June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Protein kinase signaling pathways in metastatic uveal melanoma
Author Affiliations & Notes
  • Jose M. M. Caminal
    Ophthalmology, University Hospital of Bellvitge , Hospitalet de Llobregat, Barcelona, Spain
  • Eduard Cabre
    Molecular Oncology, IDIBELL, Hospitalet de Llobregat, Barcelona, Spain
  • Antonia Vinyals
    Molecular Oncology, IDIBELL, Hospitalet de Llobregat, Barcelona, Spain
  • Montse Goma
    Pathology, University Hospital of Bellvitge, Hospitalet de Llobregat, Barcelona, Spain
  • Josep M Piulats
    Oncology, Catalan Institute of Oncology, Hospitalet de Llobregat, Barcelona, Spain
  • Mar Varela
    Pathology, University Hospital of Bellvitge, Hospitalet de Llobregat, Barcelona, Spain
  • Daniel Lorenzo
    Ophthalmology, University Hospital of Bellvitge , Hospitalet de Llobregat, Barcelona, Spain
  • Olaia Subira
    Ophthalmology, University Hospital of Bellvitge , Hospitalet de Llobregat, Barcelona, Spain
  • Maria Jose Paules
    Pathology, University Hospital of Bellvitge, Hospitalet de Llobregat, Barcelona, Spain
  • Lluis Arias
    Ophthalmology, University Hospital of Bellvitge , Hospitalet de Llobregat, Barcelona, Spain
  • Marc Rubio
    Ophthalmology, University Hospital of Bellvitge , Hospitalet de Llobregat, Barcelona, Spain
  • Estefania Cobos
    Ophthalmology, University Hospital of Bellvitge , Hospitalet de Llobregat, Barcelona, Spain
  • Pere Garcia-Bru
    Ophthalmology, University Hospital of Bellvitge , Hospitalet de Llobregat, Barcelona, Spain
  • Bruno Medeiros
    Ophthalmology, University Hospital of Bellvitge , Hospitalet de Llobregat, Barcelona, Spain
  • Noel Padron
    Ophthalmology, University Hospital of Bellvitge , Hospitalet de Llobregat, Barcelona, Spain
  • Angels Fabra
    Molecular Oncology, IDIBELL, Hospitalet de Llobregat, Barcelona, Spain
  • Footnotes
    Commercial Relationships   Jose M. Caminal, None; Eduard Cabre, None; Antonia Vinyals, None; Montse Goma, None; Josep M Piulats, None; Mar Varela, None; Daniel Lorenzo, None; Olaia Subira, None; Maria Jose Paules, None; Lluis Arias, None; Marc Rubio, None; Estefania Cobos, None; Pere Garcia-Bru, None; Bruno Medeiros, None; Noel Padron, None; Angels Fabra, None
  • Footnotes
    Support  Grants: ISCIII (PI15/1461 to JMCaminal) and the AECC (GCB15152978SOEN to AFabra)
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 3965. doi:
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      Jose M. M. Caminal, Eduard Cabre, Antonia Vinyals, Montse Goma, Josep M Piulats, Mar Varela, Daniel Lorenzo, Olaia Subira, Maria Jose Paules, Lluis Arias, Marc Rubio, Estefania Cobos, Pere Garcia-Bru, Bruno Medeiros, Noel Padron, Angels Fabra; Protein kinase signaling pathways in metastatic uveal melanoma. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3965.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Uveal Melanoma (UM) is the most common intra-ocular tumor in adults. Mutations in the Gαq family members GNAQ and GNA11 are present in 80% of UM tumors whereas the BRAF mutations are less common (15%). Consequently, Protein Kinase signaling pathways are dysregulated in UM cells and may trigger cell proliferation, survival, invasion and metastasis. Furthermore, exosomes –small nanoparticles containing RNA and proteins-, may be released by the primary tumor in order to prepare the metastatic niche in the liver before the arrival of metastatic cells. We aim to characterize the dysregulated Protein Kinase signaling pathways associated to the metastatic UM cells to identify specific targets to treat these patients.

Methods : To this aim, we used a panel of 7 UM cell lines harboring either GNAQ /GNA11 or BRAF mutations to analyse the exosome production in culture and their “resistance to anoikis”, a hallmark of circulating tumor cells. We performed an in vitro screening using a kinase inhibitor library (Screen-Well® Kinase Inhibitor Library; Enzo, Catalog # BML-2832-0100) containing inhibitors that either might block kinase activation or proteins that are upstream from their kinase target. Cell proliferation after incubation for 48h in the presence of inhibitors (10µM) was assessed by WST-1 metabolic assay and cell viability. Changes in cell morphology were detected under microscopy after staining with cristal violet. Cell cycle was analysed by flow cytometry. We confirmed the results obtained from the screening by Western Blot.

Results : The cell line SP6.5 produced the lowest amount of exosomes/106cells followed by UMA and OMM-1. In contrast, Mel270 cells and the metastatic cell lines OMM-2.3 and 2.5 resulted as the major producers of exosomes.
We found a consistent activation of Protein Kinase C (PKC), MAPK and FAK/Src in both GNAQ /GNA11 and BRAF mutant genotypes. Specific inhibitors of such pathways suppressed the corresponding signaling. Importantly, SB-203580 (MAPK inhibitor) was not effective in anoikis resistant Mel 270 GNAQm cells but significantly affected the sensitive cells. An hyperactivation of the Akt/PI3K was observed in these cells but not in UMA BRAFm cells. BAY-117082 (inhibitor of NF-kB activation) significantly decreased the survival of UMA cells resistant to the anoikis but not the sensitive ones.

Conclusions : Our study identifies activated signaling pathways and therapeutic inhibitors for metastatic UM cells.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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