Abstract 3374: BRAF and NRAS mutations in melanoma regulate the phosphorylation and inactivation of RKIP, the metastasis suppressor and immune surveillance cancer gene product
Authors: Huerta-Yepez Sara , Suhendan Ekmekcioglu , Rivera-Pazos Clara M. , Antonio-Andres Gabriela , Vega Mario I. , Baay-Guzman Guillermina , Malaponte Graziella , Baritaki Stavroula , Yeung Kam , Grimm Elizabeth , Bonavida Benjamin 
Published: February 2011

The Raf Kinase Inhibitor Protein (RKIP) is a member of the phosphatidylethanolamine binding protein family (PEBP). RKIP binds to Raf-1 and prevents the activation of the ERK 1/2 cascade. RKIP expression is diminished in many primary cancers and is absent in many metastases. RKIP overexpression inhibits experimental metastases, hence, termed a metastasis suppressor. In addition, overexpression of RKIP reverses tumor cell resistance to drug-induced apoptosis, hence, termed an immune surveillance cancer gene product. Phosphorylation of RKIP (pRKIP) at Ser 153 by PKCs dissociates RKIP from Raf-1 (inactivating RKIP) and pRKIP binds to G-protein coupled receptor kinase 2 (GRK2, a kinase that inhibits G-protein coupled receptors (GPCRs) and resulting in the inhibition of GRK2 activity and stimulation of the ERK 1/2 activity. Over 90% of melanoma express oncogene mutations in BRAF and also express mutations in NRAS. Most of the transforming activity of these mutations results from the activation of MAPK. The roles of such mutations in the regulation of oncogenes involved in metastasis and resistance are not clear. The objective of this study was to initiate investigations on the roles of above mutations on the expression of RKIP and inactive pRKIP. We hypothesized that BRAF and NRAS mutations, through their activation of PKCs, will result in the phosphorylation and inactivation of RKIP and potentiation of ERK 1/2 activation, metastasis and drug resistance. In this study, we have examined a large panel of melanoma cell lines with no mutations and with BRAF and/or NRAS mutations using a Tissue microarray technology. The expression of both RKIP and pRKIP was examined and the evaluation of protein expression was performed by IHC and analyzed by blinded observers under light microscopy. While anti-RKIP antibody detects both RKIP and pRKIP, anti-pRKIP was specific for pRKIP. The mean intensities for the expression of each protein were determined using the software Image-ProPlus 6.3. In brief, five random fields of each cell line were evaluated at 400-magnification. From the IHC staining, general density expressions of each protein in the cell lines were measured in pixels/200 μm2. The data revealed that the majority (75%) of RKIP expression was in its phosphorylated form in cell lines with BRAF and NRAS mutations. These studies suggest that in melanoma cell lines with mutations, GRK2 is inactivated through its association with pRKIP and, thus, maintaining the activating signals mediated by GPCRs. Further, pRKIP expression may potentiate the metastatic potential and drug resistance. We propose that selective inhibitors of PKCs may elevate the expression of RKIP and result in the inhibition of the metastatic potential as well as in the sensitization of the tumor cells to subtoxic therapeutic drugs. These studies are currently being examined in our laboratories.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3374.