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Departments of Pathology and Integrative Oncological Pathology, Nippon Medical School, Tokyo, JapanDepartment of Dermatology, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan
Spatiotemporal expression pattern of neuroepithelial stem cell marker nestin suggests a role in dermal homeostasis, neovasculogenesis, and tumor stroma development: a study on embryonic and adult human skin.
Nestin expression has also been reported in various neoplasms. Nestin knockdown inhibits migration, invasion, and metastasis of pancreatic cancer cells (
), suggesting that the protein could be a novel therapeutic target for tumors. In melanoma, nestin overexpression has been observed in advanced stages of the disease (
). Therefore, in this study, we used a gene silencing strategy to investigate the potential effectiveness of a nestin-targeting therapy in malignant melanomas.
Nestin was found to be strongly expressed in A375 and MeWo melanoma cells, but weakly expressed in G361 cells (Supplementary Figure S1a, b, c online and Supplementary Material online). We first suppressed nestin expression in A375 cells using short hairpin RNA (shRNA) (
). Nestin expression was lower in cells transfected with nestin shRNA (Sh) than in cells transfected with a scrambled sequence shRNA (Sc) and nontreated (wild) cells (Supplementary Figure S1d and e online), confirming the efficiency of the knockdown. Control and nestin shRNA–transfected cells exhibited similar morphology (Supplementary Figure S2a online), whereas F-actin polymerization was increased in the cytoplasm of Sh cells compared with Sc and wild cells (Supplementary Figure S2c online, arrows), which was similar to that observed in pancreatic cancer cells (
). Super-high-resolution images clearly revealed the formation of F-actin fibers in Sh cells (Supplementary Figure S2d online, arrows). Nestin and F-actin colocalized to the periphery of Sc cells (Supplementary Figure S2d online, arrowheads), but not in Sh cells. The colocalization of nestin and F-actin observed in this study suggests that nestin regulates F-actin organization in melanoma cells in a manner similar to synemin, an intermediate filament protein, which was reported to modulate actin dynamics (
Intermediate filament protein synemin contributes to the migratory properties of astrocytoma cells by influencing the dynamics of the actin cytoskeleton.
The growth of Sh cells was lower than that of Sc cells, as indicated by manual counting of cell number (Figure 1a). A WST-8 assay with Sh cells confirmed these results (Figure 1b). Nestin has been reported to regulate cell proliferation through the mitogen-activated protein kinase (MAPK) signaling pathway (
); therefore, we analyzed alterations of MAPK using Phospho-MAPK Arrays. Phosphorylated AKT was the only protein affected by nestin silencing; no inhibitory effects were observed for ERK, p38, JNK, or CREB. Decreased phosphorylation of AKT in Sh cells was confirmed by western blotting (Figure 1c). This finding suggests that AKT activation is important for nestin function.
Figure 1Cell growth and motility in nestin shRNA or nestin siRNA–transfected A375 human melanoma cells. (a) Manual cell count growth assay and (b) WST-8 cell growth assay of wild-type (Wild), scrambled sequence RNA–transfected (Sc), and nestin shRNA–transfected (Sh) A375 cells. (c) Western blot of phosphorylated AKT, total AKT, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (d) Boyden chamber assay for cell migration, and (e) cell invasion through matrigel. Bar=100μm. (f) WST-8 cell growth assay of wild, negative control siRNA–transfected (siN), and nestin siRNA–transfected (siA) A375 cells. (g) Cell migration and (h) invasion in nestin siRNA–transfected A375 cells. *P<0.05 and **P<0.01 vs. wild, Sc, or siN cells.
The effect of nestin silencing on melanoma cell migration and invasion was next analyzed using a modified Boyden chamber assay. The migration and invasion of Sh cells into the matrigel layer was less than that of Sc cells (Figure 1d and e). Next, we analyzed the effect of nestin silencing using siRNA targeting nestin (siA; Supplementary Figure S1f online). Knockdown of nestin with siRNA in A375 cells also suppressed cell growth (Figure 1f), migration (Figure 1g), and invasion (Figure 1h).
We performed a sphere-formation assay to determine in vitro tumorigenicity of cells after treatment with nestin shRNA (
). The number of spheres in Sh cells was lower than that in Sc and wild cells (Figure 2a (arrows) and b). The spheres in A375 cells expressed nestin protein (Figure 2c, right panel; green fluorescence) and higher levels of nestin mRNA than non-sphere cells (Figure 2d).
Figure 2Sphere formation and in vivo analyses in nestin shRNA–transfected A375 cells. (a) Phase-contrast images of spheres. Bar=100μm. (b) Number of spheres per well. *P<0.05 vs. scrambled sequence RNA–transfected cells (Sc). (c) Phase-contrast (left, bar=10μm) and fluorescence images of nestin (right, bar=1μm) in A375 spheres. (d) The quantitative reverse transcription-PCR analysis of nestin mRNA in sphere-forming cells and cultured cells in plates (non-sphere). *P<0.05 vs. non-sphere cells. (e) Weight of subcutaneous tumors. *P<0.05 vs. Sc cells. (f) Liver metastases in mice injected with A375 cells through the tail vein. Macroscopic images (upper panels) and immunohistochemistry of HLA in the liver (lower panel). Upper panels, bar=1mm; lower panels, bar=100μm. (g) The percentage of the metastatic tumor area in the liver. *P<0.05 vs. wild cells. Sh, nestin shRNA–transfected cells.
In a xenotransplantation experiment using nude mice, subcutaneous tumors originating from Sh cells were smaller and weighed significantly less (Figure 2e) compared with those originating from Sc cells (P<0.05). Moreover, after tail vein injection of these cells into nude mice, liver metastases were observed in Sc mice, but not in Sh mice (Figure 2f, upper panels; arrows). Immunohistochemical staining of liver sections using an anti-HLA antibody showed that the liver area occupied by the tumor was less in the Sh group than in the Sc group (Figure 2f, lower panels, arrows; and Figure 2g).
We also examined the effects of reduced nestin expression on cell proliferation, migration, and invasion using nestin-targeting siRNAs (siA and siB) in melanoma cells that express different levels of nestin (Supplementary Figure S3a online). These siRNAs reduced the expression of nestin in MeWo cells, which strongly express nestin mRNA, but did not reduce the expression in G361 cells (Supplementary Figure S3a online). The effect of nestin siRNA on cell growth was more apparent in MeWo cells compared with the other cell types (Supplementary Figure S3b online). In addition, cell movement, migration, and invasion were significantly inhibited in MeWo cells (Supplementary Figure S3c and d online), but not in G361 cells (Supplementary Figure S3c and e online), after treatment with nestin siRNA. However, these findings do not eliminate the possibility that decreased proliferative abilities have an effect on metastasis and sphere formation of melanoma cells.
We next tested the effects of nestin siRNA on the cell behavior of non-tumorigenic cells. Human epidermal melanocytes (HEMn-LP) expressed low levels of nestin (Supplementary Figure S3a online). Moreover, nestin siRNA transfection of these cells did not decrease nestin mRNA levels and had no effect on cell growth and motility (Supplementary Figure S4 online).
Tumor stem cells that possess self-renewal ability and multilineage potential have been considered to have important roles in tumor development (
). Melanoma stem cells seem to possess a strong ability to efflux Hoechst33342 dye, which is a characteristic of stem cells, and these cells express nestin (
). We found that sphere-forming melanoma cells express high levels of nestin, and that knockdown of nestin suppresses the sphere-forming ability, suggesting a relationship between nestin expression and melanoma stem–like cell functions.
In conclusion, we have found that reducing the expression of nestin in melanoma cells decreases the cell growth, migration, invasion, and sphere-forming ability in vitro and tumor growth and metastasis in vivo, in part, through alteration of the expression pattern of F-actin and regulation of the MAPK pathway. Therefore, nestin may be a therapeutic candidate for treating malignant melanoma.
ACKNOWLEDGMENTS
We thank Masahito Hagio, Tetsushi Yamamoto, Taeko Suzuki, Yoko Kawamoto, Kiyoko Kawahara, and Yuji Yanagisawa (Departments of Pathology and Integrative Oncological Pathology) for technical assistance. This work was supported by Leave a Nest, a Grant-in-Aid for Scientific Research (MA), and a Grant-in-Aid for Young Scientific Research (A, No. 22689038 to YM).
Intermediate filament protein synemin contributes to the migratory properties of astrocytoma cells by influencing the dynamics of the actin cytoskeleton.
Spatiotemporal expression pattern of neuroepithelial stem cell marker nestin suggests a role in dermal homeostasis, neovasculogenesis, and tumor stroma development: a study on embryonic and adult human skin.
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