Arend Brinkman

Bcar1/p130Cas Protein and Primary Breast Cancer: Prognosis and Response to Tamoxifen Treatment

Background: The product of the Bcar1/p130Cas (breast cancer resistance/p130Crk-associated substrate) gene causes resistance to antiestrogen drugs in human breast cancer cells in vitro. To investigate its role in clinical breast cancer, we determined the levels of Bcar1/p130Cas protein in a large series of primary breast carcinomas. Methods: We measured Bcar1/p130Cas protein in cytosol extracts from 937 primary breast carcinomas by western blot analysis. The levels of Bcar1/p130Cas protein were tested for associations and trends against clinicopathologic and patient characteristics, the lengths of relapse-free survival and overall survival (n = 775), and the efficacy of first-line treatment with tamoxifen for recurrent or metastatic disease (n = 268). Results: Bcar1/ p130Cas levels in primary tumors were associated with age/ menopausal status and the levels of estrogen receptor and progesterone receptor. In univariate survival analysis, higher Bcar1/p130Cas levels were associated with poor relapse-free survival and overall survival (both two-sided P = .04; log-rank test for trend). In multivariate analysis, a high level of Bcar1/p130Cas was independently associated with poor relapse-free survival and overall survival. The response to tamoxifen therapy in patients with recurrent disease was reduced in patients with primary tumors that expressed high

Tamoxifen resistance in breast cancer: elucidating mechanisms.

Tamoxifen has been used for the systemic treatment of patients with breast cancer for nearly three decades. Treatment success is primarily dependent on the presence of the estrogen receptor (ER) in the breast carcinoma. While about half of patients with advanced ER-positive disease immediately fail to respond to tamoxifen, in the responding patients the disease ultimately progresses to a resistant phenotype.The possible causes for intrinsic and acquired resistance have been attributed to the pharmacology of tamoxifen, alterations in the structure and function of the ER, the interactions with the tumour environment and genetic alterations in the tumour cells. So far no prominent mechanism leading to resistance has been identified.The recent results of a functional screen for breast cancer antiestrogen resistance (BCAR) genes responsible for development of tamoxifen resistance in human breast cancer cells are reviewed. Individual BCAR genes can transform estrogen-dependent breast cancer cells into estrogen-independent and tamoxifen-resistant cells in vitro. Furthermore, high levels of BCAR1/p130Cas protein in ER-positive primary breast tumours are associated with intrinsic resistance to tamoxifen treatment. These results indicate a prominent role for alternative growth control pathways independent of ER signalling in intrinsic tamoxifen resistance of ER-positive breast carcinomas.Deciphering the differentiation characteristics of normal and malignant breast epithelial cells with respect to proliferation control and regulation of cell death (apoptosis) is essential for understanding therapy response and development of resistance of breast carcinoma.

The prognostic value of BCAR1 in patients with primary breast cancer.

Purpose: BCAR1, the human homologue of the rat p130Cas protein, was identified in a functional screen for human breast cancer cell proliferation resistant to antiestrogen drugs. Here, we study the prognostic value of quantitative BCAR1 levels in a large series of breast cancer specimens. Experimental Design: A specific ELISA was developed to measure BCAR1 protein levels in 2593 primary breast tumor cytosols. Tumor levels of BCAR1 were correlated with relapse-free survival (RFS) and overall survival (OS) and compared with collected data on urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor 1 (PAI-1). Results: In tumor cytosols, BCAR1 protein levels varied between 0.02 and 23 ng/mg protein. BCAR1 levels exhibited a positive correlation with steroid hormone receptor levels, age and menopausal status, and uPA and PAI-1 levels. The level of BCAR1 (continuous or categorized as low, intermediate, or high) was inversely related with RFS and OS time. Multivariate analysis showed that BCAR1 levels contributed independently to a base model containing the traditional prognostic factors for both RFS and OS (both P < 0.0001). When added together with uPA and PAI-1 in the multivariate model, BCAR1 contributed independently of PAI-1 and was favored over uPA. Interaction tests allowed for additional analyses of BCAR1 protein levels in clinically relevant subgroups stratified by nodal and menopausal status. Conclusions: The quantitative BCAR1 protein level represents a prognostic factor for RFS and OS in primary breast cancer, independent of the traditional prognostic factors and the other novel marker PAI-1.

BCAR1/p130Cas expression in untreated and acquired tamoxifen-resistant human breast carcinomas.

High BCAR1/p130Cas expression in primary breast tumour cytosol predicts a poor chance of response recurrent disease to tamoxifen treatment in patients with oestrogen receptor (ER)–positive breast carcinomas. In this study, we assessed whether BCAR1/p130Cas expression is altered during acquisition of anti‐oestrogen resistance. BCAR1/p130Cas protein was quantitatively measured by chemiluminescent Western blot analysis in the cytosol of 34 predominantly ER+ carcinomas that initially responded to primary tamoxifen treatment and subsequently progressed (n = 22 ) or developed during adjuvant tamoxifen treatment (n = 12) and compared to 54 untreated ER+ human breast carcinomas. We did not detect significant differences in the level of BCAR1/p130Cas protein in untreated and acquired tamoxifen‐resistant carcinomas. Our results indicate that in tumour progression towards tamoxifen resistance, increase of BCAR1/p130Cas may be only one of the molecular mechanisms. Thus, high BCAR1/p130Cas protein levels appear to be a hallmark for intrinsic resistance to tamoxifen in breast carcinomas. Int. J. Cancer 89:465–468, 2000.

Breast cancer oestrogen independence mediated by BCAR1 or BCAR3 genes is transmitted through mechanisms distinct from the oestrogen receptor signalling pathway or the epidermal growth factor receptor signalling pathway.

IntroductionTamoxifen is effective for endocrine treatment of oestrogen receptor-positive breast cancers but ultimately fails due to the development of resistance. A functional screen in human breast cancer cells identified two BCAR genes causing oestrogen-independent proliferation. The BCAR1 and BCAR3 genes both encode components of intracellular signal transduction, but their direct effect on breast cancer cell proliferation is not known. The aim of this study was to investigate the growth control mediated by these BCAR genes by gene expression profiling.MethodsWe have measured the expression changes induced by overexpression of the BCAR1 or BCAR3 gene in ZR-75-1 cells and have made direct comparisons with the expression changes after cell stimulation with oestrogen or epidermal growth factor (EGF). A comparison with published gene expression data of cell models and breast tumours is made.ResultsRelatively few changes in gene expression were detected in the BCAR-transfected cells, in comparison with the extensive and distinct differences in gene expression induced by oestrogen or EGF. Both BCAR1 and BCAR3 regulate discrete sets of genes in these ZR-75-1-derived cells, indicating that the proliferation signalling proceeds along distinct pathways. Oestrogen-regulated genes in our cell model showed general concordance with reported data of cell models and gene expression association with oestrogen receptor status of breast tumours.ConclusionsThe direct comparison of the expression profiles of BCAR transfectants and oestrogen or EGF-stimulated cells strongly suggests that anti-oestrogen-resistant cell proliferation is not caused by alternative activation of the oestrogen receptor or by the epidermal growth factor receptor signalling pathway.

CITED2 and NCOR2 in anti-oestrogen resistance and progression of breast cancer

Background:Endocrine therapies of breast cancer are effective but ultimately fail because of the development of treatment resistance. We have previously revealed several genes leading to tamoxifen resistance in vitro by retroviral insertion mutagenesis. To understand the manner in which these genes yield tamoxifen resistance, their effects on global gene expression were studied and those genes resulting in a distinct gene expression profile were further investigated for their clinical relevance.Methods:Gene expression profiles of 69 human breast cancer cell lines that were made tamoxifen resistant through retroviral insertion mutagenesis were obtained using oligonucleotide arrays and analysed with bioinformatic tools. mRNA levels of NCOR2 and CITED2 in oestrogen receptor-positive breast tumours were determined by quantitative RT–PCR. mRNA levels were evaluated for association with metastasis-free survival (MFS) in 620 patients with lymph node-negative primary breast cancer who did not receive systemic adjuvant therapy, and with clinical benefit in 296 patients receiving tamoxifen therapy for recurrent breast cancer.Results:mRNA expression profiles of most tamoxifen-resistant cell lines were strikingly similar, except for the subgroups of cell lines in which NCOR2 or CITED2 were targeted by the retrovirus. Both NCOR2 and CITED2 mRNA levels were associated with MFS, that is, tumour aggressiveness, independently of traditional prognostic factors. In addition, high CITED2 mRNA levels were predictive for a clinical benefit from first-line tamoxifen treatment in patients with advanced disease.Conclusions:Most retrovirally targeted genes yielding tamoxifen resistance in our cell lines do not impose a distinctive expression profile, suggesting that their causative role in cell growth may be accomplished by post-transcriptional processes. The associations of NCOR2 and CITED2 with outcome in oestrogen receptor-positive breast cancer patients underscore the clinical relevance of functional genetic screens to better understand disease progression, which may ultimately lead to the development of improved treatment options.

Immunohistochemical study of the BCAR1/p130Cas protein in non-malignant and malignant human breast tissue

BCAR1/p130Cas is a docking protein involved in intracellular signaling pathways and in vitro resistance of estrogen-dependent breast cancer cells to antiestrogens. The BCAR1/p130Cas protein level in primary breast cancer cytosols was found to correlate with rapid recurrence of disease. A high BCAR1/p130Cas level was also associated with a higher likelihood of resistance to first-line tamoxifen treatment in patients with advanced breast cancer. Using antibodies raised against the rat p130Cas protein, we determined by immunohistochemical methods the BCAR1/p130Cas localization in primary breast carcinomas, in tumors of stromal origin, and in non-neoplastic breast tissues. The BCAR1/p130Cas protein was detected in the cytoplasm of non-malignant and neoplastic epithelial cells and in the vascular compartment of all tissue sections analyzed. Immunohistochemistry demonstrated variable intensity of BCAR1/p130Cas staining and variation in the proportion of BCAR1/p130Cas-positive epithelial tumor cells for the different breast carcinomas. Double immunohistochemical staining for BCAR1/p130Cas and estrogen receptor confirmed coexpression in non-malignant luminal epithelial cells and malignant breast tumor cells. The stromal cells in non-malignant tissues and tumor tissues as well as breast tumors of mesodermal origin did not stain for BCAR1/p130Cas. This immunohistochemical study demonstrates a variable expression of BCAR1/p130Cas in malignant and non-malignant breast epithelial cells, which may be of benefit for diagnostic purposes.

The substrate domain of BCAR1 is essential for anti-estrogen-resistant proliferation of human breast cancer cells.

To unravel the mechanisms underlying failure of endocrine therapy of breast cancer, we have previously executed a functional genetic screen and identified the adaptor protein BCAR1 to be causative for tamoxifen resistance. As a consequence of the manifold of interactions with other proteins, we characterized the contribution of individual protein domains of BCAR1 to anti-estrogen-resistant proliferation of human breast cancer cells. We took advantage of the observation that the closely related family member HEF1 was unable to support long-term anti-estrogen-resistant cell proliferation. Chimerical proteins containing defined domains of BCAR1 and HEF1 were evaluated for anti-estrogen-resistant growth. Exchange of the SH3 and C-terminal domains did not modify the capacity to support cell proliferation. Full support of anti-estrogen resistant proliferation was observed for chimerical molecules containing the central part of BCAR1. The bi-partite SRC-binding site or the Serine-rich domain did not explain the differential capacity of BCAR1. These findings indicate that the differences between BCAR1 and HEF1 with respect to support of anti-estrogen resistance reside in the substrate domain which contains multiple sites for tyrosine phosphorylation. The crucial interactions required for anti-estrogen resistance occur within the substrate domain of BCAR1. Further deciphering of these interactions may resolve the growth regulatory mechanism and provide an explanation for the observation that primary tumors with high levels of BCAR1 are likely to fail on tamoxifen therapy. This information may also help to devise alternative personalized treatment strategies with improved outcome for breast cancer patients.

Abstract P6-08-10: Mutational signatures impact the breast cancer transcriptome and distinguish mitotic from immune response pathways

A comprehensive whole genome analysis of a large breast cancer cohort of 560 cases (Nik-Zainal et al, submitted 2015) reports novel and existing DNA substitution and rearrangement signatures next a comprehensive list of events driving the breast cancer cell to its malignant potency. In the current study, we linked the observed genetic diversity to the breast cancer transcriptome for 260 cases for which whole genome and whole transcriptome data were both available. Cluster analysis of the global gene expression showed the familiar view of a coherent basal-like and a heterogeneous luminal subgroup. New and previously reported 1 subtype-specific aberrations with concordant expression changes were found in TP53, PIK3CA, PTEN, CCND1, CDH1 and GATA3, and mutations in PIK3CA, PTEN, AKT1 and AKT2 were mutually exclusive confirming they are active in the same pathway in breast cancer. Integrating the identified DNA substitutions signatures with the transcriptome, we observed that the total number of substitutions in a cancer, irrespective of substitution type, was positively associated with cell cycle regulated gene expression and with adverse outcome. In addition and more remarkably, we observed that the number substitution of two substitution signatures 2 particularly associated with immune-response specific gene expression, with increased amount of tumor infiltrating lymphocytes and with a better outcome. These two signatures comprised 1) mutations of the APOBEC-type (predominant C>G in a TCN context), and 2) mutations which lacks specific features but which are strongly associated with genetic and epigenetic inactivating aberrations in BRCA1 and BRCA2. Thus, while earlier reports 3-5 imply that the sheer number of driver events triggers an immune-response, we refine this statement by observing that substitutions of a particular type are much very effective in doing so explaining the superior outcome of cancer having these particular types of substitutions. This result also implies that purposefully augmenting T-cell reactivity against amino-acid substitutions resulting from either of these two DNA substitution types could potentially improve immunotherapies in breast cancer. 1. Comprehensive molecular portraits of human breast tumours. Nature 490, 61-70 (2012). 2. Alexandrov, L.B., et al. Signatures of mutational processes in human cancer. Nature 500, 415-421 (2013). 3. Rizvi, N.A., et al. Cancer immunology. Mutational landscape determines sensitivity to PD-1 blockade in non-small cell lung cancer. Science 348, 124-128 (2015). 4. Schumacher, T.N. & Schreiber, R.D. Neoantigens in cancer immunotherapy. Science 348, 69-74 (2015). 5. Snyder, A., et al. Genetic basis for clinical response to CTLA-4 blockade in melanoma. N Engl J Med 371, 2189-2199 (2014). Citation Format: Martens JWM, Smid M, Rodriguez-Gonzalez G, Sieuwerts AM, Prager-Van der Smissen WJC, Van Der Vlugt - Daane M, Van Galen A, Nik-Zainal S, Staaf J, Brinkman AB, Van de Vijver MJ, Richardson AL, Berentsen K, Caldas C, Butler A, Martin S, Davies HD, Debets R, Meijer-Van Gelder ME, Van Deurzen CHM, Ramakrishna MR, Ringner M, Viari A, Birney E, Borresen-Dale A-L, Stunnenberg HG, Stratton M, Foekens JA. Mutational signatures impact the breast cancer transcriptome and distinguish mitotic from immune response pathways. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-08-10.