Lung cancer (LC) is the leading cause of cancer death in the US, claiming over 160,000 lives annually. Although CT screening has been shown to be efficacious in reducing mortality, the limited access to screening programs among high-risk individuals and the high number of false positives contribute to low survival rates and increased healthcare costs. As a result, there is an urgent need for preventative therapeutics and novel interception biomarkers that would enhance current methods for detection of early-stage LC. This thesis addresses this challenge by examining the hypothesis that transcriptomic changes preceding the onset of LC can be identified by studying bronchial premalignant lesions (PMLs) and the normal-appearing airway epithelial cells altered in their presence (i.e., the PML-associated airway field of injury). PMLs are the presumed precursors of lung squamous cell carcinoma (SCC) whose presence indicates an increased risk of developing SCC and other subtypes of LC. Here, I leverage high-throughput mRNA and miRNA sequencing data from bronchial brushings and lesion biopsies to develop biomarkers of PML presence and progression, and to understand regulatory mechanisms driving early carcinogenesis. First, I utilized mRNA sequencing data from normal-appearing airway brushings to build a biomarker predictive of PML presence. After verifying the power of the 200-gene biomarker to detect the presence of PMLs, I evaluated its capacity to predict PML progression and detect presence of LC (Aim 1). Next, I identified likely regulatory mechanisms associated with PML severity and progression, by evaluating miRNA expression and gene coexpression modules containing their targets in bronchial lesion biopsies (Aim2). Lastly, I investigated the preservation of the PML-associated miRNAs and gene modules in the airway field of injury, highlighting an emergent link between the airway field and the PMLs (Aim 3). Overall, this thesis suggests a multi-faceted utility of PML-associated genomic signatures as markers for stratification of high-risk smokers in chemoprevention trials, markers for early detection of lung cancer, and novel chemopreventive targets, and yields valuable insights into early lung carcinogenesis by characterizing mRNA and miRNA expression alterations that contribute to premalignant disease progression towards LC. / 2020-01-25

Squamous cell carcinoma of the bronchus is the second most common and fatal subtype of lung cancer. In the process of squamous carcinogenesis, the normal bronchial epithelium undergoes a series of histologic transformations known as the metaplasia-dysplasia-carcinoma sequence. These intermediate histologic patterns are called premalignant lesions, and occur prior to the development of cancer. Compared to early stage cancer, survival following resection of premalignant lesions approaches 100%, highlighting the promise of lung cancer interception. However, because of our lack of understanding of the molecular events during squamous carcinogenesis, we are currently unable to predict which lesions will progress to cancer, and we do not have molecular targets for noninvasive treatment. The work in this thesis seeks to improve our understanding of the changes associated with grades of premalignant histology and progression at the level of single cells. I analyzed single cell RNA sequencing data from a cohort of 41 lesions from 26 patients, encompassing the normal-appearing bronchus, premalignant lesions, and early stage carcinoma. I described histology-associated changes in basal cells. Basal cells from low grade lesions expressed genes related to the maintenance of the normal epithelium, while basal cells from high grade lesions expressed genes related to the cell cycle and detoxification of the airway from smoking toxicants. Secondly, I identified a high grade lesion undergoing the epithelial-to-mesenchymal transition. These cells transitioned from a high grade basal cell state, lost their expression of basal cell markers, and expressed canonical EMT genes, including SPARC, FN1, and MMP2. Finally, I identified shifts in T cell subtypes and widespread expression of exhaustion markers PD-1, CTLA4, LAG3, and TIGIT co-occurring with high grade basal cells. Secondly, I leveraged our single cell data to identify gene modules associated with histology and progression in bulk RNA sequencing data. I identified a module of genes expressed in B and dendritic cells involved in antigen presentation through the MHC II pathway whose expression was decreased in progressive lesions. I also identified a module of stromal-expressed genes that were less expressed in progressive lesions, which had previously been unidentified. Associations between module expression and progression were validated in a second data set. This work improves our understanding of the signaling and interactions between cell types associated with histology and progression of premalignant lesions. These findings may be used to improve our prognostication and treatment of premalignant lesions.