JNK1和JNK2在TNBC进展和肿瘤微环境免疫调节中的双重作用揭示了一种新的治疗脆弱性

Background: Triple-negative breast cancer (TNBC), one of the most lethal breast cancer subtypes, is driven by hyperactive oncogenic pathways that promote tumor growth and suppress antitumor immunity. Aberrantly high c-Jun N-terminal kinase (JNK) signaling is emerging as a key driver of tumor progression and immune evasion; however, the distinct roles of JNK1 and JNK2 in TNBC are largely undefined. This knowledge gap constrains efforts to exploit JNK biology for therapeutic advancement. Here, we investigated how each isoform drives tumor-intrinsic behavior and remodels the tumor microenvironment (TME). We hypothesized that both JNK1 and JNK2 drive TNBC growth and promote an immunosuppressive TME, and that loss of either isoform reprograms the TME toward a more immune-permissive, antitumor state. Methods: The impact of JNK signaling inhibition on PyMT-N TNBC cell growth was evaluated pharmacologically, using the pan-JNK inhibitor JNK-IN-8, and genetically, by knocking out JNK using the CRISPR-Cas9 system. Cell growth was evaluated using CellTiter-Blue viability and clonogenic assays. Immunocompetent syngeneic C57BL/6 mice bearing JNK1-, JNK2-, or dual-knockout (KO) tumors were used to assess tumor-intrinsic roles of each isoform; in parallel, jnk1 -/- and jnk2 -/- C57BL/6 mice bearing JNK-KO tumors were used to distinguish host-dependent effects. Immune cell tumor infiltration was quantified by flow cytometry. Cytokines were profiled by cytokine arrays and validated by ELISA and qPCR. Results: JNK-IN-8 (5 μm) suppressed PyMT-N cell growth by 85.1%, while KO of JNK1, JNK2, or both reduced colony formation by 55.3%, 67.7%, and 42.0%, respectively. Intratumoral JNK1 or JNK2 KO reduced tumor growth by 44.2% and 53.4%, respectively, and dual KO produced the most profound effect. These results highlight the strong tumor-intrinsic dependence of TNBC on both isoforms. Compared with Cas9 controls, JNK1-KO and JNK2-KO tumors exhibited increases in activated CD8⁺ T cells (83.4% and 53.3%, respectively) and reductions in Tregs (37.9% and 24.9%, respectively), indicating a shift toward an immunoactive TME. Furthermore, JNK1-KO and JNK2-KO tumors showed reduced TSLP expression and increased IGF-1 expression, suggesting JNK's regulation of these cytokines. In host-dependent studies using jnk1 -/- and jnk2 -/- C57BL/6 mice, loss of either isoform reduced tumor growth, though to a lesser degree than intratumoral KO. Host JNK1 deficiency increased M1 macrophages and NK cells, while JNK2 deficiency increased CD8⁺ T cells and NK cells; both showed reduced Tregs, indicating the induction of an immunoactive TME. Conclusion : Both intratumoral JNK1 and JNK2 are critical drivers of TNBC growth and TME immunosuppression, and their loss reprograms the TME toward robust antitumor immunity, positioning JNK signaling as a compelling therapeutic target in TNBC.