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. 2014 Oct 7;21(1):97.
doi: 10.1186/s12929-014-0097-8.

Up-regulation of S100A16 expression promotes epithelial-mesenchymal transition via Notch1 pathway in breast cancer

Wenbin Zhou  1 , Hong Pan  2 , Tiansong Xia  3 , Jinqiu Xue  4 , Lin Cheng  5 , Ping Fan  6 , Yifen Zhang  7 , Weidong Zhu  8 , Yi Xue  9 , Xiaoan Liu  10 , Qiang Ding  11 , Yun Liu  12 , Shui Wang  13
Affiliations

Up-regulation of S100A16 expression promotes epithelial-mesenchymal transition via Notch1 pathway in breast cancer

Wenbin Zhou et al. J Biomed Sci. .

Abstract

Background: Our previous studies demonstrated that S100A16 promotes adipogenesis and is involved in weight gain attenuation induced by dietary calcium. Till now, the function of S100A16 in the breast cancer remains to be elucidated.

Results: In this study, we observed that S100A16 was expressed in higher levels in human breast cancer tissues compared with paired adjacent non-cancerous tissues. Further examination showed that overexpression of S100A16 in MCF-7 cells could increase cell proliferation and colony formation. One major mechanistic change was that S100A16 was able to up-regulate the transcription factors Notch1, ZEB1, and ZEB2, which had the capacities to directly repress the expression of epithelial markers E-cadherin and β-catenin but increase mesenchymal markers N-cadherin and vimentin, a characterized phenotype of epithelial-mensenchymal transition (EMT). In addition to display with morphologic change, migration and invasion were increased in S100A16 over-expressed MCF-7 cells. Importantly, knockdown of Notch1 by specific siRNA could reverse the EMT induced by S100A16 overexpression, which confirmed that Notch1 played a critical role in the process of EMT induced by S100A16.

Conclusions: All together, our data indicated that S100A16 had a potential function to regulate some embryonic transcription factors to promote EMT in breast cancer cells which may be an important target site for the therapy of breast cancer.

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Figures

Figure 1
Figure 1
S100A16 expression in tissues and cell lines. (A) qRT-PCR analysis of S100A16 expression in 20 pairs of breast cancer tissue and adjacent tissue. Of these 20 pairs of tissues, 14 showed significantly higher S100A16 mRNA expression in the cancer tissue compared with the adjacent tissue (P < 0.05). (B) A scatter plot showed the mean expression level of S100A16 mRNA in breast cancer tissue was significantly higher than that in adjacent non-cancerous tissue (*P < 0.05 by paired t-test). (C) Western blot analysis of S100A16 expression in eight breast cancer cell lines and a normal breast cell line MCF10A. It was expressed in MCF10A and five breast cancer lines including MCF-7. (D) Representative examples of immunostaining (×400). In non-cancerous tissue, few S100A16 immunostaining was observed, while the expression of S100A16 was significantly higher in cancerous tissue than non-cancerous tissue, especially in the invading front of cancerous tissue.
Figure 2
Figure 2
Up-regulation of S100A16 increased the capacities of proliferation, migration and invasion in MCF-7 cells. (A) S100A16 was transfected in MCF-7 cells. Western blot was used to measure S100A16 protein expression in control cells (MCF7-GFP) and S100A16 overexpression cells (MCF7-S100A16). (B) MTT assay showed that the cell proliferation rate was increased after S100A16 overexpression in MCF-7 cells (Bars, mean ± SD, *P < 0.05, **P < 0.01). (C) Colony formation assay confirmed that up-regulation of S100A16 markedly increased the number of cell colonies in MCF-7 cells (P < 0.05). (D, E) Transwell migration and invasion assays showed that up-regulation of S100A16 increased cell migration (D) and invasion (E) abilities compared with control cells (P < 0.05). Triplicate assays were used for each experiment (Magnification, 10×).
Figure 3
Figure 3
Overexpression of S100A16 promoted EMT in MCF-7 cells. (A) Compared with control cells (MCF7-GFP), MCF7-S100A16 cells showed spindle-like, fibroblastic morphology (10×). (B, C) qRT-PCR (B) and western blot (C) analyses showed that epithelial markers E-cadherin and β-Catenin were significantly reduced in mRNA and protein levels in MCF7-S100A16 cells compared with MCF7-GFP cells, and mesenchymal markers Vimentin and N-cadherin were significantly up-regulated in MCF7-S100A16 cells (Bars, mean ± SD, P < 0.05). (D, E) Immunofluorescence staining was used to examine the location of epithelial and mesenchymal markers. After fixation, the celluar location of E-cadherin (red), β-catenin (red), Vimentin (red) and N-cadherin (red) were analyzed by confocal microscopy. Cell nuclei were stained with DAPI (4′, 6-diamidino-2-phenylindole, blue). Immunofluorescence staining showed that both E-cadherin (red) and β-catenin (red) resided in the cell membrane and intensity of fluorescence were reduced in MCF7-S100A16 cells compared with MCF7-GFP cells (D), whereas the mesenchymal markers Vimentin (red) and N-cadherin (red) were increased by S100A16 (E).
Figure 4
Figure 4
Up-regulation of S100A16 increased Notch1, ZEB1 and ZEB 2 expression. (A, B) qRT-PCR (A) and western blot (B) analyses showed that mRNA and protein levels of Notch 1, ZEB1 and ZEB2 were significantly enhanced in MCF7-S100A16 cells compared with control cells (Bars, mean ± SD, P < 0.05). (C, D) Immunofluorescence staining was used to examine the location of two transcription factors ZEB1 and ZEB2. After fixation, the celluar location of ZEB1 (red) and ZEB2 (red) were analyzed by confocal microscopy. Cell nuclei were stained with DAPI (4′, 6-diamidino-2-phenylindole, blue). Immunofluorescence staining showed that two transcription factors ZEB1 (C) and ZEB2 (D) localized in the nucleus and the fluorescence signals were enhanced after S100A16 up-regulation.
Figure 5
Figure 5
Knockdown of Notch1 reversed EMT by decreasing ZEB1 and ZEB 2 expression. (A, B) Specific siRNA was used to knockdown Notch1 in MCF7-S100A16 cells. qRT-PCR (A) and western blot (B) analyses showed that Notch1 was successfully knockdown. ZEB1 and ZEB2 were significantly reduced in mRNA and protein levels after knockdown of Notch1. Consequently, epithelial markers E-cadherin and β-Catenin were significantly increased but mesenchymal markers Vimentin and N-cadherin were reduced. There was no variation in the expression of S100A16 influenced by Notch1 knockdown. (Bars, mean ± SD, P < 0.05). (C) MTT assay showed that knockdown of Notch1 decreased the proliferation of MCF7-S100A16 cells (Bars, mean ± SD, *P < 0.05, **P < 0.01). (D, E) Transwell migration and invasion assays showed knockdown of Notch1 decreased migration and invasion in MCF7-S100A16 cells (P < 0.05). Triplicate assays were used for each experiment (Magnification, 10×).
Figure 6
Figure 6
S100A16 activated Notch1 transcriptional activity. Dual luciferase reporter assay showed that transcriptional activities were increased in four transfected plasmids in S100A16-transduced MCF-7 cells (Bars, mean ± SD, P < 0.05). However, the region from −1496 to +76 had the strongest promoter activity. It seemed that the region from −1496 to −998 was the core part accounting for increasing promoter activity.
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