Metabolic reprogramming of macrophages: Glucose transporter 1 (GLUT1)-mediated glucose metabolism drives a proinflammatory phenotype

Glucose is a critical component in the proinflammatory response of macrophages (MΦs). However, the contribution of glucose transporters (GLUTs) and the mechanisms regulating subsequent glucose metabolism in the inflammatory response are not well understood. Because MΦs contribute to obesity-induced inflammation, it is important to understand how substrate metabolism may alter inflammatory function. We report that GLUT1 (SLC2A1) is the primary rate-limiting glucose transporter on proinflammatory-polarized MΦs. Furthermore, in high fat diet-fed rodents, MΦs in crown-like structures and inflammatory loci in adipose and liver, respectively, stain positively for GLUT1. We hypothesized that metabolic reprogramming via increased glucose availability could modulate the MΦ inflammatory response. To increase glucose uptake, we stably overexpressed the GLUT1 transporter in RAW264.7 MΦs (GLUT1-OE MΦs). Cellular bioenergetics analysis, metabolomics, and radiotracer studies demonstrated that GLUT1 overexpression resulted in elevated glucose uptake and metabolism, increased pentose phosphate pathway intermediates, with a complimentary reduction in cellular oxygen consumption rates. Gene expression and proteome profiling analysis revealed that GLUT1-OE MΦs demonstrated a hyperinflammatory state characterized by elevated secretion of inflammatory mediators and that this effect could be blunted by pharmacologic inhibition of glycolysis. Finally, reactive oxygen species production and evidence of oxidative stress were significantly enhanced in GLUT1-OEMΦs; antioxidant treatment blunted the expression of inflammatory mediators such as PAI-1 (plasminogen activator inhibitor 1), suggesting that glucose-mediated oxidative stress was driving the proinflammatory response. Our results indicate that increased utilization of glucose induced a ROS-driven proinflammatory phenotype in MΦs, which may play an integral role in the promotion of obesity-associated insulin resistance

Von Hippel-Lindau mutations disrupt vascular patterning and maturation via Notch

Von Hippel-Lindau (VHL) gene mutations induce neural tissue hemangioblastomas, as well as highly vascularized clear cell renal cell carcinomas (ccRCCs). Pathological vessel remodeling arises from misregulation of HIFs and VEGF, among other genes. Variation in disease penetrance has long been recognized in relation to genotype. We show Vhl mutations also disrupt Notch signaling, causing mutation-specific vascular abnormalities, e.g., type 1 (null) vs. type 2B (murine G518A representing human R167Q). In conditional mutation retina vasculature, Vhl-null mutation (i.e., UBCCreER/+Vhlfl/fl) had little effect on initial vessel branching, but it severely reduced arterial and venous branching at later stages. Interestingly, this mutation accelerated arterial maturation, as observed in retina vessel morphology and aberrant α-smooth muscle actin localization, particularly in vascular pericytes. RNA sequencing analysis identified gene expression changes within several key pathways, including Notch and smooth muscle cell contractility. Notch inhibition failed to reverse later-stage branching defects but rescued the accelerated arterialization. Retinal vessels harboring the type 2B Vhl mutation (i.e., UBCCreER/+Vhlfl/2B) displayed stage-specific changes in vessel branching and an advanced progression toward an arterial phenotype. Disrupting Notch signaling in type 2B mutants increased both artery and vein branching and restored arterial maturation toward nonmutant levels. By revealing differential effects of the null and type 2B Vhl mutations on vessel branching and maturation, these data may provide insight into the variability of VHL-associated vascular changes - particularly the heterogeneity and aggressiveness in ccRCC vessel growth - and also suggest Notch pathway targets for treating VHL syndrome

Combined deletion of Glut1 and Glut3 impairs lung adenocarcinoma growth.

Glucose utilization increases in tumors, a metabolic process that is observed clinically by <sup>18</sup> F-fluorodeoxyglucose positron emission tomography ( <sup>18</sup> F-FDG-PET). However, is increased glucose uptake important for tumor cells, and which transporters are implicated in vivo? In a genetically-engineered mouse model of lung adenocarcinoma, we show that the deletion of only one highly expressed glucose transporter, Glut1 or Glut3, in cancer cells does not impair tumor growth, whereas their combined loss diminishes tumor development. <sup>18</sup> F-FDG-PET analyses of tumors demonstrate that Glut1 and Glut3 loss decreases glucose uptake, which is mainly dependent on Glut1. Using <sup>13</sup> C-glucose tracing with correlated nanoscale secondary ion mass spectrometry (NanoSIMS) and electron microscopy, we also report the presence of lamellar body-like organelles in tumor cells accumulating glucose-derived biomass, depending partially on Glut1. Our results demonstrate the requirement for two glucose transporters in lung adenocarcinoma, the dual blockade of which could reach therapeutic responses not achieved by individual targeting

Myeloid SLC2a1-deficient murine model revealed macrophage activation and metabolic phenotype are fueled by GLUT1

Macrophages (MFs) are heterogeneous and metabolically flexible, with metabolism strongly affecting immune activation. A classic response to proinflammatory activation is increased flux through glycolysis with a downregulation of oxidative metabolism, whereas alternative activation is primarily oxidative, which begs the question of whether targeting glucose metabolism is a viable approach to control MF activation. We created a murine model of myeloid-specific glucose transporter GLUT1 (Slc2a1) deletion. Bone marrow-derived MFs (BMDM) from Slc2a1 M -/ - mice failed to uptake glucose and demonstrated reduced glycolysis and pentose phosphate pathway activity. Activated BMDMs displayed elevated metabolism of oleate and glutamine, yet maximal respiratory capacity was blunted in MF lacking GLUT1, demonstrating an incomplete metabolic reprogramming. Slc2a1 M -/ - BMDMs displayed a mixed inflammatory phenotype with reductions of the classically activated pro- and anti-inflammatory markers, yet less oxidative stress. Slc2a1 M -/ - BMDMs had reduced proinflammatory metabolites, whereas metabolites indicative of alternative activation-such as ornithine and polyamines-were greatly elevated in the absence of GLUT1. Adipose tissue MFs of lean Slc2a1 M -/ - mice had increased alternative M2-like activation marker mannose receptor CD206, yet lack of GLUT1 was not a critical mediator in the development of obesity-associated metabolic dysregulation. However, Ldlr-/ - mice lacking myeloid GLUT1 developed unstable atherosclerotic lesions. Defective phagocytic capacity in Slc2a1 M -/ - BMDMs may have contributed to unstable atheroma formation. Together, our findings suggest that although lack of GLUT1 blunted glycolysis and the pentose phosphate pathway, MF were metabolically flexible enough that inflammatory cytokine release was not dramatically regulated, yet phagocytic defects hindered MF function in chronic diseases

An Integrated TCGA Pan-Cancer Clinical Data Resource to Drive High-Quality Survival Outcome Analytics

For a decade, The Cancer Genome Atlas (TCGA) program collected clinicopathologic annotation data along with multi-platform molecular profiles of more than 11,000 human tumors across 33 different cancer types. TCGA clinical data contain key features representing the democratized nature of the data collection process. To ensure proper use of this large clinical dataset associated with genomic features, we developed a standardized dataset named the TCGA Pan-Cancer Clinical Data Resource (TCGA-CDR), which includes four major clinical outcome endpoints. In addition to detailing major challenges and statistical limitations encountered during the effort of integrating the acquired clinical data, we present a summary that includes endpoint usage recommendations for each cancer type. These TCGA-CDR findings appear to be consistent with cancer genomics studies independent of the TCGA effort and provide opportunities for investigating cancer biology using clinical correlates at an unprecedented scale. Analysis of clinicopathologic annotations for over 11,000 cancer patients in the TCGA program leads to the generation of TCGA Clinical Data Resource, which provides recommendations of clinical outcome endpoint usage for 33 cancer types

Measurement of the Bottom-Strange Meson Mixing Phase in the Full CDF Data Set

We report a measurement of the bottom-strange meson mixing phase \beta_s using the time evolution of B0_s -> J/\psi (->\mu+\mu-) \phi (-> K+ K-) decays in which the quark-flavor content of the bottom-strange meson is identified at production. This measurement uses the full data set of proton-antiproton collisions at sqrt(s)= 1.96 TeV collected by the Collider Detector experiment at the Fermilab Tevatron, corresponding to 9.6 fb-1 of integrated luminosity. We report confidence regions in the two-dimensional space of \beta_s and the B0_s decay-width difference \Delta\Gamma_s, and measure \beta_s in [-\pi/2, -1.51] U [-0.06, 0.30] U [1.26, \pi/2] at the 68% confidence level, in agreement with the standard model expectation. Assuming the standard model value of \beta_s, we also determine \Delta\Gamma_s = 0.068 +- 0.026 (stat) +- 0.009 (syst) ps-1 and the mean B0_s lifetime, \tau_s = 1.528 +- 0.019 (stat) +- 0.009 (syst) ps, which are consistent and competitive with determinations by other experiments.Comment: 8 pages, 2 figures, Phys. Rev. Lett 109, 171802 (2012

Designing a broad-spectrum integrative approach for cancer prevention and treatment

Targeted therapies and the consequent adoption of "personalized" oncology have achieved notablesuccesses in some cancers; however, significant problems remain with this approach. Many targetedtherapies are highly toxic, costs are extremely high, and most patients experience relapse after a fewdisease-free months. Relapses arise from genetic heterogeneity in tumors, which harbor therapy-resistantimmortalized cells that have adopted alternate and compensatory pathways (i.e., pathways that are notreliant upon the same mechanisms as those which have been targeted). To address these limitations, aninternational task force of 180 scientists was assembled to explore the concept of a low-toxicity "broad-spectrum" therapeutic approach that could simultaneously target many key pathways and mechanisms. Using cancer hallmark phenotypes and the tumor microenvironment to account for the various aspectsof relevant cancer biology, interdisciplinary teams reviewed each hallmark area and nominated a widerange of high-priority targets (74 in total) that could be modified to improve patient outcomes. For thesetargets, corresponding low-toxicity therapeutic approaches were then suggested, many of which werephytochemicals. Proposed actions on each target and all of the approaches were further reviewed forknown effects on other hallmark areas and the tumor microenvironment. Potential contrary or procar-cinogenic effects were found for 3.9% of the relationships between targets and hallmarks, and mixedevidence of complementary and contrary relationships was found for 7.1%. Approximately 67% of therelationships revealed potentially complementary effects, and the remainder had no known relationship. Among the approaches, 1.1% had contrary, 2.8% had mixed and 62.1% had complementary relationships. These results suggest that a broad-spectrum approach should be feasible from a safety standpoint. Thisnovel approach has potential to be relatively inexpensive, it should help us address stages and types ofcancer that lack conventional treatment, and it may reduce relapse risks. A proposed agenda for futureresearch is offered

Driver Fusions and Their Implications in the Development and Treatment of Human Cancers.

Gene fusions represent an important class of somatic alterations in cancer. We systematically investigated fusions in 9,624 tumors across 33 cancer types using multiple fusion calling tools. We identified a total of 25,664 fusions, with a 63% validation rate. Integration of gene expression, copy number, and fusion annotation data revealed that fusions involving oncogenes tend to exhibit increased expression, whereas fusions involving tumor suppressors have the opposite effect. For fusions involving kinases, we found 1,275 with an intact kinase domain, the proportion of which varied significantly across cancer types. Our study suggests that fusions drive the development of 16.5% of cancer cases and function as the sole driver in more than 1% of them. Finally, we identified druggable fusions involving genes such as TMPRSS2, RET, FGFR3, ALK, and ESR1 in 6.0% of cases, and we predicted immunogenic peptides, suggesting that fusions may provide leads for targeted drug and immune therapy

Perspective on Oncogenic Processes at the End of the Beginning of Cancer Genomics

The Cancer Genome Atlas (TCGA) has catalyzed systematic characterization of diverse genomic alterations underlying human cancers. At this historic junction marking the completion of genomic characterization of over 11,000 tumors from 33 cancer types, we present our current understanding of the molecular processes governing oncogenesis. We illustrate our insights into cancer through synthesis of the findings of the TCGA PanCancer Atlas project on three facets of oncogenesis: (1) somatic driver mutations, germline pathogenic variants, and their interactions in the tumor; (2) the influence of the tumor genome and epigenome on transcriptome and proteome; and (3) the relationship between tumor and the microenvironment, including implications for drugs targeting driver events and immunotherapies. These results will anchor future characterization of rare and common tumor types, primary and relapsed tumors, and cancers across ancestry groups and will guide the deployment of clinical genomic sequencing

The Cancer Genome Atlas Comprehensive Molecular Characterization of Renal Cell Carcinoma

Renal cell carcinoma(RCC) is not a single disease, but several histologically defined cancers with different genetic drivers, clinical courses, and therapeutic responses. The current study evaluated 843 RCC from the three major histologic subtypes, including 488 clear cell RCC, 274 papillary RCC, and 81 chromophobe RCC. Comprehensive genomic and phenotypic analysis of the RCC subtypes reveals distinctive features of each subtype that provide the foundation for the development of subtype-specific therapeutic and management strategies for patients affected with these cancers. Somatic alteration of BAP1, PBRM1, and PTEN and altered metabolic pathways correlated with subtype-specific decreased survival, while CDKN2A alteration, increased DNA hypermethylation, and increases in the immune-related Th2 gene expression signature correlated with decreased survival within all major histologic subtypes. CIMP-RCC demonstrated an increased immune signature, and a uniform and distinct metabolic expression pattern identified a subset of metabolically divergent (MD) ChRCC that associated with extremely poor survival