Abstract 3879: Osteopontin (SPP1/OPN) alternative splice variants and metastatic potential in esophageal adenocarcinoma.

Abstract

Abstract Objective: The incidence of esophageal adenocarcinoma (EAC) has increased over 300% in the past decades in western countries. EAC is characterized by an early metastasis, with a 5-year survival rate of only 19%, making the identification of clinically-targetable proteins critical for improving patient outcome. Osteopontin (SPP1/OPN) is a secreted glycosylated phosphoprotein that is found overexpressed in many cancer types including EAC. OPN protein functions in cell adhesion and cell migration by regulating cell-matrix interactions and cellular signaling through binding with integrin and CD44 receptors. However, little is known about the role of OPN in EAC invasion and metastasis. Methods: We analyzed the OPN gene expression in a series of 46 esophageal samples including esophageal Barrett's, low-grade dysplasia, high-grade dysplasia and adenocarcinomas mRNA using Affymetrix U133A arrays. Real-time RT-PCR was performed to validate array results and examine a larger cohort of 130 EAC samples. Tissue microarrays (TMA) were made containing 64 EACs, eight lymph node metastases, eight dysplastic and 11 none dysplastic Barrett's mucosa sections from 59 patients. Primers flanking the potential gene alternative splice regions were designed and the in-frame transcript variants were sequenced. Expression of OPN in EAC cell lines was analyzed using RT-PCR and western blot analysis. Ectopic OPN expression of splice variants and OPN knockdown in EAC cell lines have been performed to examine its roles in tumor invasion and EMT assays. Results: OPN is highly overexpressed in EAC and overexpression of the gene is associated with poorer survival in patients. Three OPN splice variants were identified with their expression either proportionally increased or differentially expressed in the primary EAC and EAC-derived cells. Highly overexpressed OPN is a transcriptional event as no OPN gene amplification was found in the corresponding EACs. In addition, abundant OPN protein was found primarily in tumor cells in the tissue sections using immunohistochemical (IHC)-TMA assays. OPN variants differ in their N-terminal thrombin fragment with either exon 6 or 5 being absent in OPN-b or OPN-c, respectively. We found that EAC cells treated with the medium containing different OPN variants demonstrated distinctive morphological phenotypes. We further identified that OPN variants played different roles in tumor cell proliferation, epithelial-mesenchymal transition (EMT), and chemosensitivity. Conclusion: Upregulation of OPN is predominant event in primary EACs and associated with poorer survival in patients. The alternative splice variants demonstrate differential tumor invasion, EMT transition and chemosensitivity in EAC cells. Citation Format: Zhuwen Wang, Kimmy Leverenz, Dafydd G. Thomas, Amy L. Myers, Andrew C. Chang, Mark B. Orringer, Thomas J. Giordano, Jules Lin, David G. Beer, Lin Lin. Osteopontin (SPP1/OPN) alternative splice variants and metastatic potential in esophageal adenocarcinoma. [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 3879. doi:10.1158/1538-7445.AM2013-3879