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The mechanisms by which deregulated nuclear factor erythroid-2-related factor 2 (NRF2) and kelch-like ECHassociated protein 1 (KEAP1) signaling promote cellular proliferation and tumorigenesis are poorly understood. Using an integrated genomics and 13 C-based targeted tracer fate association (TTFA) study, we found that NRF2 regulates miR-1 and miR-206 to direct carbon flux toward the pentose phosphate pathway (

Summary Phosphoenolpyruvate carboxykinase (PEPCK) is well known for its role in gluconeogenesis. However, PEPCK is also a key regulator of TCA cycle flux. The TCA cycle integrates glucose, amino acid and lipid metabolism depending on cellular needs. In addition, biosynthetic pathways crucial to tumor growth require the TCA cycle for the processing of glucose and glutamine derived carbons. We show here an unexpected role for PEPCK in promoting cancer cell proliferation in vitro and in vivo by increasing glucose and glutamine utilization toward anabolic metabolism. Unexpectedly, PEPCK also increased the synthesis of ribose from non-carbohydrate sources, such as glutamine, a phenomenon not previously described. Finally, we show that the effects of PEPCK on glucose metabolism and cell proliferation are in part mediated via activation of mTORC1. Taken together, these data demonstrate a role for PEPCK that links metabolic flux and anabolic pathways to cancer cell proliferation.

Activation of PPAR␥ by synthetic ligands, such as thiazolidinediones, stimulates adipogenesis and improves insulin sensitivity. Although thiazolidinediones represent a major therapy for type 2 diabetes, conflicting studies showing that these agents can increase or decrease colonic tumors in mice have raised concerns about the role of PPAR␥ in colon cancer. To analyze critically the role of this receptor, we have used mice heterozygous for Ppar␥ with both chemical and genetic models of this malignancy. Heterozygous loss of PPAR␥ causes an increase in ␤-catenin levels and a greater incidence of colon cancer when animals are treated with azoxymethane. However, mice with preexisting damage to Apc, a regulator of ␤-catenin, develop tumors in a manner insensitive to the status of PPAR␥. These data show that PPAR␥ can suppress ␤-catenin levels and colon carcinogenesis but only before damage to the APC͞␤-catenin pathway. This finding suggests a potentially important use for PPAR␥ ligands as chemopreventative agents in colon cancer.

Summary c-Myc is known to promotes glutamine usage by up-regulating glutaminase (GLS), which converts glutamine to glutamate that is catabolized in the TCA cycle. Here we report that in a number of human and murine cells and cancers, Myc induces elevated expression of glutamate-ammonia ligase (GLUL), also termed glutamine synthetase (GS), which catalyzes the de novo synthesis of glutamine from glutamate and ammonia. This is through upregulation of a Myc transcriptional target thymine DNA glycosylase (TDG), which promotes active demethylation of the GS promoter and its increased expression. Elevated expression of GS promotes cell survival under glutamine limitation, while silencing of GS decreases cell proliferation and xenograft tumor growth. Upon GS overexpression, increased glutamine enhances nucleotide synthesis and amino acid transport. These results demonstrate an unexpected role of Myc in inducing glutamine synthesis, and suggest a novel molecular connection between DNA demethylation and glutamine metabolism in Myc-driven cancers.

Peroxisomal proliferator-activated receptor (PPAR)gamma has been shown to decrease the inflammatory response via transrepression of proinflammatory transcription factors. However, the identity of PPARgamma responsive genes that decrease the inflammatory response has remained elusive. Because generation of the reactive oxygen species hydrogen peroxide (H(2)O(2)) plays a role in the inflammatory process and activation of proinflammatory transcription factors, we wanted to determine whether the antioxidant enzyme catalase might be a PPARgamma target gene. We identified a putative PPAR response element (PPRE) containing the canonical direct repeat 1 motif, AGGTGA-A-AGTTGA, in the rat catalase promoter. In vitro translated PPARgamma and retinoic X receptor-alpha proteins were able to bind to the catalase PPRE. Promoter deletion analysis revealed that the PPRE was functional, and a heterologous promoter construct containing a multimerized catalase PPRE demonstrated that the PPRE was necessary and sufficient for PPARgamma-mediated activation. Treatment of microvascular endothelial cells with PPARgamma ligands led to increases in catalase mRNA and activity. These results demonstrate that PPARgamma can alter catalase expression; this occurs via a PPRE in the rat catalase promoter. Thus, in addition to transrepression of proinflammatory transcription factors, PPARgamma may also be modulating catalase expression, and hence down-regulating the inflammatory response via scavenging of reactive oxygen species.

Despite the role of aerobic glycolysis in cancer, recent studies highlight the importance of the mitochondria and biosynthetic pathways as well. PPARγ coactivator 1α (PGC1α) is a key transcriptional regulator of several metabolic pathways including oxidative metabolism and lipogenesis. Initial studies suggested that PGC1α expression is reduced in tumors compared to adjacent normal tissue. Paradoxically, other studies show that PGC1α is associated with cancer cell proliferation. Therefore the role of PGC1α in cancer and especially carcinogenesis is unclear. Using Pgc1α-/- and Pgc1α+/+ mice we show that loss of PGC1α protects mice from azoxymethane induced colon carcinogenesis. Similarly, diethylnitrosamine induced liver carcinogenesis is reduced in Pgc1α-/- mice compared to Pgc1α+/+ mice. Xenograft studies using gain and loss of PGC1α expression demonstrated that PGC1α also promotes tumor growth. Interestingly, while PGC1α induced oxidative phosphorylation and TCA cycle gene expression, we also observed an increase in the expression of two genes required for de novo fatty acid synthesis, ACC and FASN. In addition, SLC25A1 and ACLY, which are required for the conversion of glucose in to acetyl CoA for fatty acid synthesis, were also increased by PGC1α, thus linking the oxidative and lipogenic functions of PGC1α. Indeed, using 13C stable isotope tracer analysis we show that PGC1α increased de novo lipogenesis. Importantly, inhibition of fatty acid synthesis blunted these progrowth effects of PGC1α. In conclusion, these studies show for the first time that loss of PGC1α protects against carcinogenesis and that PGC1α coordinately regulates mitochondrial and fatty acid metabolism to promote tumor growth.

A number of factors have been identified that increase the risk of HCC. Recently it has become appreciated that type II diabetes increases the risk of developing HCC. This represents a patient population that can be identified and targeted for cancer prevention. The biguanide metformin is a first line therapy for the treatment of type II diabetes where it exerts its effects primarily on the liver. A role of metformin in HCC is suggested by studies linking metformin intake for control of diabetes with a reduced risk of HCC. While a number of preclinical studies demonstrate the anticancer properties of metformin in a number of tissues, no studies have directly examined the effect of metformin on preventing carcinogenesis in the liver, one of its main sites of action. We show in these studies that metformin protected mice against chemically induced liver tumors. Interestingly, metformin did not increase AMPK activation, often shown to be a metformin target. Rather metformin decreased the expression of several lipogenic enzymes and lipogenesis. Additionally, restoring lipogenic gene expression by ectopic expression of the lipogenic transcription factor SREBP1c rescues metformin mediated growth inhibition. This mechanism of action suggests that metformin may also be useful for patients with other disorders associated with HCC where increased lipid synthesis is observed. As a whole these studies demonstrate that metformin prevents HCC and that metformin should be evaluated as a preventive agent for HCC in readily identifiable at risk patients.