喉鳞状细胞癌中与失巢凋亡相关的四基因预后特征及ANXA5的功能表征

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中文摘要

背景： 喉鳞状细胞癌（LSCC）仍然是头颈部区域最致命的恶性肿瘤之一。肿瘤转移是治疗失败的主要原因，与失巢凋亡逃逸密切相关。然而，LSCC中这一过程的分子驱动因素尚未完全阐明。本研究系统探讨了LSCC中失巢凋亡相关基因（ARGs）的预后和功能景观，重点关注其与肿瘤免疫微环境的相互作用及治疗脆弱性。
方法： 分析了TCGA-LSCC队列的RNA测序谱和临床数据，并从GeneCards和Harmonizome数据库中整理了候选失巢凋亡相关基因（ARGs）。使用limma和单变量Cox回归筛选差异表达和预后相关的ARGs。在训练集中使用LASSO-Cox和多变量Cox回归构建了四基因预后特征，并在测试集中进行内部评估。评估了所得失巢凋亡相关风险评分与生存、临床病理变量、免疫特征、通路富集和预测药物反应的关系。使用单细胞RNA测序数据表征了特征基因的细胞分布。ANXA5被优先用于实验评估，因为它在风险相关基因中具有最大的正向模型系数，显示出肿瘤相关失调，并与失巢凋亡相关过程具有生物学相关性。使用表达、细胞活力、EMT标志物、凋亡和伤口愈合试验在TU686细胞中检测了其效应。
结果： 基于ARG的特征有效地将LSCC患者分为高风险和低风险组，总生存期显著不同（p < 0.001）。高失巢凋亡相关风险评分与免疫抑制性TME相关，提示有利于免疫逃逸的环境。药物敏感性分析显示，高风险患者对某些酪氨酸激酶抑制剂更敏感，但对常规化疗药物耐药，突出了亚型特异性治疗机会。在ARGs中，ANXA5作为一个具有强预后能力的枢纽基因出现。体外试验表明，ANXA5在LSCC中发挥致癌功能。敲低ANXA5后，我们检测到喉鳞状癌细胞增殖活性受抑制、EMT特征基因差异表达、凋亡率升高和迁移能力减弱。总体而言，这些初步结果表明ANXA5可能参与调节喉鳞状癌细胞的增殖、间充质分子特征、细胞存活和迁移行为。
结论： 四基因失巢凋亡相关特征在TCGA-LSCC队列中显示出预后分层，并与肿瘤免疫微环境的差异相关。ANXA5可能代表一个候选预后生物标志物和一个具有生物学相关性的基因，有待进一步机制研究；然而，需要外部验证和额外的功能研究。

英文摘要

BACKGROUND: Laryngeal squamous cell carcinoma (LSCC) remains one of the most lethal malignancies of the head and neck region. Tumor metastasis, the primary cause of treatment failure, is intimately linked to the evasion of anoikis. Yet the molecular drivers of this process in LSCC are incompletely understood. This study systematically investigates the prognostic and functional landscape of anoikis-related genes (ARGs) in LSCC, with an emphasis on their interplay with the tumor immune microenvironment and therapeutic vulnerability.
METHODS: RNA-sequencing profiles and clinical data from the TCGA-LSCC cohort were analyzed, and candidate anoikis-related genes (ARGs) were curated from the GeneCards and Harmonizome databases. Differentially expressed and prognosis-associated ARGs were screened using limma and univariate Cox regression. A four-gene prognostic signature was constructed in the training set using LASSO-Cox and multivariate Cox regression and internally evaluated in the testing set. The resulting anoikis-related risk score was assessed in relation to survival, clinicopathological variables, immune characteristics, pathway enrichment, and predicted drug response. Single-cell RNA-sequencing data were used to characterize the cellular distribution of the signature genes. ANXA5 was prioritized for experimental evaluation because it had the largest positive model coefficient among the risk-associated genes, showed tumor-associated dysregulation, and had biological relevance to anoikis-related processes. Its effects were examined in TU686 cells using expression, cell-viability, EMT-marker, apoptosis, and wound-healing assays.
RESULTS: The ARG-based signature effectively stratified LSCC patients into high- and low-risk groups with significantly different overall survival (p < 0.001). High anoikis-related risk score correlated with an immunosuppressive TME, suggesting a permissive milieu for immune evasion. Drug sensitivity analysis revealed that high-risk patients were more responsive to certain tyrosine kinase inhibitors but resistant to conventional chemotherapeutics, highlighting subtype-specific therapeutic opportunities. Among the ARGs, ANXA5 emerged as a hub gene with strong prognostic power. In vitro assays demonstrated that ANXA5 exerts oncogenic functions in LSCC. Following ANXA5 knockdown, we detected suppressed proliferative activity, differential expression of EMT signature genes, elevated apoptosis rate and weakened migratory ability in laryngeal squamous carcinoma cells. Collectively, these preliminary results suggest that ANXA5 is likely implicated in regulating proliferation, mesenchymal molecular traits, cell survival and migratory behaviors of laryngeal squamous carcinoma cells.
CONCLUSION: The four-gene anoikis-related signature showed prognostic stratification within the TCGA-LSCC cohort and was associated with differences in the tumor immune microenvironment. ANXA5 may represent a candidate prognostic biomarker and a biologically relevant gene for further mechanistic investigation; however, external validation and additional functional studies are required.