Abstract 2457: Nestin regulates pancreatic cancer stem cell functions

Abstract

Abstract Background: Nestin, a class VI intermediate filament protein, was originally described as a neuronal stem cell/progenitor cell marker. Nestin expression has also been shown to be up-regulated in progenitor cells in muscle, testis, teeth and pancreas. In the pancreas, lineage-tracing experiments have indicated that exocrine cells are derived from Nestin-expressing progenitor cells. Moreover, activation of K-ras in the nestin cell lineage is sufficient for the initiation of premalignant pancreatic intraepithelial neoplasia lesions in mice. To examine cancer stem cell (CSC) functions, floating cultures in plates with a low-attachment surface have been widely employed. CSCs are reported to be able to grow in floating spheres in serum-free culture medium supplemented with epidermal growth factor (EGF) and/or fibroblast growth factor-2 (FGF-2), whereas non-CSCs, more differentiated non-CSCs cannot form spheres. In this study, we examined nestin expression in spheres and clarified whether nestin regulates sphere forming activity. Methods: Human pancreatic ductal adenocarcinoma (PDAC) cell lines, PANC-1, KLM-1, MIAPaCa-2 and PK-45H were used to perform sphere-forming assay in ultra-low attachment plates supplemented with EGF and FGF-2. Expression of CSC markers including CD133, CD44, CD24, epithelial specific antigen (ESA), and nestin in spheres were determined by real time RT-PCR and immunocytochemistry. Sphere-forming cells were dispersed with trypsin, and then cell growth and migration were assessed. Next, short hairpin RNA (shRNA) targeting nestin was stably transfected into PANC-1 and PK-45H, human PDAC cells, which express high levels of nestin, in order to assess the effects of decreased nestin expression on sphere-forming ability. Conversely, KLM-1 cells which express low level of nestin were stably transfected with a nestin expression vector and their sphere forming ability was then determined. Results: All the PDAC cell lines formed spheres, and all CSC markers examined were expressed in the spheres. The sphere cells showed low cell growth rates and increased cell migration as compared to non-sphere cells. Expression levels of nestin in the spheres of PDAC cells were higher than those of non-sphere cells. Nestin shRNA transfected PANC-1 and PK-45H cells showed a marked decrease in sphere number and diameter. By contrast, nestin-gene-transfected KLM-1 cells exhibited increased sphere formation. Conclusion: Nestin is a pancreatic CSC marker that promotes the sphere-forming ability of CSCs. Therefore, nestin may represent a novel therapeutic target for CSCs in PDAC. 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 2457. doi:10.1158/1538-7445.AM2011-2457