Department of General Surgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200020, P.R. China.
Oncol Rep. 2023 Jul;50(1). doi: 10.3892/or.2023.8571. Epub 2023 May 19.
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 and limiting dilution assays . 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 , and using a tumor xenograft model . 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.
叉头框蛋白 D1（FOXD1）在结直肠癌（CRC）中起着关键作用。FOXD1 的表达是 CRC 患者的独立预后因素；然而，调节细胞干性和化疗耐药性的 FOXD1 的分子机制和信号通路尚未完全阐明。本研究旨在进一步验证 FOXD1 对 CRC 细胞增殖和迁移的影响，并深入探讨 FOXD1 在 CRC 临床治疗中的潜在可能性。使用细胞计数试剂盒 8（CCK-8）和集落形成实验评估 FOXD1 对细胞增殖的影响。通过划痕愈合和 Transwell 实验评估 FOXD1 对细胞迁移的影响。通过球体形成和有限稀释实验评估 FOXD1 对细胞干性的影响。通过 Western blot 检测干性相关蛋白，富含亮氨酸重复的 G 蛋白偶联受体 5（LGR5）、OCT4、Sox2 和 Nanog，以及上皮-间充质转化相关蛋白，E-钙黏蛋白、N-钙黏蛋白和波形蛋白的表达。通过共免疫沉淀实验评估蛋白质相互关系。使用 CCK-8 和凋亡实验评估奥沙利铂耐药性，并使用肿瘤异种移植模型进行评估。通过构建 FOXD1 过表达和敲低稳定转染的结肠癌细胞株，发现 FOXD1 的过表达增加了 CRC 细胞的干性和化疗耐药性。相反，敲低 FOXD1 则产生相反的效果。这些现象是由于 FOXD1 与 β-连环蛋白直接相互作用，从而促进其核转位和下游靶基因，如 LGR5 和 Sox2 的激活。值得注意的是，用特定的 β-连环蛋白抑制剂（XAV-939）抑制该途径可削弱 FOXD1 过表达引起的作用。总之，这些结果表明，FOXD1 可能通过直接与 β-连环蛋白结合并增强 β-连环蛋白核定位来促进 CRC 细胞的干性和化疗耐药性；因此，它可能被认为是一个潜在的临床靶点。
