Abstract：
Yes-associated protein (YAP), an effector molecule of the Hippo signaling pathway, is expressed at high levels in cutaneous melanoma. However, the role of YAP in melanoma progression according to cellular localization is poorly understood. Tissues from 140 patients with invasive melanoma were evaluated by immunohistochemistry. Flow cytometry, western blotting, viability assays, wound healing assays, verteporfin treatment, and xenograft assays were conducted using melanoma cell lines B16F1 and B16F10 subjected to YapS127A transfection and siYap knockdown. Nuclear YAP localization was identified in 63 tumors (45.0%) and was more frequent than cytoplasmic YAP in acral lentiginous and nodular subtypes (P = .007). Compared with cytoplasmic YAP melanomas, melanomas with nuclear YAP had higher mitotic activity (P = .016), deeper invasion (P < .001), and more frequently metastasized to lymph nodes (P < .001) and distant organs (P < .001). Patients with nuclear YAP melanomas had poorer disease-free survival (P < .001) and overall survival (P < .001). Nuclear YAP was an independent risk factor for distant metastasis (hazard ratio: 3.206; 95% CI, 1.032-9.961; P = .044). Proliferative ability was decreased in siYapB16F1 (P < .001) and siYapB16F10 (P = .001) cells and increased in YapS127AB16F1 (P = .003) and YapS127AB16F10 (P = .002) cells. Cell cycle analysis demonstrated relative G1 retention in siYapB16F1 (P < .001) and siYapB16F10 (P < .001) cells and S retention in YapS127AB16F1 cells (P = .008). Wound healing assays showed that Yap knockdown inhibited cell invasion (siYapB16F1, P = .001; siYapB16F10, P < .001), whereas nuclear YAP promoted it (YapS127AB16F, P < .001; YapS127AB16F1, P = .017). Verteporfin, a direct YAP inhibitor, reduced cellular proliferation in B16F1 (P = .003) and B16F10 (P < .001) cells. Proliferative effects of nuclear YAP were confirmed in xenograft mice (P < .001). In conclusion, nuclear YAP in human melanomas showed subtype specificity and correlated with proliferative activity and proinvasiveness. It is expected that YAP becomes a useful prognostic marker, and its inhibition may be a potential therapy for melanoma patients.
摘要：
是相关蛋白（YAP），Hippo信号通路的效应分子，在皮肤黑色素瘤中高水平表达。然而,根据细胞定位,YAP在黑色素瘤进展中的作用尚不清楚.通过免疫组织化学评估了140例侵袭性黑色素瘤患者的组织。流式细胞术,西方印迹,活力测定，伤口愈合试验，维替泊芬治疗,使用经受YapS127A转染和siYap敲低的黑素瘤细胞系B16F1和B16F10进行异种移植测定。在63个肿瘤中发现了核YAP定位（45.0％），并且在肢端微带和结节亚型中比胞质YAP更频繁（P=0.007）。与细胞质YAP黑色素瘤相比，有核YAP的黑素瘤具有较高的有丝分裂活性（P=0.016），较深的侵袭（P<0.001），更频繁地转移到淋巴结（P<0.001）和远处器官（P<0.001）。核型YAP黑色素瘤患者的无病生存率（P<0.001）和总生存率（P<0.001）较差。核YAP是远处转移的独立危险因素(HR，3.206；95%CI，1.032-9.961；P=0.044）。siYapB16F1（P<0.001）和siYapB16F10（P=0.001）细胞的增殖能力降低，而YapS127AB16F1（P=0.003）和YapS127AB16F10（P=0.002）细胞的增殖能力升高。细胞周期分析显示，G1保留在siYapB16F1（P<0.001）和siYapB16F10（P<0.001）细胞中，S保留在YapS127AB16F1细胞中（P=0.008）。伤口愈合试验显示Yap敲低抑制细胞侵袭(siYapB16F1,P=0.001;siYapB16F10,P<0.001),虽然核YAP推广了它(YapS127AB16F，P<0.001;YapS127AB16F1,P=0.017)。Verteporfin,一种直接的YAP抑制剂,B16F1(P=0.003)和B16F10(P<0.001)细胞增殖减少。在异种移植小鼠中证实了核YAP的增殖作用(P<0.001)。总之,人黑色素瘤中的核YAP显示出亚型特异性，并与增殖活性和侵袭性相关。预计YAP成为有用的预后标志物，其抑制可能是黑色素瘤患者的潜在疗法。
