Background
Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide [
1], and evolves as a result of the accumulation of genetic and epigenetic events [
2]. Data evaluated by the International Agency for Research on Cancer (IARC) indicated that about 1.8 million new cases of CRC were diagnosed and over 860,000 CRC patients died worldwide in 2018, which account for about 10% of all cancers and 9% of all cancer-related deaths, respectively [
3]. Therefore, it is of most importance to develop sensitive biomarkers and effective treatments for patients with CRC.
Nowadays, increased attention has been paid to the research of biomarkers for diseases, which could be used for early diagnosis and predict cancer outcome [
4]. Moreover, measuring biomarkers in blood offers a simple and effective way for disease diagnosis [
5]. Among nineteen identified Wingless and Int-related protein (WNT) ligands, WNT4 is highly secretory and its secretion has been reported in wound healing, acute kidney injury, and angiogenesis [
6]. However, to our knowledge, no study has resported on exploring Wnt levels in blood. In our study, we aimed to study WNT4 levels in serum, and explored its oncogenic role in CRC.
The tumor stroma is composed of extra-cellular matrix (ECM) proteins and a variety of cell types including fibroblasts, infiltrating immune cells, and endothelial cells [
7,
8]. Fibroblasts are a major component of tumor stroma and may acquire an activated phenotype called cancer associated fibroblasts (CAFs), which specifically express markers, such as fibronectin (FN) and α-SMA [
9,
10]. CAF has been demonstrated to have the ability to modulate the tumor microenvironment to promote CRC [
11]. Cytokines and inflammatory factors derived from cancer tissue can stimulate the transformation of normal fibroblasts (NFs) into CAFs [
12]. Therefore, identifying activators of CAFs in CRC may potentially help inhibit the development of CRC.
In addition, tumor start to grow only when the endothelial cells are recruited to the tumor site and form capillaries [
13]. Previous studies have demonstrated that angiogenesis could be enhanced by the WNT4/β-catenin pathway in Human umbilical cord mesenchymal stem cells [
14]. ANG2 is a crucial gene that is vital for angiogenesis and is closely related to the functions of human umbilical vein endothelial cells (HUVECs) [
15]. However, it is not clear if WNT4 can affect angiogenesis in CRC by regulating ANG2.
The Wnt/β-catenin pathway is the “canonical” Wnt pathway, which is essential for stem cell self-renewal and maintaining homeostasis of the intestinal tract [
16]. As a member of Wnt family, WNT4 may play a role in CRC through the β-catenin-dependent pathway. Therefore, it would be interesting to elucidate whether WNT4 can promote the progression of CRC via promoting epithelial-to-mesenchymal transition (EMT) of CRC cells, activating CAFs and enhancing angiogenesis through the β-catenin-dependent pathway.
In this study, we first showed that WNT4 levels were increased in the serum of CRC patients and originated from CRC tissues, and were decreased after tumor resection. Mechanistically, we explored the effect of WNT4 on the development of CRC in vitro and in vivo, we found that WNT4 promoted EMT of CRC cells, triggered the transformation of NFs into CAFs, and induced tumors angiogenesis in a β-catenin-dependent pathway.
Materials and methods
Human colorectal tissue and serum samples
The CRC tissues and paired adjacent normal tissues were collected from 24 CRC patients undergoing surgical resection (8 were used for Western blot analysis, 2 were used for the isolation of NFs and CAFs, 4 were used for tissue culture and another 10 were used for qPCR analysis). Serum samples from 40 CRC patients and 37 healthy donors were also collected at the Renmin Hospital of Wuhan University, Wuhan, China. Informed consents were signed by those patients and healthy donors before sample collection. All works were approved by the ethics review board of Renmin Hospital of Wuhan University (Wuhan, China) and carried out in compliance with the Declaration of Helsinki of the World Medical Association.
Oncomine database analysis
Antibodies and reagents
Antibodies were obtained from the following sources: rabbit anti-WNT4 polyclonal antibody (ab91226), rabbit anti-AXIN2 monoclonal antibody (ab109307), rabbit anti-fibronectin polyclonal antibody (ab2413) and rabbit anti-β-catenin monoclonal antibody (ab32572) were obtained from Abcam (Cambridge, MA, USA); rabbit anti-ZO-1 monoclonal antibody (#13663) and rabbit anti-E-cadherin monoclonal antibody (#3195) were obtained from Cell Signaling Technology (Beverly, MA, USA); anti-rabbit and anti-mouse HRP-conjugated secondary antibodies (#31460, #31430) were obtained from Invitrogen (Carlsbad, CA, USA). Anti-fluorescein isothiocyanate labeled phalloidin (P5282) and anti-α-SMA mouse monoclonal (A5228) antibodies were obtained from Sigma-Aldrich (St. Louis, MO, USA). Recombinant Human WNT4 Protein (6076-WN-005/CF) was obtained from R&D Systems (Minneapolis, MN, USA).
Cell culture
The CRC cell lines HCT 116, LoVo and SW480 were cultured in Dulbecco’s modified Eagle medium (DMEM, Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin. HUVECs were cultured in Ham’s F-12 K (HyClone, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS, 0.1 mg/mL heparin, 0.03–0.05 mg/ml endothelial cell growth supplement (ECGs). Cells were incubated in a humidified incubator containing 5% CO2 at 37 °C.
Immunohistochemical staining
Indirect immunohistochemical staining was performed as follows. All sections were dewaxed and dehydrated, followed by antigen retrieval and blocking. Subsequently, sections were incubated with a primary antibody and HRP-conjugated secondary antibodies. A 3,3′-diaminobenzidine (DAB) kit was used for visualization, and hematoxylin was used to stain the nuclei. Finally, sections were dehydrated with alcohol and sealed with neutral resin.
Cell migration and invasion assay
The cell migration assay was performed using chambers (8.0 μm) in a 24-well plate. For migration assays, cells pretreated with 100 ng/mL WNT4 for 24 h or not were added to the upper chamber, which contained serum-free DMEM; the lower compartment was filled with DMEM medium supplemented with 10% FBS. Cells were allowed to migrate for 12 h, and those that had migrated across the filter were fixed in methanol and stained with 0.1% crystal violet. The invasion potential of those cells were determined by using a trans-well chamber (8.0 μm), coated with Matrigel (Corning, Albany, NY, USA) in DMEM medium (1:4 v/v), and the lower compartment was filled with DMEM medium supplemented with 20% FBS. Cells were incubated for 24 h before invasive cells were fixed, stained, and counted. Results were analyzed by ImageJ.
ELISA
The WNT4 concentration in serum from healthy donors, CRC patients, and conditioned medium from fresh tissues were quantitatively determined by ELISA kits (CSB-EL026137HU, CUSABIO, Wuhan, China) according to the manufacturer’s instructions. For ANG2, the protein concentrations in media from HUVEC cells were detected by a human ANG2 ELISA kit (Abcam, Cambridge, MA, USA).
Luciferase assay
A Dual-Luciferase Reporter Assay system was used to measure luciferase activities (Promega, Fitchburg, WI, USA). For TOP/FOP-Flash reporter assays, cells were divided into five groups: blank, WNT4 (100 ng/mL), DMSO, ICG-001, and ICG-001+ WNT4 (100 ng/mL). After transfection, WNT4 protein (100 ng/mL) was added 6 h before luciferase detection. The relative ratio of TOP-Flash firefly luciferase activity to pRL-TK Renilla luciferase activity was determined as the strength of the transcriptional activity.
Western blots
In brief, equal amounts of protein were separated using SDS–PAGE, and transferred onto PVDF membranes (Millipore, Burlington, MA, USA). After blocking with 5% non-fat milk in Tris-buffered saline with Tween 20 (TBST), membranes were incubated with primary antibodies at 4 °C overnight and HRP-conjugated secondary antibodies at room temperature for 60 min. Then membrane were incubated with enhanced chemiluminescence (ECL) reagents, and signals were visualized by chemiluminescent a gel imaging system.
Immunofluorescence
For immunofluorescence assays, 12-well chambers were used. Each well was seeded with 1 × 105 cells on the coverslip and fixed with 4% paraformaldehyde, treated with 0.2% Triton X-100 for 1 min and blocked with 1% bovine serum albumin (BSA). For paraffin embedded tissues, sections were dewaxed and dehydrated, followed by antigen retrieval and blocking. After incubation with primary antibodies and fluorescent-labeled secondary antibodies and Phalloidin, the cells on coverslips or tissue sections were stained with 4′,6-diamidino-2-phenylindole (DAPI). Fluorescent images were obtained using an upright Olympus fluorescence microscope (OLYMPUS BX53).
Cell transfection
Inhibition of WNT4 expression was performed by using specific siRNA constructs. The sequences of WNT4 siRNA-1, WNT4 siRNA-2 and normal control used in this study are presented in Supplementary Table
1. In brief, cells were transiently transfected with WNT4 siRNAs using Lipofectamine 2000 and were harvested for analysis of the knockdown efficiency at 48 h ours after transfection.
To establish stable WNT4-overexpressing cells, an overexpressing plasmid was constructed and packaged into a lentivirus system (WNT4-HA). Lentivirus-NC was used as the negative control (WNT4-vector). In addition, to generate cells with low expression of WNT4 (sh-WNT4) and ANG2 (sh-ANG2), short hairpin RNA (shRNA) sequences directed against WNT4 and ANG2 were transfected (Supplementary Table
1), while a scramble construct was used as a control. Non-transfected cells were screened out by treatment with 10 μg/mL puromycin (Sigma Aldrich, St Louis, MO).
Quantitative real-time PCR
TRIzol reagents (Invitrogen) was used to extract RNA from cell lines or tissues according to the manufacturer’s instructions. RT-qPCR was performed to quantify the mRNA expression using SYBR Green PCR Master Mix (Takara, Ohtsu, Japan), and miRNA expression with the NCode miRNA RT-qPCR analysis (Takara, Ohtsu, Japan). Primer sequences are presented in Supplementary Table
2.
Tumor xenograft study
To generate a xenograft model, 1 × 107 cells/200 μL were injected subcutaneously into the dorsal flank of each mouse (8-week-old, male, BALB/c-nu/nu). After 2 weeks, mice were sacrificed, and subcutaneous nodules were collected. For establishing the tumor metastasis model, 1 × 106 cells/100 μL were injected into the tail vein of each mouse (8-week-old, male, BALB/c-nu/nu). After 4 weeks, mice were sacrificed, and the weights of mice livers were recorded.
After tumor weights were recorded, all tissues were fixed with 4% paraformaldehyde. Animal experiments were approved by the Committee on the Use of Live Animals in Teaching and Research (CULATR), Renmin Hospital of Wuhan University (No:11400700326686 and No:110011111003022). Animals care was in accordance with institution guidelines.
Isolation of NFs and CAFs from CRC and normal tissues
Fresh tissues were cut into small pieces (around 1 mm3) and subjected to enzymatic digestion in DMEM supplemented with 5% FBS following the instructions of the tumor dissociation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Next, cells were filtered through a 100-μm cell strainer (Thermo Fisher Scientific, MA, USA) and resuspended and cultured in fibroblast medium (FM). Flow cytometry using a CD31-Cy7 conjugated antibody, CD45-Cy7 conjugated antibody, and CD329-Cy7 conjugated antibody was performed to confirm the absence of endothelial, immune, and epithelial cell contamination in the primary fibroblasts.
Preparation of conditioned medium
Fresh human CRC and adjacent normal tissue samples were obtained directly from the operating room. Tissues were weighed and preserved in Falcon tubes containing 10 mL of DMEM with antibiotics (100 U/mL penicillin, and 100 mg/mL streptomycin) to avoid bacterial or fungal contamination. Tissues were cut into similar sized pieces and cultured with DMEM for 12 h. Then, collected conditioned medium (CM) was collected, and filtered through 40-μm cell strainers (Thermo Fisher Scientific, MA, USA) to obtain tissue-conditioned medium and stored at − 80 °C until further use.
After the SW480 cells (vector/WNT4-HA) grew to 70–80% confluency, then cells cultured in DMEM medium (no FBS) for 24 h. CM was obtained and centrifuged (1000 rpm, 10 min), and the supernatant was filtered through a 0.22 μm filter (Beyotime Biotechnology, China) and stored at 4 °C for treating HUVECs.
The tube formation assay was performed following the manufacturer’s protocol (BD Biosciences, Franklin Lakes, NJ, USA,
https://www.bdbiosciences.com). In brief, 50 μL of growth factor reduced Matrigel (BD Biosciences company, San Jose, CA, USA) was added to each well of a precooled 96-well plate and allowed to polymerize at 37 °C. Subsequently, HUVECs or transfected HUVECs (sh-ANG2/scramble ANG2) were treated with CM from SW480 cells (vector/WNT4-HA) and pretreated with ICG-001 (10 μM) 12 h before collection. Subsequently, cell (50 μL) at a density of 1 × 10
5 cells/mL were seeded onto the matrix gel and incubated at 37 °C for 12 h. Then, cells were viewed under a microscope (OLYMPUS IX71, Japan) and imaged. The tube length was measured by ImageJ software (
https://imagej.nih.gov/ij/, MD, USA).
The protocol used for analyzing sphere formation was described previously [
17]. In brief, primary fibroblasts were labeled with PKH-26 (red) and mixed with GFP-transfected tumor cells (Scramble or sh-WNT4, WNT4-vector or WNT4-HA) at a ratio of 3:1 in an ultra-low attachment plate (Corning, Albany, NY, USA) with DMEM medium at 37 °C overnight. Typical heterospheroids were observed and counted using an inverted Olympus fluorescence microscope (OLYMPUS IX71).
Collagen matrix contraction assay
Contraction of collagen gels was performed in 96-well plates as previously reported [
18]. In brief, collagen gel (Corning, Albany, NY, USA), matrigel (Corning, Albany, NY, USA), FM, FBS, and suspended fibroblasts (with or without 400 ng/mL WNT4 pretreatment) were gently mixed. Then, 100 μl of the mixture was added to each well of a 96-well plate and allowed to solidify at 37 °C for 30 min before complete medium was added. For inhibitory experiments, both gels and media were incubated with 10 μM ICG-001.
Statistical analysis
All experiments were performed at least in triplicate. Data are presented as the mean ± standard deviation (SD). Data of the relationship of WNT4, CA199, and CEA expression with clinicopathological parameters of CRC patients are presented as the mean ± standard error of mean (SEM). Comparisons between two groups were performed by Student’s t-tests or Mann–Whitney U tests for continuous variables. Survival probabilities and recurrence rates were estimated using Kaplan–Meier survival analysis and differences between Kaplan–Meier curves were compared by the log-rank test. All statistical tests were two-sided. SPSS v17.0 (Chicago, IL, USA) was used for statistical analyses. P < 0.05 were considered statistically significant. *P < 0.05, **P < 0.01.
Discussion
WNT ligands have frequently been reported to play an important role in CRC, but the role of WNT4 on CRC has rarely been studied. In a previous study, it has been reported that WNT4 promoted the proliferation of breast cancer stem cells [
27], and promoted progression of gastric cancer [
28], which suggested a pro-carcinogenic role of WNT4. However, these studies focused on studying the expression of Wnt ligands in tissues samples [
29,
30], while the serum levels of WNT4 has not been identified previously. In the present study, we demonstrated for the first time that WNT4 was elevated in the serum of CRC patient and originated from tumor tissues by measuring the CM from colorectal tissue culture, and was decreased after tumor resection. Besides, analysis of the relationships between serum levels of WNT4 and clinicopathological characteristics showed that elevated WNT4 correlated with the advanced stage and metastasis of CRC. These findings indicated that WNT4 may be a potential biomarker for CRC. The detection of tumor-associated biomarkers in peripheral blood of cancer patients provides an opportunity to analyze the changes in tumor burden and monitor the response to treatment [
31]. The results obtained in this study suggested that WNT4 could potentially be a serum marker for diagnosis, and value the risk of metastasis for CRC.
Moreover, when compared to CEA and CA199 levels in serum, we found that serum levels of WNT4 had significantly higher sensitivity and specificity for detection of CRC. However, due to the lack of a robustly designed prospective trial, these results remain subject to further verification. Taken together, our results highlight the potential clinical significance of WNT4, and suggest that further studies are needed to investigate the diagnostic applications of WNT4 in CRC.
Although the upregulation of WNT expression has been extensively investigated in CRC and corresponding liver metastasis [
32‐
34], the role of WNT4 in CRC has rarely been explored. To investigate the effect of elevated WNT4 levels in CRC tissues, exogenous and endogenous WNT4 was used to treat CRC cells, fibroblasts and HUVECs. In our study, we demonstrated that WNT4 could promote EMT of CRC cells through the WNT4/β-catenin cascade. Cancer cells often required mesenchymal phenotype to enhance their ability of invasion and metastasis [
35], which is consistent with our finding that WNT4 could promote EMT in CRC cells to promote the invasion and migration ability both in vitro and in vivo. The canonical Wnt signalling pathway regulates target genes via stabilization of nuclear β-catenin [
36]. As evidently supported that Axin2 is a part of destruction complex and could captures and phosphorylates β-catenin in the absence of Wnt [
16,
37]. In our study, we found the interesting phenomenon that total levels of β-catenin and Axin2 protein were reduced following WNT4 knockdown in both cell lines, which may infer that WNT4 may could also regulate β-catenin through a non-canonical pathway, and Axin2 may be regulated by WNT4. Previous study also demonstrated that the putative tumor suppressor Axin2 is also upregulated in CRC, and they proved that Axin2 could upregulate the activity of Snail1(a transcriptional repressor), thereby inducing a EMT and driving metastatic activity in CRC [
38]. Thus, it would be interesting to elucidate the potential mechanism involved.
Furthermore, most studies have focused on the role of WNT ligands in cancer cells, the Wnt/β-Catenin pathway, for example, could promote gastric cancer cells [
39], non-small cell lung cancer [
40] and CRC cells [
41]. However, little attention has been paid to its possible role in the tumor microenvironment, including CAFs and endothelial cells. In a previous study, it was demonstrated that WNT2 protein could be secreted by fibroblasts to promote the progression of esophageal cancer [
42]. In this study, we firstly showed that WNT4 secreted by CRC cells could activate surrounding fibroblasts through the WNT4/β-catenin pathway. Furthermore, recent evidence suggests a significant role for EMT in fibrosis, including its involvement in tumor stroma activation [
24,
43]. In the current study, we showed that WNT4 prompted the nuclear translocation of β-catenin and increased the expression of α-SMA and fibronectin in NFs, which subsequently enhanced reinforced cell contraction and microsphere formation. We next found that WNT4 secreted by CRC cells could enhance angiogenesis in CRC. ANG2 iss a critical gene for angiogenesis, however, the correlation of ANG2 and WNT4/β-catenin has never been investigated before. In our study, we demonstrated that angiogenesis could be activated by WNT4 through the WNT4/β-catenin/ANG2 pathway, which is vital for tumor invasion and metastasis. To our knowledge, few studies have explored the effect of WNT4 on those components in the CRC microenvironment. In a previous study, it was shown that WNT4 could be released into the stroma in CRC in the form of exosomes [
44]. It has been widely reported that exosomes from CRC cells play critical role in modulating invasion, immune responses and angiogenesis [
45]. Therefore, we speculated that WNT4 could be secreted as exosomes from CRC cells and act on various components in the tumor microenvironment to promote tumor progression. Additional studies will be required to support this hypothesis.
It is worth noting that these effects of WNT4 were observed at a concentration that has been reported in previous studies [
46,
47], while no effect was observed at serum concentration. Combined with our results, we speculated that WNT4 secreted by CRC tissue mainly acted on the tumor microenvironment to create an environment that is conducive to tumor growth, while WNT4 in serum may simply act as a diagnostic or prognostic indicator for CRC.
In addition, we found that downregulation miR-497 may lead to elevated WNT4 in CRC tissues. Downregulation of miR-497 and its tumor-suppressive role have been reported in multiple cancers [
48‐
51]. Our data showed that WNT4 was a novel direct target of miR-497. However, further studies are required to confirm the regulatory effect of miR-497 on WNT4 expression and its role in the progression of CRC.
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