Hepatocyte growth factor increases the invasive potential of PC-3 human prostate cancer cells via an ERK/MAPK and Zeb-1 signaling pathway

Abstract

Hepatocyte growth factor (HGF) has been implicated in epithelial-mesenchymal transition (EMT) in numerous types of cancer. However, to the best of our knowledge, there has been no previous evidence that HGF has a role in prostate cancer. The present study aimed to investigate the effect of HGF on EMT and invasive potential, as well as the underlying molecular mechanisms, in a human prostate cancer cell line. Therefore, PC-3 cells were treated with various concentrations of HGF for varying durations. EMT-associated proteins, including E-cadherin and vimentin, were examined by western blot analysis. The effects of HGF on cell proliferation, migration, invasion and tumorigenicity were assessed using MTT, wound-healing, Transwell and soft-agar assays. Subsequently, the role of c-Met in the mediation of EMT-like changes was investigated using reverse transcription-polymerase chain reaction, western blot analysis and gene knockdown by small interfering RNA. Finally, western blot analysis was used to quantify the expression of a downstream transcription factor and extracellular signal-related kinase/mitogen activated protein kinase (ERK/MAPK) signaling pathway proteins. The results indicated that treatment with HGF induced EMT-like changes and enhanced the invasive potential of PC-3 cells. There was an increase in the expression of ERK, phosphorylated-ERK and zinc finger E-box binding homeobox-1 (Zeb-1), suggesting that EMT-like changes may be mediated through the ERK/MAPK and Zeb-1 signaling pathway. Furthermore, HGF-mediated EMT-like changes were associated with c-Met activation, and these changes were able to be blocked by c-Met knockdown. The present study demonstrated that HGF-induced EMT increased the invasive potential of PC-3 human prostate cancer cells through activating the ERK/MAPK and Zeb-1 signaling pathway.

Keywords: epithelial-mesenchymal transition; extracellular signal-related kinase/mitogen activated protein kinase signaling pathway; hepatocyte growth factor; prostate cancer; zinc finger E-box binding homeobox.

Figure 1.

HGF induces epithelial-mesenchymal transition-like changes in PC-3 cells. (A) HGF (60 ng/ml) treatment downregulates E-cadherin and upregulates vimentin in a time-dependent manner, compared with untreated PC-3 cells (control). Changes were not observed with treatemnet with 20 or 40 ng/ml HGF. (B) E-cadherin expression was restored 7 days subsequent to the withdrawal of HGF. HGF, hepatocyte growth factor.

Figure 2.

HGF treatment increases the expression of c-Met. PC-3 cells were treated with 60 ng/ml HGF, and c-Met expression was measured at the (A) messenger RNA level (treatment for 0, 12, 24 and 36 h) and (B) protein level (treatment for 36 h). HGF, hepatocyte growth factor; p, phosphorylated.

Figure 3.

HGF enhances cell proliferation, tumorigenicity, migration and invasion. (A) HGF stimulated PC-3 cell proliferation in the MTT assay. (B) HGF-treated PC-3 cells exhibited increased tumorigenicity in the soft-agar assay (t=2.773; *P<0.05 vs. HGF). (C) HGF-treated cells demonstrate increased migration potential in the wound-healing assay. (D and E) In contrast to control PC-3 cells, HGF-treated cells displayed significantly increased invasiveness in the Transwell assay (t=2.481 and 2.532; P<0.05 vs. HGF at *24 and **48 h, respectively). Magnification, ×400. Data are expressed as the mean ± standard deviation. HGF, hepatocyte growth factor; OD, optical density.

Figure 4.

ERK/mitogen activated protein kinase signaling is involved in HGF-induced epithelial-mesenchymal transition. PC-3 cells were treated with HGF (60 ng/ml) for 36 h, and ERK, p-ERK and Zeb-1 expression levels were examined by western blotting. HGF, hepatocyte growth factor; ERK, extracellular signal-related kinase; Zeb-1, zinc finger E-box binding homeobox-1; p, phosphorylated.

Figure 5.

Role of c-Met in HGF-induced EMT. (A) Western blot analysis of c-Met expression. (B) c-Met knockdown inhibits EMT-like changes induced by HGF (60 ng/ml), compared with untreated PC-3 cells and cells treated with control siRNA. (C) HGF treatment upregulates ERK, p-ERK and Zeb-1 in PC-3 cells and cells treated with control siRNA cells, but not in cells treated with c-Met siRNA. Lanes: a, PC-3 cells; b, cells treated with control siRNA; and c, cells treated with c-Met siRNA. EMT, epithelial-mesenchymal transition; siRNA, small interferingRNA; HGF, hepatocyte growth factor; ERK, extracellular signal-related kinase; Zeb-1, zinc finger E-box binding homeobox-1; p, phosphorylated.