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. 2019 Jun 27;14(6):e0218737.
doi: 10.1371/journal.pone.0218737. eCollection 2019.

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

Weiya Liu  1 , Benjamin L Woolbright  1 , Karim Pirani  1 , Ryan Didde  1 , Erika Abbott  1 , Gaurav Kaushik  2 , Paige Martin  1 , Jill Hamilton-Reeves  1 , John A Taylor 3rd  1 , Jeffrey M Holzbeierlein  1 , Shrikant Anant  2 , Eugene K Lee  1
Affiliations

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

Weiya Liu et al. PLoS One. .

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.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Glucose enhances BCa cell proliferation.
Bladder cancer cells were seeded at 1000 cells/well in 96-well plates. Cells were exposed to different glucose concentrations (25 mg/dL, 100 mg/dL, and 200 mg/dL) for 7 days. Hexosiminidase assay was performed every 24 hours. Data were analyzed from four independent experiments, each data point represents the mean ± S.E.M. * indicates significant two-tailed t-test p value of < 0.05, compared with Day.1.
Fig 2
Fig 2. PKM2 expression in bladder cancer cells.
UM-UC3, HTB-9, and HTB-5 cells were cultured in media containing different glucose concentrations (25 mg/dL, 100 mg/dL, and 200 mg/dL) for 7 days. On day 5, 6, and 7, protein was extracted and subjected to western blot analysis of PKM2 expression with β-actin as control. Columns represent the mean ± S.E.M. from three independent experiments (n = 3). *, **, and *** indicates significant two-tailed t-test p value of < 0.05, < 0.01, and < 0.001, compared with glucose concentration 25 mg/dL at day 5, 6, and 7 respectively.
Fig 3
Fig 3. Shikonin inhibits bladder cancer cell proliferation.
UM-UC3, HTB-9, and HTB-5 cells were treated with shikonin under three different glucose concentrations 25 mg/dL, 100 mg/dL, and 200 mg/dL. Data were analyzed from three independent experiments, each data point represents the mean ± S.E.M. * indicates significant two-tailed t-test p value of < 0.05, compared with vehicle-treated (DMSO) control.
Fig 4
Fig 4. Shikonin induced apoptosis of bladder cancer cells.
(A-C): 10μM shikonin treatment over 48h induced activation of Caspase-3 in HTB-9, HTB-5 and UM-UC3 cells. (D): PKM2 tetramer vs dimer formation after shikonin exposure for 72h in UM-UC3 cells. Columns depict the mean ± S.E.M. from three independent experiments (n = 3). * and ** indicates significant two-tailed t-test p value of < 0.05 and < 0.01 respectively, compared with vehicle-treated (DMSO) control.
Fig 5
Fig 5. Tumor M2-PK accurately distinguishes controls from patients with bladder cancer.
Voided urine was collected from either control volunteers or bladder cancer patients. Tumor M2-PK was performed at various dilutions on urine and M2-PK concentrations were determined (A). Receiver operator characteristic curve analysis was used to assess the capacity of M2-PK to distinguish between control and bladder cancer patients (B).
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