Table_2_SMAD4 Somatic Mutations in Head and Neck Carcinoma Are Associated With Tumor Progression.docx

As the incidence and the mortality rate of head and neck squamous cell carcinoma (HNSCC) is increasing worldwide, gaining knowledge about the genomic changes which happen in the carcinogenesis of HNSCC is essential for the diagnosis and therapy of the disease. SMAD4 (DPC4) is a tumor suppressor gene. It is located at chromosome 18q21.1 and a member of the SMAD family. Which mediates the TGF-β signaling pathway, thereby controlling the growth of epithelial cells. In the study presented here, we analyzed tumor samples by multiplex PCR-based next-generation sequencing (NGS) and found deleterious mutations of SMAD4 in 4.1% of the tumors. Knock-down experiments of endogenous and exogenous SMAD4 expression demonstrated that SMAD4 is involved in the migration and invasion of HNSCC cells. Functional analysis of a missense mutation in the MH1 domain of SMAD4 may be responsible for the loss of function in suppressing tumor progression. Missense SMAD4 mutations, therefore, could be useful prognostic determinants for patients affected by HNSCCs. This report is the first study where NGS analysis based on multiplex-PCR is used to demonstrate the imminent occurrence of missense SMAD4 mutations in HNSCC cells. The gene analysis that we performed may support the identification of SMAD4 mutations as a diagnostic marker or even as a potential therapeutic target in head and neck cancer. Moreover, the analytic strategy proposed for the detection of mutations in the SMAD4 gene may be validated as a platform to assist mutation screening.

随着全球范围内头颈部鳞状细胞癌（head and neck squamous cell carcinoma, HNSCC）的发病率与死亡率持续攀升，解析其癌变过程中发生的基因组改变，对于该疾病的诊断与治疗至关重要。SMAD4（DPC4）是一种抑癌基因，定位于18号染色体q21.1区域，属于SMAD家族成员，可介导转化生长因子-β（TGF-β）信号通路，进而调控上皮细胞的生长。本研究中，我们采用基于多重PCR技术的下一代测序（NGS）对肿瘤样本进行分析，在4.1%的肿瘤样本中检测到SMAD4的有害突变。对内源性与外源性SMAD4表达进行敲低实验的结果显示，SMAD4参与头颈部鳞状细胞癌细胞的迁移与侵袭过程。对SMAD4的MH1结构域中一处错义突变的功能分析表明，该突变可能导致其抑制肿瘤进展的功能丧失。因此，SMAD4错义突变可作为头颈部鳞状细胞癌患者的有效预后判断指标。本研究是首次采用基于多重PCR的NGS分析，证实头颈部鳞状细胞癌细胞中存在SMAD4错义突变的相关研究。我们开展的基因分析可为将SMAD4突变作为头颈部癌症的诊断标志物，乃至潜在治疗靶点提供依据。此外，本研究提出的SMAD4基因突变检测分析策略，有望作为辅助突变筛查的平台得到验证与应用。