Both high expression of pyruvate kinase M2 and vascular endothelial growth factor-C predicts poorer prognosis in human breast cancer

Abstract

Pyruvate kinase M2 (PKM2) and vascular endothelial growth factor-C (VEGF-C) have been known to play an important role in tumorigenesis and tumor progression in breast cancer. However, the association between PKM2 and VEGF-C in breast cancer remains unclear. In the present study, a total of 218 specimens from breast cancer patients and 26 paired breast tumors with adjacent normal tissues as well as two breast cancer cell lines were enrolled to investigate the correlation between PKM2 and VEGF-C. We found that PKM2 and VEGF-C mRNA levels were both significantly increasing in breast tumors compared with adjacent normal tissues. Knockdown of PKM2 mRNA expression resulted in VEGF-C mRNA and protein down-regulated as well as cell proliferation inhibited. A positive correlation between PKM2 and VEGF-C expression was identified by immunohistochemical analyses of 218 specimens of patients with breast cancer (P=0.023). PKM2 high expression was significantly correlated with histological grade (P=0.030), lymph node stage (P=0.001), besides VEGF-C high expression was significantly associated with lymphovascular invasion (P=0.012). While combined high expression of PKM2 and VEGF-C was found to be associated with worse histological grade, more lymph node metastasis, more lymphovascular invasion, shorter progression free survival (PFS), and poorer overall survival (OS) in human breast cancer. The results of the present study suggested that PKM2 expression was correlated with VEGF-C expression, and combination of PKM2 and VEGF-C levels had the better prognostic significance in predicting the poor outcome of patients with breast cancer.

Keywords: Breast cancer; pyruvate kinase M2; vascular endothelial growth factor-C.

Figure 1

PKM2 expression was correlated with VEGF-C expression and cell proliferation. A. PKM2 and VEGF-C mRNA expression elevated in breast tumors compared with paired adjacent normal tissues. B. qRT-PCR analyzed the efficacy of PKM2 knockdown by lentivirus-mediated RNAi in MCF-7 and MDA-MB-231 cell lines. C. RT-PCR analysis of VEGF-C mRNA expression in MCF-7 and MDA-MB-231 cell lines following PKM2 knockdown. D. Western blot analysis of PKM2 and VEGF-C expression in MCF-7 and MDA-MB-231 cell lines after PKM2 knockdown. E, F. Effects of PKM2 knockdown on cell proliferation was evaluated by CCK8 assays in MCF-7 and MDA-MB-231 cell lines, respectively. *Indicates P<0.05, **indicates P<0.01.

Figure 2

Immunohistochemical staining for PKM2 and VEGF-C expression in breast invasive ductal carcinoma. These two proteins were both restricted to the cytoplasm of tumor cells. A. PKM2 low expression (200×). B. PKM2 high expression (200×). C. VEGF-C low expression (200×). D. VEGF-C high expression (200×).

Figure 3

The association of combined of PKM2 and VEGF-C expression with patients’ clinicopathologic features. No significant differences in tumor stage among the three groups (A). Both high expression of PKM2 and VEGF-C had the worst histological grade (B), the most lymph node metastasis (C) and the most lymphovascular invasion (D) in the three groups. *Indicates P<0.05, **indicates P<0.01, ***indicates P<0.001.

Figure 4

Kaplan-Meier plots with log rank test of progression-free survival (PFS) and overall survival (OS). The mean time (months) of survival was showed. PFS (A) and OS (B) based on combined PKM2 and VEGF-C status. All of the 218 patients were separated into three groups according to the grouping method before: the blue line represents PKM2 and VEGF-C both low expression; the green line represents only one protein high expression; the red line represents PKM2 and VEGF-C both high expression.