The aim of the present study was to investigate possible differences in the sensitivity of HNSCC cells to known EMT regulators. Three HNSCC cell lines (UM-SCC-1, -3, -22B) and the HaCaT control keratinocyte cell line were exposed to transforming growth factor beta 1 (TGF-β1), a known EMT master regulator, and the cellular response was evaluated by real-time cell analysis (RTCA), Western blot, quantitative PCR, flow cytometry, immunocytochemistry, and the wound closure (scratch) assay. Targeted sequencing on 50 cancer-related genes was performed using the Cancer Hotspot Panel v2. Mutant, and wild typeSMAD4cDNA was used to generate recombinantSMAD4constructs for expression in mammalian cell lines. The most extensive response to TGF-β1, such as cell growth and migration, β-actin expression, or E-cadherin (CDH1) downregulation, was seen in cells with a more epithelial phenotype. Lower response correlated with higher basal p-TGFβ RII (Tyr424) levels, pointing to a possible autocrine pre-activation of these cell lines. Targeted sequencing revealed a homozygousSMAD4mutation in the UM-SCC-22B cell line. Furthermore, PCR cloning ofSMAD4cDNA from the same cell line revealed an additionalSMAD4transcript with a 14 bp insertion mutation, which gives rise to a truncated SMAD4 protein. Overexpression of this mutant SMAD4 protein in the highly epithelial control cell line HaCaT resulted in upregulation of TGF-β1 and vimentin. Consistent with previous reports, the invasive and metastatic potential of HNSCC tumor cells appears associated with the level of autocrine secretion of EMT regulators such as TGF-β1, and it could be influenced by exogenous EMT cytokines such as those derived from immune cells of the tumor microenvironment. Furthermore, mutant SMAD4 appears to be a significant contributor to the mesenchymal transformation of HNSCC cells.
本研究旨在探讨头颈部鳞状细胞癌（HNSCC）细胞对已知上皮间质转化（EMT）调控因子的敏感性差异。通过将三种HNSCC细胞系（UM-SCC-1、-3、-22B）及HaCaT对照角质形成细胞系暴露于EMT核心调控因子转化生长因子β1（TGF-β1），采用实时细胞分析、蛋白质印迹、定量PCR、流式细胞术、免疫细胞化学及划痕实验评估细胞反应。使用Cancer Hotspot Panel v2对50个癌症相关基因进行靶向测序，并利用突变型与野生型SMAD4 cDNA构建重组质粒在哺乳动物细胞系中表达。结果显示：具有较典型上皮表型的细胞对TGF-β1反应最为显著，表现为细胞生长迁移能力增强、β-肌动蛋白表达上调及E-钙黏蛋白（CDH1）下调；而反应较弱者则与较高基础水平磷酸化TGFβ RII（Tyr424）相关，提示这些细胞系可能存在自分泌预激活现象。靶向测序发现UM-SCC-22B细胞系存在SMAD4纯合突变，进一步通过PCR克隆鉴定出该细胞系存在14 bp插入突变的SMAD4转录本，可编码截短型SMAD4蛋白。在上皮特性显著对照细胞系HaCaT中过表达该突变蛋白，可诱导TGF-β1与波形蛋白表达上调。本研究证实：HNSCC肿瘤细胞的侵袭转移潜能与EMT调控因子（如TGF-β1）的自分泌水平相关，且可能受肿瘤微环境中免疫细胞来源的外源性EMT细胞因子影响；同时突变型SMAD4在HNSCC细胞间质转化过程中发挥重要作用。
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