PO.ET02.12 · 实验与分子治疗
靶向TGFBR2的非经典neddylation修饰抑制口腔鳞状细胞癌的转移潜能
Targeting the non-canonical neddylation of TGRBR2 suppresses the metastatic potentials of oral squamous cell carcinoma
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：口腔鳞状细胞癌（OSCC）是头颈部最常见的恶性肿瘤，其特点是生长迅速、侵袭性强、颈部淋巴结转移早以及总体转移率高。原发手术后辅以同步放化疗是目前大多数晚期OSCC患者的首选治疗策略。西妥昔单抗（Cetuximab），一种表皮生长因子受体（EGFR）酪氨酸激酶抑制剂（TKI），联合放疗，近期已获美国食品药品监督管理局（FDA）批准用于治疗复发或转移性OSCC。然而，在非小细胞肺癌中已有报道，转化生长因子-β（TGF-β）信号轴的替代激活会介导TKI耐药。因此，阐明TGF-β受体信号与EGFR介导的信号转导之间的串扰，对OSCC未来的精准管理可能至关重要。
方法：应用基因集富集分析（GSEA）预测蛋白neddylation抑制剂MLN4924（Pevonedistat）抑制OSCC细胞迁移和淋巴结转移的潜在机制。进行定点突变以确认TGFBR2 neddylation在促进OSCC转移潜能中的必要性。采用pulldown实验验证TGFBR2 neddylation，并采用放线菌酮追踪实验评估MLN4924处理后HSC3细胞中TGFBR2蛋白的降解。
结果：我们发现MLN4924处理在体外和体内均能有效抑制OSCC细胞的转移潜能。针对K1667和K1700残基（Lys→Arg）的定点突变证实TGFBR2 neddylation对OSCC转移进展至关重要。GSEA预测MLN4924主要抑制受处理细胞中的TGF-β信号轴。pulldown实验进一步证明MLN4924显著降低HSC3细胞中neddylation修饰的TGFBR2水平。与此一致，放线菌酮追踪分析显示MLN4924处理加速了TGFBR2降解。在挽救实验中，在TGFBR2沉默的HSC3细胞中重新表达野生型TGFBR2——而非K1667R/K1700R neddylation缺陷型突变体——显著恢复了迁移能力。此外，敲低E3连接酶c-Cbl大幅降低了TGFBR2 neddylation并损害了细胞迁移。
结论：本研究首次证明非经典的c-Cbl介导的TGFBR2 neddylation在驱动OSCC转移进展中发挥决定性作用。
查看英文原文 English abstract
Background: Oral squamous cell carcinoma (OSCC) is the most common malignant tumor in the head and neck region and is characterized by rapid growth, strong invasiveness, early cervical lymph node metastasis, and a high overall metastatic rate. Primary surgery followed by adjuvant concurrent chemoradiotherapy is currently the preferred treatment strategy for most patients with advanced OSCC. Cetuximab, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), in combination with radiotherapy, has recently been approved by the U.S. Food and Drug Administration (FDA) for the treatment of recurrent or metastatic OSCC. However, TKI resistance mediated by alternative activation of the transforming growth factor-beta (TGF-beta) signaling axis has been reported in non-small cell lung cancer. Therefore, elucidating the crosstalk between TGF-beta receptor signaling and EGFR-mediated signal transduction may be critical for the future precision management of OSCC.
Methods: Gene set enrichment analysis (GSEA) was applied to predict the potential mechanisms through which the protein neddylation inhibitor MLN4924 (Pevonedistat) suppresses cellular migration and lymph node metastasis in OSCC. Site-directed mutagenesis was performed to confirm the requirement of TGFBR2 neddylation in promoting OSCC metastatic potential. A pulldown assay was used to validate TGFBR2 neddylation, and a cycloheximide chase assay was employed to evaluate TGFBR2 protein degradation upon MLN4924 treatment in HSC3 cells.
Results: We found that MLN4924 treatment effectively suppresses the metastatic potential of OSCC cells both in vitro and in vivo. Site-directed mutagenesis targeting residues K1667 and K1700 (Lys→Arg) confirmed that TGFBR2 neddylation is essential for OSCC metastatic progression. GSEA predicted that MLN4924 mainly suppresses the TGF-beta signaling axis in treated cells. Pulldown assays further demonstrated that MLN4924 markedly reduces neddylated TGFBR2 levels in HSC3 cells. Consistently, MLN4924 treatment accelerated TGFBR2 degradation, as shown by cycloheximide chase analysis. In rescue experiments, re-expression of wild-type TGFBR2-but not the K1667R/K1700R neddylation-defective mutant-significantly restored migration in TGFBR2-silenced HSC3 cells. Moreover, knockdown of the E3 ligase c-Cbl substantially decreased TGFBR2 neddylation and impaired cell migration.
Conclusion: This study is the first to demonstrate that non-canonical c-Cbl-mediated neddylation of TGFBR2 plays a decisive role in driving OSCC metastatic progression.
利益披露 Disclosure
Y. Lin, None.