Tight junction-associated signaling pathways modulate cell proliferation in uveal melanoma.

Uveal melanoma is the most common primary intraocular malignancy in adults.1 Several histopathologic features of these tumors are indicative of disease prognosis, including epithelioid cell morphology2 and the presence of vascular looping patterns.3,4 Additionally, molecular profiling of tumors has revealed genes associated with uveal melanoma growth, invasion, and metastasis, such as the downregulation of cell communication and development genes5,6 and the expression of matrix metalloproteinases.7 Further elucidation of the molecular participants in the pathogenesis of uveal melanoma would facilitate the development of targeted diagnostic approaches and therapeutic interventions. Tight junctions (TJs) are major components of cell junctional assemblies, which play an important role in controlling the passage of solutes across epithelial barriers, such as the cornea and gastric mucosa.8 In addition to maintaining barrier function in epithelia, TJs serve as a cell signaling apparatus for multiple cell types throughout the body, contributing to the regulation of vesicular trafficking,9 cell proliferation,10 and cell motility.11 Alterations of TJ expression and function have been associated with the development of cancer through mechanisms such as the loss of contact inhibition of cell growth.12 Recently, we reported on the localization and function of a cell adhesion molecule, Bves (blood vessel epicardial substance), in corneal epithelial cells and the trabecular meshwork.13–16 Bves is an approximately 360-amino acid protein expressed in many cell types from early stages of embryonic development to adulthood. It contains an extracellular amino terminus, three transmembrane domains, and an intracellular carboxyl terminus.17 The C-terminal domain interacts with the TJ-associated protein zonula occludens 1 (ZO-1), a TJ-associated protein, through direct and indirect mechanisms.14 Alterations in Bves expression in several cell types are associated not only with profound effects on TJ formation but also on cell morphology, motility, and proliferation.14,17–21 These observations suggest that Bves may have roles that extend beyond the regulation of barrier formation in epithelia. In cancer, the TJ complex may serve a critical role in facilitating communication between the cancer cell and the tumor microenvironment and in spatially and temporally orchestrating the mechanisms underlying tumor initiation and progression.22 Although the localization and function of TJs have been partially characterized in several cancer types, including cutaneous melanoma,22 their role in uveal melanoma is unclear. In this study, we investigated the role of Bves in the proliferation of uveal melanoma. Three human uveal melanoma cell lines stably transfected to overexpress Bves were analyzed using two- and three-dimensional cell proliferation assays and cell cycle distribution assays. Additionally, cells were measured for expression of TJ proteins and for the localization and activity of the TJ-associated transcription factor ZO-1–associated nucleic acid binding protein (ZONAB).23,24 ZONAB is a Y-box transcription factor that modulates cell proliferation through its interaction with the SH3 domain of ZO-1 at TJs, through its association with cell division kinase 4 (CDK4), and through its upregulation of cell cycle entry proteins.23 Specifically, in proliferating cells, ZONAB is observed primarily in the nucleus, where it promotes proliferation through binding of CDK4 and upregulation of proliferating cell nuclear antigen (PCNA) and cyclin D1, resulting in cell cycle entry.25 However, in slowly or nonproliferating cells, nuclear levels of ZONAB are reduced. The ZONAB protein is primarily localized in the cytoplasm, where it is sequestered from the nuclear compartment through its interaction with ZO-1.24 Because Bves is associated with ZO-1 at TJs,14 the association of Bves with ZO-1/ZONAB signaling in uveal melanoma was investigated.