Mesothelin as a potential therapeutic target in human cholangiocarcinoma

Abstract

Background: Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) are the two most common primary liver cancers, yet there have been no significant advances in effective therapeutics. Mesothelin has been reported as a new therapeutic target in various types of cancer. Here, we investigated the expression of mesothelin in liver cancer and its potential role as a novel therapeutic target for immunotherapy.

Methods: HCC and CCA specimens were examined by immunohistochemistry for mesothelin expression. Protein expression was assessed by immunoblotting and flow cytometry. The SS1P immunotoxin targeting mesothelin was evaluated in the well-established CCA cell lines HuCCT1, HuH-28, KMBC, KMCH, Mz-ChA-1 and OZ.

Results: We showed strong immunochemical mesothelin staining in 33% of the surgically resected CCA specimens and 3 of 6 CCA cell lines (OZ, KMBC and KMCH). No mesothelin staining was found in HCC or normal liver tissue. Mesothelin was primarily localized to the cellular plasma membrane and the mature form (molecular weight, ~40 kDa) was expressed at a high level in CCA tissues. Moreover, 22% of CCA specimens had a high mesothelin expression level which was comparable to the CCA cell line models. Interestingly, SS1P showed very high and specific growth inhibition when added to mesothelin-expressing CCA cells with IC(50) values ranging from 0.5 to 11 ng/mL.

Conclusions: Mesothelin is overexpressed in one-third of CCA tissues. SS1P targeting mesothelin reveals a remarkable single agent activity against CCA in vitro. These findings indicate a potential for SS1P in the immunotherapeutic treatment of CCA.

Keywords: SS1P; bile duct carcinoma; cholangiocarcinoma; hepatocellular carcinoma; immunotoxin; mesothelin.

Figure 1

Summary of immunoreactivity patterns in cancer patient specimens. Immunohistochemical evaluation using the 5B2 mAb showed high (A) and low (B) expression of mesothelin in two representative CCA specimens. No staining was found in both HCC (C) and normal liver tissues (D). Each immunoreactvity pattern was confirmed by duplicate specimens from the same patient. Scale bar, 100 μm.

Figure 2

Characterization of mesothelin protein expression in liver cancers. (A) Immunoblotting analysis of mesothelin proteins in human liver cancer cell lines. Forty μg of whole cell lysate was loaded for each sample except A431 and H9 (only 2 μg of total protein was loaded). (B) Immunoblotting analysis of mesothelin proteins in cancer specimens. Forty μg of whole cell lysate was loaded for each sample. OVCAR3 (a human ovarian cancer cell line) and H9 (A431.MSLN+) were used as positive controls. A431 (MSLN-) was used as a negative control. MSLN: mesothelin (~40 kDa); Precursor: ~71 kDa.

Figure 3

Flow cytometric analysis of mesothelin protein expression on CCA cells. Cells were probed with an anti-mesothelin mAb (solid dark line) or an irrelevant isotype mAb control (light gray shading). Each cell line and its GeoMean value: HuCCT1 (6.21), HuH-28 (5.54), KMBC (108.74), KMCH (84.63), Mz-ChA-1 (25.19) and OZ (195.48). GeoMean of the isotype antibody control was about 5.0.

Figure 4

Inhibition of CCA cell proliferation by SS1P. Cancer cells (10,000 per well) were incubated with various concentrations of SS1P. Cell proliferation was measured by a WST assay as described in Materials and Methods. Each point represents the mean of triplicate experiments and the error bars indicate the standard deviation. The dashed line indicates 50% inhibition of cell viability, which is halfway between the level of viability in the absence of toxin and that in the presence of 10 μg/ml of cycloheximide. Each cell line and its IC₅₀ (ng/mL): HuCCT1 (>1000 ng/ml for both SS1P and LMB2), HuH-28 (>1000 ng/ml for both SS1P and LMB2), KMBC (6.8 ng/ml for SS1P; 440.5 ng/ml for LMB2), KMCH (10.7 ng/ml for SS1P; >1000 ng/ml for LMB2), Mz-ChA-1 (9.1 ng/ml for SS1P; >1000 ng/ml for LMB2) and OZ (0.5 ng/ml for SS1P; >1000 ng/ml for LMB2). LMB2, a PE immunotoxin control specific for CD25.