Abstract 825: Id3: Tumor suppressor of squamous cell carcinoma.

Skin cancer, the most common type of cancer with more than 1 million cases diagnosed in the US every year, occurs at least once to about half of Americans 65 years or older. The three main types of skin cancer—Basal Cell Carcinoma (BCC), Squamous Cell Carcinoma (SCC), and melanoma have mortality rates that are estimated to be 0.1%, 4.3% and 10% respectively in 2011. The Inhibitor of DNA Binding/Differentiation 3 (Id3) protein, a helix-loop-helix (HLH), dominant negative transcription factor, is one of four Id proteins found in mammals (Id1-4). The Id protein family plays a role in many cellular processes, including differentiation, proliferation, apoptosis, and angiogenesis. Previous studies in the Rosenthal lab have shown that Id3 over-expression in immortalized human keratinocytes induces apoptosis in part by activation of a Bax-caspase-9-caspase-3-dependent mitochondrial pathway via upregulation of bax promoter activity. To further elucidate the roles of Id3 in apoptosis, proliferation, and tumorigenesis, our studies focused on effects of induced Id3 expression on tumor growth in vivo. To investigate the role of Id3 in SCC, a tetracycline (Tet)-regulated expression (T-REx™) system was utilized to induce Id3 expression in A431 cells, a human malignant cervical SCC cell line. Immunoblot and RT-PCR analysis of A431-TRex-Id3 cells verified Id3 expression in the presence of Tet. When Id3 was induced by Tet in these cells in vitro, caspase-3 and -9 were activated. Cells were stably transfected with GFP to monitor tumor growth over thirty days by Maestro® in vivo imaging. Effects of Id3 on tumorigenesis in vivo was examined by subcutaneous injection of these cells onto athymic nude mice, half of which were fed with a doxycycline (Dox; a tetracycline derivative)-containing feed, leading to induced Id3 expression in the tumor, and half with a regular diet. Subcutaneously-injected A431 cells formed significantly (50%) smaller tumors upon Id3 induction. Immunofluorescent staining of tumor sections from Dox-fed mice revealed a 1.5-fold increase in active caspase-3 but no significant change in Ki-67, a proliferation marker. To investigate the mechanism of tumor shrinkage caused by Id3, microarray analysis was performed, and four genes were found to be significantly up- or down-regulated by Id3. Id3 deletion mutants were further created to study the domain(s) responsible for the decrease in tumor size. Mutation analysis showed that Id3 induces Max dimerization protein 3 (MXD3) protein expression through its HLH domain. Other candidate genes remain to be tested. The role of Id3 in SCC cell killing by cisplatin, a commonly used chemotherapeutic drug for SCC, is also currently under investigation. Our results together indicate that Id3 is a potential apoptosis inducer and therefore a therapeutic target in treating SCC. Citation Format: You-Shin Chen, Cynthia Rosenthal, Dean Rosenthal. Id3: Tumor suppressor of squamous cell carcinoma. [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 825. doi:10.1158/1538-7445.AM2013-825