RHOJ-induced chemotherapy resistance through epithelial–mesenchymal transition in drug-tolerant persister cells of head and neck cancer

• RHOJ is upregulated in DTP cells, driving chemoresistance via oxidative stress, DNA damage response, and IPO9/EpCAM-mediated EMT. • High RHOJ levels in endothelial cells and M2 macrophages promote immune evasion by impairing vascular integrity and limiting immune infiltration. • Latrunculin B sensitizes HNSCC DTP cells to chemotherapy by disrupting RHOJ-dependent cytoskeletal dynamics. • RHOJ knockdown inhibits M2 polarization and boosts M1 antitumor activity, highlighting the RHOJ/Rho kinase axis as a target to enhance immunotherapy in resistant HNSCC. Background: Building on our previous identification of TXNRD1 and RHOJ as mediators of immunotherapy resistance in head and neck squamous cell carcinoma (HNSCC), this study investigates the role of RHOJ in regulating endothelial markers and signaling pathways in drug-tolerant persister (DTP) cells, as well as its contribution to immune suppression within the resistant tumor microenvironment. Methods: We examined the role of RHOJ in regulating tumor-associated macrophage polarization and enhancing M1 antitumor activity and evaluated the therapeutic potential of targeting the RHOJ/Rho kinase axis in an HNSCC DTP mouse model. Results: RHOJ expression was significantly upregulated in HNSCC DTP cells. RHOJ contributes to chemoresistance by increasing oxidative stress and initiating the DNA damage response. RNA sequencing revealed that the IPO9/EpCAM signaling pathway positively regulates RHOJ expression. Knockdown of RHOJ with shRNA suppressed HNSCC DTP cell migration and downregulated IPO9/EpCAM signaling. Gene enrichment analysis revealed high RHOJ expression in tumor-associated endothelial cells. Treatment with the RHOJ-dependent F-actin inhibitor Latrunculin B (HY-101,848) impaired actin polymerization and increased the chemosensitivity of HNSCC DTP cells. Silencing RHOJ inhibited M2 tumor-associated macrophage activation. RHOJ overexpression promoted M2 macrophage proliferation. Conclusion: RHOJ plays a critical role in modulating immunosuppressive signaling in both tumor and endothelial cells. RHOJ promotes the function of tumor-associated endothelial cells and drives EMT through its interaction with the IPO9/EpCAM signaling pathway, thereby increasing cell survival and drug resistance. Additionally, RHOJ may limit immune cell infiltration into the tumor core and promote immune evasion by contributing to vascular abnormalities and impaired barrier function.