Foxhead box D1 promotes the partial epithelial-to-mesenchymal transition of laryngeal squamous cell carcinoma cells via transcriptionally activating the expression of zinc finger protein 532

The presence of cervical lymph node metastases has been considered as the most important adverse prognostic factor for patients with laryngeal squamous cell carcinoma (LSCC). However, the underlying mechanisms remain to be fully revealed. In this study, we explored the expression profile of <i>Foxhead box D1</i> (<i>FOXD1</i>), its association with epithelial-to-mesenchymal transition (EMT), and its downstream targets in LSCC. Bioinformatic analysis was performed based on the LSCC subset of The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma (TCGA-HSNC) and Chromatin immunoprecipitation (ChIP)-seq data from Cistrome Data Browser. LSCC cell lines AMC-HN-8 and TU212 were used for <i>in vitro</i> studies. Results showed that <i>FOXD1</i> upregulation was associated with poor prognosis of LSCC. <i>FOXD1</i> knockdown reduced N-cadherin and Vimentin expression but increased E-cadherin expression in AMC-HN-8 cells. Its overexpression showed opposite effects in TU212 cells. FOXD1 could bind to the promoter of <i>ZNF532</i> and activate its transcription. <i>ZNF532</i> overexpression enhanced the invasion of both AMC-HN-8 and TU212 cells. In comparison, its knockdown significantly impaired their invasion. <i>ZNF532</i> knockdown nearly abrogated the alterations of EMT markers caused by <i>FOXD1</i> overexpression. Its overexpression largely rescued the phenotypes caused by <i>FOXD1</i> knockdown. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that <i>ZNF532</i> correlated genes are largely enriched in extracellular matrix regulations. LSCC patients with high <i>ZNF532</i> expression (top 50%) had a significantly worse progression-free survival. In summary, this study confirmed that FOXD1 promotes partial-EMT of LSCC cells via transcriptionally activating the expression of <i>ZNF532</i>.

颈淋巴结转移（cervical lymph node metastases）已被视为喉鳞状细胞癌（laryngeal squamous cell carcinoma, LSCC）患者最重要的不良预后因子。然而其潜在分子机制仍有待全面阐明。本研究探讨了叉头框D1（Foxhead box D1, FOXD1）的表达谱、其与上皮间质转化（epithelial-to-mesenchymal transition, EMT）的关联，以及其在LSCC中的下游调控靶标。本研究基于癌症基因组图谱-头颈部鳞状细胞癌队列（The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma, TCGA-HSNC）中的LSCC子集，以及来自Cistrome数据浏览器的染色质免疫沉淀测序（Chromatin immunoprecipitation sequencing, ChIP-seq）数据开展生物信息学分析，并采用LSCC细胞系AMC-HN-8与TU212开展体外（in vitro）实验。结果显示，FOXD1高表达与LSCC患者不良预后显著相关。在AMC-HN-8细胞中敲低FOXD1可下调N-钙粘蛋白（N-cadherin）与波形蛋白（Vimentin）的表达，同时上调E-钙粘蛋白（E-cadherin）的表达；而在TU212细胞中过表达FOXD1则呈现相反效应。FOXD1可结合至ZNF532的启动子区域并激活其转录。过表达ZNF532可增强AMC-HN-8与TU212细胞的侵袭能力；反之，敲低ZNF532则显著削弱二者的侵袭特性。敲低ZNF532几乎可完全逆转FOXD1过表达所引发的上皮间质转化标志物表达变化；而过表达ZNF532则可极大挽救FOXD1敲低所导致的细胞表型改变。基因本体（Gene Ontology, GO）与京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）富集分析显示，ZNF532的相关基因主要富集于细胞外基质调控通路。ZNF532高表达（位列前50%）的LSCC患者无进展生存期显著更短。综上，本研究证实FOXD1可通过转录激活ZNF532的表达，促进LSCC细胞的部分上皮间质转化。