Cooperative interaction of MUC1 with the HGF/c-Met pathway during hepatocarcinogenesis

Abstract

Background: Hepatocyte growth factor (HGF) induced c-Met activation is known as the main stimulus for hepatocyte proliferation and is essential for liver development and regeneration. Activation of HGF/c-Met signaling has been correlated with aggressive phenotype and poor prognosis in hepatocellular carcinoma (HCC). MUC1 is a transmembrane mucin, whose over-expression is reported in most cancers. Many of the oncogenic effects of MUC1 are believed to occur through the interaction of MUC1 with signaling molecules. To clarify the role of MUC1 in HGF/c-Met signaling, we determined whether MUC1 and c-Met interact cooperatively and what their role(s) is in hepatocarcinogenesis.

Results: MUC1 and c-Met over-expression levels were determined in highly motile and invasive, mesenchymal-like HCC cell lines, and in serial sections of cirrhotic and HCC tissues, and these levels were compared to those in normal liver tissues. Co-expression of both c-Met and MUC1 was found to be associated with the differentiation status of HCC. We further demonstrated an interaction between c-Met and MUC1 in HCC cells. HGF-induced c-Met phosphorylation decreased this interaction, and down-regulated MUC1 expression. Inhibition of c-Met activation restored HGF-mediated MUC1 down-regulation, and decreased the migratory and invasive abilities of HCC cells via inhibition of β-catenin activation and c-Myc expression. In contrast, siRNA silencing of MUC1 increased HGF-induced c-Met activation and HGF-induced cell motility and invasion.

Conclusions: These findings indicate that the crosstalk between MUC1 and c-Met in HCC could provide an advantage for invasion to HCC cells through the β-catenin/c-Myc pathway. Thus, MUC1 and c-Met could serve as potential therapeutic targets in HCC.

Figure 1

Expression analysis of MUC1 and c-Met in HCC cell lines. Total cell lysates were extracted from HCC cell lines to detect MUC1 and c-Met protein expression by immunoblotting assay. Two bands were detected for MUC1 due to posttranslational modification. Precursor protein band (170 kDa) and biologically active transmembrane β-subunit (140 kDa) of c-Met were detected. Calnexin was used to verify equal protein loading and transfer.

Figure 2

Analysis of MUC1 and c-Met expression in normal and cirrhotic liver tissue, and HCC. Negative MUC1 expression in normal hepatocytes and positive MUC1 expression was localized to bile ducts (×400) (A). Cirrhotic liver tissue showed weak, diffuse cytoplasmic MUC1 staining (×400) (B). HCC displayed intense MUC1 staining (×400) (C). Each column represents histologically classified liver tissues (normal liver, cirrhotic liver, HCC) with the height representing the ratio of positive staining for MUC1 (D). c-Met expression in normal liver tissue showed very weak or no immunoreactivity (×400) (E). Cytoplasmic c-Met staining in hepatocytes in the cirrhotic liver tissue (×400) (F). HCC displayed positive cytoplasmic and strong membranous c-Met staining (×400) (G). Comparison of the ratios of positive staining for c-Met in normal liver, cirrhotic liver, and HCC tissues (H).

Figure 3

Immunohistochemical characterization of MUC1 and c-Met expression in HCC tissues relative to tumor differentiation status. Tissue sections from tumors with well (A, E), moderate (B, F), and poor (C, G) differentiation were assayed for MUC1 (A,B,C) and c-Met (E, F, G) expression by immunohistochemistry. Each column represents the ratio of positive staining for MUC1 (D) and c-Met (H) in well-, moderate-, and poorly-differentiated HCC. (E: 200X; A-D, F,G: 400X magnification).

Figure 4

Identification of MUC1 as an interaction partner for c-Met and effect of HGF on this complex. Endogenous MUC1/c-Met interaction was carried out in a total cell lysate from Mahlavu cells (A) and SNU-449 (C) using anti-c-Met antibody by IP. The immunoblotting (IB) with MUC1 showed co-precipitation of endogenous c-Met and MUC1 in unstimulated and HGF stimulated cells. Anti-c-Met antibody was probed to the membrane as a loading control. There was no detectable c-Met and MUC1 in immunoprecipitates prepared with IgG as an IP-control. MUC1 signal intensities were measured by scanning the ECL exposed films with a densitometer. The relative MUC1 intensities (mean ± S.E.) for the different treatments relative to the levels present in the untreated control sample are compared in the bar graphs. Graphs represent data obtained from Mahlavu cells (B) and SNU-449 cells (D).

Figure 5

Activation of c-Met signaling pathway by HGF administration induced p42/44-MAPK and β-catenin phosphorylation. Overnight starved Mahlavu (A, B) and SNU-449 (C, D) cells were stimulated with medium alone and with HGF for 15, 30, and 60 min. Total cell lysates were then analyzed with Western blotting. Blots were probed with anti-p-Met, anti-c-Met, anti-MUC1, p-p42/44-MAPK, p42/44-MAPK, anti-p-β-catenin, anti-β-catenin, anti-c-Myc, and anti-calnexin antibodies.

Figure 6

SU11274 selectively inhibited c-Met phosphorylation and downstream signaling. Mahlavu cells were incubated with medium alone or with SU11274 overnight. Then cells were treated with or without HGF for 60 min. Total protein lysates were analyzed by immunoblotting. Membranes were blotted with anti-p-Met, anti-c-Met, anti-MUC1 (A), anti-p-β-catenin, anti-β-catenin, anti-c-Myc (B), and anti-calnexin antibodies. SU11274 pretreated cells and control cells were seeded in the upper chamber of Boyden chambers. No HGF or 40 ng/ml HGF was added to the lower chamber. Following 24 h incubation, cells that had migrated or invaded onto the lower surface were stained and counted. Bars represent mean number ± S.E. of migrating (C) or invading (D) cells (* indicates p < 0.05, NS: not significant).

Figure 7

Inhibition of MUC1 in Mahlavu cells. Inhibition of MUC1 protein expression was demonstrated by siRNA gene silencing targeting of MUC1 mRNA. Mahlavu cells were transfected with MUC1-specific siRNA and control siRNA and analyzed using immunoblotting with anti-MUC1 antibody (A). Calnexin was blotted as internal loading control. Relative quantification of the MUC1 bands was done using ImageJ. Graph represents signals obtained from MUC1 bands. Error bars indicate SEM (*indicates p < 0.05, NS: not significant) (B). Total cell lysates from Mahlavu cells transfected with the control and MUC siRNAs and control siRNA were analyzed by immunoblotting. Membranes were blotted with anti-p-Met, anti-c-Met, anti-p-β-catenin, anti-β-catenin, anti-c-Myc and anti-calnexin antibodies (C). MUC1 knock-down cells and control cells were seeded in the upper chamber of Boyden chambers. After 24 h incubation, migrated and invaded cells were counted. Bars represent the mean ± S.E. of migrating (D) or invading (E) cell number (* indicates p < 0.05).

Figure 8

Consequences of MUC1 silencing on downstream signaling molecules. Mahlavu cells were transfected with siRNA targeted to MUC1 for 72 h followed by 60 min HGF stimulation. Controls included no treatment and MUC1 siRNA-treated cells. Total protein lysates were analyzed by immunoblotting. Membranes were blotted with anti-p-Met, anti-c-Met, anti-MUC1, anti-p-β-catenin, anti-β-catenin, anti-c-Myc, and anti-calnexin antibodies.

Figure 9

Effect of MUC1 silencing on the cellular motility and invasion. MUC1 and control siRNA treated cells were seeded in the upper chamber of Boyden chambers. Medium with or without HGF was added to the lower chamber. After 24 h incubation, migrated Mahlavu (A) and SNU-449 (C) cells and invaded Mahlavu (B) and SNU-449 (D) cells were counted. Bars represent the mean ± S.E. of migrating (A,C) or invading (B,D) cell number (* indicates p < 0.05).