Background
Hepatocellular carcinoma (HCC) is the most common malignancies worldwide with a third rank of mortality rate in all types of cancer [
1]. HBV or HCV, exposure to alcohol intoxication are major risk factors which induced hepatic inflammation and fibrosis and consequently lead to cirrhosis as observed in 80% of HCC patients [
2]. Elucidating the molecular signaling mechanisms of cirrhosis to HCC and the search for the novel therapeutic targets and treatment are of great significance in improving the overall prognosis of HCC patients.
Cartilage oligomeric matrix protein (COMP), a cartilage metabolism marker, is an extracellular matrix protein that modulates the cellular phenotype during tissue genesis and remodeling. Recently, it has been extensively studied for its pro-fibrosis potential against various internal organs, for instance, pulmonary fibrosis [
3], and liver cirrhosis [
4]. Earlier work has suggested that COMP levels in the serum of HCC patients and HCC tissues are abnormally elevated compared with healthy controls and it can be used as diagnostically in the non-invasive estimation of liver cirrhosis and HCC development [
5,
6]. Recent studies have shown that COMP promotes the progression of breast cancer, colon cancer and prostate cancer [
7‐
9]. These results suggest that COMP may be an important pro-HCC molecule, but the mechanism by which COMP plays a role in HCC still needs to be further studied.
CD36 is a traditional membrane receptor of the thrombospondin family that binds to CD36 thereby activating downstream signaling pathways involved in various cellular processes. In HCC, CD36 is abnormally high which promotes EMT process by increasing free fatty acid uptake [
10,
11]. Increasing evidences indicate that EMT contributes to tumor metastasis and chemotherapy resistance of HCC [
12‐
16]. Transcriptional repressors of epithelial gene such as Snail, Slug and Twist are essentially involved in EMT and either directly or indirectly induced by signaling from MEK/ERK or PI3K/AKT [
17]. Recent studies have found that COMP binds to CD36 and activates downstream signaling pathways and ultimately leading to the progression of liver cirrhosis [
4,
18].
In the present study, we specifically investigated the effect of COMP on the malignant behaviors of HCC by in vivo and in vitro experiments, including proliferation, migration and invasion. Notably, we discovered that activated hepatic stellate cells (HSCs)-derived COMP regulated mesenchymal gene expression and MMPs in HCC cells via CD36 and caused aberrant phosphorylation of ERK and AKT. Analysis of primary HCC serum samples supported the predictive role of COMP in patient’s survival, suggesting that COMP was a promising biomarker and an effective bioactive strategy to combat tumor-progression.
Methods
Cell culture and treatment
The HCC cell lines (MHCC-97H, HepG2, Huh7, Hep-3B, SMMC-7721) and the immortalized human liver cell line LO2 and activated hepatic stellate cells (HSCs) LX2 were purchased from the Shanghai cell bank (Shanghai Institute for Biological Science, Chinese Academy of Science, Shanghai, China) and cultured at 37 °C in a humidified atmosphere with 5% CO2 in high-glucose Dulbecco’s Modified Eagle’s Medium (DMEM; Invitrogen, NY, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA) and 1% penicillin-streptomycin. The culture medium was replaced with serum-free DMEM 12 h before treatment with 0–5 μg/ml of human rCOMP (R&D Systems, Minneapolis, MN) according to the manufacture’s recommendations.
Clinical samples and ELISA
Venous blood samples of 100 HCC patients without other complications (before the surgery) and 30 healthy volunteers were obtained from the First Affiliated Hospital of Xi’an Jiaotong University between 2015 and 2017, the serum was obtained after centrifugation at 3, 000 rpm for 15 min and stored at − 80 °C. Ethic permission was obtained from the Ethics Committee of the First Affiliated Hospital of Xi’an Jiaotong University. Written informed consent was obtained from each patient or family members.
Quantitative measurement of human COMP in serum of HCC patients were performed by human COMP ELISA kit (R&D System, Minneapolis, MN) following the manufacturer’s protocols. Briefly, serum of HCC patients was collected before the surgery, cell supernatants were collected after cultivated in serum-free medium for 24 h. A seven-point standard curve was generated for every plate and quantified using the GraphPad Prism 5.0 software (La Jolla, USA). All samples were analyzed in triplicates.
In vitro cell proliferation and plate colony-forming assays
The cell proliferation was measured using the Cell Counting Kit-8 (CCK-8) (Dojindo, Kyushu, Japan). Cells were seeded in 96-well plates at 4 × 103 cells/well and incubated overnight to allow their adhesion to the plate. Cells were treated with rCOMP in different concentrations (0, 0.8, 1, 2 and 5 μg/ml) for 12 h, 24 h, and 36 h, five parallel wells for each concentration. Ten microliters of CCK8 (Sigma-Aldrich, St. Louis, MO, USA) was added to 90ul of the medium per well and incubated at 37 °C for 4 h. The absorbance was detected at 450 nm with microplate autoreader (Bio-Rad, CA, USA).
For the colony formation assay, Hep-3B and SMMC-7721 cells in exponential growth phase were seeded into 6-well plates at a density of 1000 cells/well. After overnight incubation, cells were treated with rCOMP (0, 1, 2 and 5 μg/ml) and maintained in culture medium for 2 weeks and the medium with corresponding rCOMP concentrations was replaced every three days. The colonies were fixed with 4% methanol and stained with 0.1% crystal violet at room temperature and the number of colonies was counted. Data were collected from three independent experiments.
Wound-healing assay and Transwell migration and invasion assays
The cells were grown to a 90–100% confluence cell monolayer overnight prior to serum starvation for 8 h in 6-well plates. After wounding with a sterile pipette tip, cells were then washed with PBS to eliminate the floating cells. Cells were treated with rCOMP (0, 1, 2 and 5 μg/ml) and the wounds were photographed at time 0 h and 24 h post-wounding under a phase-contrast microscope. Cell migration was quantitated by measuring the width of the wounds. Migration rate was calculated as (%) = [W (24 h) - W (0 h)]/W (0 h). Experiments were performed with at least three times.
The migration assay was performed in a Transwell chemotaxis 24-well chamber (BD Biosciences, Franklin Lakes, NJ), 2 × 104 cells in 200 μl serum-free medium were plated in the upper chambers. For invasion assay, the basement membrane of filters was coated with 50 μl Matrigel (Matrigel; BD Biosciences, Bedford, MA). After incubation with rCOMP (0, 1, 2 and 5 μ g/ml) for 24 h, cells migrated or invaded to the lower surface of the membrane were stained with crystal violet. The result was determined by counting the stained cells using optical microscopy (200 × magnifications) in five randomly selected fields. Each experiment was carried out in triplicate wells and repeated at least three times.
Western blot analysis
Western blot analysis was performed to detected the levels of COMP (ab11056, Abcam, Cambridge, UK), CD36 (ab133625, Abcam), E-cadherin (3195, Cell Signaling Technology, Danvers, USA), N-cadherin (14,215, Cell Signaling Technology), Vimentin (5741, Cell Signaling Technology), MMP-2 (13,132, Cell Signaling Technology), MMP-9 (sc-393,859, SANTA CRUZ), Snail (ab167609, Abcam), Slug (9585, Cell Signaling Technology), Twist (ab175430, Abcam), AKT (4691, Cell Signaling Technology), P-AKT (Thr308) (13,038, Cell Signaling Technology), ERK (5013, Cell Signaling Technology), P-ERK (4370, Cell Signaling Technology), ki-67 (ab15580, Abcam), α-SMA (ab5694, Abcam), β-actin (sc-47,778, SANTA CRUZ). Cells treated with rCOMP (0, 1, 2 and 5 μg/ml) were planted in 6-well plates for 24 h or 48 h, and lysed in lysis buffer (Invitrogen). Protein concentration was determined by the BCA Kit (Pierce, IL, USA) and 40 μg of each samples was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). After incubated with primary antibodies overnight at 4 °C, the membranes were then incubated with the appropriate HRP-conjugated secondary antibody for 1 h at room temperature and visualized using the Bio-Rad Gel imaging system and analyzed by the software program as specified by Bio-Rad.
Immunohistochemical analysis (IHC) and immunofluorescence (IF) analysis
The tumor tissue sections embedded in paraffin were incubated with ki-67 (1:200), CD36 (1:200), CD36 (1:200), E-cadherin (1:200), N-cadherin (1:200) and Vimentin (1:200) antibodies.
For immunofluorescence staining, treated cells were stained with E-cadherin (1:100; Cell Signaling Technology), Vimentin (1:100; Cell Signaling Technology) overnight at 4 °C, followed by incubation with corresponding FITC-conjugated secondary antibody (Invitrogen) for 1 h at room temperature. Cells were quantified by confocal immunofluorescence microscopy (Zeiss, Oberkochen, Germany).
Cell transfections
For CD36 stable knockdown assay, lentiviral containing short hairpin RNAs specially targeting CD36 (shCD36, sense: 5’-GUACCCUGUUACUACCACAdTdT-3′, antisense: 5’-UGUGGUAGUAACAGGGUACdTdT-3′) and the scramble control short hairpin RNA (shCtl) cloned were purchased from GeneChem Corporation (Shanghai, China) and transfected into SMMC-7721 cells using Lipofectamine 2000 according to the manufacturer’s instructions. Experiments were conducted 48 h and knockdown efficiency was verified by Western blot.
For COMP knockdown assay in LX2 cells, small interfering RNA (siRNA) specific to COMP (siRNA1: sense: 5’-AGAAACUUGAGCUGUUGAUGCC-3′, antisense: 5’-GGCUAUCAAGACAGCUCAAGUUUCU-3′; siRNA2: sense: 5’-GAGACAAGATCGACGTGTGTC-3′, antisense: 5’-GACACACGTCGATCTTGTCTC-3′) and the scramble siRNA (NC siRNA) were purchased from Biomics Biotechnologies (Guangzhou, PR China). The cells were plated into 6-well plates and then transfected with 100 nM siRNA using Lipofectamine 2000 (Invitrogen, Eugene, OR, USA) according to the manufacturer’s instructions. Cells were collected for further investigation at the indicated hours after transfection.
Animal experiments
All animal experiments were conducted in compliance with ethical regulations and approved by the ethical committee of animal care of the Xi’an Jiaotong University, Xi’an, China. For the in vivo tumor formation, 10 female BALB/C nude mice aged 4 weeks (Shanghai SLAC Laboratory Animal Center of Chinese Academy of Sciences, Shanghai, China) were used to establish subcutaneous tumor model. 1
× 10
6 SMMC-7721 cells, which were used in our in vitro experiments, were suspended in 100 μl PBS and incubated with rCOMP (2 μg/ml) at 37 °C for 2 h prior to injection. Then SMMC-7721/rCOMP and SMMC-7721/PBS were subcutaneously injected into the left and right (control) flanks of mice. Tumor growth was monitored by estimating the tumor volume by the formula “a/2
× b
2”, in which a and b represents the largest and smallest diameters, respectively. The mice were sacrificed at day 7. This animal model was applied in our previous publication [
19].
For the effect of COMP on pulmonary metastasis was examined by intravenous tail veil injections experiment of 1 × 10
6 SMMC-7721 cells with or without 2 h rCOMP pre-incubation. In addition, experimental animals (
n = 6/group) received either rCOMP (1 mg/kg/d) or PBS 3 times per week by tail vein injections. The dose was calculated based on the cutoff value of COMP level in human [
20]. The mice were sacrificed 4 weeks and the lung metastases were confirmed by H&E staining.
For assessment of the function of CD36 in vivo, an orthotopic liver tumor model in nude mice was established. Briefly, 1
× 10
6 SMMC-7721-shCD36 and SMMC-7721-shCtl cells (
n = 5/group) were suspended in 100ul PBS and incubated with rCOMP for 2 h subcutaneously injected into nude mice liver as described [
21]. Subsequently, these mice were injected with rCOMP at doses of 1 mg/kg/d or PBS through tail vein, 3 times per week for the duration of the experiment. After 4 weeks the livers and lungs were collected and prepared for further analysis.
Transwell coculture system
Hep-3B or SMMC-7721/LX2 cocultures were conducted in serum-free DMEM for 24 h using transwell inserts (6.5 mm diameter with polycarbonate membrane filters containing 0.4 μM pores, Corning, NY) which allow diffusion of media components but prevent cell migration (Corning Inc., NY). HCC cells were collected for further investigation.
Statistical analysis
All the data were expressed as mean ± standard deviation (SD) of 3 independent experiments. Prism 5 and SPSS 13.0 software were applied for all statistical analyses. Pearson chi-square test, ANOVA and Student’s t-test were used for comparison between multiple or two groups. Kaplan-Meier method with Log-rank test were used for survival analysis. Univariable and multivariable survival analyses were performed by Cox proportional hazards regression model. *P < 0.05 and **P < 0.01 were taken as indicative of statistically significant difference.
Discussion
The process of viral hepatitis-cirrhosis-HCC is the main epidemiological development path of HCC in world. Most (80%) of HCCs originate from severe liver fibrosis or cirrhosis [
2]. Increased hepatic matrix stiffness results from the deposition and cross-linking of large amounts of matrix proteins, not only extensively occurs in most solid tumors but also promotes cell growth, motility, proliferation, metabolism and tumor metastasis [
25‐
28]. HSCs, multifunctional hepatic stromal cells, differentiate into fibrogenic, hyperproliferative, contractile, and migrating myofibroblasts in chronic liver disease. In fibrosis and cirrhosis, these myofibroblastic HSCs are the culprits for many abnormal ECM deposits. Several publications revealed that COMP was involved with process of cirrhosis and HCC progression. However, the exact sources and functions of COMP still remain to be fully elucidated in HCC-related literature. In this regard, we demonstrated for the first time that COMP was mainly derived from activated HSCs and dose-dependently promoted HCC growth and metastasis. COMP induced CD36-dependent activation of MEK/ERK and PI3K/AKT, and a panel of tumor-promoting factors, including EMT makers, MMP-2/9, Slug and Twist, so as to promote its tumor-promoting effects. Our data illustrated a novel signal transduction pathway for metastatic growth of HCC (Fig.
7e).
In the present study, we identified that the level of COMP was frequently elevated in the serum of HCC patients. Patients with high level of serum COMP showed more unfavorable disease parameters such as higher incidence of vascular invasion and tumor recurrence. Additionally, HCC patients with high serum COMP level had a poorer prognosis than those with low serum COMP level. These results suggest that COMP may play important oncogenic roles in HCC progression which is consistent with previous report [
5]. Cell proliferation and migration have been reported to involve various growth factors, which bind to their receptors on the cell surface to activate downstream signaling pathways, leading to cytoskeletal reorganization and stimulation of cellular motility machinery [
29]. Here the oncogenic effects of COMP on HCC pathogenesis were directly demonstrated in the current study by both in vitro and in vivo functional assays. Our finding indicated that either exogenous COMP treatment or HSCs coculture stimulated malignant behaviors, such as proliferation, invasion and migration of HCC cells. In both subcutaneous xenografts and the tail vein injection model, rCOMP group generated bigger primary tumors and more lung metastatic foci, indicating that COMP enhanced aggressive and metastatic properties of HCC. Besides, COMP did not affect cell apoptosis of HCC cell lines (data not shown). To our knowledge, this is the first report that COMP acts as a driver of HCC proliferation and metastasis.
The acquisition of invasive capabilities includes degradation of the cell matrix and turnover of cell-cell adhesion junctions [
30]. Down-regulation of E-cadherin is a significant hallmark of EMT. In our study, rCOMP treatment in HCC cells led to the up-regulation of Slug/Twist, N-cadherin and Vimentin, and repressed expression of E-cadherin and thereby triggered EMT. It has been demonstrated that MMP-2/9 can regulate the degradation of the extracellular matrix (ECM), which plays an important role in cancer metastasis [
31]. Our study also observed that COMP advanced the expression level of MMP-2/9. Therefore, this study clearly demonstrates that COMP functions as a metastasis inducer in HCC through promoting EMT via regulation of Slug/Twist and inducting matrix degradation.
Both the MER/ERK and PI3K/AKT signaling pathway are involved in the regulation of tumor cell growth, metabolism, proliferation, as well as metastasis and are frequently proved to be active in many different types of cancer [
32]. A recent report indicated that COMP could promote the process of liver fibrosis through MEK/ERK signaling pathway [
4]. In our research, both the phosphorylation level of ERK and AKT were dramatically induced by rCOMP. EMT markers, MMP-2/9 and Slug/Twist are well-known downstream regulators of MEK/ERK and PI3K/AKT signaling pathways. Therefore, as expected, the expression of these proteins were suppressed when ERK or AKT pathway were inhibited. Besides, a crosstalk between AKT and ERK signaling pathways could be observed in the results, which was consistent with other studies [
33,
34]. Taken together, our data confirmed the pro-proliferative and pro-invasive effects of COMP in HCC.
It has been shown that CD36 is up-regulated in human HCCs and involved in EMT [
10]. Thus, we found that knockdown of CD36 attenuated rCOMP-induced proliferation, migration and invasion of HCC cells. Moreover, down-regulation of CD36 led to severe inhibition of rCOMP-induced tumor growth and lung metastasis of HCC in vivo. Therefore, we conclude that CD36 is essentially required for COMP/ERK and COMP/AKT induced HCC progression. To our knowledge, this is the first report that COMP connects with CD36 to stimulate HCC metastasis, in addition to tumor proliferation and growth. However, further studies will be necessary to expand our knowledge of the relationship between COMP and CD36.
In HCC, metastatic microenvironment is consisted of hepatoma cells, activated hepatic stellate cells, extracellular matrix, and their secreted or released various cytokines to regulate tumor metastasis. These cells and matrix components interact in the presence of various cytokines and participate in the process of hepatocellular carcinoma metastasis [
35]. It has been increasingly recognized that activated HSCs act as important contributors to tumor progression [
36]. Identifying the critical pathway involved in this crosstalk could potentially improve the efficiency of treatment. In this study, we detected the expression of COMP in activated HSCs LX2 and several HCC cells and hepatocytes LO2. The result indicated that the level of COMP in LX2 cells and its supernatants was the highest. These data suggested that COMP might be primarily secreted by HSCs. To support this hypothesis, we further established hepatocytes-HSCs crosstalk to analyzing the role of COMP in HCC microenvironment. Interestingly, the MEK/ERK and PI3K/AKT pathways were activated in HCC cells by coculture of activated HSCs and hepatoma cells, along with the upregulation of their downstream factors. Furthermore, above factors were deregulated in Hep-3B and SMMC-7721 cells respond to the coculture with LX2 after knockdown of COMP. Thus, the data suggest that COMP plays an important role in the dynamic interactions between cancer cells and activated HSCs in the progression of hepatocellular carcinoma.