Unraveling the glycan-mediated molecular resistance to trastuzumab in ErbB2-dependent gastric cancer

The glycosylation landscape of the ErbB2 receptor in gastric cancer remains elusive. Here, we have comprehensively characterized the site-specific glycosylation profile of ErbB2 and disclosed a glycan-mediated mechanism of molecular resistance to the trastuzumab therapeutic monoclonal antibody.

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Unresectable gastric cancer (GC) remains a largely incurable and extremely lethal disease [1, 2]. Due to the indolent behavior of gastric neoplasms during the early stages of disease onset and development, at the moment of diagnosis, the vast majority of patients exhibits metastatic dissemination (stage IV). A significant fraction of gastric tumors harbors the overexpression and hyperactivation of the human epidermal growth factor receptor 2 (ErbB2), which constitutes membrane-bound receptor tyrosine kinase (RTK) responsible for the activation of oncogenic signaling pathways supporting malignant cell growth and proliferation [2, 3]. The humanized monoclonal antibody (mAb) trastuzumab, which has found wide clinical applicability in the treatment of ErbB2-positive breast carcinomas, was granted FDA approval for the treatment of patients harboring stage IV ErbB2-positive stage GC [3]. Unfortunately, the clinical benefits from trastuzumab remain limited in the GC setting, as virtually all treated patients exhibit either intrinsic or acquired molecular resistance to this personalized therapeutic agent [4].

            The extracellular region of ErbB2 constitutes a well-known target for extensive post-translational glycosylation, a highly regulated cellular process in which monosaccharide sugar building blocks are enzymatically assembled into structurally complex carbohydrate chains (glycans) that are then attached to specific amino acid residues of selected protein carriers [5, 6]. Glycans decorating the ectodomain of RTKs act as crucial regulators of receptor membrane dynamics, dimerization capacity, signaling potential and response to therapeutic agents, including mAbs [7]. As for ErbB2, however, the specific glycosylation profile of the receptor, and the molecular mechanisms through which aberrant glycan signatures actively regulate the acquisition of molecular resistance by ErbB2-driven GC cells to trastuzumab-induced cytotoxicity remain elusive.

            In this study, we have comprehensively characterized the structural N-glycosylation profile of ErbB2’s extracellular region. Interestingly, we came across a highly diverse and site-specific glycosylation landscape, in which complex and, in particular, terminally α2,6-sialylated N-glycan chains are restricted to two glycosylation sites occurring within the receptor’s trastuzumab-binding domain. Furthermore, we were able to confirm the expression of ErbB2 sialylated glycoforms in gastric carcinoma clinical specimens, thus validating ErbB2 as an in vivo carrier of cancer-associated sialic acid motifs. The terminal α2,6-sialylation of N-glycans is solely catalyzed by the ST6Gal1 Golgi-residing sialyltransferase, which is widely overexpressed in multiple epithelial cancers. By abrogating the activity of the ST6Gal1 enzyme in ErbB2-driven GC cells, we observed a drastic reshaping of the ErbB2 glycome, in which terminally sialylated glycans are selectively replaced by multi-fucosylated species, specifically at the trastuzumab-binding domain. ST6GAL1 K.O. sensitized ErbB2-addicted GC cells to trastuzumab-induced cytotoxicity, by reducing the receptor’s membrane turnover rate, stabilizing ErbB2-containing oligomers at the cell surface, and reducing ErbB2 and EGFR intracytoplasmic phosphorylation.

ST6Gal1 drives the molecular resistance to trstuzumab in ErbB2-addicted gastric cancer cells

            By unraveling a previously elusive mechanism of glycan-mediated molecular resistance to trastuzumab, our findings set the ground for the rational design of novel personalized therapeutic strategies directed at individuals harboring ErbB2-driven GC. Moreover, specific ErbB2 glycoforms may represent novel, yet so far overlooked, molecular predictors of trastuzumab therapeutic efficacy and GC patient clinical outcome.

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Henrique Duarte

Junior Researcher, Institute for Research and Innovation in Health, University of Porto (i3S)