Pigment epithelium-derived factor promotes tumor metastasis through an interaction with laminin receptor in hepatocellular carcinomas

Abstract

Pigment epithelium-derived factor (PEDF) has complex functions in tumor metastasis, but little is known about the roles of PEDF and its receptors in hepatocellular carcinoma (HCC). Here we found that high expression of PEDF is associated with shorter overall survival in HCC patients. Forced expression of PEDF enhanced HCC cell aggressive behavior in vitro and in vivo, whereas silencing PEDF expression reduced migration and invasion. Furthermore, PEDF expression led to changes in cell morphology and the expression of epithelial-mesenchymal transition (EMT)-related markers via ERK1/2 signaling pathway, including the upregulation of N-cadherin and slug, and the downregulation of E-cadherin in HCC cells. Our results further showed that PEDF could interact with laminin receptor (LR) and LR knockdown attenuated PEDF-induced migration, invasion and the change of EMT-related markers. More importantly, in clinical HCC specimens, we found that PEDF expression was correlated with subcellular localization of LR, and that high expression of PEDF and positive expression of LR predicted a poor prognosis. In conclusion, our results demonstrate a novel functional role of PEDF/LR axis in driving metastasis through ERK1/2-mediated EMT in HCC and provided a promising prognostic marker in HCC.

Figure 1

Clinical significance of PEDF in HCC. (a) Western blotting of PEDF expression level in HCC tissues and matched adjacent non-tumorous liver tissues. T, HCC tissues; N, adjacent non-tumorous liver tissues. (b) Statistical analysis of PEDF expression in HCC samples between HCC tissues and adjacent non-cancerous tissues. (c) IHC analysis of PEDF expression in 149 HCC samples. Representative images are shown. (d) OS analysis of HCC patients with PEDF expression (n=149)

Figure 2

PEDF promotes HCC cell migration and invasion in vitro and in vivo. (a) The in vitro migration and invasion ability of SMMC-7721, HCC-LY5 and PLC/PRF/5 cells lentivirally transduced with PEDF or with vector (pWPXL) were assessed using a transwell assay. (b) The in vitro migration and invasion ability of Huh7 and MHCC-97L cells lentivirally transduced with shPEDF were assessed using a transwell assay; shNC was used as the control. (c) The numbers of intrahepatic metastatic nodules are shown in the panels. (d) Representative images of liver metastatic nodules and lung metastatic nodules derived from SMMC-7721 cells stably overexpressing PEDF or the control are presented. The statistical analyses compare the experimental group and the control group. *P<0.05 and **P<0.01

Figure 3

PEDF positively regulates EMT in HCC cells. (a) The images show morphologic changes in PEDF-overexpressing SMMC-7721, HCC-LY5 and PLC/PRF/5 cells and in the control. (b) The expression of E-cadherin, N-cadherin, slug, pERK1/2 and ERK1/2 was detected by western blotting in PEDF-overexpressing SMMC-7721, HCC-LY5 and PLC/PRF/5 cells, and PEDF knockdown Huh7 and MHCC-97L cells, and the corresponding controls. (c) Western blotting analysis showed that ERK1/2 inhibitor U0126 (10 μM) and PD98059 (30 μM) inhibited the activation of ERK1/2 signaling induced by PEDF, as well as the EMT-related markers in SMMC-7721, HCC-LY5 and PLC/PRF/5. (d) Western blotting analysis showed that slug knockdown inhibited the change of E-cadherin and N-cadherin induced by PEDF overexpression

Figure 4

PEDF induces the change of EMT-related molecules in xenograft tissues. (a) The expression of E-cadherin, N-cadherin, slug, pERK1/2 and ERK1/2 was detected by western blotting in xenograft tissues. (b) IHC analysis of PEDF, LR, slug, N-cadherin and CD31 expression in intrahepatic metastasis tissues and lung metastasis tissues. Representative images are shown

Figure 5

PEDF can interact with LR in HCC cells. (a) Co-IP analysis of PEDF interacting with LR in Huh7 and MHCC-97L cells. (b) Co-IP analysis of PEDF interacting with LR in HCC tissues. (c) Immunofluorescence staining was used to assess the colocalization of PEDF and LR in Huh7 (overlap coefficient, means±S.E.M., 0.754±0.058; n=3) and MHCC-97L (overlap coefficient, means±S.E.M., 0.823±0.015; n=3) cells. Representative images are shown. (d) Immunofluorescence staining was used to examine the subcellular localization of LR in PEDF-overexpressing SMMC-7721, HCC-LY5 and PLC/PRF/5 cells, and their corresponding controls. Representative images are shown. (e) Western blotting analysis of LR in membrane (mem) and cytosol (cyto), respectively, in PEDF-overexpressing SMMC-7721, HCC-LY5 and PLC/PRF/5 cells

Figure 6

LR knockdown inhibits HCC cell migration and invasion in vitro, and high expression levels of LR predict a poor prognosis in HCC patients. (a,b) The in vitro migratory and invasive ability of LR knockdown Huh7 and MHCC-97L cells and their corresponding controls were detected and analyzed. The mean values from duplicate samples three independent experiments are shown in (b) (*P<0.05 and **P<0.01). (c) The expression of E-cadherin, N-cadherin, slug, pERK1/2 and ERK1/2 was detected by western blotting in Huh7 and MHCC-97L cells with LR shRNA. (d) Western blotting analysis of LR expression in HCC tissues and matched adjacent non-tumorous liver tissues. T, HCC tissues; N, adjacent non-tumorous liver tissues. (e) Statistical analysis of LR expression in HCC samples between HCC tissues and adjacent non-cancerous tissues. (f) IHC staining analysis of LR expression in 149 HCC samples. Representative images are shown. (g) The OS analysis of HCC patients with LR expression (n=149)

Figure 7

Knockdown of LR attenuates PEDF-induced HCC cell migration, invasion and EMT. (a) SMMC-7721, HCC-LY5 and PLC/PRF/5 cells overexpressing PEDF were transfected with shRNA targeting to LR as described. The migration and invasion ability were assessed using transwell assays. Cells transfected with the empty vector were used as a control (*P<0.05 and **P<0.01). (b) SMMC-7721, HCC-LY5 and PLC/PRF/5 cells overexpressing PEDF were transfected with shRNA targeting to LR as described. The expression of E-cadherin, N-cadherin, slug, pERK1/2 and ERK1/2 was detected by western blotting

Figure 8

LR and N-cadherin expression is positively correlated with PEDF in HCC tissues, indicating a poor prognosis in HCC patients. (a) Representative images of PEDF and LR in HCC tissues are shown in the upper panel. The correlation between PEDF and subcellular localization of LR in HCC tissue is shown in the lower panel. (b) Representative images of PEDF and N-cadherin in HCC tissues are displayed in the upper panel. The correlation between PEDF and N-cadherin in HCC tissues is presented in the lower panel. (c) The OS analysis of HCC patients with high expression of PEDF and positive expression of LR. (d) The OS analysis of HCC patients with high co-expression of N-cadherin and PEDF. (e) The OS analysis of HCC patients with high co-expression of N-cadherin, PEDF and positive expression of LR. (n=149)