Geoffrey D. Girnun

HIF-independent regulation of VEGF and angiogenesis by the transcriptional coactivator PGC-1|[agr]|

Ischaemia of the heart, brain and limbs is a leading cause of morbidity and mortality worldwide. Hypoxia stimulates the secretion of vascular endothelial growth factor (VEGF) and other angiogenic factors, leading to neovascularization and protection against ischaemic injury. Here we show that the transcriptional coactivator PGC-1α (peroxisome-proliferator-activated receptor-γ coactivator-1α), a potent metabolic sensor and regulator, is induced by a lack of nutrients and oxygen, and PGC-1α powerfully regulates VEGF expression and angiogenesis in cultured muscle cells and skeletal muscle in vivo. PGC-1α-/- mice show a striking failure to reconstitute blood flow in a normal manner to the limb after an ischaemic insult, whereas transgenic expression of PGC-1α in skeletal muscle is protective. Surprisingly, the induction of VEGF by PGC-1α does not involve the canonical hypoxia response pathway and hypoxia inducible factor (HIF). Instead, PGC-1α coactivates the orphan nuclear receptor ERR-α (oestrogen-related receptor-α) on conserved binding sites found in the promoter and in a cluster within the first intron of the VEGF gene. Thus, PGC-1α and ERR-α, major regulators of mitochondrial function in response to exercise and other stimuli, also control a novel angiogenic pathway that delivers needed oxygen and substrates. PGC-1α may provide a novel therapeutic target for treating ischaemic diseases.

APC-dependent suppression of colon carcinogenesis by PPARγ

Activation of PPARγ 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 PPARγ in colon cancer. To analyze critically the role of this receptor, we have used mice heterozygous for Pparγ with both chemical and genetic models of this malignancy. Heterozygous loss of PPARγ causes an increase in β-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 β-catenin, develop tumors in a manner insensitive to the status of PPARγ. These data show that PPARγ can suppress β-catenin levels and colon carcinogenesis but only before damage to the APC/β-catenin pathway. This finding suggests a potentially important use for PPARγ ligands as chemopreventative agents in colon cancer.

Transcription factor NRF2 regulates miR-1 and miR-206 to drive tumorigenesis

The mechanisms by which deregulated nuclear factor erythroid-2-related factor 2 (NRF2) and kelch-like ECH-associated protein 1 (KEAP1) signaling promote cellular proliferation and tumorigenesis are poorly understood. Using an integrated genomics and ¹³C-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 (PPP) and the tricarboxylic acid (TCA) cycle, reprogramming glucose metabolism. Sustained activation of NRF2 signaling in cancer cells attenuated miR-1 and miR-206 expression, leading to enhanced expression of PPP genes. Conversely, overexpression of miR-1 and miR-206 decreased the expression of metabolic genes and dramatically impaired NADPH production, ribose synthesis, and in vivo tumor growth in mice. Loss of NRF2 decreased the expression of the redox-sensitive histone deacetylase, HDAC4, resulting in increased expression of miR-1 and miR-206, and not only inhibiting PPP expression and activity but functioning as a regulatory feedback loop that repressed HDAC4 expression. In primary tumor samples, the expression of miR-1 and miR-206 was inversely correlated with PPP gene expression, and increased expression of NRF2-dependent genes was associated with poor prognosis. Our results demonstrate that microRNA-dependent (miRNA-dependent) regulation of the PPP via NRF2 and HDAC4 represents a novel link between miRNA regulation, glucose metabolism, and ROS homeostasis in cancer cells.

PGC1α Promotes Tumor Growth by Inducing Gene Expression Programs Supporting Lipogenesis

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 with 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 as compared with Pgc1α(+/+) mice. Xenograft studies using gain and loss of PGC1α expression showed that PGC1α also promotes tumor growth. Interestingly, while PGC1α induced oxidative phosphorylation and tricarboxylic acid 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 into acetyl-CoA for fatty acid synthesis, were also increased by PGC1α, thus linking the oxidative and lipogenic functions of PGC1α. Indeed, using stable (13)C 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.

Metformin prevents liver tumorigenesis by inhibiting pathways driving hepatic lipogenesis.

A number of factors have been identified that increase the risk of hepatocellular carcinoma (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 in which 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. Although a number of preclinical studies show 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. In addition, 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 in which increased lipid synthesis is observed. As a whole these studies show that metformin prevents HCC and that metformin should be evaluated as a preventive agent for HCC in readily identifiable at-risk patients. Cancer Prev Res; 5(4); 544–52. ©2012 AACR.

Liposomal Doxorubicin Increases Radiofrequency Ablation–induced Tumor Destruction by Increasing Cellular Oxidative and Nitrative Stress and Accelerating Apoptotic Pathways

PURPOSE To determine if oxidative and nitrative stress and/or apoptosis contribute to increased coagulation when combining radiofrequency (RF) ablation with liposomal doxorubicin. MATERIALS AND METHODS Animal care committee approval was obtained. R3230 mammary adenocarcinomas in Fischer rats were treated with either RF ablation (n = 43), 1 mg of intravenously injected liposomal doxorubicin (n = 26), or combined therapy (n = 30) and were compared with control subjects (n = 11). A subset of animals receiving combination therapy (n = 24) were treated in the presence or absence of N-acetylcysteine (NAC) administered 24 hours and 1 hour before RF ablation. Tumors were analyzed 2 minutes to 72 hours after treatment to determine the temporal range of response by using immunohistochemical staining of the apoptosis marker cleaved caspase-3, phosphorylated gammaH2AX, and HSP70 and of markers of oxidative and nitrative stress (8-hydroxydeoxyguanosine [8-OHdG], 4-hydroxynonenal [4-HNE]-modified proteins, and nitrotyrosine [NT]). Statistical analyses, including t tests and analysis of variance for comparisons where appropriate, were performed. RESULTS By 4 hours after RF ablation alone, a 0.48-mm +/- 0.13 (standard deviation) peripheral band with 57.0% +/- 7.3 cleaved caspase-3 positive cells was noted at the ablation margin, whereas a 0.73-mm +/- 0.18 band with 77.7% +/- 6.3 positivity was seen for combination therapy (P < .03 for both comparisons). Combination therapy caused increased and earlier staining for 4-HNE-modified proteins, 8-OHdG, NT, and gammaH2AX with colocalization to cleaved caspase-3 staining. A rim of increased HSP70 was identified peripheral to the area of cleaved caspase-3. Parameters of oxidative and nitrative stress were significantly inhibited by NAC 1 hour following RF ablation, resulting in decreased cleaved caspase-3 positivity (0.28-mm +/- 0.09 band of 25.9% +/- 7.4 positivity vs 0.59-mm +/- 0.11 band of 62.9% +/- 6.0 positivity, P < .001 for both comparisons). CONCLUSION Combining RF ablation with liposomal doxorubicin increases cell injury and apoptosis in the zone of increased coagulation by using a mechanism that involves oxidative and nitrative stress that leads to accelerated apoptosis.

Regression of Drug-Resistant Lung Cancer by the Combination of Rosiglitazone and Carboplatin

Purpose: Current therapy for lung cancer involves multimodality therapies. However, many patients are either refractory to therapy or develop drug resistance. KRAS and epidermal growth factor receptor (EGFR) mutations represent some of the most common mutations in lung cancer, and many studies have shown the importance of these mutations in both carcinogenesis and chemoresistance. Genetically engineered murine models of mutant EGFR and KRAS have been developed that more accurately recapitulate human lung cancer. Recently, using cell-based experiments, we showed that platinum-based drugs and the antidiabetic drug rosiglitazone (PPARγ ligand) interact synergistically to reduce cancer cell and tumor growth. Here, we directly determined the efficacy of the PPARγ/carboplatin combination in these more relevant models of drug resistant non–small cell lung cancer. Experimental Design: Tumorigenesis was induced by activation of either mutant KRAS or EGFR. Mice then received either rosiglitazone or carboplatin monotherapy, or a combination of both drugs. Change in tumor burden, pathology, and evidence of apoptosis and cell growth were assessed. Results: Tumor burden remained unchanged or increased in the mice after monotherapy with either rosiglitazone or carboplatin. In striking contrast, we observed significant tumor shrinkage in mice treated with these drugs in combination. Immunohistochemical analyses showed that this synergy was mediated via both increased apoptosis and decreased proliferation. Importantly, this synergy between carboplatin and rosiglitazone did not increase systemic toxicity. Conclusions: These data show that the PPARγ ligand/carboplatin combination is a new therapy worthy of clinical investigation in lung cancers, including those cancers that show primary resistance to platinum therapy or acquired resistance to targeted therapy.

Oncogenic Myc Induces Expression of Glutamine Synthetase through Promoter Demethylation

c-Myc is known to promote glutamine usage by upregulating 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 molecular connection between DNA demethylation and glutamine metabolism in Myc-driven cancers.

PEPCK Coordinates the Regulation of Central Carbon Metabolism to Promote Cancer Cell Growth

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.

The diverse role of the PPARγ coactivator 1 family of transcriptional coactivators in cancer

The critical role that altered cellular metabolism plays in promoting and maintaining the cancer phenotype has received considerable attention in recent years. For many years it was believed that aerobic glycolysis, also known as the Warburg Effect, played an important role in cancer. However, recent studies highlight the requirement of mitochondrial function, oxidative phosphorylation and biosynthetic pathways in cancer. This has promoted interest into mechanisms controlling these metabolic pathways. The PPARγ coactivator (PGC)-1 family of transcriptional coactivators have emerged as key regulators of several metabolic pathways including oxidative metabolism, energy homeostasis and glucose and lipid metabolism. While PGC-1s have been implicated in a number of metabolic diseases, recent studies highlight an important role in cancer. Studies show that PGC-1s have both pro and anticancer functions and suggests a dynamic role for the PGC-1s in cancer. We discuss in this review the links between PGC-1s and cancer, with a focus on the most well studied family member, PGC-1α.