Lung cancer is the third most common type of cancer and the leading cause of cancer death, in both men and women, and prognosis for lung carcinoma remains poor due to late diagnosis. While lung squamous cell carcinoma (LUSC) makes up 20-30% of all lung cancer cases, identification of genetic signatures and successful targeted therapies remain limited. An ongoing effort is being made to create an in vitro system for modeling the early stages of lung squamous carcinoma and premalignancy, which will ultimately serve as a model for drug discovery. A previous effort performed whole exome and targeted DNA sequencing to reveal the somatic mutations in endobronchial biopsies that harbored lung squamous premalignant histology. EP300 was identified as a candidate gene which may act as a driver for carcinogenesis, but remains understudied when compared to prominent oncogenic driver genes such TP53, NOTCH1, or NFE2L2 in LUSC. The p300 protein is a histone acetyltransferase that regulates gene expression by means of chromatin remodeling and has been implicated in various diseases, including cancer. My objective as part of my thesis was to first generate stable EP300 knockout (KO) clones from the NL20 bronchial epithelial cell line utilizing the CRISPR/Cas gene editing system. Using the NL20 clones and EP300 KO clones in the HBEC-3KT cell line generated in a previous effort, I then validated the knockouts at the DNA, RNA, and protein levels. Literature review was also conducted to identify possible cellular pathways that EP300 participates in and validate its role in those pathways by observing changes in downstream protein targets. Finally, I generated RNA sequencing data from the functionally validated clones to identify differentially expressed genes and cellular pathways perturbed by EP300 knockout. Through these efforts, I developed sets of gene signatures unique to each cell line and found that EP300 is associated with bronchial carcinogenesis progression and likely functions as an oncogene in LUSC.
|14 June 2023
|Mazzilli, Sarah A., Beane-Ebel, Jennifer E.
|Attribution 4.0 International, http://creativecommons.org/licenses/by/4.0/
Page generated in 0.403 seconds