Overexpression of c-Met increases the tumor invasion of human prostate LNCaP cancer cells in vitro and in vivo

Abstract

c-Met is a transmembrane tyrosine kinase receptor that may be activated by hepatocyte growth factor, an inducer of epithelial-mesenchymal transition (EMT), to regulate the associated downstream gene expression. This process is critical to cell migration in normal and pathological conditions. In the present study, the function of c-Met in the process of EMT was investigated in prostate cancer. Initially, a c-Met stable expression cell line was constructed using EMT- and c-Met-negative LNCaP prostate cancer cells. Following the identification of c-Met in the transfected cells, the changes in EMT, phosphatidylinositol 3-kinase (PI3K) and extracellular signal-regulated kinase pathway biomarkers were determined by western blot analysis. MTT, soft agar and Transwell assays, and xenograft studies were used to investigate the effects of c-Met on the proliferation, migration and tumorigenicity of LNCaP cells. The results of the present study revealed downregulation of E-cadherin and upregulation of vimentin in LNCaP-Met cells. The results demonstrated that c-Met enhanced proliferation, migration and tumorigenicity capacity when compared with LNCaP and LNCaP-pcDNA3.1 cells. Furthermore, these EMT-like changes were mediated via the PI3K and mitogen-activated protein kinase signaling pathways. The present study clearly demonstrates a crucial function for c-Met in EMT development in prostate cancer. c-Met-targeted treatment may be an effective adjuvant therapy for improving survival rates in patients with prostate cancer.

Keywords: c-Met; epithelial-mesenchymal transition; invasive potency; prostate cancer.

Figure 1

Overexpression of c-Met in LNCaP-Met cells. (A) Immunocytochemistry revealed marked red immunofluorescence staining in the membranes of LNCaP-Met cells when compared with LNCaP and LNCaP-pcDNA3.1 cells (tetramethylrhodamine staining, magnification, ×400). (B) Western blot analysis revealed the expression levels of c-Met and phospho-c-Met in (a) LNCaP-Met, (b) LNCaP and (c) LNCaP-pcDNA3.1 cells. DAPI, 4′,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 2

Changes in epithelial-mesenchymal transition-associated markers in three cell lines. The expression of E-cadherin and vimentin were determined by western blot analysis in (a) LNCaP-pcDNA3.1, (b) LNCaP and (c) LNCaP-Met cells. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 3

c-Met enhances the invasiveness of LNCaP-Met cells. (A and B) Transwell, (C) MTT and (D) soft agar assays indicated that c-Met increases the migration, proliferation and tumorigenicity of LNCaP-Met cells in contrast to the control LNCaP and LNCaP-pcDNA3.1 cells. ^*P<0.05, LNCaP-Met vs. LNCaP cells; ^**P<0.05, LNCaP-Met vs. LNCaP-pcDNA3.1 cells. OD, optical density. Crystal violet staining. Magnification, ×200.

Figure 4

Xenograft studies in mice. (A) Tumors grew more rapidly in the LNCaP-Met cell group when compared with the other two groups. (B) The average tumor weight in the LNCaP-Met cell group was the largest after eight weeks, when compared with the other groups (P<0.05).^*P<0.05, LNCaP-Met vs. LNCaP cells; ^**P<0.05, LNCaP-Met vs. LNCaP-pcDNA3.1 cells.

Figure 5

Western blot analysis of phosphoinositide 3-kinase and mitogen-activated protein kinase pathways in LNCaP-Met cells. Proteins were extracted from (a) LNCaP-pcDNA3.1, (b) LNCaP and (c) LNCaP-Met cells and the expression levels of ERK, p-ERK, AKT, p-AKT were determined. ERK, extracellular signal-regulated kinase; p, phosphorylated; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.