Tumor‐Associated Neutrophils Secrete AGR2 to Promote Colorectal Cancer Progression by Stabilizing ALYREF to Boost Snail m5C Modification

Colorectal cancer (CRC) is the second leading cause of cancer-related deaths worldwide, primarily due to distant metastasis. Tumor-associated neutrophils (TANs), a major component of the tumor microenvironment (TME), play multifaceted roles in tumor progression. However, the molecular mechanisms by which TANs regulate epithelial-mesenchymal transition (EMT) in CRC remain unclear. This study aims to investigate how TANs promote CRC progression through EMT. CRC and paired adjacent normal tissues were collected for Western Blot, IHC, and flow cytometry to evaluate ALYREF and CD66b expression. NB-4 cells were induced with all-trans retinoic acid to generate TANs, and conditioned medium (TAN-CM) was collected for co-culture with CRC cells. Malignant phenotypes of CRC cells were assessed by CCK-8, EdU, and transwell assays. The m5C modification level and stability of Snail mRNA were determined by MeRIP and actinomycin D assays. Protein and RNA interactions were examined using Co-IP and RIP. A dual-luciferase reporter assay was used to evaluate the interaction between ALYREF and the m5C site in Snail mRNA. CHX assay was performed to assess ALYREF protein stability. A subcutaneous xenograft model with neutrophil depletion was established to validate the role of the AGR2-ALYREF-Snail axis in vivo, followed by HE staining and IHC analysis of tumor tissues. The infiltration of TANs was positively correlated with ALYREF expression in CRC. TAN-secreted AGR2 stabilized ALYREF, enhancing its recognition of m5C sites in Snail mRNA, thereby increasing Snail stability and promoting EMT. Blockade of AGR2 mitigated TAN-induced CRC cell proliferation, migration, and EMT, while ALYREF overexpression partially rescued these effects. In vivo, AGR2 overexpression promoted tumor growth and EMT through neutrophil-dependent activation of the ALYREF-Snail axis. TAN-secreted AGR2 enhances ALYREF stability, thereby promoting m5C-dependent Snail mRNA stabilization and sustaining EMT and CRC progression.