Investigation of Metastatic Regulation and Therapeutic Target Discovery in Anaplastic Thyroid Cancer Using a Multigenerational Lung Metastasis Animal Model and Genome-Wide CRISPR/Cas9 Knockout Screening

Anaplastic thyroid cancer (ATC) is a rare but highly aggressive malignancy, with a five-year survival rate below 7% due to rapid progression and early metastasis. To identify genes involved in ATC metastasis and tumor survival, we utilized two complementary platforms: a spontaneous lung metastasis mouse model and a Genome-Scale CRISPR Knockout screen. The in vivo model enabled the isolation of metastatic ARO cells, allowing comparative gene expression analysis between primary tumors and metastatic lung lesions. In parallel, the GeCKO screen was performed in ARO cells using a pooled sgRNA library targeting the human genome, followed by next-generation sequencing to assess gene essentiality based on sgRNA abundance over time. Analysis from both platforms revealed several candidate genes, including IL12Rβ2, associated with metastasis, and DUT and PCBP2, which is essential for cancer cell survival.
CRISPR/Cas9-mediated knockout of each gene was performed in metastatic ARO 2 Lung-derived ATC cell lines. Functional assays, including MTT, clonogenic, and invasion assays, revealed that loss of IL12Rβ2, DUT, or PCBP2 significantly impaired cell viability, proliferation, and invasiveness. Western blot analysis further demonstrated that gene knockouts reduced activation of the PI3K/AKT and MAPK/ERK pathways and increased expression of cell cycle inhibitors p21 and p27. Notably, IL12Rβ2, DUT and PCBP2 knockouts reversed epithelial–mesenchymal transition, indicated by increased E-cadherin and decreased expression of mysenchymal markers.
These findings suggest that IL12Rβ2, DUT, and PCBP2 play essential roles in ATC progression by promoting cell survival, proliferation, and metastasis. Their depletions provide insight into the molecular drivers of ATC progression and offer potential targets for future therapeutic intervention.