Abstract：
The role of Wnt signaling in oncogenesis and drug resistance is well known. Receptor-interacting protein kinase (RIPK4) contributing to the increased activity of many signaling pathways, including Wnt/β-catenin, may be an important target for designing new drugs for metastatic melanoma, but its role in melanoma is not fully understood.
We tested the effect of genetic manipulation of RIPK4 (CRISPR/Cas9) on xenograft growth. In addition, immunohistochemistry was used to detect active β-catenin, Ki67 and necrosis in xenografts. Wnt signaling pathway activity was examined using Western blot and Top-Flash. The effect of RIPK4 knockout on melanoma cells in vitro stimulated Wnt3A on wound overgrowth, migration and invasion ability was then evaluated.
Our study showed that CRISPR/Cas9-mediated RIPK4 knockout (KO) significantly reduced tumor growth in a mouse model of melanoma, particularly of WM266.4 cells. RIPK4 KO tumors exhibited lower percentages of Ki67+ cells as well as reduced necrotic area and decreased levels of active β-catenin. In addition, we observed that RIPK4 knockout impaired Wnt3A-induced activation of LRP6 and β-catenin, as manifested by a decrease in the transcriptional activity of β-catenin in Top-Flash in both tested melanoma cell lines, A375 and WM266.4. Prolonged incubation (48 h) with Wnt3A showed reduced level of MMP9, C-myc, and increased SOX10, proteins whose transcription is also dependent on β-catenin activity. Moreover, RIPK4 knockout led to the inhibition of scratch overgrowth, migration and invasion of these cells compared to their controls.
RIPK4 knockdown inhibits melanoma tumor growth and Wnt3A stimulated migration and invasion indicating that RIPK4 might be a potential target for melanoma therapy.
We tested the effect of genetic manipulation of RIPK4 (CRISPR/Cas9) on xenograft growth. In addition, immunohistochemistry was used to detect active β-catenin, Ki67 and necrosis in xenografts. Wnt signaling pathway activity was examined using Western blot and Top-Flash. The effect of RIPK4 knockout on melanoma cells in vitro stimulated Wnt3A on wound overgrowth, migration and invasion ability was then evaluated.
Our study showed that CRISPR/Cas9-mediated RIPK4 knockout (KO) significantly reduced tumor growth in a mouse model of melanoma, particularly of WM266.4 cells. RIPK4 KO tumors exhibited lower percentages of Ki67+ cells as well as reduced necrotic area and decreased levels of active β-catenin. In addition, we observed that RIPK4 knockout impaired Wnt3A-induced activation of LRP6 and β-catenin, as manifested by a decrease in the transcriptional activity of β-catenin in Top-Flash in both tested melanoma cell lines, A375 and WM266.4. Prolonged incubation (48 h) with Wnt3A showed reduced level of MMP9, C-myc, and increased SOX10, proteins whose transcription is also dependent on β-catenin activity. Moreover, RIPK4 knockout led to the inhibition of scratch overgrowth, migration and invasion of these cells compared to their controls.
RIPK4 knockdown inhibits melanoma tumor growth and Wnt3A stimulated migration and invasion indicating that RIPK4 might be a potential target for melanoma therapy.
摘要：
目的：Wnt信号在肿瘤发生和耐药中的作用是众所周知的。受体相互作用蛋白激酶（RIPK4）有助于增加许多信号通路的活性，包括Wnt/β-catenin，可能是设计转移性黑色素瘤新药的重要目标，但其在黑色素瘤中的作用尚未完全了解。
方法：我们测试了RIPK4（CRISPR/Cas9）的遗传操作对异种移植生长的影响。此外,免疫组织化学用于检测活性β-catenin，Ki67和异种移植物坏死。使用Western印迹和Top-Flash检查Wnt信号通路活性。RIPK4敲除对黑色素瘤细胞体外模拟Wnt3A伤口过度生长的影响,然后评估迁移和入侵能力。
结果：我们的研究表明，在黑色素瘤小鼠模型中，CRISPR/Cas9介导的RIPK4基因敲除（KO）显著降低了肿瘤生长，特别是WM266.4细胞。RIPK4KO肿瘤表现出较低的Ki67+细胞百分比以及减少的坏死面积和降低的活性β-连环蛋白水平。此外,我们观察到RIPK4敲除损害Wnt3A诱导的LRP6和β-catenin的激活，正如两个测试的黑色素瘤细胞系中Top-Flash中β-catenin的转录活性降低所表明的那样，A375和WM266.4。与Wnt3A的长时间孵育（48小时）显示MMP9，C-myc水平降低，和增加的SOX10，其转录也依赖于β-连环蛋白活性的蛋白质。此外，RIPK4敲除导致划痕过度生长的抑制，与对照相比，这些细胞的迁移和侵袭。
结论：RIPK4敲低抑制黑色素瘤肿瘤的生长和Wnt3A刺激的迁移和侵袭，表明RIPK4可能是黑色素瘤治疗的潜在靶点。
方法：我们测试了RIPK4（CRISPR/Cas9）的遗传操作对异种移植生长的影响。此外,免疫组织化学用于检测活性β-catenin，Ki67和异种移植物坏死。使用Western印迹和Top-Flash检查Wnt信号通路活性。RIPK4敲除对黑色素瘤细胞体外模拟Wnt3A伤口过度生长的影响,然后评估迁移和入侵能力。
结果：我们的研究表明，在黑色素瘤小鼠模型中，CRISPR/Cas9介导的RIPK4基因敲除（KO）显著降低了肿瘤生长，特别是WM266.4细胞。RIPK4KO肿瘤表现出较低的Ki67+细胞百分比以及减少的坏死面积和降低的活性β-连环蛋白水平。此外,我们观察到RIPK4敲除损害Wnt3A诱导的LRP6和β-catenin的激活，正如两个测试的黑色素瘤细胞系中Top-Flash中β-catenin的转录活性降低所表明的那样，A375和WM266.4。与Wnt3A的长时间孵育（48小时）显示MMP9，C-myc水平降低，和增加的SOX10，其转录也依赖于β-连环蛋白活性的蛋白质。此外，RIPK4敲除导致划痕过度生长的抑制，与对照相比，这些细胞的迁移和侵袭。
结论：RIPK4敲低抑制黑色素瘤肿瘤的生长和Wnt3A刺激的迁移和侵袭，表明RIPK4可能是黑色素瘤治疗的潜在靶点。
