Genome‐Wide In Vivo RNAi Screening Identifies HOXD4 as a Tumor Metastasis Suppressor in Colorectal Cancer

ABSTRACT Metastasis remains a major therapeutic challenge in colorectal cancer, highlighting an urgent need to elucidate its underlying molecular mechanisms. In this study, an in vivo screening system integrating genome‐wide short hairpin RNA library and next‐generation sequencing identifies six candidate metastasis suppressors, among which Homeobox D4 (HOXD4) shows the most pronounced effects. Clinicopathological analyses reveal significant HOXD4 downregulation in tumor tissues relative to adjacent normal tissues, with reduced expression strongly correlating with aggressive tumor features. Functional assays demonstrate that HOXD4 depletion enhances migration, invasion, and tumorsphere formation in HCT116 cells, while ectopic HOXD4 overexpression reverses these malignant phenotypes in SW620 cells. Mechanistically, HOXD4 suppresses epithelial‐mesenchymal transition (EMT) by directly binding to the promoter of Forkhead box Q1 (FOXQ1), a key driver of EMT and stemness, and thereby transcriptionally repressing its expression. Immunohistochemistry confirms an inverse correlation between HOXD4 and FOXQ1 expression in clinical specimens. Rescue experiments substantiate that HOXD4 exerts its metastasis‐suppressing functions via FOXQ1 regulation. Collectively, these findings not only establish an efficient platform for screening tumor metastasis suppressors, but also identify HOXD4 as a master transcriptional regulator of the FOXQ1‐EMT axis, providing a promising target for metastasis interception.