gC1qR/HABP1/p32 Is a Potential New Therapeutic Target Against Mesothelioma

Abstract

Mesothelioma is an aggressive cancer of the serous membranes with poor prognosis despite combination therapy consisting of surgery, radiotherapy, and platinum-based chemotherapy. Targeted therapies, including immunotherapies, have reported limited success, suggesting the need for additional therapeutic targets. This study investigates a potential new therapeutic target, gC1qR/HABP1/p32 (gC1qR), which is overexpressed in all morphologic subtypes of mesothelioma. gC1qR is a complement receptor that is associated with several cellular functions, including cell proliferation and angiogenesis. In vitro and in vivo experiments were conducted to test the hypothesis that targeting gC1qR with a specific gC1qR monoclonal antibody 60.11 reduces mesothelioma tumor growth, using the biphasic mesothelioma cell line MSTO-211H (MSTO). In vitro studies demonstrate cell surface and extracellular gC1qR expression by MSTO cells, and a modest 25.3 ± 1.8% (n = 4) reduction in cell proliferation by the gC1qR blocking 60.11 antibody. This inhibition was specific for targeting the C1q binding domain of gC1qR at aa 76-93, as a separate monoclonal antibody 74.5.2, directed against amino acids 204-218, had no discernable effect. In vivo studies, using a murine orthotopic xenotransplant model, demonstrated an even greater reduction in MSTO tumor growth (50% inhibition) in mice treated with the 60.11 antibody compared to controls. Immunohistochemical studies of resected tumors revealed increased cellular apoptosis by caspase 3 and TUNEL staining, in 60.11 treated tumors compared to controls, as well as impaired angiogenesis by decreased CD31 staining. Taken together, these data identify gC1qR as a potential new therapeutic target against mesothelioma with both antiproliferative and antiangiogenic properties.

Keywords: complement; gC1qR/HABP1/p32; mesothelioma; monoclonal antibody therapy; therapeutic target.

Figure 1

Mesothelioma MSTO-211H cells expresses gC1qR. (A) Immunofluorescence photomicrographs of stained MSTO-211H cells in culture. Results of a typical experiment. Non-permeabilized cell monolayers were stained with non-immune rabbit IgG (NIRG) or 60.11 anti gC1qR antibody (āgC1qR) and appropriate secondary antibodies conjugated to AlexaFluor 488 (green), as well as DAPI nuclear stain (blue). Cells were imaged with a fluorescence microscope at 10× magnification. The presence of gC1qR is shown by green staining in the right image. (B) Flow cytometric analysis of MSTO-211H cell surface gC1qR expression. Non-permeabilized MSTO-211H cells in suspension were stained with non-immune mouse IgG (MOPC) or anti gC1qR antibody (āgC1qR) and appropriate secondary antibodies conjugated to AlexaFluor 488. Stained and unstained cells were analyzed. Expression of cell surface gC1qR in a typical experiment is shown by an increase in fluorescence of cells treated with anti gC1qR antibody (blue curve).

Figure 2

Mesothelioma cells shed gC1qR into the extracellular milieu. MSTO-211H cells were cultured in complete medium, and supernatants were sampled at 24-h intervals. Relative concentrations of gC1qR in the supernatants at each time point were determined via a direct ELISA. Error bars represent mean substrate absorbance ± standard deviation. n = 2 separate experiments performed in duplicate. *Significantly different results at p < 0.05.

Figure 3

Extracellular gC1qR enhances MSTO-211H cell growth in culture. Tissue culture wells were coated with gC1qR or coating buffer (control) and seeded with MSTO-211H cells. Cells were supplemented with recombinant gC1qR (5 μg/ml) as noted. Cell cultures were imaged at 24-h intervals via compound light microscopy (10× magnification). Representative images are shown. Increased cell growth is evident at 72 and 96 h in the presence of soluble or immobilized extracellular gC1qR.

Figure 4

Antibodies directed against the C1q binding site of gC1qR decrease mesothelioma cell proliferation. MSTO-211H cells was treated with 10 μg/mL monoclonal anti gC1qR antibody mAb 60.11 directed against the C1q binding region (aa 76–93) or mAb 74.5.2 directed against the high molecular weight kininogen binding domain (aa 204–218). Cell proliferation was determined via hemocytometer cell counts of viable cells after 96 h incubation. Error bars represent mean cell population ± standard deviation. n = 2 separate experiments performed in duplicate. *Significant difference from control at p = 1.33 × 10⁻⁵.

Figure 5

MSTO-211H tumors resected from control and 60.11 treated mice are histologically similar. Representative histologic (10×) images of tumors stained with hematoxylin and eosin show tightly packed MSTO-211H cells and areas of necrosis. No histologic differences in tumor morphology were apparent between control and treatment groups.

Figure 6

Therapy with 60.11 increases apoptosis and reduces neovascularization in mesothelioma. Representative histologic images (20 × original magnification) of tumors obtained from control and 60.11 treated mice stained with cleaved caspase 3, TUNEL, CD 31, and Ki 76 are shown. Positive immunohistochemical reactivity is indicated by brown stain. Quantitative analysis of staining intensity is shown in the inset. N, number of microscopic fields selected for analysis.