Metformin inhibits thyroid cancer cell growth. We sought to determine if variable glucose concentrations in medium alter the anti-cancer efficacy of metformin. Thyroid cancer cells (FTC133 and BCPAP) were cultured in high-glucose (20 mM) and low-glucose (5 mM) medium before treatment with metformin. Cell viability and apoptosis assays were performed. Expression of glycolytic genes was examined by real-time PCR, western blot, and immunostaining. Metformin inhibited cellular proliferation in high-glucose medium and induced cell death in low-glucose medium. In low-, but not in high-glucose medium, metformin induced endoplasmic reticulum stress, autophagy, and oncosis. At micromolar concentrations, metformin induced phosphorylation of AMP-activated protein kinase and blocked p-pS6 in low-glucose medium. Metformin increased the rate of glucose consumption from the medium and prompted medium acidification. Medium supplementation with glucose reversed metformin-inducible morphological changes. Treatment with an inhibitor of glycolysis (2-deoxy-d-glucose (2-DG)) increased thyroid cancer cell sensitivity to metformin. The combination of 2-DG with metformin led to cell death. Thyroid cancer cell lines were characterized by over-expression of glycolytic genes, and metformin decreased the protein level of pyruvate kinase muscle 2 (PKM2). PKM2 expression was detected in recurrent thyroid cancer tissue samples. In conclusion, we have demonstrated that the glucose concentration in the cellular milieu is a factor modulating metformin's anti-cancer activity. These data suggest that the combination of metformin with inhibitors of glycolysis could represent a new strategy for the treatment of thyroid cancer.
Athanasios Bikas, Kirk Jensen, Aneeta Patel, John Costello Jr, Dennis McDaniel, Joanna Klubo-Gwiezdzinska, Olexander Larin, Victoria Hoperia, Kenneth D Burman, Lisa Boyle, Leonard Wartofsky and Vasyl Vasko
Barbara Salani, Alberto Del Rio, Cecilia Marini, Gianmario Sambuceti, Renzo Cordera and Davide Maggi
Metformin is the first-line treatment for type 2 diabetes. Results from several clinical studies have indicated that type 2 diabetic patients treated with metformin might have a lower cancer risk. One of the primary metabolic changes observed in malignant cell transformation is an increased catabolic glucose metabolism. In this context, once it has entered the cell through organic cation transporters, metformin decreases mitochondrial respiration chain activity and ATP production that, in turn, activates AMP-activated protein kinase, which regulates energy homeostasis. In addition, metformin reduces cellular energy availability and glucose entrapment by inhibiting hexokinase-II, which catalyses the glucose phosphorylation reaction. In this review, we discuss recent findings on molecular mechanisms that sustain the anticancer effect of metformin through regulation of glucose metabolism. In particular, we have focused on the emerging action of metformin on glycolysis in normal and cancer cells, with a drug discovery perspective.
Joanna Klubo-Gwiezdzinska, Kirk Jensen, John Costello, Aneeta Patel, Victoria Hoperia, Andrew Bauer, Kenneth D Burman, Leonard Wartofsky and Vasyl Vasko
Medullary thyroid cancer (MTC) is associated with activation of mammalian target of rapamycin (mTOR) signaling pathways. Recent studies showed that the antidiabetic agent metformin decreases proliferation of cancer cells through 5′-AMP-activated protein kinase (AMPK)-dependent inhibition of mTOR. In the current study, we assessed the effect of metformin on MTC cells. For this purpose, we determined growth, viability, migration, and resistance to anoikis assays using two MTC-derived cell lines (TT and MZ-CRC-1). Expressions of molecular targets of metformin were examined in MTC cell lines and in 14 human MTC tissue samples. We found that metformin inhibited growth and decreased expression of cyclin D1 in MTC cells. Treatment with metformin was associated with inhibition of mTOR/p70S6K/pS6 signaling and downregulation of pERK in both TT and MZ-CRC-1 cells. Metformin had no significant effects on pAKT in the cell lines examined. Metformin-inducible AMPK activation was noted only in TT cells. Treatment with AMPK inhibitor (compound C) or AMPK silencing did not prevent growth inhibitory effects of metformin in TT cells. Metformin had no effect on MTC cell migration but reduced the ability of cells to form multicellular spheroids in nonadherent conditions. Immunostaining of human MTC showed over-expression of cyclin D1 in all tumors compared with corresponding normal tissue. Activation of mTOR/p70S6K was detected in 8/14 (57.1%) examined tumors. Together, these findings indicate that growth inhibitory effects in MTC cells are associated with downregulation of both mTOR/6SK and pERK signaling pathways. Expression of metformin's molecular targets in human MTC cells suggests its potential utility for the treatment of MTC in patients.
Sunmi Park, Mark C Willingham, Jun Qi and Sheue-Yann Cheng
Compelling epidemiological evidence shows a strong positive correlation of obesity with thyroid cancer. In vivo studies have provided molecular evidence that high-fat-diet-induced obesity promotes thyroid cancer progression by aberrantly activating leptin-JAK2-STAT3 signaling in a mouse model of thyroid cancer (Thrb PV/PV Pten +/ − mice). The Thrb PV/PV Pten +/ − mouse expresses a dominantly negative thyroid hormone receptor β (denoted as PV) and a deletion of one single allele of the Pten gene. The Thrb PV/PV Pten +/ − mouse spontaneously develops follicular thyroid cancer, which allows its use as a preclinical mouse model to test potential therapeutics. We recently showed that inhibition of STAT3 activity by a specific inhibitor markedly delays thyroid cancer progression in high-fat-diet-induced obese Thrb PV/PV Pten +/ − mice (HFD-Thrb PV/PV Pten +/ − mice). Further, metformin, a widely used antidiabetic drug, blocks invasion and metastasis, but not thyroid tumor growth in HFD-Thrb PV/PV Pten +/ − mice. To improve efficacy in reducing thyroid tumor growth, we treated HFD-Thrb PV/PV Pten +/ − with JQ1, a potent inhibitor of the activity of bromodomain and extraterminal domain (BET) and with metformin. We found that the combined treatment synergistically suppressed thyroid tumor growth by attenuating STAT3 and ERK signaling, resulting in decreased anti-apoptotic key regulators such as Mcl-1, Bcl-2 and survivin and increased pro-apoptotic regulators such as Bim, BAD and cleave caspase 3. Furthermore, combined treatment of JQ1 and metformin reduced cMyc protein levels to suppress vascular invasion, anaplasia and lung metastasis. These findings indicate that combined treatment is more effective than metformin alone and suggest a novel treatment modality for obesity-activated thyroid cancer.
Carolyn Algire, Mahvash Zakikhani, Marie-Jose Blouin, Jian Hua Shuai and Michael Pollak
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 0.5×106 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.
Paolo Vigneri, Francesco Frasca, Laura Sciacca, Giuseppe Pandini and Riccardo Vigneri
Diabetes and cancer are two heterogeneous, multifactorial, severe, and chronic diseases. Because of their frequency, reciprocal influences – even minor influences – may have a major impact. Epidemiological studies clearly indicate that the risk of several types of cancer (including pancreas, liver, breast, colorectal, urinary tract, and female reproductive organs) is increased in diabetic patients. Mortality is also moderately increased. Several confounding factors, having general or site-specific relevance, make it difficult to accurately assess cancer risk in diabetic patients. These factors include diabetes duration, varying levels of metabolic control, different drugs used for therapy, and the possible presence of chronic complications. Hyperinsulinemia most likely favors cancer in diabetic patients as insulin is a growth factor with pre-eminent metabolic but also mitogenic effects, and its action in malignant cells is favored by mechanisms acting at both the receptor and post-receptor level. Obesity, hyperglycemia, and increased oxidative stress may also contribute to increased cancer risk in diabetes. While anti-diabetic drugs have a minor influence on cancer risk (except perhaps the biguanide metformin that apparently reduces the risk), drugs used to treat cancer may either cause diabetes or worsen a pre-existing diabetes. In addition to the well-known diabetogenic effect of glucocorticoids and anti-androgens, an increasing number of targeted anti-cancer molecules may interfere with glucose metabolism acting at different levels on the signaling substrates shared by IGF-I and insulin receptors. In conclusion, diabetes and cancer have a complex relationship that requires more clinical attention and better-designed studies.
Yevgeniya Kushchayeva, Kirk Jensen, Kenneth D Burman and Vasyl Vasko
Repositioning of established non-cancer pharmacotherapeutic agents with well-known activity and side-effect profiles is a promising avenue for the development of new treatment modalities for multiple cancer types. We have analyzed some of the medications with mechanism of action that may have relevance to thyroid cancer (TC). Experimental in vitro and in vivo evidences, as well as results of clinical studies, have indicated that molecular targets for medications currently available for the treatment of mood disorders, sexually transmitted diseases, metabolic disorders, and diabetes may be active and relevant in TC. For instance, the derivatives of cannabis and an anti-diabetic agent, metformin, both are able to inhibit ERK, which is commonly activated in TC cells. We present here several examples of well-known medications that have the potential to become new therapeutics for patients with TC. Repositioning of established medications for the treatment of TC could broaden the scope of current therapeutic strategies. These diverse treatment choices could allow physicians to provide an individualized approach to optimize treatment for patients with TC.
K M Biernacka, R A Persad, A Bahl, D Gillatt, J M P Holly and C M Perks
The incidence of many common cancers varies between different populations and appears to be affected by a Western lifestyle. Highly proliferative malignant cells require sufficient levels of nutrients for their anabolic activity. Therefore, targeting genes and pathways involved in metabolic pathways could yield future therapeutics. A common pathway implicated in energetic and nutritional requirements of a cell is the LKB1/AMPK pathway. Metformin is a widely studied anti-diabetic drug, which improves glycaemia in patients with type 2 diabetes by targeting this pathway. We investigated the effect of metformin on prostate cancer cell lines and evaluated its mechanism of action using DU145, LNCaP, PC3 and VCaP prostate cancer cell lines. Trypan blue dye-exclusion assay was used to assess levels of cell death. Western immunoblotting was used to determine the abundance of proteins. Insulin-like growth factor-binding protein-2 (IGFBP-2) and AMPK genes were silenced using siRNA. Effects on cell morphology were visualised using microscopy. IGFBP-2 gene expression was assessed using real-time RT-PCR. With DU145 and LNCaP cells metformin alone induced cell death, but this was reduced in hyperglycaemic conditions. Hyperglycaemia also reduced the sensitivity to Docetaxel, but this was countered by co-treatment with metformin. LKB1 was required for the activation of AMPK but was not essential to mediate the induction of cell death. An alternative pathway by which metformin exerted its action was through downregulation of IGFBP-2 in DU145 and LNCaP cells, independently of AMPK. This finding could have important implications in relation to therapeutic strategies in prostate cancer patients presenting with diabetes.
Carolyn Algire, Lilian Amrein, Mahvash Zakikhani, Lawrence Panasci and Michael Pollak
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 5×105 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.
Dara Hope Cohen and Derek LeRoith
Epidemiological studies suggest a positive association between obesity and type 2 diabetes mellitus (T2D) with the risk of cancer and cancer-related mortality. Insulin resistance, hyperinsulinemia, increased levels of IGF, elevated levels of steroid and peptide hormones, and inflammatory markers appear to play a role in the connection between these different diseases. Medications, such as metformin and exogenous insulin, used to treat T2D may affect the risk of cancer and cancer-related mortality. Newer therapies targeting the insulin and IGF1 systems are being developed for use in cancer therapy.