Abstract 3341: Molecular mechanisms of hedgehog (HH) signaling in human colon carcinoma (cc)

Abstract

Abstract HH signaling is essential for normal embryonic development and tissue patterning, and absent in most adult tissues. Aberrant HH signaling is implicated in many human cancers. The classical HH signaling cascade is initiated by ligation of secretory HH molecules to their receptor, Patched, thereby releasing the inhibition on Smoothened (Smo), which then causes the activation of Gli1 and Gli2. The active Gli molecules transcriptionally modulate HH target gene expression. Non-classical pathways, including Ras/Raf/MEK/Erk and PI3K-Akt, can also activate Gli molecules in lymphomas. We have observed that inhibition of PI3K (using LY294002) or ERK (with U0126) decrease Gli1-luciferase activity (by 20% and 50%, respectively) as well as Gli1 mRNA expression in the human cc cell line, HT29, indicating the existence of a Smo-independent HH signaling axis. Inhibition of classical HH signaling using Cyclopamine, a natural inhibitor of Smo, or CUR61414, a synthetic Smo antagonist, demonstrated activity in mouse models of human cancers (basal cell carcinoma [BCC]), but with absence of activity in clinical trials. In the current study 1) the cytotoxicity of HH signaling inhibition by Cyclopamine or GANT61 (a synthetic small molecule Gli antagonist), and 2) the molecular mechanism(s) downstream of HH signaling inhibition leading to cytotoxicity, are being elucidated in human cc cell lines. Data demonstrate that GANT61 (IC50: 20-30 μM) elicits greater cytotoxicity in human cc than Cyclopamine (Annexin V/PI staining; clonogenic survival). Furthermore in HT29 cells, GANT61 increases Fas expression (a known regulator of apoptosis in cc cells), concomitantly with decreased PDGFRα expression (directly regulated by Gli1), which also regulates Fas. We previously determined that interferon-gamma (IFN-γ) promotes Fas-induced apoptosis via Fas upregulation in cc. Thus, co-treatment of HT29 with non-cytotoxic concentrations of GANT61 (10μM) + IFN-γ (100IU/ml) was synergistic in the induction of cell death, increasing both Fas receptor and Fas ligand expression at the transcriptional level. Immunoprecipitation of the Fas receptor complex demonstrated recruitment of increased FADD and decreased c-FLIP when GANT61 and IFN-γ were combined, confirming the importance of Fas in the cytotoxic response downstream of HH signaling inhibition. Data suggest in cc 1) Gli1 is regulated by both canonical HH and non-canonical PI3K and ERK pathways, 2) the importance of Gli activation downstream and independent of Smo activation as a therapeutic target, 3) activated Gli represses Fas expression via PDGFRα, and 4) Fas plays a critical role downstream of Gli1-targeted HH signaling inhibition. Supported by NCI awards CA32613 and CA108929 to JAH. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3341.