PO.ET03.06 · 实验与分子治疗
TROP2上调及其与HER2的相互作用介导trastuzumab耐药
TROP2 upregulation and interaction with HER2 mediate trastuzumab resistance
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
Trastuzumab(Herceptin)仍是HER2阳性胃癌的基石疗法，但获得性耐药严重限制了其长期疗效。鉴定耐药机制对开发下一代治疗策略至关重要。我们研究了已知致癌蛋白TROP2在介导trastuzumab耐药中的作用。建立、表征了trastuzumab耐药的HER2阳性胃癌(GC)细胞系，并与亲本细胞进行比较。在对TROP2进行基因敲低(siRNA)后，使用细胞毒性实验评估TROP2对敏感性的功能影响。使用多种实验明确评估了TROP2与HER2之间的分子相互作用，包括共免疫沉淀(Co-IP)、免疫荧光(IF)共定位、蓝色非变性PAGE(BN-PAGE)和标记转移结合实验。为界定特定的异二聚体界面，针对预测残基进行了定点诱变。
Trastuzumab耐药的胃癌细胞表现出IC50显著增加(>5倍)，并且与敏感亲本细胞相比显示出明显更高的TROP2表达。瞬时敲低TROP2表达可恢复耐药细胞系中的trastuzumab敏感性，确认了TROP2作为治疗逃逸介质的直接作用。HER2/TROP2异二聚化通过Co-IP(共免疫沉淀)、IF(免疫荧光)得到确认，BN-PAGE为HER2和TROP2异二聚化的直接相互作用提供了有力证据。针对TROP2-Asp99和HER2-Val308/Lys333界面的诱变进行结合界面测定，成功破坏了HER2与TROP2之间的物理相互作用。
TROP2上调及其随后与HER2的物理异二聚化构成了HER2阳性胃癌中对trastuzumab获得性耐药的可靶向机制。这种相互作用提示，即使HER2被药物抑制，TROP2仍可能维持关键的致癌信号传导。这些发现为开发旨在破坏TROP2-HER2复合物以增强和恢复治疗疗效的策略提供了有力的分子依据。
查看英文原文 English abstract
Trastuzumab (Herceptin) remains a cornerstone therapy for HER2-positive gastric cancer, yet acquired resistance severely limits its long-term efficacy. Identifying resistance mechanisms is critical for developing next-generation treatment strategies. We investigated the role of TROP2, a known oncogenic protein, in mediating resistance to trastuzumab. Trastuzumab-resistant HER2-positive gastric cancer (GC) cell lines were developed, characterized, and compared to parental cells. The functional impact of TROP2 on sensitivity was assessed using cytotoxicity assays following genetic knockdown (siRNA) of TROP2. The molecular interaction between TROP2 and HER2 was definitively evaluated using several assays, including co-immunoprecipitation (Co-IP), immunofluorescence (IF) co-localization, blue native PAGE (BN-PAGE), and label transfer binding assays. To define the specific heterodimer interface, site-directed mutagenesis was performed targeting predicted residues.
Trastuzumab-resistant gastric cancer cells exhibited a significant increase in IC 50 (>5-fold) and displayed markedly higher TROP2 expression compared to sensitive parental cells. Knockdown of TROP2 expression transiently can restore trastuzumab sensitivity in resistant cell lines, confirming TROP2's direct role as a mediator of therapy evasion. HER2/TROP2 heterodimerization was confirmed by Co-IP (co-immunoprecipitation), IF (immunofluorescence), and BN-PAGE provided strong evidence for the direct interaction of HER2 and TROP2 heterodimerization. Binding Interface determination in mutagenesis targeting TROP2-Asp99 and the HER2-Val308/Lys333 interface successfully disrupted the physical interaction between HER2 and TROP2.
TROP2 upregulation and its subsequent physical heterodimerization with HER2 constitutes targetable mechanism of acquired resistance to trastuzumab in HER2-positive gastric cancer. This interaction suggests that TROP2 may sustain critical oncogenic signaling even when HER2 is pharmacologically inhibited. These findings provide a strong molecular rationale for developing strategies aimed at disrupting the TROP2-HER2 complex to enhance and restore therapeutic efficacy.
利益披露 Disclosure
Y. Wang, None..
C. Lu, None..
C. Wei, None..
J. Huang, None..
W. Chan, None..
L. Chen, None..
Y. Chen, None.