Rationale: We and others had shown that microRNA-1 (miR-1) is downregulated in the non-small cell lung cancer (NSCLC) tumor microenvironment and that a low mature miR-1 level in the tumor is a predictor of shorter overall survival in lung adenocarcinoma patients. Molecular alterations within the tumor microenvironment can induce cancerization and CS is the most prevalent inducer of NSCLC. In this study we evaluated the clinical associations of miR-1 in clinical samples and its significance as an indicator of cancerization. Methods: We assessed the clinical associations of miR-1 in the tumor (T), adjacent cancer-free (AT), and distant cancer-free (DT) lung tissues of 82 NSCLC patients which were obtained by bronchoscopy and surgery. Ex-vivo cultures of normal human lung tissues and cultured human bronchial epithelial cells, immortalized human endothelial cell were exposed to increasing percentage of CS extract in the growth media and harvested after 24 hours. Mature miR-1 levels and mRNA were measured by Taqman assay and quantitative real time PCR, respectively. Human lung epithelial and endothelial fractions were isolated by Magnetic-activated cell sorting (MACS) technique. For clinical data analysis we used SAS 9.4 proc correction for spearman rank test, proc logistic for regression model analysis and Kaplan-Meier analysis for survival studies. Results: In NSCLC patients, mature miR-1 levels in the tumor had an inverse correlation with the extent of CS exposure (pack-year of smoking). Also, miR-1 levels in AT and DT samples of current smokers were significantly lower than the former smokers, suggesting that CS has a suppressive effect on miR-1 levels. We asked whether CS exposure decreases mature miR-1 levels in the lung tissue. Treatment of ex vivo human lung sample with CS specifically decreased mature miR-1 levels without altering its precursors. This change was observed in both epithelial and endothelial cells isolated from the ex-vivo lungs, as well as cultured cells. Next, we asked whether miR-1 changes in the lungs of NSCLC patients has clinical significance. We found that miR-1 levels in the tumor, AT and DT tissues follows a cancerization gradient. MiR-1 levels were lowest and showed the least variability in the tumors and became higher and more widely distributed in the AT and DT samples. Moreover, mature miR-1 levels in the AT samples had a significant association with overall survival of the patient. Conclusion:Our observations show that CS specifically downregulates mature miR-1 in the NSCLC microenvironment, and together with our previous findings strongly suggests that miR-1 downregulation is an inducer of cancerization. Citation Format: Asawari Korde, Anuradha Ramaswamy, Mark S. Godfrey, Margaret A. Pisani, Jonathan T. Puchalski, Shervin S. Takyar. Cigarette smoke downregulates mature microRNA-1 in non-small cell cancer (NSCLC) tumor microenvironment [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO012.