Defining Tumor-associated Glycans as Molecular Drivers of Malignancy and Therapeutic Resistance in ErbB2-Positive Advanced Gastric Cancer

Abstract

Advanced gastric and gastroesophageal cancer (GC) remains a worldwide health concern characterized by poor patient prognosis, mainly due to late disease diagnosis and lack of efficient therapeutic strategies. Efforts towards the comprehensive molecular profiling of tumors have revealed potential therapeutic targets, one of which is the ErbB2 receptor. ErbB2 is overexpressed in 10-20% GC cases, making patients eligible to undergo therapy with the ErbB2-specific trastuzumab (TTZ) monoclonal antibody (mAb). Unfortunately, the majority of treated patients experience molecular resistance, thereby undermining the therapeutic efficacy of this targeted agent. Importantly, ErbB2 is extensively glycosylated in its extracellular region. Protein glycosylation is the most abundant and diverse post-translational modification, and the enzymatic addition of structurally complex polysaccharide chains (glycans) to specific residues of the peptidic backbone critically regulates protein stability, conformation, and functionality. In cancer cells, however, the glycosylation process is severely deregulated and actively contributes to several of their malignant features. This project aims to: i) understand if the ErbB2 site-specific glycosylation profile actively tunes TTZ binding and, consequently, its therapeutic efficacy; ii) explore whether the acquisition of molecular resistance to TTZ is underpinned and supported by shifts in the cellular glycome. For these purposes, we are establishing a glycoengineered cell-based library with ErbB2-positive GC cell lines to decipher the mechanistic contributions of individual glycosylation signatures into the therapeutic response and acquisition of molecular resistance to TTZ. Concomitantly, we established ErbB2-addicted TTZ-resistant cell lines to study how the acquisition of molecular resistance can reshape the cellular and ErbB2-specific glycosylation profiles. GC cell lines with high ErbB2 and endogenous activation were previously selected to undergo an established pipeline of precise genome editing with previously validated gRNAs; isolation and validation of independent cell clones, and, finally, in vitro validation of their glycosylation profile by western blot (WB), immunofluorescence, and flow cytometry (FC). Surface Plasmon Resonance (SPR) was used to measure TTZ-ErbB2 affinity. A protocol of a co-culture system with TTZ is being established to measure antibody-dependent cell cytotoxicity (ADCC) in the glycoengineered cell models. Two ERBB2-amplified TTZ-resistant (TTZ-R) cell lines were established by continuous exposure to incremental dosages of TTZ, and their molecular glyco-profile is being characterized by WB, FC, and an RNAseq analysis. Two GC cell lines (NCI-N87 and OE-19) showing high ErbB2 expression and endogenous activation were selected for CRISPR/Cas9-based glycoengineering. Several genomically edited cell line clones were obtained, depicting tailored glycosylation profiles, that is, the selective abrogation of pre-determined cancer-associated glycan antigens. Successfully edited glycogenes include ST6GAL1, ST3GAL3, ST3GAL4, and FUT3. knock-out (K.O.) cell lines depict absent expression of α2,6- and α2,3-sialylation and α1,3-4-fucosylation, respectively, from the cell membrane. Glycoengineered cell models have shown increased binding affinity for TTZ compared to their wild-type (WT) counterpart. TTZ-R cell lines depict a reshaping of their glyco-transcriptome and glycome, increased ErbB2 activation, and morphological differences in comparison with parental cells. We are setting up a multidisciplinary approach that can be an important pre-clinical step to address glycan-mediated resistance to TTZ in ErbB2-positive GC. The removal of specific cancer-associated glycans from the cell and the receptor was done successfully. Furthermore, we have established TTZ-insensitive ErbB2-positive gastric cancer cell lines as invaluable models to study the molecular influence of cancer-associated glycosylation pathways in the acquisition of TTZ molecular resistance.