Decoding the Link Between XPC and Lung Cancer Susceptibility: A Study of Cigarette Smoke-Induced DNA Damage in the Setting of XPC Deficiency

Abstract

Rationale: Despite the known mutagenicity of cigarette smoke, only 10-15% of smokers will develop lung cancer in their lifetimes. What determines a smoker’s susceptibility to lung cancer is poorly understood. We identify the nucleotide excision repair (NER) protein Xero­derma pigmentosum Complementation Group C (XPC) as a tumor suppressor that may con­tribute to lung tumorigenesis when mutated and combined with cigarette smoke (CS). Micronu­clei, which are chromosome fragments and/or lagging chromosomes separated from the main nucleus, occur in many cancers, and indicate ge­nomic instability. We hypothesize that cigarette smoke and XPC knockdown will cause genomic instability that will activate the DNA Damage Response (DDR) and increase the frequency of micronuclei and nuclear aberrancies.

Methods: A human bronchial epithelial cell line (Beas-2B), and two lung adenocarcinoma cell lines (H1299 and A549) with stable XPC len­tiviral knock-down (shXPC) or control shRNA (shCtrl) were treated with cigarette smoke ex­tract (CSE) or air control (AC). DNA Damage Response (DDR) proteins were analyzed via immunoblot (Western). Micronuclei and nucle­ar aberrancies were quantified through the cyto­kinesis-block micronucleus assay (CBMT) using immunofluorescence microscopy (DAPI).

Results: Both CSE and XPC knockdown inde­pendently amplify expression of DDR proteins in H1299, Beas-2B, and A549 cell lines. Nucle­ar aberrancies increased significantly (p<0.05) with CSE in all three cell lines. Micronucleus frequency increased significantly with CSE in H1299 and Beas-2B cells (p<0.05) and with XPC knockdown in Beas-2B cells compared to shCtrl (p<0.001). XPC deficiency was associated with cell-type specific alterations in activation of the DDR.

Conclusions: We identified a previously unchar­acterized role of XPC deficiency in augmenting cigarette-smoke induced chromosomal breaks manifesting as micronuclei, particularly in non-cancerous Beas-2B cells. These findings of­fer insight into tumorigenesis in cigarette smok­ing and shed light on mechanisms of continued DNA damage in cancer cells. Future research should clarify the mechanisms of micronucleus formation in human translational specimens and pinpoint additional functions of XPC beyond