PKC-ζ mediated reduction of the extracellular vesicles-associated TGF-β1 overcomes radiotherapy resistance in breast cancer

Background Radiotherapy is widely applied in breast cancer treatment, while radiotherapy resistance is inevitable. TGF-β1 has been considered to be an endogenous factor for the development of radiotherapy resistance. As a large portion of TGF-β1 is secreted in an extracellular vesicles-associated form (TGF-β1 EV ), particularly in radiated tumors. Thus, the understanding of the regulation mechanisms and the immunosuppressive functions of TGF-β1 EV will pave a way for overcoming the radiotherapy resistance in cancer treatment. Methods The superoxide-Zinc-PKC-ζ-TGF-β1 EV pathway in breast cancer cells was 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 were recorded. Student’s t test or two-way ANOVA with correction was used for comparisons of groups. Results The radiotherapy resulted in an increased expression of the intratumoral TGF-β1 and an enhanced infiltration of the Tregs in the breast cancer tissues. The intratumoral TGF-β1 was found mainly in the extracellular vesicles associated form both in the murine breast cancer model and in the human lung cancer tissues. Furthermore, radiation induced more TGF-β1 EV secretion and higher percentage of Tregs by promoting the expression and phosphorylation of protein kinase C zeta (PKC-ζ). Importantly, we found that naringenin rather than 1D11 significantly improved radiotherapy efficacy with less side effects. Distinct from TGF-β1 neutralizing antibody 1D11, the mechanism of naringenin was to downregulate the radiation-activated superoxide-Zinc-PKC-ζ-TGF-β1 EV pathway. 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 radiotherapy resistance in the treatment of breast cancer or other cancers. Trial registration : The using of patient tissues 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 8th, 2022).