The γ-glutamyl cycle serves as an amino acids supply system in colorectal cancer organoids under chronic hypoxia.

Malignant tumors are characterized by a hypoxic microenvironment, and metabolic reprogramming is necessary to ensure energy production and oxidative stress resistance. Although the microenvironmental properties of tumors vary under acute and chronic hypoxia, studies on chronic hypoxia-induced metabolic changes are limited. In the present study, we performed a comprehensive metabolic analysis in a chronic hypoxia model using colorectal cancer (CRC) organoids, and identified an amino acid supply system through the γ-glutamyl cycle, a glutathione recycling pathway. We analyzed the metabolic changes caused by hypoxia over time and observed that chronic hypoxia resulted in an increase in 5-oxoproline and a decrease in oxidized glutathione (GSSG) compared to acute hypoxia. These findings suggest that chronic hypoxia induces metabolic changes in the γ-glutamyl cycle. Moreover, inhibition of the γ-glutamyl cycle via γ-glutamyl cyclotransferase (GGCT) and γ-glutamyl transferase 1 (GGT1) knockdown significantly reversed chronic hypoxia-induced upregulation of 5-oxoproline and several amino acids. Notably, GGT1 knockdown downregulated the intracellular levels of γ-glutamyl amino acids. Conclusively, these results indicate that the γ-glutamyl cycle serves as an amino acid supply system in CRC under chronic hypoxia, which provides fresh insight into cancer metabolism under chronic hypoxia.

Plasma and Urinary Metabolomic Analysis of Gout and Asymptomatic Hyperuricemia and Profiling of Potential Biomarkers: A Pilot Study.

Gout results from monosodium urate deposition caused by hyperuricemia, but most individuals with hyperuricemia remain asymptomatic. The pathogenesis of gout remains uncertain. To identify potential biomarkers distinguishing gout from asymptomatic hyperuricemia, we conducted a genetic analysis of urate transporters and metabolomic analysis as a proof-of-concept study, including 33 patients with gout and 9 individuals with asymptomatic hyperuricemia. The variant allele frequencies of rs72552713, rs2231142, and rs3733591, which are related to serum urate levels (SUA) and gout, did not differ between the gout and asymptomatic hyperuricemia groups. In metabolomic analysis, the levels of citrate cycle intermediates, especially 2-ketoglutarate, were higher in patients with gout than in those with asymptomatic hyperuricemia (fold difference = 1.415, p = 0.039). The impact on the TCA cycle was further emphasized in high-risk gout (SUA ≥ 9.0 mg/dL). Of note, urinary nicotinate was the most prominent biomarker differentiating high-risk gout from asymptomatic hyperuricemia (fold difference = 6.515, p = 0.020). Although urate transporters play critical roles in SUA elevation and promote hyperuricemia, this study suggests that the progression from asymptomatic hyperuricemia to gout might be closely related to other genetic and/or environmental factors affecting carbohydrate metabolism and urinary urate excretion.

Metabolic Hallmarks for Purine Nucleotide Biosynthesis in Small Cell Lung Carcinoma.

Small cell lung cancer (SCLC) has a poor prognosis, emphasizing the necessity for developing new therapies. The de novo synthesis pathway of purine nucleotides, which is involved in the malignant growth of SCLC, has emerged as a novel therapeutic target. Purine nucleotides are supplied by two pathways: de novo and salvage. However, the role of the salvage pathway in SCLC and the differences in utilization and crosstalk between the two pathways remain largely unclear. Here, we found that deletion of the HPRT1 gene, which codes for the rate-limiting enzyme of the purine salvage pathway, significantly suppressed tumor growth in vivo in several SCLC cells. We also demonstrated that HPRT1 expression confers resistance to lemetrexol (LMX), an inhibitor of the purine de novo pathway. Interestingly, HPRT1-knockout had less effect on SCLC SBC-5 cells, which are more sensitive to LMX than other SCLC cell lines, suggesting that a preference for either the purine de novo or salvage pathway occurs in SCLC. Furthermore, metabolome analysis of HPRT1-knockout cells revealed increased intermediates in the pentose phosphate pathway and elevated metabolic flux in the purine de novo pathway, indicating compensated metabolism between the de novo and salvage pathways in purine nucleotide biosynthesis. These results suggest that HPRT1 has therapeutic implications in SCLC and provide fundamental insights into the regulation of purine nucleotide biosynthesis. IMPLICATIONS: SCLC tumors preferentially utilize either the de novo or salvage pathway in purine nucleotide biosynthesis, and HPRT1 has therapeutic implications in SCLC.

Rare FGFR fusion genes in cervical cancer and transcriptome-based subgrouping of patients with a poor prognosis.

BACKGROUND: Although cervical cancer is often characterized as preventable, its incidence continues to increase in low- and middle-income countries, underscoring the need to develop novel therapeutics for this disease.This study assessed the distribution of fusion genes across cancer types and used an RNA-based classification to divide cervical cancer patients with a poor prognosis into subgroups. MATERIAL AND METHODS: RNA sequencing of 116 patients with cervical cancer was conducted. Fusion genes were extracted using StarFusion program. To identify a high-risk group for recurrence, 65 patients who received postoperative adjuvant therapy were subjected to non-negative matrix factorization to identify differentially expressed genes between recurrent and nonrecurrent groups. RESULTS: We identified three cases with FGFR3-TACC3 and one with GOPC-ROS1 fusion genes as potential targets. A search of publicly available data from cBioPortal (21,789 cases) and the Center for Cancer Genomics and Advanced Therapeutics (32,608 cases) showed that the FGFR3 fusion is present in 1.5% and 0.6% of patients with cervical cancer, respectively. The frequency of the FGFR3 fusion gene was higher in cervical cancer than in other cancers, regardless of ethnicity. Non-negative matrix factorization identified that the patients were classified into four Basis groups. Pathway enrichment analysis identified more extracellular matrix kinetics dysregulation in Basis 3 and more immune system dysregulation in Basis 4 than in the good prognosis group. CIBERSORT analysis showed that the fraction of M1 macrophages was lower in the poor prognosis group than in the good prognosis group. CONCLUSIONS: The distribution of FGFR fusion genes in patients with cervical cancer was determined by RNA-based analysis and used to classify patients into clinically relevant subgroups.

In vivo Drug Screening to Identify Anti-metastatic Drugs in Twist1a-ERT2 Transgenic Zebrafish.

Here, we present an in vivo drug screening protocol using a zebrafish model of metastasis for the identification of anti-metastatic drugs. A tamoxifen-controllable Twist1a-ERT2 transgenic zebrafish line was established to serve as a platform for the identification. By crossing Twist1a-ERT2 with xmrk (a homolog of hyperactive form of the epidermal growth factor receptor) transgenic zebrafish, which develop hepatocellular carcinoma, approximately 80% of the double transgenic zebrafish show spontaneous cell dissemination of mCherry-labeled hepatocytes from the liver to the entire abdomen and tail regions in five days, through induction of epithelial to mesenchymal transition (EMT). This rapid and high-frequency induction of cell dissemination makes it possible to perform an in vivo drug screen for the identification of anti-metastatic drugs targeting metastatic dissemination of cancer cells. The protocol evaluates the suppressor effect of a test drug on metastasis in five days, by comparing the frequencies of the fish showing abdominal and distant dissemination patterns in the test drug-treated group with those in the vehicle-treated group. Our study previously identified that adrenosterone, an inhibitor for hydroxysteroid (11-beta) dehydrogenase 1 (HSD11ß1), has a suppressor effect on cell dissemination in the model. Furthermore, we validated that a pharmacologic and genetic inhibition of HSD11ß1 suppressed metastatic dissemination of highly metastatic human cell lines in a zebrafish xenotransplantation model. Taken together, this protocol opens new routes for the identification of anti-metastatic drugs. Graphical overview Timing Day 0: Zebrafish spawning Day 8: Primary tumor induction Day 11: Chemical treatment Day 11.5: Metastatic dissemination induction in the presence of a test chemical Day 16: Data analysis.

Gastrulation Screening to Identify Anti-metastasis Drugs in Zebrafish Embryos.

Few models exist that allow for rapid and effective screening of anti-metastasis drugs. Here, we present a drug screening protocol utilizing gastrulation of zebrafish embryos for identification of anti-metastasis drugs. Based on the evidence that metastasis proceeds through utilizing the molecular mechanisms of gastrulation, we hypothesized that chemicals interrupting zebrafish gastrulation might suppress the metastasis of cancer cells. Thus, we developed a phenotype-based chemical screen that uses epiboly, the first morphogenetic movement in gastrulation, as a marker. The screen only needs zebrafish embryos and enables hundreds of chemicals to be tested in five hours by observing the epiboly progression of chemical-treated embryos. In the screen, embryos at the two-cell stage are firstly corrected and then developed to the sphere stage. The embryos are treated with a test chemical and incubated in the presence of the chemical until vehicle-treated embryos develop to the 90% epiboly stage. Finally, positive 'hit' chemicals that interrupt epiboly progression are selected by comparing epiboly progression of the chemical-treated and vehicle-treated embryos under a stereoscopic microscope. A previous study subjected 1,280 FDA-approved drugs to the screen and identified adrenosterone and pizotifen as epiboly-interrupting drugs. These were validated to suppress metastasis of breast cancer cells in mice models of metastasis. Furthermore, 11ß-hydroxysteroid dehydrogenase 1 (HSD11ß1) and serotonin receptor 2C (HTR2C), the primary targets of adrenosterone and pizotifen, respectively, promoted metastasis through induction of epithelial-mesenchymal transition (EMT). Therefore, this screen could be converted into a chemical genetic screening platform for identification of metastasis-promoting genes. Graphical abstract.

Cinnamon bark extract suppresses metastatic dissemination of cancer cells through inhibition of glycolytic metabolism.

Metastasis is responsible for approximately 90% of cancer-associated mortality and proceeds through multiple steps. Several herbal medicines are reported to inhibit primary tumor growth, but the suppressor effects of the medicines on metastasis progression are still not fully elucidated. Here we report that cinnamon bark extract (CBE) has a suppressor effect on metastatic dissemination of cancer cells. Through a phenotypic screening using zebrafish embryos, CBE was identified to interfere with the gastrulation progression of zebrafish embryos, of which the molecular mechanisms are conserved in metastasis progression. A Boyden chamber assay showed that CBE decreased cell motility and invasion of MDA-MB-231 human breast cancer cells without affecting their cell viability. Furthermore, CBE suppressed metastatic dissemination of the cells in a zebrafish xenotransplantation model. Quantitative metabolome analyses revealed that the productions of glucose-6-phosphate (G6P) and fructose 6-phosphate which are intermediate metabolites of glycolytic metabolism were interrupted in CBE-treated cells. qPCR and western-blotting analyses revealed that CBE-treated cells showed decreased expression of hexokinase 2 (HK2) which yields G6P. Pharmacological inhibition of HK2 with 2-deoxy-D-glucose suppressed cell invasion and migration of the cells without affecting their cell viability. Taken together, CBE suppresses metastatic dissemination of cancer cells through inhibition of glycolysis metabolism.

A zebrafish embryo screen utilizing gastrulation identifies the HTR2C inhibitor pizotifen as a suppressor of EMT-mediated metastasis.

Metastasis is responsible for approximately 90% of cancer-associated mortality but few models exist that allow for rapid and effective screening of anti-metastasis drugs. Current mouse models of metastasis are too expensive and time consuming to use for rapid and high-throughput screening. Therefore, we created a unique screening concept utilizing conserved mechanisms between zebrafish gastrulation and cancer metastasis for identification of potential anti-metastatic drugs. We hypothesized that small chemicals that interrupt zebrafish gastrulation might also suppress metastatic progression of cancer cells and developed a phenotype-based chemical screen to test the hypothesis. The screen used epiboly, the first morphogenetic movement in gastrulation, as a marker and enabled 100 chemicals to be tested in 5 hr. The screen tested 1280 FDA-approved drugs and identified pizotifen, an antagonist for serotonin receptor 2C (HTR2C) as an epiboly-interrupting drug. Pharmacological and genetic inhibition of HTR2C suppressed metastatic progression in a mouse model. Blocking HTR2C with pizotifen restored epithelial properties to metastatic cells through inhibition of Wnt signaling. In contrast, HTR2C induced epithelial-to-mesenchymal transition through activation of Wnt signaling and promoted metastatic dissemination of human cancer cells in a zebrafish xenotransplantation model. Taken together, our concept offers a novel platform for discovery of anti-metastasis drugs.

Metabolic Alteration in Cancer Cells by Therapeutic Carbon Ions.

BACKGROUND/AIM: Carbon-ion radiotherapy has strong antitumor effects in X-ray-resistant tumors. However, the mechanisms underlying the strong antitumor effect, especially on metabolic alterations, are not fully elucidated. This study aimed to determine the effect of therapeutic carbon ions on metabolic alterations in cancer cells. MATERIALS AND METHODS: Five human cancer cell lines were used in this study. The change in 333 metabolite levels in response to carbon ions was analyzed using gas chromatography-mass spectrometry. RESULTS: Fifty-two metabolites were commonly detected in all cell lines. The levels of five metabolites significantly changed in four or more cell lines. Three of the five metabolites (i.e., 2-ketoglutaric acid, fumaric acid and 2-hydroxyglutaric acid) were associated with the TCA cycle. TCA cycle intermediates and the downstream oncometabolite 2-hydroxyglutaric acid were up-regulated by carbon ions. CONCLUSION: We demonstrated for the first time that TCA cycle intermediates and 2-hydroxyglutaric acid are up-regulated after carbon-ion irradiation.

Upregulation of Thymidylate Synthase Induces Pemetrexed Resistance in Malignant Pleural Mesothelioma.

Malignant pleural mesothelioma (MPM) is an invasive malignancy that develops in the pleural cavity, and antifolates are used as chemotherapeutics for treating. The majority of antifolates, including pemetrexed (PMX), inhibit enzymes involved in purine and pyrimidine synthesis. MPM patients frequently develop drug resistance in clinical practice, however the associated drug-resistance mechanism is not well understood. This study was aimed to elucidate the mechanism underlying resistance to PMX in MPM cell lines. We found that among the differentially expressed genes associated with drug resistance (determined by RNA sequencing), TYMS expression was higher in the established resistant cell lines than in the parental cell lines. Knocking down TYMS expression significantly reduced drug resistance in the resistant cell lines. Conversely, TYMS overexpression significantly increased drug resistance in the parental cells. Metabolomics analysis revealed that the levels of dTMP were higher in the resistant cell lines than in the parental cell lines; however, resistant cells showed no changes in dTTP levels after PMX treatment. We found that the nucleic acid-biosynthetic pathway is important for predicting the efficacy of PMX in MPM cells. The results of chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR) assays suggested that H3K27 acetylation in the 5'-UTR of TYMS may promote its expression in drug-resistant cells. Our findings indicate that the intracellular levels of dTMP are potential biomarkers for the effective treatment of patients with MPM and suggest the importance of regulatory mechanisms of TYMS expression in the disease.

De novo deoxyribonucleotide biosynthesis regulates cell growth and tumor progression in small-cell lung carcinoma.

Deoxyribonucleotide biosynthesis from ribonucleotides supports the growth of active cancer cells by producing building blocks for DNA. Although ribonucleotide reductase (RNR) is known to catalyze the rate-limiting step of de novo deoxyribonucleotide triphosphate (dNTP) synthesis, the biological function of the RNR large subunit (RRM1) in small-cell lung carcinoma (SCLC) remains unclear. In this study, we established siRNA-transfected SCLC cell lines to investigate the anticancer effect of silencing RRM1 gene expression. We found that RRM1 is required for the full growth of SCLC cells both in vitro and in vivo. In particular, the deletion of RRM1 induced a DNA damage response in SCLC cells and decreased the number of cells with S phase cell cycle arrest. We also elucidated the overall changes in the metabolic profile of SCLC cells caused by RRM1 deletion. Together, our findings reveal a relationship between the deoxyribonucleotide biosynthesis axis and key metabolic changes in SCLC, which may indicate a possible link between tumor growth and the regulation of deoxyribonucleotide metabolism in SCLC.

Zebrafish-Based Screening Models for the Identification of Anti-Metastatic Drugs.

Metastasis, a leading contributor to the morbidity of cancer patients, occurs through a multi-step process: invasion, intravasation, extravasation, colonization, and metastatic tumor formation. Each process is not only promoted by cancer cells themselves but is also affected by their microenvironment. Given this complexity, drug discovery for anti-metastatic drugs must consider the interaction between cancer cells and their microenvironments. The zebrafish is a suitable vertebrate animal model for in vivo high-throughput screening studies with physiological relevance to humans. This review covers the zebrafish model used to identify anti-metastatic drugs.

Uptake of collagen type I via macropinocytosis cause mTOR activation and anti-cancer drug resistance.

Collagen type I (Col I) is one of the major extracellular matrix proteins in the cancer tissue. Previously, we have reported that Col I induces epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) resistance by mTOR activation through Akt and ERK1/2 independent pathway. In this study, we aimed to elucidate the molecular mechanism of Col I induced EGFR-TKI resistance. First, we demonstrated the uptake of fluorescently labeled Col I by EGFR-mutated lung cancer cell line PC-9 cells using confocal microscopy and flow cytometry. Metabolome analysis revealed that the metabolic profiles of PC-9 cells was influenced by Col I treatment. Uptake of Col I into PC-9 cells was not inhibited by MMP inhibitor, GM6001, and endocytosis inhibitors, Pitstop2 and Dyngo4a; however, macropinocytosis inhibitor EIPA prevented its uptake. Moreover, the combination of EIPA and EGFR-TKI abrogated Col I-induced EGFR-TKI resistance in PC-9 cells. Inhibition of Rac1, which is essential for micropinocytosis, also decreased the uptake of Col I in PC-9 cells and restored their sensitivity to EGFR-TKI. Thus, EGFR mutated lung cancer cells could develop EGFR-TKI resistance by Col I uptake by macropinocytosis route.

A Novel Zebrafish Model of Metastasis Identifies the HSD11ß1 Inhibitor Adrenosterone as a Suppressor of Epithelial-Mesenchymal Transition and Metastatic Dissemination.

Metastasis of cancer cells is multi-step process and dissemination is an initial step. Here we report a tamoxifen-controllable Twist1a-ERT2 transgenic zebrafish line as a new animal model for metastasis research, and demonstrate that this model can serve as a novel platform for discovery of antimetastasis drugs targeting metastatic dissemination of cancer cells. By crossing Twist1a-ERT2 with xmrk (a homolog of hyperactive form of EGFR) transgenic zebrafish, which develops hepatocellular carcinoma, approximately 80% of the double transgenic zebrafish showed spontaneous cell dissemination of mCherry-labeled hepatocytes from the liver to the entire abdomen region and the tail region. The dissemination is accomplished in 5 days through induction of an epithelial-to-mesenchymal transition. Using this model, we conducted in vivo drug screening and identified three hit drugs. One of them, adrenosterone, an inhibitor for hydroxysteroid (11-beta) dehydrogenase 1 (HSD11ß1), has a suppressor effect on cell dissemination in this model. Pharmacologic and genetic inhibition of HSD11ß1 suppressed metastatic dissemination of highly metastatic human cell lines in a zebrafish xenotransplantation model. Through downregulation of Snail and Slug, adrenosterone-treated cells recovered expression of E-cadherin and other epithelial markers and lost partial expression of mesenchymal markers compared with vehicle-treated cells. Taken together, our model offers a useful platform for the discovery of antimetastasis drugs targeting metastatic dissemination of cancer cells. IMPLICATIONS: This study describes a transgenic zebrafish model for liver tumor metastasis and it has been successfully used for identification of some drugs to inhibit metastatic dissemination of human cancer cells.

Correlation between maximum standardized uptake values on FDG-PET and microenvironmental factors in patients with clinical stage IA radiologic pure-solid lung adenocarcinoma.

OBJECTIVES: The purpose of this study was to investigate whether fluorodeoxyglucose (FDG) accumulation is associated with the expression of microenvironmental factors in radiological pure-solid lung adenocarcinoma. METHODS: We selected 50 cases involving patients with clinical stage IA radiological pure-solid lung adenocarcinoma who were examined with 18 F-FDG positron emission tomography (18 F-FDG PET) prior to surgery and whose FDG-PET maximal standardized uptake values (SUVmax) were calculated. Tumor specimens were analyzed by immunohistochemistry (IHC) for phosphorylated AKT (pAKT), glucose transporter type 1 (GLUT-1), carbonic anhydrase IX (CA IX), podoplanin-positive cancer associated fibroblasts (PDPN + CAFs), and CD204-positive tumor-associated macrophages (CD204+ TAMs). We compared the clinicopathological characteristics and the immunophenotypes between two groups with high and low SUVmax. RESULTS: A multivariate analysis revealed that SUVmax was an independently significant prognostic factor (P = .03). The 5-year overall survival (OS) and recurrence free survival (RFS) rates of the SUV max high and low groups were 68.0% versus 100% ((P = .002; OS) 54.3% versus 90.8% (P < .001; RFS)), respectively. Vascular invasion, pleural invasion, and the prevalence of solid predominant subtype tumors were more frequent in the SUVmax high group. Additionally, the expression levels of GLUT-1 and pAKT in cancer cells were significantly higher in this group (P < .001, and P < .001 respectively). Furthermore, the numbers of the tumor-promoting stromal cells, i.e., PDPN + CAFs and CD204+ TAMs, were also significantly higher in the SUVmax high group (P = .001, and P < .001 respectively). CONCLUSION: Our results indicated that a close association exists between the SUVmax and expressions of not only metabolism associated markers in cancer cells but also of tumor promoting markers in stromal cells among patients with clinical stage IA adenocarcinoma with radiologically pure-solid nodules.

Extraction of Aqueous Metabolites from Cultured Adherent Cells for Metabolomic Analysis by Capillary Electrophoresis-Mass Spectrometry.

Metabolomic analysis is a promising omics approach to not only understand the specific metabolic regulation in cancer cells compared to normal cells but also to identify biomarkers for early-stage cancer detection and prediction of chemotherapy response in cancer patients. Preparation of uniform samples for metabolomic analysis is a critical issue that remains to be addressed. Here, we present an easy and reliable protocol for extracting aqueous metabolites from cultured adherent cells for metabolomic analysis using capillary electrophoresis-mass spectrometry (CE-MS). Aqueous metabolites from cultured cells are analyzed by culturing and washing cells, treating cells with methanol, extracting metabolites, and removing proteins and macromolecules with spin columns for CE-MS analysis. Representative results using lung cancer cell lines treated with diamide, an oxidative reagent, illustrate the clearly observable metabolic shift of cells under oxidative stress. This article would be especially valuable to students and investigators involved in metabolomics research, who are new to harvesting metabolites from cell lines for analysis by CE-MS.

Targeting the Vulnerability of Glutathione Metabolism in ARID1A-Deficient Cancers.

ARID1A encodes an SWI/SNF chromatin-remodeling factor and is frequently mutated in various cancers. This study demonstrates that ARID1A-deficient cancer cells are specifically vulnerable to inhibition of the antioxidant glutathione (GSH) and the glutamate-cysteine ligase synthetase catalytic subunit (GCLC), a rate-limiting enzyme for GSH synthesis. Inhibition of GCLC markedly decreased GSH in ARID1A-deficient cancer cells, leading to apoptotic cell death triggered by excessive amounts of reactive oxygen species. The vulnerability of ARID1A-deficient cancer cells results from low basal levels of GSH due to impaired expression of SLC7A11. The SLC7A11-encoded cystine transporter supplies cells with cysteine, a key source of GSH, and its expression is enhanced by ARID1A-mediated chromatin remodeling. Thus, ARID1A-deficient cancers are susceptible to synthetic lethal targeting of GCLC.

Genetic profiling-based prognostic prediction of patients with advanced small-cell lung cancer in large scale analysis.

OBJECTIVES: Comprehensive genomic analysis of small-cell lung cancer (SCLC) revealed various genetic alterations. However, obtaining suitable samples for genetic analysis is difficult in advanced SCLC. Thus, the prognostic effect of genetic alterations on the outcome of SCLC patients has not been well investigated. Therefore, this study evaluated the effect of genetic alterations on the survival of SCLC patients. MATERIALS AND METHODS: We collected samples obtained from 220 patients with advanced SCLC before cancer treatment. Genomic DNA extracted from the samples was subjected to a 1.499 Mb-sized custom panel that captured all exons of 244 cancer-related genes, and the captured DNA was analyzed through next-generation sequencing. The associations between genetic alterations and overall survival were evaluated. RESULTS: Genetic analysis was successful in 204 samples (93%). Genetic alterations in the PI3K/AKT/mTOR pathway and inactivating mutations inTP53 and RB1 were detected in 14 (7%), 150 (74%), and 85 (42%) of the tumors. In extensive disease (ED, N = 126) patients, multivariate analysis revealed that the presence of genetic alterations in the PI3K/AKT/mTOR pathway was significantly associated with unfavorable survival [hazard ratio (HR), 2.14; 95% CI 1.02-4.06; P = 0.04]. In limited disease (LD, N = 78) patients, the presence of TP53 mutation and the absence of RB1 mutation were significantly associated with unfavorable survival (HR, 2.41; 95% CI 1.21-5.34; P = 0.01, and HR, 0.45; 95% CI 0.25-0.79; P < 0.01, respectively). CONCLUSIONS: Sequencing-based genetic profiling is feasible and useful to predict the prognosis in advanced SCLC. Genetic alterations in the PI3K/AKT/mTOR pathway, TP53 mutations and RB1 mutations were associated with prognosis in SCLC patients. The genetic alterations associated with the prognosis were different between ED-SCLC and LD-SCLC.

Metabolic Characterization of Antifolate Responsiveness and Non-responsiveness in Malignant Pleural Mesothelioma Cells.

Antifolates are a class of drugs effective for treating malignant pleural mesothelioma (MPM). The majority of antifolates inhibit enzymes involved in purine and pyrimidine synthesis such as dihydrofolate reductase (DHFR), thymidylate synthase (TYMS), and glycinamide ribonucleotide formyltransferase (GART). In order to select the most suitable patients for effective therapy with drugs targeting specific metabolic pathways, there is a need for better predictive metabolic biomarkers. Antifolates can alter global metabolic pathways in MPM cells, yet the metabolic profile of treated cells has not yet been clearly elucidated. Here we found that MPM cell lines could be categorized into two groups according to their sensitivity or resistance to pemetrexed treatment. We show that pemetrexed susceptibility could be reversed and DNA synthesis rescued in drug-treated cells by the exogenous addition of the nucleotide precursors hypoxanthine and thymidine (HT). We observed that the expression of pemetrexed-targeted enzymes in resistant MPM cells was quantitatively lower than that seen in pemetrexed-sensitive cells. Metabolomic analysis revealed that glycine and choline, which are involved in one-carbon metabolism, were altered after drug treatment in pemetrexed-sensitive but not resistant MPM cells. The addition of HT upregulated the concentration of inosine monophosphate (IMP) in pemetrexed-sensitive MPM cells, indicating that the nucleic acid biosynthesis pathway is important for predicting the efficacy of pemetrexed in MPM cells. Our data provide evidence that may link therapeutic response to the regulation of metabolism, and points to potential biomarkers for informing clinical decisions regarding the most effective therapies for patients with MPM.

Metabolic Determinants of Sensitivity to Phosphatidylinositol 3-Kinase Pathway Inhibitor in Small-Cell Lung Carcinoma.

Comprehensive genomic analysis has revealed that the PI3K/AKT/mTOR pathway is a feasible therapeutic target in small-cell lung carcinoma (SCLC). However, biomarkers to identify patients likely to benefit from inhibitors of this pathway have not been identified. Here, we show that metabolic features determine sensitivity to the PI3K/mTOR dual inhibitor gedatolisib in SCLC cells. Substantial phosphatidyl lipid analysis revealed that a specific phosphatidylinositol (3,4,5)-trisphosphate (PIP3) subspecies lipid product PIP3 (38:4) is predictive in assessing sensitivity to PI3K/mTOR dual inhibitor. Notably, we found that higher amounts of purine-related aqueous metabolites such as hypoxanthine, which are characteristic of SCLC biology, lead to resistance to PI3K pathway inhibition. In addition, the levels of the mRNA encoding hypoxanthine phosphoribosyl transferase 1, a key component of the purine salvage pathway, differed significantly between SCLC cells sensitive or resistant to gedatolisib. Moreover, complementation with purine metabolites could reverse the vulnerability to targeting of the PI3K pathway in SCLC cells normally sensitive to gedatolisib. These results indicate that the resistance mechanism of PI3K pathway inhibitors is mediated by the activation of the purine salvage pathway, supplying purine resource to nucleotide biosynthesis. Metabolomics is a powerful approach for finding novel therapeutic biomarkers in SCLC treatment.Significance: These findings identify features that determine sensitivity of SCLC to PI3K pathway inhibition and support metabolomics as a tool for finding novel therapeutic biomarkers. Cancer Res; 78(9); 2179-90. ©2018 AACR.