Abstract 618: Anti-tumor effects of Notch pathway activation in Gastrointestinal Stromal Tumors cells

Abstract

Abstract Gastrointestinal Stromal Tumors (GISTs) are the most common sarcomas of the GI tract and harbor a gain-of-function mutation of the KIT receptor tyrosine kinase. The introduction of imatinib, a specific inhibitor of KIT, has significantly extended survival of many GIST patients. Unfortunately, patients inevitably develop resistance, prompting interest in understanding GIST biology and developing new therapeutic options. Notch signaling may act as an oncogene or a tumor suppressor depending cellular context. In neuroendocrine tumors Notch signaling acts constantly as a tumor suppressor. Additionally, histone deacetylase inhibitors (HDACi) were recently shown to induce Notch signaling in neuroendocrine neoplasms. Given that GIST is of neuroectodermal origin, we hypothesized that inhibition of growth in GIST cells by HDAC inhibitors is mediated by activation of Notch signaling. To characterize Notch signaling in GIST, we assessed the expression of Notch-1, and its effector human hairy enhancer of split-1 (HES1) in human GIST cell lines (GIST-T1 and GIST 882) by quantitative RT-PCR. To determine the activity of Notch signaling in GIST, we assayed the effect of Notch activation in GIST cells by transduction with retroviral vectors to overexpress active forms of Notch1 and HES1 in GIST cells. The proportion of GFP-positive cells in unselected culture was assessed to determine the transgene's effect on cell growth. To study the effect of HDAC inhibition, we quantified viability, caspase activity, cell cycle analysis and expression of Notch pathway genes after HDAC inhibition with suberoylanilide hydroxamic acid (SAHA). Notch signaling genes (Notch1, HES1) were weakly expressed at the basal level in cells compared to other sarcomas. Notch signaling with a constitutively active, truncated form of the receptor (ICN-1) and HES1 potently induced growth arrest in GIST cell lines. The proportion of ICN-1 GFP+ cells dramatically declined after transduction compared to GFP- cells, (decrease of 69% at day 7 and 78% at day 9 after transduction). Vector empty-transduced GFP+ cells gave no change in the proportion of positive cells. SAHA caused dose dependent decrease in viability, activation of caspase 3 and 7, increase of sub-G1 population (6 fold increase at 1µM, p<0.01), as well as upregulation of Notch-1 expression (5 fold at 1µM, p<0.01). Retroviral silencing of downstream targets of Notch (dnHes-1 and dnMastermind [dnMAM]) partially rescued GIST-T1 cells from the inhibitory effects of SAHA (61% v 105%, p<0.05 for dnHes-1 and 61 v 87%, p<0.01 for dnMAM after 2 days of treatment with SAHA 0.6 µM). These results suggest that Notch signaling is a tumor suppressor in GIST cells and the activation of Notch signaling by HDAC inhibitors may represent a new strategy for treating GIST patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 618. doi:10.1158/1538-7445.AM2011-618