Norcantharidin Suppresses Colon Cancer Cell Epithelial-Mesenchymal Transition by Inhibiting the αvβ6-ERK-Ets1 Signaling Pathway

Norcantharidin (NCTD) is an efficacious anti-cancer drug that has been used in China for many years, but its underlying mechanism of action is still not fully understood. In the present study, we found that NCTD could induce morphological changes in colon cancer cells, causing a transition from a spindle-shaped morphology to a typical round or oval shape, which was indicative of a mesenchymal-epithelial transition (MET) process. Next, we investigated the mechanism by which NCTD induced the MET process. Using a transwell assay, we found that NCTD could suppress the migratory and invasive ability of colon cancer cells in a dose-dependent manner. Moreover, NCTD suppressed the expression of integrin αvβ6, MMP-3, and MMP-9 as well as the polymerization of F-actin, further supporting its suppressive effect on migratory and invasive ability. Furthermore, the expression of αvβ6, N-cadherin, vimentin and phosphorylated ERK was decreased, while the expression of E-cadherin was up-regulated. We verified that phosphorylated Ets1 was down-regulated substantially after treatment with NCTD. Taken together, our data demonstrated that NCTD could inhibit the EMT process of colon cancer cells by inhibiting the αvβ6-ERK-Ets1 signaling pathway. This study revealed part of the mechanism through which NCTD could reverse the EMT process in colon cancer.


Transwell assay for cell invasion. The invasive ability of colon cancer cells was analyzed in 24-well
Boyden chambers with polycarbonate membranes (8-μ m pore size) (Costar, Acton, USA). The membranes were pre-coated with 50 μ l of Matrigel (BD Biosciences, San Diego, USA) to form matrix barriers. Cells were resuspended in 100 μ l of serum-free medium at a concentration of 1 × 10 6 cells/ml and dispensed into the upper chamber; the lower compartments were filled with 600 μ l of medium with 10% FBS. After incubation, the cells remaining on the upper surface of the membrane were removed. Cells on the lower surface of the membrane were fixed and stained with crystal violet and counted under a light microscope at ×200 magnification.
Western blotting.  and WiDr cells were treated with various doses of NCTD for 24 h. Both adherent and floating cells were collected and frozen at − 80 °C. To detect the levels of potential signaling pathway proteins, the proteins were extracted from the cells. The protein concentration was measured using the BCA protein assay reagent, and equal amounts of protein (10 μ g) were loaded onto a 12.5% SDS-PAGE gel and electrophoresed under non-reducing conditions. After electrophoresis, the proteins were transferred to nitrocellulose membranes. Equivalent protein loading in each lane was reconfirmed by staining the nitrocellulose membrane with Ponceau using the 36-kDa GAPDH band as a reference marker. The membranes were then probed with primary polyclonal antibodies against the crucial signaling pathway factors followed by peroxidase-conjugated secondary antibodies. Proteins on the western blots were visualized using the enhanced chemiluminescence detection system according to the manufacturer's instructions. The optical density was analyzed with the Image J software.
Transcription factor activation profiling array. We used the transcription factor activation profiling plate array (synthesized by Signosis, Sunnyvale, CA) to analyze the activity of different transcription factors in HT-29 and WiDr colon cancer cells treated with NCTD for 24 h according to the manufacturer's instructions. First, nuclear proteins were extracted from HT-29 and WiDr cells using a Nuclear Extraction Kit (Signosis, Scientific RepoRts | 6:20500 | DOI: 10.1038/srep20500 Sunnyvale, CA). Then, 15 μ g of nuclear protein extract was mixed with biotin-labeled probes based on the consensus sequences of transcription factor DNA binding sites. Thus, transcription factor/probe complexes were formed. Next, we separated the transcription factor-DNA complexes from free probes, transferred the complexes to a PCR tube, and denatured the eluted probes. After separation of the bound probes from the complexes, they were hybridized with sequences complementary to the probes in the hybridization plate. Finally, the captured DNA probes were detected with streptavidin-HRP, and the signal intensity was measured based on relative light units (RLUs) using a microplate luminometer. Immunofluorescence (IF). Cells on cover slips were fixed, permeabilized and stained with tetramethylrhodamine (TRITC)-conjugated phalloidin (Sigma-Aldrich, St. Louis, USA) for 1 h. Then, 4′ ,6-diamidino-2-phenylindole (DAPI) (Beyotime) was used for nuclear staining. Cells were observed under a confocal laser microscope (Carl Zeiss, LSM780, Oberkochen, Germany). The "% decrease in F-actin" was calculated as follows: [(F-actin in untreated cells-F-actin in NCTD treated cells)/F-actin in untreated cells] ×100.

Statistical analysis.
The results were expressed as the means ± SD. Comparisons between the two groups were performed with Student's t test. Comparisons of multiple samples and rates were carried out using single-factor analysis of variance. All statistical analyses were performed using SPSS for Windows version 16.0. P values <0.05 were considered statistically significant.

NCTD treatment induced Morphological changes of colon cancer cells.
To investigate the effect of NCTD on cell morphology, we pre-tested a series of NCTD concentrations (from 0 to 60 μ mol/L) at different times. The results of these preliminary assays showed that the morphological changes associated with MET were most obvious when the cells were treated with 40 μ mol/L NCTD for 24 h. Therefore, we treated the HT-29 and WiDr colon cancer cells with 40 μ mol/L NCTD for 24 h. As shown in Fig. 1, HT-29 and WiDr colon cancer cells had a spindle-or fibroblast-like morphology prior to treatment with NCTD, but most cells exhibited a typical round or oval shape after treatment with NCTD. This morphology change was similar to what had been shown to occur during MET, which was the reverse process of EMT.

NCTD suppressed the expression of αvβ6, MMP-3, and MMP-9 and the polymerization of F-actin in colon cancers.
To evaluate the mechanism by which NCTD inhibited invasion, we treated colon cancer cells with different doses of NCTD for 24 h, and we subsequently performed RT-PCR and western blotting to detect mRNA and protein levels, respectively. The RT-PCR results showed that NCTD significantly reduced the mRNA levels of α vβ 6, MMP-3 and MMP-9. The inhibitory effect of NCTD was dose dependent (Fig. 3A). Moreover, western blotting showed that NCTD significantly reduced the expression of α vβ 6, MMP-3 and MMP-9 at the protein level, also in a dose-dependent manner (Fig. 3B). To further investigate whether NCTD could interfere with the activity of MMP-3 and MMP-9, an MMP activity assay was performed. When colon cancer cells were treated with 20, 40 and 60 μ mol/L NCTD for 24 h, the activity of MMP-3 and MMP-9 decreased substantially in a dose-dependent manner (Fig. 3C).
It is well known that cytoskeletal reorganization is essential in cell motility and tumor metastasis. Microfilaments (also referred to as actin filaments or F-actin) are helical polymers of globular actin subunits and are the thinnest fibers of the cytoskeleton. It is known that actin filaments provide mechanical support for the cell, determine the cell shape, enable cell movements, are associated with certain cell-cell junctions, and participate in cytoplasmic streaming and contraction of the cell during cytokinesis. Therefore, we investigated the organization of microfilaments in cells treated with NCTD. Immunofluorescence results (shown in Fig. 3D) revealed that NCTD could significantly induce the depolymerization of cellular microfilaments (F-actin), causing changes in cellular morphology and the redistribution of F-actin. However, there were no obvious changes in the untreated group. These results might account for the inhibitory effect of NCTD on colon cancer cell migration. The effect of NCTD on EMT-related proteins in colon cancer cells. After treatment with NCTD, colon cancer cells underwent a MET-like process, and we detected some typical MET-related molecules. We found that the expression level of the epithelial marker E-cadherin increased, while the mesenchymal markers N-cadherin and vimentin showed decreased expression (Shown in Fig. 4). All changes were dose dependent, indicating that NCTD could induce the MET process in colon cancer cells.
Classical signaling pathways involved in the cellular EMT process include the TGF-β , MAPK, Notch, Wnt, and AKT pathways. After colon cancer cells were treated with 20, 40 and 60 μ mol/L NCTD for 24 h, we detected the expression of proteins that were representative of the aforementioned classical signaling pathways. The results showed that only the protein level of p-ERK decreased, and no obvious variations in the other proteins were observed ( Fig. 4 and Supplemental Fig. 1).
NCTD induced the MET process in colon cancer cells via the αvβ6-ERK-Ets1 signaling pathway. We found that α vβ 6 promoted malignancy of colon cancer cells by binding with ERK and subsequently activating the downstream signaling pathways 5 . To investigate the mechanism by which NCTD mediated MET in colon cancer cells, we treated the colon cancer cells with peptide IK2, which specifically blocks the direct binding of β 6 and ERK. The cells were treated with different concentrations of NCTD, and a transwell assay was performed to detect their invasion capability. Interestingly, after treatment with IK2, NCTD had no effect on the invasion ability of colon cancer cells, indicating that NCTD suppressed the invasion ability of colon cancer cells by interfering with the direct connection between β 6 and ERK (Fig. 5A,B). Next, we investigated the downstream factors involved in this process. HT-29 colon cancer cells were pretreated with 40 μ mol/L NCTD for 24 h. Then, we examined downstream targets of ERK2 to observe variations in the activity of transcription factors (including c-Myc, NF-kappaB, CREB, Ets1, AP-1, Stat-3, RB, E2F-1, and Snail) in HT-29 and WiDr cells. The results showed that the activity of the transcription factor Ets1 was significantly inhibited in HT-29 cells treated with NCTD (P < 0.05), but it had no significant effect on the activity of other transcription factors (Fig. 5C). Similar results were found in WiDr cells (Fig. 5D).

Discussion
EMT is the process by which the cell phenotype changes from epithelial to mesenchymal. It is widely accepted that the EMT process plays an important role in regulating malignant epithelial tumor cell invasion, and it is the key step in distant tumor metastasis. During the process of EMT, the expression of epithelial protein markers, such as E-cadherin, decreases, and the expression of mesenchymal protein markers, such as vimentin and N-cadherin, is up-regulated 6 . Researchers have previously confirmed that EMT is involved in the invasion and metastasis process in malignant epithelial tumor tissues 7 .
The process is regulated at the transcriptional and post-transcriptional levels, and it involves many signaling pathways 8 . Researchers have found that different Smads can mediate the EMT process via the classical TGF/Smad signaling pathway in colon cancer, hepatocarcinoma, and mammary adenocarcinoma 9,10 . In addition, TGF-β can also activate TGF-β -activated kinase 1 and subsequently activate RAS homolog gene family member A (RhoA) or other factors, hence mediating the EMT process via non-Smad signaling pathways 11 . Howard 12 confirmed that Wnt could combine with the transmembrane receptor Frizzled, interfered with the degradation of β -catenin, and promoted the entry of β -catenin into the cell nucleus, which could activate Wnt target genes, including Ets, Jun, and Slug, thus promoting the EMT process. Yan 13 speculated that the EMT process in liver cancer cells was induced by hypoxia and was regulated by the PI3K-AKT signal pathway. When hepatocellular carcinoma and pancreatic cancer cells undergo EMT 14,15 , the Notch receptor, which is activated by its ligand, splits into two parts. The intracellular portion of Notch is released and enters the cell nucleus, subsequently regulating the transcription of downstream target genes, including members of the basic helix-loop-helix (bHLH), hairy and enhancer of split (HES) and HES-related inhibitory protein families as well as the zinc finger transcription factors (e.g., Snail and Slug). Secker 16 showed that the mitogen-activated protein kinase (MAPK) signaling pathway also regulated the EMT process. There are two major pathways participating in the MAPK regulation: Ras/Raf/MAPK and P38 Jun 17,18 .
Integrin is a transmembrane glycoprotein receptor consisting of a non-covalently bonded α and β subunit, and it belongs to the cell surface adhesion molecular family. α vβ 6 is a special integrin subtype that is only expressed in epithelial cells, and its major ligand is fibronectin (FN). In normal epithelial cells, the expression of α vβ 6 is rare and can hardly be detected 19 , but it increases substantially in response to injury and/or inflammation and in epithelial tumors (gastric carcinoma, colon cancer, and others) [20][21][22] . The de novo expression of integrin α vβ 6 has been shown to modulate several behaviors of colon carcinoma cells, including cell adhesion and spreading on fibronectin, proliferation in collagen gels, tumor growth, cell invasion and metastasis, and cell apoptosis, which we demonstrated throughout our 20-year investigation of integrin α vβ 6 [23][24][25][26] . Importantly, we have previously shown a direct physical linkage between ERK2 and the cytoplasmic domain of β 6, 746 EAERSKAKWQTGTNPLYRG 764 (the ERK2 binding sequence is italicized) 27 . Through the direct linkage between α vβ 6 and ERK2, integrin α vβ 6 transmits out-in signals accompanied by an increase in the phosphorylation level of ERK2, influencing many of the malignant activities of cancer cells 28 . Effective targets of phosphorylated ERK2 are found to be localized in the nucleus.
It is known that the direct linkage between α vβ 6 and ERK2 can increase the phosphorylation level of ERK2 in colorectal cancer cells, and activated ERK 1/2 can increase Ets-1 transcriptional activity by forming a signaling complex with Ets-1, which could enhance binding to the promoter region and induce transcriptional activation of genes involved in malignancy 29,30 . In this study, we found that after colon cancer cells were treated with NCTD for 24 h, the protein level of p-ERK decreased, accompanied by an increase in the epithelial marker E-cadherin and a decrease in the mesenchymal markers N-cadherin and vimentin. However, other key proteins in the EMT process, such as Notch1 and Notch3 in the Notch signaling pathway, Wnt3a and β -catenin in the WNT signaling pathway, Akt in the PI3K signaling pathway, and Smad2 and Smad3 in the TGF-β signaling pathway, showed no obvious variation. We treated the colon cancer cells with peptide IK2, specifically blocking the direct binding of β 6 and ERK. Interestingly, after treatment with IK2, NCTD had no effect on the invasion ability of these cells, indicating that NCTD inhibited the invasion ability of colon cancer cells through the direct connection between β 6 and ERK. Furthermore, the transcription factor p-ERK was investigated via an activation profiling array, which showed that only the activity of the transcription factor Ets1 was significantly inhibited in colon cancer cells treated with NCTD. Thus, we demonstrated that NCTD induced the MET process of colon cancer cells via the α vβ 6-ERK-Ets1 signaling pathway. NCTD suppressed the migration and invasion ability of colon cancer cells in a dose-dependent manner.
The Ets-1 transcription factor contains an approximately 85-amino-acid DNA-binding domain, which binds to special purine-rich DNA sequences with a core motif of GGAA/T. Ets-1 transcriptionally regulates a number Then, the expression of mean EMT-related proteins involved in classical signaling pathways was detected. The results showed that there were no significant changes in these pathways.
In our previous study of NCTD, we preliminarily demonstrated that NCTD, as an anti-cancer traditional Chinese medicine, could decrease α vβ 6 expression and inhibit ERK phosphorylation in HT-29 cells 4 . In this study, we found that NCTD could inhibit the EMT process of colon cancer cells, and it could cause colon cancer cells to switch from a spindle-shaped morphology to a round morphology. NCTD suppressed the migration and invasion ability of colon cancer cells in a dose-dependent manner. Meanwhile, the western blotting analysis and transcription factor activation profiling array confirmed that ERK and Ets1 participated in the signaling pathway. In light of these findings and our previous results, we propose that NCTD can inhibit the EMT process in colon cancer cells by interrupting the α vβ 6-ERK-Ets1 signaling pathway. However, it is possible that another signaling pathway may participate in the NCTD-dependent inhibition of the EMT process because some cancer cells do not express α vβ 6; this possibility needs to be investigated further. In addition, this study suggests that targeting any key protein in the α vβ 6-ERK-Ets1 signaling pathway, combined with NCTD treatment, may serve as a potential therapeutic strategy for epithelial cell cancers expressing integrin α vβ 6.