Dataset Information

Tumor M2-PK: A novel urine marker of bladder cancer.

ABSTRACT: Purpose: Bladder cancer is a "Warburg-like" tumor characterized by a reliance on aerobic glycolysis and expression of pyruvate kinase M2 (PKM2). PKM2 oscillates between an active tetramer and an inactive dimer. We aim to further characterize PKM2, in particular PKM2 dimer, as a urinary biomarker of bladder cancer and a potential target for treatment.

Methods: HTB-9, HTB-5, and UM-UC3 bladder cancer cells were assessed for proliferation under differential glucose levels using the hexosaminidase assay. Western blot and Blue-native analysis was performed for protein expression of PKM2. Shikonin, an herb that is known to bind and inhibit PKM2, was utilized to determine if PKM2 has a role in glucose usage and cellular proliferation in bladder cancer cells by caspase activity assay. Institutional review board approval was obtained to collect healthy control and bladder cancer patient urine samples. The ScheBo M2-PK EDTA Plasma Test was performed on urine samples to assess urine Tumor M2-PK values.

Results: The three bladder cancer cell lines tested all demonstrate statistically significant increases in proliferation when exposed to higher level of glucose (200mg/dL). Similarly, low doses of glucose (25mg/dL) result in reduced proliferation. Increased cell growth in higher glucose concentration correlated with up-regulation of PKM2 protein expression. Shikonin, a PKM2 inhibitor, reduced cell proliferation and switched PKM2 isoforms from the dimer to tetramer. Lastly, dimer PKM2 (Tumor-M2PK) levels were assessed in the urine samples from bladder cancer (Bca) patients and healthy controls. Tumor M2-PK significantly correlated with the presence of BCa in our subjects.

Conclusions: Our studies demonstrate the potential of PKM2, specifically the dimer (Tumor-M2PK) as a target of drug therapy and as a urinary marker for bladder cancer.

SUBMITTER: Liu W

PROVIDER: S-EPMC6597081 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Tumor M2-PK: A novel urine marker of bladder cancer.

Liu Weiya W Woolbright Benjamin L BL Pirani Karim K Didde Ryan R Abbott Erika E Kaushik Gaurav G Martin Paige P Hamilton-Reeves Jill J Taylor John A JA Holzbeierlein Jeffrey M JM Anant Shrikant S Lee Eugene K EK

PloS one 20190627 6

<h4>Purpose</h4>Bladder cancer is a "Warburg-like" tumor characterized by a reliance on aerobic glycolysis and expression of pyruvate kinase M2 (PKM2). PKM2 oscillates between an active tetramer and an inactive dimer. We aim to further characterize PKM2, in particular PKM2 dimer, as a urinary biomarker of bladder cancer and a potential target for treatment.<h4>Methods</h4>HTB-9, HTB-5, and UM-UC3 bladder cancer cells were assessed for proliferation under differential glucose levels using the hex ...[more]

PMID: 31246990

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Project description:BackgroundBetter prognostic outcome is closely correlated with early detection of bladder cancer. Current non-invasive urianalysis relies on simultaneously testing multiple methylation markers to achieve relatively high accuracy. Therefore, we have developed an easy-to-use, convenient, and accurate single-target urine-based DNA methylation test for the malignancy.MethodsBy analyzing TCGA data, 344 candidate markers with 424 primer pairs and probe sets synthesized were systematically screened in cancer cell lines, paired tissue specimens, and urine sediments from bladder cancer patients and normal controls. The identified marker was further validated in large case-control cohorts. Wilcoxon rank sum tests and c2 tests were performed to compare methylation levels between case-control groups and correlate methylation levels with demographic and clinical characteristics. In addition, MSP, qMSP, RT-PCR, western blot analysis, and immunohistochemistry were performed to measure levels of DNA methylation, mRNA transcription, and protein expression in cancer cell lines and tissues.ResultsA top-performing DMRTA2 marker identified was tested in both discovery and validation sets, showing similar sensitivity and specificity for bladder cancer detection. Overall sensitivity in the aggregate set was 82.9%(179/216). The specificity, from a control group consisting of patients with lithangiuria, prostatoplasia, and prostatitis, is 92.5%(468/506). Notably, the methylation assay had the highest sensitivities for tumors at stages of T1(90.4%) and T2(95.0%) compared with Ta (63.0%), T3(81.8%), and T4(81.8%). Furthermore, the test showed admirable detection rate of 80.0%(24/30) for recurring cancers. While methylation was observed in 39/54(72.2%) urine samples from patients with carcinomas of renal pelvis and ureter, it was detected at extremely low rate of 6.0%(8/133) in kidney and prostate cancers. Compared with SV-HUC-1, the normal bladder epithelial cell line, DMRTA2 was hypermethylated in 8/9 bladder cancer cell lines, consistent with the results of MSP and qMSP, but not correlated with mRNA and protein expression levels in these cell lines. Similarly, DMRTA2 immunostaining was moderate in some tissues but weak in others. Further studies are needed to address functional implications of DMRTA2 hypermethylation.ConclusionsOur data demonstrated that a single-target DNA methylation signature, mDMRTA2, could be highly effective to detect both primary and recurring bladder cancer via urine samples.

Project description:The present study aimed to explore the mechanism by which the immune landscape of the tumor microenvironment influences bladder cancer. CIBERSORT and ssGSEA analyses revealed that M2 macrophages accounted for the highest proportion from 22 subsets of tumor‑infiltrating immune cells and were enriched in higher histologic grade and higher pathologic stage bladder cancer and 'basal' subtype of muscle invasive bladder cancer (MIBC). Kaplan‑Meier survival curve analysis indicated that patients with high numbers of infiltrating M2 macrophages had worse overall and disease‑specific survival rates. RNA sequencing and immunohistochemistry results indicated that M2 macrophages were enriched in MIBC and promoted angiogenesis. M2 macrophage infiltration was higher in bladder cancer tissues with mutant TP53, RB transcriptional corepressor 1, phosphatidylinositol‑4,5‑bisphosphate 3‑kinase catalytic subunit α, lysine methyltransferase 2A, lysine demethylase 6A and apolipoprotein B mRNA editing enzyme catalytic‑polypeptide‑like, but lower in tissues with mutant fibroblast growth factor receptor 3 (FGFR3), E74‑like ETS transcription factor 3, PC4 and SFRS1 interacting protein 1 and transmembrane and coiled‑coil domains 4. In addition, M2 macrophage infiltration was lower in the tissues with amplified FGFR3, erb‑b2 receptor tyrosine kinase 2, BCL2‑like 1, telomerase reverse transcriptase and tyrosine‑3‑monooxygenase/tryptophan‑5‑monooxygenase activation protein ζ, as well as in the tissues with deleted cyclin‑dependent kinase inhibitor 2A, CREB binding protein, AT‑rich interaction domain 1A, fragile histidine triad diadenosine triphosphatase, phosphodiesterase 4D, RAD51 paralog B, nuclear receptor corepressor 1 and protein tyrosine phosphatase receptor type D. Finally, seven micro (mi) RNAs (miR‑214‑5p, miR‑223‑3p, miR‑155‑5p, miR‑199a‑3p, miR‑199b‑3P, miR‑146b‑5p, miR‑142‑5p) which were expressed differentially in at least three mutant genes and were positively correlated with M2 macrophage infiltration as well as expressed highly in high grade bladder cancer were identified. Overall, the present study concluded that M2 macrophages are the predominant tumor‑infiltrating immune cell in bladder cancer and differentially expressed miRNAs due to cancer‑specific genomic alterations may be important drivers of M2 macrophage infiltration. These findings suggested that M2 macrophage infiltration may serve as a potential immunotherapy target in bladder cancer.

Dataset Information

Tumor M2-PK: A novel urine marker of bladder cancer.

Publications

Tumor M2-PK: A novel urine marker of bladder cancer.

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