Chronic exposure to chewing tobacco induces metabolic reprogramming and cancer stem cell-like properties in esophageal epithelial cells

Tobacco in its smoke and smokeless form are major risk factors for esophageal squamous cell carcinoma (ESCC). However, molecular alterations associated with smokeless tobacco exposure are poorly understood. In the Indian subcontinent, tobacco is predominantly consumed in chewing form. An understanding of molecular alterations associated with chewing tobacco exposure is vital for identifying molecular markers and potential targets. We developed an in vitro cellular model by exposing non-transformed esophageal epithelial cells to chewing tobacco over an eight-month period. Chronic exposure to chewing tobacco led to increase in cell proliferation, invasive ability and anchorage independent growth, indicating cell transformation. Molecular alterations associated with chewing tobacco exposure were characterized by carrying out exome sequencing and quantitative proteomic profiling of parental cells and chewing tobacco exposed cells. Quantitative proteomic analysis revealed increased expression of cancer stem cell markers in tobacco treated cells. In addition, tobacco exposed cells showed the Oxidative Phosphorylation (OXPHOS) phenotype with decreased expression of enzymes associated with glycolytic pathway and increased expression of a large number of mitochondrial proteins involved in electron transport chain as well as enzymes of the tricarboxylic acid (TCA) cycle. Electron micrographs revealed increase in number and size of mitochondria. Based on these observations, we proposethat chronic exposure of esophageal epithelial cells to tobacco leads to cancer stem cell-like phenotype. These cells show the characteristic OXPHOS phenotype, which can be potentially targeted as a therapeutic strategy.