Protein Kinase C- delta Regulates Cellular Attachment, Survival, and Metastases-Associated Genes in Uveal Melanoma

Abstract

Purpose : Uveal melanoma (UM) is the most common primary intraocular malignancy in adults. It comprises about 5% of total melanomas and it is genetically and molecularly distinct than cutaneous melanoma. Somatic mutations in the GNAQ and GNA11 genes have been found in over 80% of UM signaling through PLC and ERK pathways. We have previously shown an anti-apoptotic role of Protein Kinase C-delta (PKC-d), a novel isoform of the PKC family, which is activated by diacylglycerol and phospholipids, in UM. We also demonstrated increased expression in UM eyes compared to healthy controls. In this study we focus on the morphological changes, AKT- and NFkB- cell survival mechanisms, and the modulation of genes highly associated with metastatic progress in UM after PKCD gene silencing.

Methods : We utilized 3 different UM cell lines: Mel270, OMM2.5, and Mel290. Knockdown of PKCD expression was performed by siRNA and confirmed by qPCR and Western blot analyses. Morphological changes were evaluated by microscopy imaging among different cell culture conditions, including untreated, scramble siRNA, PKCD siRNA, and the pharmacological PKC modulator Rottlerin. AKT and MDM2 signaling were evaluated by Wb and nuclear translocation of the p65 NFkB subunit by imaging flow cytometry.

Results : We observed increase in cell detachment across all UM cell lines studied after PKCD knockdown. These morphological changes were confirmed with Rottlerin. There was a reduction in cell survival mechanisms across all UM after treatment with PKCD siRNA. We found a reduction in MMP2 and CD44, which are associated with metastatic progress in cancer.

Conclusions : Our work demonstrates PKCD knockdown could reduce tumor migration as it increases cell detachment, and reduced the expression of CD44 and MMP2. In addition, we observed a reduction in cell survival in an AKT-dependent manner. Collectively, our work suggests PKC-d could be used as a potential targeted therapy for UM.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.