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Tumor Biology |
Division of Hematology/Oncology, Cedars-Sinai Medical Center, Department of Biomathematics, UCLA School of Medicine, Los Angeles, California 90048 [D. X., H. W., R. E., H. P. K.], and Saitama Cancer Center, Ina, Saitama 362, Japan [K. N.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several lines of evidence support a role for CCN molecules in tumorigenesis. Consistent with its profibrotic properties, CTGF is overexpressed in pancreatic cancers (14) and melanomas (15) . WISP-1 is strongly expressed in the fibrovascular stroma of breast tumors developing in Wnt-1 transgenic mice (6) . Moreover, forced overexpression of WISP-1 in normal rat kidney fibroblasts (NRK-49F) was sufficient to induce their transformation (16) . Recently, several studies have suggested that CYR61 was overexpressed in breast cancers and may be involved in estrogen-mediated tumor development (1, 2, 3) .
To quantify expression of CCN genes in breast cancer, we performed real-time quantitative RT-PCR based on TaqMan methodology to measure mRNA levels of four genes: CTGF, WISP-1, CYR61, and NOVH. Measurements were made from the same total RNA preparation for each of 44 primary breast tumors and seven paired normal breast tissue samples. Furthermore, we determined whether overexpression of one of the CCN genes was correlated with clinical and pathological parameters of breast cancer by univariate analysis and classification tree model.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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RNA Extraction and cDNA Synthesis
Total RNA was extracted from breast specimens by using TRIzol reagent (Life Technologies, Inc.) according to the standard protocol. The quality of the RNA samples was determined by electrophoresis through agarose gels and staining with ethidium bromide, and the 18S and 28S RNA bands were visualized under UV light. Total RNA (µg) was processed directly to cDNA by reverse transcription with Superscript II (Life Technologies, Inc.), according to the manufacturer’s protocol in a total volume of 50 µl.
Real-Time RT-PCR
Theoretical Basis.
Reactions are characterized at the point during cycling when amplification of the PCR product is first detected, rather than the amount of PCR product accumulated, after a fixed number of cycles. The parameter Ct is defined as the fractional cycle number at which the fluorescence generated by cleavage of the probe passes a fixed threshold above baseline. The CCN target message in unknown samples is quantified by measuring Ct and by using a standard curve to determine the quantity of starting target message. We also quantified transcripts of ß-actin as the endogenous RNA control, and each sample was normalized on the basis of its ß-actin content. For each experimental sample, the amount of the targets and endogenous reference is determined from the standard curve. The target amount is divided by the endogenous reference amount to obtain a normalized target value. The relative gene target expression level was also normalized to a mean value (value = 1) from seven normal breast tissue samples (calibrator). Final results, expressed as N-fold difference in CCN gene expression relative to the ß-actin and the calibrator, termed 
CCN, were determined as follows:
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Another housekeeping gene, 18S, was used as a second endogenous reference gene to determine the consistency of normalization.
Primers and Probes.
Primers and probes for the CCN and ß-actin genes (Table 1)
were designed using software PRIMER3.4
We conducted BLASTN searches against dbEST and nr (the nonredundant set of GenBank, EMBL, and DDBJ database sequences) to confirm the total gene specificity of the nucleotide sequences chosen for the primers and probes and the absence of DNA polymorphisms. To avoid amplification of contaminating genomic DNA, one of the two primers or the probe was placed at the junction between two exons or in a different exon. Primers were purchased from Life Technologies, Inc., and probes were from Perkin-Elmer Applied Biosystems.
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shows the real-time RT-PCR standard curve for the CTGF gene. A strong linear relationship between the Ct and the log of the starting copy number was always demonstrated. The efficiency of the reaction (E), calculated by the formula: E = 101/m - 1, where "m" is the slope of the standard curve, ranged from 90 to 100% in the different assays. The standard curves for WISP-1, CYR61, and NOVH were also constructed (data not shown).
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Statistical Analysis
2 test, t test, and Wilcoxon’s rank-sum test were used to study the association of each gene with single clinical factors (age, stage, tumor size, lymph node status, hormone receptors, and HER-2/neu status). A logistic regression model was developed to associate the probability of being a positive CCN marker with various clinical features. Stage and tumor size were dichotomized as stages I/II and III/IV and tumor size 
50 mm (1a/2a) and >50 mm (3a/4b). Backward procedure was used for predictor selection. Classification tree analysis was also carried out to explore the association of gene status with clinical factors. 
statistics was used to assess the relationship between all pairs of the four genes. The value, its SE, and 95% confidence interval were reported.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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3 as the cutoff point for overexpression of CCN genes at the RNA level in the breast cancers. To determine whether ß-actin is suitable for the calibrator of normalization, a second housekeeping gene, 18S, was also used as a reference calibrator for CYR61, CTGF, WISP-1, and NOVH in four normal breast and five breast tumor samples. The levels of expression of the CCN genes were comparable with those when ß-actin was used as the reference gene (data not shown).
Expression of CTGF in Primary Breast Cancers.
Among the 44 breast tumor RNA samples tested, 24 of 44 (55%) showed CTGF gene overexpression (Fig. 2A)
. Univariate analysis (Table 2A)
showed either a significant or borderline significant association between breast cancer stage, tumor size, lymph node status, and HER-2/neu status in the primary tumor, as well as age at onset of disease compared with whether the primary tumor overexpressed CTGF. Analysis of stage showed that only 7 of 27 (26%) of the patients with stage I and II breast cancer overexpressed CTGF; in contrast, all 17 (100%) of those with stage III or IV overexpressed CTGF (P = 0.001). Similarly, only 8 of 28 (29%) individuals whose primary breast cancer was 50 mm overexpressed CTGF; in comparison, all 16 of those whose primary tumor was 50 mm overexpressed CTGF (P = 0.001). Furthermore, of the 17 patients who were lymph node negative, only 5 (29%) overexpressed CTGF in their primary breast cancer. In contrast, 19 individuals overexpressed CTGF in their primary tumors among the 27 individuals who were lymph node positive (70%, P = 0.008).
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Expression of WISP-1 in Primary Breast Cancers.
Overexpression of WISP-1 was found in 20 of 44 (46%) breast cancer samples (Fig. 2B)
. The correlations of WISP-1 mRNA levels with clinical and pathological parameters were similar to those of CTGF except for age at diagnosis (Table 2B)
. A strongly significant association existed between stage, tumor size, lymph node status, and HER-2/neu status versus WISP-1 expression. For stage, only 7 of 27 (26%) cases with stage I or II overexpressed WISP-1 compared with 13 of 17 (76%) samples from patients with stage III or IV (P = 0.001) breast cancers. Concerning tumor size, only 8 of 28 (29%) primary tumors, which were 50 mm, overexpressed WISP-1, contrasted to 12 of 16 (75%) primary tumors, which were 50 mm and overexpressed WISP-1 (P = 0.003). Similarly, WISP-1 was overexpressed in 4 of 17 (24%) women having no lymph node involvement compared with 16 of 27 (59%) women having positive lymph nodes at the time of removal of the primary tumor (P = 0.02). A significant association was noted between those who were HER-2/neu positive and overexpressed WISP-1 (9 of 13, 69%) versus those who were HER-2/neu positive with low levels of WISP-1 (11 of 31, 35%; P = 0.040). Statistical analysis showed no significant difference between age (P = 0.39), ER (P = 0.09), and progesterone (P = 0.45) versus WISP-1 expression in the primary breast cancers of 44 individuals.
Expression of Cyr61 in Primary Breast Cancers.
Seventeen specimens overexpressed CYR61 among 44 samples tested (39%; Fig. 2C
). This is very similar to what we noted earlier by Northern analysis (1)
. Correlations between CYR61 expression and the clinical and pathological features of these patients is similar to those that we reported previously (Table 2C
; Ref. 1
). A significant correlation was found between elevated levels of CYR61 and advanced stage and size of the primary tumor and lymph node involvement at the time of removal of the primary tumor. Also, overexpression of CYR61 is strongly correlated with ER positivity of the primary tumor; in contrast, no correlation with ER expression was noted for either CTGF or WISP-1.
Expression of NOVH in Primary Breast Cancers.
In contrast to the other members of CCN gene family, only five (11%) primary breast tumors overexpressed NOVH among the 44 patients (Fig. 2C)
. Statistical analysis showed no difference between the clinical and pathological parameters versus level of expression of NOVH in the primary breast cancers (Table 2D)
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Classification Tree Multivariate Models of CTGF, WISP-1, and CYR61.
Classification tree analysis was also carried out to explore the association of CTGF, WISP-1, and CYR61 and clinical features of the disease (Fig. 3)
. Stage and lymph node status were the important predictors for CTGF expression (Fig. 3A)
. This model showed that 100% of stage III or IV breast cancers had high levels of CTGF. For WISP-1, the age, HER-2/neu expression, and stage were the important predictors (Fig. 3B)
. Stage I/II breast cancers with HER-2/neu positivity have an 80% chance of being WISP-1 positive, a four times higher frequency than those whose breast cancers were HER-2/neu negative. Furthermore, if the patient has an advanced breast tumor (stage III/IV) and is over age 59, the primary tumor will most likely (91%) overexpress WISP-1. Age, ER status, and stage are important predictors for CYR61 expression (Fig. 3C)
. The classification tree posits that ER is only important in women under age 60, whereas stage is only important in women over age 60. CYR61 expression was low in all these ER- breast cancers.
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shows statistical analysis, a measure of agreement/correlation, where a value of 0.5 denotes significant association and a value of 0.5 implies no significant correlation. statistical analysis showed that significant concordance occurred among CTGF, WISP-1, and CYR61 expression; however, the association of NOVH with the other three genes was not significant. Classification tree analysis was performed to study the links among CTGF, WISP-1, and CYR61 with all seven clinical and pathological parameters. This analysis showed that ER, HER-2/neu, and stage were important predictors for having prominent expression of two or three CCN proteins (Fig. 3D)
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this study, we performed real-time PCR to quantify the mRNA levels of CTGF, WISP-1, CYR61, and NOVH. RNA quantitation using real-time PCR was made even more precise and reproducible by being based on Ct values established in the early exponential phase of the PCR reaction rather than the end point measurement of the amount of accumulated PCR product. Real-time PCR has high intra-assay and interassay reproducibility and gives statistical confidence. Overexpression of CYR61 was identified in 17 of the 44 breast cancer patients (39%). This frequency is similar to what we found previously by Northern blot analysis (1)
and what others reported by immunohistochemistry (3)
. Previous studies suggested that CYR61 is involved in angiogenesis through its interaction with integrin 
ß3 (17, 18, 19)
. Integrin signals are involved in a variety of cellular activities, including cell migration, proliferation, and survival, as well as diverse biological processes, including angiogenesis and tumor progression (reviewed in Refs. 20, 21, 22
). Understanding the interaction of CYR61 with integrins may provide insight into how it might be involved in tumor development.
CTGF was identified as a mitogen found in the conditioned medium of human umbilical vein endothelial cells (23) . It encodes a protein of 349 aa with 43% sequence identity to CYR61, and all 38 cysteines in CTGF and CYR61 are completely conserved. CTGF is transcriptionally activated with rapid kinetics in fibroblasts by serum growth factors (24) and transforming growth factor-ß (25 , 26) . Moreover, CTGF has been implicated in cellular proliferation, migration, and tube formation of vascular endothelial cells in culture and angiogenesis in vivo (27 , 28) . Consistent with these properties, CTGF is often overexpressed in melanomas, sarcomas, chondrosarcomas, and pancreatic cancer cells (14 , 15 , 29 , 30) . Recently, overexpression of CTGF has also been found in acute lymphoblastic leukemia and pediatric myofibroblastic tumors (31 , 32) . Expression of CTGF in breast cancer was noted by Northern blot analysis in 7 of 11 (64%) human invasive mammary ductal carcinomas and xenografts (9) . This study examined only a small number of primary samples, no normal breast samples served as control, and the correlation between CTGF expression and clinical and pathological parameters was not analyzed. In the present study, expression of CTGF was quantified by real-time PCR, and a high level of CTGF mRNA was noted in 24 of 44 (55%) breast cancer samples. Univariate analysis showed significant association between age at diagnosis, breast cancer stage, tumor size, lymph node status, and HER-2/neu status compared with whether the primary tumor overexpressed CTGF. Considering that CTGF is closely related to CYR61 and each signal through integrins (18 , 19) , both CYR61 and CTGF may trigger downstream events via integrin signaling.
WISP-1 was identified as a gene up-regulated in Wnt-1 transformed C57 MG mouse mammary epithelial cells (6) . WISP-1 encodes a protein with a secretory signal peptide and has complete conservation of all 38 cysteine residues with those of CYR61 and CTGF. To our knowledge, expression of WISP-1 has not been studied in either normal or cancerous human breast cells. We found it was highly expressed in 20 of 44 (46%) primary breast cancers. Interestingly, statistical analysis showed a strongly significant association between stage, tumor size, lymph node status, and HER-2/neu expression versus mRNA level of WISP-1. The role of WISP-1 in breast cancer is unclear. Overexpression of WISP-1 induced morphological transformation, increased cellular saturation density, promoted growth in normal rat kidney fibroblasts, and induced tumor formation in nude mice (16) .
NOV was identified as an aberrantly expressed gene in chicken nephroblastomas induced by myeloblastosis-associated virus (33) . NOVH encodes a protein of 357 aa with a secretory signal peptide and complete conservation of the 38 cysteines found in CYR61, CTGF, and WISP-1. Whereas CTGF, WISP-1, and CYR61 were reported to act as positive regulators of cell growth, NOV provided the first example of a CCN protein with negative regulatory properties and the first example of aberrant expression being associated with tumor development (33) . Whereas expression of the full-length NOV has a growth inhibitory effect in chicken embryo fibroblasts, expression of an NH2-terminally truncated form of NOV can transform these cells (33) . However, because this truncated form lacks the secretory signal, its transforming activity is not likely mediated through interaction with a cell surface receptor. In our study, overexpression of NOVH was found in only 5 of 44 (11%) breast tumors, and no significant correlation was linked between expression of this gene and the clinical and pathological features of the breast cancers. These findings suggesting NOVH are not involved in either the development or progression of breast cancer.
In summary, our data indicate that overexpression of CTGF, WISP-1, and CYR61 may be involved in the process of breast cancer development and points to an association between expression of these proteins and clinical and pathological features of breast cancer. This comprehensive elucidation of CCN gene expression in breast tumors is an important first step to explore the mechanism and function of these genes in the development of breast cancer. Clinically, our studies showed that prominent expression of the genes coding for CTGF, WISP-1, and CYR61 is associated with an advanced stage of breast cancer at diagnosis. Future studies will attempt to determine whether measurement of these proteins at diagnosis can provide prognostic data and suggest those tumors that might be responsive to therapy. Hopefully, understanding the aberrant signaling pathways that are activated by high levels of expression of these CCN proteins may offer useful therapeutic targets.
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FOOTNOTES |
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1 Supported in part by NIH, Marcia Schwartz Trust, Thornworth Fund, and the C. and H. Koeffler Research Fund. H. P. K. is a member of the Jonsson Comprehensive Cancer Center and holds the endowed Mark Goodson Chair of Oncology Research at Cedars-Sinai Medical Center/UCLA School of Medicine. 
2 To whom requests for reprints should be addressed, at Division of Hematology/Oncology, Cedars-Sinai Medical Center, UCLA School of Medicine, Los Angeles, CA 90048. Phone: 01-310-423-7740; Fax: 01-310-423-0225; E-mail: xied{at}ucla.edu
3 The abbreviations used are: CTGF, connective tissue growth factor; RT-PCR, reverse transcription-PCR; ER, estrogen receptor; NOVH, human NOV; PR, progesterone receptor.
4 Internet address: http://www.genome.wi.nit.edu/cgi-bin/primer/primer3www.cgi.
Received 7/17/01. Accepted 10/17/01.
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, data for breast cancer patients performed in triplicate.
