PDF(Pubmed)
Abstract：
Forkhead box D1 (FOXD1) serves a critical role in colorectal cancer (CRC). FOXD1 expression is an independent prognostic factor in patients with CRC; however, the molecular mechanism and signaling pathway of FOXD1 that regulates cell stemness and chemoresistance has not been fully characterized. The aim of the present study was to further validate the effect of FOXD1 on the proliferation and migration of CRC cells, and to delve into the possible potential of FOXD1 in the clinical treatment of CRC. The effect of FOXD1 on cell proliferation was assessed using Cell Counting Kit 8 (CCK‑8) and colony formation assays. The effect of FOXD1 on cell migration was assessed by wound‑healing and Transwell assays. The effect of FOXD1 on cell stemness was assessed by spheroid formation in vitro and limiting dilution assays in vivo. The expression of stemness associated proteins, leucine rich repeat containing G protein‑coupled receptor 5 (LGR5), OCT4, Sox2 and Nanog, and epithelial‑mesenchymal transition associated proteins, E‑cadherin, N‑cadherin and vimentin, were detected by western blotting. Proteins interrelationships were assessed by a co‑immunoprecipitation assay. Oxaliplatin resistance was assessed using CCK‑8 and apoptosis assays in vitro, and using a tumor xenograft model in vivo. By constructing FOXD1 overexpression and knockdown stably transfected strains of colon cancer cells, it was revealed that the overexpression of FOXD1 increased CRC cell stemness and chemoresistance. By contrast, knockdown of FOXD1 produced the opposite effects. These phenomena were caused by the direct interaction between FOXD1 and β‑catenin, thus promoting its nuclear translocation and the activation of downstream target genes, such as LGR5 and Sox2. Notably, inhibition of this pathway with a specific β‑catenin inhibitor (XAV‑939) could impair the effects induced by the overexpression of FOXD1. In summary, these results indicated that FOXD1 may promote cell stemness and the chemoresistance of CRC by binding directly to β‑catenin and enhancing β‑catenin nuclear localization; therefore, it may be considered a potential clinical target.
摘要：
叉头盒D1(FOXD1)在结直肠癌(CRC)中起关键作用。FOXD1表达是CRC患者的独立预后因素；然而，FOXD1调节细胞干性和化疗耐药的分子机制和信号通路尚未完全明确。本研究的目的是进一步验证FOXD1对CRC细胞增殖和迁移的影响。探讨FOXD1在CRC临床治疗中的可能潜力。使用细胞计数试剂盒8(CCK‑8)和集落形成测定法评估FOXD1对细胞增殖的影响。通过伤口愈合和Transwell测定评估FOXD1对细胞迁移的影响。通过体外球状体形成和体内有限稀释测定来评估FOXD1对细胞干性的影响。干性相关蛋白的表达，富含亮氨酸的重复序列含有G蛋白偶联受体5（LGR5），OCT4，Sox2和Nanog，和上皮间质转化相关蛋白，E-cadherin,N-钙粘蛋白和波形蛋白，通过蛋白质印迹检测。通过共免疫沉淀测定法评估蛋白质的相互关系。使用CCK‑8和体外细胞凋亡测定法评估奥沙利铂耐药性，并在体内使用肿瘤异种移植模型。通过构建FOXD1过表达和敲低稳定转染的结肠癌细胞株,结果表明，FOXD1的过表达增加了CRC细胞的干细胞性和化学抗性。相比之下,FOXD1的击倒产生了相反的效果。这些现象是由FOXD1和β-catenin之间的直接相互作用引起的，从而促进其核易位和下游靶基因的激活，例如LGR5和Sox2。值得注意的是,用特异性β-连环蛋白抑制剂（XAV-939）抑制该途径可能会削弱FOXD1过表达诱导的作用。总之,这些结果表明，FOXD1可能通过直接结合β‑catenin并增强β‑catenin的核定位来促进细胞干性和CRC的化学抗性；因此，它可能被认为是一个潜在的临床目标。
