PDF file - 4209K, Supplementary Figure S3 The CAM invasion assay was performed with GFP (green) expressing UM-SCC-1-shC and -shTTP cells placed onto the upper CAM of an 11-day-old chick embryo for two days. Tissue sections were prepared and specimens were stained with Collagen IV (red) and DAPI (blue). Images were taken on Olympus BX-51 fluorescent microscope and merged. Results are representative of 3 independent experiments. White arrows point to invasive islands.
ARTICLE ABSTRACTPurpose: Invasion is the critical step in progression of a precancerous lesion to squamous cell carcinoma of the head and neck (HNSCC). Invasion is regulated by multiple proinflammatory mediators. Tristetraprolin (TTP) is an mRNA-degrading protein that regulates multiple proinflammatory mediators. TTP may serve as an excellent treatment target. Rap1 is a ras-like oncoprotein that induces critical signaling pathways. In this study, the role of rap1 in TTP-mediated invasion was investigated.Experimental Design: Using complementary approaches, we modulated TTP and altered expression of interleukin (IL)-6 and matrix metalloproteinase (MMP) 2/9, which were quantified by ELISA and zymogram. Invasion was evaluated in vitro using the oral-cancer-equivalent (OCE) three-dimensional model and in vivo in the chick chorioallantoic membrane (CAM). The role of rap1 and p38 were established using knockdown strategies.Results: Downregulation of TTP significantly increased invasion via secretion of MMP9/2 and IL-6. In the novel OCE and CAM invasion models of HNSCC, cells with downregulated TTP destroyed the basement membrane to invade the underlying connective tissue. Rap1 induces p38 mitogen-activated protein kinase (p38)-mediated inactivation of TTP. Inactive TTP enhances transcript stability via binding to the 3′-untranslated region (UTR). High IL-6 and MMP9 are prognostic for poor clinical outcomes in patients with HNSCC.Conclusions: Targeting the rap1-p38-TTP cascade is an attractive novel treatment strategy in HNSCC to concurrently suppress multiple mediators of invasion. Clin Cancer Res; 19(5); 1169–79. ©2012 AACR.