RESUMO
In a previous transcriptomic analysis of 63 ocular melanomas of the uvea, we found that expression of the PRL-3/PTP4A3 gene, encoding a phosphatase that is anchored to the plasma membrane, was associated with the risk of metastasis, and a poor prognosis. We also showed that PRL-3 overexpression in OCM-1 ocular melanoma cells significantly increased cell migration in vitro and invasiveness in vivo, suggesting a direct role for PRL-3 in the metastatic spreading of uveal melanoma. Here, we aimed to identify PRL-3 substrates at the plasma membrane involved in adhesion to the extracellular matrix. We focused on integrin ß1, which is the most highly expressed integrin in our cohort of uveal melanomas. We show that preventing PRL-3 anchorage to the plasma membrane i) abolishes PRL-3-induced migration in OCM-1 cells, ii) specifically enhances the spreading of OCM-1 cells overexpressing PRL-3, and iii) favors the maturation of large focal adhesions (FAs) containing integrin ß1 on collagen I. Knockdown experiments confirmed integrin ß1 involvement in PRL3-induced migration. We identified interactions between PRL-3 and integrin ß1, as well as with FAK P-Y397, an auto-activated form of Focal Adhesion Kinase found in FAs. We also show that integrin ß1 may be dephosphorylated by PRL-3 in its intracytoplasmic S/T region, an important motif for integrin-mediated cell adhesion. Finally, we observed that PRL-3 regulated the clustering of integrin ß1 in FAs on collagen I but not on fibronectin. This work identifies PRL-3 as a new regulator of cell adhesion structures to the extracellular matrix, and further supports PRL-3 as a key actor of metastasis in uveal melanoma, of which molecular mechanisms are still poorly understood.
Assuntos
Movimento Celular/genética , Integrina beta1/biossíntese , Melanoma/genética , Proteínas de Neoplasias/biossíntese , Proteínas Tirosina Fosfatases/biossíntese , Neoplasias Uveais/genética , Adesão Celular/genética , Linhagem Celular Tumoral , Membrana Celular/genética , Membrana Celular/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/patologia , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Integrina beta1/genética , Melanoma/patologia , Metástase Neoplásica , Proteínas de Neoplasias/genética , Proteínas Tirosina Fosfatases/genética , Neoplasias Uveais/patologiaRESUMO
PURPOSE: To study PTP4A3 phosphatase and MMP14 metalloprotease synergy in uveal melanoma aggressiveness. METHODS: Cell membrane localization of matrix metalloprotease 14 (MMP14) in uveal melanoma cells expressing protein tyrosine phosphatase A3 (PTP4A3) was assessed by flow cytometry or immunohistochemistry. The vesicular trafficking of MMP14 in the presence of PTP4A3 was evaluated in OCM-1 cells expressing either the wild-type or mutated phosphatase. Finally, MMP14 localization at the cell membrane of OCM-1 cells was impaired using RNA interference, and the PTP4A3-related migration in vitro and invasiveness in vivo of the treated cells were evaluated. RESULTS: We found that the membrane-anchored MMP14 is enriched at the cell surface of OCM-1 cells, patient-derived xenograft cells, and human primary uveal melanoma tumors expressing PTP4A3. Moreover, we show that PTP4A3 and MMP14 colocalize and that the vesicular trafficking of MMP14 is faster in the presence of active PTP4A3. Finally, we demonstrate that inhibition of MMP14 expression in uveal melanoma cells expressing PTP4A3 impairs their migration in vitro and invasiveness in vivo. CONCLUSIONS: Our observations indicate that PTP4A3 increases cell membrane accumulation of MMP14 as a result of increased cellular trafficking of the metalloprotease. We also show that downregulation of MMP14 expression reduced PTP4A3-induced cell migration and invasiveness. Taken together, our findings suggest that PTP4A3-related subcellular localization of MMP14 is an important event in metastasis induction.