Abstract
INTRODUCTION: The tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), has been extensively studied for its carcinogenic role in lung cancer. However, the involvement of N6-methyladenosine (m6A) methylation in NNK-induced carcinogenesis remains unclear. OBJECTIVES: The aim of this study is to investigatewhether NNK promotes lung carcinogenesis by dysregulating m6A methylation, focusing on the demethylase ALKBH5 and its target COL5A1. METHODS: Levels of m6A were analyzed in NNK-exposed (100 mg/L, 24 h) Beas-2B bronchial epithelial cells, NNK-induced malignant-transformed Beas-2B (2B-NNK) cells, and paired lung tumor tissues. Methylated RNA immunoprecipitation sequencing, RNA sequencing, and bioinformatics identified COL5A1 as a key target. We performed proliferation, apoptosis, migration and invasion experiments involving COL5A1 and ALKBH5 knockdown and COL5A1 stability experiments. We then overexpressed COL5A1 and knocked down ALKBH5 for phenotypic rescue experiments. RESULTS: Levels of m6A were significantly reduced in NNK-exposed Beas-2B cells, 2B-NNK cells, and human lung tumor tissues. COL5A1 exhibited hypomethylation and overexpression in 2B-NNK cells. COL5A1 knockdown suppressed 2B-NNK cell proliferation, migration, and invasion, while increasing apoptosis. The m6A demethylase ALKBH5 was upregulated in 2B-NNK cells. Its knockdown elevated global m6A levels, reduced COL5A1 mRNA stability, and reversed malignant phenotypes. COL5A1 overexpression also enhanced the proliferation, migration, and invasion abilities of 2B-NNK cells, whereas concurrent downregulation of ALKBH5 expression resulted in the restoration of these enhancements. CONCLUSION: ALKBH5 drives NNK-induced lung cancer via m6A demethylation of COL5A1, promoting tumor progression. Targeting the ALKBH5-COL5A1 axis may offer therapeutic strategies against tobacco-related cancers.