Long Intergenic Non-Coding RNAs in the Malignant Progression of Barrett's Esophagus
Incidence of esophageal adenocarcinoma (EAC) has increased at an alarming rate (>500%) in the last few decades, far exceeding any other cancer type in the United States. The prognosis for EAC patients remains poor with very limited treatment options. Although Barrett’s esophagus (BE), a columnar metaplasia of the distal-esophagus epithelium, is the only known precursor of EAC, the vast majority of BE patients however do not develop dysplasia or cancer; consequently, the factors driving progression from BE to EAC remain elusive. Our long-term objective is to elucidate the mechanisms underlying EAC progression, such that reliable biomarkers and targeted therapies can be developed for effective management of this deadly disease. Recently, using innovative RNA sequencing in BE-associated lesions, we identified two large intergenic non-coding RNAs (lincRNAs) showing marked and selective inductions in ~50% of EAC lesions. Both lincRNAs exhibited nuclear localization, and preliminary functional assessments strongly suggested these lincRNAs to play pro-tumorigenic roles during EAC progression.
Our study thus provides the first global analysis of lincRNAs in this disease, identifying two novel lincRNAs with potential oncogenic roles in esophageal carcinogenesis. Accordingly, the specific goals of the current proposal are: (AIM 1) To elucidate the function of candidate EAC-associated lincRNAs. We will generate CRISPR/Cas9-based inducible lincRNA-knockout EAC cell line models to comprehensively characterize the phenotypic effects of candidate lincRNAs using both in vitro, and in vivo xenograft experimental systems; (AIM 2) To dissect the molecular regulatory networks upstream and downstream of EAC-associated lincRNAs. We will evaluate for potential genomic/epigenomic mechanisms driving lincRNA expression in EACs; using the EAC cell line models, we will perform global gene expression microarray profiling to delineate the genes/pathways modulated by the lincRNAs; and we will use ChIRP-seq/-MS approaches to map the genomic occupancy and to identify potential gene targets and protein partners of candidate lincRNAs, thus establishing a regulatory-roadmap of candidate lincRNAs; (AIM 3) To determine the timing and stage-associated deregulations in candidate lincRNAs during EAC progression. Our preliminary findings showed both lincRNAs being induced in high-grade dysplasia (HGD), a histopathologic surrogate for EAC risk. Accordingly, we will validate and ascertain the frequency of lincRNA deregulations in these overtly pre-malignant phases of disease progression.
Additionally, we will test: whether non-dysplastic BE lesions, within close proximity to EAC, show induction of these candidate lincRNAs; and whether candidate lincRNAs show induction early-on in non-dysplastic BE/low-grade dysplasia (LGD) mucosa, derived from high-risk patients who developed cancer during follow-up. Success in these studies will uncover molecular mechanisms contributing to EAC progression, enable development of evidence-based molecular biomarkers for early cancer detection and surveillance, and open new avenues for targeted therapies in this increasingly fatal cancer.
Project 3 Key Personnel
- Project 3 PI: Kishore Guda, D.V.M., Ph.D.
- Project 3 Co-PI: Sanford Markowitz, M.D., Ph.D.
- Project 3 Co-Investigator: Amitabh Chak, M.D.
- Project 3 Co-Investigator: Joseph E. Willis, M.D.
- Project 3 Co-Investigator: Ahmad Khalil, Ph.D.
- Project 3 Co-Investigator: Vinay Varadan, Ph.D.