Semaphorin 6D drives anti-tumor type I interferon responses to reprogram the tumor microenvironment in colorectal cancer.

Colorectal cancer (CRC) persists as the third most prevalent cause of cancer-associated mortality worldwide, largely due to late diagnosis, limited treatment efficacy, and poor response to immunotherapy. However, the underlying molecular mechanisms remain incompletely characterized. This study identified Semaphorin 6D (SEMA6D) as a potential tumor suppressor that was markedly underexpressed in CRC and associated with poor prognosis. Promoter hypermethylation emerges as the primary mechanism underlying its transcriptional silencing, with notably low expression observed in CIMP-H, MSI-H, CMS4 and CMS1 subtypes. Functional experiments demonstrated that SEMA6D overexpression significantly attenuated cellular proliferation, epithelial-mesenchymal transition (EMT), and migratory capacity in vitro, while concurrently suppressing tumor growth and metastatic spread in vivo. Conversely, SEMA6D depletion induced pro-tumorigenic phenotypes. Moreover, SEMA6D enhanced anti-tumor immunity by enhancing the infiltration of CD4(+) and CD8(+) T lymphocytes into the tumor microenvironment (TME). Importantly, treatment with hypomethylating agents successfully restored SEMA6D expression and potentiated immunotherapy efficacy. Mechanistically, Plexin A4 was identified as the key receptor mediating SEMA6D signaling in CRC. Plexin A4 facilitates the interaction between SEMA6D and IRF9, promoting IRF9 activation and subsequent induction of the type I interferon signaling pathway. This cascade enhances T cell-infiltration, remodels the immunosuppressive TME, and ultimately suppresses tumor progression. Collectively, our findings establish SEMA6D as a crucial tumor suppressor epigenetically silenced in CRC. SEMA6D represents a valuable indicator for molecular classification, prognostic evaluation, and clinical decision-making in CRC. The Plexin A4/SEMA6D/IRF9 axis represents a promising therapeutic target, while the demonstrated efficacy of demethylating agents provides a strong rationale for incorporating epigenetic therapies into CRC treatment strategies, offering significant translational implications for clinical practice.