Background
Esophageal carcinoma remains one of the most aggressive malignant tumors and according to recent reports, it ranks seventh regarding incidence of cancers and sixth in mortality worldwide [
1]. Esophageal squamous cell carcinoma (ESCC) is the main histologic subtype within the scope of lower income countries, especially in parts of Asia and Africa [
2]. Due to its rapid progression towards advanced stages with strengthened capability of metastasis, early diagnosis and early treatment make sense. Despite of traditional therapies, targeted therapy emerges a more promising method for ESCC [
3]. Therefore, identification of novel genes that may function as biomarkers and developing effective agents targeting these genes are of significance to improve quality of patients with ESCC.
3,3′-Diindolylmethane (DIM) is an active metabolite of indole-3-carbinol (I3C) found in cruciferous family. Evidences show that I3C can exert its anti-tumor properties through regulating cell growth, cell cycle and division, apoptosis and metastasis mainly through aryl hydrocarbon receptor (AHR) [
4]. Meanwhile, AHR is a ligand-dependent transcription factor and has been found abnormally elevated in some tumors including breast cancer, non-small cell lung cancer, colon cancer and ovarian cancer [
5‐
8]. Once binding to a proper ligand, AHR will translocate into the nucleus forming complex with AHR nuclear translocator (ARNT) for locating on the xenobiotic response elements (XREs) region to regulate transcription of downstream genes [
9]. Our previous study have reported that both knockdown of AHR and modulation of AHR by DIM could inhibit ESCC growth, induce cell cycle arrest and promote apoptosis [
10]. Emerging evidence shows that AHR plays a crucial role in suppressing epithelial-mesenchymal transition (EMT) [
11]. EMT is a process that epithelial cells somehow obtain the properties of mesenchymal cells with the loss of tight cell-cell junction, regulation of cytoskeleton remodeling and increased capability of cell mobility to overcome the restraint of basement-membrane [
12]. EMT confers cancerous cells a perfect occasion for metastasizing to distant site with the result of accelerating disease progression. As an assistant of EMT, cytoskeleton rearrangement owns its role in facilitating cell mobility and invasion. And members of Rho GTPase family including RhoA, Rac1, Cdc42 and so on are widely involved in dynamics of actin assembly and disassembly. Among of these members, RhoA has been lucubrated with its downstream target, Rho-associated coiled-coil kinase 1 (ROCK1) and researches have elucidated RhoA/ROCK1 pathway are related to EMT process and tumor metastasis [
13,
14]. Similarly, cyclooxygenase 2 (COX2) as an inflammation-related factor and enzyme to catalyze arachidonic acid partly into prostaglandin E
2 (PGE
2), exhibits its potential role in disseminating breast cancer cells to distant organs [
15]. Meanwhile, PGE
2 could cause activation of β-catenin, an classic EMT marker of mesenchymal cells, for promoting colon cancer metastasis and COX2/PGE
2 pathway has also been involved in ovarian cancer angiogenesis [
16,
17]. What is more, once binding to a proper ligand, AHR would initiate the transcription of the most well-known targeted downstream genes which include the cytochrome P450-dependent monooxygenases family, such as CYP1A1. Occasionally, I3C has been reported to inhibit AHR binding to the COX2 promoter [
4].
Thus, our study aims to explore if DIM could modulate AHR to reverse EMT of ESCC through RhoA/ROCK1 pathway or COX2/PGE2 pathway and if these two classic pathways have some interactions synergistically to suppress EMT process and metastasis.
Methods
Cell culture and antibodies
We obtained the human ESCC cell lines TE1 and KYSE150 from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in RPMI 1640 medium with 10% Fetal Bovine Serum (FBS, Cellmax, China) at an atmosphere of 5% CO
2 in a humid condition. All antibodies used in Western blot or IHC were shown in Additional file
1: Table S1.
Immunohistochemistry (IHC) staining
Fifty samples of ESCC patients who had experienced esophagectomy at The First Hospital of China Medical University from 2011 to 2013 were collected for IHC staining. All patients were diagnosed ESCC postoperative pathologically by pathologists and all informed consent letters were signed clearly. Follow-up was conducted by calculating the overall survival (OS) time from date of surgery to date of death or endpoint. Fifty ESCC and paired normal esophagus tissue slides were stained with AHR antibody. Meanwhile, only ESCC slides were treated with RhoA and ROCK1 antibodies. We used H-score for evaluating gene expression levels by combining staining intensity and staining ratio. Staining scores were evaluated as follows: 0 (no staining), 1 (weak), 2 (moderate), 3 (strong). Staining ratio ranged from 0 to 100. H-score (0–300) was calculated through multiplying staining intensity by staining ratio. For survival analysis, cutoff was set by the median of relative protein H-score.
GEPIA (
http://gepia.cancer-pku.cn) was conducted for analyzing gene expression levels and correlations between gene A with B of esophageal carcinoma (ESCA) [
18]. We downloaded four ESCC databases GSE23400 (53 pairs), GSE38129 (30 pairs), GSE20347 (17 pairs) and GSE29001 (21 ESCC and 24 normal tissues) from GEO Datasets (
https://www.ncbi.nlm.nih.gov/geo/). Statistic analysis was conducted with R software 3.2.2. UALCAN (
http://ualcan.path.uab.edu) was used for further analyzing the relationships between AHR and clinical pathological parameters [
19].
Wound healing assay
TE1 and KYSE150 cells were treated with various concentrations of DIM or Celecoxib or PGE2 dissolved in serum-free medium. Scratches were made using 10 μl pipette tips. Images were acquired by inverted microscope (Leica, Germany) at 0, 24 and 48 h, respectively. We used Image J software (USA) for calculating the relative percentage of wound healing areas.
Transwell assay
Cells were seeded onto the upper chambers of the Transwell Chamber (8 μm pore size, Costar, USA) with 100 μl serum-free medium containing different concentrations of DIM or Celecoxib or PGE2. The lower chambers were filled with 10% FBS medium. After incubation for 24 or 48 h, the upper chambers were removed and fixed with 4% paraformaldehyde (Solarbio, China) for 30 min and then stained with 0.1% crystal violet (Beyotime, China). Images were obtained with the Leica inverted microscope.
Western blot
TE1 and KYSE150 cell lines were treated with DIM (MCE, USA) at concentrations of 0, 20, 40, 60 μM for 48 h or Celecoxib (MCE, USA) at 60 μM or PGE2 (MCE, USA) at 10 μM or Fasudil (MCE, USA) at 50 μM and 100 μM for 24 h. Cells were harvested with RIPA (Beyotime, China) lysis buffer and then underwent electrophoresis and were transferred onto PVDF membrane (Millipore, USA). Membranes were blocked with QuickBlock™ Blocking Buffer for WB (Beyotime, China) and then incubated with primary antibodies overnight at 4 °C. Secondary antibodies were used and ECL was utilized for visualization.
Cell morphology
ESCC cells were treated with DIM at 0, 20, 40 and 60 μM concentrations for 48 h. Images of cell morphology changes were captured by invented microscope (Leica, Germany).
Immunofluorescence of F-actin
ESCC cells were treated with DIM for 48 h or transfection in 12-well plates and after that, cells were washed with PBS and fixed with 4% paraformaldehyde for 30 min. We used 0.5% Triton-X100 to permeate for 15 min and Alexa Fluor 488 Phalloidin (CST, USA) for F-actin staining at a ratio of 1:20 for 30 min. DAPI (Solarbio, China) at the final concentration of 0.5 μg/ml was used for nuclei staining for 15 min. After complete wash, fluorescent images were captured by Live Cell Imaging System (BioTek, USA). Image J was used for fluorescent quantitative analysis.
Quantitative PCR (qPCR)
Total RNA was extracted from treated cells using the miRNeasy Mini Kit (QIAGEN, Germany) and quantified with spectrophotometer (BioTek, USA). cDNA was synthesized with GoScript Reverse Transcription Kit (Promega, USA). qPCR was performed with GoTaq qPCR System Kit (Promega, USA) using the 7900HT qPCR System (Applied Biosystems, USA). The primer sequences were listed in Additional file
1: Table S2.
Elisa
To detect relative expression levels of PGE2 after treatment of various concentrations of DIM dissolved in serum-free medium incubating for 48 h, ELISA was performed by collecting supernates respectively. The PGE2 released into the medium was measured using the PGE2 ELISA Kit (R&D System, USA) and all procedures were in agreement with corresponding protocols.
Cell transfection
ESCC cells were transfected with lentivirus (Genechem, China) or siRNAs (OriGene, USA). For AHR transfection, shRNA 1 sequence was used as GCATAGAGACCGACTTAAT and shRNA 2 as AACAAGATGAGTCTATTTA. For ROCK1, siRNA 1 sequences were 5′-CGGUUAGAACAAGAGGUAAAUGAAC-3′ and siRNA 2 were 5′-GGAAAUAUCAAACGAUAUGGCUGGA-3′. For PTGS2 (COX2), siRNA 1 squences were 5’GGCUAAUACUGAUAGGAGAGACUAT-3′ and siRNA 2 were 5′-GCAGCUUCCUGAUUCAAAUGAGATT-3′. Meanwhile, overexpression of AHR (OE-AHR) with lentivirus (Genechem, China) in TE1 and KYSE150 cell lines was also conducted with sequence as GAGGATCCCCGGGTACCGGTCGCCACCATGAACAGCAGCAGCGCCAACATC.
Animal study
The animal study was approved by the Animal Center of China Medical University (No.2018146). Four to six weeks old BALB/C nude male mice were purchased from Beijing Vital River Laboratory (Beijing, China) and raised in condition of SPF level. Sixteen nude mice were inoculated subcutaneously with approximately 2 × 106 TE1 cells and randomly separated into two groups (Control and DIM groups) after 1 week. Control group was fed with PBS while DIM group was treated with DIM (10 mg/kg/day). After 4 weeks of gavage, mice were sacrificed and xenograft tumors were removed to prepare for IHC staining. Image J was used for quantitive analysis.
Co-immunoprecipitation (co-IP) assay
Proteins were harvested after certain treatment and divided into two parts, one for co-IP and the other for Input assay. Approximate 200 μl protein lysates were incubated with 40 μl Protein A + G Agarose (Beyotime, China) and proper volume of antibodies (Additional file
1: Table S1) as well as Rabbit IgG antibody (Beyotime, China) at 4 °C overnight with gentle shaking. Then, samples were centrifuged at 6000 rpm at 4 °C for 3 min and washed with 1 ml pre-cooling PBS for at least five times repeatedly. About 10 μl 5 × SDS was added into each tube after resuspended with 40 μl PBS and samples were heated to 95 °C for 10 min. Finally, samples were loaded on 10% SDS-PAGE for WB analysis.
Statistical analysis
All experiments were repeated at least three times and data were shown as mean ± standard deviation (SD). SPSS 20 and GraphPad Prism 8 were used for analyzing data and creating statistical graphics. For comparing statistical significance of two groups, paired Student’s t-test was used and Spearman correlation analysis was used for comparing relationships between genes of ESCC. For comparisons of more than three groups, one-way ANOVA was used and LSD method was used for multiple comparisons. Mann-Whitney test was used for analyzing the correlations between H-scores and clinical pathological parameters. Kaplan-Meier method and Cox regression analysis were performed for ESCC prognostic analysis. A P-value less than 0.05 was considered statistical significant.
Discussion
Esophageal squamous cell carcinoma is one of the most aggressive tumors with rapid tumor progression and lower five-year survival rate. There are still no effective clinical drugs that can block the metastatic process, especially of ESCC patients at advanced stage [
20,
21]. For metastasis, a novel process is described as follows: cancer cells obtain the capability of disseminating from primary tumor site to proper distant organs with strengthened migratory and invasive abilities. Epithelial-mesenchymal transition is a complicated program that it confers epithelial cells successful transformation into mesenchymal cells with morphological change which leads to attenuated cell-cell junction, dissolved cell-matrix adhesion, rearranged cytoskeleton and enhanced invasive growth [
12]. EMT program is the vital initiation of tumor metastasis and targeting EMT as one of the most effective solutions to block ESCC progression is deserved explorations. Aryl hydrocarbon receptor is a ligand-dependent transcription factor which has been reported to have connections with tumor metastasis of thyroid carcinoma, neuroblastoma and inflammatory breast cancer [
22‐
24]. And DIM as a selective modulator of AHR, has been demonstrated it has inhibitory effects on tumor proliferation, migration and invasion [
25,
26]. Meanwhile, our previous study have demonstrated that AHR overexpression contributed to worse functional phenotypes and DIM treatment could inhibit ESCC cell growth, induce G1 phase arrest and promote cell apoptosis of TE1 cells [
10]. Therefore, we want to explore whether DIM can reverse EMT process of ESCC through modulation of AHR. As expected, DIM actually can downregulate expressions of mesenchymal cell markers including β-Catenin, Vimentin and Slug and epithelial cell marker Claudin-1 has been upregulated. Meanwhile, DIM also exerts its prohibitive effects on cell migration and invasion.
For EMT process, we have identified rearrangement of cancer cytoskeleton emphasizes its role in EMT and from experimental observation, cellular morphology of ESCC has changed from irregular long fusiform to almost round or ellipse type which implies the disruption of dynamic balance of local assembly and disassembly of cell actin filaments. Cytoskeleton regulation is the prerequisite of cell endocytosis, mobility, migration and invasion [
27]. RhoA/ROCK1 pathway has been reported to regulate actin stress fiber formation, cell-cell junction and cell-matrix adhesion by inducing MMPs production or regulating CXCR4/Akt signaling [
28,
29]. What is more, overexpression of RhoA or ROCK1 has contributed to malignant phenotype of cancers, such as ESCC [
30]. DLC1 SAM domain-binding peptides have been reported to inhibit breast cancer growth and migration through inactivation of RhoA [
31] and ROCK1 can promote migration and invasion of non small cell lung cancer by activating PTEN/PI3K/FAK pathway [
32]. Our results verify that DIM can reduce F-actin assembly through inhibition of RhoA/ROCK1 pathway with decreased transcription activities and kinase activities. Evidences show that AHR can bind to the XREs in the RhoA promoter region at the position of − 455 to − 431 bp with AHR agonist 3-MC (3-methylcholanthrene) [
33] and EGFR has been reported to promote human trophoblast cell migration through activation of RhoA and RhoC [
34]. Moreover, EGFR has been reported to interact with AHR and GPER (G protein estrogen receptor) to stimulate breast cancer progression and EGFR can bypass RhoA to activate YAP signaling to promote hepatocellular carcinoma proliferation [
35,
36]. Meanwhile, our co-IP results showed direct protein-protein interactions between AHR and EGFR as well as EGFR and RhoA. Thus, we hypothesize that DIM can modulate AHR, which leads to decreased transcription activity of RhoA/ROCK1 pathway and weakened interaction with EGFR since overexpression of AHR contributes to elevated expression levels of p-EGFR. Certainly, F-actin assembly or disassembly makes sense for cell migration with involvement in formation of two distinguishing functional states that mediate cell protrusive and contractile steps to couple with the extracellular matrix (ECM) for further generation of MMPs [
37]. As reported, vitamin C can inhibit triple-negative breast cancer metastasis by suppressive effect on formation of F-actin and lamellipodia through regulating expression of YAP1 and synaptopodin 2 [
38]; Migration and invasion enhancer 1 (MIEN1) has recently been indicated to promote cancer progression and metastasis by polymerizing G-actin and stabilizing F-actin filaments [
39].
As mentioned above, DIM can block transcriptional activity of AHR binding to the COX2 promoter. COX2/PGE
2 pathway is associated with tumor EMT and metastasis. Inhibition of COX2/PGE
2 pathway can effectively suppress tumor growth, EMT and metastasis of non small cell lung cancer or extrahepatic cholangiocarcinoma through PLA2G4A/PGE
2/STAT3 pathway [
40,
41]. Our results show that DIM can inhibit expression of COX2 and PGE
2 and COX2 selective inhibitor Celecoxib limits the capabilities of ESCC migration and invasion as well as reverses EMT process. On the contrary, directly supplying ESCC with PGE
2 promotes EMT and metastasis. Since both RhoA/ROCK1 pathway and COX2/PGE
2 pathway are able to reverse EMT and inhibit metastasis regulated by modulation of AHR by DIM, we wonder whether some interactions exist. As reported, salidroside can regulate Rho/ROCK1/NF-κB pathway to ameliorate arthritis-induced brain cognition deficits and microRNA-145 can inhibit proliferation and promote apoptosis of hepatocellular carcinoma through downregulation of ROCK1/NF-κB pathway [
42,
43]. It is well-known that NF-κB can regulate COX2/PGE2 pathway and is the upstream regulator of the pathway. Thus, we hypothesize that NF-κB is the connection of RhoA/ROCK1 and COX2/PGE2 pathways. Our co-IP assay had demonstrated that NF-κB actually had direct interactions with ROCK1 and COX2. Meanwhile, DIM can also inhibit transcription activity of NF-κB and phosphorylation level. That is to say, DIM exerts its reversal of EMT process mainly through modulation of AHR to inhibit EGFR/RhoA/ROCK1/NF-κB/COX2/PGE
2 pathway. This is the first time for us to demonstrate that AHR is the upstream gene of above pathways and COX2/PGE
2 pathway can be mediated by RhoA/ROCK1 pathway, which stands a chance to investigation of ESCC targeted therapy. Therefore, blockade of AHR as the original source of EMT process in ESCC with DIM as the modulator is of significance. Through reversal of EMT process that is currently in the limelight of keeping cancer cells in captivity, we can finally anchored tumors at primary sites to achieve success of prolonged patients’ lifespan.
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