Ton van Agthoven

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.

Identification of BCAR3 by a random search for genes involved in antiestrogen resistance of human breast cancer cells.

The antiestrogen tamoxifen is important in the treatment of hormone‐dependent breast cancer, although development of resistance is inevitable. To unravel the molecular mechanisms of antiestrogen resistance, a search for involved genes was initiated. Retrovirus‐mediated insertional mutagenesis was applied to human ZR‐75‐1 breast cancer cells. Infected cells were subjected to tamoxifen selection and a panel of resistant cell clones was established. Screening for a common integration site resulted in the identification of a novel gene designated BCAR3. Transfer of this locus by cell fusion or transfection of the BCAR3 cDNA to ZR75‐1 and MCF‐7 cells induces antiestrogen resistance. BCAR3 represents a putative SH2 domain‐containing protein and is partly homologous to the cell division cycle protein CDC48.

Relevance of Breast Cancer Antiestrogen Resistance Genes in Human Breast Cancer Progression and Tamoxifen Resistance

PURPOSE We have previously identified a set of breast cancer antiestrogen resistance (BCAR) genes causing estrogen independence and tamoxifen resistance in vitro using a functional genetic screen. Here, we explored whether these BCAR genes provide predictive value for tamoxifen resistance and prognostic information for tumor aggressiveness in breast cancer patients. PATIENTS AND METHODS mRNA levels of 10 BCAR genes (AKT1, AKT2, BCAR1, BCAR3, EGFR, ERBB2, GRB7, SRC, TLE3, and TRERF1) were measured in estrogen receptor-positive breast tumors using quantitative reverse-transcriptase polymerase chain reaction. Normalized mRNA levels were evaluated for association with progression-free survival (PFS) in 242 patients receiving tamoxifen as first-line monotherapy for recurrent disease, and with distant metastasis-free survival (MFS) in 413 lymph node-negative (LNN) primary breast cancer patients who did not receive systemic adjuvant therapy. RESULTS Concerning tamoxifen resistance, BCAR3, ERBB2, GRB7, and TLE3 mRNA levels were predictive for PFS, independent of traditional predictive factors. By combining GRB7 (or ERBB2) and TLE3 mRNA levels, patients could be classified in three subgroups with distinct PFS. For the evaluation of tumor aggressiveness, AKT2, EGFR, and TRERF1 mRNA levels were all significantly associated with MFS, independent of traditional prognostic factors. Using the combined AKT2 and EGFR mRNA status, four prognostic groups were identified with different MFS outcomes. CONCLUSION The majority of BCAR genes, which were revealed to confer tamoxifen resistance and estrogen independence in vitro by functional screening, have clinical relevance, and associate with tamoxifen resistance and/or tumor aggressiveness in breast cancer patients.

Functional screen for genes responsible for tamoxifen resistance in human breast cancer cells

Antiestrogens, such as tamoxifen, are widely used for endocrine treatment of estrogen receptor–positive breast cancer. However, as breast cancer progresses, development of tamoxifen resistance is inevitable. The mechanisms underlying this resistance are not well understood. To identify genes involved in tamoxifen resistance, we have developed a rapid screening method. To alter the tamoxifen-sensitive phenotype of human ZR-75-1 breast cancer cells into a tamoxifen-resistant phenotype, the cells were infected with retroviral cDNA libraries derived from human placenta, human brain, and mouse embryo. Subsequently, the cells were selected for proliferation in the presence of 4-hydroxy-tamoxifen (OH-TAM) and integrated cDNAs were identified by sequence similarity searches. From 155 OH-TAM-resistant cell colonies, a total of 25 candidate genes were isolated. Seven of these genes were identified in multiple cell colonies and thus cause antiestrogen resistance. The epidermal growth factor receptor, platelet-derived growth factor receptor-α, platelet-derived growth factor receptor-β, colony-stimulating factor 1 receptor, neuregulin1, and fibroblast growth factor 17 that we have identified have been described as key regulators in the mitogen-activated protein kinase pathway. Therefore, this pathway could be a valuable target in the treatment of patients with breast cancer resistant to endocrine treatment. In addition, the putative gene LOC400500, predicted by in silico analysis, was identified. We showed that ectopic expression of this gene, designated as breast cancer antiestrogen resistance 4 (BCAR4), caused OH-TAM resistance and anchorage-independent cell growth in ZR-75-1 cells and that the intact open reading frame was required for its function. We conclude that retroviral transfer of cDNA libraries into human breast cancer cells is an efficient method for identifying genes involved in tamoxifen resistance. (Mol Cancer Res 2006;4(6):379–86)

Fibroblast growth factor receptor 4 predicts failure on tamoxifen therapy in patients with recurrent breast cancer

Tamoxifen treatment of estrogen-dependent breast cancer ultimately loses its effectiveness due to the development of resistance. From a functional screen for identifying genes responsible for tamoxifen resistance in human ZR-75-1 breast cancer cells, fibroblast growth factor (FGF) 17 was recovered. The aim of this exploratory study was to assess the predictive value of FGF17 and the receptors FGFR1-4 for the type of response to tamoxifen treatment (clinical benefit) and the duration of progression-free survival (PFS) in patients with recurrent breast cancer. mRNA levels of FGF17 and FGFR1-4 were quantified by real-time reverse transcriptase PCR in 285 estrogen receptor-positive breast carcinomas with clinical follow-up. All patients had recurrent disease and were treated with tamoxifen as first-line systemic therapy for local or distant relapse. FGF17 and FGFR1-3 mRNA levels had no significant predictive value for this group of patients. However, high FGFR4 mRNA levels analyzed as a continuous log-transformed variable predicted poor clinical benefit (odds ratio=1.22; P=0.009) and shorter PFS (hazard ratio=1.18; P<0.001). In addition, in multivariable analysis, the predictive value of FGFR4 was independent from the traditional predictive factors. Our analyses show that FGFR4 may play a role in the biological response of the tumor to tamoxifen treatment. In addition, as altered expression of FGF17 causes tamoxifen resistance in vitro, the FGF signaling pathway could be a valuable target in the treatment of breast cancer patients resistant to endocrine treatment.

Characterization of BCAR4, a novel oncogene causing endocrine resistance in human breast cancer cells†

Resistance to the antiestrogen tamoxifen remains a major problem in the management of estrogen receptor‐positive breast cancer. Knowledge on the resistance mechanisms is needed to develop more effective therapies. Breast cancer antiestrogen resistance 4 (BCAR4) was identified in a functional screen for genes involved in tamoxifen resistance. BCAR4 is expressed in 27% of primary breast tumors. In patients treated with tamoxifen for metastized disease high BCAR4 mRNA levels are associated with reduced clinical benefit and progression‐free survival. Regarding tumor aggressiveness high BCAR4 mRNA levels are associated with a shorter metastasis free survival and overall survival. In the present study, we investigated the role of BCAR4 in endocrine resistance. Forced expression of BCAR4 in human ZR‐75‐1 and MCF7 breast cancer cells resulted in cell proliferation in the absence of estrogen and in the presence of various antiestrogens. Inhibition of estrogen receptor 1 (ESR1) expression with small interfering RNA (siRNA), implied that the BCAR4‐induced mechanism of resistance is independent of ESR1. Highly conserved BCAR4 homologues of rhesus monkey, green monkey, and the less conserved common marmoset gene induced tamoxifen‐resistant cell proliferation, in contrast to the distant BCAR4 homologues of bovine and rabbit. Injection of BCAR4‐expressing ZR‐75‐1 cells into nude mice resulted in rapidly growing tumors. In silico analysis showed that BCAR4 mRNA is highly expressed in human placenta and oocyte, and absent in other normal tissues. In conclusion, BCAR4 is a strong transforming gene causing estrogen‐independent growth and antiestrogen resistance, and induces tumor formation in vivo. Due to its restricted expression, BCAR4 may be a good target for treating antiestrogen‐resistant breast cancer. J. Cell. Physiol. 226: 1741–1749, 2011.

Functional identification of genes causing estrogen independence of human breast cancer cells

Endocrine treatment of breast cancer is widely applied and effective. However, in advanced disease cases, the tumors will eventually progress into an estrogen-independent and therapy-resistant phenotype. To elucidate the molecular mechanisms underlying this endocrine therapy failure, we applied retroviral insertion mutagenesis to identify the main genes conferring estrogen independence to human breast cancer cells. Estrogen-dependent ZR-75-1 cells were infected with replication-defective retroviruses followed by selection with the anti-estrogen 4-hydroxy-tamoxifen. In the resulting panel of 79 tamoxifen-resistant cell lines, the viral integrations were mapped within the human genome. Genes located in the immediate proximity of the retroviral integration sites were characterized for altered expression and their capacity to confer anti-estrogen resistance when transfected into breast cancer cells. Out of 15 candidate BCAR (breast cancer anti-estrogen resistance) genes, seven (AKT1, AKT2, BCAR1, BCAR3, EGFR, GRB7, and TRERF1/BCAR2) were shown to directly underlie estrogen independence. Our results show that insertion mutagenesis is a powerful tool to identify BCAR loci, which may provide insights into the molecular and cellular mechanisms of breast tumor progression and therapy resistance thereby offering novel targets for the development of tailor-made therapeutical and prevention strategies.

Development of malignant germ cells - the genvironmental hypothesis.

Human germ cell tumors are of interest because of their epidemiology, clinic and patho-biology. Histologically, they are subdivided into various elements, with similarities to embryogenesis. Recent insight triggered development of a higher order division into five types of human germ cell tumors. In the context of male germ cells, only three are relevant; Type I: teratomas and yolk sac tumors of neonates and infants; Type II: seminomas and nonseminomas of (predominantly) adolescents and adults; and Type III: spermatocytic seminomas of the elderly. Various animal models, both occurring spontaneous or induced, are reported, of which their relevance is still a matter of debate. Recent multidisciplinary studies have led to a significant increase in our understanding of the parameters involved in the earliest pathogenetic steps of human germ cells tumors, particularly the seminomas and nonseminomas (Type II). This paper will discuss a number of interesting insights into the normal and aberrant regulation of germ cell development, resulting in the so-called genvironmental hypothesis. This assumes a subtle interaction between environmental- and (epi)genetic parameters, resulting in clinical/phenotypical characteristics. These influence signaling pathways and thereby developmental processes, including gonadal development, germ cell proliferation, maturation and apoptosis. In the case of a disturbed physiology, either due to the germ cell itself, or the micro-environment, embryonic germ cells, during a specific window of sensitization, might be blocked in their maturation, resulting in carcinoma in situ or gonadoblastoma, the precursors of seminomas and nonseminomas. The level of testicularization of the gonad determines the histological composition of the precursor. These insights will allow a better definition of individuals at risk of developing a germ cell malignancy, and allow a better selection of scientific approaches to elucidate the corresponding pathogenesis.

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.

Selective recruitment of breast cancer anti-estrogen resistance genes and relevance for breast cancer progression and tamoxifen therapy response.

Although endocrine treatment of breast cancer is effective and common practice, in advanced disease the development of resistance is nearly inevitable. To get more insight into individual genes that account for resistance against hormonal agents, we have executed functional genetic screens and subsequently evaluated the clinical relevance of several identified genes with respect to tumor aggressiveness and tamoxifen resistance in estrogen receptor-positive patients. Estrogen-dependent human breast cancer cells were transduced with different retroviral cDNA expression libraries and subjected to selective cultures with various anti-estrogens. From a total of 264 resistant cell clones, 132 different genes were recovered by PCR. By applying stringent selection criteria, we identified 15 breast cancer anti-estrogen resistance (BCAR) genes individually yielding resistance. BCAR genes were recovered with differential frequencies for the diverse culture conditions and anti-estrogen drugs. Analysis of the relation of BCAR genes (EIF1, FBXL10, HRAS, NRG1, PDGFRA, PDGFRB, RAD21, and RAF1) with tamoxifen treatment in patients with advanced disease showed significant association with clinical benefit and progression-free survival for EIF1 and PDGFRA mRNA levels. Furthermore, PDGFRA and HRAS mRNA levels were significantly associated with tumor aggressiveness in lymph node-negative patients who had not received adjuvant systemic therapy. In conclusion, our functional genetic screens showed that BCAR genes differ in their ability to confer resistance towards distinct anti-estrogens. Based on the clinical relevance of several BCAR genes, further studies are warranted to characterize the underlying mechanisms, which may ultimately lead to the development of novel treatments and more individualized management of breast cancer patients.