A proteomic atlas of glypican-3 interacting partners: Identification of alpha-fetoprotein and other extracellular proteins as potential immunotherapy targets in liver cancer

Antibody and cell-based therapeutics targeting cell surface receptors have emerged as a major class of immune therapeutics for treating cancer. However, the number of cell surface targets for cancer immunotherapy remains limited. Glypican-3 (GPC3) is a cell surface proteoglycan and an oncofetal antigen. In this study, we report a large-scale tumor-associated GPC3 co-immunoprecipitation (CoIP)-proteomic study using liver cancer xenograft tumors in mice. We identified 153 GPC3-associated proteins through mass spectrometry. To identify potential drug targets, we categorized GPC3-associated proteins based on their subcellular locations using UniProt annotations, with a focus on extracellular proteins. Additionally, we annotated differentially expressed proteins in hepatocellular carcinoma (HCC) versus non-tumor liver samples based on the literature, analyzed expression levels in tumor versus normal tissues using TCGA and GTEx databases via GEPIA, and identified prognostic liver cancer markers according to GEPIA. Among GPC3-associated proteins, Immunoglobulin Superfamily Member 1 (IGSF1), alpha-fetoprotein (AFP), FAT Atypical Cadherin 1 (FAT1), Formin 1 (FMN1), and Guanylate Cyclase 2C (GUCY2C), were identified as potential therapeutic targets. Furthermore, we validated the direct protein interaction between GPC3 and AFP through immunoprecipitation with purified proteins and through co-localization imaging using immunofluorescence microscopy. This study provides large proteomic datasets related to GPC3-associated proteins, enhancing our understanding of glypican biology in cancer cells and offering a new approach to identifying immunotherapy targets.

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