International Journal of Gynecological Pathology:
Pathology of the Upper Genital Tract: Original Articles
Inverse Expression of Cystein-rich 61 (Cyr61/CCN1) and Connective Tissue Growth Factor (CTGF/CCN2) in Borderline Tumors and Carcinomas of the Ovary
Bartel, Frank Ph.D.; Balschun, Katharina M.D.; Gradhand, Elise M.D.; Strauss, Hans G. M.D.; Dittmer, Jürgen Ph.D.; Hauptmann, Steffen M.D.
Institute of Pathology (F.B., E.G., S.H.)
Department of Gynecology (H.G.S., J.D.), Martin Luther University Halle-Wittenberg, Halle (Saale)
Institute of Pathology (K.B.), University of Kiel, Kiel, Germany
F.B. and K.B. have contributed equally to the results of this study.
The authors declare no conflicts of interest.
Address correspondence and reprint requests to Steffen Hauptmann, MD, Institute of Pathology, Hospital of Dueren, Roonstr. 30, D-52309 Dueren, Germany. E-mail: email@example.com.
Members of the CCN [cystein-rich 61 (Cyr61)/connective tissue growth factor (CTGF)/nephroblastoma (NOV)] protein family are involved in the regulation of cellular proliferation, apoptosis, and migration and are also assumed to play a role in carcinogenesis. Therefore, we performed a retrospective study to investigate the immunohistochemical expression of both Cyr61 and CTGF in 92 borderline tumors (BOTs) and 107 invasive carcinomas of the ovary (IOCs). To determine their diagnostic and prognostic value, we correlated protein expression with clinicopathologic factors including overall and disease-free survival. Cyr61 and CTGF were found to be inversely expressed in both BOTs and IOCs, with a stronger expression of Cyr61 in IOCs. Moreover, Cyr61 was found to be preferentially expressed in high-grade serous carcinomas, whereas CTGF was found more frequently in low-grade serous carcinomas. Weak Cyr61 levels correlated with both low estrogen receptor and p53 expression (P=0.038, P=0.04, respectively). However, no association was observed between CTGF, estrogen receptor, and p53 expression levels in IOCs. Regarding prognosis, Cyr61 was found to be of no value, but the loss of CTGF was found to be associated with a poor prognosis in multivariate analysis of overall (relative risk 2.8; P=0.050) and disease-free (relative risk 2.3; P=0.031) survival. Cyr61 and CTGF are inversely expressed in BOTs and IOCs, and loss of CTGF independently indicates poor prognosis in IOCs.
The CCN family of growth factors (cysteine-rich 61=Cyr61, connective tissue growth factor=CTGF, nephroblastoma=NOV) currently comprises six 30 to 40-kD cysteine-rich proteins and consists of 6 members that belong to matricellular proteins of the extracellular matrix (ECM) 1–3. They do not play a structural role; rather, they modulate cell-ECM interactions 4. CCN proteins are characterized by 4 nearly identical domain structures and they participate in various biologic processes such as organ development during embryogenesis, wound healing, angiogenesis, and fibrosis 4. They are involved in the regulation of diverse cellular activities, including proliferation, adhesion, migration, and apoptosis 1–3, and they also play a role in carcinogenesis. Cyr61 and CTGF are expressed in mesenchymal cells including fibroblasts and endothelial cells. They are involved in the regulation of angiogenesis (mainly Cyr 61) and ECM remodeling (mainly CTGF). Information on the expression of these 2 proteins in epithelial ovarian tumors is sparse. Therefore, we performed a retrospective immunohistochemical study on a series of borderline tumors (BOTs) and invasive ovarian carcinomas (IOCs), and correlated the expression of Cyr61 and CTGF with the clinical outcome of the patients. We were also interested in determining whether the expression pattern could aid in the differential diagnosis of epithelial ovarian tumors.
MATERIAL AND METHODS
In this retrospective study, paraffin-embedded tissue samples from 199 patients with surface epithelial ovarian tumors diagnosed at the Institute of Pathology, Martin-Luther-University Halle-Wittenberg between 1996 and 2005 were selected on the basis of the availability of tissue. The tumors were classified according to the WHO guidelines (reviewed in 5). Tissue specimens encompassed 107 ovarian carcinomas and 92 BOTs. The tumor stage was determined according to the criteria of the International Federation of Gynecology and Obstetrics (FIGO). Follow-up information was obtained for 105 IOC patients and for 71 BOT patients by inquiry from the tumor registry and local population registry.
Cell Culture and Western Blot Analysis
Ovarian carcinoma cell lines derived from serous carcinomas (CAOV-42, OAW-42, OVCAR-3, and SKOV-3) and 1 clear-cell carcinoma (ES-2) were grown in 75-cm2 cell culture flasks (Falcon) in Dulbecco’s modified eagle medium supplemented with 10% fetal calf serum. Nonmalignant ovarian surface epithelium human ovarian surface epithelium (HOSE) cells were maintained in MCDB 105 and medium 199 (1:1) plus 15% fetal calf serum. For cell lysis, cells were trypsinized, harvested by centrifugation, and boiled for 10 min in 200 µL Laemmli buffer. An aliquot of the lysate containing 50 µg protein was mixed with 5 µL lithium dodecyl sulfate sample buffer and 2 µL NuPAGE-reducing agent (Invitrogen) and Laemmli buffer in a final volume of 20 µL. The mixture was boiled for 10 min and loaded onto a 10% polyacrylamide sodium dodacyl sulfate gel (NuPAGE). After blotting the proteins onto a nitrocellulose membrane using the Transblot Semi Dry Transfer Cell (Bio-Rad), the membrane was rinsed with phosphate-buffered saline (PBS) and blocked in blocking buffer (5 g nonfat milk dissolved in 100 mL PBS and 100 µL Tween 20) for 1 hr at room temperature followed by incubation with either anti-Cyr61 (rabbit polyclonal, H-78, 1 µg/mL, Santa Cruz), anti-CTGF (mouse monoclonal, clone 88430, 1 µg/mL, R&D Systems), or anti-β-actin (1:1000) in blocking buffer for 2 hr at room temperature. After washing with PBS/0.1% Tween 20, the membrane was incubated with an anti-mouse or anti-rabbit horseraddish peroxidase conjugate (1:5000) in blocking buffer for 90 min. For detection, ECL Western blotting detection reagent and ECL hyperfilm (Amersham) were used.
Immunohistochemistry was performed using neutral-buffered formalin-fixed and paraffin-embedded tissue according to standard procedures. For detection of Cyr61, a rabbit polyclonal antibody (H-78; Santa Cruz, CA), and for CTGF a monoclonal antibody (clone 88430; R&D Systems, Minneapolis, MN), was used. For antigen retrieval, slides were treated with sodium citrate buffer, pH 6.0, in a microwave oven for 4×5 min. Primary antibodies were diluted 1:50 for Cyr61 in PBS and 1:200 for CTGF in target retrieval solution (pH 9, DAKO) and incubated for 30 min at 37°C. After washing with PBS for Cyr61 and target retrieval solution for CTGF, slides were sequentially incubated with a biotinylated secondary antibody and a streptavidin-horseradish peroxidase conjugate. Colorimetric development was performed using aminoethylcarbazol (AEC, Zytomed, Berlin, Germany). Fallopian tube and corpus luteum slides were carried in every trial as positive controls. The expression of the estrogen receptor (ER) was analyzed using the SP1 monoclonal antibody (LabVision Corp., Fremont CA), diluted 1:500, and the expression of the p53 protein was analyzed using the DO-7 mouse monoclonal antibody (DAKO). Whole slides from all 199 ovarian epithelial tumors were stained.
Evaluation of Immunohistochemical Staining
Immunohistochemical staining was independently evaluated by 2 investigators (K.B., S.H.), blinded to the clinical data. For semiquantitative analysis of the target expression within tumor cells, an immunoreactive score (IRS) 6 was applied. Scoring the number of stained tumor cells and the staining intensity and multiplying these scores yielded the IRS for each sample. The scores for the number of stained tumor cells and for the staining intensities were as follows: 0=no cells stained, 1=<10% of cells stained, 2=11% to 50% of cells stained, 3=51% to 80% of cells stained, 4=81% to 100% of cells stained; staining intensity: 1=weak, 2=moderate, 3=strong. An IRS between 0 and 3, between 4 and 6, or between 8 and 12 was designated as weak, moderate, or strong expression, respectively. The target expression within the tumor stroma was also evaluated and scored as negative, expressed, or strongly expressed. In cases where the investigators calculated different scores, a multihead microscope session was held to achieve consensus.
We compared the correlation between Cyr61 and CTGF expression levels by a Mann-Whitney U test. Using a χ2 test for trends, the statistical correlation between clinicopathologic factors and target expression was assessed. The probability of differences in overall survival and disease-free survival (DFS) were determined as a function of time by a Kaplan-Meier test with significance probing by applying log-rank test. We used multivariate regression analysis based on the Cox proportional hazard model to test the independence of these parameters to predict overall survival and disease-free survival. P values of <0.05 were considered as significant. For all statistical procedures, SPSS v15.0.1 software was used.
We have investigated 107 patients with IOCs and 92 patients with BOTs. The median age of the IOC patients was 64 yr, and their clinicopathologic data are shown in Table 1. All patients were treated surgically according to the guidelines of the Arbeitsgemeinschaft Gynäkologische Onkologie (Working Group Gynecological Oncology, AGO). Eighty-three of 107 (87%) received chemotherapy (21 cisplatinum, 62 cisplatinum, and paclitaxel). Other types of chemotherapy were given to 2 patients. Thirteen patients refused chemotherapy and information was not available for 4 patients. Five patients were not treated systemically because of FIGO IA stage. Follow-up information was available from 87 IOC patients. The median follow-up time was 37.9 mo (range, 1–109 mo). Sixty-four patients suffered from recurrent disease, and 42 patients died of disease. Seven patients died of uncertain cause and they were included in the survival analysis. Six patients died of other causes; these cases were censored for the overall survival analysis. The mean DFS time was 15 mo and the 5-yr disease-free survival rate was 17%. The mean overall survival time was 56 mo and the 5-yr overall survival rate was 47.2%.
The median age of patients with BOTs was 48 yr, and their clinicopathologic data are shown in Table 2. Fifteen BOT patients obtained chemotherapy (7 cyclophosphamide, 4 cisplatinum and paclitaxel, 3 cisplatinum). Within the BOT cohort, survival data of 72 patients were available. The median follow-up time was 73 mo (range, 0–144 mo). Five women with serous BOT suffered from recurring disease. Seven patients with BOT died. Two women died of progressive disease (low-grade serous carcinoma) and 1 died of uncertain cause. These 3 patients were included in the survival analysis, but not the remaining 4 patients, who died of other causes. The 5-yr overall survival rate was 96.7%. The mean disease-free survival time was 68 mo (range, 0–144 mo), and the 5-yr disease-free survival rate was 94.6%.
For statistical analyses, we have grouped FIGO stages into 3 categories: (1) organ-confined disease with intraovarian tumors (FIGO IA+B), which was present in 11.2% (12/107) of IOCs and 35.2% (25/71) of BOTs; (2) ovarian surface involvement and pelvic disease (FIGO IC+II), which was present in 25.2% (27/107) of IOCs and 19.7% (14/71) of BOTs; and (3) advanced disease (FIGO III+IV), which was present in 63.6% (68/107) of IOCs and 45.1% (32/71) of BOTs.
Expression of Cyr61 and CTGF in Cell Lines
Cyr61 was expressed in HOSE cells and in all carcinoma cell lines (ES-2, OAW-42, OVCAR-3, SKOV-3), except Caov-42. The immunoblot revealed a single band at 48 kD (Fig. 1). CTGF was also detectable in all carcinoma cell lines with a single band at 38 kDa, but the protein amount was much lower compared with Cyr61. HOSE cells did not express any CTGF.
Cyr61 Expression in Normal Ovaries, Fallopian Tube, and Surface Epithelial Ovarian Tumors
The normal ovarian surface epithelium did not express Cyr61, whereas the fibroblastic ovarian stroma and all 3 cell types of fallopian tube epithelium were strongly positive with a cytoplasmic pattern (Fig. 2). Eighty-three percent (76/92) of BOTs expressed Cyr61 only weakly, with the remaining 17% of cases showing a more intense expression (Table 2 and Figs. 2, 3). A reverse picture was seen in IOCs. We found a strong expression in 13%, intermediate expression intensity in 56%, and low expression or negativity in 31% of cases (Table 1 and Figs. 2, 3). The different expression of Cyr61 in BOTs and IOCs was highly significant (Fig. 3, P<0.0001 Mann-Whitney U nonparametric test). There was no correlation between Cyr61 expression and any clinicopathologic parameter in BOTs (Table 2).
In IOCs, the expression of Cyr61 was correlated with FIGO stage, serous type, residual tumor, ascites, lymph node involvement, histologic grade, number of mitosis, and ER and p53 positivity (Table 1). None of the mucinous, endometrioid, or clear-cell carcinomas were strongly Cyr61 positive. Despite the preferential expression of Cyr61 in IOCs with prognostic unfavorable features, we did not find any significant correlation with overall (P=0.588; Fig. 4A) and DFS (P=0.173; Fig. 4B).
CTGF Expression in Normal Ovaries, Fallopian Tube, and Surface Epithelial Ovarian Tumors
Normal ovarian surface epithelium, the fibroblastic ovarian stroma, the ciliated and secretory cells of the fallopian tube epithelium, and the luteinized cells of the corpus luteum strongly expressed CTGF. Sixty percent (55/92) of BOTs maintained the strong epithelial CTGF positivity, and in 37% (34/92), the expression was still moderate. Only 3% (3/92) of BOTs had a weak CTGF expression. There was no significant correlation between CTGF expression and clinicopathologic factors within the BOT cohort (Table 2).
In IOC, a strong CTGF expression was maintained in only 22.5% (24/107). Forty percent (43/107) of IOC expressed CTGF at an intermediate level, and 37.5% (40/107) were CTGF-negative or only focally positive. The difference between CTGF expression in BOTs and IOCs was statistically significant (Tables 1 and 2, Figs. 2, 3, P<0.0001, Mann-Whitney U nonparametric test). There was no difference between the expression level of fibroblasts and endothelial cells within the normal ovarian and the tumor stroma. In nonserous IOCs, we observed a tendency toward a higher CTGF expression, and an inverse correlation between mitotic activity and CTGF expression (Table 1). Moreover, a loss of CTGF expression was significantly associated with a reduced overall (Fig. 5A, P=0.019) and disease-free survival (Fig. 5, P=0.026). Other prognostic factors in univariate analysis (log-rank test, Table 3) were residual tumor (P<0.0001), FIGO stage (P=0.0001), Silverberg grading (P=0.012), histologic type (P=0.015), and age of the patient (P=0.045). There was no association between CTGF, ER, and p53 expression (Table 1). In a multivariate analysis, the strongest risk factors were the residual tumor with an estimated relative risk for overall and disease-free survival of 4.6 and 3.4 mo, respectively (P=0.003), and stage of disease for disease-free survival with a relative risk of 1.7 (P=0.041). Interestingly, a loss or weak expression of CTGF was found to be an indicator for poor outcome (Table 4) with an estimated relative risk of 2.3 for recurrence (P=0.031) and of 2.8 for tumor-related death (P=0.050).
The aim of this study was to investigate the expression of Cyr61 and CTGF, 2 members of the CCN matricellular protein family, in a series of BOTs and IOCs to correlate their expression with clinicopathologic parameters and survival data. The CCN proteins are widely distributed in the human tissue and involved in various biologic phenomena; however, less is known about their physiological and pathologic functions and precise mechanisms of action 4.
Under physiological conditions, Cyr61 is important for normal embryonic development and wound healing. Its essential role in angiogenesis has been demonstrated by knockout mice showing lethal vascular defects 7. Brigstock 1 had shown previously that Cyr61 and CTG promote endothelial cell growth, migration, adhesion, and survival in vitro, and that they can also regulate the activity of angiogenic molecules such as vascular endothelial growth factor and basic fibroblast growth factor along with other molecules of the ECM such as collagens, matrix metalloproteases, and tissue inhibitors of matrix metalloproteases. In addition, CCN proteins modulate the signals of several essential proteins such as integrins, bone morphogenetic protein, Wnt, and Notch 8. In the normal ovary, Cyr61 is expressed in luteinized granulosa cells, consistent with the vascular changes during the development and preservation of the corpus luteum 1.
In contrast, CTGF is rather involved in the regulation of ECM synthesis. This has been shown by heterozygous deletion experiments, leading to immediate death after birth due to defects in matrix organization and skeletal development 9. CTGF is expressed in adult human heart, brain, placenta, liver, muscle, kidney, and lung 10. In this study, CTGF was found to be highly expressed in normal surface ovarian epithelial cells and BOTs, whereas its expression was low in IOCs. Moreover, the loss of CTGF was associated with worse prognosis in IOC patients. A comparable prognostic effect has also been reported for other tumor entities such as in breast, lung, oral squamous carcinoma 11, pancreatic carcinoma, gastric carcinoma, and colorectal carcinoma 12–15. However, the reverse is true for malignant melanoma 16, glioblastoma 17 and esophageal cancer 18, breast carcinoma 19,20, and diverse mesenchymal tumors, that is, rhabdomyosarcoma 21 and chondrosarcoma 22. In these tumor types, a strong CTGF expression is correlated with a poor prognosis.
The biologic effect of CTGF seems to be tumor-type specific, resembling the situation with transforming growth factor (TGF)-β. Like TGFβ, CTGF also promotes proliferation of mesenchymal cells and suppresses that of normal epithelial cells 11,23. This suppressive effect is maintained in some carcinoma cell lines, getting lost or even being reversed in other tumor cells, depending on the downstream signaling available within the cell. In contrast, the protein may have an anti-angiogenic effect by direct binding of vascular endothelial growth factor to the thrombospondin-1-module of CTGF 24. Therefore, tumors with low CTGF levels may have an advantage by the loss of suppression of angiogenesis.
The situation with Cyr61 is in some aspects inverse to CTGF. We found that normal ovarian surface epithelial cells and BOTs do not or only weakly express Cyr61. IOCs of endometroid and clear-cell type and low-grade serous carcinomas also do not produce substantial amounts of Cyr61. In contrast, high levels of Cyr61 protein were found in most of the IOCs of high-grade serous, transitional, and mixed type and in undifferentiated carcinomas. To our knowledge, this is the first report about Cyr61 protein expression in IOCs. There is only 1 report on Cyr61 mRNA expression in IOCs, detecting the mRNA in 63% of cases 25, which is comparable to our data (Cyr61 protein expression in 69% of the cases). Surprisingly, Cyr61 expression was not associated with decreased DFS in our patient cohort, despite its preferential expression in IOCs with adverse prognostic features. This is probably due to the limited number of patients in our study because in other carcinomas such as those of the breast, endometrium, pancreas, and stomach, high expression of Cyr61 was found to be associated with advanced and aggressive disease 15,26–29. The prognostic influence of Cyr61 could be related to 3 properties of the molecule. First, Cyr61 is associated with a chemoresistant phenotype as shown in many in vitro studies 30. The molecular background of this association is related to αvβ3-integrin, which is inducible by Cyr61 and acts as its receptor 31. Ligation of αvβ3-integrin by Cyr61 activates phosphoinositide3-kinase/Akt and ERK/mitogen-activated protein kinase pathways, which prevent apoptosis 2. A second important point is its angiogenic activity. Several reports have described a relationship between the expression of Cyr61 and tumor angiogenesis in human malignancies 32 and mice 2,25. A third point is the stimulating effect of Cyr61 on tumor cell motility and invasiveness as shown for gastric cancer cells 15. Another interesting finding is that the expression of Cyr61 in ovarian (and breast) carcinoma cells is estrogen-dependent 25,29 because we have shown that ER-expressing IOCs behave differently. Finally, Gery et al. 25 observed that estrogen increased Cyr61 mRNA and protein levels in ovarian cancer cells . The identical positive association between estrogenic action and a consecutive Cyr61 expression was also observed in ER-positive MCF-7 breast cancer cells 33. Moreover, Sampath et al. 34 demonstrated in MCF-7 human breast cancer cells an upregulation of Cyr61 by epidermal growth factor. In conclusion, the levels of Cyr61 were found to be higher in ER-positive and epidermal growth factor receptor-positive breast carcinomas, compared with ER-negative carcinomas.
It is well accepted in the literature that the matricellular proteins Cyr61 and CTGF play a key role in tumorigenesis in several malignancies. The controversial results and interpreted functionalities in diverse studies of various tumor entities may depend on their tumor/tissue-specific roles and the changing tumor microenvironment with a resulting complex interaction between these target proteins and further factors and cofactors (such as cytokines and growth factors). For example, hypoxia alters the CCN gene expression, resulting in reduced Cyr61 and CTGF mRNA expression in CRC cell lines (HT29 and Caco-2) under hypoxic conditions 35. In the same recently published study, however, an increase in CTGF protein levels correlated with an advanced tumor/TNM stage 35. In contrast, there was no correlation between Cyr61 and clinicopathologic parameters. Unfortunately, their investigations were based on a limited sample size (39 patients with colorectal cancer), and no survival analyses were performed. Therefore, no direct prognostic effect of CTGF in colorectal cancer could be seen. CCN proteins are considered to influence tumor progression and regression in several ways such as cell survival or apoptosis, angiogenesis, and by modulating different signalings pathways such as TGF-β, the insulin-like growth factor system, and Wnt.
In summary, although belonging to the same family, Cyr61 and CTGF have completely different expression patterns in surface epithelial ovarian tumors, and exert different biologic effects. Expression of Cyr61 is linked to a more aggressive phenotype of ovarian carcinomas, and the loss of CTGF is associated with poor prognosis.
The authors thank Dr Schmidt (Tumor registry) and Ms. Westhusen (local population registry) for providing data. The excellent technical assistance of Ms. Beer and Ms. Krell is gratefully acknowledged. Furthermore, we thank Dr Wolfgang Schmitt and Martin Köbel for helpful discussions.
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Ovarian carcinoma; Ovarian borderline tumor; Cyr61; CTGF; Prognosis; Immunohistochemistry
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