Introduction

Cellular communication network factor 4 (CCN4/WISP1) is a secreted matricellular protein that stimulates metastasis in multiple malignancies but has an unclear impact on phenotypic changes in melanoma. Recent data using cells edited via a double-nickase CRISPR/Cas9 approach suggest that CCN4/WISP1 stimulates invasion and metastasis of melanoma cells. While these data also suggest that loss of CCN4/WISP1 increases cell proliferative, the CRISPR approach used may be an alternative explanation rather than the loss of gene function.

Methods

To test whether CCN4/WISP1 also influences the proliferative phenotype of melanoma cells, we used mouse melanoma models and knocked out Ccn4 using a homology-directed repair CRISPR/Cas9 system to generate pools of Ccn4-knockout cells. The resulting edited cell pools were compared to parental cell lines using an ensemble of in vitro and in vivo assays.

Results

In vitro assays using knockout pools supported previous findings that CCN4/WISP1 promoted an epithelial–mesenchymal-like transition in melanoma cells and stimulated invasion and metastasis. While Ccn4 knockout also enhanced cell growth in optimal 2D culture conditions, the knockout suppressed certain cell survival signaling pathways and rendered cells less resistant to stress conditions. Tumor cell growth assays at sub-optimal conditions in vitro, quantitative analysis of tumor growth assays in vivo, and transcriptomics analysis of human melanoma cell lines were also used to quantify changes in phenotype and generalize the findings.

Conclusions

In addition to stimulating invasion and metastasis of melanoma cells, the results suggested that CCN4/WISP1 repressed cell growth and simultaneously enhanced cell survival.

中文翻译：

---

细胞通信网络因子 4 (CCN4/WISP1) 将黑色素瘤细胞从脆弱的增殖状态转变为弹性转移状态。

介绍

细胞通讯网络因子 4 (CCN4/WISP1) 是一种分泌的基质细胞蛋白，可刺激多种恶性肿瘤的转移，但对黑色素瘤的表型变化的影响尚不清楚。最近使用通过双切口酶 CRISPR/Cas9 方法编辑的细胞的数据表明，CCN4/WISP1 刺激黑色素瘤细胞的侵袭和转移。虽然这些数据还表明 CCN4/WISP1 的缺失会增加细胞增殖，但使用的 CRISPR 方法可能是另一种解释，而不是基因功能的缺失。

方法

为了测试 CCN4/WISP1 是否也影响黑色素瘤细胞的增殖表型，我们使用小鼠黑色素瘤模型并使用同源定向修复 CRISPR/Cas9 系统敲除Ccn4以生成Ccn4敲除细胞池。使用一组体外和体内测定法将所得编辑的细胞池与亲本细胞系进行比较。

结果

使用敲除池的体外测定支持先前的发现，即 CCN4/WISP1 促进黑色素瘤细胞中的上皮-间充质样转变并刺激侵袭和转移。虽然Ccn4敲除还增强了最佳 2D 培养条件下的细胞生长，但敲除抑制了某些细胞存活信号通路并使细胞对压力条件的抵抗力降低。体外次优条件下的肿瘤细胞生长试验、体内肿瘤生长试验的定量分析以及人黑色素瘤细胞系的转录组学分析也用于量化表型变化并概括研究结果。

结论

除了刺激黑色素瘤细胞的侵袭和转移外，结果表明 CCN4/WISP1 抑制细胞生长并同时增强细胞存活。