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 IC50 (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. Citation Format: Yuan-Liang Wang, Chi-Huan Lu, Cheng-Yen Wei, Jye-Yu Huang, Woan-Eng Chan, Lu-Tzu Chen, Ya-Chi Chen. TROP2 upregulation and interaction with HER2 mediate trastuzumab resistance abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2972.
Wang et al. (Fri,) studied this question.