Abstract LB-183: Suppression of colon carcinogenesis by targeting Notch signaling.

Abstract

Abstract Background and aims: Recent studies have shown that aberrant Notch signaling contributes to the pathogenesis of colorectal cancer (CRC). Notch signaling can be abrogated in cells by treatment with γ-secretase inhibitors (GSI) that suppress Notch signaling via inhibition of Notch cleavage. This effect is associated with suppression of cell proliferation in several human cancer cell lines. However, the potential therapeutic benefits of Notch pathway inhibitors, including GSIs, on colon carcinogenesis are still unresolved. In the present study, the effects of GSI treatment and its underlying mechanisms in colon carcinogenesis were investigated both in vitro and in vivo. We also evaluated the expression level of Krüppel-like factor 4 (KLF4), a downstream repression target of Notch signaling, in human polyps. Methods: Human colon cancer cell lines HCT116 (Wt and p21−/−) and SW480 were treated with 0-100 μM GSI, N-[N-3,5-Difluorophenacetyl]-L-alanyl-S-phenylglycine Methyl Ester (DAPM), for either 48 or 72 hours to evaluate the effects of DAPM on cell proliferation and protein expression by the MTT assay and Western blotting analysis, respectively. For the mouse study, male A/J mice were injected with azoxymethane (AOM, 10 mg/kg, bw) once a week for 6 weeks. Nine weeks after the last AOM injection, the presence of tumors in the distal colon was confirmed by colonoscopic imaging. Mice were then treated with 1 mg/kg bw of DAPM or vehicle control by ip injection every other day for a total of 4 weeks. Tumor frequency and size was determined 14 weeks after the last injection of AOM. In addition, human specimens were obtained from eighteen patients undergoing routine screening colonoscopy at the University of Connecticut Health Center and screened for the presence of KLF4 and p21 expression. Results: DAPM suppressed cell proliferation and induced the expression of KLF4 and p21 in human colon cancer cells. Interestingly, p21−/− HCT116 cells were largely resistant to the suppressive effects of DAPM on cell proliferation compared with the parental cells, indicating a key role for p21 activity in the response to DAPM. The frequency of both large (≥ 4 mm) and small (< 1 mm) colon tumors was significantly reduced by DAPM treatment (reduced by 60% and 33%, respectively) in AOM-treated mice. Furthermore, colon tumors in the DAPM-treated mice displayed induction of KLF4 and p21 expression accompanied by reduced Ki-67 staining compared to controls. Notably, in human colon tumor biopsies, KLF4 and p21 expression were present within serrated hyperplastic polyps, but the levels of both proteins were markedly reduced within tubular adenomas. Conclusion: Our results suggest that inhibition of Notch signaling by DAPM provides a potential chemopreventive strategy for patients with tubular adenomas, in part through activation of the KLF4-p21 axis. Citation Format: Shingo Miyamoto, Daniel W. Rosenberg. Suppression of colon carcinogenesis by targeting Notch signaling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-183. doi:10.1158/1538-7445.AM2013-LB-183