Carolyn Algire

Metformin blocks the stimulative effect of a high-energy diet on colon carcinoma growth in vivo and is associated with reduced expression of fatty acid synthase

The molecular mechanisms responsible for the association of obesity with adverse colon cancer outcomes are poorly understood. We investigated the effects of a high-energy diet on growth of an in vivo colon cancer model. Seventeen days following the injection of 5x10(5) MC38 colon carcinoma cells, tumors from mice on the high-energy diet were approximately twice the volume of those of mice on the control diet. These findings were correlated with the observation that the high-energy diet led to elevated insulin levels, phosphorylated AKT, and increased expression of fatty acid synthase (FASN) by the tumor cells. Metformin, an antidiabetic drug, leads to the activation of AMPK and is currently under investigation for its antineoplastic activity. We observed that metformin blocked the effect of the high-energy diet on tumor growth, reduced insulin levels, and attenuated the effect of diet on phosphorylation of AKT and expression of FASN. Furthermore, the administration of metformin led to the activation of AMPK, the inhibitory phosphorylation of acetyl-CoA carboxylase, the upregulation of BNIP3 and increased apoptosis as estimated by poly (ADP-ribose) polymerase (PARP) cleavage. Prior work showed that activating mutations of PI3K are associated with increased AKT activation and adverse outcome in colon cancer; our results demonstrate that the aggressive tumor behavior associated with a high-energy diet has similar effects on this signaling pathway. Furthermore, metformin is demonstrated to reverse the effects of the high-energy diet, thus suggesting a potential role for this agent in the management of a metabolically defined subset of colon cancers.

Metformin attenuates the stimulatory effect of a high-energy diet on in vivo LLC1 carcinoma growth

We investigated the effects of metformin on the growth of lewis lung LLC1 carcinoma in C57BL/6J mice provided with either a control diet or a high-energy diet, previously reported to lead to weight gain and systemic insulin resistance with hyperinsulinemia. Forty-eight male mice were randomized into four groups: control diet, control diet+metformin, high-energy diet, or high-energy diet+metformin. Following 8 weeks on the experimental diets, selected groups received metformin in their drinking water. Three weeks following the start of metformin treatment, mice were injected with 0.5x10(6) LLC1 cells and tumor growth was measured for 17 days. By day 17, tumors of mice on the high-energy diet were nearly twice the volume of those of mice on the control diet. This effect of diet on tumor growth was significantly attenuated by metformin, but metformin had no effect on tumor growth of the mice on the control diet. Metformin attenuated the increased insulin receptor activation associated with the high-energy diet and also led to increased phosphorylation of AMP kinase, two actions that would be expected to decrease neoplastic proliferation. These experimental results are consistent with prior hypothesis-generating epidemiological studies that suggest that metformin may reduce cancer risk and improve cancer prognosis. Finally, these results contribute to the rationale for evaluation of the anti-neoplastic activity of metformin in hyperinsulinemic cancer patients.

Metformin Reduces Endogenous Reactive Oxygen Species and Associated DNA Damage

Pharmacoepidemiologic studies provide evidence that use of metformin, a drug commonly prescribed for type II diabetes, is associated with a substantial reduction in cancer risk. Experimental models show that metformin inhibits the growth of certain neoplasms by cell autonomous mechanisms such as activation of AMP kinase with secondary inhibition of protein synthesis or by an indirect mechanism involving reduction in gluconeogenesis leading to a decline in insulin levels and reduced proliferation of insulin-responsive cancers. Here, we show that metformin attenuates paraquat-induced elevations in reactive oxygen species (ROS), and related DNA damage and mutations, but has no effect on similar changes induced by H202, indicating a reduction in endogenous ROS production. Importantly, metformin also inhibited Ras-induced ROS production and DNA damage. Our results reveal previously unrecognized inhibitory effects of metformin on ROS production and somatic cell mutation, providing a novel mechanism for the reduction in cancer risk reported to be associated with exposure to this drug. Cancer Prev Res; 5(4); 536–43. ©2012 AACR.

Are Metformin Doses Used in Murine Cancer Models Clinically Relevant

An important mechanism of action of metformin as an antidiabetic drug involves inhibition of hepatic gluconeogenesis, but the molecular basis for this is controversial. A recent perspective (He and Wondisford, 2015) argued that direct action of the drug on AMPK is of key importance and occurs at clinically relevant concentrations of ∼70 μM. However, that review cited conflicting studies, which concluded either that AMPK-independent mechanisms are important in metformin action, or that AMPK activation plays a role but is secondary to energetic stress resulting from inhibition of oxidative phosphorylation by the drug.

Metformin abolishes increased tumor 18F-2-fluoro-2-deoxy-D-glucose uptake associated with a high energy diet

Insulin regulates glucose uptake by normal tissues. Although there is evidence that certain cancers are growth-stimulated by insulin, the possibility that insulin influences tumor glucose uptake as assessed by 18F-2-Fluoro-2-Deoxy-d-Glucose Positron Emission Tomography (FDG-PET) has not been studied in detail. We present a model of diet-induced hyperinsulinemia associated with increased insulin receptor activation in neoplastic tissue and with increased tumor FDG-PET image intensity. Metformin abolished the diet-induced increases in serum insulin level, tumor insulin receptor activation and tumor FDG uptake associated with the high energy diet but had no effect on these measurements in mice on a control diet. These findings provide the first functional imaging correlate of the well-known adverse effect of caloric excess on cancer outcome. They demonstrate that, for a subset of neoplasms, diet and insulin are variables that affect tumor FDG uptake and have implications for design of clinical trials of metformin as an antineoplastic agent.

Effects of castration on insulin levels and glucose tolerance in the mouse differ from those in man

Plasma insulin concentration is increased in prostate cancer patients during androgen deprivation therapy (ADT) and hyperinsulinemia has been associated with aggressive prostate cancer behavior. To investigate the possible role of castration‐induced hyperinsulinemia as a mechanism that may attenuate the beneficial effects of ADT in patients with prostate cancer, a murine model would be useful. We therefore investigated long‐term metabolic effects of castration in several mouse models.

Abstract 223: Comparison of human-specific versus cross-reactive Complex I inhibitor for in vivo tumor efficacy

Mitochondria are both key regulators of energy supply and apoptotic cell death. The mitochondrial electron transport chain (ETC) consists of four enzyme complexes that transfer electrons from NADH to oxygen. During electron transfer, the ETC (Complex I to IV) pumps protons into the inter-membrane space, generating a gradient across the inner mitochondrial membrane that is used by Complex V to drive ATP synthesis. Recent publications have shown that tumor cells harboring specific mutations (LKB1, mIDH and others) are more sensitive to Complex I inhibition, compared to cells that do not have these mutations. We have identified an optimized human/mouse cross-reactive Complex I inhibitor that allows profiling of Complex I inhibitors in pharmacological models. We have pursued different approaches based on the literature, an unbiased screen and in-house results generated with the human-specific Complex I inhibitor BAY 872243 to identify sensitive in vivo tumor models. However, using the cross-reactive Complex I inhibitor we were unable to identify sensitive models apart from weakly sensitive LKB1-deficient tumors (A549, G361) when animals were treated at maximum tolerated dose (MTD). In addition, all approaches for combination therapy failed to improve efficacy in vivo. Direct comparison of human-specific Complex I inhibitor BAY 87-2243 and cross-reactive inhibitor BAY179 in a sensitive LKB1-deficient melanoma model, G361, demonstrated that inhibition of Complex I specifically in the tumor is a valid approach as it results in tumor growth inhibition of ∼50%. However, cross-reactive compounds do not reach exposures at MTD to generate comparable effects. Citation Format: Sven Christian, Carolyn Algire, Wolfgang Schwede, Jeffrey S. Mowat, Alexander Ehrmann, Stephan Menz, Marcus Bauser, Andrea Haegebarth. Comparison of human-specific versus cross-reactive Complex I inhibitor for in vivo tumor efficacy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 223.

Abstract 317: 3D high-content screening for the identification of compounds that target cells in dormant tumor spheroid regions

Cancer cells in poorly vascularized tumor regions need to adapt to an unfavorable metabolic microenvironment. As distance from supplying blood vessels increases, oxygen and nutrient concentrations decrease and cancer cells react by stopping cell cycle progression and becoming dormant. As cytostatic drugs mainly target proliferating cells, cancer cell dormancy is considered as a major resistance mechanism to this class of anti-cancer drugs. Therefore, substances that target cancer cells in poorly vascularized tumor regions have the potential to enhance cytostatic-based chemotherapy of solid tumors. With three-dimensional growth conditions, multicellular tumor spheroids (MCTS) reproduce several parameters of the tumor microenvironment, including oxygen and nutrient gradients as well as the development of dormant tumor regions and therefore represent a promising model system for discovery of phenotypes specifically expressed in stressed, hypoxic and nutrient-depleted conditions. We here show the setup of a 3D cell culture compatible high-content screening system for the identification of substances that specifically target cells in inner MCTS core regions, while cells in outer MCTS regions or in 2D cell culture remain unaffected. This setup was used in a pilot screen to identify tool compounds and subsequently in a larger screening approach covering approximately 500000 compounds from the Bayer substance library. The resulting hit list could be subdivided into two different categories based on the identification of two distinct phenotypes. Mode of action studies identified the first category as inhibitors of the mitochondrial respiratory chain, being critically dependent on extracellular glucose concentrations. On the other hand the second hit category could be identified as modifiers of fatty acid metabolism. The identified hits offer the opportunity for further characterization in vivo, particularly in combination with drugs targeting the well perfused, proliferating tumor areas, such as chemotherapy. Taken together the data presented here show for the first time a high-content based screening setup on 3D tumor spheroids for the identification of substances that specifically induce cell death in inner tumor spheroid core regions. This validates the approach to use 3D cell culture screening systems to identify substances that would not be detectable by 2D based screening in otherwise similar culture conditions. Citation Format: Carsten Wenzel, Sven Christian, Carolyn Algire, Wolfgang Schwede, Roland Neuhaus, Judith Guenther, Ningshu Liu, Sebastian Raese, Karsten Parczyk, Stefan Prechtl, Patrick Steigemann. 3D high-content screening for the identification of compounds that target cells in dormant tumor spheroid regions. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 317. doi:10.1158/1538-7445.AM2015-317

Abstract 1126: Differential effects of metformin and phenformin vs. other complex 1 inhibitors in vitro and in vivo

Biguanides, such as metformin and phenformin, are currently under investigation for their potential use as anti-neoplastic therapy. Recent publications suggest that both metformin and phenformin exert effects through inhibition of Complex 1 in the electron transport chain. We investigated the effects of metformin and phenformin compared to rotenone, in vitro, and known Complex 1 inhibitor BAY 872243, in vitro and in vivo. As expected, rotenone and BAY 872243 showed strong inhibition of Complex I in cell-based and enzymatic assays with IC50 values in the nanomolar range. The high affinity binding to Complex I was also reflected by induction of cellular reactive oxygen species (ROS) with EC50 values in the nanomolar range. In contrast, the biguanides neither inhibited Complex I in cell-based and biochemical assays, nor led to an induction of ROS at concentrations up to 300 μM. In vivo exposure analysis shows that at the maximal tolerated dose (100 mg/kg QD i.p for phenformin and 350 mg/kg i.p QD for metformin), neither metformin nor phenformin had plasma exposure levels over the IC50 for proliferation in vitro. Recent reports have suggested that biguanides, via the inhibition of Complex 1 and subsequent reduction in oxygen consumption, can be used to re-oxygenate tumor areas prior to radiation therapy. Pimonidazole staining demonstrated that metformin and phenformin effectively eliminated hypoxic regions in NCI-H460 xenografts in a time course dependent manner that reflected the exposure. In contrast to the biguanides, BAY 87-2243 effectively eliminated hypoxic regions up to 24 hours post compound administration. Finally, both phenformin and metformin had minimal effects on inhibition of tumor growth, even in LKB1-deleted xenografts which have been reported to be especially sensitive to biguanides. In conclusion, our in vitro experiments on the mode of action of biguanides raise questions as whether the in vivo effects on hypoxic tumor regions are related to direct inhibition of Complex I. Citation Format: Carolyn Algire, Alexander Ehrmann, Sven Christian, Roland Neuhaus, Stephan Menz, Wolfgang Schwede, Michael Haerter, Andrea Haegebarth. Differential effects of metformin and phenformin vs. other complex 1 inhibitors in vitro and in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1126. doi:10.1158/1538-7445.AM2015-1126

Abstract LB-169: Metformin reduces somatic cell mutation rate by inhibiting mitochondrial ROS production: relevance to cancer prevention

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Pharmacoepidemiological studies provide evidence that use of metformin, a drug commonly prescribed for type II diabetes, is associated with a substantial reduction in cancer risk. Experimental models show that metformin inhibits the growth of certain neoplasms by cell autonomous mechanisms such as activation of AMP kinase with secondary inhibition of protein synthesis, or by an indirect mechanism involving reduction in gluconeogenesis leading to a decline in insulin levels and reduced proliferation of insulin-responsive cancers. Here we show that metformin attenuates paraquat-induced elevations in reactive oxygen species (ROS), DNA damage and mutations, but has no effect on similar changes induced by H202, indicating a reduction in endogenous ROS production. Metformin also inhibited Ras-induced ROS production and DNA damage. Our results reveal previously unrecognized inhibitory effects of metformin on ROS production and somatic cell mutation, providing a novel mechanism for the reduction in cancer risk associated with exposure to this drug. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-169. doi:1538-7445.AM2012-LB-169