Glutamine Anabolism Plays a Critical Role in Pancreatic Cancer by Coupling Carbon and Nitrogen Metabolism.

Glutamine is thought to play an important role in cancer cells by being deaminated via glutaminolysis to α-ketoglutarate (aKG) to fuel the tricarboxylic acid (TCA) cycle. Supporting this notion, aKG supplementation can restore growth/survival of glutamine-deprived cells. However, pancreatic cancers are often poorly vascularized and limited in glutamine supply, in alignment with recent concerns on the significance of glutaminolysis in pancreatic cancer. Here, we show that aKG-mediated rescue of glutamine-deprived pancreatic ductal carcinoma (PDAC) cells requires glutamate ammonia ligase (GLUL), the enzyme responsible for de novo glutamine synthesis. GLUL-deficient PDAC cells are capable of the TCA cycle but defective in aKG-coupled glutamine biosynthesis and subsequent nitrogen anabolic processes. Importantly, GLUL expression is elevated in pancreatic cancer patient samples and in mouse PDAC models. GLUL ablation suppresses the development of KrasG12D-driven murine PDAC. Therefore, GLUL-mediated glutamine biosynthesis couples the TCA cycle with nitrogen anabolism and plays a critical role in PDAC.

Autophagy maintains tumour growth through circulating arginine.

Autophagy captures intracellular components and delivers them to lysosomes, where they are degraded and recycled to sustain metabolism and to enable survival during starvation1-5. Acute, whole-body deletion of the essential autophagy gene Atg7 in adult mice causes a systemic metabolic defect that manifests as starvation intolerance and gradual loss of white adipose tissue, liver glycogen and muscle mass1. Cancer cells also benefit from autophagy. Deletion of essential autophagy genes impairs the metabolism, proliferation, survival and malignancy of spontaneous tumours in models of autochthonous cancer6,7. Acute, systemic deletion of Atg7 or acute, systemic expression of a dominant-negative ATG4b in mice induces greater regression of KRAS-driven cancers than does tumour-specific autophagy deletion, which suggests that host autophagy promotes tumour growth1,8. Here we show that host-specific deletion of Atg7 impairs the growth of multiple allografted tumours, although not all tumour lines were sensitive to host autophagy status. Loss of autophagy in the host was associated with a reduction in circulating arginine, and the sensitive tumour cell lines were arginine auxotrophs owing to the lack of expression of the enzyme argininosuccinate synthase 1. Serum proteomic analysis identified the arginine-degrading enzyme arginase I (ARG1) in the circulation of Atg7-deficient hosts, and in vivo arginine metabolic tracing demonstrated that serum arginine was degraded to ornithine. ARG1 is predominantly expressed in the liver and can be released from hepatocytes into the circulation. Liver-specific deletion of Atg7 produced circulating ARG1, and reduced both serum arginine and tumour growth. Deletion of Atg5 in the host similarly regulated [corrected] circulating arginine and suppressed tumorigenesis, which demonstrates that this phenotype is specific to autophagy function rather than to deletion of Atg7. Dietary supplementation of Atg7-deficient hosts with arginine partially restored levels of circulating arginine and tumour growth. Thus, defective autophagy in the host leads to the release of ARG1 from the liver and the degradation of circulating arginine, which is essential for tumour growth; this identifies a metabolic vulnerability of cancer.

A YY1-dependent increase in aerobic metabolism is indispensable for intestinal organogenesis.

During late gestation, villi extend into the intestinal lumen to dramatically increase the surface area of the intestinal epithelium, preparing the gut for the neonatal diet. Incomplete development of the intestine is the most common gastrointestinal complication in neonates, but the causes are unclear. We provide evidence in mice that Yin Yang 1 (Yy1) is crucial for intestinal villus development. YY1 loss in the developing endoderm had no apparent consequences until late gestation, after which the intestine differentiated poorly and exhibited severely stunted villi. Transcriptome analysis revealed that YY1 is required for mitochondrial gene expression, and ultrastructural analysis confirmed compromised mitochondrial integrity in the mutant intestine. We found increased oxidative phosphorylation gene expression at the onset of villus elongation, suggesting that aerobic respiration might function as a regulator of villus growth. Mitochondrial inhibitors blocked villus growth in a fashion similar to Yy1 loss, thus further linking oxidative phosphorylation with late-gestation intestinal development. Interestingly, we find that necrotizing enterocolitis patients also exhibit decreased expression of oxidative phosphorylation genes. Our study highlights the still unappreciated role of metabolic regulation during organogenesis, and suggests that it might contribute to neonatal gastrointestinal disorders.

Therapeutic starvation and autophagy in prostate cancer: a new paradigm for targeting metabolism in cancer therapy.

BACKGROUND: Autophagy is a starvation induced cellular process of self-digestion that allows cells to degrade cytoplasmic contents. The understanding of autophagy, as either a mechanism of resistance to therapies that induce metabolic stress, or as a means to cell death, is rapidly expanding and supportive of a new paradigm of therapeutic starvation. METHODS: To determine the effect of therapeutic starvation in prostate cancer, we studied the effect of the prototypical inhibitor of metabolism, 2-deoxy-D-glucose (2DG), in multiple cellular models including a transfected pEGFP-LC3 autophagy reporter construct in PC-3 and LNCaP cells. RESULTS: We found that 2DG induced cytotoxicity in PC-3 and LNCaP cells in a dose dependent fashion. We also found that 2DG modulated checkpoint proteins cdk4, and cdk6. Using the transfected pEGFP-LC3 autophagy reporter construct, we found that 2DG induced LC3 membrane translocation, characteristic of autophagy. Furthermore, knockdown of beclin1, an essential regulator of autophagy, abrogated 2DG induced autophagy. Using Western analysis for LC3 protein, we also found increased LC3-II expression in 2DG treated cells, again consistent with autophagy. In an effort to develop markers that may be predictive of autophagy, for assessment in clinical trials, we stained human prostate tumors for Beclin1 by immunohistochemistry (IHC). Additionally, we used a digitized imaging algorithm to quantify Beclin1 staining assessment. These data demonstrate the induction of autophagy in prostate cancer by therapeutic starvation with 2DG, and support the feasibility of assessment of markers predictive of autophagy such as Beclin1 that can be utilized in clinical trials. Prostate 68: 1743-1752 (c) 2008 Wiley-Liss, Inc. These data demonstrate the induction of autophagy in prostate cancer by therapeutic starvation with 2DG, and support the feasibility of assessment of markers predictive of autophagy such as Beclin1 that can be utilized in clinical trials.

A sesquiterpenelactone from Inula britannica induces anti-tumor effects dependent on Bcl-2 phosphorylation.

BACKGROUND: The over-expression of the anti-apoptotic protein Bcl-2 in cancer is associated with resistance to chemotherapeutic drugs. The phosphorylation of Bcl-2 is one mechanism by which anti-microtubule agents, such as paclitaxel or docetaxel, may inactivate Bcl-2. Although initially active in clinical studies, current anti-microtubule agents are only temporarily effective and the discovery of new agents is warranted. MATERIALS AND METHODS: We isolated and identified two known sesquiterpenelactones, O, O-diacetylbritannilactone (OODABL) and O-acetylbritaanilactone (OABL) from the flowers of the medicinal plant Inula britannica and studied their mechanism of anti-tumor effects. To determine the biological significance of Bcl-2 phosphorylation, we used a baby rat kidney (BRK-p53) cell line that was transformed with EIA and a temperature-sensitive mutant p53. The BRK-p53 cell line was transfected with either a vector with wild type Bcl-2 or a vector in which Bcl-2 had mutations in the paclitaxel phosphorylation sites (pcDNA3.1 V5/His Bcl-2 S70, 87A). RESULTS: OODABL and OABL induced phosphorylation of Bcl-2 in breast, ovary and prostate cancer cell lines and induced G2/M cell cycle arrest. Using the BRK cells with mutant Bcl-2 (BRK-Bcl-2-mt) and control (BRK-Bcl-2-wt), we found that OODABL induced phosphorylation of Bcl-2 at sites similar to paclitaxel. Phosphorylation of Bcl-2 was important for OODABL-induced cytotoxicity, since the abrogation of phosphorylation in BRK-Bcl-2-mt cells decreased OODABL-induced cytotoxicity. CONCLUSION: We concluded that OODABL is cytotoxic in multiple tumor cell lines, and the cytotoxicity is dependent, at least in part, on the phosphorylation of Bcl-2.