Abstract
Background: The nephroblastoma overexpressed (NOV) gene, which belongs to the cysteine-rich, angiogenic inducer 61/connective tissue growth factor/nephroblastoma overexpressed (CCN) family, is located in the 8q24 region and promotes migration and invasiveness in several types of malignancies. We explored the clinical significance of NOV expression in colorectal cancer (CRC). Materials and Methods: NOV expression in CRC specimens and CRC cell lines were evaluated. The association between the clinicopathlogical factors and NOV mRNA expression of tumor tissues was assessed in 126 patients with CRC. We assessed the relationships between NOV expression and overall survival in public databases. We performed overexpression experiments in vitro. Results: CRC specimens and CRC cell lines showed high NOV expression. High NOV mRNA expression was correlated with poorer overall survival and higher Union for International Cancer Control (UICC) T factor. In public databases, high NOV expression was associated with poorer prognoses. Overexpression of NOV promoted invasiveness of CRC cells. Conclusion: NOV may be an indicator of poor prognosis and a therapeutic target in CRC.
Colorectal cancer (CRC) is one of the most common types of cancer worldwide, and it is ranked third in terms of cancer-related deaths, with the majority attributable to distant metastasis (1). Although the 5-year survival rate for patients with local CRC is 80-90%, for those with distant metastasis, it is only 10-20% (2). Therefore, identification of genes that are involved in cancer progression and metastasis is important for improving prognosis of patients with CRC.
The nephroblastoma overexpressed (NOV) gene belongs to the cysteine-rich, angiogenic inducer 61/connective tissue growth factor/nephroblastoma overexpressed (CCN) gene family and is located in the 8q24 region. It encodes a secreted protein that interacts with the extracellular matrix and controls various cellular functions (3, 4). Earlier studies have demonstrated that NOV protein modulates the migration and invasiveness of various cancer cells, and overexpression of NOV is related to poor prognosis in osteosarcoma, higher rates of metastasis in melanoma, and tumor invasiveness of prostate cancer and Ewing's sarcoma (5-10). It has been reported that chromosomal region 8q24 is significant in focal amplification in CRC. Furthermore, we previously reported that oncogenic Single Nucleotide Polymorphisms and copy number amplification of chromosome 8q24 region are associated with a poor prognosis in CRC (11-13).
There is little information regarding NOV gene expression and its clinicopathological significance in CRC, and the molecular mechanism by which NOV affects human CRC is essentially unknown. We postulated that NOV might induce tumor progression and be involved with poor prognosis in CRC, since the NOV gene also belongs to the 8q24 region. In the current study, we assessed the magnitude of NOV expression in patients with CRC, and we discuss how NOV likely contributes to CRC progression.
Materials and Methods
Clinical samples. A total of 126 colorectal cancer samples were obtained during surgery. These samples were used in accordance with the Institutional Ethical Guidelines of Kyushu University after obtaining written informed consent. All patients underwent resection of the primary tumor at the Kyushu University Hospital and affiliated hospitals between 1992 and 2002. None of the patients with the Union for International Cancer Control (UICC) stage III disease received adjuvant chemotherapy. All patients were clearly identified as having colorectal cancer based on the clinicopathological findings. The median and mean follow-up durations were 36.12 and 48.36 months, respectively.
CRC cell line culture. Human CRC cells (WiDr, COLO320, RKO, CAR1, LOVO, DLD-1, HCT116, COLO205 and SW480) were provided by the Japanese Cancer Research Bank (Tokyo, Japan). Cell lines were maintained in RPMI-1640 medium, Dulbecco's modified Eagle's medium or minimum essential medium supplemented with 10% fetal bovine serum and antibiotics. All cells were cultured at 37°C in a humidified atmosphere containing 5% CO2.
Quantitative real-time polymerase chain reaction (qRT-PCR). Gene-specific oligonucleotide primers were designed for PCR. The following primers were used: NOV: 5’-CAGCAACCAGACT GGCATC’ (sense) and 5’-GAATTTGCAGCTTGGCTGA-3’ (antisense); glyceraldehyde-3-phosphate dehydrogenase; (GAPDH): 5’-TTGGTATCGT GGAAGGACTCA-3’ (sense) and 5’-TGTC ATCATATTTGGCAGGTT-3’ (antisense). PCR amplification was performed in a LightCycler 480 instrument (Roche Applied Science, Basel, Switzerland) using LightCycler 480 Probes Master kit (Roche Applied Science). NOV mRNA amplification conditions consisted of initial denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 10 s, annealing at 62°C (60°C for other genes) for 10 s, and elongation at 67°C (65°C for other genes) for 10 s.
Establishment of a stable NOV-transfected colon cancer cell line. To generate lentiviral vectors of NOV expression, we amplified the insert (full-length human NOV; NM_002514.3) by PCR from human reference cDNA. Lentiviruses were produced by transient transfection of HEK293T cells with pCMV-VSV-G-RSV-Rev, pCAG-HIVgp and either CSII-CMV-NOV or CSII-CMV-MCS (empty) plasmid DNAs (5’-Nhe1 and 3’-Xba1 sites) plus Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer's protocol.
Immunoblotting analysis. Total protein was extracted from NOV-expressing cells and mock cells. Aliquots of total protein (40 μg) were electrophoresed in 10% polyacrylamide gels and then electrophoresed and electroblotted on pure nitrocellulose membranes (Trans-Blot Transfer Medium; Bio-Rad Laboratories, Hercules, California, United States of America) at 0.4 A for 120 min. NOV protein was detected with rabbit polyclonal antibody (ab78014; Abcam, Cambridge, UK) diluted 1:2,000. NOV protein levels were normalized to the level of β-actin protein by using a monoclonal antibody for detection at a 1:1,000 dilution (Cytoskeleton, Denver, CO, USA).
Cell proliferation and invasion assays. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (Roche Applied Science) was used to evaluate cell proliferation. The BD BioCoat Tumor Invasion System (8 micron pores; BD Bioscience, San Jose, CA, USA) was used to evaluate invasive capacity following the manufacturer's protocol. Invasive cells that migrated through the membrane were evaluated in a fluorescence plate reader at excitation/emission wavelengths of 485/535 nm. Each independent experiment was performed three times.
Immunohistochemical analysis. Colorectal carcinoma tissues were surgically removed, embedded in paraffin and sectioned (5 μm sections). The sections were then stained with hematoxylin and eosin (H&E) for histological analysis. Immunohistochemical analysis was applied to determine the localization of NOV. A polyclonal rabbit antibody to NOV (ab78014, 1:200; Abcam) was used as the primary antibody.
Statistical analysis. Data from qRT-PCR analyses were assessed using JMP 5 software (JMP, Cary, NC, USA). The relationships between NOV mRNA expression levels and clinicopathological factors were analyzed using the Student's t-test, the chi-square test and ANOVA. Overall survival curves were plotted using the Kaplan–Meier method measured from the day of surgery, and the log-rank test was applied for comparison. All differences were statistically significant at the level of p<0.05.
Meta-analysis. We obtained CRC expression profiles and survival information rates from the Cancer Genome Atlas database (TCGA) including 593 CRC samples and analyzed the association between NOV mRNA expression and probability of survival (14).
Results
Expression of NOV in CRC. We first evaluated NOV protein expression in resected CRC specimens by immunohistochemical analysis in order to confirm that NOV was overexpressed in CRC. NOV staining was remarkably strong in the cytoplasm of cancer cells in CRC tissues (Figure 1A). Moreover, we examined NOV expression in nine CRC cell lines (WiDr, COLO320, RKO, CAR1, LOVO, DLD-1, HCT116, COLO205 and SW480). COLO205 and SW480 had relatively weak NOV expression, while there was strong NOV expression in the other seven cell lines (Figure 1B).
NOV mRNA expression and clinicopathological characteristics. To assess the clinical significance of NOV mRNA expression in CRC cases, NOV mRNA levels were analyzed in 126 resected primary CRC samples by qRT-PCR. We divided the 126 patients with CRC into a high NOV mRNA expression group (n=63) and a low NOV mRNA expression group (n=63) in tumor tissues according to the median NOV mRNA expression level. Clinicopathological factors were then analyzed in the high-and the low-NOV mRNA expression groups (Table I). The high-NOV mRNA expression group had a significantly greater UICC T factor and serosal invasion compared with the low-NOV mRNA expression group. With regard to overall survival, patients with high-NOV mRNA expression had a significantly poorer prognosis than those with low NOV mRNA expression (p=0.037; Figure 2A). Furthermore, we analyzed overall survival for a public data set of TCGA, including 593 CRC samples. CRC samples were also divided into two groups according to NOV mRNA expression by the minimum p-value approach (15). According to our results, overall survival in the high-NOV mRNA expression group was significantly poorer than that in the low-NOV mRNA expression group (p=0.032, Figure 2B).
Ectopic NOV expression enhanced the invasiveness of CRC cells. To explore the biological role of NOV in CRC, we performed overexpression experiments in COLO205 and SW480, cell lines that showed relatively low expression levels of NOV. We confirmed that both NOV mRNA expression and NOV protein expression in cells transfected with NOV cDNA were higher than those in cells with empty plasmid (Figure 3A). We examined whether cell proliferation was altered in cancer cells transfected with NOV cDNA. Overexpression of NOV did not promote cell proliferation of COLO205 and SW480 (Figure 3B). Next, we investigated whether NOV promoted the invasive capacity of CRC. Invasion assays showed that overexpression of NOV promoted the invasive properties of COLO205 and SW480 (Figure 3C).
Expression of nephroblastoma overexpressed (NOV) in a colorectal carcinoma (CRC) specimen and CRC cell lines. A: Immunohistochemistry of NOV expression in a representative sample of CRC. Most staining occurred in the cytoplasm of CRC cells; original magnification, ×40 (left), ×100 (right). B: Western blotting showed that COLO205 and SW480 exhibited relatively weak NOV protein expression, while the other cell lines had strong NOV expression.
Discussion
NOV encodes a secreted protein that interacts with the extracellular matrix and regulates many cellular functions. It has been reported that NOV promotes tumor progression and metastasis in many solid cancer types (5-10). NOV was demonstrated to alter cell adhesion and induce tumor progression by regulating integrin expression in melanoma (6). In prostate cancer, NOV is up-regulated and promotes migration capacity in vitro and facilitates tumor growth in vivo (7). Elevated NOV expression also increases migration and invasion in Ewing's sarcoma (8).
NOV is located in the region of chromosome 8q24 that contains several genes associated with cancer progression including proto-oncogene c-MYC (7). We previously reported that the chromosome region 8q24 contains significant focal amplification that is associated with a poor prognosis in CRC (11-13). Copy number amplification of NOV has been identified in 5% of CRCs, and that might lead to up-regulation of NOV expression in CRC (14). Therefore, NOV overexpression might be related to the amplification of the 8q24 region.
In the present study, we demonstrated that NOV protein was expressed both in tumor tissues of patients with CRC and in CRC cell lines. Moreover, we showed that a high level of NOV mRNA in tumor tissues from patients with CRC was significantly associated with a poor prognosis for survival and UICC T factor. Moreover, our in vitro results revealed that overexpression of NOV protein promoted invasive capacity but not tumor proliferation. Our data agree with previous studies that demonstrated that NOV modulated cell motility and invasiveness (5-7), but did not affect tumor growth (8). Based on the analysis of clinical data and in vitro studies, we suggest that NOV might be a useful prognostic indicator as well as a significant factor that promotes progression of CRC.
NOV is reportedly a putative ligand of integrin receptor. Integrins can induce the activation of downstream signaling genes such as v-src avian sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (SRC), integrin-linked kinase (ILK) and mitogen-activated protein kinase kinase kinase (MAP3K5). These signaling pathways regulate tumor cell progression in various types of solid cancer, including CRC (16-19). Furthermore, it has also been reported that NOV may affect tumor progression via NOTCH1 pathways (20). In CRC, NOTCH1 modulates carcinogenesis and progression by activating nuclear factor-kappa B signaling and is associated with a poor prognosis (21). It is suggested that NOV might also contribute to tumor progression through NOTCH1 in CRC. Thus, we propose that up-regulation of NOV might promote invasion of CRC cells by activating diverse pathways responsible for tumor progression.
In conclusion, our data suggest that NOV may be a prognostic indicator and a therapeutic target in CRC. However, the function of NOV has not been clearly elucidated, and further studies are needed to determine the mechanisms through which NOV exerts its tumor-promoting effects in CRC.
Relationships between expression of nephroblastoma overexpressed (NOV) and clinicopathological factors. The 126 patients with colorectal cancer were divided into two groups according to NOV mRNA expression in cancer tissue. High NOV expression was associated with poor prognostic factor serosal invasion (p=0.004).
Kaplan–Meier survival curves for patients with colorectal carcinoma (CRC) according to the level of nephroblastoma overexpressed (NOV) mRNA expression in two cohorts. A: A total of 126 CRC cases were categorized into two groups according to the NOV mRNA expression. The duration of survival for patients in the high-expression group was significantly less than that for patients in the low-expression group (p=0.037). B: Association of NOV mRNA expression in primary CRC and overall survival in the TCGA cohort including 593 CRC samples (p=0.0032).
Overexpression of nephroblastoma overexpressed (NOV) enhanced invasive properties of human colorectal carcinoma (CRC) cells. A: Quantitative real-time polymerase chain reaction (left) and western blotting (right) show that transfection of NOV cDNA increased NOV mRNA and protein expression. B: Overexpression of NOV had no influence on cell proliferation of COLO205 and SW480 cells. C: Overexpression of NOV promoted the invasive properties of COLO205 and SW480 cells. *p<0.05.
Acknowledgements
This research used the super-computing resource provided by Human Genome Center, the Institute of Medical Science, the University of Tokyo (http://sc.hgc.jp/shirokane.html). We thank K. Oda, M. Kasagi, S. Kono, M. Aoyagi, and T. Kawano for their excellent technical assistance. This work was supported in part by the following grants and foundations: CREST, Japan Science and Technology Agency (JST); Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Science Research (grant nos. 25430111, 25461953, 25861199, and 25861200); Japan Science and Technology Agency (JSTA) A-step (grant no. AS242Z03987P); and the Founding Program for Next Generation World-leading Researchers (grant no. LS094).
Footnotes
Conflicts of interest
The Authors declare no conflicts of interest.
- Received September 8, 2015.
- Revision received October 6, 2015.
- Accepted October 8, 2015.
- Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
