A disintegrin and metalloproteinase 8 induced epithelial‐mesenchymal transition to promote the invasion of colon cancer cells via TGF‐β/Smad2/3 signalling pathway

Abstract A disintegrin and metalloproteinase 8 (ADAM8) protein is a multi‐domain transmembrane glycoprotein which involves in extracellular matrix remodelling, cell adhesion, invasion and migration. ADAM8 and epithelial‐mesenchymal transition (EMT) play an important role in tumour invasion has been well established. However, the interaction between ADAM8 and EMT has remained unclear. The data of colon cancer patients obtained from TCGA (The Cancer Genome Atlas) and GTEx (Genotype‐Tissue Expression Project) were analysed by the bioinformatics research method. The expression of ADAM8 in colon cancer cells was up‐regulated and down‐regulated by transfecting with the expression plasmid and small interfering RNA, respectively. Transwell invasion assay, immunohistochemistry, immunocytochemistry, Western blotting and qRT‐PCR were utilized to study the effect of ADAM8 on colon cancer cell's EMT and its related mechanisms. Analysis of TCGA and GTEx data revealed that ADAM8 was linked to poor overall survival in colon cancer patients. Besides, ADAM8 was correlated with multiple EMT biomarkers (E‐cadherin, N‐cadherin, Vimentin, Snail2 and ZEB2). In vitro, we also proved that the up‐regulation of ADAM8 could promote EMT effect and enhance the invasive ability of colon cancer cells. On the contrary, the down‐regulation of ADAM8 in colon cancer cells attenuated these effects above. Further studies suggested that ADAM8 modulated EMT on colon cancer cells through TGF‐β/Smad2/3 signalling pathway. Our research suggested that ADAM8 could be a potential biomarker for the prognosis of colon cancer and induced EMT to promote the invasion of colon cancer cells via activating TGF‐β/Smad2/3 signalling pathway.


| INTRODUC TI ON
Colon cancer is a common digestive tract malignant tumour with high morbidity and mortality, which is a serious threat to human health. 1 Although many different tumour biomarkers and treatments have emerged in recent years, there has been no obvious improvement in overall survival of colon cancer patients. Metastasis and recurrence are the main causes of death in colon cancer patients. 2 Therefore, it is very urgent to find more effective tumour biomarkers and therapeutic targets. Epithelial-mesenchymal transition (EMT) is crucial for the promotion of cell metastasis in malignant tumours. It is marked by the loss of epithelial cell characteristics and the acquisition of interstitial cell characteristics. 3 In EMT, polar epithelium cells could be converted into mesenchymal cells which move freely between the matrices. Extensive researches have shown that once colon cancer cells develop EMT, their ability to invade and metastasize will be greatly enhanced. [4][5][6] ADAM8, as one of the most important members of the ADAM family, has attracted increasing attention due to its close relationship with tumorigenesis, development and prognosis. ADAM8 is a multi-domain transmembrane glycoprotein which involves in extracellular matrix remodelling, cell-cell interactions and various aspects of carcinogenesis. Several studies have shown that ADAM8 is highly expressed in a variety of malignant tumour tissues, such as colon cancer, glioma, lung cancer, liver cancer, pancreatic cancer and gastric cancer, and promotes tumour invasion and metastasis. [7][8][9][10][11][12][13][14] While it is known ADAM8 and EMT play important roles in tumour invasion and metastasis, no prior studies have investigated the correlation between ADAM8 and EMT. In the present study, by analysing human colon cancer's data from TCGA and GTEx databases, we not only demonstrated that ADAM8 was closely related to poor prognosis in patients with colon cancer, but also found the correlation between ADAM8 and EMT-related biomarkers. In addition, we found that ADAM8 could induce EMT to promote colon cancer cell invasion via activating TGF-β/Smad2/3 signalling pathway. The importance and innovation of this study are to explore the correlation between ADAM8 and EMT in colon cancer cells and to highlight the pivotal role of ADAM8 in colon cancer cells' invasion and EMT. Therefore, these findings will contribute significantly to the determination of ADAM8 as an effective biomarker and therapeutic target for colon cancer.

| Public data analysis
All the work on colon cancer's data from TCGA and GTEx database was carried out by using the online tool Gene Expression Profiling Interactive Analysis (GEPIA, http://gepia.cance rpku.cn/index. html). 15 GEPIA performed survival analysis on gene expression levels and required a log-rank test for hypothesis assessment. GEPIA also performed paired gene Pearson correlation analysis between ADAM8 and EMT-related biomarkers by using colon cancer's data from TCGA and/or GTEx database.

| Stably transfected cell lines
HCT116 cells with lower endogenous ADAM8 expression levels (as shown in Figure 4A) were selected for transfecting with ADAM8 overexpression plasmid (Flag-ADAM8; Sigma-Aldrich) or empty vector (pCDNA3.1; Sigma-Aldrich), and Lipofectamine 2000 (Invitrogen) used as the transfection reagent according to the manufacturer's protocol. After 24 hours, cells were treated with 600 mg/mL G418 (Invitrogen). The transfection efficiency on cell lines with ADAM8 overexpression plasmid or empty vector was evaluated by Western blotting and qRT-PCR.

| RNA interference
Small interfering RNA (siRNA) oligonucleotides which were specific to ADAM8 and Smad2/3 obtained from RiboBio Co., Ltd. SW480 cells with higher endogenous ADAM8 expression (as shown in Figure 4A) were cultured in six-well plates until 60% confluence and transfected with 50 nmol/L of the indicated siRNA using riboFECT™ CP Reagent (RiboBio) according to the manufacturer's instructions.
The efficiency of gene knockdown was analysed by Western blotting and qRT-PCR after 48 hours.

| Cell invasion assay
The invasive ability of tumour cells was detected by Transwell invasion assay. While medium supplemented with 10% FBS was placed in the lower chamber, 1 × 10 5 cells were seeded in upper cell culture insert containing 8.0 μm pore size membrane (Corning) and cultured in FBS-free medium. A diluted extracellular matrix gel (Corning) was coated on the membrane of cell culture insert for the invasion assay.
Following incubation for 48 hours, the invaded cells below the membrane was fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The invaded cells were counted and photographed by using inverted microscope.

| Western blotting
Total protein was isolated from the colon cancer cells and determined the concentration by using Coomassie protein assay (Thermo Fisher Scientific). Protein sample from the previous step was separated by SDS-PAGE and transferred to polyvinylidene difluoride membrane. After blocking for 30 minutes in skim milk at room temperature, the membrane was incubated with specific primary antibody overnight at 4°C. The next day, the membrane was

| TMA, IHC staining and evaluation
The Tissue microarray (TMAs) were constructed using an automated TMA instrument (ALPHELYS, Plaisir). Immunohistochemistry (IHC) staining was performed on the TMA slides. All tissue specimens were fixed in 4% formaldehyde solution, embedded into paraffin and cut in 4-μm sheets. The tissue slides dewaxed with xylene, dehydrated by using gradient alcohol and retrieved antigen by

| RNA isolation and quantitative polymerase chain reaction
Total RNA extracted with TRIzol reagent (Invitrogen) according to the manufacturer's protocol. Complementary DNA was reversetranscribed using a reverse transcription kit (Takara). Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed with SYBR Green Master Mix Kit (Thermo Fisher Scientific) by using ABI 7500 System (Thermo Fisher Scientific). β-actin used as a control for the normalization of gene expression. The 2 −∆∆CT method was utilized to quantify the fold change. 13 All primers were synthesized by Sangon Biotechnology Co., Ltd and listed in Table 1.

| Statistical analysis
Statistical analysis was performed with SPSS 25.0 (SPSS Inc) and Prism software (GraphPad). Pearson's chi-squared test and Fisher's exact test were applied for categorical variables, and continuous variables were analysed by the Student's t test. All statistical analyses were two-sided, and P < .05 was considered statistically significant. All values represent the means ± SD.

| High level of ADAM8 associated with poor prognosis in colon cancer patients
To evaluate the level of ADAM8 in colon cancer tissues and adjacent normal mucosa tissues, the expression of ADAM8 protein and mRNA was examined in a group of 30 colon cancer tissues paired with adjacent normal mucosa tissues by IHC and qRT-PCR. IHC results revealed that specific ADAM8 staining was detected in the cytoplasm and cell membrane of epithelial cells ( Figure 1A,B). The IHC staining scores of ADAM8 in colon cancer tissues were higher than that of adjacent normal mucosa tissues (3.13 ± 0.27 vs 1.17 ± 0.18, respectively; P < .001). Furthermore, the mRNA level of ADAM8 at cancer tissues was significantly higher than that of adjacent normal tissues (1.54 ± 0.17 vs 0.94 ± 0.08, respectively; P = .0012; Figure 1C).
The online tool GEPIA was applied to analyse colon cancer patients' data from TCGA and GTEx databases. The results showed that patients with high ADAM8 expression had poorer overall survival than patients with low ADAM8 expression (P = .029, HR = 1.7; Figure 1D). There were no significant differences in disease-free survival (P = .33, HR = 1.3; Figure 1E) and no correlation between The level of ADAM8 associated with poor prognosis in colon cancer patients. A, Representative image of ADAM8 expression in colon cancer tissues was detected by IHC. Scale bars were indicated. B, Heat map showed the IHC staining score of ADAM8 protein in normal mucosal tissue and tumour tissues. C, Expression of ADAM8 mRNA detected by qRT-PCR in normal mucosal tissue and tumour tissues. D, Overall survival and E, disease-free survival, GEPIA performed survival analysis and required a log-rank test for hypothesis assessment. F, The level of ADAM8 was presented in different tumour stages. ADAM8, a disintegrin and metalloprotease 8; mRNA, messenger RNA; qRT-PCR, quantitative reverse transcription-polymerase chain reaction; *P < .05; **P < .01; ***P < .001; ns, no significant the level of ADAM8 and the tumour pathological stage (F = 1.59, Pr = 0.193; Figure 1F). However, in our validation cohort, we performed IHC staining assay in TMAs of 97 colon cancer samples. We pooled the level of ADAM8 with patients' clinical-pathologic factors for statistical analysis. The results showed that the level of ADAM8 in colon cancer was significantly related to patients' AJCC stage, depth of invasion, N stage and distant metastasis (P < .05; Table 2).

| Up-regulation of ADAM8 induced EMT and enhanced the invasive ability of colon cancer cells
In the analysis about public database, it was shown that ADAM8 as-   In brief, these results indicated that the down-regulation of ADAM8 attenuated EMT and colon cancer cell invasion.

| ADAM8 modulated EMT by activating TGF-β/ Smad2/3 signalling pathway
It was well known that TGF-β could mediate the canonical Smad Importantly, we demonstrated that the treatment with BK-1361 or the knockdown of ADAM8 in SW480 cells attenuated the Smad2/3 binding to the promoter of E-cadherin via using ChIP-qPCR assay ( Figure 6F). In a word, these results indicated that ADAM8 was involved in the regulation of EMT processes in colon cancer cells through activating TGF-β/Smad2/3 signalling pathway.

| D ISCUSS I ON
EMT was considered to be the initial step of tumour cell invasion. This canonical pathway could also activate integrin-linked kinase (ILK), thereby phosphorylate GSK-3β and Akt, leading to β-catenin nuclear translocation and activation of other transcription factors, which induce EMT of epithelial cells. 38,39 Thus, ADAM8 induced EMT in colon cancer cells through TGF-β/Smad2/3 signalling pathway, and integrins were the key mediators between the interaction of ADAM8 and TGF-β.
In conclusion, our study indicated that ADAM8 was an important biomarker for the prognosis of colon cancer and induced EMT to promote the invasion of colon cancer cells via activating TGF-β/ Smad2/3 signalling pathway. The present study elucidated the specific mechanism of ADAM8 promoting colon cancer cell invasion and laid the groundwork for future research of ADAM8 as a promising therapeutic target for colon cancer.

ACK N OWLED G EM ENTS
We sincerely appreciate the editor and reviewers for their constructive comments.

CO N FLI C T O F I NTE R E S T
The authors have no conflict of interest to declare.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data sets used in the current study are available from the corresponding author on reasonable request.