Ornithine aminotransferase supports polyamine synthesis in pancreatic cancer.

There is a need to develop effective therapies for pancreatic ductal adenocarcinoma (PDA), a highly lethal malignancy with increasing incidence1 and poor prognosis2. Although targeting tumour metabolism has been the focus of intense investigation for more than a decade, tumour metabolic plasticity and high risk of toxicity have limited this anticancer strategy3,4. Here we use genetic and pharmacological approaches in human and mouse in vitro and in vivo models to show that PDA has a distinct dependence on de novo ornithine synthesis from glutamine. We find that this process, which is mediated through ornithine aminotransferase (OAT), supports polyamine synthesis and is required for tumour growth. This directional OAT activity is usually largely restricted to infancy and contrasts with the reliance of most adult normal tissues and other cancer types on arginine-derived ornithine for polyamine synthesis5,6. This dependency associates with arginine depletion in the PDA tumour microenvironment and is driven by mutant KRAS. Activated KRAS induces the expression of OAT and polyamine synthesis enzymes, leading to alterations in the transcriptome and open chromatin landscape in PDA tumour cells. The distinct dependence of PDA, but not normal tissue, on OAT-mediated de novo ornithine synthesis provides an attractive therapeutic window for treating patients with pancreatic cancer with minimal toxicity.

Adaptation of pancreatic cancer cells to nutrient deprivation is reversible and requires glutamine synthetase stabilization by mTORC1.

Pancreatic ductal adenocarcinoma (PDA) is a lethal, therapy-resistant cancer that thrives in a highly desmoplastic, nutrient-deprived microenvironment. Several studies investigated the effects of depriving PDA of either glucose or glutamine alone. However, the consequences on PDA growth and metabolism of limiting both preferred nutrients have remained largely unknown. Here, we report the selection for clonal human PDA cells that survive and adapt to limiting levels of both glucose and glutamine. We find that adapted clones exhibit increased growth in vitro and enhanced tumor-forming capacity in vivo. Mechanistically, adapted clones share common transcriptional and metabolic programs, including amino acid use for de novo glutamine and nucleotide synthesis. They also display enhanced mTORC1 activity that prevents the proteasomal degradation of glutamine synthetase (GS), the rate-limiting enzyme for glutamine synthesis. This phenotype is notably reversible, with PDA cells acquiring alterations in open chromatin upon adaptation. Silencing of GS suppresses the enhanced growth of adapted cells and mitigates tumor growth. These findings identify nongenetic adaptations to nutrient deprivation in PDA and highlight GS as a dependency that could be targeted therapeutically in pancreatic cancer patients.

Author Correction: Tumours with PI3K activation are resistant to dietary restriction.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Ablation of insulin receptor substrates 1 and 2 suppresses Kras-driven lung tumorigenesis.

Non-small-cell lung cancer (NSCLC) is a leading cause of cancer death worldwide, with 25% of cases harboring oncogenic Kirsten rat sarcoma (KRAS). Although KRAS direct binding to and activation of PI3K is required for KRAS-driven lung tumorigenesis, the contribution of insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF1R) in the context of mutant KRAS remains controversial. Here, we provide genetic evidence that lung-specific dual ablation of insulin receptor substrates 1/2 (Irs1/Irs2), which mediate insulin and IGF1 signaling, strongly suppresses tumor initiation and dramatically extends the survival of a mouse model of lung cancer with Kras activation and p53 loss. Mice with Irs1/Irs2 loss eventually succumb to tumor burden, with tumor cells displaying suppressed Akt activation and strikingly diminished intracellular levels of essential amino acids. Acute loss of IRS1/IRS2 or inhibition of IR/IGF1R in KRAS-mutant human NSCLC cells decreases the uptake and lowers the intracellular levels of amino acids, while enhancing basal autophagy and sensitivity to autophagy and proteasome inhibitors. These findings demonstrate that insulin/IGF1 signaling is required for KRAS-mutant lung cancer initiation, and identify decreased amino acid levels as a metabolic vulnerability in tumor cells with IR/IGF1R inhibition. Consequently, combinatorial targeting of IR/IGF1R with autophagy or proteasome inhibitors may represent an effective therapeutic strategy in KRAS-mutant NSCLC.

Critical role for arginase 2 in obesity-associated pancreatic cancer.

Obesity is an established risk factor for pancreatic ductal adenocarcinoma (PDA). Despite recent identification of metabolic alterations in this lethal malignancy, the metabolic dependencies of obesity-associated PDA remain unknown. Here we show that obesity-driven PDA exhibits accelerated growth and a striking transcriptional enrichment for pathways regulating nitrogen metabolism. We find that the mitochondrial form of arginase (ARG2), which hydrolyzes arginine into ornithine and urea, is induced upon obesity, and silencing or loss of ARG2 markedly suppresses PDA. In vivo infusion of 15N-glutamine in obese mouse models of PDA demonstrates enhanced nitrogen flux into the urea cycle and infusion of 15N-arginine shows that Arg2 loss causes significant ammonia accumulation that results from the shunting of arginine catabolism into alternative nitrogen repositories. Furthermore, analysis of PDA patient tumors indicates that ARG2 levels correlate with body mass index (BMI). The specific dependency of PDA on ARG2 rather than the principal hepatic enzyme ARG1 opens a therapeutic window for obesity-associated pancreatic cancer.Obesity is an established risk factor for pancreatic ductal adenocarcinoma (PDA). Here the authors show that obesity induces the expression of the mitochondrial form of arginase ARG2 in PDA and that ARG2 silencing or loss results in ammonia accumulation and suppression of obesity-driven PDA tumor growth.

Starved epithelial cells uptake extracellular matrix for survival.

Extracellular matrix adhesion is required for normal epithelial cell survival, nutrient uptake and metabolism. This requirement can be overcome by oncogene activation. Interestingly, inhibition of PI3K/mTOR leads to apoptosis of matrix-detached, but not matrix-attached cancer cells, suggesting that matrix-attached cells use alternate mechanisms to maintain nutrient supplies. Here we demonstrate that under conditions of dietary restriction or growth factor starvation, where PI3K/mTOR signalling is decreased, matrix-attached human mammary epithelial cells upregulate and internalize ß4-integrin along with its matrix substrate, laminin. Endocytosed laminin localizes to lysosomes, results in increased intracellular levels of essential amino acids and enhanced mTORC1 signalling, preventing cell death. Moreover, we show that starved human fibroblasts secrete matrix proteins that maintain the growth of starved mammary epithelial cells contingent upon epithelial cell ß4-integrin expression. Our study identifies a crosstalk between stromal fibroblasts and epithelial cells under starvation that could be exploited therapeutically to target tumours resistant to PI3K/mTOR inhibition.

Autophagy is required for glucose homeostasis and lung tumor maintenance.

UNLABELLED: Macroautophagy (autophagy hereafter) recycles intracellular components to sustain mitochondrial metabolism that promotes the growth, stress tolerance, and malignancy of lung cancers, suggesting that autophagy inhibition may have antitumor activity. To assess the functional significance of autophagy in both normal and tumor tissue, we conditionally deleted the essential autophagy gene, autophagy related 7 (Atg7), throughout adult mice. Here, we report that systemic ATG7 ablation caused susceptibility to infection and neurodegeneration that limited survival to 2 to 3 months. Moreover, upon fasting, autophagy-deficient mice suffered fatal hypoglycemia. Prior autophagy ablation did not alter the efficiency of non-small cell lung cancer (NSCLC) initiation by activation of oncogenic Kras(G12D) and deletion of the Trp53 tumor suppressor. Acute autophagy ablation in mice with preexisting NSCLC, however, blocked tumor growth, promoted tumor cell death, and generated more benign disease (oncocytomas). This antitumor activity occurred before destruction of normal tissues, suggesting that acute autophagy inhibition may be therapeutically beneficial in cancer. SIGNIFICANCE: We systemically ablated cellular self-cannibalization by autophagy in adult mice and determined that it is dispensable for short-term survival, but required to prevent fatal hypoglycemia and cachexia during fasting, delineating a new role for autophagy in metabolism. Importantly, acute, systemic autophagy ablation was selectively destructive to established tumors compared with normal tissues, thereby providing the preclinical evidence that strategies to inhibit autophagy may be therapeutically advantageous for RAS-driven cancers.

Pten-null tumors cohabiting the same lung display differential AKT activation and sensitivity to dietary restriction.

PTEN loss is considered a biomarker for activated phosphoinositide 3-kinase (PI3K)/AKT, a pathway frequently mutated in cancer, and was recently shown to confer resistance to dietary restriction. Here, we show that Pten loss is not sufficient to drive AKT activation and resistance to dietary restriction in tumors with low growth factor receptor levels. We describe a murine Pten-null Kras-driven lung cancer model that harbors both dietary restriction-resistant, higher-grade, bronchiolar tumors with high AKT activity, and dietary restriction-sensitive, lower-grade, alveolar tumors with low AKT activity. We find that this phenotype is cell autonomous and that normal bronchiolar cells express higher levels of insulin-like growth factor-I receptor (IGF-IR) and of ectonucleoside triphosphate diphosphohydrolase 5 (ENTPD5), an endoplasmic reticulum enzyme known to modulate growth factor receptor levels. Suppression of ENTPD5 is sufficient to decrease IGF-IR levels and sensitize bronchiolar tumor cells to serum in vitro and to dietary restriction in vivo. Furthermore, we find that a significant percentage of human non-small cell lung carcinomas (NSCLC) have low AKT activity despite PTEN loss.

Functional genomics reveal that the serine synthesis pathway is essential in breast cancer.

Cancer cells adapt their metabolic processes to drive macromolecular biosynthesis for rapid cell growth and proliferation. RNA interference (RNAi)-based loss-of-function screening has proven powerful for the identification of new and interesting cancer targets, and recent studies have used this technology in vivo to identify novel tumour suppressor genes. Here we developed a method for identifying novel cancer targets via negative-selection RNAi screening using a human breast cancer xenograft model at an orthotopic site in the mouse. Using this method, we screened a set of metabolic genes associated with aggressive breast cancer and stemness to identify those required for in vivo tumorigenesis. Among the genes identified, phosphoglycerate dehydrogenase (PHGDH) is in a genomic region of recurrent copy number gain in breast cancer and PHGDH protein levels are elevated in 70% of oestrogen receptor (ER)-negative breast cancers. PHGDH catalyses the first step in the serine biosynthesis pathway, and breast cancer cells with high PHGDH expression have increased serine synthesis flux. Suppression of PHGDH in cell lines with elevated PHGDH expression, but not in those without, causes a strong decrease in cell proliferation and a reduction in serine synthesis. We find that PHGDH suppression does not affect intracellular serine levels, but causes a drop in the levels of α-ketoglutarate, another output of the pathway and a tricarboxylic acid (TCA) cycle intermediate. In cells with high PHGDH expression, the serine synthesis pathway contributes approximately 50% of the total anaplerotic flux of glutamine into the TCA cycle. These results reveal that certain breast cancers are dependent upon increased serine pathway flux caused by PHGDH overexpression and demonstrate the utility of in vivo negative-selection RNAi screens for finding potential anticancer targets.

Tumours with PI3K activation are resistant to dietary restriction.

Dietary restriction delays the incidence and decreases the growth of various types of tumours, but the mechanisms underlying the sensitivity of tumours to food restriction remain unknown. Here we show that certain human cancer cell lines, when grown as tumour xenografts in mice, are highly sensitive to the anti-growth effects of dietary restriction, whereas others are resistant. Cancer cells that form dietary-restriction-resistant tumours carry mutations that cause constitutive activation of the phosphatidylinositol-3-kinase (PI3K) pathway and in culture proliferate in the absence of insulin or insulin-like growth factor 1. Substitution of an activated mutant allele of PI3K with wild-type PI3K in otherwise isogenic cancer cells, or the restoration of PTEN expression in a PTEN-null cancer cell line, is sufficient to convert a dietary-restriction-resistant tumour into one that is dietary-restriction-sensitive. Dietary restriction does not affect a PTEN-null mouse model of prostate cancer, but it significantly decreases tumour burden in a mouse model of lung cancer lacking constitutive PI3K signalling. Thus, the PI3K pathway is an important determinant of the sensitivity of tumours to dietary restriction, and activating mutations in the pathway may influence the response of cancers to dietary restriction-mimetic therapies.

Ablation in mice of the mTORC components raptor, rictor, or mLST8 reveals that mTORC2 is required for signaling to Akt-FOXO and PKCalpha, but not S6K1.

The mTOR kinase controls cell growth, proliferation, and survival through two distinct multiprotein complexes, mTORC1 and mTORC2. mTOR and mLST8 are in both complexes, while raptor and rictor are part of only mTORC1 and mTORC2, respectively. To investigate mTORC1 and mTORC2 function in vivo, we generated mice deficient for raptor, rictor, or mLST8. Like mice null for mTOR, those lacking raptor die early in development. However, mLST8 null embryos survive until e10.5 and resemble embryos missing rictor. mLST8 is necessary to maintain the rictor-mTOR, but not the raptor-mTOR, interaction, and both mLST8 and rictor are required for the hydrophobic motif phosphorylation of Akt/PKB and PKCalpha, but not S6K1. Furthermore, insulin signaling to FOXO3, but not to TSC2 or GSK3beta, requires mLST8 and rictor. Thus, mTORC1 function is essential in early development, mLST8 is required only for mTORC2 signaling, and mTORC2 is a necessary component of the Akt-FOXO and PKCalpha pathways.

LXRS and FXR: the yin and yang of cholesterol and fat metabolism.

Liver X receptors (LXRs) and farnesoid X receptor (FXR) are nuclear receptors that function as intracellular sensors for sterols and bile acids, respectively. In response to their ligands, these receptors induce transcriptional responses that maintain a balanced, finely tuned regulation of cholesterol and bile acid metabolism. LXRs also permit the efficient storage of carbohydrate- and fat-derived energy, whereas FXR activation results in an overall decrease in triglyceride levels and modulation of glucose metabolism. The elegant, dual interplay between these two receptor systems suggests that they coevolved to constitute a highly sensitive and efficient system for the maintenance of total body fat and cholesterol homeostasis. Emerging evidence suggests that the tissue-specific action of these receptors is also crucial for the proper function of the cardiovascular, immune, reproductive, endocrine pancreas, renal, and central nervous systems. Together, LXRs and FXR represent potential therapeutic targets for the treatment and prevention of numerous metabolic and lipid-related diseases.

LXRs regulate the balance between fat storage and oxidation.

Despite the well-established role of liver X receptors (LXRs) in regulating cholesterol homeostasis, their contribution to lipid homeostasis remains unclear. Here we show that LXR null mice are defective in hepatic lipid metabolism and are resistant to obesity when challenged with a diet containing both high fat and cholesterol. This phenotype is dependent on the presence of dietary cholesterol and is accompanied by the aberrant production of thyroid hormone in liver. Interestingly, the inability of LXR-/- mice to induce SREBP-1c-dependent lipogenesis does not explain the LXR-/- phenotype, since SREBP-1c null mice are not obesity resistant. Instead, the LXR-/- response is due to abnormal energy dissipation resulting from uncoupled oxidative phosphorylation and ectopic expression of uncoupling proteins in muscle and white adipose. These studies suggest that, by selectively sensing the cholesterol component of a lipid-rich diet, LXRs govern the balance between storage and oxidation of dietary fat.

ECM-induced gap junctional communication enhances mammary epithelial cell differentiation.

The relationship between gap junctional intercellular communication (GJIC) and mammary cell (CID-9) differentiation in vitro was explored. CID-9 cells differentiate and express beta-casein in an extracellular matrix (ECM)- and hormone-dependent manner. In response to interaction with the ECM, cells in culture modulated the expression of their gap junction proteins at the transcriptional and post-translational levels. In the presence of EHS-matrix, connexins (Cx)26, 32 and 43 localized predominantly to the plasma membrane, and enhanced GJIC [as measured by Lucifer Yellow (LY) dye transfer assays] was noted. Inhibition of GJIC of cells on EHS-matrix with 18 alpha glycyrrhetinic acid (GA) resulted in reversible downregulation of beta-casein expression. In the presence of cAMP, cells cultured on plastic expressed beta-casein, upregulated Cx43 and Cx26 protein levels and enhanced GJIC. This was reversed in the presence of 18 alpha GA. cAMP-treated cells plated either on a non-adhesive PolyHEMA substratum or on plastic supplemented with function-blocking anti-beta 1 integrin antibodies, maintained beta-casein expression. These studies suggest that cell-ECM interaction alone may induce differentiation through changes in cAMP levels and formation of functional gap junctions. That these events are downstream of ECM signalling was underscored by the fact that enhanced GJIC induced partial differentiation in mammary epithelial cells in the absence of an exogenously provided basement membrane and in a beta 1-integrin- and adhesion-independent manner.