Snail contributes to the maintenance of stem cell-like phenotype cells in human pancreatic cancer

Abstract

Snail, a potent repressor of E-cadherin expression, plays a key role in epithelial-to-mesenchymal transition (EMT) in epithelial cancer. Recently, EMT and stemness programs are found linked together. In the current study, the expression of Snail and its contribution to cancer stem cell (CSC) marker expression, invasiveness, self-renewal, clonogenicity, and tumorigenicity of pancreatic cancer cells were studied. Our results showed that Snail was highly expressed in CSC(high) cell line Panc-1. Stable, short hairpin RNA (shRNA)-mediated Snail knockdown decreased invasion in Panc-1 cells, in line with increased E-cadherin expression and its translocation from the nucleus to the membrane. Snail silencing in Panc-1 also inhibited CSC marker ALDH expression, together with decreased sphere and colony forming capacity, which was highly consistent with the expression of stem cell associated transcription factors like Sox2 and Oct4. In mouse xenograft models, knockdown of Snail led to a reduced number of tumor-bearing mice and a reduced average size of tumors, which had a stronger membrane staining of E-cadherin and lighter staining of Oct4. Collectively, these findings implicate Snail is required for the maintenance of stem cell-like phenotype in pancreatic cancer, and inhibition of Snail could be an efficient strategy to treat pancreatic cancer by targeting CSCs.

Figure 1. Differences of epithelial-mesenchymal features and CSC properties in Panc-1 and BxPC-3 cells.

A. Morphology of Panc-1 and BxPC-3 cells and their spheres. Note that Panc-1 cells have more spindle-shaped mesenchymal populations and can form more and larger spheres. ** P<0.01 compared with BxPC-3. B. ALDH activity in Panc-1 and BxPC-3 cells. Dot plots of cells analyzed by flow cytometry for ALDH activity. Cells were treated with Aldefluor substrate in the presence or absence of ALDH inhibitor DEAB. After treatment, the samples were analyzed by flow cytometry for the presence of ALDH^high cells. The values presented are the averages of three independent experiments. C. Real-time RT-PCR quantifing Snail, Slug, Twist1, ZEB1, and ZEB2 mRNA expression in Panc-1 and BxPC-3 cells. Bar graphs show the ratio of the expression level in Panc-1 cells to that in BxPC-3 cells. ** P<0.01.

Figure 2. Changes of epithelial-mesenchymal makers after Snail silencing in Panc-1 cells.

A. Snail mRNA expression of stable shSnail-expressing (S1, S2) and negative control shRNA-expressing (NC) Panc-1 clones. Values are the averages and standard deviations of triplicate measurements. **P<0.01 compared with NC. B. Western blot showing epithelial-mesenchymal markers Snail, Slug, Twist1, ZEB1, ZEB2, E-cadherin and vimentin in Panc-1 cells transfected with lentivirus-mediated negative control shRNA (Panc-1/NC) or shSnail (Panc-1/shSnail). GAPDH was used as loading control. C. Quantification of protein levels of Snail, Slug, Twist1, ZEB1, ZEB2, E-cadherin and vimentin in Panc-1/NC and Panc-1/shSnail cells. * P<0.05, **P<0.01.

Figure 3. Changes of epithelial-mesenchymal features after Snail silencing in Panc-1 cells.

A. Morphological alterations of Panc-1 cells after Snail silencing indicate a change in the cellular growth pattern from a mesenchymal towards an epithelial phenotype. B. Immunofluorescence staining for the expression and cellular localization of E-cadherin in stable clones of Panc-1/NC and Panc-1/shSnail. Nuclear DNA was detected by DAPI staining. Stable Snail knockdown leads to an increased expression of E-cadherin and its translocation from the nucleus to the membrane. C. Snail silencing inhibits Panc-1 invasiveness in in vitro Matrigel invasion assays. **P<0.01 compared with Panc-1/NC. Data shown here are the mean ± SD of three experiments.

Figure 4. Snail is crucial for properties attributed to cancer stem cells.

A. Snail silencing decreases ALDH^high population in Panc-1 cells. Dot plots of cells analyzed by flow cytometry for ALDH activity. The values presented are the averages of three independent experiments. B. Snail silencing significantly inhibits the ability of sphere formation with serial passaging and the capability of the clonogenicity in Panc-1 cells. Representative pictures of colony are shown above the column diagram. Similar experiments were repeated three times. ** P<0.01, compared with Panc-1/NC. C. Western blot analysis of cell extracts from Panc-1/NC and Panc-1/shSnail cells for Bmi1, Nanog, Sox2, and Oct4 expression. GAPDH was used as a loading control. D. Quantification of protein levels of Bmi1, Nanog, Sox2, and Oct4 in Panc-1/NC and Panc-1/shSnail cells. * P<0.05, **P<0.01 compared with Panc-1/NC.

Figure 5. Validation of the role of Snail in tumorigenicity in mouse.

A. Graphical representation of growth rates of subcutaneous xenograft tumors using cells of Panc-1/NC and Panc-1/shSnail. 2×10⁵ of each cell was transplanted in eight mice per group. All mice in the Panc-1/NC group, while only 2 mice in the Panc-1/shSnail group had tumor formation. B. Expression of Snail, E-cadherin, and Oct4 in xenograft tumors. Representative examples of Snail, E-cadherin, and Oct4 expression determined by immunohistochemistry. Strongly positive Snail expression is present in the nucleus and cytoplasm of Panc-1/NC tumors (a) but not in Panc-1/shSnail tumors (b). Expression of E-cadherin is weak and heterogeneous in the Panc-1/NC tumors (c), but more intensive in the membrane of Panc-1/shSnail tumors (d). Lower level of Oct4 expression is present in the Panc-1/shSnail tumors (f), as compared to that of Panc-1/NC tumors (e). Scale bars: 10 µm.