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. 2012 Aug;1(1):5-16.
doi: 10.1002/cam4.4. Epub 2012 Jun 8.

Overexpression of snail induces epithelial-mesenchymal transition and a cancer stem cell-like phenotype in human colorectal cancer cells

Fan Fan  1 , Shaija Samuel, Kurt W Evans, Jia Lu, Ling Xia, Yunfei Zhou, Eric Sceusi, Federico Tozzi, Xiang-Cang Ye, Sendurai A Mani, Lee M Ellis
Affiliations

Overexpression of snail induces epithelial-mesenchymal transition and a cancer stem cell-like phenotype in human colorectal cancer cells

Fan Fan et al. Cancer Med. 2012 Aug.

Abstract

Epithelial-mesenchymal transition (EMT) is a critical process providing tumor cells with the ability to migrate and escape from the primary tumor and metastasize to distant sites. Recently, EMT was shown to be associated with the cancer stem cell (CSC) phenotype in breast cancer. Snail is a transcription factor that mediates EMT in a number of tumor types, including colorectal cancer (CRC). Our study was done to determine the role of Snail in mediating EMT and CSC function in CRC. Human CRC specimens were stained for Snail expression, and human CRC cell lines were transduced with a retroviral Snail construct or vector control. Cell proliferation and chemosensitivity to oxaliplatin of the infected cells were determined by the MTT (colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Migration and invasion were determined in vitro using modified Boyden chamber assays. EMT and putative CSC markers were analyzed using Western blotting. Intravenous injection of tumor cells was done to evaluate their metastatic potential in mice. Snail was overexpressed in human CRC surgical specimens. This overexpression induced EMT and a CSC-like phenotype in human CRC cells and enhanced cell migration and invasion (P < 0.002 vs. control). Snail overexpression also led to an increase in metastasis formation in vivo (P < 0.002 vs. control). Furthermore, the Snail-overexpressing CRC cells were more chemoresistant to oxaliplatin than control cells. Increased Snail expression induces EMT and the CSC-like phenotype in CRC cells, which enhance cancer cell invasion and chemoresistance. Thus, Snail is a potential therapeutic target in metastatic CRC.

Keywords: Cancer stem cells; EMT; Snail; colorectal cancer; migration.

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Figures

Figure 1
Figure 1
Snail expression in human CRC surgical specimens and human CRC cell lines. (A) Snail protein expression was assessed in human primary and metastatic CRC specimens by immunohistochemical analysis. Representative images are shown. (B) Western blotting showed that all the studied CRC cell lines expressed Snail.
Figure 2
Figure 2
Snail overexpression in human CRC cell–lines increased cell migration and invasion. (A) Snail overexpression induced the expression of EMT markers in HT29 (left) and HCT116 (right) cells. (B) Snail overexpression led to EMT-like phenotypic changes in HT29 (left) and HCT116 (right) cells. (C) Using Boyden chamber migration assays, we found that both HT29/Snail (left) and HCT116/Snail (right) cells had increased number of migrating cells per HPF compared with control cells (*P < 0.001). (D) Using the modified Boyden chamber invasion assays, we found that HT29/Snail cells (left) had an increased number of invading cells per HPF compared with control cells (*P < 0.001). Similar results were seen in the other tested cell line, HCT116 (right, *P < 0.002).
Figure 3
Figure 3
CRC cells overexpressing Snail acquire the stem cell-like phenotype. (A) HT29 cells overexpressing Snail (left) had higher expression of CD133 and CD44 protein than control cells in Western blot analysis. HCT116/Snail (right) cells demonstrated similar results. (B and C) Flow cytometry analysis was used to confirm the results obtained by Western blotting. Overexpression of Snail in HT29 (left) and HCT116 (right) had higher CD133 and CD44 expression than their respective control (*P < 0.001). (D) HT29 cells (left) overexpressing Snail demonstrated an increase in sphere formation when compared to control cells (*P < 0.001). HCT116/Snail cells (right) also formed a significantly higher number of spheres per well than control cells (*P < 0.03).
Figure 4
Figure 4
CRC cells overexpressing Snail become chemoresistant. (A) MTT assays were used to evaluate the oxaliplatin sensitivity of the Snail-overexpressing cells. Both control and Snail-transfected cells were exposed to oxaliplatin for 72 h, and the IC50 was calculated. The IC50 of HT29/Snail (left) was more than a log-fold higher than that of control cells (*P < 0.001). HCT116/Snail (right) cells had similar results (*P < 0.001). (B) Transient transfection of CRC cells with a siRNA against Snail significantly decreased expression of Snail in both HT29 (left) and HCT116 (right) cells in Western blot analysis. (C) Cell viability was significantly inhibited in both the HT29/siSnail (left) and HCT116/siSnail (right) cell lines under oxaliplatin treatment when compared to the respective control cells (*P < 0.001). (D) The number of apoptotic cells following oxaliplatin treatment was significantly lower in HT29/Snail cells than in HT29/control cells (*P < 0.001). (E) Western blot analysis revealed decreased expression of proapoptotic markers (cleaved caspase 3 and cleaved PARP) in HT29/Snail cells following oxaliplatin treatment when compared with the control cells.
Figure 5
Figure 5
Overexpression of Snail in human CRC cells increased metastasis. (A) Luciferase-labeled HT29/control and HT29/Snail cells were injected into the tail veins of athymic nude mice. At the time of euthanasia (6 weeks and 5 days later), the group injected with HT29/Snail cells demonstrated significantly higher luciferase activity than did the control group (*P < 0.05). The bottom panel shows images of luciferase activity in all mice from each group. (B) When compared to control, overexpression of Snail in human CRC cells led to a higher average number of metastases (*P < 0.002) in mice.
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