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. 2016 Aug 30;7(35):57066-57076.
doi: 10.18632/oncotarget.10974.

Twist1-induced epithelial-mesenchymal transition according to microsatellite instability status in colon cancer cells

Bo Young Oh  1 , So-Young Kim  2 , Yeo Song Lee  2 , Hye Kyung Hong  2 , Tae Won Kim  3 , Seok Hyung Kim  4 , Woo Yong Lee  1   3 , Yong Beom Cho  1   3   5
Affiliations

Twist1-induced epithelial-mesenchymal transition according to microsatellite instability status in colon cancer cells

Bo Young Oh et al. Oncotarget. .

Abstract

Colorectal cancer (CRC) with microsatellite instability (MSI) may exhibit impaired epithelial-mesenchymal transition (EMT), but little is known about the underlying mechanisms of this phenomenon. In this study, we investigated the role of Twist1 and its downstream signaling cascades in EMT induction according to MSI status. To investigate the effects of Twist1 on EMT induction according to MSI status, MSS LS513 and MSI LoVo colon cancer cell lines, which overexpress human Twist1, were generated. Twist1-induced EMT and its downstream signaling pathways were evaluated via in vitro and in vivo experiments. We found that Twist1 induced EMT markers and stem cell-like characteristics via AKT signaling pathways. Twist1 induced activation of AKT and suppression of glycogen synthase kinase (GSK)-3β, which resulted in the activation of β-catenin, increasing CD44 expression. In addition, Twist1 activated the AKT-induced NF-κB pathway, increasing CD44 and CD166 expression. Activation of both the AKT/GSK-3β/β-catenin and AKT/NF-κB pathways occurred in MSS LS513 cells, while only the AKT/GSK-3β/β-catenin pathway was activated in MSI LoVo cells. In conclusion, Twist1 induces stem cell-like characteristics in colon cancer cell lines related to EMT via AKT signaling pathways, and those pathways depend on MSI status.

Keywords: Twist1; colorectal cancer; epithelial to mesenchymal transition; microsatellite instability.

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Conflict of interest statement

Conflicts of Interest: None declared.

Figures

Figure 1
Figure 1. Twist1 induced EMT in MSS LS513 and MSI LoVo colon cancer cell lines
EMT induction was more prominent in MSS LS513 compared to MSI LoVo cells. A. Cellular morphologic changes depending on Twist1 overexpression are shown at 40x magnification. Scale bar: 200 μm. B. Expression of E-cadherin, β-catenin, and vimentin depending on Twist1 overexpression was analyzed by western blot. β-actin was used as a loading control. C. Transcriptional levels of E-cadherin, β-catenin, and vimentin depending on Twist1 overexpression were analyzed by real-time PCR. D. Cell migration depending on Twist1 overexpression was assessed by transwell migration chamber assay. E. Cell invasiveness depending on Twist1 overexpression was assessed by transwell migration chamber assay. Columns = mean ± SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 2
Figure 2. Twist1 activated the AKT signaling pathway in MSS LS513 and MSI LoVo colon cancer cell lines
The AKT/GSK-3β/β-catenin pathway was activated in MSS LS513 and MSI LoVo cells, and the AKT/NF-κB pathway was activated in MSS LS513 cells. A. Expression of molecules involved in the AKT/GSK-3β/β-catenin pathway and AKT/NF-κB pathway depending on Twist1 overexpression was analyzed by Western blot. B. Subcellular localization of β-catenin and NF-κB depending on Twist1 overexpression was analyzed in each cell line. C. Transcriptional levels of c-Myc, a target of β-catenin, depending on Twist1 overexpression were analyzed via real-time PCR. D. Inhibition of NF-κB by quinacrine suppressed invasiveness of Twist1-overexpressing MSS LS513 cells. Representative images of invading cells are shown (upper panel), and the mean fluorescence intensities (MFIs) of invaded areas under the various conditions are presented in bar graphs (lower panel). Columns = mean ± SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 3
Figure 3. Twist1 enhanced colony formation in MSS LS513 cells compared to in MSS LoVo cells
A. The ability of cells to self-renew was assessed via colony formation. The white rectangle in the spherical formation image denotes a region shown at 20x magnification. Scale bar: 200 μm. B. Cell growth was quantified based on the number and size of colonies. Columns = mean ± SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 4
Figure 4. Twist1 elevated the level of cancer stem cell markers in MSS LS513 and MSI LoVo colon cancer cell lines
Expression of CD44 was higher in MSS LS513 and MSI LoVo cells, and expression of CD166 was higher in MSS LS513 cells. A. Expression of CD44 and CD166 depending on Twist1 overexpression was analyzed by FACS. B. Expression of CD44 and CD166 depending on Twist1 overexpression was analyzed by immunofluorescent staining; original magnification, 400x. C. Transcriptional levels of CD44 and CD166 depending on Twist1 overexpression were analyzed by real-time PCR. D. Expression of CD44 variant isoforms (CD44v), CD44 standard isoforms (CD44s), and CD166 depending on Twist1 overexpression was analyzed via immunoblot. Columns = mean ± SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 5
Figure 5. Invasion assay was performed by adding neutralizing antibodies against CD44 and CD166 in MSS LS513 and MSI LoVo colon cancer cell lines
The inhibiting effects of neutralizing antibodies were marked in MSS LS513 cells, but were minimal in MSI LoVo cells. A. Representative images of invading cells are shown. B. The mean fluorescence intensities (MFIs) of invaded areas under the various conditions are presented. Columns = mean ± SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 6
Figure 6. Twist1-induced tumorigenesis in xenografts varied according to MSI status
Tumorigenesis was more prominent in Twist1-overexpressing MSS LS513 tumors compared to GFP-expressing tumors. In contrast, tumorigenesis was not increased in Twist1-overexpressing LoVo cells compared to GFP-expressing LoVo cells. Injected cell counts = 5 × 106/100 μl.
Figure 7
Figure 7. A proposed model for a Twist1-induced EMT signaling pathway according to MSI status is presented
In MSS LS513 cells, the AKT/GSK-3β/β-catenin pathway and AKT/NF-κB pathway activated. In contrast, in MSI LoVo cells, only the AKT/GSK-3β/β-catenin pathway activated.
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