Biological functions and role of CCN1/Cyr61 in embryogenesis and tumorigenesis in the female reproductive system (Review).

Cysteine-rich angiogenic inducer 61 (CCN1/Cyr61) was originally revealed to act as a growth factor-inducible immediate-early gene, present in serum and platelet-derived growth factor-stimulated mouse BALB/c 3T3 cells ( 1 ). As a member of CCN family, it was first cloned by O'Brien in 1990 via differential hybridization screening of a cDNA library from BALB/c 3T3 clone A31 cells ( 2 ). The CCN family is composed of six members including CCN1/Cyr61, connective tissue growth factor, nephroblastoma overexpressed, Wnt-1-induced secreted protein (WISP)-1, WISP-2 and WISP-3 ( 3 ). CCN1/Cyr61 is a basic secreted protein localized on the cell surface, cell interior and the extracellular matrix (ECM). CCN1/Cyr61 targets cells including fibroblasts, epithelial cells, endothelial cells, smooth muscle cells and neurons through integrins and heparin sulfate proteoglycan (HSPG) receptors. CCN1/Cyr61 exhibits a variety of functions in differing target cells. The biological properties of CCN1/Cyr61 in the regulation of cell survival, proliferation, differentiation, migration, adhesion and synthesis of ECM have been demonstrated to be important in the progression of embryogenesis and tumorigenesis in the female reproductive system.

Mammalian reproduction is a complex process involving ovulation, fertilization, embryo development and implantation and the failure of any process may result in sterility, malformations or adverse pregnancy outcomes. Previous studies demonstrated that CCNl/Cyr61 is expressed on the corpus luteum (CL) ( 31 ), conceptus and endometrium in early pregnancy ( 32 ), trophoblastic origin of the placenta ( 33 ), midterm fetal smooth muscle vessel walls of the arterial circulatory system ( 34 ), endothelial cells of vessels surrounding the cloaca, embryonic hippocampal neurons ( 35 ) and mesodermal and ectodermal mesenchymal cells ( 28 ). It contributes to the process of embryogenesis including the implantation ( 36 ), fetal neovascularization ( 34 ), fetal brain development ( 35 ), fetal cartilaginous development ( 28 ) and fetal-maternal cross-talk ( 32 ). The abnormal expression of CCNl/Cyr61 may result in pre-eclampsia, epispadias, embryonic death resulting from disability of allantoic fusion and the disturbance of vascular dysplasia ( 34 , 37 ). CL, which is a glandular-like structure rich in blood vessels and exhibits a key role in mammalian reproduction. The development of the follicle into the CL involves neovascularization, and previous studies have demonstrated that CCNl/Cyr61 may mediate this process ( Fig. 2 ). Zhang et al ( 31 ) suggested that CCNl/Cyr61 is expressed in the CL of cows and the expression is upregulated at days 4, 6, 10 and 16 of CL. It was therefore hypothesized that the upregulation may be associated with the angiogenesis of the CL. A further study conducted by Romereim et al ( 38 ) demonstrated that the CCNl/Cyr61 gene is highly expressed in small luteal cells isolated from cows. These findings support the hypothesis that CCNl/Cyr61 is important in the development of the CL. However, the expression pattern, regulation and function of CCNl/Cyr61 in human CLs still remains unclear and requires further investigation. Implantation is an important process during the early development of embryos. CCNl/Cyr61 has previously been suggested to act as a mediator for embryo implantation. It has previously been detected to be upregulated in the invasive extravillous trophoblasts and unclosed endometrial luminal epithelium surrounding the embryo ( 39 ). Pre-eclampsia is one of the common complications of pregnancy, which originates in the placenta and results in complication of maternal and fetal health ( 40 ). The hypothesis of the etiology has been recognized as insufficient invasion of trophoblast cells into the decidua and the obstruction of maternal spiral artery remodeling ( 41 ). This leads to an inadequate blood flow in the placenta and may result in the inhibition of placental development and intrauterine growth restrictions ( 42 ). Recombinant CCNl/Cyr61 protein-stimulated trophoblast cells demonstrate a stronger invasion ability compared with cells without the stimulus ( 43 ). Chen et al ( 33 ) revealed that CCNl/Cyr61 mRNA expression levels in placentas of pre-eclampsia cases are decreased compared with than those in normal pregnancies. Furthermore, Kipkeew et al ( 36 ) indicated that CCNl/Cyr61 and CCN3 promote the migration capability of trophoblast cells by activating FAK and Akt kinase. This evidence verified the hypothesis of a negative correlation between the expression of CCNl/Cyr61 and pre-eclampsia. These findings may act as a research foundation for the early diagnosis and prediction of pre-eclampsia through screening for expression of CCNl/Cyr61. Zhang et al ( 44 ) suggested that that micro (mi)RNA 155 contributes to pre-eclampsia development by downregulating CCNl/Cyr61. MiRNAs are relatively stable and the identification of circulating miRNAs involved in promoting the development of pre-eclampsia by regulating the expression of CCNl/Cyr61 in the plasma of pregnant woman, may aid in the clinical prediction of pre-eclampsia. CCNl/Cyr61 is additionally important in the maintenance of the pregnancy and the embryonic development. Klein et al ( 32 ) demonstrated that CCNl/Cyr61 is co-expressed on the conceptus and endometrium in the early stage of pregnancy and involved the maintenance of the early pregnancy. It contributes to the cross-talk of the conceptus and endometrium through affecting the proliferation and differentiation of endometrial and trophectoderm cells and stimulation of endometrial-angiogenesis. Furthermore, previous studies have demonstrated that CCNl/Cyr61 regulates neovascularization and matrix remodeling in the process of embryogenesis. CCNl/Cyr61 gene knock-out mice often suffer from vascular anomalies of placenta and embryonic fatalities ( 34 ). The expression of CCNl/Cyr61 may be detected in endothelial cells of vessels surrounding the cloaca and the umbilical cord on gestational days 10 and 11.5 in mice, which suggests it may be involved in the development of human epispadias ( 37 ). In addition, the expression of CCNl/Cyr61 in fetal hippocampal neurons has been demonstrated to be increased compared with mature hippocampal neurons, which suggests it may exhibit an important role during development of the embryonic brain ( 35 ). Overall, CCNl/Cyr61 is important role in the female reproductive system. It was previously demonstrated that CCNl/Cyr61 acts as a tumor-suppressor or promoter in different types of tumors depending on the complex external environment ( 45 – 47 ). CCNl/Cyr61 is involved in the occurrence and development of tumors through its role in tumor angiogenesis, tumor cell proliferation and apoptosis and tumor metastasis ( 25 , 47 – 49 ). An overview of the expression and function of CCNl/Cyr61 in female reproductive tumors is presented in Table II ( 50 – 61 ). Further research and understanding of the function of CCNl/Cyr61 in different types of cancer may provide a promising basis for cancer prognosis and therapy. Uterine leiomyoma (UL) is the most common benign tumor of the female reproductive system, which is hormone dependent and results from smooth muscle cell proliferation ( 62 ). Molecular genetic studies have provided compelling evidence that abnormally expressed genes have an important influence on UL occurrence ( 63 , 64 ). CCNl/Cyr61 expression in UL is downregulated when compared with matched normal uterine smooth muscle cells, suggesting it may exhibit a tumor-suppressor role ( 50 ). Di Tommaso et al ( 51 ) revealed that estrogen upregulates the expression of the CCNl/Cyr61 gene in myometrium tissue, however has no influence on the expression of the CCNl/Cyr61 gene in fibroid tissue, detected via gene expression analysis. The results indicated that the expression of CCNl/Cyr61 may not be regulated by the estrogen receptor in uterine fibroids. Fundamental research was conducted by Wallace et al ( 52 ) to determine if hypoxia induced CCNl/Cyr61 secretion through the Endothelin-A (ET A ) receptor in UL. However, the results identified that blockade of the ETA receptor demonstrated no significant influence on the increase of CCNl/Cyr61 expression under normoxic or hypoxic conditions and the secretion of CCNl/Cyr61 may not be regulated by the ETA receptor in UL. The exact mechanism still remains unclear and further investigation is required in the future. Endometriosis (EMT) is one of the most common diseases of obstetrics and gynecology, and commonly associates with infertility in women of child-bearing age ( 65 – 67 ). The specific pathogenesis of EMT remains to be fully elucidated. Although EMT is a frequently occurring benign disease, it is characterized by malignant behaviors including recurrence and metastasis ( 68 , 69 ). At present, various pharmacotherapies and other methods have been proposed to treat EMT ( 70 , 71 ). However, their usage has been limited due to strong and obvious adverse effects ( 65 , 72 ). It was previously demonstrated that the expression of the CCNl/Cyr61 gene is controlled by estrogen and is enhanced in endometria of women suffering from EMT and in endometriotic lesions, suggesting that CCNl/Cyr61 may act as a useful maker gene for EMT ( 53 ). A further study identified that estrogen promotes the expression of the CCNl/Cyr61 gene during the early stages of EMT-like establishment and the CCNl/Cyr61 gene contributes to regulation of cell proliferation and the generation of vasculature in the development of lesions, using the conditional knockout mice lacking uterine CCNl/Cyr61 ( 54 ). Furthermore, Gashaw et al ( 73 ) demonstrated that the expression level of CCNl/Cyr61 is upregulated by hypoxia-inducible factor 1-α, through use of the benign endometrial cell line. The study suggests that targeting CCNl/Cyr61 signaling during the early stages of lesion establishment may provide a novel treatment method for EMT. Endometrial cancer (EC) is a common malignant gynecological tumor, however non-invasive diagnostic tools for early and accurate diagnosis are still limited ( 74 ). Hormonal dysregulation is believed to contribute to the etiology of EC ( 75 ). Previous studies demonstrated that long-term replacement estrogen therapy is associated with a type I EC and progestational hormone is not sufficient to prevent this ( 56 ). Estrogen may upregulate the expression of CCNl/Cyr61 and current data indicate that there is a significant increase of CCNl/Cyr61 expression in the endometrium of women with polycystic ovarian syndrome and premalignant lesions. Hence, measures to detect the expression of CCNl/Cyr61 may result in its use as an early diagnostic marker of type I EC cancer However, Chien et al ( 55 ) demonstrated that the expression level of Cyr61 is decreased in endometrial tumors compared with normal endometrium. At present, little is known regarding the antithetical function of CCNl/Cyr61 in EC. Previous findings from Witek et al ( 76 ) revealed that CCNl/Cyr61 genes expressed in all cancer grades and their expressions levels are potentially correlated with the survival of patients. Therefore, detecting the expression level of CCNl/Cyr61 may act as a novel non-invasive diagnostic tool for early and accurate diagnosis of EC. Cervical cancer is one of the most common gynecological tumors occurring in women worldwide, with >190,000 newly diagnosed cases per year ( 77 ). The human papillomavirus infection is an important factor in the development of cervical cancer, and vaccination against it provides an effective way to prevent development of cervical cancer. However, the pathogenesis underlying cervical cancer remains unknown, and its incidence is increasing each year. Xie et al ( 58 ) demonstrated that CCNl/Cyr61 gene expression is downregulated in cervical cancer compared with normal tissues and its expression level is inversely correlated with miR-205 expression. miR-205 has been reported to contribute to the development of cervical cancer and regulate cell proliferation and migration in human cervical cancer cells. The identified inverse expression pattern between CCNl/Cyr61 and miR-205 suggests that CCNl/Cyr61 may be one of the target genes of miR-205. In addition, by performing genome-wide expression analysis of miRNAs, in addition to investigation of mRNAs in cervical cancer, Joy et al ( 57 ) demonstrated that CCNl/Cyr61 is a key gene involved in vascularization of tumors and suggests its expression level is negatively associated with the level of hsa-miR-221. The roles of CCNl/Cyr61 in human cervical cancer may provide novel insights into carcinogenesis and the clinical diagnosis. Ovarian cancer is the leading cause of gynecological malignancy-associated mortalities worldwide and the symptoms are insidious in onset ( 78 , 79 ). The search for novel biomarkers for early diagnosis in ovarian carcinoma may provide effective medical support. Lee et al ( 60 ) demonstrated that CCNl/Cyr61 promotes ovarian cancer cell proliferation and inhibits apoptosis by regulating p53 and NF-κB expression through the phosphoinositide-3-kinase/AKT Serine/Threonine kinase/mechanistic target of rapamycin signaling pathways. Bartel et al ( 59 ) revealed that CCNl/Cyr61 is preferentially expressed in high grade serous carcinomas. It was demonstrated that CCNl/Cyr61 expression is significantly upregulated in ovarian epithelial carcinoma tissue compared with benign ovarian tumor tissue samples and the levels of CCNl/Cyr61 are associated with lymph node metastases ( 61 ). CCNl/Cyr61 may therefore be useful in targeted diagnosis and therapy in ovarian epithelial carcinoma.

The present review systematically clarified the biological functions of CCN1/Cyr61 and its associated roles in the female reproductive system, including embryogenesis and tumorigenesis. CCNl/Cyr61 may regulate numerous cellular activities, angiogenesis and chondrogenesis. It is important in the reproduction and the occurrence and development of tumors in the reproductive system. However, the underlying mechanism that results in CCNl/Cyr61 acting as a tumor-suppressor or tumor-promoter in different types reproductive system tumors, remains to be fully elucidated, due to the complicated external environment, including complex crosstalk of intracellular molecules and multi-module activated signal transduction pathways.