High glucose enhances the metastatic potential of tongue squamous cell carcinoma via the PKM2 pathway

Abstract

Previous evidence has indicated an increased cancer risk in individuals with diabetes mellitus (DM). The aim of this study was to investigate the relationship between DM (high glucose) and tongue squamous cell carcinoma (TSCC) and how high glucose mediated the metastatic potential of TSCC. The relationship between DM and TSCC was assessed in a retrospective study. The role and its mechanism of high glucose on the proliferation, metastatic potential of TSCC were investigated in vitro and in vivo. The prevalence rate of DM in patients with TSCC was 12.84%, which was significantly higher than that (9.7%) in the general population in China. Although no significant difference was observed in the overall survival (OS) rate, TSCC patients with DM have a 1.38-fold increase in relative risk affecting 5-year OS compared to patients without DM. High glucose enhanced the TSCC cell proliferation, migration, invasion and upregulated PKM2 (pyruvate kinase M2) expression. Whereas, these effect was abolished after knockdown of PKM2 in TSCC cells. High glucose promoted tumour growth and lung metastasis of TSCC in a DM animal model. Our results confirm DM as a risk factor for the development of TSCC. High glucose enhances the metastatic potential of TSCC through stimulation of the PKM2 pathway.

Keywords: PKM2; high glucose; metastasis; tongue squamous cell carcinoma.

Figure 1. High glucose promotes the migration and invasiveness of TSCC in vitro

(A-C) Compared with normal glucose (5.6mM), high glucose (11.1mM, 16.7mM or 25.0mM) significantly promoted the migration (A), invasion (B) and proliferation (C) of UM1 cells in a concentration - dependent manner. (D) The protein levels of PKM2, SOD2, Vimentin, Slug and pERK1/2 were obvious increased in UM1 cells cultured with high glucose (11.1mM, 16.7mM or 25.0mM), but the protein level of E-cadherin was decreased. SOD2 activity (E) and H₂O₂ production (F) were significantly increased in UM1 cells cultured with high glucose (11.1mM, 16.7mM or 25.0mM). ^*P<0.05 compared with UM1 cells cultured with 5.6mM glucose.

Figure 2. The gene expression patterns of TSCC cells cultured with normal glucose (5.6mM) and high glucose (16.7mM) determined by microarray analysis

(A) 14/40 of the glycolytic related gene (GO0006096, glycolysis) were upregulated in TSCC cells cultured in 16.7mM glucose compared with those TSCC cells cultured in 5.6mM glucose, including PKM2. The expression of SOD2 [GO0000302, response to reactive oxygen species (B)], Slug [GO0001837, epithelial to mesenchymal transition (C)] were also found to be upregulated in 16.7mM glucose cultured TSCC cells than in 5.6mM glucose cultured TSCC cells.

Figure 3. High glucose mediated the migration and invasiveness of TSCC through PKM2 pathway

(A) The protein levels of PKM2, SOD2, Vimentin, Slug and pERK1/2 were decreased in UM1 cells after knockdown of PKM2 in TSCC cells cultured with 16.7 mM glucose, but the protein level of E-cadherin was decreased. PKM2 knockdown inhibited the migration (B) and invasion (C) abilities, cell proliferation (D), and the SOD2 activity (E) and H₂O₂ production (F) in UM1 cells cultured with 16.7 mM glucose. ^*P<0.05 compared with UM1 cells cultured with 16.7mM glucose and transfected with control siRNA.

Figure 4. High glucose promotes tumour growth of TSCC in vivo

(A-B) CAL27 cells were inoculated subcutaneously into BALC/C nude mice model of DM. Tumour growth was significantly slower in the non-DM group relative to the DM group. (C-D) PKM2 expression was obviously increased in TSCC xenografts from DM group compared with the non-DM group detected by IHC. Scale bar: 50 μm.

Figure 5. High glucose promotes tumour metastasis of TSCC in vivo

UM1 cells were injected into the tail veins of DM and non-DM nude mice. The DM mice exhibited a significantly increased number of metastatic nodules relative to the non-DM group. Scale bar: 50 μm.