Does Tumor FDG-PET Avidity Represent Enhanced Glycolytic Metabolism in Non-Small Cell Lung Cancer?

BACKGROUND: In non-small cell lung cancer (NSCLC), 18fluoro-2-deoxyglucose-positron emission tomography (FDG-PET) assists in diagnosis, staging, and evaluating treatment response. One variable of FDG-PET, the maximum standard uptake value (SUVm), is considered an objective measure of glucose uptake. However, little is known about the fate of glucose in FDG-avid lung tumors in vivo. This study used stable glucose isotope tracing to determine whether the SUVm predicts glycolytic metabolism or other glucose fates in tumors. METHODS: In this prospective Institutional Review Board-approved clinical trial, 52 untreated potentially resectable confirmed NSCLC patients underwent FDG-PET computed tomography. During the surgical procedure, the patients were infused with 13C-labeled glucose. Blood, tumor, and normal lung samples were analyzed by mass spectrometry to determine 13C enrichment in glycolytic intermediates. These values were compared with clinical variables, including SUVm, maximum tumor diameter, stage, grade, and MIB-1/Ki67 proliferation index. RESULTS: For each patient, 13C enrichment in each metabolite was compared between tumor and adjacent lung. Although all tumors metabolized glucose, SUVm did not correlate with glycolytic intermediate labeling. Rather, SUVm correlated with markers indicating the use of other respiratory substrates, including lactate, and with the proliferation index. CONCLUSIONS: SUVm does not correlate with glycolytic metabolism in human NSCLC but does correlate with the proliferation index, suggesting that SUVm predicts glucose use by pathways other than glycolysis. These pathways may offer alternative therapeutic targets, including biosynthetic pathways required for cell proliferation. The research techniques in this study offer the opportunity to understand the relationships between SUVm, tumor metabolism, and therapeutic vulnerabilities in human NSCLCs.

Insights into transcriptional silencing and anti-silencing in Shigella flexneri: a detailed molecular analysis of the icsP virulence locus.

Transcriptional silencing and anti-silencing mechanisms modulate bacterial physiology and virulence in many human pathogens. In Shigella species, many virulence plasmid genes are silenced by the histone-like nucleoid structuring protein H-NS and anti-silenced by the virulence gene regulator VirB. Despite the key role that these regulatory proteins play in Shigella virulence, their mechanisms of transcriptional control remain poorly understood. Here, we characterize the regulatory elements and their relative spacing requirements needed for the transcriptional silencing and anti-silencing of icsP, a locus that requires remotely located regulatory elements for both types of transcriptional control. Our findings highlight the flexibility of the regulatory elements' positions with respect to each other, and yet, a molecular roadblock docked between the VirB binding site and the upstream H-NS binding region abolishes transcriptional anti-silencing by VirB, providing insight into transcriptional anti-silencing. Our study also raises the need to re-evaluate the currently proposed VirB binding site. Models of transcriptional silencing and anti-silencing at this genetic locus are presented, and the implications for understanding these regulatory mechanisms in bacteria are discussed.

Lactate Metabolism in Human Lung Tumors.

Cancer cells consume glucose and secrete lactate in culture. It is unknown whether lactate contributes to energy metabolism in living tumors. We previously reported that human non-small-cell lung cancers (NSCLCs) oxidize glucose in the tricarboxylic acid (TCA) cycle. Here, we show that lactate is also a TCA cycle carbon source for NSCLC. In human NSCLC, evidence of lactate utilization was most apparent in tumors with high 18fluorodeoxyglucose uptake and aggressive oncological behavior. Infusing human NSCLC patients with 13C-lactate revealed extensive labeling of TCA cycle metabolites. In mice, deleting monocarboxylate transporter-1 (MCT1) from tumor cells eliminated lactate-dependent metabolite labeling, confirming tumor-cell-autonomous lactate uptake. Strikingly, directly comparing lactate and glucose metabolism in vivo indicated that lactate's contribution to the TCA cycle predominates. The data indicate that tumors, including bona fide human NSCLC, can use lactate as a fuel in vivo.

Metabolic Heterogeneity in Human Lung Tumors.

Non-small cell lung cancer (NSCLC) is heterogeneous in the genetic and environmental parameters that influence cell metabolism in culture. Here, we assessed the impact of these factors on human NSCLC metabolism in vivo using intraoperative (13)C-glucose infusions in nine NSCLC patients to compare metabolism between tumors and benign lung. While enhanced glycolysis and glucose oxidation were common among these tumors, we observed evidence for oxidation of multiple nutrients in each of them, including lactate as a potential carbon source. Moreover, metabolically heterogeneous regions were identified within and between tumors, and surprisingly, our data suggested potential contributions of non-glucose nutrients in well-perfused tumor areas. Our findings not only demonstrate the heterogeneity in tumor metabolism in vivo but also highlight the strong influence of the microenvironment on this feature.

TCA Cycle and Mitochondrial Membrane Potential Are Necessary for Diverse Biological Functions.

Mitochondrial metabolism is necessary for the maintenance of oxidative TCA cycle function and mitochondrial membrane potential. Previous attempts to decipher whether mitochondria are necessary for biological outcomes have been hampered by genetic and pharmacologic methods that simultaneously disrupt multiple functions linked to mitochondrial metabolism. Here, we report that inducible depletion of mitochondrial DNA (ρ(ο) cells) diminished respiration, oxidative TCA cycle function, and the mitochondrial membrane potential, resulting in diminished cell proliferation, hypoxic activation of HIF-1, and specific histone acetylation marks. Genetic reconstitution only of the oxidative TCA cycle function specifically in these inducible ρ(ο) cells restored metabolites, resulting in re-establishment of histone acetylation. In contrast, genetic reconstitution of the mitochondrial membrane potential restored ROS, which were necessary for hypoxic activation of HIF-1 and cell proliferation. These results indicate that distinct mitochondrial functions associated with respiration are necessary for cell proliferation, epigenetics, and HIF-1 activation.

Metabolic plasticity maintains proliferation in pyruvate dehydrogenase deficient cells.

BACKGROUND: Pyruvate dehydrogenase (PDH) occupies a central node of intermediary metabolism, converting pyruvate to acetyl-CoA, thus committing carbon derived from glucose to an aerobic fate rather than an anaerobic one. Rapidly proliferating tissues, including human tumors, use PDH to generate energy and macromolecular precursors. However, evidence supports the benefits of constraining maximal PDH activity under certain contexts, including hypoxia and oncogene-induced cell growth. Although PDH is one of the most widely studied enzyme complexes in mammals, its requirement for cell growth is unknown. In this study, we directly addressed whether PDH is required for mammalian cells to proliferate. RESULTS: We genetically suppressed expression of the PDHA1 gene encoding an essential subunit of the PDH complex and characterized the effects on intermediary metabolism and cell proliferation using a combination of stable isotope tracing and growth assays. Surprisingly, rapidly dividing cells tolerated loss of PDH activity without major effects on proliferative rates in complete medium. PDH suppression increased reliance on extracellular lipids, and in some cell lines, reducing lipid availability uncovered a modest growth defect that could be completely reversed by providing exogenous-free fatty acids. PDH suppression also shifted the source of lipogenic acetyl-CoA from glucose to glutamine, and this compensatory pathway required a net reductive isocitrate dehydrogenase (IDH) flux to produce a source of glutamine-derived acetyl-CoA for fatty acids. By deleting the cytosolic isoform of IDH (IDH1), the enhanced contribution of glutamine to the lipogenic acetyl-CoA pool during PDHA1 suppression was eliminated, and growth was modestly suppressed. CONCLUSIONS: Although PDH suppression substantially alters central carbon metabolism, the data indicate that rapid cell proliferation occurs independently of PDH activity. Our findings reveal that this central enzyme is essentially dispensable for growth and proliferation of both primary cells and established cell lines. We also identify the compensatory mechanisms that are activated under PDH deficiency, namely scavenging of extracellular lipids and lipogenic acetyl-CoA production from reductive glutamine metabolism through IDH1.

In vivo analysis of lung cancer metabolism: nothing like the real thing.

Cancer cells exhibit altered metabolism compared with that of the surrounding tissue. There is hope that these reprogrammed metabolic pathways in tumors hold the key to advances for both cancer imaging and therapy. Translation of observations in cultured cancer cells to live tumors, however, has proven to be highly complex, and robust methods to analyze metabolic activity in primary human tumors are sorely needed. In this issue of the JCI, Sellers et al. use perioperative administration of isotope-labeled glucose to lung cancer patients to differentiate metabolic pathways between tumors and benign lung. They identify pyruvate carboxylation, a reaction that enables glucose-derived carbon to replenish TCA cycle intermediates, as a key component of anabolic metabolism in tumor cells.

Glutamine oxidation maintains the TCA cycle and cell survival during impaired mitochondrial pyruvate transport.

Alternative modes of metabolism enable cells to resist metabolic stress. Inhibiting these compensatory pathways may produce synthetic lethality. We previously demonstrated that glucose deprivation stimulated a pathway in which acetyl-CoA was formed from glutamine downstream of glutamate dehydrogenase (GDH). Here we show that import of pyruvate into the mitochondria suppresses GDH and glutamine-dependent acetyl-CoA formation. Inhibiting the mitochondrial pyruvate carrier (MPC) activates GDH and reroutes glutamine metabolism to generate both oxaloacetate and acetyl-CoA, enabling persistent tricarboxylic acid (TCA) cycle function. Pharmacological blockade of GDH elicited largely cytostatic effects in culture, but these effects became cytotoxic when combined with MPC inhibition. Concomitant administration of MPC and GDH inhibitors significantly impaired tumor growth compared to either inhibitor used as a single agent. Together, the data define a mechanism to induce glutaminolysis and uncover a survival pathway engaged during compromised supply of pyruvate to the mitochondria.

Heptanol application to the mouse round window: a model for studying cochlear lateral wall regeneration.

OBJECTIVE: Identify cells supporting cochlear lateral wall regeneration. STUDY DESIGN: Prospective controlled trial. SETTING: Laboratory. Human presbyacusis occurs, in part, secondary to age-related degeneration of cochlear lateral wall structures such as the stria vascularis and spiral ligament fibrocytes. This degeneration is likely linked to the diminished regenerative capacity of lateral wall cells with age. While lateral wall regeneration is known to occur after an acute insult, this process remains poorly understood and the cells capable of self-replication unidentified. We hypothesized that spiral ligament fibrocytes constitute these proliferative cells. SUBJECTS AND METHODS: To test the hypothesis, an acute ototoxic insult was created in 65 normal-hearing, young adult mice via cochlear exposure to heptanol. Sacrifice occurred at 1 to 60 days posttreatment. Auditory brainstem responses, 5-ethynyl-2'-deoxyuridine assay, and immunostaining were used to assess regeneration. RESULTS: Posttreatment hearing thresholds were elevated in nearly all treated mice. Selective fibrocyte apoptosis and strial injury were observed at the time of peak hearing loss around 1 to 7 days posttreatment. Cellular proliferation was detected in the region of type II fibrocytes during this time. Hearing thresholds plateaued at 7 days posttreatment followed by a significant recovery of both hearing and morphologic appearance. Permanent outer hair cell degeneration was observed. CONCLUSIONS: Heptanol application to the round window of young adult mice is a rapid, selective, and reliable technique for investigating proliferation in the cochlear lateral wall. The data indirectly showed that spiral ligament fibrocytes may be the proliferative cells of the cochlear lateral wall. Further studies of this process are needed.

Glutamine and cancer: cell biology, physiology, and clinical opportunities.

Glutamine is an abundant and versatile nutrient that participates in energy formation, redox homeostasis, macromolecular synthesis, and signaling in cancer cells. These characteristics make glutamine metabolism an appealing target for new clinical strategies to detect, monitor, and treat cancer. Here we review the metabolic functions of glutamine as a super nutrient and the surprising roles of glutamine in supporting the biological hallmarks of malignancy. We also review recent efforts in imaging and therapeutics to exploit tumor cell glutamine dependence, discuss some of the challenges in this arena, and suggest a disease-focused paradigm to deploy these emerging approaches.

Characterization of the ospZ promoter in Shigella flexneri and its regulation by VirB and H-NS.

OspZ is an effector protein of the type III secretion system in Shigella spp. that downregulates the human inflammatory response during bacterial infection. The ospZ gene is located on the large virulence plasmid of Shigella. Many genes on this plasmid are transcriptionally repressed by the nucleoid structuring protein H-NS and derepressed by VirB, a DNA-binding protein that displays homology to the plasmid partitioning proteins ParB and SopB. In this study, we characterized the ospZ promoter and investigated its regulation by H-NS and VirB in Shigella flexneri. We show that H-NS represses and VirB partially derepresses the ospZ promoter. H-NS-mediated repression requires sequences located between -731 and -412 relative to the beginning of the ospZ gene. Notably, the VirB-dependent derepression of ospZ requires the same VirB binding sites as are required for the VirB-dependent derepression of the divergent icsP gene. These sites are centered 425 bp upstream of the ospZ gene but over 1 kb upstream of the icsP transcription start site. Although these VirB binding sites lie closer to ospZ than icsP, the VirB-dependent increase in ospZ promoter activity is lower than that observed at the icsP promoter. This indicates that the proximity of VirB binding sites to Shigella promoters does not necessarily correlate with the level of VirB-dependent derepression. These findings have implications for virulence gene regulation in Shigella and other pathogens that control gene expression using mechanisms of transcriptional repression and derepression.

Two promoters and two translation start sites control the expression of the Shigella flexneri outer membrane protease IcsP.

The Shigella flexneri outer membrane protease IcsP proteolytically cleaves the actin-based motility protein IcsA from the bacterial surface. The icsP gene is monocistronic and lies downstream of an unusually large intergenic region on the Shigella virulence plasmid. In silico analysis of this region predicts a second transcription start site 84 bp upstream of the first. Primer extension analyses and beta-galactosidase assays demonstrate that both transcription start sites are used. Both promoters are regulated by the Shigella virulence gene regulator VirB and both respond similarly to conditions known to influence Shigella virulence gene expression (iron concentration, pH, osmotic pressure, and phase of growth). The newly identified promoter lies upstream of a Shine-Dalgarno sequence and second 5'-ATG-3', which is in frame with the annotated icsP gene. The use of either translation start site leads to the production of IcsP capable of proteolytically cleaving IcsA. A bioinformatic scan of the Shigella genome reveals multiple occurrences of in-frame translation start sites associated with putative Shine-Dalgarno sequences, immediately upstream and downstream of annotated open reading frames. Taken together, our observations support the possibility that the use of in-frame translation start sites may generate different protein isoforms, thereby expanding the proteome encoded by bacterial genomes.