Multi-Omics Analysis to Characterize Cigarette Smoke Induced Molecular Alterations in Esophageal Cells.

Aafaque Ahmad Khan,Ravi Gupta,Malini Manoharan,Bipin Nair,Aditi Chatterjee,Krishna Patel,Anjali Kumari,David Sidransky,Arati Khanna-Gupta,Rekha V Kumar,Manjusha Biswas,Niraj Babu,Vishalakshi Nanjappa,Coral Karunakaran,Harsha Gowda,Saktivel Murugan,Rohit Gupta,Kiran K Mangalaparthi,Prashant Kumar,Shankargouda Patil,Hitendra S Solanki

doi:10.3389/fonc.2020.01666

Abstract

Though smoking remains one of the established risk factors of esophageal squamous cell carcinoma, there is limited data on molecular alterations associated with cigarette smoke exposure in esophageal cells. To investigate molecular alterations associated with chronic exposure to cigarette smoke, non-neoplastic human esophageal epithelial cells were treated with cigarette smoke condensate (CSC) for up to 8 months. Chronic treatment with CSC increased cell proliferation and invasive ability of non-neoplastic esophageal cells. Whole exome sequence analysis of CSC treated cells revealed several mutations and copy number variations. This included loss of high mobility group nucleosomal binding domain 2 (HMGN2) and a missense variant in mediator complex subunit 1 (MED1). Both these genes play an important role in DNA repair. Global proteomic and phosphoproteomic profiling of CSC treated cells lead to the identification of 38 differentially expressed and 171 differentially phosphorylated proteins. Bioinformatics analysis of differentially expressed proteins and phosphoproteins revealed that most of these proteins are associated with DNA damage response pathway. Proteomics data revealed decreased expression of HMGN2 and hypophosphorylation of MED1. Exogenous expression of HMGN2 and MED1 lead to decreased proliferative and invasive ability of smoke exposed cells. Immunohistochemical labeling of HMGN2 in primary ESCC tumor tissue sections (from smokers) showed no detectable expression while strong to moderate staining of HMGN2 was observed in normal esophageal tissues. Our data suggests that cigarette smoke perturbs expression of proteins associated with DNA damage response pathways which might play a vital role in development of ESCC.

Highlights

In United States, more than 50% of esophageal cancer deaths are attributed to smoking [1]
In vitro invasion assay using Matrigel showed that Het-1A cells showed an increase in their invasive ability after chronic treatment with cigarette smoke condensate (CSC) (Figure 1B)
Since chronic treatment with CSC resulted in increased proliferation and invasive ability of non-neoplastic esophageal cells, we examined the expression of Bcl-2 family proteins in cells treated with CSC for a period of 8 months (Het-1A-Smoke-8M)

Summary

Introduction

In United States, more than 50% of esophageal cancer deaths are attributed to smoking [1]. Studies have shown that carcinogens in cigarette smoke lead to p53 mutations, aberrant cell cycle regulation and are associated with DNA damage, which plays key role in development of cancer [2, 5, 6]. Persistent miscoding of adducts compromises DNA repair systems resulting in accumulation of mutations in the genome. Alterations at the genomic, transcriptomic and proteomic level has been reported in both smokers and non-smokers [13,14,15]. Despite these early studies, molecular alterations associated with cigarette smoke exposure that leads to malignancy are poorly understood

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Conclusion