A green platform to study cancer glyco-signatures

Trastuzumab (Tz) is the first therapeutic drug FDA-approved to treat ErbB2+ gastric cancers (GC). ErbB2 undergoes extensive glycosylation, which tightly controls receptor cell surface dynamics and activation. Also, cancer-associated glycosylation (TACAs) at the ErbB2 Tz-binding domain actively drive the resistance of GC cells to Tz cytotoxicity, undermining its clinical efficacy. So far, the structural heterogeneity of ErbB2 glycans has hindered the unequivocal identification and validation of Tz-sensitive/resistant TACAs, and the clinical implementation of glycan-sensitive biomarkers for the prognostic and therapeutic stratification of ErbB2+GC patients. Therefore, the role played by specific ErbB2 TACAs in Tz acquired resistance requires further clarification. This has been holdup by several biotechnological hurdles, including efficient synthesis of tailored glycoconjugates. Plants are well suited for the transient expression of glycoproteins without the need for genetic transformation. In terms of glycosylation, plants offer the advantage of a limited N-glycan processing repertoire, thus enabling a flexible stepwise overexpression of glycosyltransferases required to tailor glycans. We aim to elucidate how distinct ErbB2 glycans tune the Tz binding affinity and function, thus validating them as molecular gatekeepers of Tz therapeutic response in ErbB2+ GC. We hypothesize that plant “synthetic glycans” can be exploited to crack the ErbB2 sugar code underlying molecular processes that lead to GC therapy resistance. We will take advantage of plant’s tolerance for glycoengineering to produce ErbB2 mimicking GC glyco-signatures and identify Tz-sensitive/resistant ErbB2 glycoforms. Glycoform-specific ErbB2-Tz affinity will be orthogonally validated in cancer cell settings by CRISPR/Cas9-based precise glycogene editing. The functionality of ErbB2 TACAs will be comprehensively characterized, including receptor subcellular localization; expression levels; oligomerization capacity and activation/signaling threshold in the presence of Tz. Synthesis of homogenous glycans represents a major bottleneck in structure/function correlation studies. Using plants as bioreactors is a promising innovative and sustainable approach to produce and validate ErbB2 glycoforms to study glycan-dependent interactions. The proposed multivalent strategy will disclose mechanisms underlying resistance of ErbB2+ GC to antibody induced-toxicity and propose selected ErbB2 TACAs as novel stratifying biomarkers in the advanced GC clinical setting. Such studies can guide the design of novel personalized and more efficient targeted therapeutic agents, capable of overcoming glycan-mediated molecular resistance.