Gene Summary

Gene:NOV; nephroblastoma overexpressed
Aliases: CCN3, NOVh, IBP-9, IGFBP9, IGFBP-9
Summary:The protein encoded by this gene is a small secreted cysteine-rich protein and a member of the CCN family of regulatory proteins. CNN family proteins associate with the extracellular matrix and play an important role in cardiovascular and skeletal development, fibrosis and cancer development. [provided by RefSeq, Feb 2009]
Databases:OMIM, VEGA, HGNC, Ensembl, GeneCard, Gene
Protein:protein NOV homolog
Source:NCBIAccessed: 27 February, 2015


What does this gene/protein do?
Show (9)

Cancer Overview

Research Indicators

Publications Per Year (1990-2015)
Graph generated 27 February 2015 using data from PubMed using criteria.

Literature Analysis

Mouse over the terms for more detail; many indicate links which you can click for dedicated pages about the topic.

  • Growth Substances
  • Bone Cancer
  • Protein Isoforms
  • Intercellular Signaling Peptides and Proteins
  • Cell Movement
  • Immunohistochemistry
  • Cultured Cells
  • Cell Division
  • Promoter Regions
  • Transfection
  • Kidney Cancer
  • Rhabdomyosarcoma
  • Down-Regulation
  • Immediate-Early Proteins
  • Liver Cancer
  • Pregnancy
  • Protein Binding
  • Oncogene Proteins, Viral
  • Wilms Tumour
  • Messenger RNA
  • Cell Proliferation
  • Breast Cancer
  • Chromosome 8
  • Cancer Gene Expression Regulation
  • rac1 GTP-Binding Protein
  • Western Blotting
  • Proto-Oncogene Proteins
  • Pancreatic Cancer
  • Connective Tissue Growth Factor
  • Neoplasm Proteins
  • NOV
  • S100 Proteins
  • Oligonucleotide Array Sequence Analysis
  • WT1
  • Signal Transduction
  • Up-Regulation
  • Cell Differentiation
  • Cysteine-Rich Protein 61
  • Cell Adhesion
  • Cancer RNA
Tag cloud generated 27 February, 2015 using data from PubMed, MeSH and CancerIndex

Specific Cancers (6)

Data table showing topics related to specific cancers and associated disorders. Scope includes mutations and abnormal protein expression.

Note: list is not exhaustive. Number of papers are based on searches of PubMed (click on topic title for arbitrary criteria used).

Latest Publications: NOV (cancer-related)

Chen PC, Cheng HC, Wang J, et al.
Prostate cancer-derived CCN3 induces M2 macrophage infiltration and contributes to angiogenesis in prostate cancer microenvironment.
Oncotarget. 2014; 5(6):1595-608 [PubMed] Free Access to Full Article Related Publications
Tumor-associated macrophages (TAMs) are M2-polarized macrophages that infiltrate the tumor microenvironment and promote tumorigenesis. However, the mechanisms by which TAMs modulate prostate cancer (PCa) growth are poorly understood. Here, we found that expression of Nephroblastoma Overexpressed (NOV/CCN3) is upregulated in PCa cells and correlated with M2 macrophage infiltration. RAW264.7 macrophage migration was induced by conditioned media (CM) from various PCa cells in proportion to the cellular level of CCN3 expression and was inhibited by an anti-CCN3 neutralizing antibody. CCN3 and PCaCM treatment skewed RAW264.7 cell differentiation from an M1 phenotype to an M2 phenotype. PCa-derived CCN3 induced focal adhesion kinase (FAK)/Akt/NF-κB signaling in RAW264.7 cells, which resulted in VEGF expression and subsequently increased tube formation in endothelial progenitor cells. Finally, PCa-secreted CCN3 stimulated RAW264.7 cells and promoted angiogenesis in the chick chorioallantoic membrane assay (CAM), and increased tumor growth and tumor-associated angiogenesis in a PCa xenograft mouse model. Our results indicate that PCa-secreted CCN3 can recruit macrophages and skew their differentiation to an M2 phenotype. In turn, CCN3-stimulated macrophages contribute to VEGF-dependent angiogenesis. This study reveals a novel mechanism by which TAMs enhance PCa angiogenesis and identifies a potential therapeutic target for PCa.

Chen J, Gao Y, Xu B, et al.
NOV is upregulated and promotes migration and invasion in bladder cancer.
Tumour Biol. 2014; 35(7):6749-55 [PubMed] Related Publications
NOV is pro-tumourigenic via epithelial-mesenchymal transition (EMT) in several malignancies but is not studied in bladder cancer (BCa). Whether NOV is responsible for bladder carcinogenesis and the underlying mechanism is unclear. Using immunohistochemical staining, we quantified expressions of NOV, pS6, Vimentin and E-cadherin in 66 bladder cancer and 10 normal bladder urothelium samples. EMT was profiled by EMT index (EMTi) calculated as the ratio of Vimentin to E-cadherin. In vitro and in vivo studies were carried out to profile the role of NOV in the tumourigenesis of BCa. NOV was upregulated in bladder cancer compared to normal tissue, and its expression was correlated to pS6 and EMTi. Expression of NOV was higher in recurrent and multiple tumours and was increased with progression of tumour grade. NOV expression was also higher in BCa cell lines. Silence of NOV attenuated EMT, decreased invasion and migration of BCa cells. Silence of NOV also inhibited xenograft tumour growth and decreased tumour EMT. NOV is pro-tumourigenic in bladder cancer especially in nonmuscle-invasive entities (NMIBC). NOV may promote carcinogenesis via promotion of EMT and association with increased mTOR activity.

Zhang T, Zhao C, Luo L, et al.
The clinical and prognostic significance of CCN3 expression in patients with cervical cancer.
Adv Clin Exp Med. 2013 Nov-Dec; 22(6):839-45 [PubMed] Related Publications
BACKGROUND: CCN3 plays important roles in growth, differentiation, angiogenesis and adhesion. Recently, the role of CCN3 in human carcinogenesis has become an area of great interest. However, little is known about the function of CCN3 in human cervical cancer.
OBJECTIVES: The aim of this study was to investigate the expression profile of CCN3 in cervical cancer and to assess its clinical significance.
MATERIAL AND METHODS: In this study, qRT-PCR, immunohistochemistry and Western blotting analysis were used in the detection of CCN3 mRNA and protein expression, both in cervical cancer and in corresponding normal tissue, respectively. The data was correlated with clinicopathological features. A survival analysis was performed to assess the prognostic significance.
RESULTS: CCN3 mRNA was overexpressed in cervical cancer tissue when compared with corresponding normal tissue, as was CCN3 protein. Upregulation of CCN3 was significantly associated with the stage of the disease (P = 0.017) and with lymph node involvement (P = 0.006). Using the Kaplan-Meier analysis, a comparison of survival curves of low vs. high expressers of CCN3 revealed a highly significant difference in human cervical cancer tissue (P = 0.021), which suggests that overexpression of CCN3 is associated with a poorer prognosis.
CONCLUSIONS: The results of the current study suggest that CCN3 may play an important role in cervical carcinogenesis and therefore may have potential as a biomarker for prognosis and as a therapeutic target in cervical cancer.

Suresh S, McCallum L, Crawford LJ, et al.
The matricellular protein CCN3 regulates NOTCH1 signalling in chronic myeloid leukaemia.
J Pathol. 2013; 231(3):378-87 [PubMed] Free Access to Full Article Related Publications
Deregulated NOTCH1 has been reported in lymphoid leukaemia, although its role in chronic myeloid leukaemia (CML) is not well established. We previously reported BCR-ABL down-regulation of a novel haematopoietic regulator, CCN3, in CML; CCN3 is a non-canonical NOTCH1 ligand. This study characterizes the NOTCH1–CCN3 signalling axis in CML. In K562 cells, BCR-ABL silencing reduced full-length NOTCH1 (NOTCH1-FL) and inhibited the cleavage of NOTCH1 intracellular domain (NOTCH1-ICD), resulting in decreased expression of the NOTCH1 targets c-MYC and HES1. K562 cells stably overexpressing CCN3 (K562/CCN3) or treated with recombinant CCN3(rCCN3) showed a significant reduction in NOTCH1 signalling (> 50% reduction in NOTCH1-ICD, p < 0.05).Gamma secretase inhibitor (GSI), which blocks NOTCH1 signalling, reduced K562/CCN3 colony formation but increased that of K562/control cells. GSI combined with either rCCN3 or imatinib reduced K562 colony formation with enhanced reduction of NOTCH1 signalling observed with combination treatments. We demonstrate an oncogenic role for NOTCH1 in CML and suggest that BCR-ABL disruption of NOTCH1–CCN3 signalling contributes to the pathogenesis of CML.

Cui L, Xie R, Dang S, et al.
NOV promoted the growth and migration of pancreatic cancer cells.
Tumour Biol. 2014; 35(4):3195-201 [PubMed] Related Publications
NOV, a member of the CCN (Cyr61, CTGF and NOV) family, is involved in diverse biological processes, such as cell adhesion, proliferation and angiogenesis. However, its function in pancreatic cancer remains poorly understood. Here, we found that the expression of NOV was up-regulated in pancreatic cancer tissues. Moreover, over-expression of NOV in pancreatic cancer cells promoted cell proliferation and migration, while knock down the expression of NOV impaired the tumorigenecity of pancreatic cancer cells in vitro and in vivo. Mechanistically, NOV induced epithelial-mesenchymal transition (EMT) and regulated the expression of multiple EMT marker. Taken together, our study suggested the important role of NOV in pancreatic cancer and NOV might be an important therapeutic target.

Chen PC, Cheng HC, Tang CH
CCN3 promotes prostate cancer bone metastasis by modulating the tumor-bone microenvironment through RANKL-dependent pathway.
Carcinogenesis. 2013; 34(7):1669-79 [PubMed] Related Publications
Bone metastasis in patient with advanced-stage prostate cancer, the most commonly diagnosed malignancy in Western countries, increases the risk of intractable bone pain. The nephroblastoma overexpressed (NOV/CCN3) gene, a member of the CCN gene family, is responsible for the secretion of CCN3, a matrix-associated protein involved in many cellular functions. However, the role of CCN3 in prostate cancer metastasis to bone is poorly understood. CCN3 was found to be highly expressed in bone metastasis patients and positively correlated with malignancy in human prostate cancer cells. Prostate cancer conditioned medium-induced osteoclast differentiation was inhibited by neutralizing antibody against CCN3. Specifically, CCN3 was found to induce osteoclastogenesis through the receptor activator of NF-κB ligand (RANKL)-dependent pathway, and the focal adhesion kinase/Akt/p38/NF-κB signal pathway was found to be involved in CCN3-mediated receptor activator of NF-κB expression and RANKL-dependent osteoclastogenesis. In contrast, osteoblasts were observed to play an important role in osteoclast differentiation by paracrine manner, with treatment of osteoblasts with CCN3 found to change the RANKL (osteoclastogenesis):OPG (antiosteoclastogenesis) ratio. Compared with parental PC3 cells, highly invasive PC3-I3 cells markedly enhanced osteoclast activity and bone metastasis in vivo. These results indicate that CCN3 can be used as a novel therapeutic target in the prevention of bone metastasis of prostate cancer.

Wu L, Runkle C, Jin HJ, et al.
CCN3/NOV gene expression in human prostate cancer is directly suppressed by the androgen receptor.
Oncogene. 2014; 33(4):504-13 [PubMed] Free Access to Full Article Related Publications
Androgen receptor (AR) has essential roles during prostate cancer progression. With genome-wide AR-binding sites mapped to high resolution, studies have recently reported AR as a transcriptional repressor. How AR inhibits gene expression and how this contributes to prostate cancer, however, are incompletely understood. Through meta-analysis of microarray data, here we nominate nephroblastoma overexpressed (NOV) as a top androgen-repressed gene. We show that NOV is directly suppressed by androgen through the AR. AR occupies the NOV enhancer and communicates with the NOV promoter through DNA looping. AR activation recruits the polycomb group protein EZH2, which subsequently catalyzes histone H3 lysine 27 tri-methylation around the NOV promoter, thus leading to repressive chromatin remodeling and epigenetic silencing. Concordantly, AR and EZH2 inhibition synergistically restored NOV expression. NOV is downregulated in human prostate cancer wherein AR and EZH2 are upregulated. Functionally, NOV inhibits prostate cancer cell growth in vitro and in vivo. NOV reconstitution reverses androgen-induced cell growth and NOV knockdown drives androgen-independent cell growth. In addition, NOV expression is restored by hormone-deprivation therapies in mice and prostate cancer patients. Therefore, using NOV as a model gene we gained further understanding of the mechanisms underlying AR-mediated transcriptional repression. Our findings establish a tumor-suppressive role of NOV in prostate cancer and suggest that one important, but previously underestimated, manner by which AR contributes to prostate cancer progression is through inhibition of key tumor-suppressor genes.

Chen PC, Lin TH, Cheng HC, Tang CH
CCN3 increases cell motility and ICAM-1 expression in prostate cancer cells.
Carcinogenesis. 2012; 33(4):937-45 [PubMed] Related Publications
Nephroblastoma overexpressed (NOV or CCN3) is a secreted matrix-associated protein that belongs to the CCN gene family and is involved in many cellular functions, including growth, differentiation and adhesion. The effect of CCN3 on human prostate cancer cells, however, is unknown. Here, we have shown that CCN3 increased cell migration and intercellular adhesion molecule-1 (ICAM-1) expression in prostate cancer cells. In addition, expression of CCN3 was positively correlated with both cell migration and ICAM-1 expression in human prostate cancer cells. CCN3 activated a signal transduction pathway that included αvβ3 integrin, integrin-linked kinase (ILK), Akt and nuclear factor-kappaB (NF-κB). Reagents that inhibit specific components of this pathway each diminished the ability of CCN3 to effect cell migration and ICAM-1 expression. Moreover, CCN3 increased binding of p65 to an NF-κB-binding element in the ICAM-1 promoter. Finally, knockdown of CCN3 expression markedly inhibited cell migration, tumor growth in bone and bone metastasis. Taken together, our results indicate that CCN3 enhances the migration of prostate cancer cells by increasing ICAM-1 expression through a signal transduction pathway that involves αvβ3 integrin, ILK, Akt and NF-κB. CCN3 thus represents a promising new target for treating prostate cancer.

Brisbin AG, Asmann YW, Song H, et al.
Meta-analysis of 8q24 for seven cancers reveals a locus between NOV and ENPP2 associated with cancer development.
BMC Med Genet. 2011; 12:156 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Human chromosomal region 8q24 contains several genes which could be functionally related to cancer, including the proto-oncogene c-MYC. However, the abundance of associations around 128 Mb on chromosome 8 could mask the appearance of a weaker, but important, association elsewhere on 8q24.
METHODS: In this study, we completed a meta-analysis of results from nine genome-wide association studies for seven types of solid-tumor cancers (breast, prostate, pancreatic, lung, ovarian, colon, and glioma) to identify additional associations that were not apparent in any individual study.
RESULTS: Fifteen SNPs in the 8q24 region had meta-analysis p-values < 1E-04. In particular, the region consisting of 120,576,000-120,627,000 bp contained 7 SNPs with p-values < 1.0E-4, including rs6993464 (p = 1.25E-07). This association lies in the region between two genes, NOV and ENPP2, which have been shown to play a role in tumor development and motility. An additional region consisting of 5 markers from 128,478,000 bp - 128,524,000 (around gene POU5F1B) had p-values < 1E-04, including rs6983267, which had the smallest p-value (p = 6.34E-08). This result replicates previous reports of association between rs6983267 and prostate and colon cancer.
CONCLUSIONS: Further research in this area is warranted as these results demonstrate that the chromosomal region 8q24 may contain a locus that influences general cancer susceptibility between 120,576 and 120,630 kb.

Yang W, Wagener J, Wolf N, et al.
Impact of CCN3 (NOV) glycosylation on migration/invasion properties and cell growth of the choriocarcinoma cell line Jeg3.
Hum Reprod. 2011; 26(10):2850-60 [PubMed] Related Publications
BACKGROUND: Recently we have shown that the matricellular CCN3 protein expressed in invasive extravillous trophoblast cells (EVTs) is decreased in early-onset pre-eclampsia and is regulated by oxygen tension. Pathogenesis of pre-eclampsia relies on a shallow invasion of EVTs into the spiral arteries, which leads to hypoxia accompanied by uteroplacental insufficiency. Here we investigated the function of glycosylated and non-glycosylated CCN3 protein on cell growth as well as migration and invasion properties of the malignant trophoblast cell line Jeg3 which is a widely used model for the invasive trophoblast.
METHODS AND RESULTS: Stable transfection of Jeg3 choriocarcinoma cells with full length CCN3 resulted in high expression of secreted glycosylated and cellular non-glycosylated CCN3. These cells revealed significantly reduced growth in cell numbers combined with a significantly increased migratory and invasive capacity. Matrix metalloprotease (MMP)-2 and MMP-9 activities were enhanced dependent on CCN3 expression, which could be confirmed by CCN3 knockdown studies. Using recombinant glycosylated and non-glycosylated CCN3, we revealed that CCN3 decreased growth in Jeg3 cell numbers independent of its glycosylation status, whereas only non-glycosylated CCN3 was able to enhance migration and invasion properties.
CONCLUSIONS: The present results suggest that CCN3 protein regulates the decrease in Jeg3 cell numbers independent of its glycosylation status, whereas migratory and invasive properties are influenced only by non-glycosylated CCN3. An impaired balance in the expression of glycosylated and non-glycosylated CCN3 could contribute to the shallow invasion of EVTs observed in pre-eclampsia.

Zuo HL, Peng EL, Zhao HX, et al.
[Expression of NOV and BNIP3 gene in mouse myelomonocytic leukemia and its significance].
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2011; 19(2):293-7 [PubMed] Related Publications
This study was aimed to investigate the expression level of NOV and BNIP3 mRNA in mice myelomonocytic leukemia (AML-M(4)) and its significance. The mice were inoculated intravenously with myelomonocytic leukemia cells of WEHI-3, and divided randomly into chemotherapy group and control (untreated) group. Bone marrow samples were then collected from both groups at different times. The NOV and BNIP3 mRNA expression were detected by TaqMan quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), and the relationship between these expression levels and clinical significance in leukemia incidence and progression were analyzed with β-actin as the housekeeping gene. The results showed that the mean values of NOV and BNIP3 increased gradually from 2 weeks after inoculation and achieved highest level at death in control group. Expression level of NOV increased from 1.85E-05 before inoculation to 3.57E-02 at death (p < 0.05), and BNIP3 from 3.44E-03 to 3.48E-02. While 2 gene expression in the chemotherapy group decreased quickly to 2.51E-05 and 1.58E-03 (p < 0.05) respectively after chemotherapy, which were close to the level before inoculation (p > 0.05). The 2 gene expressions again rose at relapse, and difference of expression level between 2 group at death were no statistically significant (p > 0.05). It is concluded that the expression of NOV and BNIP3 in leukemia AML-M(4) is significantly higher than that in normal controls, of which high level expression is an important factor in the development of leukemia. Close relation between the therapeutic effect and expression level of these two genes suggests the great value in prognostic evaluation and MRD detection.

Ouellet V, Tiedemann K, Mourskaia A, et al.
CCN3 impairs osteoblast and stimulates osteoclast differentiation to favor breast cancer metastasis to bone.
Am J Pathol. 2011; 178(5):2377-88 [PubMed] Free Access to Full Article Related Publications
Bone is a preferred site for breast cancer metastasis, causing pain, fractures, spinal cord compressions, and hypercalcemia, all of which can significantly diminish the patient's quality of life. We identified CCN3 as a novel factor that is highly expressed in bone metastatic breast cancer cells from a xenograft mouse model and in bone metastatic lesions from patients with breast cancer. We demonstrate that CCN3 overexpression enhances the ability of weakly bone metastatic breast cancer cells to colonize and grow in the bone without altering their growth in the mammary fat pad. We further demonstrated that human recombinant CCN3 inhibits osteoblast differentiation from primary bone marrow cultures, leading to a higher receptor activator of NF-κB ligand (RANKL)/osteoprotegerin (OPG) ratio. In conjunction with its ability to impair osteoblast differentiation, we uncovered a novel role for CCN3 in promoting osteoclast differentiation from RANKL-primed monocyte precursors. CCN3 exerts its pro-osteoclastogenic effects by promoting calcium oscillations and nuclear factor of activated T cells c1 (NFATc1) nuclear translocation. Together, these results demonstrate that CCN3 regulates the differentiation of bone resident cells to create a resorptive environment that promotes the formation of osteolytic breast cancer metastases.

Zhang Y, Wang C
Nephroblastoma overexpressed (NOV/CCN3) gene: a paired-domain-specific PAX3-FKHR transcription target that promotes survival and motility in alveolar rhabdomyosarcoma cells.
Oncogene. 2011; 30(32):3549-62 [PubMed] Free Access to Full Article Related Publications
The CCN (Cy61, CTGF and NOV) family of proteins is a group of matricellular biomolecules involved in both physiological and pathological processes. Elevated expression of the CCN3 (also known as NOV, Nephroblastoma overexpressed) gene has been detected in clinical samples of the skeletal muscle cancer rhabdomyosarcoma, with the highest expression found in the alveolar subtype (aRMS). Over 80% of aRMSs are characterized by a chromosomal translocation-derived fusion transcription factor PAX3-FKHR. In this study, we linked elevated CCN3 levels in aRMS cells to PAX3-FKHR expression. We found reduced CCN3 levels in aRMS cells following small interfering RNA knockdown of PAX3-FKHR, and increased CCN3 levels in C2 myoblasts following ectopic expression of PAX3-FKHR. Promoter, electrophoretic mobility shift assay and chromatin immunoprecipitation analyses confirmed that the CCN3 gene was a direct target for PAX3-FKHR transcriptional activation through a paired-domain DNA sequence in the first intron of the CCN3 gene. To determine the function of CCN3, we showed that knockdown and ectopic expression of CCN3 decreased survival and increased differentiation in aRMS cells, respectively. In addition, we found that exogenously supplied CCN3 protein promoted aRMS cell adhesion, migration and Matrigel invasion. Taken together, data from this study have (1) provided a mechanistic basis for the CCN3 overexpression in aRMS cells, and (2) identified CCN3 as an autocrine/paracrine factor that contributes to the aggressive behavior of aRMS cells, perhaps through a positive feedback loop. Thus, CCN3 may be an attractive target for therapeutic intervention in aRMS.

Tzeng HE, Chen JC, Tsai CH, et al.
CCN3 increases cell motility and MMP-13 expression in human chondrosarcoma through integrin-dependent pathway.
J Cell Physiol. 2011; 226(12):3181-9 [PubMed] Related Publications
Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. Chondrosarcoma shows a predilection for metastasis to the lungs. CCN3, also called nephroblastoma overexpressed gene (NOV), regulates proliferation and differentiation of cancer cells. However, the effect of CCN3 on migration activity in human chondrosarcoma cells is mostly unknown. Here, we found that CCN3 increased the migration and expression of matrix metalloproteinase (MMP)-13 in human chondrosarcoma cells (JJ012 cells). αvβ3 or αvβ5 monoclonal antibody (mAb), phosphatidylinositol 3-kinase (PI3K) inhibitors (Ly294002 and wortmannin) and Akt inhibitor inhibited the CCN3-induced increase of the migration and MMP-13 upregulation of chondrosarcoma cells. CCN3 stimulation increased the phosphorylation of focal adhesion kinase (FAK), PI3K, and Akt. In addition, NF-κB inhibitors also suppressed the cell migration and MMP-13 expression enhanced by CCN3. Moreover, CCN3 increased NF-κB luciferase activity and binding of p65 to the NF-κB element on the MMP-13 promoter. Taken together, our results indicate that CCN3 enhances the migration of chondrosarcoma cells by increasing MMP-13 expression through the αvβ3/αvβ5 integrin receptor, FAK, PI3K, Akt, p65, and NF-κB signal transduction pathway.

Bohr W, Kupper M, Hoffmann K, Weiskirchen R
Recombinant expression, purification, and functional characterisation of connective tissue growth factor and nephroblastoma-overexpressed protein.
PLoS One. 2010; 5(12):e16000 [PubMed] Free Access to Full Article Related Publications
The CCN family of proteins, especially its prominent member, the Connective tissue growth factor (CTGF/CCN2) has been identified as a possible biomarker for the diagnosis of fibrotic diseases. As a downstream mediator of TGF-β1 signalling, it is involved in tissue scarring, stimulates interstitial deposition of extracellular matrix proteins, and promotes proliferation of several cell types. Another member of this family, the Nephroblastoma-Overexpressed protein (NOV/CCN3), has growth-inhibiting properties. First reports further suggest that these two CCN family members act opposite to each other in regulating extracellular matrix protein expression and reciprocally influence their own expression when over-expressed. We have established stable HEK and Flp-In-293 clones as productive sources for recombinant human CCN2/CTGF. In addition, we generated an adenoviral vector for recombinant expression of rat NOV and established protocols to purify large quantities of these CCN proteins. The identity of purified human CCN2/CTGF and rat CCN3/NOV was proven by In-gel digest followed by ESI-TOF/MS mass spectrometry. The biological activity of purified proteins was demonstrated using a Smad3-sensitive reporter gene and BrdU proliferation assay in permanent cell line EA•hy 926 cells. We further demonstrate for the first time that both recombinant CCN proteins are N-glycosylated.

Huang CY, Lee CY, Chen MY, et al.
Nephroblastoma overexpressed gene (NOV) enhances cell motility and COX-2 upregulation of human osteosarcoma involves αvβ5 integrin, ILK and AP-1-dependent pathways.
Biochem Pharmacol. 2011; 81(5):577-85 [PubMed] Related Publications
Osteosarcoma is characterized by a high malignant and metastatic potential. Cyclooxygenase (COX)-2, the inducible isoform of prostaglandin synthase, has been implicated in tumor metastasis. Nephroblastoma overexpressed gene (NOV), also called CCN3, was regulated proliferation and differentiation of cancer cells. However, the effect of NOV on migration activity and COX-2 expression in human osteosarcoma cells is mostly unknown. Here we found that NOV increased the migration and expression of COX-2 in human osteosarcoma cells. αvβ5 monoclonal antibody (mAb), integrin-linked kinase (ILK) and Akt inhibitor reduced the NOV-enhanced the migration and COX-2 up-regulation of osteosarcoma cells. NOV stimulation increased the ILK kinase activity and phosphorylation of Akt. In addition, c-Jun siRNA also antagonized the NOV-mediated migration and COX-2 expression. Moreover, NOV enhanced the AP-1 binding activity and promoter activity. Taken together, these results suggest that the NOV acts through αvβ5 integrin to activate ILK and Akt, which in turn activates c-Jun and AP-1, resulting in the activations of COX-2 and contributing the migration of human osteosarcoma cells.

Pasmant E, Ortonne N, Rittié L, et al.
Differential expression of CCN1/CYR61, CCN3/NOV, CCN4/WISP1, and CCN5/WISP2 in neurofibromatosis type 1 tumorigenesis.
J Neuropathol Exp Neurol. 2010; 69(1):60-9 [PubMed] Related Publications
The hallmark of neurofibromatosis type 1 is the development of dermal and plexiform neurofibromas. Neurofibromatosis type 1 patients with plexiform neurofibromas are at risk of developing malignant peripheral nerve sheath tumors. We applied a 22,000-oligonucleotide microarray transcriptomic approach to a series of plexiform neurofibromas in comparison with dermal neurofibromas, and results were confirmed with real-time quantitative reverse transcription-polymerase chain reaction. Thirteen genes were upregulated and 10 were downregulated in plexiform neurofibromas. The upregulated genes mainly encode molecules involved in cell adhesion, extracellular matrix, fibrogenesis, and angiogenesis. Several CCN gene family members were dysregulated in neurofibromatosis type 1 tumorigenesis; the angiogenic gene CCN1/CYR61 was specifically upregulated in the plexiform neurofibromas; CCN4/WISP1 was upregulated, and CCN3/NOV and CCN5/WISP2 were downregulated in paired comparisons of plexiform neurofibroma and malignant peripheral nerve sheath tumor from the same patients. CCN1 and CCN3 proteins were detected by immunohistochemistry in neurofibromatosis type 1-associated tumors. Upregulation of S100A8, S100A9, and CD36 was also observed and suggests a role of this pathway in inflammation-associated genesis of plexiform neurofibromas. In summary, a limited number of pathways are potentially involved in plexiform neurofibroma development. Some of the genes identified, particularly CCN1, might be useful diagnostic or prognostic markers or form the basis for novel therapeutic strategies.

Sin WC, Tse M, Planque N, et al.
Matricellular protein CCN3 (NOV) regulates actin cytoskeleton reorganization.
J Biol Chem. 2009; 284(43):29935-44 [PubMed] Free Access to Full Article Related Publications
CCN3 (NOV), a putative ligand for integrin receptors, is tightly associated with the extracellular matrix and mediates diverse cellular functions, including cell adhesion and proliferation. CCN3 has been shown to negatively regulate growth although it promotes migration in a cell type-specific manner. In this study, overexpression of CCN3 reduces growth and increases intercellular adhesion of breast cancer cells. Interestingly, CCN3 overexpression also led to the formation of multiple pseudopodia that are enriched in actin, CCN3, and vinculin. Breast cancer cells preincubated with exogenous CCN3 protein also induced the same phenotype, indicating that secreted CCN3 is sufficient to induce changes in cell morphology. Surprisingly, extracellular CCN3 is internalized to the early endosomes but not to the membrane protrusions, suggesting pseudopodia-enriched CCN3 may derive from a different source. The presence of an intracellular variant of CCN3 will be consistent with our finding that the cytoplasmic tail of the gap junction protein connexin43 (Cx43) associates with CCN3. Cx43 is a channel protein permitting intercellular communication to occur. However, neither the channel properties nor the protein levels of Cx43 are affected by the CCN3 protein. In contrast, CCN3 proteins are down-regulated in the absence of Cx43. Finally, we showed that overexpression of CCN3 increases the activity of the small GTPase Rac1, thereby revealing a pathway that links Cx43 directly to actin reorganization.

Perbal B, Lazar N, Zambelli D, et al.
Prognostic relevance of CCN3 in Ewing sarcoma.
Hum Pathol. 2009; 40(10):1479-86 [PubMed] Related Publications
Ewing sarcoma is a highly aggressive malignant bone tumor occurring preferentially in children and young adults. At present, only clinical features, such as patient age, presence of clinically evident metastases at diagnosis, and poor response to neoadjuvant chemotherapy, are widely accepted as prognostic indicators in Ewing sarcoma. In this study, we assessed the prognostic value of CCN3 (Nov), a matricellular protein that play crucial roles in bone formation. Polyclonal antibodies directed against each of the different CCN3 modules were used to identify variant CCN3 proteins in tumors and to draw potential relationships between the expression of these variants and the outcome of patients with Ewing sarcoma. Our results confirmed that expression of the full-length CCN3 in Ewing sarcoma is associated to a worse prognostic. Furthermore, we report a possible relationship between the expression of a CCN3 protein lacking an internal module (von Willebrand factor type C) and sensitivity to radiotherapy. We hypothesize that the increased level of variant CCN3 in the tumor cells reduces their tumorigenic potential and results in better outcome.

Vallacchi V, Rodolfo M
Regulatory role of CCN3 in melanoma cell interaction with the extracellular matrix.
Cell Adh Migr. 2009 Jan-Mar; 3(1):7-10 [PubMed] Free Access to Full Article Related Publications
It is increasingly clear that melanoma cells modify their environment not only through the release of growth factors (GFs) and cytokines that have autocrine or paracrine effects and strongly modulate the immune response, but also by secreting proteins that become structural or transient components of the extracellular matrix (ECM). Melanoma cell secreted proteins play a significant role in cell-ECM interactions, helping tumor cells to invade neighbouring stroma, disseminate and survive in other tissue contexts. CCN3/NOV (nephroblastoma overexpressed) is a matricellular protein that belongs to the CCN family of proteins containing six members in humans. Its structure consists of modules related to functional domains previously identified in major regulatory proteins: insulin-like growth factor-binding protein (IGFBP), von Willebrand factor type C repeats (VWC), thrombospondin type 1 repeats, and secreted regulatory factors containing cysteine knot motifs. Extensive studies have indicated that the biological properties of CCN3 are dependent upon the cellular context, and its role in melanoma seems to recapitulate cell context functions.

Ghayad SE, Vendrell JA, Bieche I, et al.
Identification of TACC1, NOV, and PTTG1 as new candidate genes associated with endocrine therapy resistance in breast cancer.
J Mol Endocrinol. 2009; 42(2):87-103 [PubMed] Related Publications
Cross-resistance to molecules used in endocrine therapy is among the main challenges in the treatment of estrogen receptor-alpha (ERalpha) positive breast cancer. In this study, we used two different cell models of resistance to anti-estrogens: MVLN/CL6.7 cells and VP229/VP267 cells selected after exposure to tamoxifen respectively in vitro and in vivo to characterize a phenotype rarely observed, i.e. acquisition of cross-resistance to the pure ER antagonist fulvestrant. As MVLN/CL6.7 cells and VP229/VP267 cell lines are original and valuable models of cross-resistance to tamoxifen and fulvestrant, we examined candidate genes using a RTQ-PCR strategy to identify new biomarkers of endocrine resistance. Out of the 26 candidate genes tested, 19 displayed deregulation of expression at the basal level in at least one of the two resistant cell lines. Eight genes (TACC1, NOV, PTTG1, MAD2L1, BAK1, TGFB2, BIRC5, and CCNE2) were significantly overexpressed in samples from ER-positive breast cancer patients who relapsed after tamoxifen treatment (n=24) compared with samples from patients who did not (n=24). Five genes (TACC1, NOV, PTTG1, BAK1, and TGFB2) were correlated with significantly shorter relapse-free survival (univariate analysis). Finally, we identified TACC1 and a three-gene expression signature (TACC1, NOV, and PTTG1) as independent prognostic markers (multivariate analysis). Aberrant mRNA and protein levels of TACC1, NOV, and PTTG1 were also observed under tamoxifen and/or fulvestrant exposure in resistant CL6.7 cells compared with their respective control MVLN cells. In conclusion, our data identify TACC1, NOV, and PTTG1 as promising new markers that could be used in the clinical management of ER-positive breast cancer patients.

Böhlig L, Metzger R, Rother K, et al.
The CCN3 gene coding for an extracellular adhesion-related protein is transcriptionally activated by the p53 tumor suppressor.
Cell Cycle. 2008; 7(9):1254-61 [PubMed] Related Publications
The CCN3 protein (Nov, Nephroblastoma overexpressed) is a member of the CCN family (Cyr61, CTGF, Nov) of growth regulators and exerts antiproliferative properties. We show here that the tumor suppressor p53 transcriptionally upregulates the CCN3 gene. p53 is an important transcription factor contributing to cell cycle arrest and apoptosis after cell damage through the regulation of numerous target genes. We show that CCN3 mRNA and protein are upregulated following p53 expression. A DNA binding-deficient p53 mutant fails to regulate CCN3. CCN3 protein is located in the perinuclear space after induction and is also exported to the extracellular matrix. Furthermore, the CCN3 promoter is inducible by p53 and the response element is located in the first exon of the CCN3 gene. Chromatin immunoprecipitations show that p53 binds to the CCN3 promoter in vivo. As CCN3 was shown to inhibit cell growth, transcriptional induction by p53 may serve as an antiproliferative signal in the extracellular matrix. Furthermore, CCN3 depletion was also reported to reduce collagen type IV-dependent adhesion of melanocytes. Thus, elevated levels of CCN3 protein regulated by p53 might influence cell adhesion.

Perbal B, Zuntini M, Zambelli D, et al.
Prognostic value of CCN3 in osteosarcoma.
Clin Cancer Res. 2008; 14(3):701-9 [PubMed] Related Publications
PURPOSE: Osteosarcoma, the most common bone tumor, lacks prognostic markers that could distinguish patients before therapy and drive treatment choices. We assessed the prognostic value of CCN1, CCN2, and CCN3 genes, involved in fundamental biological processes.
EXPERIMENTAL DESIGN: Expression of CCN1, CCN2, and CCN3 was measured by quantitative PCR in 45 newly diagnosed osteosarcomas. Cancer-specific survival was estimated using the Kaplan-Meier method. Associations with osteoblastic differentiation and/or drug response genes were assessed in tumor cells using Spearman correlation and Fisher's exact tests.
RESULTS: CCN1 and CCN2 expression was associated with genes involved in commitment of mesenchymal stem cells toward osteoblasts and in early phases of osteoblastic differentiation (RUNX family genes; cadherin 4, 11, and 13; jun and fos; collagen I and SPARC). Although CCN3 is barely expressed in normal proliferating osteoblasts and mesenchymal stem cells, its expression was generally high in osteosarcoma and its level of expression did not correlate with any specific osteoblastic differentiation genes. High expression of CCN3 significantly correlated with worse prognosis in osteosarcoma. This may be only partly explained by the association with the expression of multidrug resistance-related protein 1 and 4, two ATP-binding cassette transporters that also acted as predictors of worse outcome in our study.
CONCLUSIONS: Our study showed temporal and coordinated expression of CCN1, CCN2, and CCN3 genes during osteoblastic differentiation and highlighted significant differences between human normal and osteosarcoma cell differentiation in vitro. CCN1 and CCN2 expression shows no prognostic relevance in osteosarcoma. In contrast, assessment for CCN3 expression levels at diagnosis may represent a useful molecular tool to early identification of patients with different prognosis.

Vallacchi V, Daniotti M, Ratti F, et al.
CCN3/nephroblastoma overexpressed matricellular protein regulates integrin expression, adhesion, and dissemination in melanoma.
Cancer Res. 2008; 68(3):715-23 [PubMed] Related Publications
CCN3/nephroblastoma overexpressed belongs to the CCN family of genes that encode secreted proteins associated with the extracellular matrix (ECM) and exert regulatory effects at the cellular level. Overexpression of CCN3 was shown in metastatic melanoma cells compared with cells of the primary tumor from the same patient. Analysis of short-term cultures from 50 primary and metastatic melanomas revealed a heterogeneous expression pattern of both the 46-kDa full-length cytoplasmic/secreted protein and the 32-kDa nuclear-truncated form. The different protein expression patterns were not associated with gene alterations or polymorphisms. Like the metastatic cells expressing high levels of the 46-kDa CCN3, cells transfected to overexpress CCN3 showed increased adhesion to ECM proteins, whereas inhibition of CCN3 expression by small interfering RNA decreased adhesion to laminin and vitronectin. CCN3 overexpression induced increased expression of laminin and vitronectin integrin receptors alpha 7 beta 1 and alpha v beta 5 by increasing their mRNA production. Moreover, CCN3 secreted by melanoma cells acted as an adhesion matrix protein for melanoma cells themselves. Analysis of CCN3 protein expression with respect to melanoma progression detected the protein in all visceral metastases tested and in most nodal metastases from relapsing patients but in only a few nodal metastases from nonrelapsing patients and cutaneous metastases. Consistently, xenotransplantation in immunodeficient mice showed a higher metastatic potential of melanoma cells overexpressing CCN3. Together, these data indicate a role for CCN3 in melanoma cell interaction with the ECM by regulating integrin expression, resulting in altered cell adhesion and leading melanoma progression to aggressive disease.

Croci S, Landuzzi L, Nicoletti G, et al.
Expression of connective tissue growth factor (CTGF/CCN2) in a mouse model of rhabdomyosarcomagenesis.
Pathol Oncol Res. 2007; 13(4):336-9 [PubMed] Related Publications
Connective tissue growth factor (CTGF/CCN2) is a cysteine-rich matricellular protein that belongs to the CCN (CYR61, CTGF, NOV) protein family. It is highly expressed by human rhabdomyosarcoma cells and sustains their survival. In this study we investigated CCN2 expression in a mouse model of spontaneous rhabdomyosarcomagenesis that combines HER-2/neu oncogene activation and p53 oncosuppressor gene inactivation (BALB-p53neu mice). Murine rhabdomyosarcoma cells showed a 4-26 fold increase in CCN2 mRNA expression regarding to normal thigh muscle. Moreover, they expressed CCN2 protein at levels comparable to human rhabdomyosarcoma cells. Therefore BALBp53neu mice might be useful for the evaluation of the role played by CCN2 in rhabdomyosarcoma in vivo.

Subramaniam MM, Lazar N, Navarro S, et al.
Expression of CCN3 protein in human Wilms' tumors: immunohistochemical detection of CCN3 variants using domain-specific antibodies.
Virchows Arch. 2008; 452(1):33-9 [PubMed] Related Publications
We aimed to detect truncated CCN3 protein variants in formalin-fixed paraffin-embedded samples of eight Wilms' tumors using anti-K19M and novel domain-specific antibodies, anti-NH2, anti-NH3, anti-NH4, and anti-NH5 raised against C-terminal (CT) domain and modules 1, 2, 3, and 4 of the CCN3 protein, respectively. In Wilms' tumors, all the domain antibodies except anti-NH4 exhibited both nuclear and cytoplasmic staining in blastema as well as primitive tubules. NH4 was detected only in the cytoplasm of tumor cells. Normal fetal kidneys revealed mainly cytoplasmic immunoreactivity for all antibodies in tubules and glomeruli, except for K19 and NH5, which showed some nuclear staining. Our data suggest expression of a truncated nuclear CCN3 variant lacking the thrombospondin type-1-like domain and cytoplasmic full-length CCN3 protein in Wilms' tumor cells. In addition, normal fetal kidneys express mainly full-length protein mostly localized to cytoplasm. Truncated CCN3 protein in Wilms' tumor cells may provide evidence for its tumorigenic role in these tumors. Uniform NH5 staining compared to variable expression of K19M indicates that using NH5 is a better approach for detecting the CT domain of CCN3 protein in archival specimens. Thus, the domain-specific antibodies represent valuable tools for detecting CCN3 protein variants in normal and neoplastic kidneys.

Sin WC, Bechberger JF, Rushlow WJ, Naus CC
Dose-dependent differential upregulation of CCN1/Cyr61 and CCN3/NOV by the gap junction protein Connexin43 in glioma cells.
J Cell Biochem. 2008; 103(6):1772-82 [PubMed] Related Publications
Gap junctions form channels that allow exchange of materials between cells and are composed of transmembrane protein subunits called connexins. While connexins are believed to mediate cellular signaling by permitting intercellular communication to occur, there is also increasing evidence that suggest connexins may mediate growth control via a junction-independent mechanism. Connexin43 (Cx43) is the most abundant gap junction protein found in astrocytes, and gliomas exhibit reduced Cx43 expression. We have previously observed that restoration of Cx43 levels in glioma cells led to increased expression of CCN3 (NOV) proteins. We now report that overexpression of Cx43 in C6-glioma cells (C6-Cx43) also upregulates the expression of CCN1 (Cyr61). Both CCN1 and CCN3 belong to the Cyr61/Connective tissue growth factor/Nephroblastoma-overexpressed (CCN) family of secretory proteins. The CCN proteins are tightly associated with the extracellular matrix and have important roles in cell proliferation and migration. CCN1 promotes growth in glioma cells, as shown by the increased proliferation rate of CCN1-overexpressing C6 cells. In addition to its effect on cell growth, CCN1 also increased the motility of glioma cells in the presence of extracellular substrates such as fibronectin. Gliomas expressing high levels of Cx43 preferentially upregulated CCN3 which resulted in reduced growth rate. CCN3 could also be observed in Cx43 gap junction plaques in confluent C6-Cx43H culture at the stationary phase of their growth. Our results suggest that the dissimilar growth characteristics between high and low Cx43 expressors may be due to differential regulation of CCN3 by varying levels of Cx43.

Fukunaga-Kalabis M, Martinez G, Telson SM, et al.
Downregulation of CCN3 expression as a potential mechanism for melanoma progression.
Oncogene. 2008; 27(18):2552-60 [PubMed] Related Publications
Coculture of human melanocytes with keratinocytes upregulates CCN3, a matricellular protein critical to maintenance of normal homeostasis of melanocytes in the skin. CCN3 affects two fundamental features of melanocyte physiology: it inhibits melanocyte proliferation and stimulates their adhesion to the basement membrane. Here we report that expression of CCN3 is downregulated in advanced melanomas. Aggressive melanoma cell lines did not respond to treatment with CCN3 inducers, such as interleukin-1beta (IL-1beta), while less aggressive melanoma cell lines responded similarly to melanocytes. Immunostaining analyses revealed that CCN3 was present in melanoma cells close to the epidermal-dermal interface, but not in melanoma cells that had invaded deep into the dermis or had metastasized to lymph nodes. Contrary to our expectations, overexpression of CCN3 in 1205Lu metastatic melanoma cells did not affect their adhesion to collagen IV. However, CCN3 decreased the transcription and activation of matrix metalloproteinases and suppressed the invasion of 1205Lu melanoma cells. These results suggest that the lack of CCN3 in advanced melanoma cells contributes to their invasive phenotype. Whereas major matricellular proteins, such as osteopontin, tenascin or secreted protein acidic and rich in cysteine (SPARC), are strongly upregulated in melanoma cells; CCN3 is the first member of this family that is downregulated.

Doghman M, Arhatte M, Thibout H, et al.
Nephroblastoma overexpressed/cysteine-rich protein 61/connective tissue growth factor/nephroblastoma overexpressed gene-3 (NOV/CCN3), a selective adrenocortical cell proapoptotic factor, is down-regulated in childhood adrenocortical tumors.
J Clin Endocrinol Metab. 2007; 92(8):3253-60 [PubMed] Related Publications
CONTEXT: Childhood adrenocortical tumors (ACTs) have a fetal adrenal phenotype and overexpress steroidogenic factor-1 (SF-1). Nephroblastoma overexpressed (NOV)/cysteine-rich protein 61/connective tissue growth factor/nephroblastoma overexpressed gene-3 mRNA is significantly down-regulated in childhood ACTs.
OBJECTIVE: The objective of the study was to measure NOV protein levels in childhood ACTs and characterize NOV expression regulation and biological function in human adrenocortical cells.
DESIGN AND SETTING: Protein extracts from ACT and normal adrenal cortex samples, human adrenocortical carcinoma H295R, primary adrenocortical tumors and fetal adrenal cultures, tissue culture supernatants, and cell lysates from H295R cells overexpressing SF-1 in an inducible fashion were used.
MAIN OUTCOME MEASURES: NOV protein levels were measured by enzyme-linked immunoassay and immunoblot. Transient transfection assays were used to study the activity of NOV promoter. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling, caspase assays, and flow cytometry were used to assess the proapoptotic activity of NOV on cells in culture.
RESULTS: NOV mRNA and protein expression is lower in childhood ACTs than in normal adrenal cortex. No significant difference was observed between adenomas and carcinomas. SF-1 overexpression down-regulates NOV at the transcriptional level. NOV has a selective proapoptotic activity toward human adrenocortical cells. The C-terminal domain of NOV is responsible for its proapoptotic effect. NOV protein is expressed in DAX-1-positive human fetal adrenal cells.
CONCLUSIONS: NOV is a selective proapoptotic factor for human adrenocortical cells. Reduced expression of NOV in ACTs may play an important role in the process of childhood ACT tumorigenesis, accounting at least in part for the defect of apoptotic regression of the fetal adrenal that has been proposed to be responsible for tumor formation.

Kabasawa Y, Katsube K, Harada H, et al.
A male infant case of lipofibromatosis in the submental region exhibited the expression of the connective tissue growth factor.
Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2007; 103(5):677-82 [PubMed] Related Publications
PURPOSE: We encountered a case of lipofibromatosis in a 21-month-old male patient and examined its histopathologic properties.
METHODS: We examined morphological aspects of the tumor and immunohistochemical patterns.
RESULTS: Tumor proliferation was infiltrative, which did not show apparent encapsulation. Positive immunoreactivity was found for CD-34, CD-99, Ki-67, and connective tissue growth factor/CCN2 in the fibrous region, S-100 in the adipose region, and Notch1 stain was observed in the eccrine sweat gland cells juxtaposed to the tumor adipose tissue, but no reactivity for Bcl-2, alphaSMA, Notch 2-4, CCN1, and CCN3.
CONCLUSIONS: The clinical process and immunohistochemical pattern of this case was consistent with the criteria of reported lipofibromatosis. Specific expression of CCN2 might be significant for the development of the tumor.

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