RKIP regulates differentiation-related features in melanocytic cells

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. Overall design: Human melanocytes were transduced with RKIP shRNA Lentiviral Particles (sc-36430-V, 2MOI) or Control shRNA Lentiviral Particles (sc-108080 2MOI).

Raf激酶抑制蛋白（Raf Kinase Inhibitor Protein, RKIP）已被广泛证实为侵袭性肿瘤表型相关关键信号通路的抑制剂，且在多种癌症中表达水平下调。然而目前针对黑色素瘤中的RKIP及其在正常细胞中的功能，相关研究仍较为匮乏。本研究检测了RKIP在原代黑素细胞与恶性黑色素瘤细胞中的功能，并评估了其诊断与预后价值。免疫组化（Immunohistochemistry, IHC）分析显示，痣组织中RKIP的表达水平显著高于美国癌症联合委员会（American Joint Committee on Cancer, AJCC）第8版分期的I-II期早期黑色素瘤活检样本。增殖实验、划痕愈合实验及包被胶原的Transwell实验结果显示，RKIP参与调控黑色素瘤细胞的迁移能力，但不影响其增殖活性：RKIP蛋白水平与原代及转移性黑色素瘤细胞的迁移能力呈负相关，且未改变其他实验参数。RNA测序（RNA Sequencing）结果表明，在原代黑素细胞中敲低内源性RKIP会引发细胞分化相关进程的表达失调，包括与上皮间质转化（Epithelial-Mesenchymal Transition, EMT）密切相关的基因（如ZEB1、THY-1）的表达异常。值得注意的是，NANOG被鉴定为多数失调基因的潜在转录调控因子，且RKIP能够抑制NANOG启动子的激活。综上，本研究数据证实RKIP可作为早期黑色素瘤的诊断标志物。此外，本研究结果表明，RKIP通过调控与细胞迁移紧密相关的基因，参与维持黑素细胞的分化状态，同时也解释了肿瘤中RKIP表达常呈进行性下调的现象。整体实验设计：将人黑素细胞分别转导RKIP短发夹RNA（short hairpin RNA, shRNA）慢病毒颗粒（sc-36430-V，2MOI）或对照shRNA慢病毒颗粒（sc-108080，2MOI）。