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Vanessa Marie Morales, Ryan Yates, Jordan J Toutounchian, Mercy W Kibe, Duane Miller, Youde Jiang, Jena J Steinle, Pia R. Mendoza, Hans E Grossniklaus, Matthew W Wilson; Modulation of Cytoskeletal Adaptor Proteins in Uveal Melanoma to Control Metastasis. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3578.
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Approximately 50% of Uveal Melanoma (UM) patients will be diagnosed with liver metastases within 5-years of diagnosis. Micro-metastases are present in the liver as early as 2-years before diagnosis of the intraocular malignancy. Recent work from our laboratory suggests that inhibiting the upregulation of cytoskeletal signaling could offer potential therapeutic targets for metastasis control. In this study we tested in vitro the hypothesis that paxillin, a key cytoskeletal adaptor protein, regulates UM metastasis to the liver by upregulation of the alpha-4 (α4) subunit in VLA-4 regulating UM proliferation, migration and invasion.
To test this hypothesis we combined transcriptional analysis, flow cytometry, cellular and molecular biology techniques. Flow cytometry analysis allowed us to measure the effects of paxillin inhibition on cell surface protein expression, actin polymerization and cellular proliferation. We performed the scratch-wound assay to investigate cellular migration and Western blot analyses for protein expression and phosphorylation of paxillin.
Transcriptional and protein analyses showed evidence of a pattern of extracellular matrix- and adhesion-associated genes being differentially expressed in primary- and metastatic- UM tumor cell lines (Primary UM cell lines: Mel 270, 92.1; Metastatic UM cell lines: OMM 1, OMM 2.5). Targeting of paxillin through inhibition of pY118 reduced cellular proliferation in primary, not metastatic UM, by more than 40%. It also affected cytoskeletal fragmentation and cellular migration. Inhibition of paxillin pY118 significantly increased cytoskeletal fragmentation in primary UM (Mel 270, 92.1) with a p=0.0007. Cellular migration was significantly reduced in UM cell lines with a p<0.001.
Our in vitro results identified paxillin as a potential therapeutic target to arrest UM cellular proliferation and migration. It also revealed differences in the effects of targeting paxillin among primary UM that metastasize and those who do not. Paxillin inhibition might be a potential candidate for adjuvant therapy in high risk UM patients.
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