Genome-wide CRISPR Screening Identifies the Pivotal Role of ANKRD42 in Colorectal Cancer Metastasis Through EMT Regulation

Abstract

Abstract Background: Colorectal cancer (CRC), a pervasive and lethal malignancy of the gastrointestinal cancer, imposes significant challenges due to the occurrence of distant metastasis in advanced stages. Understanding the intricate regulatory mechanisms driving CRC distant metastasis is of paramount importance. Methods: To faithfully recapitulate CRC liver metastasis, we innovatively employed in vivo CRISPR-Cas9 screening with a spleen-injected liver metastasis mouse model. Through comprehensive screening of a whole-genome sgRNA library, we identified pivotal regulatory genes facilitating CRC liver metastasis. Experimental validation of ANKRD42, a key hit, was performed using migration and invasion assays, supported by an advanced in vivo spleen-injected liver metastasis model. Patient-Derived Organoid (PDO) models derived from liver metastases elucidated the impact of ANKRD42 perturbation on the expression profile of genes associated with the epithelial-mesenchymal transition (EMT) process in tumorigenesis. Results: Analysis of the TCGA database and our clinical cohorts unveiled heightened ANKRD42 expression in metastases. At the cellular level, the attenuation of ANKRD42 impaired the migration and invasion processes of tumor cells. In vivo experiments further validated these observations, highlighting the diminished liver metastatic capacity of tumor cells upon ANKRD42 knockdown. To unravel the specific mechanisms by which ANKRD42 regulates CRC distant metastasis, we leveraged PDO models. Depleting ANKRD42 in PDOs sourced from liver metastases precipitated the downregulation of pivotal genes linked to EMT, including CDH2 and SANI2, thereby effectively suppressing tumor metastasis. Conclusions: ANKRD42 emerges as a pivotal driver of distant metastasis in CRC, as unveiled by genome-wide CRISPR-Cas9 screening. Significantly elevated expression levels of ANKRD42 were notably observed within metastases across our cohort. Functionally, ANKRD42 intricately regulates the EMT process, thereby potentiating CRC's ability for distant metastatic spread. This study not only establishes a conceptual framework but also identifies potential therapeutic avenues for advanced-stage distant metastasis in CRC patients