Breast Cancer
2001; Elsevier BV; Volume: 276; Issue: 17 Linguagem: Inglês
10.1074/jbc.m009755200
ISSN1083-351X
AutoresDong Xie, Carl W. Miller, James O’Kelly, Kei Nakachi, Akiko Sakashita, Jonathan W. Said, Jeffrey Gornbein, H. Phillip Koeffler,
Tópico(s)Wnt/β-catenin signaling in development and cancer
ResumoTo identify genes involved in breast cancer, polymerase chain reaction-selected cDNA subtraction was utilized to construct a breast cancer-subtracted library. Differential screening of the library isolated the growth factor-inducible immediate-early geneCyr61, a secreted, cysteine-rich, heparin binding protein that promotes endothelial cell adhesion, migration, and neovascularization. Northern analysis revealed that Cyr61was expressed highly in the invasive breast cancer cell lines MDA-MB-231, T47D, and MDA-MB-157; very low levels were found in the less tumorigenic MCF-7 and BT-20 breast cancer cells and barely detectable amounts were expressed in the normal breast cells, MCF-12A. Univariate analysis showed a significant or borderline significant association between Cyr61 expression and stage, tumor size, lymph node positivity, age, and estrogen receptor levels. Interestingly, expression of Cyr61 mRNA increased 8- to 12-fold in MCF-12A and 3- to 5-fold in MCF-7 cells after 24- and 48-h exposure to estrogen, respectively. Induction of Cyr61mRNA was blocked by tamoxifen and ICI182,780, inhibitors of the estrogen receptor. Stable expression of Cyr61 cDNA under the regulation of a constitutive promoter in MCF-7 cells enhanced anchorage-independent cell growth in soft agar and significantly increased tumorigenicity and vascularization of these tumors in nude mice. Moreover, overexpression of Cyr61 in MCF-12A normal breast cells induced their tumor formation and vascularization in nude mice. In summary, these results suggest that Cyr61 may play a role in the progression of breast cancer and may be involved in estrogen-mediated tumor development. To identify genes involved in breast cancer, polymerase chain reaction-selected cDNA subtraction was utilized to construct a breast cancer-subtracted library. Differential screening of the library isolated the growth factor-inducible immediate-early geneCyr61, a secreted, cysteine-rich, heparin binding protein that promotes endothelial cell adhesion, migration, and neovascularization. Northern analysis revealed that Cyr61was expressed highly in the invasive breast cancer cell lines MDA-MB-231, T47D, and MDA-MB-157; very low levels were found in the less tumorigenic MCF-7 and BT-20 breast cancer cells and barely detectable amounts were expressed in the normal breast cells, MCF-12A. Univariate analysis showed a significant or borderline significant association between Cyr61 expression and stage, tumor size, lymph node positivity, age, and estrogen receptor levels. Interestingly, expression of Cyr61 mRNA increased 8- to 12-fold in MCF-12A and 3- to 5-fold in MCF-7 cells after 24- and 48-h exposure to estrogen, respectively. Induction of Cyr61mRNA was blocked by tamoxifen and ICI182,780, inhibitors of the estrogen receptor. Stable expression of Cyr61 cDNA under the regulation of a constitutive promoter in MCF-7 cells enhanced anchorage-independent cell growth in soft agar and significantly increased tumorigenicity and vascularization of these tumors in nude mice. Moreover, overexpression of Cyr61 in MCF-12A normal breast cells induced their tumor formation and vascularization in nude mice. In summary, these results suggest that Cyr61 may play a role in the progression of breast cancer and may be involved in estrogen-mediated tumor development. Breast cancer is the most common form of malignancy and the second-leading cause of cancer-related death among women in the United States. The nature of the cellular and molecular changes that lead to breast cancer remains poorly defined. Several of the aberrant tumor suppressor genes that have been identified, include BRCA1,BRCA2 (1Miki Y. Swensen J. Shattuck-Eidens D. Futreal A.P. Harshman K. Tavtigian S. Liu Q. Cochran C. Bennett M.L. 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Oncol. 1996; 14: 737-744Crossref PubMed Scopus (1265) Google Scholar, 6Fantl V. Richards M.A. Smith R. Lammie G.A. Johnstone G. Allen D. Gregory W. Peters G. Dickson C. Barnes D.M. Eur. J. Cancer. 1990; 26: 423-429Abstract Full Text PDF PubMed Scopus (119) Google Scholar, 7Ramachandra S. Machin L. Ashley S. Monaghan P. Gusterson B.A. J. Pathol. 1990; 161: 7-14Crossref PubMed Scopus (124) Google Scholar, 8Ross J.S. Fletcher J.A. Semin. Cancer Biol. 1999; 9: 125-138Crossref PubMed Scopus (170) Google Scholar), but only 15–30% of invasive breast cancers show increased expression of these genes.We set out to isolate differentially expressed genes in human breast cancer. Suppression subtractive hybridization (SSH)1 and differential screening (9Diatchenko L. Lau Y.C. Campbell A.P. Chenchik A. Moqadam F. Huang B. Lukyanov S. Lukyanov K. Gurskaya N. Sverdlov E.D. Sieber P.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6025-6030Crossref PubMed Scopus (2727) Google Scholar) identified genes highly expressed in the carcinoma cell line MDA-MB-231 and either absent or minimally expressed in the normal breast cell line MCF-12A. Cyr61 was one of the genes isolated from the screening of the subtracted cDNA library. This gene codes for a growth factor-inducible, immediate-early gene first identified in murine fibroblasts (10Lau L.F. Nathans D. EMBO J. 1985; 4: 3145-3151Crossref PubMed Scopus (366) Google Scholar). Cyr61 is a secreted, cysteine-rich, heparin-binding protein that associates with the extracellular matrix. Purified Cyr61 protein has been reported to mediate cell adhesion, stimulate chemotaxis, augment growth factor-induced DNA synthesis, enhance cell survival, and induce angiogenesis in vivo (10Lau L.F. Nathans D. EMBO J. 1985; 4: 3145-3151Crossref PubMed Scopus (366) Google Scholar, 11Lau L.F. Nathans D. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1182-1186Crossref PubMed Scopus (638) Google Scholar, 12Kireeva M.L. Mo F.-E. Yang G.P. Lau L.F. Mol. Cell. Biol. 1996; 16: 1326-1334Crossref PubMed Scopus (303) Google Scholar). Because these characteristics may foster the progression of breast cancer, we studied this gene in detail and found it to be highly expressed in some invasive breast cancer cell lines and 36% of primary breast tumors. Furthermore, characterization of the oncogenic activity of Cyr61demonstrated that forced expression of Cyr61 enhanced MCF-7 cell growth in soft agar and promoted tumor growth in both normal breast and breast cancer cells in nude mice.MATERIALS AND METHODSSSH and Differential ScreeningSSH was performed by using the PCR-Select cDNA subtraction kit (CLONTECH). Tester double-stranded cDNA was synthesized from 2 μg of poly(A)+ RNA isolated from the breast cancer cell line, MDA-MB-231; and driver cDNA was made from 2 μg of RNA from the normal breast cell line, MCF-12A. The subtracted library was differentially screened with 32P-labeled probes synthesized as first-strand cDNA from tester and driver. The differential clones were picked and confirmed by Northern analysis.Cell CultureThe cell lines MCF-12A, MCF-10A, MDA-MB-231, MCF-7, MDA-MB-157, MDA-MB-436, BT-474, BT-20, ZR-75–1, and T47D were obtained from the American Type Culture Collection (Rockville, MD). MCF-12A and MCF-10A normal breast lines were maintained in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 medium (Life Technologies, Inc.), 20 ng/ml epidermal growth factor, 100 ng/ml cholera toxin, 0.01 mg/ml insulin, 500 ng/ml hydrocortisone, and 5% horse serum; MCF-7 was cultured in Dulbecco's modified Eagle's medium (Life Technologies, Inc.); MDA-MB-231, MDA-MB-436, MDA-MB-157, BT-474, BT-20, and T47D were grown in RPMI 1640 (Life Technologies, Inc.). Media were supplemented with 10% fetal calf serum (Gemini Bio-Products, Calabasas, CA), 10 units/ml penicillin-G, 10 mg/ml streptomycin (Gemini Bio-Products). All cells were incubated at 37 °C in 5% CO2. In experiments in which the effects of estrogen were studied, MCF-12A and MCF-7 cells were first cultured in phenol red free medium with charcoal-treated newborn calf serum. Cells were then treated with estradiol (1 × 10−9 m; Sigma Chemical Co.) for different durations. In the experiments in which antiestrogens were examined, cells were pretreated with tamoxifen for 6 h (1 × 10−7 m; Sigma) or ICI182,780 (1.5 × 10−7 m; Tocris Cooksson Inc., Ballwin, MO) before estradiol treatment.Proteins and AntibodiesRecombinant human Cyr61 protein was purified from anEscherichia coli host strain (BL21) programmed for synthesis of the Cyr61 protein via pGEX-5X-2 expression vector (Amersham Pharmacia Biotech). Anti-Cyr61 antibodies were prepared from polyclonal rabbit antisera raised against a GST-Cyr61 fusion protein. Monoclonal antibodies to CD31 were obtained from DADO Corp. (Carpinteria, CA).RNA Preparation and Northern AnalysisTotal RNA was isolated from cell lines and patient tissue by using TRIzol reagent (Life Technologies, Inc.) according to the standard protocol. Cyr61 cDNA probe was labeled with [32P]dCTP by using a random primer (Life Technologies, Inc.). Total cellular RNA was separated on 1.2% formaldehyde-agarose gels and was immobilized on a Hybond-N+ membrane by standard capillary transfer and UV cross-linking. The membrane was hybridized with the Cyr61 probe by standard protocol and was rehybridized with a 32P-labeled glyceraldehyde-3-phosphate dehydrogenase cDNA to confirm equal loading of the samples.Cell Transfection and Soft Agar AssaysThe expression vector pcDNA61 was constructed by placing full-length human Cyr61 cDNA into the pcDNA3.1 eukaryotic expression vector containing the neomycin gene under the control of the same promoter (Invitrogen). The constructs were transfected into MCF-12A and MCF-7 cells by using LipofectAMINE, and transfectants were selected for G418 resistance (400 and 450 μg/ml, respectively). The selected clones were confirmed by Northern analysis. For clonogenic assay, cells were plated into 24-well flat-bottomed plates using a two-layer soft agar system with a total of 1 × 103 cells/well in a volume of 400 μl/well, as described previously (13Munker R. Norman A. Koeffler H.P. J. Clin. Invest. 1986; 78: 424-430Crossref PubMed Scopus (172) Google Scholar). After 14 days of incubation, the colonies were counted and measured. All experiments were done at least three times using triplicate plates per experimental point.Cell Migration AssaysCell migration assays were performed according to the protocol from Chemicon (Temecula, CA). 5 × 104 cells were added to the top of each modified Boyden chamber (10-μm thickness and 8-μm pores) containing polycarbonate membranes (6.5-mm diameter) coated on the underside of the membrane with 10 μg/ml vitronectin and with the lower chamber containing 500 μl of migration buffer (medium with 0.5% bovine serum albumin). Cells were allowed to migrate to the underside of the top chamber for 4–8 h. The migratory cells attached to the bottom surface of the membrane were stained with 0.1% crystal violet in 0.1 m borate, pH 9.0, and 2% ethanol for 20 min at room temperature. The stained cells were extracted by using extraction buffer (Chemicon). The number of migratory cells per membrane was determined by absorbance at 550 nm.Tumorigenicity AssayStably transfected MCF-12A/61 and MCF-12A/V cells (1.0 × 106 cells/flank) and MCF-7/61 and MCF-7/V cells (5 × 104 cell/flank) were injected subcutaneously into 8-week-old female nude mice. Each animal was injected at two sites in the flanks. The resulting tumors were measured once a week, and tumor volume (mm3) was calculated by using the standard formula: length × width × height × 0.5236. Tumors were harvested 6 weeks after injection and individually weighed before fixation. Data were presented as both tumor volume (mean ± S.D.) and tumor weight (mean ± S.D.). Statistical analysis was performed with software (GraphPad, San Diego, CA) using the Student'st test.Real-time Quantitative PCRQuantitative PCR analysis was performed using the TaqMan PCR Core Reagent kit (PE Biosystems). cDNA of breast cancer samples were diluted, and real-time PCR was performed following the protocol. Her2/neu-specific primers were 5′-ACAGTGGCATCTGTGAGCTG and 5′-CCCACGTCCGTAGAAAGGTA. The TaqMan probe for Her2/neu was 5′-CCAGCCCTGGTCACCTACAACACAG. β-Actin was used for normalization, and β-actin-specific primers were 5′-GATCATTGCTCCTCCTGAGC and 5′-ACTCCTGCTTGCTGATCCAC. The TaqMan probe for β-actin was 5′-CTCGCTGTCCACCTTCCAGCAGAT.Statistical AnalysisUnivariateChi square methods were used to compare stage, tumor size category, and node status category in Cyr61-positiveversus Cyr61-negative individuals. Age, ER levels, and PgR levels were compared using t tests and Wilcoxon rank sum tests. The kappa statistic was used to assess concordance among stage, tumor size category, and node status and is reported with its standard error.MultivariateThe simultaneous relationship between the six predictors and Cyr61 was modeled using classification tree methods. Comparison of stage versus tumor size showed that these two variables are proxies for each other (kappa = 0.94 ± 0.05, observed agreement = 42/44 = 95%). Therefore, tumor size was not included as a candidate in the multivariate analysis. The concordance between stage and lymph node status was only moderate (kappa = 0.54 ± 0.12). A logistic regression analysis was also carried out but gave poor results and is therefore not reported.RESULTSCyr61 Is Highly Expressed in Some Breast Cancer Cell Lines and Primary Breast TumorsUsing a differential screening technique (see “Materials and Methods”), we identified 36 genes highly expressed in breast cancer cell line MDA-MB-231 as compared with normal breast cell line MCF-12A (data not shown). The genes each displayed 6-fold or greater expression in MDA-MB-231 than in MCF-12A, as determined using Northern blots and densitometric analysis (Fig.1). Cyr61, a growth factor-inducible immediate-early gene, is one of these differentially expressed genes. Cyr61 expression was examined in a panel of normal breast and breast cancer cell lines. Northern analysis showed that Cyr61 mRNA was prominently expressed in the highly invasive and tumorigenic breast cancer cell lines MDA-MB-231, MDA-MB-436, MDA-MB-157, BT-474, and T47D; it was expressed at a low level in the less tumorigenic tumor cell lines MCF-7, BT-20, and ZR-57–1 and was barely detectable in the normal breast cell lines, MCF-10A and MCF-12A (Fig.2 A).Figure 2Expression of Cyr61 in human breast cancer cell lines and primary breast cancer samples.Cellular RNA was extracted, subjected to electrophoresis (10 μg of total RNA/lane for cell lines and 5 μg of total RNA/lane for primary tissue), Northern blotted, and probed with 32P-labled Cyr61 cDNA. A, MCF-10A and MCF-12A are normal breast cell lines; LNCaP is a prostate cancer cell line; the others are breast cancer cell lines. B,N1 and N15 are normal breast tissue; all other samples are from primary breast tumors.View Large Image Figure ViewerDownload (PPT)To determine the pattern of Cyr61 expression in primary breast tumors, RNAs were isolated from quick-frozen breast samples obtained at initial surgery from 44 individuals with breast cancer (Table I). Each breast carcinoma and matching normal breast tissue was confirmed histologically. Expression of Cyr61 was easily detectable in sixteen of 44 (36%) primary breast cancer samples (Table I), but levels were negligible in normal breast tissues as shown by Northern analysis (Fig.2 B, representative autoradiogram of Northern blot).Table IClinic information of the breast cancer samplesCase no.AgeStageT 1-aT, tumor size.N 1-bN, lymph node status.ERPgRHer2/neu 1-cHer2/neu expression was quantitatively measured by real-time PCR. Breast cancer cells BT-474 and MCF-7 were used as overexpressor and low expressor controls, respectively. We define overexpression (+) when expression of Her2/neu was 24 folds or higher than the expression of Her2/neu in MCF-7 cells.overexpressionCyr61 expressionyearsfmol/g172IIIa3a1a4228−+270IIIa3a200++366IIIb4b1b610−+471II2a1b270140++581II2a1a00−+661IIIb4b1b2351−+765IIIb4b200++870IIIb4b200−+968IIIb4b0320200−+1042II2a1a120370−+1151IIIa3a072260++1266II1a1a310500++1341II2a1a32470++1455II2a1b16053−+1562IIIa3a1b1300++1681IIIb4b3930−+1763II2a1b00−−1860I1a063160−−1942I1a020210+−2082II2a1bNM 1-dNM, not measured.NM−−2145II2a000−−2252II2a1b00+−2343I1a000−−2450II2a1a00−−2553II2a011035−−2648II2a0110270−−2738IIIa3a1a00+−2862II2a0240360−−2952II2a1a00−−3044I1a0150180−−3155IIIb4b0170850+−3252II2a0026+−3346II2a000−−3469II2a0180340−−3552IIIb4b300−−3650II2a02470−−3750II2a1b8.616−−3848II2a1b00−−3934IIIb4b200−−4071IV3a300+−4153II2a000−−4260II2a01236−−4357II2a1b00−−4457IIIa3a200−−All breast cancer samples were diagnosed as invasive ductal carcinomas, except case no. 19: ductal carcinoma in situ and no. 24: medullary carcinoma.1-a T, tumor size.1-b N, lymph node status.1-c Her2/neu expression was quantitatively measured by real-time PCR. Breast cancer cells BT-474 and MCF-7 were used as overexpressor and low expressor controls, respectively. We define overexpression (+) when expression of Her2/neu was 24 folds or higher than the expression of Her2/neu in MCF-7 cells.1-d NM, not measured. Open table in a new tab We analyzed the Her2/neu status for the clinical samples by performing real-time PCR using Her2/neu-specific primers. Breast cancer cell lines BT-474 and MCF-7 were used as high expressor and low expressor controls, respectively. Her2/neu was highly expressed in 7 of 16 (44%) Cyr61-positive samples compared with 6 of 28 (21%) Cyr61-negative samples, suggesting Cyr61expression is positively correlated with Her2/neuexpression (Table I).Univariate analysis (Tables II and III) showed either significant or borderline significant association between breast cancer stage, tumor size, lymph node status, ER levels in the primary tumor as well as age at onset of disease compared with whether the primary tumor expressed Cyr61 on Northern blot. For analysis of stage, only 6 of 28 (21%) of women having stages I and II were positive for Cyr61; in contrast, 10 of 16 (63%) of women having stages IIIA, IIIB, or IV expressed Cyr61 (p < 0.006). Similarly, only 6 of 28 (21%) individuals with primary breast cancer size of either 1A (≤2 cm in diameter) or 2A (≤5 cm) had tumors that were Cyr61-positive; in comparison, 10 of 16 (63%) of individuals with a stage of either 3A or 4B (≥5 cm) breast cancer had primary tumors that expressed Cyr61 (p = 0.006). Furthermore, of the 17 patients who were lymph node-negative, only two were Cyr61-positive (12%) compared with 14 Cyr61-positive tumors among the 27 individuals who were lymph node-positive (52%, p = 0.01). The median and mean ages of patients who were Cyr61-negative, were 52 and 53 (±10 S.D.), respectively, compared with a median and mean age of 66 and 64 (±11.7 S.D.) in those who were Cyr61-positive (p = 0.003). Tumors that were Cyr61-negative had a mean ER score of 40 fmol/g (±69 S.D.), whereas those that were Cyr61-positive had a mean ER score of 102 fmol/g (±110 S.D.) (p = 0.03). No statistical difference in the mean progesterone (PgR) values was noted in those that were Cyr61-negative (mean 94.56 fmol/g) versus those that were Cyr61-positive (mean 129.5 fmol/g) (p = 0.55).Table IIUnivariate associations with Cyr61-positive breast cancer discrete variablesn + 2-aNumber of breast cancer sample.Cyr61-positive (%)p valueStage 2-bStage or tumor size; tumor stage and size of primary breast cancer.I/II286 (21%)III/IV1610 (63%)0.006Tumor size 2-bStage or tumor size; tumor stage and size of primary breast cancer.1a/2a286 (21%)3a/4b1610 (63%)0.006Lymph node 2-cLymph node: axillary lymph node involvement.Negative172 (12%)Positive2714 (52%)0.0102-a Number of breast cancer sample.2-b Stage or tumor size; tumor stage and size of primary breast cancer.2-c Lymph node: axillary lymph node involvement. Open table in a new tab Table IIIUnivariate associations with Cyr61-positive breast cancer continuous variablesCyr61n 3-aNumber of breast cancer samples.Min 3-bMinimum value.MedianMeanMax 3-cMaximum value.S.D.S.E.pvalueAge 3-dAge (years) at diagnosis of breast cancer.Negative283452538210.21.9Positive164166648111.72.90.003ERNegative270040.324069.913.5Positive16066.5102.1320110.327.60.030PgRNegative270094.6850187.136.0Positive16040129.5500177.644.40.5503-a Number of breast cancer samples.3-b Minimum value.3-c Maximum value.3-d Age (years) at diagnosis of breast cancer. Open table in a new tab Our classification tree multivariate model predicted that, if the patient at initial diagnosis was less than 57 years of age and had a breast cancer with an ER of less than 24 fmol/g, Cyr61 would be negative (%Cyr61-positive, 0/17 = 0%). If the individual was less than 57 years old and had an ER greater than 24 fmol/g, the model predicted that Cyr61 would be positive (%Cyr61-positive, 4/4 = 100%). If age was greater than 57 and the stage was I or II, the model predicted that Cyr61 would be negative (3/10 = 30% Cyr61-positive); if the age of the patient was greater than 57 and had either stage IIIA or IIIB, Cyr61 was predicted to be positive (9/9 (100%) were Cyr61-positive). This model has an observed sensitivity of 81% and observed specificity of 86% and gave an overall observed correct classification of 84% correctly classified. According to the tree model, when predicting who is Cyr61-positive, ER is only important in younger women under the age of 58, whereas stage is only important in older women over the age of 57. In summary, the model posits that the proportion who have Cyr61-positive breast cancer increases with age, stage, and ER level.Expression of Cyr61 Is Modulated through the Estrogen Receptor Pathway in MCF-12A and MCF-7 CellsPrevious studies have shown that Cyr61 is inducible in the uterus by estrogen treatment in ovariectomized rats (14Rivera-Gonzalez R. Petersen D.N. Tkalcevic G. Thompson D.D. Brown T.A. J. Steroid Biochem. Mol. Biol. 1998; 64: 13-24Crossref PubMed Scopus (68) Google Scholar). Furthermore, the correlation ofCyr61 expression in ER+ breast cancers observed in our experiments suggested a potential interaction between the estrogen receptor pathway and expression of Cyr61. To determine whether expression of Cyr61 was regulated by estrogen and estrogen blockade, the estrogen-responsive normal breast cells, MCF-12A, and breast cancer cells, MCF-7, were harvested at different times after estrogen and antiestrogen treatment. Expression of Cyr61 mRNA was induced at 16 h after estradiol (10−9 m) treatment and reached maximum (8- to 12-fold) levels at 48 h and started to decrease slightly after 72 h in the MCF-12A cells (Fig.3 A). Induction of expression of Cyr61 mRNA was inhibited when either tamoxifen or ICI182,780 (estrogen receptor antagonists, 10−7 m) were added to these cultures. A parallel induction (3- to 5-fold) of Cyr61 mRNA occurred in MCF-7 cells after estrogen treatment (10−9 m), which was detectable at 8 h, reached a plateau at 24 h, and decreased at 72 h. The induction was completely blocked by either tamoxifen or ICI182,780 (Fig. 3 B).Figure 3Modulation of Cyr61expression in MCF-12A and MCF-7 cells by estrogen and an estrogen receptor antagonist. Normal breast cell line MCF-12A and breast cancer cell line MCF-7 were treated with β-estradiol (1 × 10−9 m) for different durations; cellular RNA was extracted, subjected to electrophoresis (10 μg of total RNA/lane), Northern blotted, and probed with 32P-labledCyr61 and glyceraldehyde-3-phosphate dehydrogenase cDNAs. Maximum induction of Cyr61 mRNA expression was observed at 48 h for MCF-12A (A) and 24 h for MCF-7 (B). Induction of Cyr61 expression was blocked by pretreatment of the cells with the estrogen receptor antagonists, tamoxifen (1.0 × 10−7 m) or ICI182,780 (1.5 × 10−7 m).View Large Image Figure ViewerDownload (PPT)Cyr61 Promotes Cell Proliferation in Soft Agar and Stimulates Cell Migration of Breast Cell LinesPrevious studies indicated that Cyr61 promoted DNA synthesis and cell proliferation of mesenchymal cells from the limb (15Wong M. Kireeva M.L. Kolesnikova T.V. Lau L.F. Dev. Biol. 1997; 192: 492-508Crossref PubMed Scopus (132) Google Scholar) and stimulated cell migration in fibroblasts (12Kireeva M.L. Mo F.-E. Yang G.P. Lau L.F. Mol. Cell. Biol. 1996; 16: 1326-1334Crossref PubMed Scopus (303) Google Scholar, 16Kolesnikova T.V. Lau L.F. Oncogene. 1998; 16: 747-754Crossref PubMed Scopus (68) Google Scholar). To study if similar activities occurred in breast cells, both normal breast cells (MCF-12A) and breast tumor cells (MCF-7) were stably transfected with pcDNA61 containing either full-length cDNA of Cyr61 (MCF-12A/61 and MCF-7/61) or empty vector pcDNA3.1 (MCF-12A/V and MCF-7/V) as control. As expected,Cyr61 was highly expressed in the MCF-12A/61 and MCF-7/61 but not in the MCF-12/V and MCF-7/V transfected cells as shown by Northern studies (data not shown) and Western analysis (Fig.4 A). The MCF-7/61 cells expressing the Cyr61 vector formed significantly more colonies in soft agar (mean, 2.2- ± 0.6-fold more colonies;p < 0.05) than MCF-7/V cells harboring the empty vector or MCF-7 control cells (Fig. 4 B). The MCF-7/61 colonies also were substantially larger than the MCF-7/V and MCF-7 colonies (data not shown). The results indicated that forced expression of Cyr61 promoted anchorage-independent clonogenic growth of MCF-7 cells. Neither MCF-12A/V nor MCF-12A/61 formed colonies after 4 weeks.Figure 4Formation of colonies in soft agar by MCF-7, MCF/V, and MCF-7/61 cells. Expression of Cyr61 protein by stably transfected MCF-7 (MCF-7/61) cells as shown by Western analysis. Equal loading was shown by the internal nonspecific bands at 90 kDa (A). The MCF/V and MCF-7/61 cell lines were stably transfected with either the empty pcDNA3.1 vector or theCyr61 expression vector, respectively. The MCF-7/61 clone was selected for high expression of Cyr61. B, cells (1.0 × 103/well) were seeded in soft agar for 2 weeks, and colonies were enumerated. Each experiment was performed in triplicate, and the results represent the mean ± S.D. of three experiments. C, MCF-7/V and MCF-7/61 cells (1.0 × 103/well) were seeded in soft agar with either estradiol (E2, 10−9 m) or E2 (10−9 m) and tamoxifen (TAM, 10−7 m) for 2 weeks, and colonies were counted. Each experiment was performed in triplicate, and the results represent the mean ± S.D. of three experiments.View Large Image Figure ViewerDownload (PPT)To assess the effects of estrogen and tamoxifen on anchorage-independent growth of MCF-7 cells, clonogenic proliferation of MCF-7/V and MCF-7/61 in soft agar containing either estradiol (10−9 m) or estradiol and tamoxifen (10−7 m) was evaluated. Estrogen treatment significantly (p < 0.05) enhanced colony formation of both MCF-7/V and MCF-7/61 cells, and tamoxifen blocked the estrogen-stimulated colony formation in both of these cell types (Fig.4 C).To determine whether enhanced expression of Cyr61 influenced cell migration of MCF-12A and MCF-7 cells, migration assays of MCF-12A/V, MCF-12A/61, MCF-7/V, and MCF-7/61 were performed in vitronectin-coated Boyden chambers. As shown in Fig.5, both MCF-12A/61 and MCF-7/61 cells (Cyr61 stably transfected cell lines) had significantly (p < 0.05) increased migration compared with the empty-vector-transfected MCF-12A/V and MCF-7/V cells in vitronectin-coated Boyden chambers.Figure 5Cyr61 stimulates cell migration in MCF-12A and MCF-7 cells. Cells (5 × 104) were placed into either bovine serum albumin-coated (control) or vitronectin-coated Boyden chambers. Cells were allowed to migrate for 4–8 h and quantified as described in QCM-VN (Chemicon, Temecula, CA). The number of cells that migrated through the membrane was determined by absorbance at 550 nm. Each bar represents the mean ± S.D. of triplicate experiments.View Large Image Figure ViewerDownload (PPT)Cyr61 Promotes Tumor Growth and Vascularization in Nude MiceOn the basis of our in vitro studies indicating that overexpression of Cyr61 promotes anchorage-independent clonogenic proliferation in soft agar and cell migration, we investigated the effect of expression of Cyr61 on tumor development and neovascularization by comparing tumor formation of MCF-12/V and MCF-12A/6 cells, as well as MCF-7/V and MCF-7/61 cells, in nude mice. These cells were injected subcutaneously into 8-week-old nude mice, and tumor growth was measured once a week. Tumors from the normal breast cells expressing Cyr61 (MCF-12A/61) first became apparent 3 weeks after injection, and all of the mice developed tumors ranging from 0.6 to 1.4 g at 6 weeks after injection (Figs.6 A, 6B, and 7A). In contrast, the control mice that received MCF-12A/V cells containing empt
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