Deregulation of MUC4 in gastric adenocarcinoma: potential pathobiological implication in poorly differentiated non-signet ring cell type gastric cancer

Abstract

MUC4 is a large, heavily glycosylated transmembrane mucin, that is implicated in the pathogenesis of various types of cancers. To date, no extensive study has been done to check the expression and functional significance of MUC4 in different types of gastric adenocarcinomas. Here, we report the expression profile of MUC4 in gastric adenocarcinomas and its function in poorly differentiated gastric non-signet ring cell carcinoma (non-SRCC) type cells. Immunohistochemical analysis using tissue microarray (TMA) showed a significant difference in MUC4 expression between normal adjacent (n = 45) and gastric adenocarcinoma (n = 83; P < 0.001). MUC4 expression was not associated with tumour type, stage or with the degree of differentiation. To gain further insight into the significance of MUC4 expression in gastric non-SRCC cells, MUC4 was ectopically expressed in AGS, a poorly differentiated gastric non-signet ring cell line. The MUC4 overexpressing cells (AGS-MUC4) showed a significant increase (P < 0.005) in cell motility and a decrease in cellular aggregation as compared with the vector-transfected cells. Furthermore, in vivo tumorigenicity analysis revealed that animals transplanted with the MUC4 overexpressing cells (AGS-MUC4) had a greater incidence of tumours (83%) in comparison to empty vector control (17%). In addition, the expression of MUC4 resulted in enhanced expression of total cellular ErbB2 and phosphorylated ErbB2. In conclusion, our results showed that MUC4 is overexpressed in gastric adenocarcinoma tissues, and that it has a role in promoting aggressive properties in poorly differentiated gastric non-SRCC cells through the activation of the ErbB2 oncoprotein.

Figure 1

Immunohistochemical analysis of gastric tissues for MUC4 expression by using gastric cancer tissue microarray (TMA) slides. Tissue sections were stained for MUC4 using anti-MUC4 monoclonal antibody followed by biotinylated secondary antibody incubation and streptavidin peroxidase 3,3′-diaminobenzidine-chromogen detection. All the sections were examined under microscope and the immunoreactivity was judged by dark brown staining. (A) Representative picture of stained gastric normal adjacent tissues showing no visible MUC4 staining. (B) Representative picture of gastric adenocarcinoma tissues showing diffused MUC4 staining. All sections were counter stained with haematoxylin. In all the top panels, original magnification is × 10 and in bottom panels, original magnification is × 40.

Figure 2

Western blot analysis of MUC4 expression in gastric cancer cell lines. Total protein lysates from AGS, KATOIII and MKN45 gastric cancer cells were prepared. Protein lysate from FG (pancreatic cancer cell line) cells was taken as a positive control. Protein lysates were electrophoretically resolved on 2% agarose gel. Resolved proteins were transferred onto PVDF membrane and probed with MUC4 MAb (8G7) and detected using Amersham HRP-conjugated secondary antibody and ECL kit. Immunoblot of β-actin, obtained from 10% SDS–PAGE/Western, was used as an internal control to correct for the loading variation.

Figure 3

Expression of MUC4 in AGS and its derived sublines: AGS-vector (empty vector transfected) and AGS-MUC4 (MUC4-transfected) cells. (A) Western blot analysis: total protein lysates were prepared from the subconfluent cells. A total of 20 μg protein from cell extracts was electrophoretically resolved on 2% Agarose gel. Resolved proteins were transferred onto PVDF membrane and probed with MUC4 MAb (8G7). Protein from FG (pancreatic cancer cell line) cells was taken as a positive control. (B) Expression analysis of MUC4 using confocal microscopy: Cells were grown at a low density on sterilised cover slips; after methanol fixation, slides were incubated with MUC4 MAb (8G7), followed by FITC-conjugated secondary antibody, and were observed under a ZEISS confocal laser-scanning microscope (magnification, × 630).

Figure 4

Phenotypic changes of AGS-MUC4 cells compared with AGS-vector cells. (A) Cell motility assay: MUC4 expression correlates with the cell motility. Cells (1 × 10⁶) were plated in the top chamber of noncoated polyethylene teraphthalate membranes and incubated for 20 h. Cells that transversed the membranes were stained with a Diff-Quick cell staining kit. The number of cells transversing the membrane was determined by averaging 10 random fields of view at × 100 and expressed as the average number of cells/field of view and is the average of two independent experiments. Mean±s.e.; n=20; ^*P<0.005. Cell motility was significantly (P<0.005) increased in MUC4-transfected AGS cells. (B) Aggregation assay: drops of medium (20 μl each) containing 500 cells/drop were pipetted onto the inner surface of the lid of a Petri dish. After overnight incubation at 37°C, the lid of the Petri dish was inverted and photographed using a Nikon TS100 inverted tissue culture microscope at × 40 magnification. An increased cellular aggregation observed in AGS-MUC4 cells.

Figure 5

Effect of MUC4 expression on ErbB2 expression and phosphorylation. (A) Western blot analysis: a total of 20 μg of protein from AGS-derived cell lines, were resolved by SDS-PAGE and transferred to PVDF membrane and probed with antibodies against, ErbB2, Phospho-tyr 1248 ErbB2, and β-actin. MUC4-transfected AGS cells showed an increased level of total and phosphorylated ErbB2 in comparison to vector-transfected cells.