RIPK4 downregulation impairs Wnt3A-stimulated invasiveness via Wnt/β-catenin signaling in melanoma cells and tumor growth in vivo,Cellular Signalling

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RIPK4 downregulation impairs Wnt3A-stimulated invasiveness via Wnt/β-catenin signaling in melanoma cells and tumor growth in vivo
Cellular Signalling ( IF 4.4 ) Pub Date : 2023-10-21 , DOI: 10.1016/j.cellsig.2023.110938
Norbert Wronski ₁ , Ewelina Madej ₂ , Maja Grabacka ₃ , Anna A Brożyna ₄ , Agnieszka Wolnicka-Glubisz ₂

Affiliation

Purpose

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.

Methods

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.

Results

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.

Conclusion

RIPK4 knockdown inhibits melanoma tumor growth and Wnt3A stimulated migration and invasion indicating that RIPK4 might be a potential target for melanoma therapy.

中文翻译：

RIPK4 下调通过黑色素瘤细胞中的 Wnt/β-catenin 信号传导损害 Wnt3A 刺激的侵袭性和体内肿瘤生长

目的

Wnt 信号传导在肿瘤发生和耐药性中的作用是众所周知的。受体相互作用蛋白激酶 (RIPK4) 有助于增加包括 Wnt/β-catenin 在内的许多信号通路的活性，可能是设计治疗转移性黑色素瘤新药的重要靶点，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) 基因操作对异种移植物生长的影响。此外，免疫组织化学用于检测异种移植物中的活性β-catenin、Ki67和坏死。使用蛋白质印迹和 Top-Flash 检查 Wnt 信号通路活性。然后评估敲除RIPK4对体外刺激Wnt3A的黑色素瘤细胞对伤口过度生长、迁移和侵袭能力的影响。

结果

我们的研究表明，CRISPR/Cas9 介导的 RIPK4 敲除 (KO) 显着降低了黑色素瘤小鼠模型中的肿瘤生长，尤其是 WM266.4 细胞的肿瘤生长。 RIPK4 KO 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 敲除损害了 Wnt3A 诱导的 LRP6 和 β-catenin 激活，表现为两种测试的黑色素瘤细胞系 A375 和 WM266.4 中 Top-Flash 中 β-catenin 转录活性的降低。与 Wnt3A 长时间孵育（48 小时）显示 MMP9、C-myc 水平降低，SOX10 水平增加，SOX10 是其转录也依赖于 β-catenin 活性的蛋白质。此外，与对照组相比，RIPK4 敲除导致这些细胞的划痕过度生长、迁移和侵袭受到抑制。