Diffuse mesothelin expression leads to worse prognosis through enhanced cellular proliferation in colorectal cancer

Abstract

Mesothelin (MSLN) is a glycophosphatidylinositol (GPI)-linked cell surface protein that is highly expressed in several types of malignant tumor, including malignant pleural mesothelioma, ovarian cancer and pancreatic adenocarcinoma. Recently, a comprehensive immunohistochemical study using MN-1 monoclonal antibody identified a significant number of colorectal tumors in which MSLN was expressed. However, the clinicopathological profiles and survival of patients with MSLN-positive colorectal cancer have not been fully analyzed. In the current study, the expression of MSLN in 270 primary and 44 metastatic colorectal tumors was immunohistochemically analyzed to determine the clinical usefulness of MSLN immunohistochemistry and to identify potential candidates for future anti-MSLN therapy. In vitro experiments using colon cancer cell lines were performed to investigate the biological significance of MSLN expression in tumors. The results of univariate analyses identified a significant correlation between MSLN expression and females (P=0.0042). Furthermore, an inverse correlation between MSLN expression and solid/sheet-like proliferation (P=0.014) was also revealed. Additionally, overall survival was significantly shorter in patients with diffuse luminal/membranous expression of MSLN (P=0.018). Multivariable Cox hazards regression analysis revealed diffuse MSLN expression (hazard ratio, 2.26; 95% confidence interval, 1.04-4.91; P=0.039) as a potential risk factor. When comparing primary CRCs and the metastasis of each, a weakly positive correlation was identified for MSLN positivity (% positive cells; R=0.484; P<0.0001). The in vitro experiments revealed a positive role for MSLN in colon cancer cell proliferation. Thus, MSLN immunohistochemistry may be useful in the prognostication of patients with CRC. The results demonstrated that significant numbers of patients with MSLN-positive CRC exhibiting metastasis could be targeted by anti-MSLN therapies.

Keywords: cellular proliferation; colorectal cancer; immunohistochemistry; mesothelin; prognosis.

Figure 1.

MSLN immunohistochemistry in colon cancer tissue. (A) Case of tubular adenocarcinoma. Diffuse and luminal MSLN expression was detected. (B) Case of poorly differentiated adenocarcinoma exhibiting solid/sheet-like proliferation with undetectable MSLN expression. Left, low-power magnification; right, high-power magnification. Scale bar, 500 µm. MSLN, mesothelin.

Figure 2.

Overall survival of patients with CRC classified according to MSLN expression. (A) Kaplan-Meier curves for patients with colorectal tumors grouped by diffuse luminal/membranous or partial/negative MSLN expression. Kaplan-Meier curves for (B) male and (C) female patients with colorectal tumors grouped by diffuse luminal/membranous or partial/negative MSLN expression. CRC, colorectal cancer; MSLN, mesothelin.

Figure 3.

Expression of MSLN in cultured colon cancer cells. MSLN was expressed in four of seven colon cancer cell lines with no association with sex. MSLN, mesothelin; F, female; M, male.

Figure 4.

Forced-expression of MSLN upregulates colon cancer cell proliferation. (A) Immunoblot analyses of CW-2 and HCT-116 cells with stable expression of MSLN or its control, LacZ. (B) Colon cancer cells with forced MSLN expression exhibited significantly higher rates of cellular proliferation compared with their controls in serum-reduced conditions (CW-2, 1% serum; HCT-116, 5% serum). The expression of (C) p-ERK, (D) anchorage-independent cell proliferation and (E) CCNA expression in CW-2 and HCT-116 cells with or without MSLN is presented. Experiments were performed in triplicate. Data are presented as the mean ± standard deviation. **P<0.01 vs. LacZ group. MSLN, mesothelin; CCNA, cyclin A.

Figure 5.

MSLN knockdown downregulates colon cancer cell proliferation. (A) Immunoblot analyses of COLO205 and SW48 cells transfected with siRNAs. Significantly decreased (B) cellular proliferation, (C) anchorage-independent cell proliferation and (D) CCNA expression were detected in MSLN-downregualted CRC cells. Experiments were performed in triplicate. Data are presented as the mean ± standard deviation; *P<0.05 vs. siControl group. MSLN, mesothelin; siRNA, small interfering RNA; CCNA, cyclin A; CRC, colorectal cancer.

Figure 6.

CRC cases with diffuse MSLN expression exhibited a significantly higher rate of CCNA labeling. A total of 21 CRC patients with diffuse MSLN expression and 30 arbitrarily selected cases with negative or partial MSLN expression were compared according to CCNA labeling indices. MSLN, mesothelin; CRC, colorectal cancer; CCNA, cyclin A.