Gastroesophageal reflux disease and Barrett's esophagus, common disorders in Veteran patients, are the major risk factors for esophageal adenocarcinoma, a lethal tumor whose frequency has increased more than six-fold in the past few decades. The development of therapies to prevent or cure this lethal tumor has been hampered by limited understanding of the molecular events underlying the neoplastic progression of Barrett's metaplasia. During carcinogenesis in Barrett's esophagus, genetic alterations accumulate that enable the cells to provide their own growth signals, avoid growth inhibitory signals, resist apoptosis, replicate without limit, synthesize new blood vessels, invade adjacent organs and metastasize. These physiologic "hallmarks" of cancer, which were described by Hanahan and Weinberg in 2000, can be acquired by normal cells through disruptions in surprisingly few key growth regulatory pathways. Recently, cancer-related inflammation has been proposed as another physiologic hallmark of cancer that can be established through two pathways: 1) an extrinsic pathway in which disorders such as reflux esophagitis cause tissue inflammation that contributes to carcinogenesis, and 2) an intrinsic pathway in which the precancerous cells acquire genetic abnormalities that produce an inflammatory tumor microenvironment. Both pathways can converge on certain key downstream targets such as IL-6, a potent inflammatory cytokine that is known to promote cellular growth, and STAT3, a transcription factor that has a key role in IL-6 signal transduction. Chronic esophageal inflammation due to reflux esophagitis is widely regarded as the extrinsic pathway for promoting carcinogenesis in Barrett's esophagus, but little attention has been directed at the intrinsic inflammatory pathway and the role of IL-6/STAT3 signaling in the malignant progression of Barrett's metaplasia. Studies on the early molecular events that transform Barrett's metaplasia into adenocarcinoma have been hampered by the lack of appropriate model systems. During our last cycle of Merit Review funding, we addressed this deficiency by establishing cultures of non- neoplastic Barrett's epithelial (BAR-T) cells that we immortalized through the forced expression of telomerase. Using a combination of p53 knockdown and forced expression of oncogenic H- RasG12V, we have induced the malignant transformation of those BAR-T cells and, in the process, established a series of Barrett's epithelial cell lines with well defined genetic alterations representing various stages in the transition from benign Barrett's metaplasia to cancer. Our preliminary data demonstrate that the transformed Barrett's cells express IL-6 mRNA and exhibit a significant increase in IL-6 protein secretion. Moreover, we have found that IL-6 signaling causes an increase in phospho-STAT3 levels in the transformed cells, and that activation of the IL-6/STAT3 pathway endows the cells with the ability to resist apoptosis and to secrete angiogenesis-promoting factors. We propose to use our unique series of Barrett's epithelial cell lines to explore the role of the IL-6/STAT3 pathway in driving the malignant transformation of Barrett's esophagus. We hypothesize that activation of the IL-6/STAT3 signaling pathway plays a key role in driving the neoplastic transformation of Barrett's metaplasia. Based on our preliminary data, the objectives of our study are to delineate the mechanism(s) whereby p53 inactivation and Ras activation result in activation of the IL-6/STAT3 signaling pathway in Barrett's epithelial cells, to determine the effects of activation and inactivation of the IL-6/STAT3 pathway on the level of neoplastic transformation in Barrett's cell lines, and to correlate p53 inactivation and Ras pathway activation with the activation of the IL-6/STAT3 pathway during the neoplastic progression of Barrett's esophagus in vivo using biopsy specimens from patients with non- dysplastic Barrett's esophagus, dysplastic Barrett's esophagus (low-grade and high-grade), and Barrett's adenocarcinoma.
The Potential Impact on Veterans Health Care is that our studies will identify key molecular pathways that can be targeted to prevent cancer from developing in our many Veteran patients who have Barrett's esophagus, and to treat those who already have cancers in Barrett's esophagus.