USP16 promotes M2 polarization of macrophages in colorectal cancer by activating the Notch pathway via inducing the deubiquitination of E2F1.

Tumor-associated macrophages (TAMs) polarized to the M2 phenotype play a pivotal role in fostering an immunosuppressive tumor microenvironment (TME) that drives colorectal cancer (CRC) progression. Ubiquitin-specific peptidase 16 (USP16) is a deubiquitinating enzyme, yet its function in CRC-associated macrophage polarization remains undefined. The present study demonstrated that USP16 was a critical promoter of M2 macrophage polarization in CRC. We found that USP16 silencing in macrophages skewed polarization towards the antitumor M1 phenotype, enhanced phagocytic capacity, and increased lysosomal enzyme activity. Conversely, USP16 overexpression promoted M2 polarization and suppressed M1 characteristics. Mechanistically, USP16 directly interacted with and deubiquitinated the transcription factor E2F1, thereby stabilizing it. Stabilized E2F1 transcriptionally activated Notch1, a key regulator of the Notch signaling pathway. This USP16/E2F1/Notch1 axis was essential for driving M2 polarization, as silencing either E2F1 or Notch1 abrogated the effects of USP16 overexpression. Furthermore, conditioned medium from USP16-overexpressing macrophages significantly enhanced the proliferation and invasion of CRC cells, effects that were dependent on this molecular axis. In conclusion, our findings identify a novel USP16/E2F1/Notch1 signaling circuit that orchestrates protumorigenic M2 macrophage polarization in CRC, highlighting USP16 as a potential therapeutic target for reprogramming the immunosuppressive TME and improving CRC treatment.