Resistance to trastuzumab in HER2-positive breast cancer

HER2-positive (HER2+) breast cancer represents approximately 20% of all breast cancer cases and relies on overproduction of the HER2 receptor for growth and survival. This HER2 dependency has been exploited with the monoclonal antibody trastuzumab, targeting the extracellular domain of HER2. Despite the clinical success of trastuzumab, resistance can arise and remains a major obstacle in effective treatment of HER2+ breast cancer. This thesis aims to elucidate the mechanisms underlying trastuzumab resistance and to identify strategies to overcome it, with a particular focus on immune-mediated effects and glycosylation. To address this, we first established and characterized new in vitro models for studying trastuzumab resistance. In vitro, trastuzumab inhibits HER2 tumorigenic signaling, however, in vivo, binding of trastuzumab to HER2 on breast cancer cells results in recognition by natural killer (NK) cells and activation of antibody-dependent cellular cytotoxicity (ADCC). Recognizing the limitations of monoculture systems, we established a physiologically relevant co-culture model incorporating NK cells engineered to express CD16, the Fc receptor critical for ADCC. This model enabled direct assessment of trastuzumab-dependent immune-mediated killing and revealed striking differences in sensitivity compared to monoculture assays, highlighting the importance of immune context in therapeutic response. Using these models alongside xenograft experiments, this work identifies two major resistance mechanisms. First, trastuzumab treatment induces metabolic adaptations, including upregulation of the glycolytic regulator PFKFB3. Elevated PFKFB3 rewires tumor metabolism and reduces susceptibility to NK cell–mediated killing, while its silencing restores trastuzumab sensitivity. Second, analysis of patient data from the ISPY2 trial highlights specific glycosylation-related genes associated with clinical response. Indeed, we validate glycosylation as a key driver of resistance to trastuzumab. Silencing of a glycogene alters the glycoprofile of the cancer cells and re-sensitizes them to trastuzumab-dependent cytotoxicity mediated by NK cells. Therapeutically, this glycogene can be targeted using an inhibitor, which similarly modifies the cancer cell glycosylation and restores sensitivity to trastuzumab Overall, this thesis presents advanced immune-competent models and identifies metabolic rewiring and glycosylation changes as key drivers of trastuzumab resistance. These insights provide new biomarkers and therapeutic opportunities to improve the effectiveness of HER2targeted treatments.