Tetraspanin CD63 acts as a pro-metastatic factor via β-catenin stabilization. Homo sapiens

The tetraspanin CD63 is implicated in pro-metastatic signaling pathways, but so far, it is unclear how CD63 levels affect the tumor cell phenotype. Here, we investigated the effect of CD63 modulation in different metastatic tumor cell lines. In vitro, knock down of CD63 induced a more epithelial-like phenotype concomitant with increased E-cadherin expression, downregulation of its repressors Slug and Zeb1, and decreased N-cadherin. In addition, β-catenin protein was markedly reduced, negatively affecting expression of the target genes MMP-2 and PAI-1. β-catenin inhibitors mimicked the epithelial phenotype induced by CD63 knock down. Inhibition of β-catenin upstream regulators PI3K/AKT or GSK3β could rescue the mesenchymal phenotype underlining the importance of the β-catenin pathway in CD63-regulated cell plasticity. CD63 knock down-induced phenotypical changes correlated with a decrease of experimental metastasis, while CD63 overexpression enhanced the tumor cell-intrinsic metastatic potential. Taken together, our data show that CD63 is a crucial player in the regulation of the tumor cell-intrinsic metastatic potential by affecting cell plasticity. Overall design: Stable knock down of CD63 was performed in SKOV3ipL ovarian carcinoma cell line using 2 shRNAs lentiviral constructs (sh49 and sh51), and as a negative control, a shNT construct. Parental cells, control shNT and the 2 shCD63 cell lines were seeded 24 hours prior to RNA isolation on a 10 cm dish, labelling and hybridization on microarrays. One color experiment with 2 biological replicates of the 4 experimental conditions: SKOV3ipL, SKOV3ipL_shNT, SKOV3ipL_sh49 and SKOV3ipL_sh51.

四跨膜蛋白CD63（tetraspanin CD63）参与促转移信号通路，但截至目前，学界尚未明确CD63的表达水平如何调控肿瘤细胞表型。本研究针对不同转移性肿瘤细胞系中CD63的调控效应展开系统探究。体外实验结果显示，CD63敲低（knock down）可诱导细胞向更典型的上皮样表型转化，伴随E-钙粘蛋白（E-cadherin）表达上调、其转录抑制因子Slug与Zeb1的表达下调，以及N-钙粘蛋白（N-cadherin）水平降低。此外，β-连环蛋白（β-catenin）的蛋白表达量显著降低，进而对靶基因MMP-2与PAI-1的表达产生负向调控作用。β-连环蛋白抑制剂可模拟CD63敲低所诱导的上皮样表型。抑制β-连环蛋白的上游调控因子磷脂酰肌醇3-激酶/蛋白激酶B（PI3K/AKT）或糖原合成激酶3β（GSK3β）可挽救细胞的间充质表型，这一结果凸显了β-连环蛋白通路在CD63调控的细胞可塑性中的核心作用。CD63敲低所诱导的表型变化与实验性转移能力的降低相关，而CD63过表达则会增强肿瘤细胞固有的转移潜能。综上，本研究数据表明，CD63通过调控细胞可塑性，是肿瘤细胞固有转移潜能调控过程中的关键因子。整体实验设计：以SKOV3ipL卵巢癌细胞系为研究模型，采用2种短发夹RNA（shRNA）慢病毒载体（sh49与sh51）构建CD63稳定敲低细胞系，并以shNT载体作为阴性对照。将亲本细胞、阴性对照shNT细胞以及2种CD63敲低细胞系接种于10cm培养皿中，在RNA提取前24小时完成接种，随后进行芯片标记与杂交实验。本实验为单通道芯片实验，包含4组实验条件的2个生物学重复：SKOV3ipL细胞、SKOV3ipL_shNT细胞、SKOV3ipL_sh49细胞以及SKOV3ipL_sh51细胞。