Tumor-associated CALD1 splicing variant driven by hnRNPA1 promotes colorectal cancer metastasis

Abstract

Alternative splicing (AS) expands transcriptomic and proteomic diversity and is increasingly implicated in colorectal cancer (CRC) progression, yet the functional relevance of individual AS isoforms and the splicing regulators directing their generation remain poorly understood. RNA sequencing was conducted on five paired CRC tumor and adjacent normal tissue samples to profile the AS landscape and identify differentially spliced transcripts. Candidate isoform expression was verified by RT-PCR, quantitative RT-PCR, and immunoblotting. CALD1 isoform functions were assessed by gain-/loss-of-function assays in vitro and in vivo. To dissect the upstream splicing control and downstream protein interactions of CALD1S, we employed RNA immunoprecipitation, minigene splicing assays, RNA pull-down, co-immunoprecipitation, and mass spectrometry-based proteomics. Clinical relevance was evaluated using public datasets and independent CRC patient cohorts. Widespread AS aberrations were observed in CRC, with 1,048 significantly altered events identified. Among these, a short CALD1 isoform lacking exons 3b and 4 (hereafter termed CALD1S) was preferentially expressed in tumor tissues. Elevated CALD1S expression significantly correlated with advanced tumor stage, lymph node metastasis, and poorer patient survival. Functional assays demonstrated that CALD1S promoted CRC cell migration and invasion in vitro and enhanced liver metastatic dissemination in vivo, while exerting negligible impact on cell proliferation. Mechanistically, the splicing factor hnRNPA1 bound CALD1 pre-mRNA and promoted skipping of exons 3b and 4, thereby augmenting CALD1S biogenesis. Restoring CALD1S expression partially rescued the impaired migratory and invasive phenotypes induced by hnRNPA1 depletion. Furthermore, MYH9 was identified as a CALD1S-interacting protein and its knockdown attenuated CALD1S-driven cell migration. Clinically, CALD1 and MYH9 protein levels exhibited a strong positive correlation in lymph node-positive CRC tumors, and a higher combined CALD1/MYH9 expression was associated with unfavorable prognosis. Our study delineates an hnRNPA1-CALD1S-MYH9 regulatory axis that couples aberrant AS to CRC metastatic progression. These findings support CALD1S as a potential prognostic biomarker for disease progression in CRC.