Background
With an estimated 455,800 new cases and 400,200 deaths each year, oesophageal cancer is the sixth leading cause of cancer death and the eighth most common cancer worldwide [
1]. Oesophageal squamous cell carcinoma (ESCC) is the predominant histological type both in China and around the world. Despite advancements in population screening and standardized multidisciplinary treatment over the last four decades [
2], our previous report showed that ESCC remains the fourth leading cause of cancer-related death in China [
3], with a dismal 5-year survival rate of only 20.9% [
4]. The poor outcome of patients is primarily attributed to the high rate of ESCC metastasis, including both regional lymph node and further distant metastases [
5]. Therefore, it’s vital to identify the underlying molecular mechanisms that drive progression, especially metastasis, of ESCC for predicting patients’ prognosis and improving rational design of personalized medicine.
Collagen triple helix repeat containing 1 (CTHRC1) is a secreted glycoprotein that can reduce collagen matrix deposition and promote the mobility of fibroblasts and smooth muscle cells [
6‐
8]. Indeed, overexpression of CTHRC1, which has been reported in various malignancies, is suggested to serve as an independent prognostic factor [
9‐
12]. Recently, a germline mutation in CTHRC1 gene was identified to be associated with Barrett’s oesophagus and oesophageal adenocarcinoma [
13], and high CTHRC1 expression in ESCC was revealed by expression profiling studies involving a small number of cases [
14,
15]. However, these findings should be confirmed in studies including larger groups and the cellular function and clinical implications of CTHRC1 in ESCC need to be resolved.
In this study, we sought to confirm in a much larger cohort aberrant elevated expression of CTHRC1 and to investigate its association with clinicopathological characteristics in ESCC. To assess the effect of CTHRC1 on malignant phenotypes of ESCC cells in vitro and in vivo, we then established multiple cell lines with stable depletion or overexpression of CTHRC1. Furthermore, we defined the underlying signalling pathways and transcription factors that depend on CTHRC1 activation and are responsible for ESCC progression.
Methods
Patients and tissue specimens
The study design and use of clinical samples were approved by the Ethics Committee of Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College. A total of 204 formalin-fixed and paraffin-embedded (FFPE) ESCC tissue samples were obtained with informed consent and agreement from the biobank of Cancer Hospital of Chinese Academy of Medical Sciences. From 2000 to 2008, specimens were surgically resected from patients with stage I-III ESCC and who did not receive preoperative treatment. The clinicopathological characteristics of these patients are summarized in Table
1. Five tissue microarrays (TMAs) were constructed by incorporating one representative core of each tissue. The microarrays contained 204 primary ESCC tumour tissues, 169 of which were accompanied by adjacent non-tumour epithelial tissues.
Table 1
Correlations between CTHRC1 levels in ESCC tissues and clinicopathological characteristics of patients with ESCC
Age | ≤60 | 49 | 67 | 0.128 |
>60 | 28 | 60 |
Gender | Male | 62 | 106 | 0.593 |
Female | 15 | 21 |
Tobacco use | No | 27 | 45 | 0.957 |
Yes | 50 | 82 |
Alcohol use | No | 30 | 46 | 0.695 |
Yes | 47 | 81 |
Family history | No | 65 | 106 | 0.858 |
Yes | 12 | 21 |
Location | Upper/Middle | 39 | 60 | 0.637 |
Lower | 38 | 67 |
Histology grade | G1/G2 | 59 | 94 | 0.677 |
G3 | 18 | 33 |
T stage | T1/T2/T3 | 44 | 54 |
0.043
|
T4 | 33 | 73 |
Lymph node metastasis | No | 49 | 60 |
0.023
|
Yes | 28 | 67 |
TNM stage | I/II | 39 | 44 |
0.024
|
III | 38 | 83 |
FRA-1 | Low | 22 | 28 | 0.116 |
High | 45 | 97 |
Snail1 | Low | 39 | 53 | 0.100 |
High | 33 | 73 |
MMP14 | Low | 45 | 58 |
0.022
|
High | 26 | 67 |
Cyclin D1 | Low | 53 | 66 |
0.018
|
High | 24 | 61 |
Immunohistochemistry and scoring
Immunohistochemistry (IHC) was performed as previously described [
16], using anti-CTHRC1 (ab192778, Abcam, USA), anti-FRA-1 (TA500624S, Origene, USA), anti-cyclin D1 (2978, CST, USA), anti-snail1 (TA500316S, Origene, USA) and anti-MMP14 (ab51047, Abcam, USA) antibodies. Slides were evaluated independently by two pathologists (S.S. & X.F.). The staining intensity was graded as 0 (negative), 1 (low), 2 (moderate) or 3 (high), and the proportion of staining was evaluated as 0 (negative), 1 (<10%), 2 (10–50%), 3 (51–80%), or 4 (>80%). The intensity and proportion scores were multiplied to generate the IHC index. The expression level was considered as low (IHC index < 6), and as high (IHC index ≥ 6).
Cell culture
All cell lines used in this study were regularly authenticated by short tandem repeat (STR) profiling. KYSE510, KYSE30, KYSE450, KYSE180 and KYSE70 cells were cultured in RPMI 1640 medium supplemented with 10% foetal bovine serum, 100 UI/ml penicillin and 100 UI/ml streptomycin (Gibco, USA). Het1a, a non-malignant immortalized human oesophageal squamous cell line, was cultured in BEGM (Bronchial Epithelial Cell Growth) medium (Lonza, USA). All cell lines were maintained in a humidified incubator at 37 °C and 5%CO2.
Transfection and stable cell line establishment
Small interfering RNA (SiRNA; Dharmacon, USA) and plasmid transfections were performed using Lipofectamine RNAiMAX Transfection Reagent and Lipofectamine 3000 (Invitrogen, USA), respectively. For silencing of CTHRC1, two short hairpin RNA (shRNA) oligonucleotides (5’-GCTATCTGGGTTGGTACTTGTTTCAAGAGAACAAGTACCAACCCAGATAGCTT-3’ and 5’-GCTTCTACTGGATGGAATTCATTCAAGAGATGAATTCCATCCAGTAGAAGCTT-3’) were cloned into the pLKD-CMV-R&PR-U6-shRNA vector (Heyuan, China). The negative control (NC) sequence was 5’-TGTTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAGAACTT-3’. For overexpression, the coding DNA sequence (CDS) of CTHRC1 was cloned into the pLenti-EF1a-EGFP-P2A-Puro-CMV-MCS vector (Heyuan, China); the empty vector was used as the negative control. Lentivirus packaging and purification and cell infection were carried out with ViraPowerTM Lentiviral Expression Systems (Invitrogen, USA) according to the manufacturer’s instructions. Cells were selected using medium containing 1.5 μg/ml puromycin (Sigma-Aldrich, USA). The efficiency of knockdown and overexpression were confirmed by real-time polymerase chain reaction (PCR) and western blot.
RNA interference (RNAi) screening
KYSE30, KYSE510 and KYSE70 cells were plated in 96-well plates and transfected in triplicate with on-target plus smartpool siRNA (Dharmacon, USA). After 72 h, the cells were stained with 4’,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, Germany). Then the samples were imaged using a high content screening system (Operetta) and analysed using Harmony 3.1 software.
Real-time PCR (RT-PCR)
RT-PCR was performed as previously described [
17]. The primers used are listed in Additional file
1: Table S1.
Western blot
Whole cell lysates were prepared using RIPA buffer supplemented with protease and phosphatase inhibitor cocktail (Thermo, USA) and culture supernatants were concentrated using Microcon centrifugal filters (Millipore, USA). Western blot was performed as previously described [
17]. Primary antibodies against the following proteins were used: CTHRC1 (ab192778, Abcam, USA), p-c-Raf (9427, CST, USA), p-MEK1/2 (9154, CST, USA), p-ERK1/2 (4370, CST, USA), ERK1/2 (4695, CST, USA), p-FRA-1 (5841, CST, USA), FRA-1 (5281, CST, USA), cyclinD1 (2978, CST, USA), snail1 (3879, CST, USA), and MMP14 (13130, CST, USA). α − Tubulin (T9026, Sigma-Aldrich, USA) was used as a loading control.
Cell proliferation and colony formation assays
Cell proliferation and colony formation assays were performed as previously described [
18]. Cell proliferation was assessed using Cell Counting Kit-8 (CCK8). Images of the colony formation assay results were scanned and the clone number was determined using GeneSys software (Genecompany, China).
Boyden chamber Transwell assay
For invasion and migration assays, we used 24-well Boyden chambers precoated with or without Matrigel matrix (Corning, USA), respectively. The experiments were performed as previously described [
19].
All mice used in this study received humane care, and all animal experiments were performed in accordance with the guidelines approved by the Institutional Animal Care and Use Committee of Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College. BALB/c-nu mice and non-obese diabetic (NOD)-SCID mice (female, 4–5 weeks old) were purchased from Huafukang (Beijing, China). For the xenograft model, shRNA- or vector-transfected KYSE510 cells and CTHRC1- or vector-transfected KYSE450 cells were injected into the right dorsal flanks of BALB/c-nu mice (5 × 106 cells per animal, 8 mice per group). Tumour formation was monitored every 5 days by measuring tumour size with a calliper. The tumour volume was calculated using the formula: V = (L × W2)/2. After 4 weeks, all mice were sacrificed, and the tumours were excised and weighed. For the lung metastasis model, shRNA- or vector-transfected KYSE510 cells and CTHRC1- or vector-transfected KYSE450 cells were injected into NOD-SCID mice through the tail vein (1 × 106 cells per animal, 8 mice per group). Ten weeks later, the mice were sacrificed, and the lungs were excised and fixed with Bouin’s solution followed by embedding in paraffin for haematoxylin and eosin (H&E) staining. The number of lung surface metastatic nodules was evaluated by gross and microscopic examination.
RNA sequencing
RNA sequencing was performed using KYSE510-shCTHRC1 and KYSE510-vector cells. Total RNA extraction, quality analysis, cDNA library preparation and sequencing were performed at Novogene (Beijing, China). Raw RNA sequences were mapped to the GRCh37.hg19 genome based on TopHat and assembled using Cufflinks. Relative transcript levels are expressed as “fragments per kilobase of transcript per million mapped” (FPKM). Differentially expressed genes (DEGs) were identified using Cuffdiff. To verify the RNA sequencing data, we assessed the transcriptional level of twenty genes using RT-PCR (Additional file
2: Table S2).
Pyrosequencing assay
The pyrosequencing assay was conducted by QIAGEN Translational Medicine Co., Ltd. (Suzhou, China). The primers used were as follows: F-5’-AGGATAGAGGGGGTTATAAAAAGA-3’ and R-5’- ACTCTAACACATTACAAAACCTTACA-3’.
Statistical analysis
Statistical analyses were performed using Prism GraphPad version 6.0 (GraphPad Software Inc., San Diego, USA). Correlations between mRNA expression levels were analysed using Pearson’s correlation coefficient. A chi square test was performed to determine the association between clinicopathological variables and CTHRC1 expression. Survival analysis was carried out using a log-rank test. A Cox proportional hazards model was used to identify independent prognostic factors. The significance of differences between groups was analysed using two-tailed Student’s t-test and the results are expressed as the mean ± SD. Differences were considered significant when P < 0.05. *P < 0.05 and **P < 0.01.
Discussion
This is the first study to present a comprehensive set of clinical and experimental evidence establishing CTHRC1 as an oncogenic factor that facilitates ESCC tumour progression and metastasis, resulting in poor prognosis. These data indicate that CTHRC1 may serve as a potential prognostic biomarker and treatment target in ESCC.
We also investigated the possible regulation mechanism of
CTHRC1 in ESCC. Treatment with a demethylation agent (5-aza-dC) markedly elevated CTHRC1 expression in most ESCC cell lines, which was in agreement with previous reports [
16,
29,
30]. A pyrosequencing assay revealed a CpG site (cg07757887, -1220 bp in the
CTHRC1 genomic region) hypomethylated in ESCC tumour tissues, which has not been previously reported as being related to cancer. Although demethylation of the
CTHRC1 genomic region (-391 to +4 bp) in gastric cancer cells [
30], in the first exon in colon cancer [
16], and at -628 to -269 of the promoter region in hepatocellular carcinoma [
29] has been reported, we did not find significant demethylation at those CpG sites in ESCC tumour tissues. Therefore, methylation of the CpG site involved in regulating
CTHRC1 may vary in different types of cancer.
Previous reports have suggested that other mechanisms may be involved in regulation of CTHRC1, such as TGF-β and Wnt3a pathway activation in gastric and oral squamous cell carcinoma, respectively [
30,
31]. In addition, CTHRC1 was reported to be regulated by microRNA and long noncoding RNAs, such as let-7b and MALAT-1 [
32,
33], which might explain the oncogenic role of MALAT-1 in ESCC [
34]. Evidence to date supports the hypothesis that CTHRC1 integrates multiple pro-aggressiveness signalling pathways.
We also reveal for the first time that CTHRC1 exerts its effect on ESCC progression mainly through the Raf/MEK/ERK pathway, with dependence on the induction and activation of FRA-1, a FOS family transcription factor that binds to JUN-family proteins to form the AP-1 complex [
35]. Transcriptional induction and post-translational stabilization of FRA-1 via MEK/ERK signalling increases the abundance of FRA-1, which has been causally linked to more aggressive behaviours of multiple cancer cell types [
36‐
40], but not through a CTHRC1-dependent pathway.
There has been accumulating evidence for the significant role of MEK/ERK pathway in cancer development [
41‐
44]. In accordance with the results of our study, the MEK/ERK pathway has been related to CTHRC1 in pancreatic cancer, without identification of any downstream effectors [
45]. Another study suggested that CTHRC1 upregulated MMP9 via ERK activation in colorectal cancer [
16]; however, alteration in
MMP9 expression was not indicated in our RNA sequencing data. Through transcriptome sequencing and extensive step-by-step
in vitro analyses, we identified Cyclin D1 and snail1 as major downstream effectors of FRA-1, accounting for the CTHRC1-mediated regulation of proliferation and motility in ESCC cells. The most prominent function of snail1 in cancer cells is to induce the epithelial-mesenchymal transition (EMT) [
46,
47], and it was recently reported that CTHRC1 upregulated snail1 to induce EMT by activating the Wnt/β-catenin signalling pathway in epithelial ovarian cancer [
48]. Interestingly, we did not observe any meaningful alteration in β-catenin expression or in hallmarks of EMT [
49], namely, E-Cadherin and vimentin, after CTHRC1 knockdown in ESCC cell lines (Additional file
5: Figure S3), suggesting that an alternative hypothesis is needed to explain findings for ESCC. Indeed, a few recent studies invoked other possible mechanisms by which snail1 could regulate cell migration and invasion, such as MMP14-mediated pro-invasive and metastatic activities [
26‐
28]. However, the respective upstream mechanisms were not elucidated. Here, we not only show that MMP14 can be upregulated by snail1 activation, but also demonstrate it under regulation of CTHRC1/MAPK/MEK/ERK/FRA-1 signalling in ESCC.
It should be acknowledged that there was one limitation related to this study: we did not clarify how CTHRC1 activates the MAPK/MEK/ERK pathway. It was recently demonstrated that EGFR inhibitors attenuated the promoting effect of CTHRC1 on epithelial ovarian cancer invasion and that phosphorylation of EGFR and ERK1/2 was reduced in CTHRC1-silenced ovarian cancer cells [
50]. Since CTHRC1 is a secreted protein, it is worth investigating in future studies whether CTHRC1 acts as a ligand of EGFR to activate the MAPK/MEK/ERK pathway in ESCC.
Conclusions
In summary, our findings reveal that CTHRC1 plays a pivotal oncogenic role in ESCC proliferation, invasion, and metastasis by upregulating cyclin D1, snail1 and MMP14 through the Raf/MEK/ERK/FRA-1 pathway. Patients with high expression of CTHRC1 are possible candidates for biologic agents that affect the oncogenic circuit we found in ESCC, such as MEK1/2 inhibitors and CDK inhibitors. Additionally, the newly elucidated clinical implications of CTHRC1 in our cohort support its use as a potential prognostic marker for ESCC patients.
Acknowledgements
We thank Ms. Meihua Xiong, Ms. Fang Zhou, and Ms. Jing Zhang of the Lab of the Department of Thoracic Surgery for administrative and technical help.