RESUMO
Background: The aim of this study was to investigate the effect of DNA methylation of Fork Head Box O3 (FOXO3a) on the process of epithelial-mesenchymal transition (EMT) in non-small cell lung cancer (NSCLC). Methods: The expressions of FOXO3a, DNA methyltransferase 1 (DNMT1), METTL3, and EMT-related proteins (E-cadherin and N-cadherin) were measured. The influence of 5-Aza-dC and DNMT1 on the methylation level in the promoter region of FOXO3a was examined through the application of methylation-specific PCR (MSP). Chromatin immunoprecipitation (ChIP) was employed to detect binding between DNMT1 and the FOXO3a promoter. Methylated RNA immunoprecipitation (MeRIP) was utilized to evaluate the level of DNMT1 N6-methyladenosine (m6A) methylation. The assessment of cell viability and invasion abilities of A549 cells was performed using Cell Counting Kit-8 (CCK-8) and Transwell assays, respectively. NSCLC xenograft mouse models were established by subcutaneously injected treated A549 cells into nude mice. Results: The expression levels of DNMT1 and DNA methylation level FOXO3a were found to be significantly increased, whereas FOXO3a expression was considerably decreased in NSCLC cell lines and NSCLC tumor tissues. Both 5-Aza-dC treatment and DNMT1 knockdown resulted in the down-regulation of DNA methylation levels of FOXO3a while simultaneously up-regulating the expression of FOXO3a. A ChIP assay demonstrated that DNMT1 has the ability to bind to the promoter region of FOXO3a. Furthermore, the knockdown of DNMT1 promoted E-cadherin expression, but inhibited expression of N-cadherin, cell viability, and invasion ability. However, the knockdown of FOXO3a hindered the effect of DNMT1 knockdown on EMT, cell viability, and invasion ability of A549 cells. This was evidenced by decreased E-cadherin expression and increased N-cadherin expression, as well as increased cell viability and invasion ability. Increased expression of DNMT1 resulted from m6A methylation of DNMT1, which was mediated by METTL3. Overexpression of DNMT1 decreased of E-cadherin expression while increased N-cadherin expression, cell viability, and invasion ability in METTL3-shRNA treated A549 cells. In xenograft mouse models, DNMT1 knockdown significantly reduced tumor volumes and tumor weight. DNMT1 knockdown upregulated the expression of FOXO3a and E-cadherin, while downregulated N-cadherin expression in vivo. Conclusion: METTL3-mediated m6A methylation of DNMT1 up-regulates FOXO3a promoter methylation, thereby promoting the progression of NSCLC.
RESUMO
BACKGROUND: Patients with locally advanced (stage III) non-small cell lung cancer (NSCLC) demonstrate broad anatomic heterogeneity with modest survival benefits. Immune checkpoint inhibitors (ICIs) have shown survival benefit in metastatic NSCLC. We conducted this study to evaluate the efficacy and safety of neoadjuvant nivolumab in combination with chemotherapy in the treatment of this population. METHODS: We retrospectively evaluated patients with locally advanced NSCLC receiving neoadjuvant nivolumab and chemotherapy (paclitaxel with carboplatin) followed by surgery at our institution from January 2019 to January 2020. RESULTS: A total of 46 eligible patients, 26 males, and 20 females were diagnosed with NSCLC in a stage IIIA (30 cases) and IIIB (16 cases) to receive neoadjuvant treatment. The treatment was well tolerated with just 7 (15.2%) typical immune-related adverse events (hyperthyroidism, hyperglycemia, and rash) recorded. A total of 45 patients underwent surgical resection, and 43 (95.6%) of them achieved a R0 resection. No major surgical complications were observed. There was a complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD) in 2 (4.3%), 26 (56.5%), 17 (37.0%), 6 (26.1%), and 1 (2.2%) patients. Eight patients resulted in a major pathological response (MPR) (17.4%) and 24 patients had a pathological complete response (pCR) (52.2%). At the time of data cutoff (June 1, 2021), the median follow-up period was 15.5 months (IQR 3.9-29) and 27 (60%) of 45 patients who had tumor resection were progression free. At 24 months, progression-free survival was 45.8% and overall survival was 79.9%. CONCLUSION: Nivolumab plus paclitaxel and carboplatin could be a potential neoadjuvant regimen for patients with locally advanced NSCLC, rendering a potentially lethal disease to one that is curable.
