin (version 8.0), with a particular focus on comparing the profiles of Phenformin and Re-Phen. The study revealed that Re-Phen has a significantly extended half-life compared to Phenformin (9.13 hr vs 2.11 hr), indicating prolonged systemic retention. Additionally, Re-Phen demonstrated an increased area under the curve (AUC), suggesting greater systemic exposure at equivalent dosing (90.55 hr*ng/ml vs 7.13 hr*ng/ml). This could imply enhanced bioavailability or altered metabolic clearance of Re-Phen compared to its parent compound. The two-Compartment of Re-Phe, provide a deeper understanding of its pharmacokinetic characteristics. In conclusion, Re-Phen not only demonstrates superior anticancer efficacy in vitro but also exhibits a more favorable pharmacokinetic profile in vivo. The extended half-life and increased AUC of Re-Phen suggest the potential of offering improved therapeutic efficacy in pancreatic cancer treatment. These promising findings warrant further investigation and could position Re-Phen as a promising therapeutic agent for the pharmacological management of pancreatic cancer. Citation Format: Amir Mohammad Gholizadeh, Lu Dai, Fatima Dagher, Chiyi Xiong, Chun Li, Diana S-L Chow. Pharmacokinetic profiling of Re-Phenformin in vivo: A novel approach in pancreatic cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7170.

Cytotoxic effects of Phenformin on ovarian cancer cells: expression of HIF-1α and PDK1 in the hypoxic microenvironment.

Today, many anticancer drugs are used clinically for ovarian cancer, one of the leading causes of cancer-related deaths in women. Phenformin is an antidiabetic drug of the biguanide class. It improves the antiproliferative activity in cancer cells. Hypoxia is an important component associated with ovarian cancer and its tumor microenvironment. The aim of this study was to investigate the anticancer effects of Phenformin in SKOV-3 human ovarian cancer cells under hypoxic conditions. SKOV-3 human ovarian cancer cells treated with different doses of Phenformin (0.5 mM, 1 mM, 2 mM, 5 mM) for 24 hours were subjected to WST-1 cell viability assay and Annexin V apoptosis assay. A dose-dependent decrease in cell viability with Phenformin treatment was observed. In addition, Phenformin activated percentage of apoptotic SKOV-3 cancer cells in a dose-dependent manner. In this study, Cobalt(II) chloride (CoCl2) treatment leads to increased hypoxia-inducible factor-1alpha (HIF-1α) expression and Phenformin can recover hypoxic condition. HIF-1α protein expression was significantly correlated with cell viability assay and apoptosis assay. We also found that Phenformin inhibits expression of phosphoinositide-dependent kinase 1 (PDK1) in SKOV-3 ovarian cancer cells. The ability to migrate to cancer cells was significantly reduced in a dose-dependent manner with Phenformin. This data demonstrates that Phenformin treatment can induce apoptosis and inhibit proliferation in ovarian cancer cells under hypoxic conditions. The findings reveal that HIF-1α is a new target for the treatment of ovarian cancer.

Phenformin increases early hematopoietic progenitors in the Jak2V617F murine model.

Myeloproliferative neoplasms (MPN) are disorders characterized by an alteration at the hematopoietic stem cell (HSC) level, where the JAK2 mutation is the most common genetic alteration found in classic MPN (polycythemia vera, essential thrombocythemia, and primary myelofibrosis). We and others previously demonstrated that metformin reduced splenomegaly and platelets counts in peripheral blood in JAK2V617F pre-clinical MPN models, which highlighted the antineoplastic potential of biguanides for MPN treatment. Phenformin is a biguanide that has been used to treat diabetes, but was withdrawn due to its potential to cause lactic acidosis in patients. We herein aimed to investigate the effects of phenformin in MPN disease burden and stem cell function in Jak2V617F-knockin MPN mice. In vitro phenformin treatment reduced cell viability and increased apoptosis in SET2 JAK2V67F cells. Long-term treatment with 40mg/kg phenformin in Jak2V617F knockin mice increased the frequency of LSK, myeloid progenitors (MP), and multipotent progenitors (MPP) in the bone marrow. Phenformin treatment did not affect peripheral blood counts, spleen weight, megakaryocyte count, erythroid precursors frequency, or ex vivo clonogenic capacity. Ex vivo treatment of bone marrow cells from Jak2V617F knockin mice with phenformin did not affect hematologic parameters or engraftment in recipient mice. Phenformin increased the percentages of LSK, MP, and MPP populations, but did not reduce disease burden in Jak2V617F-knockin mice. Additional studies are necessary to further understand the effects of phenformin on early hematopoietic progenitors.

Differential substrate use in EGF- and oncogenic KRAS-stimulated human mammary epithelial cells.

Many metabolic phenotypes in cancer cells are also characteristic of proliferating nontransformed mammalian cells, and attempts to distinguish between phenotypes resulting from oncogenic perturbation from those associated with increased proliferation are limited. Here, we examined the extent to which metabolic changes corresponding to oncogenic KRAS expression differed from those corresponding to epidermal growth factor (EGF)-driven proliferation in human mammary epithelial cells (HMECs). Removal of EGF from culture medium reduced growth rates and glucose/glutamine consumption in control HMECs despite limited changes in respiration and fatty acid synthesis, while the relative contribution of branched-chain amino acids to the TCA cycle and lipogenesis increased in the near-quiescent conditions. Most metabolic phenotypes measured in HMECs expressing mutant KRAS were similar to those observed in EGF-stimulated control HMECs that were growing at comparable rates. However, glucose and glutamine consumption as well as lactate and glutamate production were lower in KRAS-expressing cells cultured in media without added EGF, and these changes correlated with reduced sensitivity to GLUT1 inhibitor and phenformin treatment. Our results demonstrate the strong dependence of metabolic behavior on growth rate and provide a model to distinguish the metabolic influences of oncogenic mutations and nononcogenic growth.

Nanog maintains stemness of Lkb1‐deficient lung adenocarcinoma and prevents gastric differentiation

Growing evidence supports that LKB1‐deficient KRAS‐driven lung tumors represent a unique therapeutic challenge, displaying strong cancer plasticity that promotes lineage conversion and drug resistance. Here we find that murine lung tumors from the KrasLSL‐G12D/+; Lkb1flox/flox (KL) model show strong plasticity, which associates with up‐regulation of stem cell pluripotency genes such as Nanog. Deletion of Nanog in KL model initiates a gastric differentiation program and promotes mucinous lung tumor growth. We find that NANOG is not expressed at a meaningful level in human lung adenocarcinoma (ADC), as well as in human lung invasive mucinous adenocarcinoma (IMA). Gastric differentiation involves activation of Notch signaling, and perturbation of Notch pathway by the γ‐secretase inhibitor LY‐411575 remarkably impairs mucinous tumor formation. In contrast to non‐mucinous tumors, mucinous tumors are resistant to phenformin treatment. Such therapeutic resistance could be overcome through combined treatments with LY‐411575 and phenformin. Overall, we uncover a previously unappreciated plasticity of LKB1‐deficient tumors and identify the Nanog‐Notch axis in regulating gastric differentiation, which holds important therapeutic implication for the treatment of mucinous lung cancer.

The Combination of Loss of ALDH1L1 Function and Phenformin Treatment Decreases Tumor Growth in KRAS-Driven Lung Cancer.

Lung adenocarcinoma cells express high levels of ALDH1L1, an enzyme of the one-carbon pathway that catalyzes the conversion of 10-formyltetrahydrofolate into tetrahydrofolate and NAD(P)H. In this study, we evaluated the potential of ALDH1L1 as a therapeutic target by deleting the Aldh1l1 gene in KrasLA2 mice, a model of spontaneous non-small cell lung cancer (NSCLC). Reporter assays revealed KRAS-mediated upregulation of the ALDH1L1 promoter in human NSCLC cells. Aldh1l1−/− mice exhibited a normal phenotype, with a 10% decrease in Kras-driven lung tumorigenesis. By contrast, the inhibition of oxidative phosphorylation inhibition using phenformin in Aldh1l1−/−; KrasLA2 mice dramatically decreased the number of tumor nodules and tumor area by up to 50%. Furthermore, combined treatment with pan-ALDH inhibitor and phenformin showed a decreased number and area of lung tumors by 70% in the KrasLA2 lung cancer model. Consistent with this, previous work showed that the combination of ALDH1L1 knockdown and phenformin treatment decreased ATP production by as much as 70% in NSCLS cell lines. Taken together, these results suggest that the combined inhibition of ALDH activity and oxidative phosphorylation represents a promising therapeutic strategy for NSCLC.

MA17.02 Identify Vulnerable Pathways and Improve Treatment Outcomes in LKB1-Deficient Lung Tumors

The LKB1 tumor suppressor is inactivated in about 20% of non-small cell lung cancers (NSCLC) by mutations. Cancer cells with LKB1 deficiency exert complex effects on signal transduction and transcriptional regulation, which may cause these cells more susceptible to certain therapies comparing to cells with intact LKB1 function. Phenformin, an antidiabetic medicine from the biguanides class, has shown activities against NSCLC. Phenformin as a single agent has been shown to reduce tumor burden and prolonged survival in Kras;Lkb1 compound mutant mice but not Kras;p53 mice, suggesting specific activities in tumor with LKB1 deficiency. Currently patients with unresectable locally advanced NSCLC are treated standardly with concurrent chemoradiotherapy followed by checkpoint inhibitor, durvalumab. In this project, we test treatment sensitivity to radiotherapy and/or phenformin in lung cancer cells with intact or deficient LKB1. Human lung cancer cell lines described below were used in (1) clonogenic survival assays as well as (2) generating tumor xenograft on nude mice for tumor growth delay experiments. A549, HCC15 and Calu-1 cell lines obtained from ATCC were cultured in RPMI1640 containing 5% FBS, without antibiotics. A549 cells (LKB1 deficient, TP53 WT and KRAS mutated) or HCC15 (LKB1 deficient, TP53 mutated and KRAS WT) were transfected with empty vector or WT LKB1 or LKB1-K78I plasmids; Calu-1 (LKB1 WT, KRAS mutated and p53 deleted) transfected with empty vector or LKB1 CRIPR KO were generated as described previously. A549 cells with transfected WT-LKB1 were significantly more resistant to ionizing radiation (IR) induced cell kill (8.7% survival at 8 Gy) comparing to cells transfected with empty vector (3.7%) or kinase-dead LKB1 genes (4.2%). Similarly, HCC15 cells with transfected WT-LKB1 are significantly more resistant to IR induced cell kill (7.5%) comparing to cells transfected with empty vector (4.1%) or kinase-dead LKB1 genes (3.4%). Calu-1 cells harbor WT LKB1, and it is significantly more resistant to IR induced cell kill (8.3%) comparing to their counterpart with LKB1 KO (Calu-1 transfected with LKB1 CRIPR KO) (4.1%). When A549 cells were pretreated with 30 μM phenformin prior to, during and after IR, there was no change in survival in cells transfected with WT LKB1; however there was significant further reduction in survival in cells transfected with empty vector (LKB1 deficient). Xenograft tumors were generated in nude mice with A549 cells with the above genetic alterations. There was significant further tumor growth delay in those with A549 with deficient LKB1 comparing to those with A549 with WT LKB1 gene add-back. This tumor growth delay was further enhanced when these mice were treated with oral phenformin prior to, during and after IR treatment, confirming the in vitro experimental results. Human lung cancer with deficient LKB1 are more sensitive to ionizing radiation in vitro and in vivo. This was regardless of the TP53 or KRAS mutation status. A549 cells with deficient LKB1 were also more sensitive to phenformin treatment. Phenformin treatment further sensitized LKB1 deficient lung cancer cells to IR. This suggested that LKB1 can serve as a predictive biomarker to triage patient treatments.

Ampk regulates IgD expression but not energy stress with B cell activation

Ampk is an energy gatekeeper that responds to decreases in ATP by inhibiting energy-consuming anabolic processes and promoting energy-generating catabolic processes. Recently, we showed that Lkb1, an understudied kinase in B lymphocytes and a major upstream kinase for Ampk, had critical and unexpected roles in activating naïve B cells and in germinal center formation. Therefore, we examined whether Lkb1 activities during B cell activation depend on Ampk and report surprising Ampk activation with in vitro B cell stimulation in the absence of energy stress, coupled to rapid biomass accumulation. Despite Ampk activation and a controlling role for Lkb1 in B cell activation, Ampk knockout did not significantly affect B cell activation, differentiation, nutrient dynamics, gene expression, or humoral immune responses. Instead, Ampk loss specifically repressed the transcriptional expression of IgD and its regulator, Zfp318. Results also reveal that early activation of Ampk by phenformin treatment impairs germinal center formation but does not significantly alter antibody responses. Combined, the data show an unexpectedly specific role for Ampk in the regulation of IgD expression during B cell activation.

SPARC Inhibits Metabolic Plasticity in Ovarian Cancer.

The tropism of ovarian cancer (OvCa) to the peritoneal cavity is implicated in widespread dissemination, suboptimal surgery, and poor prognosis. This tropism is influenced by stromal factors that are not only critical for the oncogenic and metastatic cascades, but also in the modulation of cancer cell metabolic plasticity to fulfill their high energy demands. In this respect, we investigated the role of Secreted Protein Acidic and Rich in Cysteine (SPARC) in metabolic plasticity of OvCa. We used a syngeneic model of OvCa in Sparc-deficient and proficient mice to gain comprehensive insight into the paracrine effect of stromal-SPARC in metabolic programming of OvCa in the peritoneal milieu. Metabolomic and transcriptomic profiling of micro-dissected syngeneic peritoneal tumors revealed that the absence of stromal-Sparc led to significant upregulation of the enzymes involved in glycolysis, TCA cycle, and mitochondrial electron transport chain (ETC), and their metabolic intermediates. Absence of stromal-Sparc increased reactive oxygen species and perturbed redox homeostasis. Recombinant SPARC exerted a dose-dependent inhibitory effect on glycolysis, mitochondrial respiration, ATP production and ROS generation. Comparative analysis with human tumors revealed that SPARC-regulated ETC-signature inversely correlated with SPARC transcripts. Targeting mitochondrial ETC by phenformin treatment of tumor-bearing Sparc-deficient and proficient mice mitigated the effect of SPARC-deficiency and significantly reduced tumor burden, ROS, and oxidative tissue damage in syngeneic tumors. In summary, our findings provide novel insights into the role of SPARC in regulating metabolic plasticity and bioenergetics in OvCa, and shines light on its potential therapeutic efficacy.

Treatment of Pancreatic Cancer Patient-Derived Xenograft Panel with Metabolic Inhibitors Reveals Efficacy of Phenformin.

Purpose: To identify effective metabolic inhibitors to suppress the aggressive growth of pancreatic ductal adenocarcinoma (PDAC), we explored the in vivo antitumor efficacy of metabolic inhibitors, as single agents, in a panel of patient-derived PDAC xenograft models (PDX) and investigated whether genomic alterations of tumors correlate with the sensitivity to metabolic inhibitors.Experimental Design: Mice with established PDAC tumors from 6 to 13 individual PDXs were randomized and treated, once daily for 4 weeks, with either sterile PBS (vehicle) or the glutaminase inhibitor bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES), transaminase inhibitor aminooxyacetate (AOA), pyruvate dehydrogenase kinase inhibitor dichloroacetate (DCA), autophagy inhibitor chloroquine (CQ), and mitochondrial complex I inhibitor phenformin/metformin.Results: Among the agents tested, phenformin showed significant tumor growth inhibition (>30% compared with vehicle) in 5 of 12 individual PDXs. Metformin, at a fivefold higher dose, displayed significant tumor growth inhibition in 3 of 12 PDXs similar to BPTES (2/8 PDXs) and DCA (2/6 PDXs). AOA and CQ had the lowest response rates. Gene set enrichment analysis conducted using the baseline gene expression profile of pancreatic tumors identified a gene expression signature that inversely correlated with phenformin sensitivity, which is in agreement with the phenformin gene expression signature of NIH Library of Integrated Network-based Cellular Signatures (LINCS). The PDXs that were more sensitive to phenformin showed a baseline reduction in amino acids and elevation in oxidized glutathione. There was no correlation between phenformin response and genetic alterations in KRAS, TP53, SMAD4, or PTENConclusions: Phenformin treatment showed relatively higher antitumor efficacy against established PDAC tumors, compared with the efficacy of other metabolic inhibitors and metformin. Phenformin treatment significantly diminished PDAC tumor progression and prolonged tumor doubling time. Overall, our results serve as a foundation for further evaluation of phenformin as a therapeutic agent in pancreatic cancer. Clin Cancer Res; 23(18); 5639-47. ©2017 AACR.

Phenformin inhibits growth and epithelial-mesenchymal transition of ErbB2-overexpressing breast cancer cells through targeting the IGF1R pathway.

Reports suggest that metformin, a popular anti-diabetes drug, prevents breast cancer through various systemic effects, including insulin-like growth factor receptor (IGFR) regulation. Although the anti-cancer properties of metformin have been well-studied, reports on a more bioavailable/potent biguanide, phenformin, remain sparse. Phenformin exerts similar functional activity to metformin and has been reported to impede mammary carcinogenesis in rats. Since the effects of phenformin on specific breast cancer subtypes have not been fully explored, we used ErbB2-overexpressing breast cancer cell and animal models to test the anti-cancer potential of phenformin. We report that phenformin (25–75 μM) decreased cell proliferation and impaired cell cycle progression in SKBR3 and 78617 breast cancer cells. Reduced tumor size after phenformin treatment (30 mg/kg/day) was demonstrated in an MMTV-ErbB2 transgenic mouse syngeneic tumor model. Phenformin also blocked epithelial-mesenchymal transition, decreased the invasive phenotype, and suppressed receptor tyrosine kinase signaling, including insulin receptor substrate 1 and IGF1R, in ErbB2-overexpressing breast cancer cells and mouse mammary tumor-derived tissues. Moreover, phenformin suppressed IGF1-stimulated proliferation, receptor tyrosine kinase signaling, and epithelial-mesenchymal transition markers in vitro. Together, our study implicates phenformin-mediated IGF1/IGF1R regulation as a potential anti-cancer mechanism and supports the development of phenformin and other biguanides as breast cancer therapeutics.

Aldehyde dehydrogenase inhibition combined with phenformin treatment reversed NSCLC through ATP depletion.

Among ALDH isoforms, ALDH1L1 in the folate pathway showed highly increased expression in non-small-cell lung cancer cells (NSCLC). Based on the basic mechanism of ALDH converting aldehyde to carboxylic acid with by-product NADH, we suggested that ALDH1L1 may contribute to ATP production using NADH through oxidative phosphorylation. ALDH1L1 knockdown reduced ATP production by up to 60% concomitantly with decrease of NADH in NSCLC. ALDH inhibitor, gossypol, also reduced ATP production in a dose dependent manner together with decrease of NADH level in NSCLC. A combination treatment of gossypol with phenformin, mitochondrial complex I inhibitor, synergized ATP depletion, which efficiently induced cell death. Pre-clinical xenograft model using human NSCLC demonstrated a remarkable therapeutic response to the combined treatment of gossypol and phenformin.

Abstract A53: Dual inhibition of mTOR in hepatocellular carcinoma: Autophagy friend or foe?

Abstract Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world and ranks second in cancer deaths. HCC is associated with chronic liver damage due to viral infection, excess alcohol, and most recently, non-alcoholic hepatitis (NASH), linked to obesity. Despite the recent success of the multikinase inhibitor sorafenib in HCC, for those patients with advanced stage disease the prognosis remains poor. A promising target in the treatment of HCC is mTOR, which is hyperactivated in 50% of HCCs. However, the FDA-approved mTOR-allosteric inhibitor, RAD001 has failed as a single agent in HCC, most likely due to its incomplete suppression of mTOR Complex 1 (mTORC1). Consistent with this finding we recently showed that RAD001 had minimal effects on the proliferation of HCC cells in culture, but when combined with BEZ235, a PI3K/mTOR-ATP site competitive inhibitor, synergistically blocked the growth of HCC tumor cells in culture and caused tumor regression in a mouse model approximating human HCC with bad prognosis (Elnakat et al., 2012). Further studies revealed the down regulation of a number of genes implicated in autophagy, which acts as a tumor suppressor in liver. This led to the finding that RAD001/BEZ235 causes the induction of autophagy in culture and induced mitophagy in tumors. Despite the finding that RAD001/BEZ235 caused tumor regression in vivo, it is argued that a small population of human CD133+ HCC stem-like cells (HSCs) is protected by mTOR inhibitors. In contrast, the biguanides, phenformin and metformin, also potent inhibitors of mTORC1, are reported to selectively suppress the proliferation of human cancer stem cells (CSC). These effects are argued to be through inhibition of mitochondrial oxidative phosphorylation, blocking ATP production, activating AMPK, and inducing autophagy. Given the ability of biguanides to inhibit mTORC1 and to selectively inhibit the proliferation of CSCs, we set out to determine its effects on tumor progression, using human HCC cell lines in a mouse orthotopic model, as compared to the RAD001/BEZ235. In parallel, we analyzed the effect of either drug treatment on whole cell energy consumption and autophagy. In our preliminary studies, similar to our results in the syngeneic mouse model, we found that orthotopic human HCC tumor progression is strongly impaired by RAD001/BEZ235, and also by phenformin as a single agent. RAD001/BEZ235 treated tumors show on-target effects as decreased phosphorylation of RPS6 and 4E-BP1, and lower proliferation indices. Similar on-target effects are observed after phenformin treatment. In vitro, we found that RAD001/BEZ235 inhibits mitochondrial respiration (25-40%), with mass decreasing (10-20%), but that mitochondria conserve their coupled vs uncoupled ATP oxygen consumption ratio, with no apparent compensatory shift towards glycolysis. In contrast, phenformin induces a greater decrease in mitochondrial respiration (80%) and a more rapid loss of mitochondrial mass (30-40%), with only 50% of mitochondrial oxygen consumption being coupled to ATP production, and a stronger induction of ROS. This strong mitochondrial impairment leads to higher glycolitic capacity, but is not sufficient to prevent cell death. Combining mTOR inhibition and mitochondrial complex 1 targeting results in even a greater decrease in mitochondrial function (>80%), with the same uncoupling and mass loss seen in phenformin treatment alone. Finally, using either a cargo-based or flux autophagic assay, implied, unexpectedly, that the benefits of phenformin in suppressing tumor progression are independent of autophagy, but consistent with mitochondrial dysfunction. Note: This abstract was not presented at the conference. Citation Format: Sonia Pereira Da Veiga, Carol Mercer, Ge Xuemei, Hala Elnakat Thomas, Sara Kozma, George Thomas. Dual inhibition of mTOR in hepatocellular carcinoma: Autophagy friend or foe? [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A53.

Genetic Disruption of Lactate/H+ Symporters (MCTs) and Their Subunit CD147/BASIGIN Sensitizes Glycolytic Tumor Cells to Phenformin

Rapidly growing glycolytic tumors require energy and intracellular pH (pHi) homeostasis through the activity of two major monocarboxylate transporters, MCT1 and the hypoxia-inducible MCT4, in intimate association with the glycoprotein CD147/BASIGIN (BSG). To further explore and validate the blockade of lactic acid export as an anticancer strategy, we disrupted, via zinc finger nucleases, MCT4 and BASIGIN genes in colon adenocarcinoma (LS174T) and glioblastoma (U87) human cell lines. First, we showed that homozygous loss of MCT4 dramatically sensitized cells to the MCT1 inhibitor AZD3965. Second, we demonstrated that knockout of BSG leads to a decrease in lactate transport activity of MCT1 and MCT4 by 10- and 6-fold, respectively. Consequently, cells accumulated an intracellular pool of lactic and pyruvic acids, magnified by the MCT1 inhibitor decreasing further pHi and glycolysis. As a result, we found that these glycolytic/MCT-deficient cells resumed growth by redirecting their metabolism toward OXPHOS. Third, we showed that in contrast with parental cells, BSG-null cells became highly sensitive to phenformin, an inhibitor of mitochondrial complex I. Phenformin addition to these MCT-disrupted cells in normoxic and hypoxic conditions induced a rapid drop in cellular ATP-inducing cell death by "metabolic catastrophe." Finally, xenograft analysis confirmed the deleterious tumor growth effect of MCT1/MCT4 ablation, an action enhanced by phenformin treatment. Collectively, these findings highlight that inhibition of the MCT/BSG complexes alone or in combination with phenformin provides an acute anticancer strategy to target highly glycolytic tumors. This genetic approach validates the anticancer potential of the MCT1 and MCT4 inhibitors in current development.

Abstract 2102: Phenformin down-regulates mammary aromatase expression via the AMPK pathway in humanized aromatase expressing ERBB2 mice

Abstract Local mammary estrogen production may be vital for breast cancer development because about 70% of breast cancers occur in postmenopausal women. Aromatase is a key enzyme essential for estrogen biosynthesis, and aromatase inhibitors (AIs) are the most effective endocrine treatment for estrogen-responsive breast cancer in postmenopausal women. However, they are also accompanied a number of severe side-effects on bone and joints. Many researchers are currently focusing on developing the novel mammary-specific AIs to prevent or treat breast cancer to minimize the side effects of treatment. Phenformin, an antidiabetic drug, lowers serum glucose levels via activating the AMP-activated protein kinase (AMPK) pathway and reduces tumor burden and prolongs survival in non-small cell lung cancers. In vitro studies show that activated AMPK inhibits human mammary adipose tissue aromatase expression via suppression of binding of cAMP-responsive element binding protein (CREB) to aromatase promoters I.3/II, leading to decreased estrogen formation. We hypothesized that phenformin will be an effective adipose-specific AI for prevention and treatment of breast cancer. However, one of the major obstacles to define in vivo effects of phenformin on mammary aromatase expression is the lack of suitable mouse models because aromatase expression, present in the human breast, is absent in the mouse mammary gland. We developed a unique humanized aromatase mouse model (Aromhum) to mimic the human aromatase expression pattern and physiology in the mouse. We observed that tumorigenesis was accelerated in the mice overexpressing ERBB2 that were crossed with Aromhum mice (AE mice). Phenformin treatment (i.p., 70 mg/kg /day for 14 days) significantly decreased blood glucose levels by 37% in AE mice as compared to vehicle treatment. Phosphorylation of CREB, a downstream effector of the AMPK pathway, which mediates aromatase expression, was significantly decreased. Furthermore, after phenformin administration, mammary aromatase mRNA expression in AE mice was significantly decreased by 58% and estrogen response genes (SUSD3 and cyclin D1) were also reduced by about 65%. Real-time PCR showed that mammary AMPKα1 mRNA expression is 10-fold higher compared with AMPKα2 mRNA. Moreover, phenformin treatment significantly decreased AMPKα1 mRNA levels by 53% but not AMPKα2 mRNA expression in AE mice. Interestingly, daily oral phenformin treatment for 7 days significantly reduced mammary tumor volume of AE mice by 76%. In conclusion, phenformin decreased mammary aromatase expression and possible estrogen production, and reduced tumorigenesis via upregulation of the AMPK pathway in breast tissue of AE mice. This study provides justification for future testing of the adipose-specific preventive and therapeutic potential of the metabolic drug phenformin in postmenopausal breast cancer with minimal side-effects. Citation Format: Hong Zhao, Robert T. Chatterton, Timothy Prajka, Lin Li, Serdar E. Bulun. Phenformin down-regulates mammary aromatase expression via the AMPK pathway in humanized aromatase expressing ERBB2 mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2102. doi:10.1158/1538-7445.AM2014-2102

Abstract 1216: Effects of phenformin in LKB1/KRAS mutated non-small cell lung cancer patient derived xenograft

Abstract Metformin is an oral antidiabetic drug of the biguanide class. Several epidemiological and case-control studies reported that diabetics using metformin may have lower cancer risk in comparison to those using other antidiabetic medications. Recent studies in experimental models suggest that another biguanide, phenformin, has higher anticancer activity compared with metformin. The aim of this study was to evaluate the effects of metformin and phenformin in Non-Small Cell Lung Cancer (NSCLC) models. In particular, the effects of these compounds in LKB1 or LKB1/KRAS mutated compared to wild type models have been investigated. NSCLC cell lines (A549, H460, H1299, LT73) have been treated with either metformin or phenformin to evaluate in vitro dose/response depending on the different mutational status of KRAS and LKB1 genes. The effects of the two biguanide have been investigated also in vivo models, exploiting the panel of Patient Derived Xenografts (PDXs) directly generated from NSCLC patients in our Institute. In vitro experiments indicated that cell lines with already impaired metabolism (LKB1 and/or KRAS mutated) were more sensitive to phenformin treatment. Interestingly, treatments of these cell lines with r phenformin resulted in a 50% decrease in the amount of CD133+ Tumor Initiating Cells (TIC). Moreover, high doses of phenformin (LD75) did not result in the increase of TIC previously observed after Cisplatin treatment. In vivo, early treatments (Tumor Volume = 50-100mm3) of an LKB1 mutated PDX model resulted in a significant reduction of tumor volume (300 ± 50 mm3 in phenformin treated vs 750 ± 100 mm3 in vehicle treated tumors) already after three weeks of treatment (100mg/Kg/day). Importantly, no regrowth was observed in responding tumors even after the end of treatments. No effects were observed in LKB1 wild type PDX treated with phenformin. Taken together, these preliminary data indicate a selective action of phenformin on LKB1 mutated lung adenocarcinoma models in vitro and in vivo. Interestingly, this compound seems to be more active in TIC than normal chemotherapeutic drugs (i.e. cisplatin). Effects of biguanide on TIC could be the cause of the lack of tumor regrowth after treatment, thus making these compounds potentially useful in counteracting disease recurrence and/or metastasis formation. Further investigations are needed to elucidate the different effects of metformin and phenformin in double mutated (LKB1 and KRAS) PDXs models. Citation Format: Massimo Moro, Luca Roz, Roberto Caserini, Ugo Pastorino, Gabriella Sozzi. Effects of phenformin in LKB1/KRAS mutated non-small cell lung cancer patient derived xenograft. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1216. doi:10.1158/1538-7445.AM2014-1216

ER Stress‐independent Noncanonical UPR Activation under Phenformin Treatment

Activation of unfolded protein response (UPR) is associated with disruption of homeostasis in the endoplasmic reticulum and has been implicated in the pathogenesis of many human diseases including obesity and type‐2 diabetes. However, whether metabolic pathways can directly regulate UPR independently of misfolded protein accumulation and ER stress remains unclear. In a test of various metabolic drugs, we show that activation of UPR sensors can be uncoupled from the ER signals. First, treatment with the antidiabetic drug phenformin activates UPR sensors IRE1α and PERK even in the absence of protein synthesis. Second, phenformin treatment strongly activates a PERK mutant only residing in the cytosol. Mechanistically, kinase activities of both IRE1α and PERK are required for the phenformin effect. Moreover, AMPK activation is required but not sufficient to initiate IRE1α and PERK pathways, suggesting the involvement of additional factor(s) under phenformin treatment. Interestingly, activation of the IRE1α, but not the PERK pathway, may partially mediate the cytotoxic effect of phenformin. Together, these findings demonstrate the existence of a noncanonical UPR where activating signals are derived from the cytosol, adding another layer of complexity to the UPR activation under metabolic stress.

Targeting tumor metabolism: biguanides as anti-neoplastic agents

e14592 Background: During the process of malignant transformation, the tumor cell adopts a new form of metabolism, characterized by aerobic glycolysis and altered TCA cycle flux, that enable it to meet the energetic and biosynthetic demands of proliferation. This shift to anabolic metabolism results from oncogene-driven activation of signaling pathways, and therefore represents a potential therapeutic target that lies downstream of these genetic events. Methods: We have characterized the proliferative and metabolic effects of phenformin, a member of the biguanide family of compounds used in the treatment of diabetes, on the bcr-abl expressing K562 erythroleukemia cell line, and compared the resulting phenotype to those of imatinib and rapamycin, two targeted agents used in the treatment of malignant disease. Results: Phenformin induced the most profound growth inhibition of K562 cells, in a manner distinct from the targeted agents. Phenformin treatment eliminated the mitochondrial contribution to anabolic metabolism, through inhibition of Complex I of the Electron Transport Chain, as demonstrated by reduced oxygen consumption and intracellular ATP levels. Glutamine metabolism was also inhibited, consistent with TCA cycle inhibition as a secondary effect. The phenformin-induced mitochondrial dysfunction made K562 cells dependent upon glycolysis for survival. In contrast, the growth arrest resulting from imatinib treatment was associated with a complete reversion from the anabolic phenotype, as demonstrated by dramatically decreased glucose and glutamine consumption. Rapamycin, a poor inhibitor of K562 proliferation, decreased glycolysis but left mitochondrial function intact. Conclusions: Our results confirm the importance of metabolism to the proliferation of malignant cells, thereby validating anabolic metabolism's potential for therapeutic intervention. The direct inhibition of tumor cell metabolism by phenformin warrants further clinical study as a promising new approach to cancer therapy. No significant financial relationships to disclose.

Comparison of gene expression changes induced by biguanides in db/db mice liver

Large-scale clinical studies have shown that the biguanide drug metformin, widely used for type 2 diabetes, to be very safe. By contrast, another biguanide, phenformin, has been withdrawn from major markets because of a high incidence of serious adverse effects. The difference in mode of action between the two biguanides remains unclear. To gain insight into the different modes of action of the two drugs, we performed global gene expression profiling using the livers of obese diabetic db/db mice after a single administration of phenformin or metformin at levels sufficient to cause a significant reduction in blood glucose level. Metformin induced modest expression changes, including G6pc in the liver as previously reported. By contrast, phenformin caused changes in expression level of many additional genes. We used a knowledge-based bioinformatic analysis to study the effects of phenformin. Differentially expressed genes identified in this study constitute a large gene network, which may be related to cell death, inflammation or wound response. Our results suggest that the two biguanides show a similar hypoglycemic effect in db/db mice, but phenformin induces a greater stress on the liver even a short time after a single administration. These findings provide a novel insight into the cause of the relatively high occurrence of serious adverse effect after phenformin treatment.