Dystroglycan expression is frequently reduced in human breast and colon cancers and is associated with tumor progression

Abstract

Dystroglycan (DG) is an adhesion molecule responsible for crucial interactions between extracellular matrix and cytoplasmic compartment. It is formed by two subunits, alpha-DG (extracellular) and beta-DG (transmembrane), that bind to laminin in the matrix and dystrophin in the cytoskeleton, respectively. In this study we evaluated by Western blot analysis the expression of DG in a series of human cancer cell lines of various histogenetic origin and in a series of human primary colon and breast cancers. Decreased expression of DG was observed in most of the cell lines and in both types of tumors and correlated with higher tumor grade and stage. Analysis of the mRNA levels suggested that expression of DG protein is likely regulated at a posttranscriptional level. Evaluation of alpha-DG expression by immunostaining in a series of archival cases of primary breast carcinomas confirmed that alpha-DG expression is lost in a significant fraction of tumors (66%). Loss of DG staining correlated with higher tumor stage (P = 0.022), positivity for p53 (P = 0.033), and high proliferation index (P = 0.045). A significant correlation was also observed between loss of alpha-DG and overall survival (P = 0.013 by log-rank test) in an univariate analysis. These data indicate that DG expression is frequently lost in human malignancies and suggest that this glycoprotein might play an important role in human tumor development and progression.

Figure 1.

A: Levels of expression of α- and β-DG in a series of human normal and cancer cell lines as assessed by Western blot analysis. B: RT-PCR analysis of DG gene expression in the same cell lines amplifying β-actin (as internal control) with DG. In lane 1 the DNA molecular marker standard is shown. The sizes of the specific RT-PCR products were 550 bp for β-actin and 485 bp for DG.

Figure 2.

Levels of expression of α- and β-DG in representative cases of primary colon carcinomas (T) and paired normal adjacent mucosa (M) as assessed by Western blot analysis.

Figure 3.

Box-plot of relative density values of α-DG (A and B) and β-DG (C and D) expression in primary colon carcinomas stratified according with tumor stage (A and C) and grade (B and D). The values shown represent the average of four experiments as shown in Figure 2 ▶ . The intensities of each of the bands on the Western blots were quantitated and adjusted according with the value of the internal positive control represented by total cell extract of the SW620 cells (not shown). For each group, the bottom and top edges of the box are the 25th and 75th percentiles, respectively. Median values are shown by the lines within the boxes. The circles indicate the minimum and maximum values, respectively. Whiskers are obtained by not including the data points in the extreme 20% of the observed values. Within each set (A–D) groups of values not sharing the same letter were significantly different (P < 0.05).

Figure 4.

A: α-DG expression was assayed by Western blot analysis in a series (1 to 7) of selected primary colon cancer samples. Cell lysate from the SW620 cell line was also included for comparison (first lane). B: Expression of DG mRNA was evaluated in the same tumors by RT-PCR (see Figure 1 ▶ ). Values of ratios of DG to β-actin are shown in the associated graphs.

Figure 5.

Immunohistochemical staining of α-DG in representative cases of breast carcinomas. A: Immunostaining shows a clear membranous staining in normal ductal epithelial cells. B: Representative case of ductal carcinoma with positive membranous staining. C: Loss of α-DG expression in high-grade ductal carcinoma (left). Normal lobules (right) display a pagetoide invasion with positive staining in the residual normal ducts but not in the infiltrating neoplastic cells. Original magnifications: ×100 (A and B); ×200 (C); ×800 (insets in A and B).

Figure 6.

Kaplan-Meier curves for disease-free (A) and overall (B) survival in 100 patients who underwent surgery for breast carcinoma stratified according to α-DG expression.