The mechanisms through which tumor cells genetically lose antigenicity and evade immune checkpoints remain largely elusive. Here, we report that tissue-specific expression of the human long-noncoding RNA LINK-A in mouse mammary glands initiated metastatic mammary gland tumors, which phenotypically resembled human triple-negative breast cancer (TNBC). LINK-A expression facilitated crosstalk between phosphatidylinositol-(3,4,5)-trisphosphate and inhibitory G-protein–coupled receptor (GPCR) pathways, attenuating protein kinase A (PKA)-mediated phosphorylation of the E3 ubiquitin ligase TRIM71. Consequently, LINK-A expression enhanced K48–polyubiquitination-mediated degradation of the antigen peptide-loading complex (PLC) and intrinsic tumor suppressors Rb and p53. Treatment with LINK-A-locked nucleic acids or GPCR antagonists stabilized the PLC components, Rb, and p53, and sensitized mammary gland tumors to immune checkpoint blockers (ICBs). Importantly, PD-1 blockade-resistant TNBC patients exhibited elevated LINK-A levels and downregulated PLC components. Hence, we demonstrated lncRNA-dependent downregulation of antigenicity and intrinsic tumor suppression, which may provide the basis for developing a therapeutic regimen of combinational immunotherapy and effective early prevention for TNBCs.

Purpose: The perturbation of metabolic pathways in high-grade bladder cancer has not been investigated. We aimed to identify a metabolic signature in high-grade bladder cancer by integrating unbiased metabolomics, lipidomics, and transcriptomics to predict patient survival and to discover novel therapeutic targets. Experimental Design: We performed high-resolution liquid chromatography mass spectrometry (LC-MS) and bioinformatic analysis to determine the global metabolome and lipidome in high-grade bladder cancer. We further investigated the effects of impaired metabolic pathways using in vitro and in vivo models. Results: We identified 519 differential metabolites and 19 lipids that were differentially expressed between low-grade and high-grade bladder cancer using the NIST MS metabolomics compendium and lipidblast MS/MS libraries, respectively. Pathway analysis revealed a unique set of biochemical pathways that are highly deregulated in high-grade bladder cancer. Integromics analysis identified a molecular gene signature associated with poor patient survival in bladder cancer. Low expression of CPT1B in high-grade tumors was associated with low FAO and low acyl carnitine levels in high-grade bladder cancer, which were confirmed using tissue microarrays. Ectopic expression of the CPT1B in high-grade bladder cancer cells led to reduced EMT in in vitro, and reduced cell proliferation, EMT, and metastasis in vivo. Conclusions: Our study demonstrates a novel approach for the integration of metabolomics, lipidomics, and transcriptomics data, and identifies a common gene signature associated with poor survival in patients with bladder cancer. Our data also suggest that impairment of FAO due to downregulation of CPT1B plays an important role in the progression toward high-grade bladder cancer and provide potential targets for therapeutic intervention.

Background The first global lipidomic profiles associated with urothelial cancer of the bladder (UCB) and its clinical stages associated with progression were identified. Objective To identify lipidomic signatures associated with survival and different clinical stages of UCB. Design, setting, and participants Pathologically confirmed 165 bladder-derived tissues (126 UCB, 39 benign adjacent or normal bladder tissues). UCB tissues included Ta (n = 16), T1 (n = 30), T2 (n = 43), T3 (n = 27), and T4 (n = 9); lymphovascular invasion (LVI) positive (n = 52) and negative (n = 69); and lymph node status N0 (n = 28), N1 (n = 11), N2 (n = 9), N3 (n = 3), and Nx (n = 75). Results and limitations UCB tissues have higher levels of phospholipids and fatty acids, and reduced levels of triglycerides compared with benign tissues. A total of 59 genes associated with altered lipids in UCB strongly correlate with patient survival in an UCB public dataset. Within UCB, there was a progressive decrease in the levels of phosphatidylserine (PS), phosphatidylethanolamines (PEs), and phosphocholines, whereas an increase in the levels of diacylglycerols (DGs) with tumor stage. Transcript and protein expression of phosphatidylserine synthase 1, which converts DGs to PSs, decreased progressively with tumor stage. Levels of DGs and lyso-PEs were significantly elevated in tumors with LVI and lymph node involvement, respectively. Lack of carcinoma in situ and treatment information is the limitation of our study. Conclusions To date, this is the first study describing the global lipidomic profiles associated with UCB and identifies lipids associated with tumor stages, LVI, and lymph node status. Our data suggest that triglycerides serve as the primary energy source in UCB, while phospholipid alterations could affect membrane structure and/or signaling associated with tumor progression. Patient summary Lipidomic alterations identified in this study set the stage for characterization of pathways associated with these altered lipids that, in turn, could inform the development of first-of-its-kind lipid-based noninvasive biomarkers and novel therapeutic targets for aggressive urothelial cancer of the bladder.

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Smoking is a major risk factor for the development of Bladder Cancer (BLCA); however, the functional consequences of the carcinogens in tobacco smoke and BLCA-associated metabolic alterations remains poorly defined. We assessed the metabolic profiles in BLCA smokers and non-smokers, and identified the key alterations in their metabolism. Liquid Chromatography – Mass Spectrometry (LC-MS), and bioinformatic analysis were performed to determine the metabolome associated with BLCA smokers and were further validated in cell line models. Smokers with BLCA were found to have elevated levels of methylated metabolites, polycyclic aromatic hydrocarbons (PAHs), DNA adducts and DNA damage. DNA methyltransferase 1 (DNMT1) expression was significantly higher in smokers than non-smokers with BLCA. An integromics approach, using multiple patient cohorts, revealed strong associations between smokers and high-grade BLCA. In vitro exposure to the tobacco smoke carcinogens, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and benzo[a]pyrene (BaP) led to increase in levels of methylated metabolites, DNA adducts, and extensive DNA damage in BLCA cells. Co-treatment of BLCA cells with these carcinogens and the methylation inhibitor 5-aza-2′-deoxycytidine (AZA) rewired the methylated metabolites, DNA adducts, DNA damage. These findings were confirmed through the isotopic labeled metabolic flux analysis. Screens using smoke associated metabolites and DNA adducts could provide robust biomarkers and improve individual risk prediction in BLCA smokers. Non-invasive predictive biomarkers that can stratify the risk of developing BLCA in smokers could aid in early detection and treatment.

BACKGROUND: African Americans (AAs) experience a disproportionally high rate of bladder cancer (BLCA) deaths even though their incidence rates are lower than those of other patient groups. Using a metabolomics approach, this study investigated how AA BLCA may differ molecularly from European Americans (EAs) BLCA, and it examined serum samples from patients with BLCA with the aim of identifying druggable metabolic pathways in AA patients. METHODS: Targeted metabolomics was applied to measure more than 300 metabolites in serum samples from 2 independent cohorts of EA and AA patients with BLCA and healthy EA and AA controls via liquid chromatography-mass spectrometry, and this was followed by the identification of altered metabolic pathways with a focus on AA BLCA. A subset of the differential metabolites was validated via absolute quantification with the Biocrates AbsoluteIDQ p180 kit. The clinical significance of the findings was further examined in The Cancer Genomic Atlas BLCA data set. RESULTS: Fifty-three metabolites, mainly related to amino acid, lipid, and nucleotide metabolism, were identified that showed significant differences in abundance between AA and EA BLCA. For example, the levels of taurine, glutamine, glutamate, aspartate, and serine were elevated in serum samples from AA patients versus EA patients. By mapping these metabolites to genes, this study identified significant relations with regulators of metabolism such as malic enzyme 3, prolyl 3-hydroxylase 2, and lysine demethylase 2A that predicted patient survival exclusively in AA patients with BLCA. CONCLUSIONS: This metabolic profile of serum samples might be used to assess risk progression in AA BLCA. These first-in-field findings describe metabolic alterations in AA BLCA and emphasize a potential biological basis for BLCA health disparities. Cancer 2019;125:921-932.

High-grade Bladder Cancer (BLCA) represents the most aggressive and treatment-resistant cancer that renders the patients with poor survival. However, only a few biomarkers have been identified for the detection and treatment of BLCA. Recent studies show that ganglioside GD2 can be used as cancer biomarker and/or therapeutic target for various cancers. Despite its potential relevance in cancer diagnosis and therapeutics, the role of GD2 is unknown in BLCA. Here, we report for the first time that high-grade BLCA tissues and cell lines have higher expression of GD2 compared to low-grade by high-resolution Mass Spectrometry. The muscle invasive UMUC3 cell line showed high GD2, mesenchymal phenotype, and cell proliferation. Besides, we have shown the cancer stem cells (CSC) property (CD44hiCD24lo) of GD2+ UMUC3 and J82 cells. Also, the evaluation of lipid metabolism in GD2+ BLCA cell lines revealed higher levels of Phosphatidylinositol (PI), Phosphatidic acid (PA), Cardiolipin (CL) and lower levels of Phosphatidylserine (PS), plasmenyl-phosphatidylethanolamines (pPE), plasmenyl-phosphocholines (pPC), sphingomyelins (SM), triglycerides (TGs) and N-Acetylneuraminic acid. These findings are significantly correlated with the tissues of BLCA patients. Based on this evidence, we propose that GD2 may be used as an effective diagnostic and therapeutic target for aggressive BLCA.

Background: The current system to predict the outcome of smokers with bladder cancer is insufficient due to complex genomic and transcriptomic heterogeneities. This study aims to identify serum metabolite-associated genes related to survival in this population. Methods: We performed LC/MS-based targeted metabolomic analysis for >300 metabolites in serum obtained from two independent cohorts of bladder cancer never smokers, smokers, healthy smokers, and healthy never smokers. A subset of differential metabolites was validated using Biocrates absoluteIDQ p180 Kit. Genes associated with differential metabolites were integrated with a publicly available cohort of The Cancer Genome Atlas (TCGA) to obtain an intersecting signature specific for bladder cancer smokers. Results: Forty metabolites (FDR < 0.25) were identified to be differential between bladder cancer never smokers and smokers. Increased abundance of amino acids (tyrosine, phe-nylalanine, proline, serine, valine, isoleucine, glycine, and asparagine) and taurine were observed in bladder cancer smokers. Integration of differential metabolomic gene signature and transcriptomics data from TCGA cohort revealed an intersection of 17 genes that showed significant correlation with patient survival in bladder cancer smokers. Importantly, catechol-O-methyltransferase, iodotyrosine deiodinase, and tubulin tyrosine ligase showed a significant association with patient survival in publicly available bladder cancer smoker datasets and did not have any clinical association in never smokers. Conclusions: Serum metabolic profiling of bladder cancer smokers revealed dysregulated amino acid metabolism. It provides a distinct gene signature that shows a prognostic value in predicting bladder cancer smoker survival. Impact: Serum metabolic signature-derived genes act as a predictive tool for studying the bladder cancer progression in smokers.