Pyruvate kinase M2 is a novel diagnostic marker and predicts tumor progression in human biliary tract cancer - PubMed (original) (raw)

Pyruvate kinase M2 is a novel diagnostic marker and predicts tumor progression in human biliary tract cancer

Dipok Kumar Dhar et al. Cancer. 2013.

Abstract

Background: The early diagnosis of biliary tract cancer (BTC) remains challenging, and there are few effective therapies. This study investigated whether the M2 isotype of pyruvate kinase (M2-PK), which serves as the key regulator of cellular energy metabolism in proliferating cells, could play a role in the diagnosis and therapy of BTC.

Methods: Plasma and bile M2-PK concentrations were measured by enzyme-linked immunosorbent assay in 88 patients with BTC, 79 with benign biliary diseases, and 17 healthy controls. M2-PK expression was assayed in a BTC tissue array by immunohistochemistry. The role of M2-PK in tumor growth, invasion, and angiogenesis was evaluated in BTC cell lines by retrovirus-mediated M2-PK transfection and short hairpin RNA silencing techniques.

Results: Sensitivity (90.3%) and specificity (84.3%) of bile M2-PK for malignancy were significantly higher than those for plasma M2-PK and serum carbohydrate antigen 19-9. M2-PK expression was specific for cancer cells and correlated with microvessel density. M2-PK positivity was a significant independent prognostic factor by multivariable analysis. Transfection of M2-PK in a negatively expressed cell line (HuCCT-1 cells) increased cell invasion, whereas silencing in an M2-PK-positive cell line (TFK cells) decreased tumor nodule formation and cellular invasion. A significant increase in endothelial tube formation was noted when supernatants from M2-PK-transfected cells were added to an in vitro angiogenesis assay, whereas supernatants from silenced cells negated endothelial tube formation.

Conclusions: Bile M2-PK is a novel tumor marker for BTC and correlates with tumor aggressiveness and poor outcome. Short hairpin RNA-mediated inhibition of M2-PK indicates the potential of M2-PK as a therapeutic target.

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Figures

Figure 1

Plasma (A) and bile (B) M2-PK levels of healthy controls, patients with benign biliary conditions and biliary tract cancer are shown. Pairwise comparison of the pM2-PK and bM2-PK values of each individual patient with biliary tract cancer(C). Comparison of ROC curves of pM2-PK and bM2-PK and CA19-9 is shown (D).

Figure 2

Survival stratified by M2-PK (left) and Ca19-9 (right) are shown. Patients with high M2-PK had significantly worse overall survival than those with low M2-PK.

Figure 3

Double immunohistochemistry for M2-PK (brown colour) and CD34 (red colour) in representative sections of BTC showing weak M2-PK expression with few blood vessels(A), strong M2-PK expression with numerous blood vessels (B, yellow arrows), and negative expression in peritumoral lymphocytes (C, arrows) (x200). Tumours with strong M2-PK expression were characterized by larger number of blood vessels (D).

Figure 4

Strong M2-PK expression was noted in all cell lines except HuCCT(A). M2-PK western blot of HuCCT following transfection with (right 3 lanes) or without (2nd lane) full length M2-PK (B). M2-PK western blot of mock-treated TFK cells and TFK cells silenced with shRNAs (clones G, R and Y) (C). Cell proliferation rates of mock-treated HuCCT, M2-PK transfected HuCCT, mock-treated TFK and M2-PK silenced TFK cells (D). MTS assay showed increased cell proliferation in M2-PK transfected HuCCT cells and significant growth inhibition in M2-PK silenced cells (D). Anti-proliferative effect of different shRNA clones are shown; all clones except clone B had significant growth inhibition (E).

Figure 5

3D spheroids formation of parental HuCCT (A), M2-PK-transfected HuCCT (B), parental TFK (C) and M2-PK-silenced TFK (D) in the AlgiMatrix 3D cell culture system. Mean number of spheroids/high power field is shown (E).

Figure 6

Invasive potential of wild-type HuCCT (A), M2-PK-transfected HuCCT (B), wild type TFK (C) and M2-PK-silenced TFK (D). Figures A-D show the cells which migrated through the membrane. (E) Bars represent the mean ± SD.

Figure 7

Capillary formation in the presence of suramin (A), recombinant VEGF (B), cell culture supernatant of wild-type HuCCT (C), supernatant of M2-PK-transfected HuCCT (D), supernatant of wild-type TFK (E) and M2-PK silenced TFK (F). (G) Bars represent the mean ± SD.

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Pyruvate kinase M2 is a novel diagnostic marker and predicts tumor progression in human biliary tract cancer - PubMed (original) (raw)