Cytotoxic activity of immunotoxin SS1P is modulated by TACE-dependent mesothelin shedding

Abstract

Mesothelin is a cell-surface tumor-associated antigen expressed in several human cancers. The limited expression of mesothelin on normal tissues and its high expression in many cancers make it an attractive candidate for targeted therapies using monoclonal antibodies, immunoconjugates, and immunotoxins. Mesothelin is actively shed from the cell surface and is present in the serum of patients with malignant mesothelioma, which could negatively affect the response to these therapies. We have found that mesothelin sheddase activity is mediated by a TNF-α converting enzyme (TACE), a member of the matrix metalloproteinase/a disintegrin and metalloprotease family. We showed that EGF and TIMP-3 act through TACE as endogenous regulators of mesothelin shedding. We also found that reducing shedding significantly improved the in vitro cytotoxicity of immunotoxin SS1P, which targets mesothelin and is currently in clinical trials for the treatment of patients with mesothelioma and lung cancer. Our findings provide a mechanistic understanding of mesothelin shedding and could help improve mesothelin-based targeted therapies.

Figure 1

The determination of shed mesothelin C-terminal sequence. A. SDS-PAGE profile of shed mesothelin preparations from A431/H9 supernatant and ascites of a patient with mesothelioma. Bands in frame are shed mesothelin and subjected to the sequencing. B. The C-terminal cleavage sites of mesothelin.

Figure 2

Mesothelin shedding is inhibited by broad-spectrum MMP/ADAM inhibitors. A. A431/H9 cells harvested by trypsinization were incubated with PI-PLC (1 U/mL) or PC-PLC (10 U/mL) for 1 h at 37°C. After wash with FACS buffer, cells were stained by Alexa488-labeled SS1P (2 μg/mL). The fluorescence signal was measured by flow cytometer. Cells without SS1P-Alexa488 staining were used as the negative control. Cells incubated with Hank’s buffer and stained by SS1P-Alexa488 were used as positive control. B and C. A431/H9 cells were seeded into 96-well plate and incubated with phospholipase inhibitor (B) or pan-MMP/ADAM inhibitors (C), including U73122 (10 μM), ET-18-OCH3 (10 μM), sPLA2-IIA inhibitor I (50 μg/mL), D609 (250 μg/mL), neomycin (1 mg/mL), GM6001 (20 μM), batimastat (20 μM), marimastat (20 μM) and camostat (20 μM). After 48 h, mesothelin in cell medium was measured by ELISA. DMSO-treated cell was negative control. The results were normalized with cell number in each well.

Figure 3

TACE is a candidate as mesothelin sheddase. A and B. A431/H9 cells with fresh medium (1×10⁵ cells/well in a 96-well plate) were incubated with TIMPs (5 μg/mL), EGF (50 ng/mL), PMA (10 μM) or EGF plus Iressa (1 μM) for 5 h. Shed mesothelin in the cell supernatant was measured by ELISA. Negative and positive controls are PBS and GM6001 (20 μM). C. Tumor cells isolated from the ascites of five mesothelioma patients were examined for TACE expression with western blot. Cells from patients NCI-M-02, 05, 10, 11 and 13 were included.

Figure 4

TACE is involved in mesothelin shedding. A431/H9 cells were transfected with TACE siRNA (Hs_ADAM17_7; 50 nM). After 48 h, the expression of TACE was examined by western blot (A). The mesothelin shedding was checked by incubating transfected cells in fresh medium for 5 h (B). The cells were stained by Alexa488-labeled SS1P (2.5 μg/mL) to measure mesothelin expression level on cell surface (C). D. The SS1P cytotoxicity on transfected cells. Two days after siRNA transfection, SS1P with serial dilutions was added for a short incubation period (2 h). After another 48 h, WST-8 reagent was added to determine IC₅₀s. Mock and luciferase siRNA-transfected cells were negative controls. The error bars represent the standard deviation of triplicate wells. The experiments were repeated at least 3 times. A typical result is shown.

Figure 5

MMP/ADAM inhibitor regulates mesothelin-targeting process. A431/H9 cells were treated with GM6001 (20 μM) for 48 h. They were then stained with SS1P-Alexa488 (2.5 μg/mL) to determine cell-surface expression of mesothelin (A). Filled area, cells without staining; dash line, DMSO treatment; solid line, GM6001 treatment. B. The measurement of SS1P internalization rates on DMSO and GM6001-treated cells. C and D. Cytotoxicity of cycloheximide and SS1P on treated cells. Control is DMSO-treated cells.