Comparative Proteomics Analysis Identifies Cdc42-Cdc42BPA Signaling as Prognostic Biomarker and Therapeutic Target for Colon Cancer Invasion,Journal of Proteome Research

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Comparative Proteomics Analysis Identifies Cdc42-Cdc42BPA Signaling as Prognostic Biomarker and Therapeutic Target for Colon Cancer Invasion
Journal of Proteome Research ( IF 3.8 ) Pub Date : 2017-11-07 00:00:00 , DOI: 10.1021/acs.jproteome.7b00550
Hui-Fang Hu ₁ , Wen Wen Xu ₂ , Yang Wang ₁ , Can-Can Zheng ₁ , Wei-Xia Zhang ₁ , Bin Li ₁ , Qing-Yu He ₁

Affiliation

Metastasis is one of the major causes of treatment failure in the patients with colon cancer. The aim of our study is to find key proteins and pathways that drive invasion and metastasis in colon cancer. Eight rounds of selection of cancer cells invading through matrigel-coated chamber were performed to obtain highly invasive colon cancer sublines HCT116-I8 and RKO-I8. Stable Isotope Labeling by Amino Acids in Cell Culture technology was used to identify the differently expressed proteins, and the proteomics data were analyzed by ingenuity pathway analysis. PAK1-PBD immunoprecipitation combined with Western blot were carried out to determine Cdc42 activity, and qRT-PCR and Western blot were used to determine gene expression. The functional role of Cdc42BPA and Cdc42 pathway in colon cancer invasion was studied by loss-of-function experiments including pharmacological blockade, siRNA knockdown, chamber invasion, and WST-1 assays. Human colon cancer tissue microarray was analyzed by immunohistochemistry for overexpression of Cdc42BPA and its correlation with clinicopathological parameters and patient survival outcomes. HCT116-I8 and RKO-I8 cells showed significantly stronger invasive potential as well as decreased E-cadherin and increased vimentin expressions compared with parental cells. The differently expressed proteins in I8 cells compared with parental cells were identified. Bioinformatics analysis of proteomics data suggested that Cdc42BPA protein and Cdc42 signaling pathway are important for colon cancer invasion, which was confirmed by experimental data showing upregulation of Cdc42BPA and higher expression of active GTP-bound form of Cdc42 in HCT116-I8 and RKO-I8 cells. Functionally, pharmacological and genetic blockade of Cdc42BPA and Cdc42 signaling markedly suppressed colon cancer cell invasion and reversed epithelial mesenchymal transition process. Furthermore, compared with adjacent normal tissues, Cdc42BPA expression was significantly higher in colon cancer tissues and further upregulated in metastatic tumors in lymph nodes. More importantly, Cdc42BPA expression was correlated with metastasis and poor survival of the patients with colon cancer. This study provides the first evidence that Cdc42BPA and Cdc42 signaling are important for colon cancer invasion, and Cdc42BPA has potential implications for colon cancer prognosis and treatment.

中文翻译：

比较蛋白质组学分析确定Cdc42-Cdc42BPA信号为结肠癌浸润的预后生物标志物和治疗靶标

转移是结肠癌患者治疗失败的主要原因之一。我们研究的目的是发现驱动结肠癌浸润和转移的关键蛋白质和途径。进行了八轮选择通过基质胶涂层腔侵袭的癌细胞的选择，以获得高度侵袭性的结肠癌亚系HCT116-I8和RKO-I8。利用细胞培养技术中的氨基酸稳定同位素标记技术来鉴定差异表达的蛋白质，并通过创造力途径分析对蛋白质组学数据进行分析。进行了PAK1-PBD免疫沉淀与Western blot的结合，以确定Cdc42的活性，并使用qRT-PCR和Western blot来确定基因的表达。通过功能丧失实验研究了Cdc42BPA和Cdc42途径在结肠癌侵袭中的功能作用，这些实验包括药理学封锁，siRNA敲低，室侵袭和WST-1分析。通过免疫组织化学分析了人类结肠癌组织微阵列中Cdc42BPA的过表达及其与临床病理参数和患者生存结果的相关性。与亲代细胞相比，HCT116-I8和RKO-I8细胞显示出显着更强的侵袭潜能以及E-钙黏着蛋白减少和波形蛋白表达增加。鉴定了与亲代细胞相比在I8细胞中不同表达的蛋白质。蛋白质组学数据的生物信息学分析表明，Cdc42BPA蛋白和Cdc42信号通路对结肠癌的侵袭非常重要，实验数据证实了Cdc42BPA的上调和Hct116-I8和RKO-I8细胞中Cdc42的活性GTP结合形式的高表达的实验数据证实了这一点。在功能上，Cdc42BPA和Cdc42信号传导的药理和遗传阻滞作用显着抑制了结肠癌细胞的侵袭并逆转了上皮间质转化过程。此外，与邻近的正常组织相比，Cdc42BPA在结肠癌组织中的表达明显更高，并且在淋巴结转移性肿瘤中进一步上调。更重要的是，Cdc42BPA表达与结肠癌患者的转移和生存不良相关。这项研究提供了第一个证据，即Cdc42BPA和Cdc42信号对于结肠癌的侵袭很重要，而Cdc42BPA对结肠癌的预后和治疗具有潜在的影响。

更新日期：2017-11-08

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