PKC-ζ is in charge of Extracellular Vesicles-associated TGF-β1 secretion for radiotherapy resistance in breast cancer

Background: Radiotherapy is widely applied to breast cancer treatment, while the resistance to radiotherapy is inevitable. TGF-β1 has been considered to be an endogenous factor for RT resistance. As a large portion of TGF-β1 is secreted associating with the extracellular vesicles (TGF-β1 EV ), to understand the regulation mechanisms and the immunosuppressive function of TGF-β1 EV in the radiated tumors will pay a way for overcoming radiotherapy resistance. Methods: Based on TCGA database of 958 samples, the intratumoral TGF-β1 expression and the Tregs production were compared between patients received and unreceived radiotherapy. The superoxide-Zinc-PKC-ζ-TGF-β1 EV pathway in breast cancer cells were identified through sequence alignments of different PKC isoforms, speculation and experimental confirmation. A series of functional and molecular studies were performed by quantitative real-time PCR, western blot and flow cytometry analysis. Mice survival and tumor growth was determined using observation of the animals and tumor growth measurement. Student’s t test or type II ANOVA with correction was used for comparisons of groups. Results: The radiotherapy brought the increased intratumoral TGF-β1 expression and the Tregs production in the breast cancer tissues, and patients with higher TGF-β1 expression are associated with a poor survival. The increased intratumoral TGF-β mainly exists in the extracellular vesicles associated form both in the murine breast cancer model and in the human lung cancer tissues. Furthermore, radiation induced the more secretion of the TGF-β1 EV and the higher percentage of Tregs by promoting protein kinase C zeta (PKC-ζ) expression and phosphorylation. Importantly, we found that naringenin rather than 1D11 significantly improve radiotherapy efficacy with low side effects. The underlying mechanism of naringenin is via downregulating radiation activated the superoxide-Zinc-PKC-ζ-TGF-β1 EV pathway, which is distinct from TGF-β1 neutralizing antibody 1D11. Conclusions: The superoxide-zinc-PKC-ζ-TGF-β1 EV release pathway was elucidated to induce the accumulation of Tregs resulting in radiotherapy resistance in the TME. Therefore, targeting PKC-ζ to counteract TGF-β1 EV function could represent a novel strategy to overcome radioresistance in breast cancer treatment or other cancers. Trial registration Using of tissues from patients with malignant Non-Small Cell Lung Cancer (NSCLC) was approved by the ethics committees at Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China (NCC2022C-702, from June 8 th , 2022).