Abstract
Raf Kinase Inhibitor Protein (RKIP) has been extensively reported as an inhibitor of key signaling pathways involved in the aggressive tumor phenotype and shows decreased expression in several types of cancers. However, little is known about RKIP in melanoma or regarding its function in normal cells. We examined the role of RKIP in both primary melanocytes and malignant melanoma cells and evaluated its diagnostic and prognostic value. IHC analysis revealed a significantly higher expression of RKIP in nevi compared with early-stage (stage I–II, AJCC 8th) melanoma biopsies. Proliferation, wound healing, and collagen-coated transwell assays uncovered the implication of RKIP on the motility but not on the proliferative capacity of melanoma cells as RKIP protein levels were inversely correlated with the migration capacity of both primary and metastatic melanoma cells but did not alter other parameters. As shown by RNA sequencing, endogenous RKIP knockdown in primary melanocytes triggered the deregulation of cellular differentiation-related processes, including genes (i.e., ZEB1, THY-1) closely related to the EMT. Interestingly, NANOG was identified as a putative transcriptional regulator of many of the deregulated genes, and RKIP was able to decrease the activation of the NANOG promoter. As a whole, our data support the utility of RKIP as a diagnostic marker for early-stage melanomas. In addition, these findings indicate its participation in the maintenance of a differentiated state of melanocytic cells by modulating genes intimately linked to the cellular motility and explain the progressive decrease of RKIP often described in tumors.
Highlights
Raf Kinase Inhibitor Protein (RKIP), known as phosphatidylethanolamine-binding protein1 (PEBP1), was originally described as an inhibitor of the MAPK and ERK1/2 pathway [1] latter studies revealed its additional role as a regulator of other signaling cascades including the G-protein-coupled receptor (GPCR), glycogen synthase kinase 3β (GSK3β) and nuclear factorCancers 2020, 12, 1451; doi:10.3390/cancers12061451 www.mdpi.com/journal/cancersCancers 2020, 12, 1451 kappa B (NF-κB)-driven ones [2]
Nevi samples exhibited higher positivity for RKIP staining compared with the whole cohort of melanoma samples (Figure 1b); 94% of nevi samples were positive for RKIP whereas only 51% of melanoma cases presented positive staining
Prior to molecular and cellular behavior analysis, we evaluated the expression of RKIP in three primary melanocyte cells and eleven melanoma cell lines by RT-qPCR and Western Blot (Figure 2)
Summary
Raf Kinase Inhibitor Protein (RKIP), known as phosphatidylethanolamine-binding protein1 (PEBP1), was originally described as an inhibitor of the MAPK and ERK1/2 pathway [1] latter studies revealed its additional role as a regulator of other signaling cascades including the G-protein-coupled receptor (GPCR), glycogen synthase kinase 3β (GSK3β) and nuclear factorCancers 2020, 12, 1451; doi:10.3390/cancers12061451 www.mdpi.com/journal/cancersCancers 2020, 12, 1451 kappa B (NF-κB)-driven ones [2]. Raf Kinase Inhibitor Protein (RKIP), known as phosphatidylethanolamine-binding protein. 1 (PEBP1), was originally described as an inhibitor of the MAPK and ERK1/2 pathway [1] latter studies revealed its additional role as a regulator of other signaling cascades including the G-protein-coupled receptor (GPCR), glycogen synthase kinase 3β (GSK3β) and nuclear factor. RKIP plays a role in essential processes such as differentiation, proliferation, and cell survival. Regarding melanoma, published data report the role of RKIP regulating cell proliferation, migration and invasion capability [12,13,14], by mechanisms leading to NFκB and. There is little known on the pathways regulated by RKIP in normal melanocytes nor on its role in the malignant transformation of this type of cells
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