Pyruvate kinase M2 deregulation enhances the metastatic potential of tongue squamous cell carcinoma

Abstract

Pyruvate kinase M2 (PKM2) has been verified to correlate with the prognosis of many types of cancer. However, its role in the development and metastasis of tongue squamous cell carcinoma (TSCC) remains unclear. The immunohistochemistry (IHC) results confirmed that PKM2 is overexpressed in patients with TSCC. PKM2 up-regulation was related to lymph node metastasis and associated with reduced overall survival. According to the microarray analysis and Western blots, PKM2 expression was up-regulated in TSCC cells with enhanced metastatic potential. PKM2 knockdown inhibited cell migration and invasion, reduced SOD2 (manganese superoxide dismutase) activity and the intracellular H₂O₂ level, and inhibited tumour growth and lung metastasis in vivo. PKM2 overexpression promoted cell migration and invasion, and increased SOD2 activity and the intracellular H₂O₂ level. Moreover, miR-138 directly targeted PKM2 and inhibited PKM2 expression. Thus, PKM2 deregulation plays an important role in TSCC and may serve as a biomarker of metastatic potential or as a therapeutic target in patients with TSCC. PKM2, a miR-138 target gene, enhances the metastatic potential of TSCC through the SOD2-H₂O₂ pathway.

Keywords: manganese superoxide dismutase; metastasis; miR-138; pyruvate kinase M2; tongue squamous cell carcinoma.

Figure 1. PKM2 deregulation in the development of TSCC and patient prognosis

PKM2 expression in normal tongue tissues (A) and TSCC tissues (B) was analysed by IHC. (C) Box plots present a comparison of PKM2 expression in normal tongue tissue samples and tissue samples from TSCC cases with different tumour stages, different clinical stages, and with or without lymph node metastasis. Each box represents the 25th to the 75th percentile of the observations, and the line in the middle of the box represents the median. (D) Kaplan–Meier plots of the 5-year OS in patient groups as defined by PKM2 expression. The differences in survival rates are statistically significant between the high and low PKM2 expression groups (P=0.012). The numbers of cases in the high PKM2 expression group and low PKM2 expression group were 79 and 58, respectively. Scale bar: 50 μm.

Figure 2. PKM2 overexpression promotes TSCC cell migration and invasion

(A) Western blot analysis showing the levels of the PKM2, pERK, ERK and EMT marker (E-cad, Vimentin, and Slug) proteins in UM2 cells infected with a lentiviral construct containing the PKM2 cDNA (LV-PKM2) or with the control lentiviral construct (Vector). PKM2 overexpression promoted UM2 cell migration (B) and invasion (C). PKM2 overexpression also resulted in increased UM2 cell proliferation (D), SOD2 expression and activity (E) and H₂O₂ production (F). *P<0.05.

Figure 3. PKM2 knockdown inhibits TSCC cell migration and invasion

(A) Western blot results showing the levels of the PKM2, pERK, ERK and EMT marker (E-cad, Vimentin, and Slug) proteins in UM1 cells transfected with the PKM2 siRNA or control siRNA. PKM2 knockdown inhibited the migration (B) and invasion (C) of UM1 cells. PKM2 knockdown also inhibited UM1 cell proliferation (D), SOD2 expression and activity (E) and H₂O₂ production (F). *P<0.05.

Figure 4. The miR-138-PKM2pathwayregulates TSCC cell migration/invasion

(A) Predicted target sequences for miR-138 are located in the 3’-UTR of PKM2 mRNA. (B) Dual luciferase reporter assays were performed to evaluate the target genes of miR-138. Following transfection with pGL-PKM2 for 24 h, luciferase activity was significantly reduced in cells treated with miR-138 mimics relative to cells treated with control mimics (*P<0.05 vs blank). After the seed region of the target site was mutated (pGL-PKM2 m), the effects of miR-138 on luciferase activity were abolished. (C, D) Western blot results showing the levels of the PKM2, SOD2, pERK, ERK and EMT marker (E-cad, Vimentin,and Slug) proteins in TSCC cells transfected with the miR-138 mimic (C) or miR-138 LNA (D).

Figure 5. PKM2 knockdown inhibits TSCC xenograft growth and lung metastasis in vivo

(A) CAL27 cells stably infected with lentiviral constructs containing control shRNA or PKM2 shRNA were subcutaneously injected into nude mice. Xenografts were measured every 3 days with a calliper, and the resulting growth curves demonstrated that PKM2 knockdown significantly inhibited CAL27 xenograft growth. (B) UM1 cells stably infected with lentiviral constructs containing control shRNA or PKM2 shRNA were injected into the tail vein of nude mice. TSCC metastasis to the lung was assessed 8 weeks after injection. The histopathological analysis of lung metastasis (magnification 400×) and metastatic lung nodules are shown. PKM2 expression in xenografts (A) and lung metastases (B) was analysed using IHC. Scale bar: 50 μm. *P<0.05.

Figure 6. PKM2 regulates TSCC cell migration/invasion through miR-138 and the SOD2-H₂O₂ pathway

The pathways for miR-138-regulated EMT had been revealed in our previous reports [–16, 21, 22, 27].