Frédéric Bost

The antidiabetic drug metformin exerts an antitumoral effect in vitro and in vivo through a decrease of cyclin D1 level

Metformin is a widely used antidiabetic agent, which regulates glucose homeostasis through inhibition of liver glucose production and an increase in muscle glucose uptake. Recent studies suggest that metformin may reduce the risk of cancer, but its mode of action in cancer remains not elucidated. We investigated the effect of metformin on human prostate cancer cell proliferation in vitro and in vivo. Metformin inhibited the proliferation of DU145, PC-3 and LNCaP cancer cells with a 50% decrease of cell viability and had a modest effect on normal prostate epithelial cell line P69. Metformin did not induce apoptosis but blocked cell cycle in G0/G1. This blockade was accompanied by a strong decrease of cyclin D1 protein level, pRb phosphorylation and an increase in p27kip protein expression. Metformin activated the AMP kinase pathway, a fuel sensor signaling pathway. However, inhibition of the AMPK pathway using siRNA against the two catalytic subunits of AMPK did not prevent the antiproliferative effect of metformin in prostate cancer cells. Importantly, oral and intraperitoneal treatment with metformin led to a 50 and 35% reduction of tumor growth, respectively, in mice bearing xenografts of LNCaP. Similar, to the in vitro study, metformin led to a strong reduction of cyclin D1 protein level in tumors providing evidence for a mechanism that may contribute to the antineoplastic effects of metformin suggested by recent epidemiological studies.

Metformin, independent of AMPK, induces mTOR inhibition and cell-cycle arrest through REDD1

Metformin is a widely prescribed antidiabetic drug associated with a reduced risk of cancer. Many studies show that metformin inhibits cancer cell viability through the inhibition of mTOR. We recently showed that antiproliferative action of metformin in prostate cancer cell lines is not mediated by AMP-activated protein kinase (AMPK). We identified REDD1 (also known as DDIT4 and RTP801), a negative regulator of mTOR, as a new molecular target of metformin. We show that metformin increases REDD1 expression in a p53-dependent manner. REDD1 invalidation, using siRNA or REDD1(-/-) cells, abrogates metformin inhibition of mTOR. Importantly, inhibition of REDD1 reverses metformin-induced cell-cycle arrest and significantly protects from the deleterious effects of metformin on cell transformation. Finally, we show the contribution of p53 in mediating metformin action in prostate cancer cells. These results highlight the p53/REDD1 axis as a new molecular target in anticancer therapy in response to metformin treatment.

Metformin in Cancer Therapy: A New Perspective for an Old Antidiabetic Drug?

Metformin is the most widely used antidiabetic drug in the world, and there is increasing evidence of a potential efficacy of this agent as an anticancer drug. First, epidemiological studies show a decrease in cancer incidence in metformin-treated patients. Second, metformin decreases insulin resistance and indirectly reduces insulin level, a beneficial effect because insulin promotes cancer cell growth. Third, several reports outline a direct inhibitory effect of metformin on cancer cell growth and an antitumoral action. Finally, metformin activates the AMP activated protein kinase (AMPK) pathway, a major sensor of the energetic status of the cell, which has been proposed as a promising therapeutic target in cancer. Mol Cancer Ther; 9(5); 1092–99. ©2010 AACR.

Targeting Cancer Cell Metabolism: The Combination of Metformin and 2-Deoxyglucose Induces p53-Dependent Apoptosis in Prostate Cancer Cells

Targeting cancer cell metabolism is a new promising strategy to fight cancer. Metformin, a widely used antidiabetic agent, exerts antitumoral and antiproliferative action. In this study, the addition of metformin to 2-deoxyglucose (2DG) inhibited mitochondrial respiration and glycolysis in prostate cancer cells leading to a severe depletion in ATP. The combination of the two drugs was much more harmful for cancer cells than the treatment with metformin or 2DG alone, leading to 96% inhibition of cell viability in LNCaP prostate cancer cells. In contrast, a moderate effect on cell viability was observed in normal prostate epithelial cells. At the cellular level, the combination of metformin and 2DG induced p53-dependent apoptosis via the energy sensor pathway AMP kinase, and the reexpression of a functional p53 in p53-deficient prostate cancer cells restored caspase-3 activity. In addition to apoptosis, the combination of metformin and 2DG arrested prostate cancer cells in G(2)-M. This G(2)-M arrest was independent of p53 and correlated with a stronger decrease in cell viability than obtained with either drug. Finally, metformin inhibited 2DG-induced autophagy, decreased beclin 1 expression, and triggered a switch from a survival process to cell death. Our study reinforces the growing interest of metabolic perturbators in cancer therapy and highlights the potential use of the combination of metformin and 2DG as an anticancerous treatment.

Hypoxia decreases insulin signaling pathways in adipocytes

OBJECTIVE—Obesity is characterized by an overgrowth of adipose tissue that leads to the formation of hypoxic areas within this tissue. We investigated whether this phenomenon could be responsible for insulin resistance by studying the effect of hypoxia on the insulin signaling pathway in adipocytes. RESEARCH DESIGN AND METHODS—The hypoxic signaling pathway was modulated in adipocytes from human and murine origins through incubation under hypoxic conditions (1% O2) or modulation of hypoxia-inducible factor (HIF) expression. Insulin signaling was monitored through the phosphorylation state of several key partners of the pathway and glucose transport. RESULTS—In both human and murine adipocytes, hypoxia inhibits insulin signaling as revealed by a decrease in the phosphorylation of insulin receptor. In 3T3-L1 adipocytes, this inhibition of insulin receptor phosphorylation is followed by a decrease in the phosphorylation state of protein kinase B and AS160, as well as an inhibition of glucose transport in response to insulin. These processes were reversible under normoxic conditions. The mechanism of inhibition seems independent of protein tyrosine phosphatase activities. Overexpression of HIF-1α or -2α or activation of HIF transcription factor with CoCl2 mimicked the effect of hypoxia on insulin signaling, whereas downregulation of HIF-1α and -2α by small interfering RNA inhibited it. CONCLUSIONS—We have demonstrated that hypoxia creates a state of insulin resistance in adipocytes that is dependent upon HIF transcription factor expression. Hypoxia could be envisioned as a new mechanism that participates in insulin resistance in adipose tissue of obese patients.

Retinoic acid activation of the ERK pathway is required for embryonic stem cell commitment into the adipocyte lineage.

Mouse embryonic stem (ES) cells are pluripotent cells that differentiate into multiple cell lineages. The commitment of ES cells into the adipocyte lineage is dependent on an early 3-day treatment with all-trans retinoic acid (RA). To characterize the molecular mechanisms underlying this process, we examined the contribution of the extracellular-signal-regulated kinase (ERK) pathway. Treatment of ES cell-derived embryoid bodies with RA resulted in a prolonged activation of the ERK pathway, but not the c-Jun N-terminal kinase, p38 mitogen-activated protein kinase or phosphoinositide 3-kinase pathways. To investigate the role of ERK activation, co-treatment of RA with PD98059, a specific inhibitor of the ERK signalling pathway, prevented both adipocyte formation and expression of the adipogenic markers, adipocyte lipid-binding protein and peroxisome-proliferator-activated receptor gamma. Furthermore, we show that ERK activation is required only during RA treatment. PD98059 does not interfere with the commitment of ES cells into other lineages, such as neurogenesis, myogenesis and cardiomyogenesis. As opposed to the controversial role of the ERK pathway in terminal differentiation, our results clearly demonstrate that this pathway is specifically required at an early stage of adipogenesis, corresponding to the RA-dependent commitment of ES cells.

p38 Mitogen‐Activated Protein Kinase Activity Commits Embryonic Stem Cells to Either Neurogenesis or Cardiomyogenesis

Mouse embryonic stem (ES) cells can be differentiated, in vitro into a variety of cell types including cardiac cells and neurons. This process is strictly controlled by the potent morphogen retinoic acid (RA). At a concentration of 10−7 M, RA induces ES cell differentiation into neurons and, conversely, inhibits cardiomyogenesis. We found that p38 mitogen‐activated protein kinase (p38MAPK) activity peaked spontaneously, between day 3 and day 5, during ES cell differentiation and that RA completely inhibited this peak of activity. In contrast to wild‐type cells, which required RA treatment, p38α −1− ES cells differentiated spontaneously into neurons and did not form cardiomyocytes. Moreover, inhibition of the peak of p38MAPK activity by a specific inhibitor, PD169316, committed ES cells into the neuronal lineage and blocked cardiomyogenesis. By genetic and biochemical approaches, we demonstrate that, in two different ES cell lines, the control of p38MAPK activity constitutes an early switch, committing ES cells into either neurogenesis (p38 off) or cardiomyogenesis (p38 on).

Apelin and APJ regulation in adipose tissue and skeletal muscle of type 2 diabetic mice and humans

Apelin, an adipocyte-secreted factor upregulated by insulin, is increased in adipose tissue (AT) and plasma with obesity. Apelin was recently identified as a new player in the control of glucose homeostasis. However, the regulation of apelin and APJ (apelin receptor) expression in skeletal muscle in relation to insulin resistance or type 2 diabetes is not known. Thus we studied apelin and APJ expression in AT and muscle in different mice models of obesity and in type 2 diabetic patients. In insulin-resistant high-fat (HF)-fed mice, apelin and APJ expression were increased in AT compared with control. This was not the case in AT of highly insulin-resistant db/db mice. In skeletal muscle, apelin expression was similar in control and HF-fed mice and decreased in db/db mice. APJ expression was decreased in both HF-fed and db/db mice. Control subjects and type 2 diabetic patients were subjected to a hyperinsulinemic-euglycemic clamp, and tissues biopsies were obtained before and at the end of the clamp. There was no significant difference in basal apelin and APJ expression in AT and muscle between control and diabetic patients. However, apelin plasma levels were significantly increased in diabetic patients. During the clamp, hyperinsulinemia increased apelin and APJ expression in AT of control but not in diabetic subjects. In muscle, only APJ mRNA levels were increased in control but also in diabetic patients. Taken together, these data show that apelin and APJ expression in mice and humans is regulated in a tissue-dependent manner and according to the severity of insulin resistance.

Concise Review: Regulation of Embryonic Stem Cell Lineage Commitment by Mitogen‐Activated Protein Kinases

Embryonic stem (ES) cells can give rise, in vivo, to the ectodermal, endodermal, and mesodermal germ layers and, in vitro, can differentiate into multiple cell lineages, offering broad perspectives in regenerative medicine. Understanding the molecular mechanisms governing ES cell commitment is an essential challenge in this field. The mitogen‐activated protein kinase (MAPK) pathways extracellular signal‐regulated kinase (ERK), c‐Jun amino‐terminal kinase (JNK), and p38MAPK are able to regulate ES commitment from early steps of the process to mature differentiated cells. Whereas the ERK pathway inhibits the self‐renewal of ES cells, upon commitment this pathway is involved in the development of extraembryonic tissues, in early mesoderm differentiation, and in the formation of mature adipocytes; p38MAPK displays a large spectrum of action from neurons to adipocytes, and JNK is involved in both ectoderm and primitive endoderm differentiations. Furthermore, for a given pathway, several of these effects are isoform‐dependent, revealing the complexity of the cellular response to activation of MAPK pathways. Regarding tissue regeneration, the potential outcome of systematic analysis of the function of different MAPKs in different ES cell differentiation programs is discussed.

The combination of metformin and 2-deoxyglucose inhibits autophagy and induces AMPK-dependent apoptosis in prostate cancer cells

Targeting cancer cell metabolism is a new promising strategy to fight cancer. Metformin, a widely used antidiabetic agent, and 2-deoxyglucose (2DG) drastically affect cancer cell metabolism. Recently, we showed that the combination of the two drugs was much more harmful for cancer cells than the treatment with metformin or 2DG alone. At the cellular level, this combination leads to p53- and AMPK-dependent apoptosis. Furthermore, we showed that metformin inhibits 2DG-induced autophagy, decreases beclin 1 expression and triggers a switch from a survival process to cell death.