Mechanistic Investigation of Resistance to SHR-A1811 and T-DXd, the Third Generation of HER2-ADC, in Breast Cancer

Background: In recent years, the therapeutic landscape for cancer patients has undergone a profound transformation with the emergence of third-generation HER2-targeted ADCs, including T-DXd and SHR-A1811. Nevertheless, recurrence and metastasis are inevitable for most patients, and the mechanisms driving resistance to these ADCs remain poorly understood. Against this backdrop, the present study seeks to elucidate the resistance mechanisms of breast cancer cells to SHR-A1811 and T-DXd, while identifying potential targets to enhance sensitivity to these agents. Methods: Two resistant cell lines-SHR-A1811-resistant and T-DXd-resistant-were established from the parental breast cancer JIMT-1 cell line via successive drug administration. To identify alterations in protein expression profiles, label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) and single-cell RNA sequencing (scRNA-seq) were employed. Validation of the findings was performed using Western blotting, RNA interference, transfection, and the CellTiter-Glo (CTG) Luminescent Cell Viability Assay. Results: A total of 5,664 differentially expressed proteins were quantitatively identified via proteomic analysis comparing SHR-A1811/T-DXd-resistant cells with their sensitive counterparts. Integrated proteomic and scRNA-seq analyses revealed significant up-regulation of CD44 and PLOD2 in resistant cells. Subsequent validation studies confirmed that CD44 expression was substantially higher in both resistant cells relative to their sensitive counterparts, and CD44 knockdown enhanced the sensitivity of the resistant cells to the ADCs. Conclusions: Our findings demonstrate that CD44 is a critical factor in the development of resistance to SHR-A1811 and T-DXd, with potential utility as both a resistance biomarker for third-generation HER2-ADCs in breast cancer and a therapeutic target to overcome such resistance.