High-Throughput Screening Reveals New Glutaminase Inhibitor Molecules.

The glutaminase (GLS) enzyme hydrolyzes glutamine into glutamate, an important anaplerotic source for the tricarboxylic acid cycle in rapidly growing cancer cells under the Warburg effect. Glutamine-derived α-ketoglutarate is also an important cofactor of chromatin-modifying enzymes, and through epigenetic changes, it keeps cancer cells in an undifferentiated state. Moreover, glutamate is an important neurotransmitter, and deregulated glutaminase activity in the nervous system underlies several neurological disorders. Given the proven importance of glutaminase for critical diseases, we describe the development of a new coupled enzyme-based fluorescent glutaminase activity assay formatted for 384-well plates for high-throughput screening (HTS) of glutaminase inhibitors. We applied the new methodology to screen a ∼30,000-compound library to search for GLS inhibitors. The HTS assay identified 11 glutaminase inhibitors as hits that were characterized by in silico, biochemical, and glutaminase-based cellular assays. A structure-activity relationship study on the most promising hit (C9) allowed the discovery of a derivative, C9.22, with enhanced in vitro and cellular glutaminase-inhibiting activity. In summary, we discovered a new glutaminase inhibitor with an innovative structural scaffold and described the molecular determinants of its activity.

GLS2 is protumorigenic in breast cancers.

Many types of cancers have a well-established dependence on glutamine metabolism to support survival and growth, a process linked to glutaminase 1 (GLS) isoforms. Conversely, GLS2 variants often have tumor-suppressing activity. Triple-negative (TN) breast cancer (testing negative for estrogen, progesterone, and Her2 receptors) has elevated GLS protein levels and reportedly depends on exogenous glutamine and GLS activity for survival. Despite having high GLS levels, we verified that several breast cancer cells (including TN cells) express endogenous GLS2, defying its role as a bona fide tumor suppressor. Moreover, ectopic GLS2 expression rescued cell proliferation, TCA anaplerosis, redox balance, and mitochondrial function after GLS inhibition by the small molecule currently in clinical trials CB-839 or GLS knockdown of GLS-dependent cell lines. In several cell lines, GLS2 knockdown decreased cell proliferation and glutamine-linked metabolic phenotypes. Strikingly, long-term treatment of TN cells with another GLS-exclusive inhibitor bis-2'-(5-phenylacetamide-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES) selected for a drug-resistant population with increased endogenous GLS2 and restored proliferative capacity. GLS2 was linked to enhanced in vitro cell migration and invasion, mesenchymal markers (through the ERK-ZEB1-vimentin axis under certain conditions) and in vivo lung metastasis. Of concern, GLS2 amplification or overexpression is linked to an overall, disease-free and distant metastasis-free worse survival prognosis in breast cancer. Altogether, these data establish an unforeseen role of GLS2 in sustaining tumor proliferation and underlying metastasis in breast cancer and provide an initial framework for exploring GLS2 as a novel therapeutic target.

Retraction notice to "Obesity-Induced Increase in Tumor Necrosis Factor-α Leads to Development of Colon Cancer in Mice" Gastroenterology 2012;143:741-753.e4.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the Editor-in-Chief and Deputy Editor-in-Chief following an investigation into the data that were presented in several figures within the article. A number of images used in this article are believed to be duplicated images. The authors stated that they inadvertently inserted images of the wrong blots in several of the figures, resulting in the duplications; however, they did not address all of the concerns raised. Because the editors were no longer confident in the conclusions of the article based on these incorrect data, a decision was made to retract the paper. All authors have been notified of this decision. The University of Campinas (UNICAMP) in São Paulo, Brazil was contacted regarding these concerns, but to date the journal has received no response.

Unsaturated fatty acids as high-affinity ligands of the C-terminal Per-ARNT-Sim domain from the Hypoxia-inducible factor 3α.

Hypoxia-inducible transcription factors (HIF) form heterodimeric complexes that mediate cell responses to hypoxia. The oxygen-dependent stability and activity of the HIF-α subunits is traditionally associated to post-translational modifications such as hydroxylation, acetylation, ubiquitination, and phosphorylation. Here we report novel evidence showing that unsaturated fatty acids are naturally occurring, non-covalent structural ligands of HIF-3α, thus providing the initial framework for exploring its exceptional role as a lipid sensor under hypoxia.

RETRACTED: Obesity-induced increase in tumor necrosis factor-α leads to development of colon cancer in mice.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Editor-in-Chief and Deputy Editor-in-Chief following an investigation into the data that were presented in several figures within the article. A number of images used in this article are believed to be duplicated images. The authors stated that they inadvertently inserted images of the wrong blots in several of the figures, resulting in the duplications; however, they did not address all of the concerns raised. Because the editors were no longer confident in the conclusions of the article based on these incorrect data, a decision was made to retract the paper. All authors have been notified of this decision. The University of Campinas (UNICAMP) in São Paulo, Brazil was contacted regarding these concerns, but to date the journal has received no response.

Metformin amplifies chemotherapy-induced AMPK activation and antitumoral growth.

PURPOSE: Metformin is a widely used antidiabetic drug whose anticancer effects, mediated by the activation of AMP-activated protein kinase (AMPK) and reduction of mTOR signaling, have become noteworthy. Chemotherapy produces genotoxic stress and induces p53 activity, which can cross-talk with AMPK/mTOR pathway. Herein, we investigate whether the combination of metformin and paclitaxel has an effect in cancer cell lines. EXPERIMENTAL DESIGN: Human tumors were xenografted into severe combined immunodeficient (SCID) mice and the cancer cell lines were treated with only paclitaxel or only metformin, or a combination of both drugs. Western blotting, flow cytometry, and immunohistochemistry were then used to characterize the effects of the different treatments. RESULTS: The results presented herein show that the addition of metformin to paclitaxel leads to quantitative potentialization of molecular signaling through AMPK and a subsequent potent inhibition of the mTOR signaling pathway. Treatment with metformin and paclitaxel resulted in an increase in the number of cells arrested in the G(2)-M phase of the cell cycle, and decreased the tumor growth and increased apoptosis in tumor-bearing mice, when compared with individual drug treatments. CONCLUSION: We have provided evidence for a convergence of metformin and paclitaxel induced signaling at the level of AMPK. This mechanism shows how different drugs may cooperate to augment antigrowth signals, and suggests that target activation of AMPK by metformin may be a compelling ally in cancer treatment.

Epidemiological and molecular mechanisms aspects linking obesity and cancer.

About 25% of cancer cases globally are due to excess weight and a sedentary lifestyle. These results are alarming, as the world knows a pandemic of obesity and, in consequence, insulin resistance. Obesity may increase risk for various cancers by several mechanisms, including increasing sex and metabolic hormones, and inflammation. Here, we present a review of epidemiological and molecular evidences linking obesity and cancer--particularly colorectal, post-menopausal breast, endometrial, pancreatic, high grade prostate, hepatocellular, gallbladder, kidney and esophageal adenocarcinoma. The expected striking increase in the incidence of cancer in the near future related to obesity turns the knowledge of this field of great impact as it is needed to the development of strategies to prevent and treat this disease.

Epidemiological and molecular mechanisms aspects linking obesity and cancer: [review] / Mecanismos epidemiológicos e moleculares que associam obesidade e câncer: [revisão]

About 25 percent of cancer cases globally are due to excess weight and a sedentary lifestyle. These results are alarming, as the world knows a pandemy of obesity and, in consequence, insulin resistance. Obesity may increase risk for various cancers by several mechanisms, including increasing sex and metabolic hormones, and inflammation. Here, we present a review of epidemiological and molecular evidences linking obesity and cancer - particularly colorectal, post-menopausal breast, endometrial, pancreatic, high grade prostate, hepatocellular, gallbladder, kidney and esophageal adenocarcinoma. The expected striking increase in the incidence of cancer in the near future related to obesity turns the knowledge of this field of great impact as it is needed to the development of strategies to prevent and treat this disease.

Aproximadamente 25 por cento dos casos de câncer são decorrentes do excesso de peso e do modo de vida sedentário. Esses resultados são alarmantes, pois o mundo vive uma pandemia de obesidade e, consequentemente, de resistência à insulina. A obesidade pode aumentar o risco de vários tipos de câncer por diversos mecanismos, incluindo aumento dos hormônios sexuais e metabólicos, e de inflamação. Neste trabalho, apresentamos uma revisão das evidências epidemiológicas e moleculares que relacionam a obesidade ao câncer - em particular aos cânceres colorretal, mamário na pós-menopausa, endometrial, pancreático, prostático avançado, hepatocelular, de bexiga, renal e esofágico. O aumento esperado da incidência de câncer relacionado à obesidade em um futuro próximo torna o conhecimento dessa área de grande importância, uma vez que este é fundamental para o desenvolvimento de estratégias preventivas e terapêuticas para a doença.

Antineoplastic effect of rapamycin is potentiated by inhibition of IRS-1 signaling in prostate cancer cells xenografts.

Proper activation of phosphoinositide 3-kinase-Akt pathway is critical for the prevention of tumorigenesis. Recent data have characterized a negative feedback loop, wherein mammalian target of rapamycin (mTOR) blocks additional activation of the Akt/mTOR pathway through inhibition insulin receptor substrate 1 (IRS-1) function. However, the potential of IRS-1 inhibition during rapamycin treatment has not been examined. Herein, we show that IRS-1 antisense oligonucleotide and rapamycin synergistically antagonize the activation of mTOR in vivo and induced tumor suppression, through inhibition of proliferation and induction of apoptosis, in prostate cancer cell xenografts. These data demonstrate that the addition of agents that blocks IRS-1 potentiate the effect of mTOR inhibition in the growth of prostate cancer cell xenografts.

A central role for neuronal AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) in high-protein diet-induced weight loss.

OBJECTIVE: A high-protein diet (HPD) is known to promote the reduction of body fat, but the mechanisms underlying this change are unclear. AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) function as majors regulators of cellular metabolism that respond to changes in energy status, and recent data demonstrated that they also play a critical role in systemic energy balance. Here, we sought to determine whether the response of the AMPK and mTOR pathways could contribute to the molecular effects of an HPD. RESEARCH DESIGN AND METHODS: Western blotting, confocal microscopy, chromatography, light microscopy, and RT-PCR assays were combined to explore the anorexigenic effects of an HPD. RESULTS: An HPD reduced food intake and induced weight loss in both normal rats and ob/ob mice. The intracerebroventricular administration of leucine reduced food intake, and the magnitude of weight loss and reduction of food intake in a leucine-supplemented diet are similar to that achieved by HPD in normal rats and in ob/ob mice, suggesting that leucine is a major component of the effects of an HPD. Leucine and HPD decrease AMPK and increase mTOR activity in the hypothalamus, leading to inhibition of neuropeptide Y and stimulation of pro-opiomelanocortin expression. Consistent with a cross-regulation between AMPK and mTOR to control food intake, our data show that the activation of these enzymes occurs in the same specific neuronal subtypes. CONCLUSIONS: These findings provide support for the hypothesis that AMPK and mTOR interact in the hypothalamus to regulate feeding during HPD in a leucine-dependent manner.