FOXD1 Promotes Cell Growth and Metastasis by Activation of Vimentin in NSCLC.

BACKGROUND/AIMS: Forkhead box D1 (FOXD1) has a well-established role in early embryonic development and organogenesis and functions as an oncogene in several cancers. However, the clinical significance and biological roles of FOXD1 in non-small cell lung cancer (NSCLC) remain largely unknown.METHODS: A total of 264 primary NSCLC tissue samples were collected. The expression levels of FOXD1 in these samples were examined by immunohistochemical staining. The expression of FOXD1 was knocked down by lentiviral shRNA. The relative expression of FOXD1 was determined by qRT-PCR, Western blotting and immunofluorescence image. The functional roles of FOXD1 in NSCLC were demonstrated cell viability CCK-8 assay, colony formation, cell invasion and migration assays, and cell apoptosis assay in vitro. In vivo mouse xenograft and metastasis models were used to assess tumorigenicity and metastatic ability. The Chi-square test was used to assess the correlation between FOXD1 expression and the clinicopathological characteristics. Survival curves were estimated by Kaplan-Meier method and compared using the log-rank test. The Cox proportional hazards model was used for univariate and multivariate analyses.RESULTS: We determined that higher levels of FOXD1 were present in NSCLC tissues, especially in metastatic NSCLC tissues. FOXD1 was also higher in all NSCLC cells compared with normal human bronchial epithelial cells. A higher expression level of FOXD1 was associated with malignant behavior and poor prognosis in NSCLC patients. Knockdown of FOXD1 significantly inhibited proliferation, migration, and invasion in vitro and tumor growth and metastasis in vivo, and it increased the apoptosis rates of NSCLC cells. Mechanistic analyses revealed that FOXD1 expressed its oncogenic characteristics through activating Vimentin in NSCLC. Multivariate Cox regression analysis indicated that FOXD1 was an independent prognostic factor both for overall survival (OS) and disease-free survival (DFS) in NSCLC patients.CONCLUSION: Our results indicated that FOXD1 might be involved in the development and progression of NSCLC as an oncogene, and thereby might be a potential therapeutic target for NSCLC patients.

背景/目的：叉头框D1（FOXD1）在早期胚胎发育和器官发生中具有明确的作用，并且在几种癌症中作为癌基因发挥功能。然而，FOXD1在非小细胞肺癌（NSCLC）中的临床意义和生物学作用在很大程度上仍然未知。 方法：共收集了264例原发性NSCLC组织样本。通过免疫组织化学染色检测这些样本中FOXD1的表达水平。利用慢病毒shRNA敲低FOXD1的表达。通过实时定量聚合酶链反应（qRT - PCR）、蛋白质印迹法和免疫荧光图像确定FOXD1的相对表达。通过细胞活力CCK - 8测定、集落形成、细胞侵袭和迁移测定以及体外细胞凋亡测定来证明FOXD1在NSCLC中的功能作用。利用体内小鼠异种移植和转移模型评估致瘤性和转移能力。卡方检验用于评估FOXD1表达与临床病理特征之间的相关性。通过卡普兰 - 迈耶（Kaplan - Meier）方法估计生存曲线，并使用对数秩检验进行比较。考克斯比例风险模型用于单变量和多变量分析。 结果：我们确定NSCLC组织中存在较高水平的FOXD1，特别是在转移性NSCLC组织中。与正常人支气管上皮细胞相比，所有NSCLC细胞中的FOXD1也更高。FOXD1的较高表达水平与NSCLC患者的恶性行为和不良预后相关。敲低FOXD1在体外显著抑制增殖、迁移和侵袭，在体内抑制肿瘤生长和转移，并增加NSCLC细胞的凋亡率。机制分析表明，FOXD1在NSCLC中通过激活波形蛋白（Vimentin）发挥其致癌特性。多变量考克斯回归分析表明，FOXD1是NSCLC患者总生存期（OS）和无病生存期（DFS）的独立预后因素。 结论：我们的结果表明，FOXD1可能作为癌基因参与NSCLC的发生和发展，因此可能是NSCLC患者的一个潜在治疗靶点。