Mullerian Inhibiting Substance Inhibits the Growth of Ocular and Metastatic Uveal Melanoma Cells

Abstract

Purpose:: Uveal melanoma is the most common primary intraocular tumor and a high percentage of patients with large tumors develop liver metastases. Currently there are no effective treatments for patients with metastatic disease, which is universally fatal. Mullerian Inhibiting Substance (MIS), a member of the TGF-ß super-family, plays a critical role in sexual development by triggering regression of the female reproductive tissues in males by activating apoptosis and/or cell cycle arrest. Previous experiments demonstrated MIS inhibits female reproductive tract tumors in vitro and in vivo. Over ten years ago it was unexpectedly discovered that MIS inhibited primary uveal melanoma cells. Recently, a unique MIS type II receptor gene was identified. In addition, the MIS gene was cloned and biologically active recombinant protein was produced. The following experiments were performed to determine the molecular basis for the MIS response in primary uveal melanoma cells and determine if liver metastases also respond to MIS.

Methods:: Ten primary and two metastatic uveal melanoma cell lines were examined. MIS receptor type II expression was determined by Western blots and immunofluorescent staining of primary and metastatic uveal melanoma cell lines utilizing a mouse monoclonal anti-human MIS RII antibody. Prostate and ovarian cancer cells (LnCaP and OVCAR 8) as well as COS cells transfected with the human MIS receptor were used as positive controls. Tumor cell proliferation was determined in cultures with or without 10ug/ml of recombinant MIS. At the end of the incubation, cell proliferation and survival was assessed by a standard MTT assay.

Results:: 5/10 primary tumor cells and 2/2 metastatic tumor cells expressed high levels of the MIS Receptor type II as determined by Western blots. Immunofluorescent staining confirmed expression of the receptors on the surface of the uveal melanoma cells. Four cell lines (two primary, two metastatic) positive for the MIS RII receptor were cultured in the presence or absence of 10ug/ml of MIS for 5-7 days. At the end of the incubation, cell growth was inhibited from 40-80% (n=5, p<0.005).

Conclusions:: Uveal melanoma cells express the MIS Receptor type II and respond to recombinant human MIS by triggering the inhibition of cellular proliferation. MIS receptors were expressed on both primary and metastatic liver melanoma cells, implying this recombinant protein may be effective in preventing growth of metastatic tumors.

Keywords: melanoma • growth factors/growth factor receptors • inhibitory receptors