The diagnostic and prognostic potential of the EGFR/MUC4/MMP9 axis in glioma patients

Abstract

Glioblastoma is the most aggressive form of brain cancer, presenting poor prognosis despite current advances in treatment. There is therefore an urgent need for novel biomarkers and therapeutic targets. Interactions between mucin 4 (MUC4) and the epidermal growth factor receptor (EGFR) are involved in carcinogenesis, and may lead to matrix metalloproteinase-9 (MMP9) overexpression, exacerbating cancer cell invasiveness. In this study, the role of MUC4, MMP9, and EGFR in the progression and clinical outcome of glioma patients was investigated. Immunohistochemistry (IHC) and immunofluorescence (IF) in fixed tissue samples of glioma patients were used to evaluate the expression and localization of EGFR, MMP9, and MUC4. Kaplan-Meier survival analysis was also performed to test the prognostic utility of the proteins for glioma patients. The protein levels were assessed with enzyme-linked immunosorbent assay (ELISA) in serum of glioma patients, to further investigate their potential as non-invasive serum biomarkers. We demonstrated that MUC4 and MMP9 are both significantly upregulated during glioma progression. Moreover, MUC4 is co-expressed with MMP9 and EGFR in the proliferative microvasculature of glioblastoma, suggesting a potential role for MUC4 in microvascular proliferation and angiogenesis. The combined high expression of MUC4/MMP9, and MUC4/MMP9/EGFR was associated with poor overall survival (OS). Finally, MMP9 mean protein level was significantly higher in the serum of glioblastoma compared with grade III glioma patients, whereas MUC4 mean protein level was minimally elevated in higher glioma grades (III and IV) compared with control. Our results suggest that MUC4, along with MMP9, might account for glioblastoma progression, representing potential therapeutic targets, and suggesting the 'MUC4/MMP9/EGFR axis' may play a vital role in glioblastoma diagnostics.

Figure 1

EGFR, MMP9, and MUC4 protein expression in glioma tissue biopsies. Immunohistochemistry was performed on 60 glioma tissue sections with EGFR, MMP9, and MUC4 antibodies. (A) The slides were categorized into three scores according to the percentage of area and intensity of staining. Score 2 represents the highest expression, magnification, 20 ×. (B) Table representing how the scores are distributed according to the percentage of expressing cells and the expression intensity. (C) Stacked bar graphs representing the distribution (contingency) of scores in grade II, grade III, and grade IV glioma for EGFR, MMP9, and MUC4 (N = 60 cases; 20 per grade) . Statistical differences were assessed with the Chi² test (95% confidence interval). For EGFR, there was no statistical significance (ns = non-significant). For MMP9 and MUC4, the higher rate of expression was observed in higher glioma grades (higher levels in GBM compared to III and II). Significance is indicated on the graph as: *Denotes p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001.

Figure 2

EGFR, MUC4, and MMP9 localization in GBM. (A) Example of one GBM case with extended cytoplasmic EGFR expression. Note the total absence of EGFR-expressing cells in the proliferative microvasculature (white arrows). Magnification. 40 ×. (B) Example of one GBM case displaying dispersed cytoplasmic expression of MUC4. MUC4 is expressed in the endothelial wall (inner layer) of GBM microvasculature (red arrows) and in neoplastic cells, but not in the outer layers (white arrows and black dotted lines). Magnification, 20 ×. (C) Example of one GBM case displaying dispersed cytoplasmic expression of MMP9 in neoplastic areas showing pleomorphism. Magnification, 20 ×. (D, E) Representative images showing specific and restricted expression of MMP9 in cells located inside vessels, resembling immune cells, in one GBM patient; the surrounding neoplastic cells are negative for MMP9. Magnification, 40 ×. (F) Representative image showing MMP9 strong expression in microvascular proliferative structure (glomeruli-shaped) in one GBM patient. Purple arrows indicate MMP9-expressing cells; red blood cells (RBC) localization is indicated by red arrows Magnification, 40 ×. (G) Example of a GBM area showing expression of MUC4 in cancer cells. Magnification, 20 ×. (H, I) Images showing MUC4 expression in endothelial walls of microvascular proliferative areas (purple arrows). Two examples (from two different GBM patients) are shown. RBC red blood cells. Magnification, 20 ×.

Figure 3

MMP9 and MUC4 expression in tumor vascular cells of glioblastoma tissues. MMP9 (A) and MUC4 (B) were expressed in vascular cells using immunohistochemistry and co-localized individually with the vascular/angiogenesis marker CD31 in double Immunofluorescence. RBC red blood cells, white arrows indicate co-localization. Magnification, 20–40 ×.

Figure 4

MMP9, MUC4, and EGFR co-localization in glioblastoma tissues. (A) MMP9 and MUC4 concomitant vascular expression in representative glioblastoma case. Co-localization of both proteins is observed in the vascular areas with double immunofluorescence. (B) EGFR and MUC4 concomitant expression in vascular areas in representative case of glioblastoma observed by immunohistochemistry and immunofluorescence. Co-localization of both proteins is observed with double IF. RBC red blood cells, white arrows indicate co-localization. Magnification, 20–40 ×.

Figure 5

Survival analyses in glioma patients. Comparison of overall survival (OS) (A) between two MMP9 tissue expression groups (low and high) in glioma patients. High expression of MMP9 predicts worse outcome than low expression, but the difference is not significant; (B) between two MUC4 tissue expression groups (low and high) in glioma patients. High expression of MUC4 predicts worse outcome than low expression, but the difference is not significant. (C) MUC4-MMP9 combined high expression significantly predicts worse survival in glioma patients as assessed by the log-rank test for trend (p = 0.018). MUC4-L/MMP9-L patients have better survival than MUC4-H/MMP9-H patients as assessed by log-rank test (Mantel Cox, p = 0.023). (D) MUC4/MMP9/EGFR expression significantly predicts worse survival in glioma patients. Triple low patients (with low expression for the three markers) have better survival than double high patients (with high expression of two out of the three markers) as assessed by log-rank test (Mantel Cox, p = 0.004).

Figure 6

MMP9 and MUC4 protein serum levels determined by ELISA immunoassays. Scatter graphs showing two forms of MMP9 (active form and pro-form) (A), MMP9 bound to TIMP1 (B), and MUC4 (C) individual concentration values (with means) of non-glioma (CTRL: controls), glioma grade (III), and glioblastoma (GBM) patients. N = 10 for each group (30 patients in total). MMP9 protein levels (ng/ml) and MUC4 protein levels (ng/ml) measured in serum, were normalized based on the total protein; results are given as ng/ml per mg/ml of total protein. The different forms of MMP9 measured in each assay are represented in (A) and (B), and MUC4 structure and cleavage site is represented in (C). Bars represent protein mean levels. MMP9 two forms protein levels as well as the MMP9:TIMP1 complex are significantly higher in the GBM than in grade III group (Tukey’s multiple comparison test, p = 0.022 and 0.018, respectively). MUC4 values are represented on a stretched Y axis in (C). Higher concentrations of MUC4 are found in malignant glioma grades compared with controls but the difference does not reach significance. NIDO Nidogen-like domain, AMOP Adhesion-associated domain, and VWD Von Willbrand factor domain.