Identification of the JNK-Active Triple-Negative Breast Cancer Cluster Associated with an Immunosuppressive Tumor Microenvironment

Background: Although an immunosuppressive tumor microenvironment (TME) is key for tumor progression, the molecular characteristics associated with the immunosuppressive TME remain unknown in triple-negative breast cancer (TNBC). Our previous functional proteomic study of TNBC tumors identified that C-JUN N-Terminal kinase (JNK) pathway-related molecules were enriched in a cluster associated with the inflammatory pathway. However, the role of the JNK pathway in the TNBC TME is still unclear. Methods: Transcriptomic analysis was conducted using The Cancer Genome Atlas datasets. The effect of JNK-IN-8, a covalent pan-JNK inhibitor, on TNBC tumor growth, lung metastasis, and the TME was measured in TNBC syngeneic mouse models (n = 13 per group). Tumor (n = 43) or serum (n = 46) samples from TNBC patients were analyzed using multiplex immunohistochemistry or Luminex assay. All statistical tests were 2-sided. Results: CIBERSORT analysis revealed that TNBC patients with high phosphorylated JNK level (n = 47) had more regulatory T cell (Treg) infiltration than those with a low phosphorylated JNK level (n = 47) (P =. 02). Inhibition of JNK signaling statistically significantly reduced tumor growth (P <. 001) and tumor-infiltrating Tregs (P =. 02) while increasing the infiltration of CD8+ T cells in TNBC mouse models through the reduction of C-C motif ligand 2 (CCL2). Tumor-Associated macrophages were the predominant cells secreting CCL2, and inhibition of JNK signaling reduced CCL2 secretion of human primary macrophages. Moreover, in patients with TNBC (n = 43), those with high levels of CCL2+ tumor-Associated macrophages had more Treg and less CD8+ T cell infiltration (P =. 04), and the serum CCL2 level was associated with poor overall survival (hazard ratio = 2.65, 95% confidence interval = 1.29 to 5.44, P =. 008) in TNBC patients (n = 46). Conclusions: The JNK/C-JUN/CCL2 axis contributes to TNBC aggressiveness via forming an immunosuppressive TME and can offer novel therapeutic strategies for TNBC.