Abstract 1918: Type 2 cGMP-dependent protein kinase activates antineoplastic signaling in the colon

Abstract

Abstract Signaling through cGMP has emerged as a potentially important suppressor of tumorigenesis in the colon but the mechanisms are poorly defined. Studies of guanylyl-cyclase (GCC) knockout mice indicate that cGMP signaling inhibits proliferation in the colon epithelium and that AKT may have an important role. Type-2 cGMP-dependent protein kinase (PKG2) is a central effector of cGMP in the colon epithelium and likely mediates the suppressive signaling. An important growth-inhibitory effect of AKT is the inactivation of forkhead transcription factors (FoxO). FoxO proteins are tumor suppressors that regulate growth-inhibitory and pro-apoptotic target gene expression but these proteins have not previously been examined in the intestinal tract. The present study sought to determine whether PKG2 can activate FoxO in colon cancer cells and in the colon epithelium. Activation of PKG2 in LS174T colon cancer cells inhibited cell proliferation but did not significantly affect apoptosis. Suggestive of a mechanism, it was found that PKG2 inhibited the activity of AKT but not ERK in these cells. Activation of PKG2 also significantly increased luciferase reporter activity driven by FoxO-responsive elements in LS174T cells. In support of FoxO activation, PKG2 also increased the expression of several FoxO target genes, including catalase, GADD45, and p27kip. Treatment of colon explants with 8Br-cGMP also activated FoxO target gene expression at both RNA and protein levels, and this was associated with reduced redox stress. The regulation of FoxO by cGMP in vivo most likely requires PKG2, because target gene expression was reduced in the colon mucosa of Prkg2-/- mice compared to wild type siblings. Since FoxO has not previously been studied in the colon, we first examined expression using IHC. These data showed that FoxO3a is the most prominent isoform, and was concentrated almost exclusively at the lumenal border. While FoxO1 showed negligible staining, FoxO4 was observed deeper in the crypt. In order to determine whether cGMP could activate FoxO in vivo, the PDE-5 inhibitor vardenafil (Levitra™) was used. Mice treated with vardenafil showed a dramatic mobilization of FoxO3a to the nucleus of lumenal epithelial cells. Analysis of the colon mucosa from treated animals showed increased levels of FoxO target genes and reduced redox stress. This was likely to be mediated by FoxO3a since vardenafil did not significantly affect the localization of FoxO1 or FoxO4. Taken together these data define a novel signaling pathway in the colon epithelium, where PKG2 leads to activation of FoxO. The established tumor suppressor role of FoxO proteins highlights the potential therapeutic role for cGMP signaling in colon cancer prevention. Citation Format: Allison Bridges, Bianca Islam, Sarah Sharman, Rui Wang, Subbaramiah Sridhar, Darren Douglas Browning. Type 2 cGMP-dependent protein kinase activates antineoplastic signaling in the colon. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1918. doi:10.1158/1538-7445.AM2015-1918