Differential expression of CCN family members CYR611, CTGF and NOV in gastric cancer and their association with disease progression
- Authors:
- Published online on: September 8, 2016 https://doi.org/10.3892/or.2016.5074
- Pages: 2517-2525
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Copyright: © Li et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Gastric cancer (GC) is the fourth most commonly diagnosed cancer in males and the fifth in females worldwide, with over 751,600 new cases and 723,100 deaths estimated to have occurred in 2012 (1). The highest incidence rates are found in Eastern Asia (particularly in Korea, Mongolia, Japan and China), Central and Eastern Europe, and South America, and the lowest incidences are noted in Northern America and most parts of Africa. The incidence is twice higher in males than in females (1).
The tumor microenvironment plays a pivotal role in tumourigenesis and subsequent dissemination of cancer cells by coordinating morphological transformation of cancer cells and their proliferation, survival and invasion (2,3). Recent reviews have highlighted a profound role played by a group of proteins in the tumour microenvironment which belong to the CCN family. CCN is an acronym for cysteine-rich protein 61 (CYR61), connective tissue growth factor (CTGF) and nephroblastoma overexpressed (NOV) which comprises CYR61, CTGF, NOV and another three members i.e. Wnt1-inducible signalling pathway protein (WISP)-1, -2 and -3 (4–6).
CCNs are matricellular proteins and can be secreted into the extracellular matrix. CCN3 can be detected in the culture medium, extracellular matrix (ECM) and at the cell membrane (7–9). The CCN family proteins have been preserved during evolution which can be found in vertebrates including zebra fish, chickens, mice, rats and humans. CCN proteins generally have an N-terminal secretory signal peptide and four functional domains: an insulin-like growth factor binding protein domain (IGFBP); a Von Mayebrand factor type C (VWC) domain; a thrombospondin type-1 repeat module (TSP-1) and a cysteine knot domain (CT). The two N-terminal domains are separated from the two C-domains by a linker with a variable sequence of amino acids. However, CCN5 does not have the CT domain (10).
An elevated expression of CCN1 has been demonstrated in a variety of malignancies including ovarian, prostate, breast and colorectal cancers (11–14). Similarly, an overexpression of CTGF has been noted in breast and colorectal cancer, oesophageal squamous cell carcinoma and pancreatic cancer (15–18), while increased expression of NOV has been shown in prostate and cervical cancers (19,20). In contrast to these findings, reduced expression of these three CCNs has also been observed in certain solid tumours. For example, CYR61 expression is reduced in endometrial cancer and lung cancer (21,22). A reduced expression of CYR61 has also been seen in advanced GC (23). CTGF can act as an inhibitor in lung cancer by suppressing proliferation of non-small cell lung cancer cells (24). A similar inhibitory effect on the proliferation of cancer cells has also been evident for the NOV protein in gliobastoma and malignant adrenocortical tumour cells (25,26). This suggests that the expression and function of CCNs in malignancies can be organ- or tissue-specific.
Early studies have demonstrated a profound role played by CCNs in fibrosis which has been reviewed most recently by Riser et al (6). Their involvement in fibrotic disorder stimulated research interest concerning their implication in a specific histologic type of gastric carcinoma, i.e. scirrhous carcinoma which is well known for a vast fibrous stroma, rapid and invasive growth and poor prognosis. Tanaka et al identified a novel variant of WISP1 that was highly expressed in scirrhous carcinomas. This variant lacks the von Mayebrand type C module and is named WISP1v. WISP1v can induce transformation and promote proliferation and invasion of GC cells through both autocrine and paracrine pathways (27). Tanaka et al also reported a loss of function mutation of WISP3 with a frequency of 10–20% in microsatellite unstable gastric carcinoma. This mutation resulted in a truncated variant of WISP3 that was lacking the TSP-1 and CT domains and was unable to suppress the invasiveness of GC cells (28). Although reduced expression of CYR61 was reported in advanced GC which was inversely correlated with the expression of MMP-7 (23), most studies demonstrated a positive role played by CYR61 in GC by promoting invasion, metastasis and also tumour-associated angiogenesis (29,30). CYR61 promotes GC cell invasion through hypoxia-inducing factor-1α (HIF-1α)-dependent upregulation of plasminogen activator inhibitor-1 (PAI-1) (31).
Elevated expression of CYR61 and CTGF has been observed in GC which is associated with lymph node metastasis, however the expression of NOV in GC is yet to be revealed. The present study aimed to determine the expression of these three CCNs in a cohort of GC tumours, in particular, by dissecting the role played by NOV in GC.
Materials and methods
Cell lines and culture conditions
Human GC cell lines AGS and HGC27 were purchased from the European Collection of Cell Cultures (ECACC; Salisbury, UK) and incubated at 37°C, with 5% CO2 and 95% humidity. The wild-type cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (PAA Laboratories Ltd., Somerset, UK), penicillin, streptomycin and amphotericin B.
Human gastric tissues
Gastric adenocarcinoma and Siewert type III gastrooesophageal junction adenocarcinoma tissues (n=245) with matched adjacent background tissues (n=158) were immediately collected after surgical resection at the Beijing Cancer Hospital with informed consent from the patients. All patients underwent surgery without any prior treatment. The tissue samples were stored at −80°C at the Tissue Bank of Peking University Oncology School with a record of the relevant clinical and histopathological data. All protocols were reviewed and approved by the Beijing Cancer Hospital Research Ethics Committee (MTA10062009).
RNA isolation, reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qPCR)
RNA was extracted from confluent cells in a 25 cm2 flask using total RNA isolation (TRI) reagent (Sigma-Aldrich, Dorset, UK). Fresh frozen tissues were also first homogenised in the TRI reagent. First strand of cDNA was synthesised from 1 µg RNA using a first-strand DNA synthesis kit (Bio-Rad, Hemel Hempstead, UK). Quantitative analysis of NOV mRNA expression in GC tissues was performed using Amplifluor™-based real-time PCR, in which a 6-carboxy-fluorescine-tagged Uniprimer™ (Biosearch Technologies, Inc.) was used as a probe along with a pair of specific primers with an addition of a Z-sequence (actgaacctgaccgtaca) to the 5′-end of the reverse primer. The quality of cDNA samples was verified using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a housekeeping gene. All the primer sequences are listed in Table I.
Quantitative analyses of transcript expression of CYR61, CTGF and NOV in human GC
Following the real-time PCR quantification of each gene transcripts, the number of samples with valid data for each individual genes were: 322 samples for NOV, 252 samples for CYR61 and 320 samples for CTGF. These cohorts were composed of 230 men (71.4%) and 92 women (28.6%) in the NOV cohort, 180 men (71.4%) and 72 women (28.6%) in the CYR61 cohort, and 228 men (71.3%) and 92 women (28.7%) in the CTGF cohort. Data are shown in Tables II–IV.
Construction of the ribozyme transgene targeting human NOV and the establishment of corresponding stable transfectants
Anti-human NOV hammerhead ribozymes were designed using the Zuker RNA mFold program (Zuker 2003). The ribozymes and full-length coding sequence of human NOV were cloned into a pEF6/V5-His TOPO vector (Invitrogen, Paisley, UK). The verified ribozyme transgenes, NOV expression vectors and empty plasmids were transfected into AGS and HGC27 cells, respectively, using an Easyjet Plus electroporator (EquiBio, Kent, UK). After one week of selection with 5 µg/ml blasticidin, the selected cells were maintained in DMEM with 0.5 µg/ml blasticidin.
Western blot analysis
The protein concentrations in the cell lysates were determined using the DC protein assay kit (Bio-Rad) and the ELx800 spectrophotometer (Bio-Tek, Winooski, VT, USA). Proteins were probed with the anti-NOV antibody (1:2,000; Abcam, Ltd., Cambridge, UK) and anti-GAPDH antibody (1:2,000; Santa Cruz Biotechnology, Inc.) as housekeeping gene control, followed by a peroxidase-conjugated secondary antibody (1:2,000; Sigma). Protein bands were visualised using a chemiluminescence detection kit (Luminata, Millipore Ltd., Watford, UK) and photographed using Syngene imager (Syngene International Ltd., Bangalore, India).
In vitro cell growth assay
Cells (3,000) were seeded into 96-well plates in normal culture medium. The cells were incubated over a period of up to 4 days, and were then fixed with 4% formaldehyde followed by staining with 0.5% crystal violet. The crystal violet was dissolved in 10% acetic acid prior to a colorimetric detection of cell density at a wavelength of 540 nm using the ELx800 spectrophotometer.
In vitro invasion assay
The in vitro invasion assay was previously described (32). All the culture plate inserts containing 8-µm pores were pre-coated with 50 µg of Matrigel (BD Bioscience, Oxford, UK) and air-dried. Cells (30,000) were seeded to each well after a 40-min rehydration of the Matrigel. After an incubation of 72 h, cells that had migrated through the matrix to the other side of the insert were fixed and stained. The cell number was determined.
Cell-matrix adhesion assay
The cell-matrix adhesion assay was conducted as previously described (32). The 96-well culture plate was pre-coated with 5 µg of Matrigel and air-dried. Following the rehydration, 30,000 cells were seeded to each well. After an incubation of 40 min, non-adherent cells were washed-off using phosphate-buffered saline (PBS). The adherent cells were counted after a fixation and staining using crystal violet.
Wound healing assay
The assay was performed following a previously described procedure (32). In brief, the monolayer of cells was scraped with a 10 µl pipette tip. The migration of cells was photographed using a time-lapse image system (EVOS, Life Technologies Ltd., Paisley, UK).
Statistical analysis
Statistical analyses were performed using SPSS (version 11; SPSS, Inc., Chicago, IL, USA). Mann-Whitney U test and t-test were used for non-parametric and normally distributed data, respectively, including research data from the clinical cohort and cell-based experiments. Kaplan-Meier survival analysis was also performed using SPSS statistical software. Differences were considered to be statistically significant at p<0.05.
Results
Increased expression of NOV in human GC
Transcript levels of CYR61, CTGF and NOV were determined in the GC cohort using real-time PCR, respectively. The results showed that NOV expression was significantly upregulated in gastric tumours compared to normal tissue (p=0.009) (Table II). An increased expression of NOV in GC was associated with local invasion. The transcript levels of NOV were higher in the tumours with more advanced local invasion. According to the tumor-node-metastasis (TNM) staging, T4 tumours which invaded the serosa or adjacent structures expressed higher levels of NOV transcripts, p=0.0026 vs. T1. Tumours classified as T3 and T4 which invaded beyond subserosal connective tissues expressed higher levels of NOV transcripts in comparison with tumours (T1 and T2) with less local invasion (p=0.0013). According to the overall TNM staging, stage I GCs exhibited lower expression levels of NOV, p=0.016 vs. stage II, p=0.0017 vs. stage III, and p=0.0007 vs. tumours of stage II-IV. Notably, we found that high-moderately differentiated tumours exhibited significantly lower levels of NOV expression in comparison with moderately and/or poorly differentiated tumours. However, no association was observed for lymph node and distant metastases.
CYR61 is upregulated in GCs
CYR61 mRNA levels were significantly elevated in the GC tissues compared to levels in the non-cancerous tissues (p=0.0019), particularly in paired tissues (p=0.0013) (Table III). However, the expression of CYR61 transcripts appeared to be lower in more advanced tumours (stage III and IV) according to the TNM staging, although it did not reach a statistically significant level. According to clinical outcomes, tumours with distant metastases had lower expression levels of CYR61 (p=0.055) compared with that of patients who remained disease-free. No association was observed between CYR61 expression and differentiation and local invasion.
Higher expression of CTGF in GC and the involvement in local invasion
Although no statistically significant differences were noted for CTGF mRNA levels in GCs compared with adjacent normal gastric tissues, a higher transcript level of CTGF in GCs was positively associated with local invasion (T4 vs. T1, p<0.001; T3 vs. T1, p=0.031; Table IV). However, there were no other significant correlations between CTGF mRNA levels and other clinical parameters.
Expression of CYR61, CTGF and NOV and survival of patients with GC
Kaplan-Meier survival curves revealed that GC patients with a low CYR61 transcript level had longer overall survival (OS) (p=0.018) and disease-free survival (DFS) (p=0.015) than those with a higher CYR61 transcript level (Fig. 1). Transcript levels of CTGF and NOV exhibited no correlation with either OS or DFS when individually analysed; however, analysis of the combination of CYR61 and CTGF showed that patients with lower transcript levels of these two genes had longer OS (p=0.033) and DFS (p=0.025). Similarly, analysis of combined CYR61, CTGF and NOV showed that patients with higher transcript levels of all three genes had a poorer OS (p=0.027) and DFS (p=0.021) compared to patients with lower expression of all these genes.
Knockdown and overexpression of NOV in GC cells
The expression profile of NOV in AGS and HGC27 cell lines was assessed using RT-PCR (Fig. 2A). For assessing the effect of NOV on cellular functions, overexpression of NOV was performed in the AGS cells which had an almost undetectable level of NOV as determined using PCR, while knockdown of NOV was carried out in the HGC27 cells which highly expressed NOV. The overexpression and knockdown of NOV in AGS and HGC27 transfectants were confirmed using RT-PCR (Fig. 2B) and western blotting (Fig. 2C).
Effect of NOV knockdown and overexpression on cell growth in vitro
Overexpression of NOV increased the growth of AGS cells over the periods of 3 (p<0.01) and 4 days (p<0.001) compared with the control cells. An opposite effect was observed in the HGC27 NOV-knockdown cells over the periods of 3 (p<0.01) and 4 days (p<0.01) (Fig. 3).
Effect of NOV knockdown and overexpression on the invasion, adhesion and migration of GC cells
Overexpression of NOV resulted in increased invasion in the AGS cells, while NOV knockdown exhibited reduced invasiveness in the HGC27 cells (p<0.001) (Fig. 4). Knockdown and overexpression of NOV did not enhance or reduce the adhesion of the GC cells to Matrigel in comparison with the control cells. A wound healing assay was employed to determine the influence of NOV on cell migration. There was no obvious effect observed in our experiments (data not shown).
Discussion
This is the first study to assess the role played by NOV (CCN3) in gastric cancer (GC). In the present study, we determined the expression of NOV, along with CYR61 and CTGF transcripts in a cohort of human GCs and paired adjacent normal gastric tissues. Increased expression of NOV was observed in the GC tissues compared with the expression noted in the adjacent normal gastric tissues. Increased expression was found to be associated with local invasion and poorer differentiation. Elevated expression of NOV in GC is consistent with observations in other cancers. For example, overexpression of NOV is evident in prostate cancer and cervical cancer and is involved in tumourigenesis and disease progression (19,20). Overexpression of NOV in cervical cancers was found to be associated with lymph node metastases and poorer prognosis of cervical cancer patients (20). This suggests that NOV is positively involved in tumourigenesis and disease progression by regulating morphological transformation and the invasiveness of GC cells. Our previous study showed reduced expression of NOV in breast cancer which was associated with poor prognosis and mortality of the disease (15). Differential expression patterns and roles can be played by NOV in different malignancies which can be organ- or tissue-specific.
In the present study, we also determined the transcript levels of CYR61 and CTGF in GC samples. Similarly, overexpression of CYR61 transcripts was noted in GC although no difference was observed for the expression of CTGF. No association was clearly evident for elevated expression of CYR61 and disease progression in the present cohort of GC cases. Overexpression of CYR61 has been observed in many different cancers, including prostate, breast, ovarian, endometrial and colorectal carcinomas (11,12,33–35). In contrast to the elevated expression of CYR61 in these malignancies, reduced expression has also been noted in various types of cancers, in particular GC which is associated with local invasion (23). In the present study, a trend of reduced expression of CYR61 was also noted in the more advanced GCs appearing to be in line with observations made by Maeta et al (23). A controversy may still exist for its role in GC as experimental evidence using GC cell lines indicates that CYR61 can promote invasion, metastasis and angiogenesis in GC (29–31). The present study also showed a link between higher CYR61 expression and poorer survival outcomes of patients with GC. This was also reflected in the survival analysis of the combined expression of CYR61 with CTGF, or CTGF and NOV (Fig. 1). CTGF and NOV alone or combined did not exhibit any correlation with survival (data not shown). This suggests that a more profound role is played by CYR61 in GC which is also supported by studies of this molecule in GC (30,36). In a comparison with NOV expression and corresponding association between elevated NOV expression and local invasion and poorer differentiation of GCs, the increased expression CYR61 exhibited little implication with these clinicopathological features of the disease apart from its correlation with poor prognosis. A larger cohort of GC tissue samples may help to clarify this. Better understanding of the molecular mechanisms of GC may shed light on this issue in the near future.
Although the expression of CTGF transcripts in the present cohort of GCs was not different from its expression in the paired adjacent normal gastric tissues, its expression was increased in more invasive tumours. This tends to concur with observations from other studies focusing on this molecule and its role in GC. Higher expression of CTGF in GC exhibits involvement in local lymph node metastasis and also peritoneal metastasis (37,38). Suppression of CTGF inhibits the growth and invasion of GC cells, and also their peritoneal dissemination (39). In addition to its role in invasion and metastases, CTGF also promotes angiogenesis to facilitate tumour growth (40).
In addition to the evaluation of NOV, CYR61 and CTGF transcripts in the GC samples, we further examined the impact of NOV on cellular functions in GC cell lines. The two GC cell lines examined in the present study exhibited differential expression of NOV, where NOV was highly expressed in the HGC27 cells and almost absent in the AGS cells. This allowed us to establish contrasting models, i.e. overexpression of NOV in AGS cells and knockdown of NOV expression in HGC27 cells, for examining the consequent effect on the cellular functions. NOV overexpression promoted the in vitro proliferation of AGS cells while the knockdown resulted in reduced proliferation of the HGC27 cells. A similar effect was observed in regards to the invasion of these two GC cell lines. These results indicate a positive role played by NOV in promoting proliferation and invasion of GC which is consistent with its increased expression in the GC tumour samples. Increased expression of NOV has also been observed in other malignancies, such as cervical and prostate cancer (19,20). Certainly, an inhibitory effect on cellular functions has also been noted for NOV in a variety of cancer cells. For example, NOV had an anti-proliferative effect on glioblastoma cells by interfering with S/G2 transition of the cell cycle leading to an accumulation at the S phase (25). Gap junction protein connexin 43 was also found to be involved in its inhibitory effect on the proliferation of glioma cells (41). NOV has also been reported to decrease the transcription and activation of matrix metalloproteinases and suppress the invasion of melanoma cells (42). However, our in vitro experimental data indicate that NOV promotes both proliferation and invasion of GC cells. Further investigation may shed light on the underlying molecular mechanisms for such a differential impact.
In summary, the expression of NOV and CYR61 was increased in GC. The elevated expression of CYR61 was associated with poorer survival, and NOV promoted the proliferation and invasion of GC cells.
Acknowledgments
The authors thank Cancer Research Wales for supporting the present study. Dr Li and Dr Gao are recipients of the Chinese Medical Research Scholarship of Cardiff University.
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