Glucose and glutamine metabolism in relation to mutational status in NSCLC histological subtypes.
ABSTRACT: BACKGROUND:Both hypoxia and oncogenic mutations rewire tumor metabolism. In this study, glucose and glutamine metabolism-related markers were examined in stage I - resectable stage IIIA non-small cell lung cancer (NSCLC). Furthermore, expression of metabolism-related markers was correlated with mutational status to examine mutations associated with rewired tumor metabolism. METHODS:Mutation analysis was performed for 97 tumors. Glucose and glutamine metabolism-related marker expression was measured by immunofluorescent staining (protein) and qPCR (mRNA) (n = 81). RESULTS:Glutamine metabolism-related markers were significantly higher in adeno- than squamous cell NSCLCs. Glucose transporter 1 (GLUT1) protein expression was higher in solid compared to lepidic adenocarcinomas (P?
Project description:Efforts to target glutamine metabolism for cancer therapy have focused on the glutaminase isozyme GLS. The importance of the other isozyme, GLS2, in cancer has remained unclear, and it has been described as a tumor suppressor in some contexts. Here, we report that GLS2 is upregulated and essential in luminal-subtype breast tumors, which account for >70% of breast cancer incidence. We show that GLS2 expression is elevated by GATA3 in luminal-subtype cells but suppressed by promoter methylation in basal-subtype cells. Although luminal breast cancers resist GLS-selective inhibitors, we find that they can be targeted with a dual-GLS/GLS2 inhibitor. These results establish a critical role for GLS2 in mammary tumorigenesis and advance our understanding of how to target glutamine metabolism in cancer.
Project description:We identified a p53 target gene, phosphate-activated mitochondrial glutaminase (GLS2), a key enzyme in conversion of glutamine to glutamate, and thereby a regulator of glutathione (GSH) synthesis and energy production. GLS2 expression is induced in response to DNA damage or oxidative stress in a p53-dependent manner, and p53 associates with the GLS2 promoter. Elevated GLS2 facilitates glutamine metabolism and lowers intracellular reactive oxygen species (ROS) levels, resulting in an overall decrease in DNA oxidation as determined by measurement of 8-OH-dG content in both normal and stressed cells. Further, siRNA down-regulation of either GLS2 or p53 compromises the GSH-dependent antioxidant system and increases intracellular ROS levels. High ROS levels following GLS2 knockdown also coincide with stimulation of p53-induced cell death. We propose that GLS2 control of intracellular ROS levels and the apoptotic response facilitates the ability of p53 to protect cells from accumulation of genomic damage and allows cells to survive after mild and repairable genotoxic stress. Indeed, overexpression of GLS2 reduces the growth of tumor cells and colony formation. Further, compared with normal tissue, GLS2 expression is reduced in liver tumors. Thus, our results provide evidence for a unique metabolic role for p53, linking glutamine metabolism, energy, and ROS homeostasis, which may contribute to p53 tumor suppressor function.
Project description:We have previously identified solute-linked carrier family A1 member 5 (SLC1A5) as an overexpressed protein in a shotgun proteomic analysis of stage I non-small cell lung cancer (NSCLC) when compared with matched controls. We hypothesized that overexpression of SLC1A5 occurs to meet the metabolic demand for lung cancer cell growth and survival.To test our hypothesis, we first analyzed the protein expression of SLC1A5 in archival lung cancer tissues by immunohistochemistry and immunoblotting (N = 98) and in cell lines (N = 36). To examine SLC1A5 involvement in amino acid transportation, we conducted kinetic analysis of l-glutamine (Gln) uptake in lung cancer cell lines in the presence and absence of a pharmacologic inhibitor of SLC1A5, gamma-l-Glutamyl-p-Nitroanilide (GPNA). Finally, we examined the effect of Gln deprivation and uptake inhibition on cell growth, cell-cycle progression, and growth signaling pathways of five lung cancer cell lines.Our results show that (i) SLC1A5 protein is expressed in 95% of squamous cell carcinomas (SCC), 74% of adenocarcinomas (ADC), and 50% of neuroendocrine tumors; (ii) SLC1A5 is located at the cytoplasmic membrane and is significantly associated with SCC histology and male gender; (iii) 68% of Gln is transported in a Na(+)-dependent manner, 50% of which is attributed to SLC1A5 activity; and (iv) pharmacologic and genetic targeting of SLC1A5 decreased cell growth and viability in lung cancer cells, an effect mediated in part by mTOR signaling.These results suggest that SLC1A5 plays a key role in Gln transport controlling lung cancer cells' metabolism, growth, and survival.
Project description:Many tumor cells are fueled by altered metabolism and increased glutamine (Gln) dependence. We identify regulation of the L-glutamine carrier proteins SLC1A5 and SLC38A2 (SLC1A5/38A2) by the ubiquitin ligase RNF5. Paclitaxel-induced ER stress to breast cancer (BCa) cells promotes RNF5 association, ubiquitination, and degradation of SLC1A5/38A2. This decreases Gln uptake, levels of TCA cycle components, mTOR signaling, and proliferation while increasing autophagy and cell death. Rnf5-deficient MMTV-PyMT mammary tumors were less differentiated and showed elevated SLC1A5 expression. Whereas RNF5 depletion in MDA-MB-231 cells promoted tumorigenesis and abolished paclitaxel responsiveness, SLC1A5/38A2 knockdown elicited opposing effects. Inverse RNF5(hi)/SLC1A5/38A2(lo) expression was associated with positive prognosis in BCa. Thus, RNF5 control of Gln uptake underlies BCa response to chemotherapies.
Project description:The altered metabolism of tumors has been considered a target for anticancer therapy. However, the relationship between distinct tumor-initiating lesions and anomalies of tumor metabolism in vivo has not been addressed. We report that MYC-induced mouse liver tumors significantly increase both glucose and glutamine catabolism, whereas MET-induced liver tumors use glucose to produce glutamine. Increased glutamine catabolism in MYC-induced liver tumors is associated with decreased levels of glutamine synthetase (Glul) and the switch from Gls2 to Gls1 glutaminase. In contrast to liver tumors, MYC-induced lung tumors display increased expression of both Glul and Gls1 and accumulate glutamine. We also show that inhibition of Gls1 kills cells that overexpress MYC and catabolize glutamine. Our results suggest that the metabolic profiles of tumors are likely to depend on both the genotype and tissue of origin and have implications regarding the design of therapies targeting tumor metabolism.
Project description:Malignant tumors reprogram cellular metabolism to support cancer cell proliferation and survival. Although most cancers depend on a high rate of aerobic glycolysis, many cancer cells also display addiction to glutamine. Glutamine transporters and glutaminase activity are critical for glutamine metabolism in tumor cells. We found that the receptor tyrosine kinase EphA2 activated the TEAD family transcriptional coactivators YAP and TAZ (YAP/TAZ), likely in a ligand-independent manner, to promote glutamine metabolism in cells and mouse models of HER2-positive breast cancer. Overexpression of EphA2 induced the nuclear accumulation of YAP and TAZ and increased the expression of YAP/TAZ target genes. Inhibition of the GTPase Rho or the kinase ROCK abolished EphA2-dependent YAP/TAZ nuclear localization. Silencing YAP or TAZ substantially reduced the amount of intracellular glutamate through decreased expression of SLC1A5 and GLS, respectively, genes that encode proteins that promote glutamine uptake and metabolism. The regulatory DNA elements of both SLC1A5 and GLS contain TEAD binding sites and were bound by TEAD4 in an EphA2-dependent manner. In patient breast cancer tissues, EphA2 expression positively correlated with that of YAP and TAZ, as well as that of GLS and SLC1A5 Although high expression of EphA2 predicted enhanced metastatic potential and poor patient survival, it also rendered HER2-positive breast cancer cells more sensitive to glutaminase inhibition. The findings define a previously unknown mechanism of EphA2-mediated glutaminolysis through YAP/TAZ activation in HER2-positive breast cancer and identify potential therapeutic targets in patients.
Project description:OBJECTIVE:It is well established that the liver-specific miR-122, a bona fide tumor suppressor, plays a critical role in lipid homeostasis. However, its role, if any, in amino acid metabolism has not been explored. Since glutamine (Gln) is a critical energy and anaplerotic source for mammalian cells, we assessed Gln metabolism in control wild type (WT) mice and miR-122 knockout (KO) mice by stable isotope resolved metabolomics (SIRM) studies. METHODS:Six-to eight-week-old WT and KO mice and 12- to 15-month-old liver tumor-bearing mice were injected with [U-13C5,15N2]-L-Gln, and polar metabolites from the liver tissues were analyzed by nuclear magnetic resonance (NMR) imaging and ion chromatography-mass spectrometry (IC-MS). Gln-metabolism was also assessed in a Gln-dependent hepatocellular carcinoma (HCC) cell line (EC4). Expressions of glutaminases (Gls and Gls2) were analyzed in mouse livers and human primary HCC samples. RESULTS:The results showed that loss of miR-122 promoted glutaminolysis but suppressed gluconeogenesis in mouse livers as evident from the buildup of 13C- and/or 15N-Glu and decrease in glucose-6-phosphate (G6P) levels, respectively, in KO livers. Enhanced glutaminolysis is consistent with the upregulation of expressions of Gls (kidney-type glutaminase) and Slc1a5, a neutral amino acid transporter in KO livers. Both Gls and Slc1a5 were confirmed as direct miR-122 targets by the respective 3'-UTR-driven luciferase assays. Importantly, expressions of Gls and Slc1a5 as well as glutaminase activity were suppressed in a Gln-dependent HCC (EC4) cell line transfected with miR-122 mimic that resulted in decreased 13C-Gln, 13C-á-ketoglutarate, 13C-isocitrate, and 13C-citrate levels. In contrast, 13C-phosphoenolpyruvate and 13C-G6P levels were elevated in cells expressing ectopic miR-122, suggesting enhanced gluconeogenesis. Finally, The Cancer Genome Atlas-Liver Hepatocellular Carcinoma (TCGA-LIHC) database analysis showed that expression of GLS is negatively correlated with miR-122 in primary human HCCs, and the upregulation of GLS RNA is associated with higher tumor grade. More importantly, patients with higher expressions of GLS or SLC1A5 in tumors exhibited poor survival compared with those expressing lower levels of these proteins. CONCLUSIONS:Collectively, these results show that miR-122 modulates Gln metabolism both in vitro and in vivo, implicating the therapeutic potential of miR-122 in HCCs that exhibit relatively high GLS levels.
Project description:<h4>Background</h4>In recent years, metabolic reprogramming has been identified as a hallmark of cancer. Accumulating evidence suggests that glutamine metabolism plays a crucial role in oncogenesis and the tumor microenvironment. In this study, we aimed to perform a systematic and comprehensive analysis of six key metabolic node genes involved in the dynamic regulation of glutamine metabolism (referred to as GLNM regulators) across 33 types of cancer.<h4>Methods</h4>We analyzed the gene expression, epigenetic regulation, and genomic alterations of six key GLNM regulators, including <i>SLC1A5</i>, <i>SLC7A5</i>, <i>SLC3A2</i>, <i>SLC7A11</i>, <i>GLS</i>, and <i>GLS2</i>, in pan-cancer using several open-source platforms and databases. Additionally, we investigated the impacts of these gene expression changes on clinical outcomes, drug sensitivity, and the tumor microenvironment. We also attempted to investigate the upstream microRNA-mRNA molecular networks and the downstream signaling pathways involved in order to uncover the potential molecular mechanisms behind metabolic reprogramming.<h4>Results</h4>We found that the expression levels of GLNM regulators varied across cancer types and were related to several genomic and immunological characteristics. While the immune scores were generally lower in the tumors with higher gene expression, the types of immune cell infiltration showed significantly different correlations among cancer types, dividing them into two clusters. Furthermore, we showed that elevated GLNM regulators expression was associated with poor overall survival in the majority of cancer types. Lastly, the expression of GLNM regulators was significantly associated with PD-L1 expression and drug sensitivity.<h4>Conclusions</h4>The elevated expression of GLNM regulators was associated with poorer cancer prognoses and a cold tumor microenvironment, providing novel insights into cancer treatment and possibly offering alternative options for the treatment of clinically refractory cancers.
Project description:A key hallmark of cancer, altered metabolism, is central to cancer pathogenesis and therapy resistance. Robust glutamine metabolism is among cellular processes regulating tumor progression and responsiveness to therapy in a number of cancers, including melanoma and breast cancer. Among mechanisms underlying the increase in glutamine metabolism in tumors is enhanced glutamine uptake mediated by the glutamine transporters, with SLC1A5 (also known as ASCT2) shown to play a predominant role. Correspondingly, increased SLC1A5 expression coincides with poorer survival in patients with breast cancer and melanoma. Therefore, we performed an image-based screen to identify small molecules that are able to prevent the localization of SLC1A5 to the plasma membrane without impacting cell shape. From 7,000 small molecules, nine were selected as hits, of which one (IMD-0354) qualified for further detailed functional assessment. IMD-0354 was confirmed as a potent inhibitor of glutamine uptake that attained sustained low intracellular glutamine levels. Concomitant with its inhibition of glutamine uptake, IMD-0354 attenuated mTOR signaling, suppressed two- and three-dimensional growth of melanoma cells, and induced cell-cycle arrest, autophagy, and apoptosis. Pronounced effect of IMD-0354 was observed in different tumor-derived cell lines, compared with nontransformed cells. RNA-sequencing analysis identified the unfolded protein response, cell cycle, and response (DNA damage response pathways) to be affected by IMD-0354. Combination of IMD-0354 with GLS1 or LDHA inhibitors enhanced melanoma cell death. <i>In vivo</i>, IMD-0354 suppressed melanoma growth in a xenograft model. As a modulator of glutamine metabolism, IMD-0354 may serve as an important therapeutic and experimental tool that deserves further examination.