DNA methylation patterns in bladder tumors of African American patients point to distinct alterations in xenobiotic metabolism.

Racial/ethnic disparities have a significant impact on bladder cancer outcomes with African American patients demonstrating inferior survival over European-American patients. We hypothesized that epigenetic difference in methylation of tumor DNA is an underlying cause of this survival health disparity. We analyzed bladder tumors from African American and European-American patients using reduced representation bisulfite sequencing (RRBS) to annotate differentially methylated DNA regions. Liquid chromatography-mass spectrometry (LC-MS/MS) based metabolomics and flux studies were performed to examine metabolic pathways that showed significant association to the discovered DNA methylation patterns. RRBS analysis showed frequent hypermethylated CpG islands in African American patients. Further analysis showed that these hypermethylated CpG islands in patients are commonly located in the promoter regions of xenobiotic enzymes that are involved in bladder cancer progression. On follow-up, LC-MS/MS revealed accumulation of glucuronic acid, S-adenosylhomocysteine, and a decrease in S-adenosylmethionine, corroborating findings from the RRBS and mRNA expression analysis indicating increased glucuronidation and methylation capacities in African American patients. Flux analysis experiments with 13C-labeled glucose in cultured African American bladder cancer cells confirmed these findings. Collectively, our studies revealed robust differences in methylation-related metabolism and expression of enzymes regulating xenobiotic metabolism in African American patients indicate that race/ethnic differences in tumor biology may exist in bladder cancer.

Large-scale profiling of serum metabolites in African American and European American patients with bladder cancer reveals metabolic pathways associated with patient survival.

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.

Recent advances in the metabolomic study of bladder cancer.

INTRODUCTION: Metabolomics is a chemical process, involving the characterization of metabolites and cellular metabolism. Recent studies indicate that numerous metabolic pathways are altered in bladder cancer (BLCA), providing potential targets for improved detection and possible therapeutic intervention. We review recent advances in metabolomics related to BLCA and identify various metabolites that may serve as potential biomarkers for BLCA. Areas covered: In this review, we describe the latest advances in defining the BLCA metabolome and discuss the possible clinical utility of metabolic alterations in BLCA tissues, serum, and urine. In addition, we focus on the metabolic alterations associated with tobacco smoke and racial disparity in BLCA. Expert commentary: Metabolomics is a powerful tool which can shed new light on BLCA development and behavior. Key metabolites may serve as possible markers of BLCA. However, prospective validation will be needed to incorporate these markers into clinical care.

The IL6/JAK/STAT3 signaling axis is a therapeutic vulnerability in SMARCB1-deficient bladder cancer.

SMARCB1 loss has long been observed in many solid tumors. However, there is a need to elucidate targetable pathways driving growth and metastasis in SMARCB1-deficient tumors. Here, we demonstrate that SMARCB1 deficiency, defined as genomic SMARCB1 copy number loss associated with reduced mRNA, drives disease progression in patients with bladder cancer by engaging STAT3. SMARCB1 loss increases the chromatin accessibility of the STAT3 locus in vitro. Orthotopically implanted SMARCB1 knockout (KO) cell lines exhibit increased tumor growth and metastasis. SMARCB1-deficient tumors show an increased IL6/JAK/STAT3 signaling axis in in vivo models and patients. Furthermore, a pSTAT3 selective inhibitor, TTI-101, reduces tumor growth in SMARCB1 KO orthotopic cell line-derived xenografts and a SMARCB1-deficient patient derived xenograft model. We have identified a gene signature generated from SMARCB1 KO tumors that predicts SMARCB1 deficiency in patients. Overall, these findings support the clinical evaluation of STAT3 inhibitors for the treatment of SMARCB1-deficient bladder cancer.

Thioredoxin reductase is a major regulator of metabolism in leukemia cells.

Despite the fact that AML is the most common acute leukemia in adults, patient outcomes are poor necessitating the development of novel therapies. We identified that inhibition of Thioredoxin Reductase (TrxR) is a promising strategy for AML and report a highly potent and specific inhibitor of TrxR, S-250. Both pharmacologic and genetic inhibition of TrxR impairs the growth of human AML in mouse models. We found that TrxR inhibition leads to a rapid and marked impairment of metabolism in leukemic cells subsequently leading to cell death. TrxR was found to be a major and direct regulator of metabolism in AML cells through impacts on both glycolysis and the TCA cycle. Studies revealed that TrxR directly regulates GAPDH leading to a disruption of glycolysis and an increase in flux through the pentose phosphate pathway (PPP). The combined inhibition of TrxR and the PPP led to enhanced leukemia growth inhibition. Overall, TrxR abrogation, particularly with S-250, was identified as a promising strategy to disrupt AML metabolism.

Measurement of methylated metabolites using Liquid Chromatography-Mass Spectrometry and its biological application.

Methylation aberrations play an important role in many metabolic disorders including cancer. Methylated metabolites are direct indicators of metabolic aberrations, and currently, there is no Liquid chromatography - Mass spectrometry (LC-MS) based method available to cover all classes of methylated metabolites at low detection limits. In this study, we have developed a method for the detection of methylated metabolites, and it's biological application. In this approach, we used a HILIC based HPLC with MS to measure methylated organic acids, amino acids, and nucleotides. These metabolites were separated from each other by their hydrophobic interactions and analyzed by targeted metabolomics of single reaction monitoring by positive and negative mode of electrospray ionization. These metabolites were quantified, and the interday reproducibility was <10% relative standard deviation. Furthermore, by applying this method, we identified high levels of methylated metabolites in bladder cancer cell lines compared to benign cells. In vitro treatment of cancer cells with methylation inhibitor, 5- aza-2'-deoxycytidine showed a decrease in these methylated metabolites. This data indicates that HPLC analysis using this HILIC based method could be a powerful tool for measuring methylated metabolites in biological specimens. This method is rapid, sensitive, selective, and precise to measure methylated metabolites.

Serum Metabolic Profiling Identified a Distinct Metabolic Signature in Bladder Cancer Smokers: A Key Metabolic Enzyme Associated with Patient Survival.

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, phenylalanine, 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.

Multi-omics Integration Analysis Robustly Predicts High-Grade Patient Survival and Identifies CPT1B Effect on Fatty Acid Metabolism in Bladder Cancer.

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.