Abstract 1421: The stem cell marker nestin is a novel therapeutic target to suppress tumor growth and invasion of malignant melanoma
Authors: Akiyama Michiko , Kawahara Kiyoko , Matsuda Yoko , Suzuki Taeko , Takayama Ryoko , Yamamoto Tetsushi , Ishiwata Toshiyuki , Kawana Seiji , Naito Zenya 
Published: June 2012

Abstract
BACKGROUND: Nestin, a class VI intermediate filament protein, was first described as a neural stem cell/progenitor cell marker expressed during central nervous system development. Recently, nestin was detected in various neoplasms such as glioblastomas, gastrointestinal stromal tumors, prostate cancers, breast cancers, pancreatic cancers and malignant melanomas. An abundance of nestin has been identified in metastatic sites and the dermal parts of nodular melanomas, and the expression level of nestin has relevance to poor prognosis in malignant melanomas. These findings suggest that nestin plays important roles in the invasion of melanoma cells. Furthermore, recent studies have shown that nestin is one of the cancer stem cell markers in melanomas. Therefore, we hypothesized that nestin may be a novel therapeutic target for malignant melanomas. In this study, we used a silencing strategy to clarify the effectiveness of nestin-targeting therapy in malignant melanomas. METHODS: To determine the expression patterns of nestin, we performed immunohistochemical analysis of nestin in human melanoma tissues. Next, we stably knocked down nestin expression in A375 human malignant melanoma cells by short hairpin RNA (shRNA) transfection in vitro. We also transfected sham vectors to A375 cells as negative controls. Cell proliferation, invasion, morphology, and sphere-forming ability were compared with nestin-shRNA-transfected cells (Sh) and sham cells (Sc). RESULTS: Immunohistochemical staining demonstrated that nestin was strongly expressed in melanoma cells in the invasive front of human melanoma tissues. Reduced expression level of nestin mRNA and protein in Sh cells was confirmed by quantitative RT-PCR, western blot and immunocytochemical analyses. Sh and Sc cells did not show characteristic morphological differences; however, stress fiber formation of F-actin was markedly increased in Sh cells. Cell growth of Sh cells was slower than that of Sc cells at 48 hrs as determined by cell counts and MTT assay. On the cell invasion assay using a modified Boyden chamber coated with matrigel on the inner surface of the inserts, the invasion of Sh cells was significantly attenuated in comparison with that of Sc cells. Sphere-forming ability using ultra-low attachment plates supplemented with EGF and FGF-2 showed that Sh cells formed fewer spheres than Sc cells. CONCLUSION: Nestin is expressed in melanoma cells at the invasive front of melanoma tissues, and decreased expression level of nestin inhibits the growth and invasion of melanoma cells. Furthermore, nestin regulates the functions of cancer stem cells of malignant melanoma as determined by sphere-forming ability. Our findings imply that nestin is a promising novel therapeutic target in malignant melanomas.
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 1421. doi:10.1158/1538-7445.AM2011-1421