CAST as a Tumor-Specific Suppressor in Colorectal Cancer: Discovery Through Integrative Analysis of Unannotated Isoforms, Proteomics, and Epigenetic Regulation

Colorectal cancer (CRC) remains a leading cause of cancer-related mortality worldwide, yet the molecular mechanisms driving tumor initiation and progression are incompletely understood. Here, we integrate single-cell long-read sequencing, proteomics, and DNA methylation data to identify tumor-specific targets from unannotated isoforms in CRC. Analysis of 29,429 isoforms across 3,262 single cells revealed 3,338 novel isoforms, comprising 2,779 Novel In Catalog (NIC) and 559 Novel Not in Catalog (NNC). Cross-referencing with 68 normal tissues from GTEx identified 3 tumor-specific candidates absent from normal colon. Among these, CAST (Calpastatin) emerged as a tumor-specific suppressor, with a 63.1-fold higher expression in tumor (2.62) versus normal colon (0.03). CAST protein was confirmed in 100 tumor samples by mass spectrometry (mean expression: 0.22). Mutational analysis of 100 CRC samples revealed 8 deleterious mutations, including 2 nonsense, 2 splice-site, 2 missense, 1 frameshift, and 1 in-frame variant, consistent with a tumor-suppressor role. Promoter methylation analysis of 393 tumor samples showed that 89.3% (351/393) were hypomethylated ({beta} < 0.1), whereas only 1.5% (6/393) were hypermethylated ({beta} > 0.3), indicating demethylation-mediated reactivation. Furthermore, CAST expression was negatively correlated with promoter methylation and regulated by the miR-200 family (miR-200c: 13.48, miR-141: 10.13, miR-429: 8.09). Our findings establish CAST as a tumor-specific suppressor in CRC and provide a generalizable framework for discovering therapeutic targets from unannotated isoforms.