The CCN family: a new stimulus package

The CCN family comprises cysteine-rich 61 (CYR61/CCN1), connective tIssue growth factor (CTGF/CCN2), nephroblastoma overexpressed (NOV/CCN3), and Wnt-induced secreted proteins-1 (WISP-1/CCN4), -2 (WISP-2/CCN5) and -3 (WISP-3/CCN6). These proteins stimulate mitosis, adhesion, apoptosis, extracellular matrix production, growth arrest and migration of multiple cell types. Many of these activities probably occur through the ability of CCN proteins to bind and activate cell surface integrins. Accumulating evidence supports a role for these factors in endocrine pathways and endocrine-related processes. To illustrate the broad role played by the CCN family in basic and clinical endocrinology, this Article highlights the relationship between CCN proteins and hormone action, skeletal growth, placental angiogenesis, IGF-binding proteins and diabetes-induced fibrosis.     

Involvement of multiple CCN family members in platelets that support regeneration of joint tissues

Objectives: Platelet-rich plasma (PRP) has been widely used to enhance the regeneration of damaged joint tissues, such as osteoarthritic and rheumatoid arthritic cartilage. The aim of this study is to clarify the involvement of all of the CCN family proteins that are crucially associated with joint tissue regeneration.

Methods: Cyr61-CTGF-NOV (CCN) family proteins in human platelets and megakaryocytic cells were comprehensively analyzed by Western blotting analysis. Production of CCN family proteins in megakaryocytes in vivo was confirmed by immunofluorescence analysis of mouse bone marrow cells. Effects of CCN family proteins found in platelets on chondrocytes were evaluated by using human chondrocytic HCS-2/8 cells.

Results: Inclusion of CCN2, a mesenchymal tissue regenerator, was confirmed. Of note, CCN3, which counteracts CCN2, was newly found to be encapsulated in platelets. Interestingly, these two family members were not detectable in megakaryocytic cells, but their external origins were suggested. Furthermore, we found for the first time CCN5 and CCN1 that inhibits ADAMTS4 in both platelets and megakaryocytes. Finally, application of a CCN family cocktail mimicking platelets onto HCS-2/8 cells enhanced their chondrocytic phenotype.

Conclusions: Multiple inclusion of CCN1, 2 and 3 in platelets was clarified, which supports the harmonized regenerative potential of PRP in joint therapeutics.     

A comparative study of the expression of Wnt-1, WISP-1, survivin and cyclin-D1 in colorectal carcinoma

Background and aims It is well accepted that activation of Wnt signalling occurs in colorectal carcinoma (CRC), but the correlation amongst the various proteins involved in primary tumours are still unclear. The expression of the inducer of this pathway, Wnt-1, and the downstream effectors, WISP-1, cyclin-D1 and survivin proteins, was compared in a series of CRC tissues with the apparently normal adjacent tissues to determine the relationship of these proteins.Patients and methods Formalin-fixed, paraffin-embedded tissue samples of 47 CRCs surgically resected at the Kuala Lumpur Hospital (KLH) between 1999 and 2000 were used. Immunohistochemical staining with monoclonal antibodies against cyclin-D1 and survivin and polyclonal antibodies against Wnt-1 and WISP-1 was performed. Results of immunohistochemistry were analysed for correlation between biomolecules and histopathological data of the patients.Results Of the 47 CRCs, 26 (55.3%), 15 (31.9%), 5 (10.6%) and 28 (59.6%) of the tumours exhibited positivity for Wnt-1, WISP-1, cyclin D1 and survivin, respectively. A lower percentage of the 40 apparently normal adjacent tissues were found to be positive for Wnt-1 (7, 17.5%), WISP-1 (±5, 12.5%) and survivin (13, 32.5%), but cyclin D1 was not detected in any of them. Interestingly, the total scores of Wnt-1, WISP-1 and survivin were significantly higher in CRC tissues (p=0.001, 0.034 and 0.044, respectively). Using the Spearman rank correlation test, a positive linear relationship was found between total Wnt-1 score with total WISP-1 score (rho=0.319, p=0.003) and total survivin score (rho=0.609, p=<0.001). The expression of WISP-1 in the CRC tissues was found to be positively correlated with patients older than 60 years old (p=0.011). In addition, nuclear cyclin-D1 expression was found to be associated with poorly differentiated CRC tissues (p<0.001, Table 5) and right-sided CRC tumour (p=0.019, Table 6). Total WISP-1 score was associated with well-differentiated CRC tissues (p=0.029).Conclusions Overexpression and interplay between Wnt-1, WISP-1, survivin and cyclin-D1 may play a role in tumorigenesis, possibly by promoting cell cycle checkpoint progression, accelerating cell growth and inhibiting apoptosis. Our data may provide useful information towards the search for potent therapeutic targets towards the development of novel treatment strategies for CRC.     

Cyr61/CTGF/Nov family proteins in gastric carcinogenesis

Gastric cancer(GC)is the second leading cause of cancer-related death.The poor survival rate may reflect the relatively aggressive tumor biology of GC.Recently,the importance of the tumor microenvironment in carcinogenesis has emerged.In the tumor microenvironment,tumor cells and the surrounding stromal cells aberrantly secrete matricellular proteins capable of modulating carcinogenesis and regulating metastasis.The Cyr61/CTGF/Nov(CCN)proteins are a family of matricellular proteins with variable roles in many physiological and pathological processes.The evidence suggests that CCN family proteins contribute to GC carcinogenic processes.Here,we briefly review recent research on the effects of CCN family proteins in GC carcinogenesis and the development of new targeted agents in this field.     

WISP genes are members of the connective tissue growth factor family that are up-regulated in Wnt-1-transformed cells and aberrantly expressed in human colon tumors

Wnt family members are critical to many developmental processes, and components of the Wnt signaling pathway have been linked to tumorigenesis in familial and sporadic colon carcinomas. Here we report the identification of two genes, WISP-1 and WISP-2, that are up-regulated in the mouse mammary epithelial cell line C57MG transformed by Wnt-1, but not by Wnt-4. Together with a third related gene, WISP-3, these proteins define a subfamily of the connective tissue growth factor family. Two distinct systems demonstrated WISP induction to be associated with the expression of Wnt-1. These included (i) C57MG cells infected with a Wnt-1 retroviral vector or expressing Wnt-1 under the control of a tetracyline repressible promoter, and (ii) Wnt-1 transgenic mice. The WISP-1 gene was localized to human chromosome 8q24.1–8q24.3. WISP-1 genomic DNA was amplified in colon cancer cell lines and in human colon tumors and its RNA overexpressed (2- to >30-fold) in 84% of the tumors examined compared with patient-matched normal mucosa. WISP-3 mapped to chromosome 6q22–6q23 and also was overexpressed (4- to >40-fold) in 63% of the colon tumors analyzed. In contrast, WISP-2 mapped to human chromosome 20q12–20q13 and its DNA was amplified, but RNA expression was reduced (2- to >30-fold) in 79% of the tumors. These results suggest that the WISP genes may be downstream of Wnt-1 signaling and that aberrant levels of WISP expression in colon cancer may play a role in colon tumorigenesis.     

CCN family proteins and angiogenesis: from embryo to adulthood

The CCN family is a novel class of extracellular signal modulators that has been recently established. Typical members are composed of four conserved modules connected tandem, each of which is rich in cysteines and highly interactive with other molecules. The mammalian CCN family consists of six members, most of which have been described as multifunctional factors for the developmental process of mesenchymal tissue including blood vessel formation/induction. Particularly, the angiogenic properties of the three classical members, CCN1, 2 and 3 have so far been characterized, and their physiological and pathological significance has thus been indicated. Recent research has uncovered a unique mechanism regarding these proteins in promoting and/or modulating developmental, physiological and pathological angiogenic events. Namely, CCN proteins exert their ability to drive angiogenesis, not by stimulating a particular behavior of a particular type of cells, but by manipulating the cell communication networks that integrate most of the associated molecules/cells toward angiogenesis. In this article, the role of the CCN proteins in a variety of angiogenic events as an organizer of microenvironmental cell society is comprehensively described, together with a brief summary of the recent findings on each CCN family member relevant to angiogenesis including cardiovascular development and diseases.     

WNT1 inducible signaling pathway protein 3, WISP-3, a novel target gene in colorectal carcinomas with microsatellite instability.

BACKGROUND & AIMS: Microsatellite instability (MSI) is the phenotype of colorectal carcinomas with defect mismatch repair. Genes with repetitive sequences within their coding regions are targets for mutations in these tumors. We have evaluated 2 novel candidate genes for potential involvement in development of MSI colorectal carcinomas and compared them with alterations in known target genes. METHODS: The MSI status was determined by multiplex polymerase chain reactions (PCRs) of 5-17 markers in a Norwegian series of 275 colorectal carcinomas. All MSI tumors were analyzed for gene mutations using fluorescence PCR followed by capillary electrophoresis. Two novel candidate genes, WNT1-inducible signaling pathway protein 3 (WISP-3) and caspase-1, and 9 known target genes were analyzed. RESULTS: Thirteen percent of the tumors were MSI-high (H) and 12% were MSI-low (L). Thirty-three of 37 MSI-H vs. 1 of 34 MSI-L tumors showed mutations in the target genes (P < 0.001). WISP-3 was mutated in 31% of the MSI-H tumors. The frequencies of frameshift mutations in the known target genes were comparable with other studies. CONCLUSIONS: The relative high frequency of mutation, higher than those seen for other known target genes, the predicted truncation of the protein product, and the homology with WISP-1 and WISP-2, 2 proteins induced downstream of WNT1 signaling, strongly suggest WISP-3 as a novel target in development of MSI-H colorectal carcinomas.     

The angiogenic factor cysteine-rich 61 (CYR61, CCN1) supports vascular smooth muscle cell adhesion and stimulates chemotaxis through integrin alpha(6)beta(1) and cell surface heparan sulfate proteoglycans

Cysteine-rich 61 (CYR61, CCN1) is a heparin-binding, extracellular, matrix-associated protein of the cysteine-rich 61/nephroblastoma family, which also includes connective tissue growth factor, nephroblastoma overexpressed, Wnt-induced secreted protein-1 (WISP-1), WISP-2, and WISP-3. CYR61 induces angiogenesis in vivo and supports cell adhesion, promotes cell migration, and enhances growth factor-stimulated mitogenesis in fibroblasts and endothelial cells. Although the expression of CYR61 has been observed in arterial walls, its function in vascular smooth muscle cells (VSMCs) has not been examined to date. Here we show that purified CYR61 supports VSMC adhesion in a dose-dependent, saturable manner through integrin alpha(6)beta(1) with an absolute requirement of cell surface heparan sulfate proteoglycans. In addition, CYR61 induces VSMC chemotaxis, but not chemokinesis, through integrin alpha(6)beta(1) and heparan sulfate proteoglycans. Heparin-binding defective CYR61 mutants are unable to support VSMC adhesion but can still induce chemotaxis at a reduced level. Following balloon angioplasty in rat carotid artery, CYR61 protein level is elevated in the media and neointima of the injured vessel by d 4 post angioplasty, peaks from d 7 to 14, and remains high for at least 28 d. These data demonstrate the activities of CYR61 in VSMCs, identify the receptors that mediate its functions, and show that CYR61 is synthesized in arterial smooth muscle walls during proliferative restenosis. Together, these results implicate CYR61 as a novel factor that modulates the responses of VSMCs to vascular injury.     

Stress granules in colorectal cancer: Current knowledge and potential therapeutic applications

Stress granules(SGs) represent important non-membrane cytoplasmic compartments, involved in cellular adaptation to various stressful conditions(e.g., hypoxia, nutrient deprivation, oxidative stress). These granules contain several scaffold proteins and RNA-binding proteins, which bind to mRNAs and keep them translationally silent while protecting them from harmful conditions. Although the role of SGs in cancer development is still poorly known and vary between cancer types, increasing evidence indicate that the expression and/or the activity of several key SGs components are deregulated in colorectal tumors but also in pre-neoplastic conditions(e.g., inflammatory bowel disease), thus suggesting a potential role in the onset of colorectal cancer(CRC). It is therefore believed that SGs formation importantly contributes to various steps of colorectal tumorigenesis but also in chemoresistance. As CRC is the third most frequent cancer and one of the leading causes of cancer mortality worldwide, development of new therapeutic targets is needed to offset the development of chemoresistance and formation of metastasis. Abolishing SGs assembly may therefore represent an appealing therapeutic strategy to re-sensitize colon cancer cells to anti-cancer chemotherapies. In this review, we summarize the current knowledge on SGs in colorectal cancer and the potential therapeutic strategies that could be employed to target them.     

CCN proteins: multifunctional signalling regulators

CONTEXT: Although little is known as yet about the processes that coordinate cell-signalling pathways, matrix proteins are probably major players in this type of global control. The CCN (cyr61, ctgf, nov) proteins are an important family of matricellular regulatory factors involved in internal and external cell signalling. This family participates in angiogenesis, chondrogenesis, and osteogenesis, and they are probably involved in the control of cell proliferation and differentiation. STARTING POINT: Runping Gao and David Brigstock (Hepatol Res 2003; 27: 214-20) recently showed that CCN2 (CTGF, connective tissue growth factor) is a cell-adhesion factor for hepatic stellate cells. On exposure to transforming growth factor beta, hepatic stellate cells produce distinct CCN2 isoforms. Gao and Brigstock assign to CCN2 module 3 the capacity to mediate binding to low-density-lipoprotein receptor-related protein (LRP), which was previously reported to interact with CCN2 and to be involved in various types of signalling. They also establish that CCN2 binding to LRP is heparin dependent and that module 4 of CCN2 promotes LRP-independent adhesion of hepatic stellate cells. The differential binding of CCN2 isoforms to LRP highlights the importance of functional interactions between individual modules, and reinforces the concept that different module combinations might confer agonistic or antagonistic activities. WHERE NEXT? It is essential to understand how the distinct configuration of the various CCN isoform affects their biological activities and bioavailability, and to explore the mechanisms and the regulatory processes involved in the production of truncated CCN isoforms. A better understanding of the structural basis for their multifunctionality is a prerequisite to wider use of CCN proteins in molecular medicine.     

The opposing effects of CCN2 and CCN5 on the development of cardiac hypertrophy and fibrosis

CCN family members are matricellular proteins with diverse roles in cell function. The differential expression of CCN2 and CCN5 during cardiac remodeling suggests that these two members of the CCN family play opposing roles during the development of cardiac hypertrophy and fibrosis. We aimed to evaluate the role of CCN2 and CCN5 in the development of cardiac hypertrophy and fibrosis. In isolated cardiomyocytes, overexpression of CCN2 induced hypertrophic growth, whereas the overexpression of CCN5 inhibited both phenylephrine (PE)- and CCN2-induced hypertrophic responses. Deletion of the C-terminal (CT) domain of CCN2 transformed CCN2 into a CCN5-like dominant negative molecule. Fusion of the CT domain to the Carboxy-terminus of CCN5 transformed CCN5 into a CCN2-like pro-hypertrophic molecule. CCN2 transgenic (TG) mice did not develop cardiac hypertrophy at baseline but showed significantly increased fibrosis in response to pressure overload. In contrast, hypertrophy and fibrosis were both significantly inhibited in CCN5 TG mice. CCN2 TG mice showed an accelerated deterioration of cardiac function in response to pressure overload, whereas CCN5 TG mice showed conserved cardiac function. TGF-β-SMAD signaling was elevated in CCN2 TG mice, but was inhibited in CCN5 TG mice. CCN2 is pro-hypertrophic and -fibrotic, whereas CCN5 is anti-hypertrophic and -fibrotic. CCN5 lacking the CT domain acts as a dominant negative molecule. CCN5 may provide a novel therapeutic target for the treatment of cardiac hypertrophy and heart failure.     

A novel integrin alpha5beta1 binding domain in module 4 of connective tissue growth factor (CCN2/CTGF) promotes adhesion and migration of activated pancreatic stellate cells

BACKGROUND: Connective tissue growth factor (CCN2) is upregulated in pancreatic fibrosis and desmoplastic pancreatic tumours. CCN2 interacts with integrin alpha5beta1 on pancreatic stellate cells (PSC) in which it stimulates fibrogenesis, adhesion, migration, and proliferation. AIM: To determine the structural domain(s) in CCN2 that interact with integrin alpha5beta1 to regulation PSC functions. METHODS: Primary activated rat PSC were tested for their adherence to isoforms of CCN2 comprising modules 1-4 (CCN2(1-4)), modules 3-4 (CCN2(3-4)), module 3 alone (CCN2(3)), or module 4 alone (CCN2(4)). Adhesion studies were performed in the presence of EDTA, divalent cations, anti-integrin alpha5beta1 antibodies, CCN2 synthetic peptides, or heparin, or after pretreatment of the cells with heparinase, chondroitinase, or sodium chlorate. CCN2 integrin alpha5beta1 binding was analysed in cell free systems. The ability of CCN2(1-4), CCN2(3-4), or CCN2(4) to stimulate PSC migration was evaluated in the presence of anti-integrin alpha5beta1 or heparin. RESULTS: PSC adhesion was stimulated by CCN2(1-4), CCN2(3-4), or CCN2(4) and supported by Mg2+ but not Ca2+. CCN2(4) supported PSC adhesion or migration were blocked by anti-integrin alpha5beta1 antibodies or by treatment of cells with heparinase or sodium chlorate. A direct interaction between CCN2(4) and integrin alpha5beta1 was demonstrated in cell free assays. The sequence GVCTDGR in module 4 mediated the binding between CCN2(4) and integrin alpha5beta1 as well as CCN2(4) mediated PSC adhesion and migration. CONCLUSIONS: A GVCTDGR sequence in module 4 of CCN2 is a novel integrin alpha5beta1 binding site that is essential for CCN2 stimulated functions in PSC and which represents a new therapeutic target in PSC mediated fibrogenesis.     

Differential expression of the CCN family members Cyr61, CTGF and Nov in human breast cancer

The CCN family members cysteine-rich 61 (Cyr61/CCN1), connective tissue growth factor (CTGF/CCN2) and nephroblastoma over-expressed (Nov/CCN3) play diverse roles in cells, are known to regulate cell growth, adhesion, matrix production and migration and are involved in endocrine-regulated pathways in various cell types. The role of these molecules in cancer remains controversial. In a cohort of 122 human breast tumours (together with 32 normal breast tissues) we have analysed the expression of all three CCN members at the mRNA and protein levels. Significantly higher levels of Cyr61 (P = 0.02), but low levels of CTGF and Nov, were seen in tumour tissues compared with normal tissues. Significantly raised levels of Cyr61 were associated with poor prognosis (P = 0.02), nodal involvement (P = 0.03) and metastatic disease (P = 0.016). Patients who died of breast cancer also had high levels of Cyr61. In contrast, CTGF in patients with poor prognosis (P = 0.021), metastasis (P = 0.012), local recurrence (P = 0.0024) and mortality (P = 0.0072) had markedly reduced levels. Similar to CTGF, low levels of Nov were also seen in patients with poor prognosis and mortality and with significantly decreased survival (P = 0.033 and P = 0.0146, respectively). This result was fully supported by immunohistochemical analysis of frozen sectioned tissues. While fibroblasts and endothelial cells generally expressed good levels of all three CCN proteins, highly invasive MDA MB 231 cells expressed lower levels of CTGF and Nov, but higher levels of Cyr61, than the less invasive MCF-7. It is concluded that members of the CCN family are differentially expressed and may play important but contrasting roles in the progressive nature of human breast cancer. While Cyr61 appears to act as a factor stimulating aggressiveness, CTGF and Nov may act as tumour suppressors.     

Potential role of TRIM3 as a novel tumour suppressor in colorectal cancer (CRC) development

Objective Colorectal cancer (CRC) is the third leading cause of cancer-related mortality in the United States. Recent cancer genome-sequencing efforts and complementary functional studies have led to the identification of a collection of candidate ‘driver’ genes involved in CRC tumorigenesis. Tripartite motif (TRIM3) is recently identified as a tumour suppressor in glioblastoma but this tumour-suppressive function has not been investigated in CRC. Material and methods In this study, we investigated the potential role of TRIM3 as a tumour suppressor in CRC development by manipulating the expression of TRIM3 in two authentic CRC cell lines, HCT116 and DLD1, followed by various functional assays, including cell proliferation, colony formation, scratch wound healing, soft agar, and invasion assays. Xenograft experiment was performed to examine in vivo tumour-suppressive properties of TRIM3. Results Small-interfering RNA (siRNA) mediated knockdown of TRIM3 conferred growth advantage in CRC cells. In contrast, overexpression of TRIM3 affected cell survival, cell migration, anchorage independent growth and invasive potential in CRC cells. In addition, TRIM3 was found to be down-regulated in human colon cancer tissues compared with matched normal colon tissues. Overexpression of TRIM3 significantly inhibited tumour growth in vivo using xenograft mouse models. Mechanistic investigation revealed that TRIM3 can regulate p53 protein level through its stabilisation. Conclusions TRIM3 functions as a tumour suppressor in CRC progression. This tumour-suppressive function is exerted partially through regulation of p53 protein. Therefore, this protein may represent a novel therapeutic target for prevention or intervention of CRC.     

Emerging roles of non-coding RNAs in colorectal cancer oxaliplatin resistance and liquid biopsy potential

Colorectal cancer (CRC) is one of the most common malignancies of the digestive tract,with the annual incidence and mortality increasing consistently.Oxaliplatinbased chemotherapy is a preferred therapeutic regimen for patients with advanced CRC.However,most patients will inevitably develop resistance to oxaliplatin.Many studies have reported that non-coding RNAs (ncRNAs),such as microRNAs,long non-coding RNAs,and circular RNAs,are extensively involved in cancer progression.Moreover,emerging evi...     

The aminoterminal insulin-like growth factor (IGF) binding domain of IGF binding protein-3 cannot be functionally substituted by the structurally homologous domain of CCN3

IGF binding proteins (IGFBPs) are a family of structurally homologous proteins that bind IGFs with high affinities and can modulate IGF activity. The IGF binding site has been shown to comprise residues in both the aminoterminal and carboxyterminal domains. In recent years several proteins including members of the CCN (connective tissue growth factor, Cyr61, and nephroblastoma overexpressed) family were recognized as having structural homology in their aminoterminal domains to the IGFBPs. Despite their low or undetectable IGF binding ability, a proposal was made to rename them as IGFBP-related proteins. To test whether the aminoterminal domain of a CCN protein can fulfill the high-affinity IGF binding function of an IGFBP, we created a chimera in which the aminoterminal domain of IGFBP-3 was substituted with the aminoterminal domain of CCN3 (previously known as Nov). The CCN3-IGFBP-3 chimera bound IGFs and inhibited IGF activity very weakly, similar to CCN3 itself. Although structurally similar, the aminoterminal domain of CCN3 is unable to replace the aminoterminal domain of IGFBP-3 in forming a high-affinity IGF-binding site. These results argue against a direct role of CCN3 in the regulation of IGF bioavailability and indicate that the nomenclature of IGFBP-related proteins (which implies functional relationship to the classical IGFBPs) is inappropriate for CCN proteins.