Abstract 3077: Krüppel-like factor 5 and its connection to Sonic Hedgehog signaling in Barrett's esophagus and esophageal adenocarcinoma

Abstract

Abstract Aims: We sought to determine whether KLF5 was involved, at either phenotypic or functional levels, in the genesis and progression of Barrett's esophagus (BE) to esophageal adenocarcinoma (EAC), and whether this involvement was related to sonic hedgehog (SHH) signaling. In human adults, KLF5 is expressed in the less differentiated cells of the intestinal crypts, where its activity promotes cell proliferation, contributes to intestinal epithelial homeostasis, and is necessary for villus formation. KLF5 has been found to be highly active in a number of other cancer types, including gastric cancer; moreover, the known role of KLF5 in intestinal epithelial differentiation may contribute to metaplasia and dysplasia in BE and EAC development. We also sought to ascertain whether KLF5 activity was connected to the sonic hedgehog (SHH) pathway, since this pathway is already known to be highly active in BE and EAC. Methods and results: Quantitative reverse transcriptase PCR (qRT-PCR) performed on RNA obtained from BE cell lines (GhTRT and QhTRT) and EAC cell lines (SKGT4 and OE33) revealed that KLF5 is underexpressed in BE cell lines and overexpressed in EAC cell lines relative to normal primary squamous esophageal epithelial cells (HEEpic). Moreover, qRT-PCR performed on clinical BE and EAC tissue specimens showed the same pattern, with downregulation in BE tissues and upregulation in EAC tissues (relative to matched normal esophageal control tissues). Next, we determined KLF5 functional effects in EAC in vitro. The EAC cell line SKGT4 was transfected with KLF5 siRNA, and WST-1 cell proliferation assays revealed significantly lower cell proliferation at 72 hours (p<0.00005) and 96 hours (p<0.05) after transfection, relative to mock-transfected SKGT4. We then assayed KLF5 downstream effects on esophageal adenocarcinogenesis as well as SHH signaling in vitro. qRT-PCR performed on KLF5-silenced SKGT4 RNA revealed significantly diminished expression (relative to mock-transfected SKGT4) of the following known EAC-related genes: CDX1 (p<0.005), VIL1 (p<0.05), MUC2 (p<0.005), and MUC5ac (p<0.0005); moreover, GLI1, a known SHH pathway target gene, was significantly downregulated by KLF5 inhibition (p<0.005), while PTCH1, a known SHH pathway-inhibitory gene, was significantly upregulated (p<0.05). Conclusion: These tissue phenotypic and in vitro functional data identify KLF5 as a potential oncogene in esophageal adenocarcinogenesis. KLF5 is a potential therapeutic and/or chemopreventive target that can be evaluated in future studies of EAC or BE, such as with metformin, which is known to inhibit KLF5 activity. We also identify a potential new connection between KLF5 and SHH pathway activity in the development of EAC. This potential connection deserves further exploration in other malignancies, as well as in physiologic functions such as human development or adult stem cell maintenance. Citation Format: Christopher K. Ng, Yulan Cheng, John M. Abraham, Stephen J. Meltzer. Krüppel-like factor 5 and its connection to Sonic Hedgehog signaling in Barrett's esophagus and esophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3077.