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 Kras in the Nestin cell lineage is sufficient for the initiation of premalignant pancreatic intraepithelial neoplasia lesions in mice. We have reported that Nestin was expressed in 30% of human pancreatic ductal adenocarcinoma (PDAC) cases, and Nestin expression in PDAC positively correlated with nerve involvement and invasion of the peripancreatic tissue margin. Therefore, we hypothesized that Nestin may play an important role in the migration and invasive potential of PDAC cells. In this study, we used a silencing strategy to clarify the roles of Nestin in human pancreatic cancer cells. Methods: An expression vector carrying a short hairpin RNA (shRNA) targeting Nestin was stably transfected into PANC-1 and PK-45H human pancreatic cancer cells, which express high levels of Nestin. Changes in the morphology and alignment of actin filaments were analyzed using phase-contrast images and immunocytochemistry. Effects of decreased expression of Nestin on cell growth, migration in scratch and Boyden chamber assays, invasion through matrigel, and cell adhesion on extracellular matrices were examined. Differences in mRNA levels of selected signaling molecules were examined by PCR arrays. Results: Targeting Nestin with shRNA caused a marked decrease in Nestin mRNA and protein levels. Nestin shRNA-transfected cells (Nes-sh cells) exhibited a sheet-like appearance with tight cell-cell adhesion, and increased expression of filamentous (F)-actin by comparison with sham-transfected cells, whereas anchorage-dependent and -independent cell growth and cell-extracellular matrix adhesion of Nes-sh cells did not differ from those of sham cells. By contrast, migration and invasion of Nes-sh cells were markedly attenuated. To clarify the mechanisms underlying this observation, we analyzed differences in selected signaling molecules by PCR arrays that could determine the expression of mRNA closely related to tumor metastasis. The gene expressing the greatest value in Nes-sh cells was E-cadherin. Real-time PCR and western blotting confirmed that E-cadherin expression was increased in Nes-sh cells by comparison with sham cells. Conclusion: Nestin participates in the regulation of pancreatic cancer cell migration and invasion, in part, by altering F-actin and E-cadherin expression. These observations suggest that Nestin may modulate the migration of Nestin-positive progenitor cells during pancreatic development, and may serve as a novel target for suppression of invasion and metastasis in pancreatic cancer.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 408.
- ©2010 American Association for Cancer Research
Volume 70, Issue 8 Supplement
