Harnessing NOPE: A key oncofetal protein driving beta-catenin signaling through peptidic antigen receptor macrophages in hepatocellular carcinoma

Background: Tumor cells utilize the embryonic stem cell (ESC) pathway, which enhances cell plasticity, to evade targeted therapies. Oncofetal proteins, reactivated in tumors and regulating ESC properties, act as markers for liver tumor-initiating cells (T-ICs). Glypican-3 (GPC-3) is a promising therapeutic target for hepatocellular carcinoma (HCC), but the limited identification of functionally significant cell surface oncofetal proteins restricts clinical application. Identifying novel oncofetal proteins with specific T-IC functions is crucial for developing strategies to combat tumor recurrence and drug resistance in HCC. Methods: Integrated OMICs analysis identified liver T-IC populations with increased expression in TCGA datasets. We used AP-MS and stable isotope labeling coupled with SILAC to identify NOPE's binding partners. The functional role of NOPE in liver T-IC regulation was assessed through various cancer stem cell assays. GSEA and beta-catenin pathway analysis elucidated the molecular pathways involved. We targeted NOPE-expressing HCC cells with a peptidic chimeric antigen receptor macrophage approach (pCAR-Ms). Results: Through a comprehensive analysis of our in-house T-IC OMICS profiling datasets in clinical HCC samples, alongside a model of human hepatocyte differentiation, we identified IGDCC4, also known as NOPE, as being highly expressed in liver T-ICs and shows particular expression pattern of oncofetal protein. Consistently, Gene Ontology Biological Process (GOBP) and Chemical and Genetic Perturbation (CGP) analyses revealed that pathways related to stem cell differentiation were enriched in NOPE-high HCC patients. We observed a stepwise increase in NOPE expression from normal liver to cirrhosis and to progressive HCC stages, with NOPE overexpression correlating with poor overall patient survival. In MHCC-97L cells, NOPE overexpression enhanced T-IC features, including self-renewal, tumorigenicity, cell invasiveness, expression of liver T-IC markers, and resistance to lenvatinib treatment. Using AP-MS combined SILAC analysis, we identified PABPC1 as a direct binding partner of NOPE, a finding confirmed through co-immunoprecipitation (co-IP) analysis. The NOPE/PABPC1 complex regulates cell state and drug resistance by directly affecting the beta-catenin signaling pathway, potentially through its impact on YB-1 mRNA stability. We have developed a novel pCAR-Ms with designed peptides specifically targeting NOPE-expressing HCC cells. Our pCAR-Ms demonstrated specificity and potency in inducing phagocytosis of NOPE-expressing HCC cells. Conclusions: We identified NOPE as a functional oncofetal protein that promotes liver cancer stemness by activating the beta-catenin signaling pathway, which can be targeted by a novel pCAR-Ms approach.