RUNX1 Directly Activates WNT2 to Orchestrate Tumor‐Associated Macrophage Reprogramming in Colorectal Cancer

Tumor-associated macrophages (TAMs), particularly the M2 subtype, foster immune suppression and metastasis in colorectal cancer (CRC). Although WNT2 is upregulated in several cancers and RUNX1 is an oncogenic transcription factor in solid tumors, whether a RUNX1-WNT2 axis orchestrates M2 polarization and CRC progression has been unclear. We profiled RUNX1 and WNT2 expressions and correlations with M2 markers in TCGA_COAD/READ using GEPIA and validated findings in 36 paired CRC and adjacent tissues by qRT-PCR, Western blotting, and immunohistochemistry. Functional effects of WNT2 were tested in CRC cell lines (SW480, SW620) using CCK-8, colony formation, and Transwell assays after shRNA knockdown. A Transwell co-culture with PMA-differentiated THP-1 macrophages assessed polarization by flow cytometry (CD86, CD206) and Western blotting (CD68, MAC2, CD20), alongside ELISAs for CCL2, CSF1, and IL-10. RUNX1 regulation of WNT2 was examined by JASPAR/UCSC prediction, ChIP-qPCR, and dual-luciferase reporter assays. In vivo, we evaluated tumor growth (subcutaneous xenografts) and lung metastasis (tail-vein injection) following WNT2 knockdown and performed rescue studies using an IL-10 neutralizing antibody or the CSF1R inhibitor BLZ945. WNT2 was significantly overexpressed in CRC versus normal tissues and positively associated with M2-TAM signatures. WNT2 knockdown curtailed CRC cell viability, clonogenicity, migration, and invasion, reduced secretion of CCL2/CSF1/IL-10 and shifted THP-1 macrophages from an M2-like (CD206⁺) toward an M1-like (CD86⁺) phenotype. RUNX1 expression correlated with WNT2; RUNX1 loss decreased WNT2, while overexpression increased it. ChIP and reporter assays demonstrated direct RUNX1 binding and transcriptional activation of the WNT2 promoter. In vivo, WNT2 silencing reduced tumor volume and weight, increased CD86⁺ and decreased CD206⁺ macrophages in tumors, and diminished lung metastatic burden. Furthermore, blocking M2 polarization (anti-IL-10) or depleting macrophages (BLZ945) mitigated WNT2-driven tumor growth and M2 infiltration. RUNX1 directly activates WNT2 to promote cytokine production and M2 macrophage polarization, thereby accelerating CRC growth and metastasis. These findings suggest that targeting the RUNX1-WNT2 axis and its downstream macrophage programs may offer a therapeutic strategy and potential biomarker framework for CRC.