Faculty Bibliography

Metastatic disease of the skin can be difficult to diagnose, particularly when lesions occur in unusual anatomic locations. We report the case of an 80-year-old woman with a history of anal squamous cell carcinoma (SCC) who developed genital ulcers. Biopsy of the lesions revealed features consistent with metastatic SCC. Cutaneous metastases are an infrequent cause of genital ulcerations, and it is important for physicians to consider this entity when evaluating genital ulcers in patients with prior malignancies.

Oncogenic Myc alters mitochondrial metabolism, making it dependent on exogenous glutamine (Gln) for cell survival. Accordingly, Gln deprivation selectively induces apoptosis in MYC-overexpressing cells via unknown mechanisms. Using MYCN-amplified neuroblastoma as a model, we identify PUMA, NOXA, and TRB3 as executors of Gln-starved cells. Gln depletion in MYC-transformed cells induces apoptosis through ATF4-dependent, but p53-independent, PUMA and NOXA induction. MYC-transformed cells depend on both glutamate-oxaloacetate transaminase and glutamate dehydrogenase to maintain Gln homeostasis and suppress apoptosis. Consequently, either ATF4 agonists or glutaminolysis inhibitors potently induce apoptosis in vitro and inhibit tumor growth in vivo. These results reveal mechanisms whereby Myc sensitizes cells to apoptosis, and validate ATF4 agonists and inhibitors of Gln metabolism as potential Myc-selective cancer therapeutics.

UNLABELLED: Recently, there has been a renewed interest in the study of tumor metabolism above and beyond the Warburg effect. Studies on cancer cell metabolism have provided evidence that tumor-specific activation of signaling pathways, such as the upregulation of the oncogene myc, can regulate glutamine uptake and its metabolism through glutaminolysis to provide the cancer cell with a replacement of energy source. METHODS: We report a convenient procedure to prepare l-[5-(11)C]-glutamine. The tracer was evaluated in 9L and SF188 tumor cells (glioma and astrocytoma cell lines). The biodistribution of l-[5-(11)C]-glutamine in rodent tumor models was investigated by dissection and PET. RESULTS: By reacting (11)C-cyanide ion with protected 4-iodo-2-amino-butanoic ester, the key intermediate was obtained in good yield. After hydrolysis with trifluoroacetic and sulfonic acids, the desired optically pure l-[5-(11)C]-glutamine was obtained (radiochemical yield, 5% at the end of synthesis; radiochemical purity, >95%). Tumor cell uptake studies showed maximum uptake of l-[5-(11)C]-glutamine reached 17.9% and 22.5% per 100 mug of protein, respectively, at 60 min in 9L and SF188 tumor cells. At 30 min after incubation, more than 30% of the activity appeared to be incorporated into cellular protein. Biodistribution in normal mice showed that l-[5-(11)C]-glutamine had significant pancreas uptake (7.37 percentage injected dose per gram at 15 min), most likely due to the exocrine function and high protein turnover within the pancreas. Heart uptake was rapid, and there was 3.34 percentage injected dose per gram remaining at 60 min after injection. Dynamic small-animal PET studies in rats bearing xenografted 9L tumors and in transgenic mice bearing spontaneous mammary gland tumors showed a prominent tumor uptake and retention. CONCLUSION: The data demonstrated that this tracer was favorably taken up in the tumor models. The results suggest that l-[5-(11)C]-glutamine might be useful for probing in vivo tumor metabolism in glutaminolytic tumors.

Citrate is a critical metabolite required to support both mitochondrial bioenergetics and cytosolic macromolecular synthesis. When cells proliferate under normoxic conditions, glucose provides the acetyl-CoA that condenses with oxaloacetate to support citrate production. Tricarboxylic acid (TCA) cycle anaplerosis is maintained primarily by glutamine. Here we report that some hypoxic cells are able to maintain cell proliferation despite a profound reduction in glucose-dependent citrate production. In these hypoxic cells, glutamine becomes a major source of citrate. Glutamine-derived alpha-ketoglutarate is reductively carboxylated by the NADPH-linked mitochondrial isocitrate dehydrogenase (IDH2) to form isocitrate, which can then be isomerized to citrate. The increased IDH2-dependent carboxylation of glutamine-derived alpha-ketoglutarate in hypoxia is associated with a concomitant increased synthesis of 2-hydroxyglutarate (2HG) in cells with wild-type IDH1 and IDH2. When either starved of glutamine or rendered IDH2-deficient by RNAi, hypoxic cells are unable to proliferate. The reductive carboxylation of glutamine is part of the metabolic reprogramming associated with hypoxia-inducible factor 1 (HIF1), as constitutive activation of HIF1 recapitulates the preferential reductive metabolism of glutamine-derived alpha-ketoglutarate even in normoxic conditions. These data support a role for glutamine carboxylation in maintaining citrate synthesis and cell growth under hypoxic conditions.

UNLABELLED: Changes in gene expression, metabolism, and energy requirements are hallmarks of cancer growth and self-sufficiency. Upregulation of the PI3K/Akt/mTor pathway in tumor cells has been shown to stimulate aerobic glycolysis, which has enabled (18)F-FDG PET tumor imaging. However, of the millions of (18)F-FDG PET scans conducted per year, a significant number of malignant tumors are (18)F-FDG PET-negative. Recent studies suggest that several tumors may use glutamine as the key nutrient for survival. As an alternative metabolic tracer for tumors, (18)F-(2S,4R)4-fluoroglutamine was developed as a PET tracer for mapping glutaminolytic tumors. METHODS: A series of in vitro cell uptake and in vivo animal studies were performed to demonstrate tumor cell addiction to glutamine. Cell uptake studies of this tracer were performed in SF188 and 9L glioblastoma tumor cells. Dynamic small-animal PET studies of (18)F-(2S,4R)4-fluoroglutamine were conducted in 2 animal models: xenografts produced in F344 rats by subcutaneous injection of 9L tumor cells and transgenic mice with M/tomND spontaneous mammary gland tumors. RESULTS: In vitro studies showed that both transformed 9L and SF188 tumor cells displayed a high rate of glutamine uptake (maximum uptake, approximately 16% dose/100 mug of protein). The cell uptake of (18)F-(2S,4R)4-fluoroglutamine by SF188 cells is comparable to that of (3)H-L-glutamine but higher than that of (18)F-FDG. The tumor cell uptake can be selectively blocked. Biodistribution and PET studies showed that (18)F-(2S,4R)4-fluoroglutamine localized in tumors with a higher uptake than in surrounding muscle and liver tissues. Data suggest that certain tumor cells may use glutamine for energy production. CONCLUSION: The results support that (18)F-(2S,4R)4-fluoroglutamine is selectively taken up and trapped by tumor cells. It may be useful as a novel metabolic tracer for tumor imaging.

A versatile synthetic route to prepare all four stereoisomeric 4-fluoro-glutamines was developed by exploiting a Passerini three-component reaction. The skeleton of 4-substituted glutamine derivatives was efficiently constructed. Subsequent four-step reactions, highlighted by a "neutralized" TASF fluorination, provided the desired products with high yields and excellent optical purity. The optically pure fluorine-18 labeled 4-fluoroglutamines were also successfully prepared using either a 18-crown-6/KHCO(3) or K[222]/K(2)CO(3) catalysis system. Preliminary cell uptake and inhibition studies using the 9L tumor cells and SF188(Bcl-xL) tumor cells (a glutamine addicted tumor derived from glioblastoma) provided strong evidence for their potential application in conjunction with positron emission tomography (PET) for in vivo imaging of tumors, which use glutamine as an alternative energy source.

Most cancers depend on a high rate of aerobic glycolysis for their continued growth and survival. Paradoxically, some cancer cell lines also display addiction to glutamine despite the fact that glutamine is a nonessential amino acid that can be synthesized from glucose. The high rate of glutamine uptake exhibited by glutamine-dependent cells does not appear to result solely from its role as a nitrogen donor in nucleotide and amino acid biosynthesis. Instead, glutamine plays a required role in the uptake of essential amino acids and in maintaining activation of TOR (target of rapamycin) kinase. Moreover, in many cancer cells, glutamine is the primary mitochondrial substrate and is required for maintenance of mitochondrial membrane potential and integrity and for support of the NADPH production needed for redox control and macromolecular synthesis.

The somatic mutations in cytosolic isocitrate dehydrogenase 1 (IDH1) observed in gliomas can lead to the production of 2-hydroxyglutarate (2HG). Here, we report that tumor 2HG is elevated in a high percentage of patients with cytogenetically normal acute myeloid leukemia (AML). Surprisingly, less than half of cases with elevated 2HG possessed IDH1 mutations. The remaining cases with elevated 2HG had mutations in IDH2, the mitochondrial homolog of IDH1. These data demonstrate that a shared feature of all cancer-associated IDH mutations is production of the oncometabolite 2HG. Furthermore, AML patients with IDH mutations display a significantly reduced number of other well characterized AML-associated mutations and/or associated chromosomal abnormalities, potentially implicating IDH mutation in a distinct mechanism of AML pathogenesis.

Mammalian cells fuel their growth and proliferation through the catabolism of two main substrates: glucose and glutamine. Most of the remaining metabolites taken up by proliferating cells are not catabolized, but instead are used as building blocks during anabolic macromolecular synthesis. Investigations of phosphoinositol 3-kinase (PI3K) and its downstream effector AKT have confirmed that these oncogenes play a direct role in stimulating glucose uptake and metabolism, rendering the transformed cell addicted to glucose for the maintenance of survival. In contrast, less is known about the regulation of glutamine uptake and metabolism. Here, we report that the transcriptional regulatory properties of the oncogene Myc coordinate the expression of genes necessary for cells to engage in glutamine catabolism that exceeds the cellular requirement for protein and nucleotide biosynthesis. A consequence of this Myc-dependent glutaminolysis is the reprogramming of mitochondrial metabolism to depend on glutamine catabolism to sustain cellular viability and TCA cycle anapleurosis. The ability of Myc-expressing cells to engage in glutaminolysis does not depend on concomitant activation of PI3K or AKT. The stimulation of mitochondrial glutamine metabolism resulted in reduced glucose carbon entering the TCA cycle and a decreased contribution of glucose to the mitochondrial-dependent synthesis of phospholipids. These data suggest that oncogenic levels of Myc induce a transcriptional program that promotes glutaminolysis and triggers cellular addiction to glutamine as a bioenergetic substrate.

Under hypoxic conditions, cells suppress energy-intensive mRNA translation by modulating the mammalian target of rapamycin (mTOR) and pancreatic eIF2alpha kinase (PERK) pathways. Much is known about hypoxic inhibition of mTOR activity; however, the cellular processes activating PERK remain unclear. Since hypoxia is known to increase intracellular reactive oxygen species (ROS), we hypothesized that hypoxic ROS regulate mTOR and PERK to control mRNA translation and cell survival. Our data indicate that although exogenous ROS inhibit mTOR, eIF2alpha, and eEF2, mTOR and eEF2 were largely refractory to ROS generated under moderate hypoxia (0.5% O(2)). In direct contrast, the PERK/eIF2alpha/ATF4 integrated stress response (ISR) was activated by hypoxic ROS and contributed to global protein synthesis inhibition and adaptive ATF4-mediated gene expression. The ISR as well as exogenous growth factors were critical for cell viability during extended hypoxia, since ISR inhibition decreased the viability of cells deprived of O(2) and growth factors. Collectively, our data support an important role for ROS in hypoxic cell survival. Under conditions of moderate hypoxia, ROS induce the ISR, thereby promoting energy and redox homeostasis and enhancing cellular survival.