Acta Histochemica 115 (2013) 795–802 Contents lists available at ScienceDirect Acta Histochemica jou rn al homepage: www.elsevier.de/acthis Raf kinase inhibitor protein (RKIP) and phospho-RKIP expression in melanomas Venera Cardile a , Grazia Malaponte b , Carla Loreto c , Massimo Libra b , Silvia Caggia a , Francesca Maria Trovato d , Giuseppe Musumeci c,∗ a Department of Bio-medical Sciences, Section of Physiology, University of Catania, Viale A. Doria 6, 95125 Catania, Italy b Department of Bio-medical Sciences, Section of Pathology and Oncology, University of Catania, Via Androne 81, 95123 Catania, Italy c Department of Bio-medical Sciences, Division of Anatomy and Histology, University of Catania, Via S. Sofia 87, 95123 Catania, Italy d Department of Internal Medicine and Systemic Diseases, University of Catania, Via S. Sofia 78, 95123 Catania, Italy a r t i c l e i n f o Article history: Received 17 February 2013 Accepted 10 March 2013 Keywords: Cancer marker Melanoma Metastasis RKIP pRKIP a b s t r a c t Melanoma, a cancer notorious for its high potential to metastasize, arises from melanocytes, cells dedi- cated to melanin production and located in the basal layer of the epidermis. Raf-1 kinase inhibitor protein (RKIP) is an inhibitory molecule that down-regulates the effects of the Ras/Raf/MEK/ERK signaling path- way. The aim of this study was to examine the expression of RKIP and pRKIP in melanomas at different stages. We evaluated the RKIP and pRKIP protein by immunohistochemistry in control skin, pigmented nevi and melanomas, and through Western blotting in human normal melanocytes and in four different melanoma-derived cell lines (WM35, A375, M14, and A2058). Our results demonstrated a correlation between the expression of RKIP and pRKIP, and metastatic ability in melanoma cells. This raises the possibility to analyze both RKIP and pRKIP in all melanomas. Down-regulation of both RKIP and pRKIP expression could represent a useful marker of metastatic melanoma. On the contrary for non-metastatic melanoma, especially in Clark I and II, low RKIP and high pRKIP expression could be indicative. In con- clusion, the observed negative correlation of the RKIP and pRKIP expression in metastatic melanomas indicates that expression of these proteins may become a prognostic marker for the progression of human cutaneous melanoma. We propose that the investigation of both RKIP and pRKIP may provide a useful tool indicative for metastatic or non-metastatic melanoma in different Clark’s level melanomas. Further studies are required to verify the molecular background of the observed RKIP and pRKIP variations. © 2013 Elsevier GmbH. All rights reserved. Introduction Cancer is a disease caused by uncontrolled cell growth and pro- liferation. Melanoma, a cancer notorious for its high potential to metastasize, arises from melanocytes, cells dedicated to melanin production and located in the basal layer of the epidermis. It rep- resents 2–3% of malignant tumors in the United States of America and Northern Europe (Lin et al., 2010). Melanoma remains one of the most aggressive of human malignancies. However, research to date suggests that not all melanomas are created equal. Several molecular pathways, specific to melanoma subtypes, have been described and these findings have been translated into clinical ben- efit (Ko et al., 2010). Moreover, cancer mortality often results from metastatic disease and is not the direct effect of the primary tumor. With advances in cancer treatment, control of the primary tumor can be managed by multimodal therapy, however, metastatic dis- ease is frequently refractory to the same therapeutic approaches and has a very poor prognosis, with a median survival rate of 6 ∗ Corresponding author. E-mail address: g.musumeci@unict.it (G. Musumeci). months (Miller and Mihm, 2006). Thus, identification of biomark- ers that could more accurately predict future metastasis from the state of the primary tumor would be invaluable for guiding therapy. Raf-1 kinase inhibitor protein (RKIP) is a cytosolic protein, member of the phosphatidyl ethanolamine binding protein (PEBP) family, originally identified as an interacting partner of Raf-1 and a negative regulator of the mitogen-activated protein kinase cas- cade initiated by Raf-1 (Yeung et al., 1999; Nottage and Siu, 2002; O’Neill and Kolch, 2004). RKIP is an inhibitory molecule that down- regulates the effects of the Ras/Raf/MEK/ERK signaling pathway (Yeung et al., 2000; Schuierer et al., 2004). Phosphorylation of RKIP at serine-153 by protein kinase C abolishes RKIP’s inhibition of Raf-1 (Corbit et al., 2003) and converts it to an inhibitor of the G- protein coupled receptor kinase, GRK-2, thus facilitating cross-talk between the EGF and GPCR signaling pathways (Lorenz et al., 2003). Furthermore, RKIP functions as a metastatic tumor suppressor have been studied in prostate (Keller et al., 2004) and breast cancers (Dangi-Garimella et al., 2009), in which it sensitizes cancer cells to drug induced apoptosis and regulates the integrity of the cell cycle via the spindle checkpoint (Eves et al., 2006; Baritaki et al., 2009a). In mammals, RKIP may be involved in regulating the partitioning of chromosomes and mitosis progression through RKIP binding to 0065-1281/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.acthis.2013.03.003