Malignant Non-small Cell Lung Cancer Research Articles

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

BackgroundRadiotherapy 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-β1EV), particularly in radiated tumors. Thus, the understanding of the regulation mechanisms and the immunosuppressive functions of TGF-β1EV will pave a way for overcoming the radiotherapy resistance in cancer treatment.MethodsThe superoxide-Zinc-PKC-ζ-TGF-β1EV 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.ResultsThe 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-β1EV 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-β1EV pathway.ConclusionsThe superoxide-zinc-PKC-ζ-TGF-β1EV release pathway was elucidated to induce the accumulation of Tregs, resulting in radiotherapy resistance in the TME. Therefore, targeting PKC-ζ to counteract TGF-β1EV 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).

P3.01-064 The Overexpression and Cleavage of SASH1 by Caspase-3 Stimulates Cell Death in Lung Cancer Cells: Topic: Apoptosis in Lung Cancer

SASH1 (SAM and SH3 domain-containing protein 1) is a recently identified gene with tumor suppressor properties and has a role in induction of apoptosis. Previous work has shown that 90 % of lung cancer cell lines have a decrease in SASH1 mRNA levels (Zeller et al., 2003), however little characterization of SASH1 function in lung cancer has been undertaken. We evaluated SASH1 expression in transformed normal and malignant non-small cell lung cancer cell lines. We also utilized cell based assays to study the effects of altered SASH1 levels on cell survival and proliferation. Identification of a novel SASH1 targeting drug was performed through connectivity mapping. SASH1 protein expression was down regulated in two of the five lung cancer cell lines compared to immortalized normal bronchial epithelial cells. Prognoscan assessment identified decreased SASH1 mRNA expression reduced patient survival. The depletion of SASH1 in lung cells resulted in a significant increase in cellular proliferation in cancer lung cells. Connectivity mapping predicted the drug Chloropyramine would lead to an increase in SASH1 expression. We demonstrated that Chloropyramine upregulates SASH1 in malignant cell lines. In keeping with this we have demonstrated the Chloropyramine inhibited lung cancer proliferation in vitro. We also explored the role of SASH1 in apoptosis. Following ultraviolet light exposure SASH1 is cleaved by Caspase-3. The C-terminal fragment of SASH1 then translocates from the cytoplasm to the nucleus where it associates with chromatin. The overexpression of wild type SASH1 or cleaved SASH1 amino acids 231-1247 leads to an increase in apoptosis, however loss of the SASH1 cleavage site and/or nuclear translocation prevents this initiation of apoptosis. Mechanistically SASH1 cleavage is required for the translocation of the transcription factor NF-κB to the nucleus. The use of the NF-κB inhibitor DHMEQ demonstrated that the effect of SASH1 on apoptosis was dependent on NF-κB, indicating a co-dependence between SASH1 and NF-κB for this process. We have shown that SASH1 contributes to apoptosis via a NF-κB-dependent mechanism. Agents that upregulate SASH1, such as chloropyramine or SASH1 gene therapy, are potential novel approaches to the management of NSCLC in the future.