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The tumor microenvironment (TME) of pancreatic ductal adenocarcinoma (PDAC) is composed of a dense stromal compartment and is poorly vascularized, resulting in limited nutrient delivery. As a result, PDAC cells must adapt to cope with the metabolic stresses brought on by TME nutrient limitation. In this article, we first review recent studies that have provided quantitative measurements of nutrient levels in the PDAC TME. These studies have provided a new understanding of the nutrient limitations and metabolic stresses that occur in PDAC. We next discuss the adaptive strategies employed by PDAC in response to TME nutrient limitation. We propose that PDAC adaptations to metabolic stress can be generalized into four categories: (a) cutting down on metabolic costs by recycling metabolites and suppressing nonessential processes, (b) upregulating biosynthetic pathways to meet TME metabolic demands, (c) supporting essential metabolic processes with alternative fuel sources, and (d) dampening antiproliferative and cell death responses that nutrient limitation normally triggers. Improving our understanding of the nutrient limitations within the TME, and the adaptations cells employ to cope with these stresses, provides a more complete picture of PDAC biology and reveals new opportunities for therapeutic targeting of this disease.

The roles for glycolytic and respiratory metabolism in supporting in vivo tumor growth in different contexts are not well understood. In this issue of PLOS Biology, a new study reveals that primary and metastatic tumors demonstrate divergent metabolic requirements.

Nutrient availability affects cancer cell metabolism and therapeutic responses Understanding the molecular basis of cancer has led to a revolution in how cancers are classified and treated. Subsets of patients benefit from precision-medicine drugs that target growth-promoting signaling networks, but not all cancer patients respond to these approaches ( 1 ). Precision medicine is largely built on the assumption that cancer cell–intrinsic factors, such as genetic mutations or epigenetic identity, determine which pathways and processes are required in cells and thus response to therapies. However, in many cases, the presence of particular genetic lesions is insufficient to identify patients that will respond to a drug ( 1 ). For instance, standard cell culture models have not identified the subsets of cancer patients that respond to most conventional chemotherapies ( 1 ). Nevertheless, these chemotherapy drugs remain standard of care for many cancers and, in some cases, contribute to curative regimens. Emerging data suggest that beyond cell-intrinsic factors, nutrient availability in the tumor microenvironment can also influence drug response. This highlights the importance of understanding the microenvironmental factors that dictate which cellular processes are essential for disease progression and ultimately how tumors respond to treatments that target these processes.

Cancer cell metabolism is heavily influenced by microenvironmental factors, including nutrient availability. Therefore, knowledge of microenvironmental nutrient levels is essential to understand tumor metabolism. To measure the extracellular nutrient levels available to tumors, we utilized quantitative metabolomics methods to measure the absolute concentrations of >118 metabolites in plasma and tumor interstitial fluid, the extracellular fluid that perfuses tumors. Comparison of nutrient levels in tumor interstitial fluid and plasma revealed that the nutrients available to tumors differ from those present in circulation. Further, by comparing interstitial fluid nutrient levels between autochthonous and transplant models of murine pancreatic and lung adenocarcinoma, we found that tumor type, anatomical location and animal diet affect local nutrient availability. These data provide a comprehensive characterization of the nutrients present in the tumor microenvironment of widely used models of lung and pancreatic cancer and identify factors that influence metabolite levels in tumors. In the body, cancer cells can rely on different nutrients than normal cells, and they can use these nutrients in a different way. What cancer cells consume also depends on what is available in their immediate environment. In a tumor, cells grab nutrients from the ‘interstitial’ fluid that surrounds them, but what is present in this liquid may vary within tumors arising in different locations. Understanding what nutrients are ‘on the menu’ in specific tumors would help to target diseased cells while sparing healthy ones, but this knowledge has been difficult to obtain. To investigate this, Sullivan et al. used a technique called mass spectrometry to measure the amounts of 120 nutrients present in the interstitial fluid of mouse pancreas and lung tumors. Different levels of nutrients were found in the two types of tumors, and analyses showed that what was present in the interstitial fluid depended on the type of cancer cells, where the tumor was located, and what the animals ate. This suggests that cancer cells may have different needs because they are limited in what they have access to. It remains to be seen whether the nutrients levels found in mouse tumors are the same as those in humans. Armed with this knowledge, it may then be possible to feed cancer cells grown in the laboratory with the nutrient menu that they would have access to in the body. This could help identify new cancer treatments.

Cancers have an altered metabolism, and there is interest in understanding precisely how oncogenic transformation alters cellular metabolism and how these metabolic alterations can translate into therapeutic opportunities. Researchers are developing increasingly powerful experimental techniques to study cellular metabolism, and these techniques have allowed for the analysis of cancer cell metabolism, both in tumors and in ex vivo cancer models. These analyses show that, while factors intrinsic to cancer cells such as oncogenic mutations, alter cellular metabolism, cell-extrinsic microenvironmental factors also substantially contribute to the metabolic phenotype of cancer cells. These findings highlight that microenvironmental factors within the tumor, such as nutrient availability, physical properties of the extracellular matrix, and interactions with stromal cells, can influence the metabolic phenotype of cancer cells and might ultimately dictate the response to metabolically targeted therapies. In an effort to better understand and target cancer metabolism, this Review focuses on the experimental evidence that microenvironmental factors regulate tumor metabolism, and on the implications of these findings for choosing appropriate model systems and experimental approaches.

Many mammalian cancer cell lines depend on glutamine as a major tri-carboxylic acid (TCA) cycle anaplerotic substrate to support proliferation. However, some cell lines that depend on glutamine anaplerosis in culture rely less on glutamine catabolism to proliferate in vivo. We sought to understand the environmental differences that cause differential dependence on glutamine for anaplerosis. We find that cells cultured in adult bovine serum, which better reflects nutrients available to cells in vivo, exhibit decreased glutamine catabolism and reduced reliance on glutamine anaplerosis compared to cells cultured in standard tissue culture conditions. We find that levels of a single nutrient, cystine, accounts for the differential dependence on glutamine in these different environmental contexts. Further, we show that cystine levels dictate glutamine dependence via the cystine/glutamate antiporter xCT/SLC7A11. Thus, xCT/SLC7A11 expression, in conjunction with environmental cystine, is necessary and sufficient to increase glutamine catabolism, defining important determinants of glutamine anaplerosis and glutaminase dependence in cancer. Cancer cells need to consume certain nutrients in order to grow, and some cancer drugs work by affecting the ability of the cells to use these nutrients. For decades researchers have grown cancer cells in petri dishes with standard nutrient formulations (also known as tissue culture), but the nutrients available to cancer cells in tissue culture are not the same as those found in the body. Cancer cells growing in tissue culture consume large amounts of a nutrient called glutamine. These cells die when exposed to a class of drugs called glutaminase inhibitors that prevent them from processing glutamine. However, when these same cancer cells grow as tumors in animals, they process less glutamine and are not affected by glutaminase inhibitors. So what differences are there between growing cancer cells in tumors and tissue culture that explain this different reliance on glutamine? Muir et al. reasoned that changing the levels of nutrients available to cancer cells might change what these cells consume, and so grew human cancer cells in cow serum (which has a similar nutrient content to blood in humans and other mammals). Indeed, these cells consumed less glutamine and responded to glutaminase inhibitors in a way that is similar to how tumors in the body respond to these drugs. Comparing the nutrient content of cow serum and typical tissue culture formulations revealed that high levels of the nutrient cystine cause the cells to rely more on glutamine. The results presented by Muir et al. suggest that cancer cells in tumors could be made to consume more glutamine and that this would make them sensitive to glutaminase inhibitors – a possibility that will be studied in future work. Exposing cultured cancer cells to nutrient levels closer to those found in the body may also better predict how tumor cells use nutrients and respond to some treatments.

Conditional power combines the findings of a partially completed study with assumptions about the future. The goal is to estimate the probability that the eventual study result will be incompatible with a criterion value, such as acceptable risk or the null hypothesis. Some history and motivation for conditional power calculations are provided, with examples illustrating the application to drug safety studies. This is an expository article suggesting that conditional power, which is well-established in clinical trials research, also has application to observational studies. The utility may be highest in regulatory settings where resources are limited and interim decisions have to be made accurately in the shortest possible time.

The Orm family proteins are conserved integral membrane proteins of the endoplasmic reticulum that are key homeostatic regulators of sphingolipid biosynthesis. Orm proteins bind to and inhibit serine:palmitoyl-coenzyme A transferase, the first enzyme in sphingolipid biosynthesis. In Saccharomyces cerevisiae, Orm1 and Orm2 are inactivated by phosphorylation in response to compromised sphingolipid synthesis (e.g., upon addition of inhibitor myriocin), thereby restoring sphingolipid production. We show here that protein kinase Ypk1, one of an essential pair of protein kinases, is responsible for this regulatory modification. Myriocin-induced hyperphosphorylation of Orm1 and Orm2 does not occur in ypk1▵ cells, and immunopurified Ypk1 phosphorylates Orm1 and Orm2 robustly in vitro exclusively on three residues that are known myriocin-induced sites. Furthermore, the temperature-sensitive growth of ypk1ts ypk2▵ cells is substantially ameliorated by deletion of ORM genes, confirming that a primary physiological role of Ypk1-mediated phosphorylation is to negatively regulate Orm function. Ypk1 immunoprecipitated from myriocin-treated cells displays a higher specific activity for Orm phosphorylation than Ypk1 from untreated cells. To identify the mechanism underlying Ypk1 activation, we systematically tested several candidate factors and found that the target of rapamycin complex 2 (TORC2) kinase plays a key role. In agreement with prior evidence that a TORC2-dependent site in Ypk1(T662) is necessary for cells to exhibit a wild-type level of myriocin resistance, a Ypk1(T662A) mutant displays only weak Orm phosphorylation in vivo and only weak activation in vitro in response to sphingolipid depletion. Additionally, sphingolipid depletion increases phosphorylation of Ypk1 at T662. Thus, Ypk1 is both a sensor and effector of sphingolipid level, and reduction in sphingolipids stimulates Ypk1, at least in part, via TORC2-dependent phosphorylation.

Plasma membrane lipid composition must be maintained during growth and under environmental insult. In yeast, signaling mediated by TOR Complex 2 (TORC2)-dependent protein kinase Ypk1 controls lipid abundance and distribution in response to membrane stress. Ypk1, among other actions, alleviates negative regulation of L-serine:palmitoyl-CoA acyltransferase, upregulating production of long-chain base precursors to sphingolipids. To explore other roles for TORC2-Ypk1 signaling in membrane homeostasis, we devised a three-tiered genome-wide screen to identify additional Ypk1 substrates, which pinpointed both catalytic subunits of the ceramide synthase complex. Ypk1-dependent phosphorylation of both proteins increased upon either sphingolipid depletion or heat shock and was important for cell survival. Sphingolipidomics, other biochemical measurements and genetic analysis demonstrated that these modifications of ceramide synthase increased its specific activity and stimulated channeling of long-chain base precursors into sphingolipid end-products. Control at this branch point also prevents accumulation of intermediates that could compromise cell growth by stimulating autophagy. Cells are enclosed by a plasma membrane that separates and protects each cell from its environment. These membranes are made of a variety of proteins and fatty molecules called lipids, which are carefully organized throughout the membrane. When cells experience stresses such as heat or excessive pressure, the plasma membrane changes to help protect the cell. In particular, more of a group of lipids called sphingolipids are incorporated into the membrane under stress conditions. In yeast cells, a protein called Ypk1 plays an important role in protecting the cell from stress. Ypk1 controls the activity of a number of proteins that are responsible for balancing the amounts of different types of lipids in cell membranes. The combined action of these Ypk1-dependent proteins leads to the remodelling of the cell membrane to protect against stress. While several proteins that work with Ypk1 are known, some of the changes that serve to protect the plasma membrane cannot be explained by the action of these proteins alone. To provide a more comprehensive picture of how Ypk1 helps cells to respond to changes in the environment, Muir et al. developed a new approach that combines biochemical, genetic and bioinformatics techniques to survey the yeast genome for proteins that could be Ypk1 targets. Muir et al. first produced a list of potential candidate proteins by searching for proteins with features similar to known Ypk1 targets, and then considered those that are known to be involved in processes that also involve Ypk1. To filter the potential targets further, Muir et al. performed experiments in yeast cells to see which proteins prevented normal cell growth if they were over-produced. Further experiments investigating which of these proteins interact with Ypk1 when purified identified 12 new proteins that are most likely targets of the Ypk1 protein. Two of these newly identified Ypk1 target proteins form part of an enzyme complex called ceramide synthase, which produces a family of waxy lipid molecules from which more complex sphingolipids are built. Muir et al. discovered that during stress, Ypk1 enhances the activity of the ceramide synthase enzyme, which increases lipid production and the amount of sphingolipid deposited in the cell membrane. If this process is interrupted at any stage, cells struggle to survive under stress conditions. The other candidate proteins identified by Muir et al. remain to be validated and characterized as Ypk1 targets. Nevertheless, the techniques used have conclusively identified some new Ypk1 targets and could also be applied to similar searches for proteins targeted in other biological processes.

During tumorigenesis, the high metabolic demand of cancer cells results in increased production of reactive oxygen species. To maintain oxidative homeostasis, tumor cells increase their antioxidant production through hyperactivation of the NRF2 pathway, which promotes tumor cell growth. Despite the extensive characterization of NRF2-driven metabolic rewiring, little is known about the metabolic liabilities generated by this reprogramming. Here, we show that activation of NRF2, in either mouse or human cancer cells, leads to increased dependency on exogenous glutamine through increased consumption of glutamate for glutathione synthesis and glutamate secretion by xc- antiporter system. Together, this limits glutamate availability for the tricarboxylic acid cycle and other biosynthetic reactions creating a metabolic bottleneck. Cancers with genetic or pharmacological activation of the NRF2 antioxidant pathway have a metabolic imbalance between supporting increased antioxidant capacity over central carbon metabolism, which can be therapeutically exploited.