Affiliations of authors: S. van der Flier, A. Brinkman, E. M. Kok, L. C. J. Dorssers (Department of Pathology/Division of Molecular Biology), M. P. Look, M. E. Meijer-van Gelder, J. G. M. Klijn, J. A. Foekens (Department of Medical Oncology/Division of Endocrine Oncology), Josephine Nefkens Institute, University Hospital Rotterdam, The Netherlands.
Correspondence to: Lambert C. J. Dorssers, Ph.D., Department of Pathology/Division of Molecular Biology, Josephine Nefkens Institute, University Hospital Rotterdam, Rm. Be 432, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands (e-mail: dorssers{at}bidh.azr.nl).
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ABSTRACT |
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INTRODUCTION |
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Endocrine therapies have been developed to block the effects of estrogen on cancer cells or to reduce serum estrogen levels. Tamoxifen is an antiestrogenic compound that is widely used as an adjuvant therapy for patients with ER-positive breast tumors (6). The decision to use endocrine therapy for advanced breast cancer is based on a number of predictive factors, including ER or progesterone receptor (PgR) status, age, menopausal status, disease-free interval, and site of relapse. About 50% of the patients with advanced breast cancer receiving endocrine treatment have an objective response or stable disease (no change for >6 months). The rest of the patients have a short period of stable disease or immediate progressive disease (7). The latter patients are considered to have intrinsic-resistant disease. However, almost all of the responding patients eventually experience disease recurrence (i.e., acquired resistance). Tamoxifen resistance may result from multiple mechanisms, and a number of causes have been suggested, including (epi)genetic alterations, resulting in ER mutants and splice variants (8-10), and alteration of absorption, distribution, and metabolism of tamoxifen (11,12). Elucidation of the mechanism(s) of tamoxifen resistance is, therefore, a comprehensive challenge in cancer biology.
To identify genes involved in hormone-independent growth, we used the technique of retroviral insertional mutagenesis and ZR-75-1 cells, a human breast cancer cell line. With this system, we have identified three loci responsible for breast cancer antiestrogen resistance (BCAR) (13-15). The first genomic locus linked to the antiestrogen drug-resistant phenotype of the cells that we identified was the BCAR1 locus (13). Characterization of the BCAR1 gene indicated that it is the human homologue of the rat-docking protein also known as p130 Crk-associated substrate (p130Cas) (16). p130Cas, which contains an Src homology (SH) 3 domain, a proline-rich region, and a substrate domain containing multiple SH2-binding motifs, was identified as a major substrate for protein tyrosine phosphorylation in v-Src- and v-Crk-transformed cells (17,18) and has been implicated in a variety of biologic processes, including cell adhesion (19,20), cell migration (21-23), growth factor stimulation (24), cytokine receptor engagement (25), and bacterial infection (26,27). Cell fusion-mediated transfer of the BCAR1 locus and transfection of BCAR 1/p130Cas complementary DNA into ZR-75-1 cells demonstrated that overexpression of Bcar1/p130Cas results in sustained cell proliferation in the presence of antiestrogens (16). These observations prompted us to investigate the clinical relevance of Bcar1/p130Cas expression by measuring Bcar1/p130Cas protein in specimens of human breast carcinoma and testing these data against various characteristics of the patients and their disease for clinically relevant associations.
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MATERIALS AND METHODS |
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Our study design was approved by the medical ethical committee of the University Hospital Rotterdam, The Netherlands.
Nonmalignant breast tissues. Breast tissue without specific abnormalities from 10 women who underwent reduction mammoplasty was used to study Bcar1/p130Cas levels in normal tissue. Three 50-µm cryosections from these tissues in TS buffer (i.e., 10 mM Tris-HCl [pH 7.5] and 1% sodium dodecyl sulfate [SDS]) were sonicated for 10 seconds and boiled for 10 minutes. Lysates were cleared by centrifugation at 12 000g for 10 minutes at room temperature, and total protein concentrations were determined with the BCA protein assay (Pierce Chemical Co., Rockford, IL). Bcar1/p130Cas levels were measured with the western blotting assay described below.
Primary breast carcinoma. Bcar1/p130Cas levels were determined in cytosol preparations (as described below) from 937 primary breast tumors collected from 1978 through 1988. Selection of samples was based on the availability of stored cytosol extracts (in liquid nitrogen), which remained after routine ER and PgR analyses. Of the 937 patients, 775 (modified mastectomy = 408 patients; breast-conserving treatment = 367 patients) were eligible for analysis of relapse-free survivors and overall survival according to the criteria described elsewhere (28). Exclusion criteria were patient tissue that was taken from a biopsy specimen only (i.e., inoperable stage T4 tumors or tissue that was not obtained from the primary breast tumor) and patients with insufficient follow-up documentation. Patients who were referred to our institute more than 100 days after primary surgery and patients with distant metastasis at the time of primary surgery [M1 patients; staging according to the International Union Against Cancer TNM [tumor-node-metastasis] classification (29)] were excluded from analyses of relapse-free survival and overall survival, but they were not necessarily excluded from the analysis of response. In fact, we used 36 such patients in our analysis of the response to tamoxifen therapy in addition to those who were included according to the criteria described below.
Tumor size, lymph node status, and differentiation grade were gathered from the reports of
regional pathologists. The differentiation grade is not based on a central pathologic review of all
tumor samples and, thus, reflects daily practice. The median age of the 775 patients at surgery
was 57 years (range, 27-90 years). Radiotherapy was given to 87% of the patients at one
or more of the following sites: the breast/thoracic wall of 549 patients and/or the axilla of 296
patients and/or the lymph node areas other than the axilla of 328 patients. Adjuvant chemotherapy
(mainly, cyclophosphamide, methotrexate, and 5-fluorouracil [CMF]) was given to
144 patients, and adjuvant hormonal therapy, either alone (38 patients) or in combination with
CMF (14 patients), was given to 52 patients. All patients were routinely examined every 3-6
months during the first 5 years of follow-up and once a year thereafter. Of the 775 patients
included in the study for analysis of relapse-free survival and overall survival, 442 (57%)
showed evidence of relapse during follow-up, and 372 (48%) died. At the end of the study,
403 patients were alive, with a median follow-up of 105 months (range, 18-175 months). The
following characteristics at surgery are shown in Table 1: tumor size,
lymph node status, differentiation grade of the tumor, age, menopausal status, ER status, and PgR
status.
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Preparation of Cytosolic Extracts From Tumors
Cryopreserved (liquid nitrogen) breast tumor specimens were pulverized with a microdismembrator as recommended by the European Organization for Research and Treatment of Cancer (EORTC) for determining cytosolic levels of the ER and PgR proteins (35). The resulting tissue powder was suspended in EORTC receptor buffer (10 mM K2HPO4, 1.5 mM K2EDTA, 3 mM sodium azide, 10 mM monothioglycerol, and 10% [vol/vol] glycerol [pH 7.5]). The homogenate was centrifuged at 100 000g for 30 minutes at 40 °C, and the supernatant fraction (cytosol extract) was obtained and stored in liquid nitrogen until assayed. Total protein levels in the extracts were determined by use of the Bradford dye-binding assay (Bio-Rad Protein Assay; Bio-Rad Laboratories, Inc., Hercules, CA), with human serum albumin (Kabi Vitrum Diagnostica, Stockholm, Sweden) as the standard. ER and PgR levels were determined by a ligand-binding assay or an enzyme immunoassay as described previously (36). The cutoff point used to classify tumors as ER or PgR positive was 10 fmol of receptor protein/mg of cytosol protein.
Western Blot Assay for Bcar1/p130Cas
To determine Bcar1/p130Cas levels in tissue, 5 µg of cytosolic or total extract protein
from tumor tissue, nonmalignant breast tissue, or a cell line standard was boiled in loading buffer
(i.e., 10 mM Tris-HCl [pH 6.8], 1 mM EDTA, 10% glycerol,
2% SDS, 2.5% 2-mercaptoethanol, and 0.00025% bromophenol blue) and
electrophoretically separated in SDS-6% polyacrylamide gel. Each gel also contained
prestained SDS-polyacrylamide gel electrophoresis molecular weight markers (Bio-Rad
Laboratories) and lysate from the Bcar1/p130Cas-overexpressing human cell line 4A12 (16) in two concentrations as a standard. Proteins were transferred to a
poly(vinylidene difluoride) membrane (Hybond P; Amersham Pharmacia Biotech,
Buckinghamshire, U.K.) with a semidry transblotter (Owl Scientific Inc., Woburn, MA). After an
overnight blocking step at 4 °C in TBST (i.e., 100 mM Tris-HCl [pH
7.4], 1.5 M NaCl, and 0.05% Tween 20) containing 0.6% bovine
serum albumin, the blots were incubated with a 1 : 5000 dilution of anti-p130Cas monoclonal
antibody generated against rat p130Cas (Transduction Laboratories, Lexington, KY) for 1 hour.
This procedure was followed by incubation with a 1 : 10 000 dilution of a horseradish
peroxidase-conjugated rabbit anti-mouse antibody (Dako, Glostrup, Denmark) for 1 hour.
Immunocomplexes were visualized by chemiluminescence (enhanced chemiluminescence western
blotting detection reagents; Amersham Pharmacia Biotech) and exposure to blue-light-sensitive
x-ray screen film (CEA, Strängnäs, Sweden). To determine Bcar1/p130Cas protein
levels, several exposure times (45 seconds, 90 seconds, 3 minutes, and 6 minutes) were used for
each blot, and a Bcar1/p130Cas standard (two concentrations) was used as reference.
Bcar1/p130Cas protein levels were scored visually by three investigators (S. van der Flier, A.
Brinkman, and L. C. J. Dorssers) who were not aware of the clinical data. The consensus score
was divided into the four following groups: none = no Bcar1/p130Cas protein expression
detected on both short and long exposures of the film (e.g., see Fig. 1, lanes T1 and T9); low = no signal on a short exposure and a weak signal on a
long exposure (Fig. 1,
lane T4); intermediate = a weak signal on a
short exposure and a clear signal on a long exposure (Fig. 1,
lanes T2, T3,
and T8); and high = a clear signal on a short exposure (Fig. 1,
lanes T5, T6, and T7).
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The strength of the association between Bcar1/p130Cas levels in cytosolic extracts from tumors (treated as an ordinal variable) and other prognostic variables (patient or tumor characteristics treated as grouping variables) was tested with the Mann-Whitney U test or the Kruskal-Wallis test. To test for trends, a nonparametric test for trend across ordered groups as developed by Cuzick (37) was used. All P values are two-sided. Two-sided P values of less than .05 were considered to be statistically significant. Relapse-free survival and overall survival probabilities were calculated by the actuarial method of Kaplan and Meier (38). A local disease recurrence (n = 442), distant spread (n = 313), or contralateral breast cancer (n = 37) was considered an event for relapse-free survival. For the analysis of relapse-free survival, patients who died without a reported disease recurrence were censored at their date of death. Both univariate and multivariate analyses were performed with the Cox proportional hazard model. For missing values, separate dummy variables were created. The assumption of proportional hazards was verified graphically. Relative hazard ratios were calculated and presented with 95% confidence intervals. For multivariate analysis, a basic model was introduced, including the classical prognostic factors (age, menopausal status, tumor size, number of positive lymph nodes, ER status, PgR status, differentiation grade, and systemic adjuvant therapy). Age and menopausal status combined were treated as one variable. The prognostic importance of Bcar1/p130Cas expression was tested by adding Bcar1/p130Cas to the basic model. The likelihood ratio test in the univariate Cox regression model was used to test for differences, and the log-rank test for trend was used to evaluate trends. The relation with response to therapy was examined with logistic regression analysis. For analysis of the time to disease progression after the start of tamoxifen treatment, the Cox proportional hazard model was used. All statistical analyses were performed with Stata Statistical Software (release 5.0; Stata Corporation, College Station, TX).
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RESULTS |
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The anti-p130Cas monoclonal antibody used recognizes the human
Bcar1/p130Cas protein with an apparent molecular mass of 116 kd, as
estimated by SDS-polyacrylamide gel electrophoresis (16). By
analyzing subcellular fractions, we retained the bulk of Bcar1/p130Cas
protein in routinely prepared cytosolic extracts (Fig. 1), thus
enabling the screening of a large number of breast carcinomas for
Bcar1/p130Cas expression. As shown by western blot analysis, levels of
Bcar1/p130Cas protein differed in different tumors (Fig. 1
).
Bcar1/p130Cas protein expression was also evaluated in nonmalignant
breast tissue from 10 women. In contrast to malignant tissue,
Bcar1/p130Cas protein was not detected in any nonmalignant breast
tissue analyzed (Fig. 1
). To study the possible relationship with
relapse-free survival and overall survival and response to first-line
tamoxifen treatment, the expression levels of Bcar1/p130Cas from breast
tumors were divided into four groups (none, low, intermediate, and
high) based on the intensity of the western blot bands.
Relationship of Bcar1/p130Cas Levels With Patient and Tumor Characteristics
We compared the levels of Bcar1/p130Cas protein in cytosolic
extracts from 775 tumors with patient and tumor characteristics (Table
1). Thirty-three percent of the tumors had no Bcar1/p130Cas protein,
39% had low Bcar1/p130Cas expression, 20% had intermediate
expression, and 8% had high Bcar1/p130Cas expression. Intermediate and
high levels of Bcar1/p130Cas were more frequently found in tumors from
older patients (P = .006). No statistically significant
association of Bcar1/p130Cas levels with tumor size or lymph node
status was observed. Reduced levels of Bcar1/p130Cas expression (P= .01) were statistically significantly associated with poorly
differentiated tumors. Higher Bcar1/p130Cas levels were statistically
significantly associated with ER- and PgR-positive tumors
(P<.001). Expression levels of Bcar1/p130Cas were similar
in tumors from patients who did not receive adjuvant treatment or who
received adjuvant hormonal therapy or chemotherapy with or without
hormonal therapy (
2 = 9.79; df = 6; P= .13).
Bcar1/p130Cas and Relapse-Free Survival and Overall Survival
We next studied the relationship between the level of Bcar1/p130Cas
protein in primary tumors and the length of relapse-free survival. We
found a statistically significant association of high Bcar1/p130Cas
levels with poor relapse-free survival (log-rank test for trend;
2 = 4.38; df = 1; P = .04) and poor
overall survival (log-rank test for trend;
2 = 4.01;
df = 1; P = .04). Patients with low and intermediate
levels of Bcar1/p130Cas protein in their primary tumor had similar
periods of relapse-free survival (Fig. 2)
. On the
basis of this observation, we decided to combine these levels for
further analyses. Multivariate analysis was used to evaluate the
possible independent prognostic importance of Bcar1/p130Cas expression
in analyses of relapse-free survival and overall survival (Table
2)
. We introduced a basic model that included age,
menopausal status, tumor size, number of positive lymph nodes, and the
dichotomized biochemical parameters ER and PgR status. When we added
the Bcar1/p130Cas level as an ordinal variable to the basic models for
relapse-free survival and overall survival, we found that its
expression level contributed to the prognostic information provided by
the classical prognostic factors (Table 2
). Thus, in patients with
primary breast cancer, the level of Bcar1/p130Cas protein is an
independent predictor of early recurrence and death.
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Next, we investigated a possible relationship between the level of
Bcar1/p130Cas protein in the tumor and the efficacy of response to
tamoxifen as first-line systemic therapy for recurrent or metastatic
disease. Of the 268 patients who were treated with tamoxifen, 136
(51%) responded, with a median time to disease progression of 16
months. The responses to tamoxifen were similar in the subgroups with
no (53%), low (50%), or intermediate (58%) levels of Bcar1/p130Cas
protein in their tumors, whereas the response in patients with a high
level of Bcar1/p130Cas in their tumors was lower (33%). However, the
objective response was similar in all subgroups. The observed lower
response rate in the high Bcar1/p130Cas subgroup is thus a reflection
of a reduced number of patients with long-term stable disease. The
tumors with high Bcar1/p130Cas levels were predominantly ER positive
(29 [97%] of 30 tumors); fewer patients in the other subgroups had
ER-positive tumors (approximately 85%). Again, the subgroups with low
or intermediate Bcar1/p130Cas expression were combined. When analysis
was performed on 230 ER-positive tumors only, there was a statistically
significant association between a poor response to tamoxifen treatment
and high expression of Bcar1/p130Cas (high = 34%; none = 61%; P= .02). In the multivariate analysis for response to treatment,
Bcar1/p130Cas levels were corrected for menopausal status, relapse-free
interval, and ER status (as a dichotomized variable). Independent
predictors of poor response were premenopausal status, short
disease-free interval, ER-negative status of the tumor, and high levels
of Bcar1/p130Cas (Table 3). In a separate analysis,
we included ER status as a continuous variable (log transformed) and
found that a high level of Bcar1/p130Cas expression, compared with no
expression, was still associated with a poor response to tamoxifen
(P = .01). However, these results should be interpreted with
some caution because we did not correct for multiple comparisons. An
explanation for the observed differences between univariate and
multivariate analyses could be the strong relation between an
ER-positive status of the tumors and high Bcar1/p130Cas expression,
which was corrected in the multivariate analysis.
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DISCUSSION |
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Analysis of the expression of Bcar1/p130Cas protein in primary breast cancer has led to two distinct observations. First, relatively high expression of Bcar1/p130Cas in primary breast tumors is associated with a rapid recurrence of the disease. The risk of relapse was 62% greater for patients whose tumors had high levels of Bcar1/p130Cas than for patients whose tumors had undetectable levels of Bcar1/p130Cas protein. Multivariate analysis showed that a high level of Bcar1/p130Cas in the primary tumor was associated with an early relapse, independent of the classical prognostic parameters. Consequently, a high level of Bcar1/p130Cas protein in primary tumor extracts (compared with no detectable Bcar1/p130Cas in their primary tumor) is statistically significantly associated with a 65% reduced overall survival of patients. These results indicate that tumors with high-level expression behave more aggressively than tumors with low-level or undetectable expression. This association may be explained biologically by the involvement of Bcar1/p130Cas in cellular migration and invasion (21-23). These properties suggest a higher risk of early dissemination of individual cells from the subgroup of tumors with high-level expression of Bcar1/p130Cas.
Second, high-level expression of Bcar1/p130Cas in the primary tumors (compared with tumors with no detectable Bcar1/p130Cas expression) is associated with a poorer response to first-line tamoxifen treatment. This observation is consistent with our findings with the in vitro model system; i.e., Bcar1/p130Cas overexpression resulted in antiestrogen resistance in cell lines (13,16). Tamoxifen generally induces an objective response or stable disease in about half of the patients with ER-positive primary tumors and recurrent or metastatic disease (39,40). The other patients who have ER-positive primary tumors experience immediate disease progression because of intrinsic resistance to tamoxifen. In our study, 67% of the patients with a relatively high level of Bcar1/p130Cas protein in their primary tumors did not respond to tamoxifen. This finding may explain some of the clinically observed intrinsic resistance to tamoxifen. Because these tumors are generally ER positive and the patients are, thus, eligible for tamoxifen therapy, Bcar1/p130Cas levels could be used by physicians to determine which patients may not respond to tamoxifen. Therapeutic decisions may benefit from more precise quantification of Bcar1/p130Cas protein levels in primary tumors. Because several lines of evidence show that development of resistance to antiestrogen agents is not always associated with the loss of ER expression in the tumor cells (41-43), the association of the high levels of Bcar1/p130Cas in ER-positive primary tumors that have a poor response to tamoxifen suggests the possibility of an alternative pathway to bypass ER-mediated cell proliferation.
Despite many reports on Bcar1/p130Cas function in various cell systems, few clues are available about the pathway leading to the altered proliferation control in our cells (44,45). Changes in adhesion and migration may affect cell proliferation, but the precise pathway in breast epithelial cells is yet unclear. Bcar1/p130Cas protein can be phosphorylated on tyrosine residues and can associate with many proteins that contain SH2 domains (44,46,47). In addition, the SH3 domain of Bcar1/p130Cas may also associate with other proteins through proline-containing target sequences (17,48,49). Bcar1/p130Cas serves as an adapter protein to contact proteins that participate in various cellular processes and is dependent on the availability, activation status, and affinity of its partners. These interactions are part of a cascade of signaling events leading to the regulation of gene expression and the regulation of the cell cycle. Because Bcar1/p130Cas appears to have a role in the progression of clinical breast cancer, elucidation of this critical pathway could contribute valuable information for the development of new treatment strategies for antiestrogen-resistant breast cancer.
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NOTES |
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We thank Henk Portengen for his excellent assistance with the tumor bank. We also thank Ton van Agthoven and Jos Veldscholte for their stimulating discussion and Professor Dr. D. Bootsma for his support.
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REFERENCES |
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---|
1 Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G, Umesono K, et al. The nuclear receptor superfamily: the second decade. Cell 1995;83:835-9.[Medline]
2 Freedman LP. Increasing the complexity of coactivation in nuclear receptor signaling. Cell 1999;97:5-8.[Medline]
3
Kuiper GG, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson
JA. Cloning of a novel receptor expressed in rat prostate and ovary. Proc Natl Acad Sci U
S A 1996;93:5925-30.
4 Mosselman S, Polman J, Dijkema R. ERß: identification and characterization of a novel human estrogen receptor. FEBS Lett 1996;392:49-53.[Medline]
5
Katzenellenbogen BS, Korach KS. A new actor in the estrogen
receptor dramaenter ER-ß. Endocrinology 1997;138:861-2.
6 Early Breast Cancer Trialists' Collaborative Group. Tamoxifen for early breast cancer: an overview of the randomised trials. Lancet 1998;351:1451-67.[Medline]
7 Hayward JL, Carbone PP, Heuson JC, Kumaoka S, Segaloff A, Rubens RD. Assessment of response to therapy in advanced breast cancer: a project of the Programme on Clinical Oncology of the International Union Against Cancer, Geneva, Switzerland. Cancer 1977;39:1289-94.[Medline]
8 Horwitz KB. Mechanisms of hormone resistance in breast cancer. Breast Cancer Res Treat 1993;26:119-30.[Medline]
9 Chan CM, Dowsett M. A novel estrogen receptor variant mRNA lacking exons 4 to 6 in breast carcinoma. J Steroid Biochem Mol Biol 1997;62:419-30.[Medline]
10 Dowsett M, Daffada A, Chan CM, Johnston SR. Oestrogen receptor mutants and variants in breast cancer. Eur J Cancer 1997;33:1177-83.[Medline]
11 Osborne CK, Coronado E, Allred DC, Wiebe V, DeGregorio M. Acquired tamoxifen resistance: correlation with reduced breast tumor levels of tamoxifen and isomerization of trans-4-hydroxytamoxifen. J Natl Cancer Inst 1991;83:1477-82.[Abstract]
12 Johnston SR, Haynes BP, Smith IE, Jarman M, Sacks NP, Ebbs SR, et al. Acquired tamoxifen resistance in human breast cancer and reduced intra-tumoral drug concentration. Lancet 1993;342:1521-2.[Medline]
13 Dorssers LC, Van Agthoven T, Dekker A, Van Agthoven TL, Kok EM. Induction of antiestrogen resistance in human breast cancer cells by random insertional mutagenesis using defective retroviruses: identification of bcar-1, a common integration site. Mol Endocrinol 1993;7:870-8.[Abstract]
14 Dorssers LC, Veldscholte J. Identification of a novel breast-cancer-anti-estrogen-resistance (BCAR2) locus by cell-fusion-mediated gene transfer in human breast-cancer cells. Int J Cancer 1997;72:700-5.[Medline]
15
Van Agthoven T, Van Agthoven TL, Dekker A, Van der Spek
PJ, Vreede L, Dorssers LC. Identification of BCAR3 by a random search for genes involved in
antiestrogen resistance of human breast cancer cells. EMBO J 1998;17:2799-808.
16 Brinkman A, van der Flier, Kok EM, Dorssers LC. BCAR1, a human homologue of the adapter protein p130Cas and antiestrogen resistance in breast cancer cells. J Natl Cancer Inst 2000;92:000-000]
17 Sakai R, Iwamatsu A, Hirano N, Ogawa S, Tanaka T, Mano H, et al. A novel signaling molecule, p130, forms stable complexes in vivo with v-Crk and v-Src in a tyrosine phosphorylation-dependent manner. EMBO J 1994;13:3748-56.[Abstract]
18
Nakamoto T, Sakai R, Ozawa K, Yazaki Y, Hirai H. Direct
binding of C-terminal region of p130Cas to SH2 and SH3 domains of Src kinase. J Biol Chem 1996;271:8959-65.
19 Vuori K, Hirai H, Aizawa S, Ruoslahti E. Introduction of p130cas signaling complex formation upon integrin-mediated cell adhesion: a role for Src family kinases. Mol Cell Biol 1996;16:2606-13.[Abstract]
20
Polte TR, Hanks SK. Complexes of focal adhesion kinase (FAK)
and Crk-associated substrate (p130Cas) are elevated in cytoskeleton-associated
fractions following adhesion and Src transformationrequirements for Src kinase activity
and FAK proline-rich motifs. J Biol Chem 1997;272:5501-9.
21
Cary LA, Han DC, Polte TR, Hanks SK, Guan JL. Identification
of p130Cas as a mediator of focal adhesion kinase-promoted cell migration. J
Cell Biol 1998;140:211-21.
22
Klemke RL, Leng J, Molander R, Brooks PC, Vuori K, Cheresh
DA. CAS/Crk coupling serves as a "molecular switch" for induction of cell
migration. J Cell Biol 1998;140:961-72.
23
Tamura M, Gu J, Takino T, Yamada KM. Tumor suppressor
PTEN inhibition of cell invasion, migration, and growth: differential involvement of focal adhesion
kinase and p130Cas. Cancer Res 1999;59:442-9.
24
Ojaniemi M, Vuori K. Epidermal growth factor modulates
tyrosine phosphorylation of p130Cas. Involvement of phosphatidylinositol
3'-kinase and actin cytoskeleton.J Biol Chem 1997;272:25993-8.
25
Ingham RJ, Krebs DL, Barbazuk SM, Turck CW, Hirai H,
Matsuda M, et al. B cell antigen receptor signaling induces the formation of complexes containing
the Crk adapter proteins. J Biol Chem 1996;271:32306-14.
26
Black DS, Bliska JB. Identification of p130Cas as a
substrate of Yersinia YopH (Yop51), a bacterial protein tyrosine phosphatase that translocates
into mammalian cells and targets focal adhesions. EMBO J 1997;16:2730-44.
27
Persson C, Carballeira N, Wolf-Watz H, Fallman M. The PTPase
YopH inhibits uptake of Yersinia, tyrosine phosphorylation of p130Cas and FAK,
and the associated accumulation of these proteins in peripheral focal adhesions. EMBO J 1997;16:2307-18.
28 Foekens JA, Look MP, Bolt-de Vries J, Meijer-van Gelder ME, van Putten WL, Klijn JG. Cathepsin-D in primary breast cancer: prognostic evaluation involving 2810 patients. Br J Cancer 1999;79:300-7.[Medline]
29 Sherman CD, Hossfeld DK. Breast cancer. In: Hossfeld DK, Sherman CD, Lover RR, Bosch FX, editors. Manual of clinical oncology. Geneva (Switzerland): International Union Against Cancer; 1990. p. 253-71.
30 Nicholson S, Sainsbury JR, Halcrow P, Chambers P, Farndon JR, Harris AL. Expression of epidermal growth factor receptors associated with lack of response to endocrine therapy in recurrent breast cancer. Lancet 1989;1:182-5.[Medline]
31 Ravdin PM, Green S, Dorr TM, McGuire WL, Fabian C, Pugh RP, et al. Prognostic significance of progesterone receptor levels in estrogen receptor-positive patients with metastatic breast cancer treated with tamoxifen: results of a prospective Southwest Oncology Group study. J Clin Oncol 1992;10:1284-91.[Abstract]
32 Foekens JA, Look MP, Peters HA, Van Putten WL, Portengen H, Klijn JG. Urokinase-type plasminogen activator and its inhibitor PAI-1: predictors of poor response to tamoxifen therapy in recurrent breast cancer. J Natl Cancer Inst 1995;87:751-6.[Abstract]
33 Foekens JA, Portengen H, Look MP, Van Putten WL, Thirion B, Bontenbal M, et al. Relationship of PS2 with response to tamoxifen therapy in patients with recurrent breast cancer. Br J Cancer 1994;70:1217-23.[Medline]
34 Robertson JF, Willsher PC, Cheung KL, Blamey RW. The clinical relevance of static disease (no change) category for 6 months on endocrine therapy in patients with breast cancer. Eur J Cancer 1997;33:1774-9.[Medline]
35 EORTC Breast Cancer Cooperative Group. Revision of the standards for the assessment of hormone receptors in human breast cancer; report of the second E.O.R.T.C. Workshop, held on 16-17 March, 1979, in the Netherlands Cancer Institute. Eur J Cancer 1980;16:1513-5.[Medline]
36 Foekens JA, Portengen H, Van Putten WL, Peters HA, Krijnen HL, Alexieva-Figusch J, et al. Prognostic value of estrogen and progesterone receptors measured by enzyme immunoassays in human breast tumor cytosols. Cancer Res 1989;49:5823-8.[Abstract]
37 Cuzick J. A Wilcoxon-type test for trend. Stat Med 1985;4:87-90.[Medline]
38 Kaplan EL, Meier P. Nonparametric estimation from incomplete observations. J Am Statist Assoc 1958;53:457-81.
39 Horowitz KB, McGuire WL. Predicting response to endocrine therapy in human breast cancer: a hypothesis. Science 1975;189:726-7.[Medline]
40 Jaiyesimi IA, Buzdar AU, Decker DA, Hortobagyi GN. Use of tamoxifen for breast cancer: twenty-eight years later. J Clin Oncol 1995;13:513-29.[Abstract]
41 Encarnacion CA, Ciocca DR, McGuire WL, Clark GM, Fuqua SA, Osborne CK. Measurement of steroid hormone receptors in breast cancer patients on tamoxifen. Breast Cancer Res Treat 1993;26:237-46.[Medline]
42 Lykkesfeldt AE, Larsen SS, Briand P. Human breast cancer cell lines resistant to pure anti-estrogens are sensitive to tamoxifen treatment. Int J Cancer 1995;61:529-34.[Medline]
43 Larsen SS, Madsen MW, Jensen BL, Lykkesfeldt AE. Resistance of human breast-cancer cells to the pure steroidal anti-estrogen ICI 182,780 is not associated with a general loss of estrogen-receptor expression or lack of estrogen responsiveness. Int J Cancer 1997;72:1129-36.[Medline]
44 Schlaepfer DD, Broome MA, Hunter T. Fibronectin-stimulated signaling from a focal adhesion kinase-c-Src complex: involvement of the Grb2, p130cas, and Nck adaptor proteins. Mol Cell Biol 1997;17:1702-13.[Abstract]
45
Schlaepfer DD, Jones KC, Hunter T. Multiple Grb2-mediated
integrin-stimulated signaling pathways to ERK2 mitogen-activated protein kinase: summation of
both c-Src- and focal adhesion kinase-initiated tyrosine phosphorylation events. Mol Cell
Biol 1998;18:2571-85.
46 Burnham MR, Harte MT, Richardson A, Parsons JT, Bouton AH. The identification of p130cas-binding proteins and their role in cellular transformation. Oncogene 1996;12:2467-72.[Medline]
47
Astier A, Avraham H, Manie SN, Groopman J, Canty T,
Avraham S, et al. The related adhesion focal tyrosine kinase is tyrosine-phosphorylated after
ß1-integrin stimulation in B cells and binds to p130cas. J Biol Chem 1997;272:228-32.
48 Garton AJ, Burnham MR, Bouton AH, Tonks NK. Association of PTP-PEST with the SH3 domain of p130cas; a novel mechanism of protein tyrosine phosphatase substrate recognition. Oncogene 1997;15:877-85.[Medline]
49 Ohba T, Ishino M, Aoto H, Sasaki T. Interaction of two proline-rich sequences of cell adhesion kinase ß with SH3 domains of p130Cas-related proteins and a GTPase-activating protein, Graf. Biochem J 1998;330:1249-54.[Medline]
Manuscript received June 7, 1999; revised October 19, 1999; accepted November 3, 1999.
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