BCAR4 induces antioestrogen resistance but sensitises breast cancer to lapatinib.

BACKGROUND: High BCAR4 and ERBB2 mRNA levels in primary breast cancer associate with tamoxifen resistance and poor patient outcome. We determined whether BCAR4 expression sensitises breast cancer cells to lapatinib, and identifies a subgroup of patients who possibly may benefit from ERBB2-targeted therapies despite having tumours with low ERBB2 expression. METHODS: Proliferation assays were applied to determine the effect of BCAR4 expression on lapatinib treatment. Changes in cell signalling were quantified with reverse-phase protein microarrays. Quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) of ERBB2 and BCAR4 was performed in 1418 primary breast cancers. Combined BCAR4 and ERBB2 mRNA levels were evaluated for association with progression-free survival (PFS) in 293 oestrogen receptor-α (ER)-positive patients receiving tamoxifen as first-line monotherapy for recurrent disease. RESULTS: BCAR4 expression strongly sensitised ZR-75-1 and MCF7 breast cancer cells to the combination of lapatinib and antioestrogens. Lapatinib interfered with phosphorylation of ERBB2 and its downstream mediators AKT, FAK, SHC, STAT5, and STAT6. Reverse transcriptase-PCR analysis showed that 27.6% of the breast cancers were positive for BCAR4 and 22% expressed also low levels of ERBB2. The clinical significance of combining BCAR4 and ERBB2 mRNA status was underscored by the finding that the group of patients having BCAR4-positive/ERBB2-low-expressing cancers had a shorter PFS on tamoxifen treatment than the BCAR4-negative group. CONCLUSION: This study shows that BCAR4 expression identifies a subgroup of ER-positive breast cancer patients without overexpression of ERBB2 who have a poor outcome and might benefit from combined ERBB2-targeted and antioestrogen therapy.

Relevance of BCAR4 in tamoxifen resistance and tumour aggressiveness of human breast cancer.

BACKGROUND: Breast cancer anti-oestrogen resistance 4 (BCAR4) was identified in a search for genes involved in anti-oestrogen resistance in breast cancer. We explored whether BCAR4 is predictive for tamoxifen resistance and prognostic for tumour aggressiveness, and studied its function. METHODS: BCAR4 mRNA levels were measured in primary breast tumours, and evaluated for association with progression-free survival (PFS) and clinical benefit in patients with oestrogen receptor (ERα)-positive tumours receiving tamoxifen as first-line monotherapy for advanced disease. In a separate cohort of patients with lymph node-negative, ERα-positive cancer, and not receiving systemic adjuvant therapy, BCAR4 levels were evaluated for association with distant metastasis-free survival (MFS). The function of BCAR4 was studied with immunoblotting and RNA interference in a cell model. RESULTS: Multivariate analyses established high BCAR4 mRNA levels as an independent predictive factor for poor PFS after start of tamoxifen therapy for recurrent disease. High BCAR4 mRNA levels were associated with poor MFS and overall survival, reflecting tumour aggressiveness. In BCAR4-expressing cells, phosphorylation of v-erb-b2 erythroblastic leukaemia viral oncogene homolog (ERBB)2, ERBB3, and their downstream mediators extracellular signal-regulated kinase 1/2 and v-akt murine thymoma viral oncogene homolog (AKT) 1/2, was increased. Selective knockdown of ERBB2 or ERBB3 inhibited proliferation, confirming their role in BCAR4-induced tamoxifen resistance. CONCLUSION: BCAR4 may have clinical relevance for tumour aggressiveness and tamoxifen resistance. Our cell model suggests that BCAR4-positive breast tumours are driven by ERBB2/ERBB3 signalling. Patients with such tumours may benefit from ERBB-targeted therapy.

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.

Cellular origin of microRNA-371a-3p in healthy males based on systematic urogenital tract tissue evaluation.

BACKGROUND: The microRNA-371a-3p (miR-371a-3p) has been reported to be an informative liquid biopsy (serum and plasma) molecular biomarker for both diagnosis and follow-up of patients with a malignant (testicular) germ cell tumor ((T)GCT). It is expressed in all histological cancer elements, with the exception of mature teratoma. However, normal testis, semen, and serum of males with a disrupted testicular integrity without a TGCT may contain miR-371a-3p levels above threshold, of which the cellular origin is unknown. OBJECTIVES: Therefore, a series of relevant tissues (frozen and formalin-fixed paraffin-embedded (FFPE), when available) from the complete male urogenital tract (i.e., kidney to urethra and testis to urethra) and semen was investigated for miR-371a-3p levels using targeted quantitative RT-PCR (qRT-PCR). MATERIALS AND METHODS: In total, semen of males with normospermia (n = 11) and oligospermia (n = 3) was investigated, as well as 88 samples derived from 32 different patients. The samples represented one set of tissues related to the entire male urogenital tract (11 anatomical locations), three sets for 10 locations, and four sets for six locations. RESULTS: All testis parenchyma (n = 17) cases showed low miR-371a-3p levels. Eight out of 14 (57%) semen samples showed detectable miR-371a-3p levels, irrespective of the amount of motile spermatozoa, but related to sperm concentration and matched Johnsen score (Spearman's rho correlation coefficient 0.849 and 0.871, p = 0.000, respectively). In all other tissues investigated, miR-371a-3p could not be detected. DISCUSSION: This study demonstrates that the miR-371a-3p in healthy adult males is solely derived from the germ cell compartment. CONCLUSIONS: The observation is important in the context of applying miR-371a-3p as molecular liquid biopsy biomarker for diagnosis and follow-up of patients with malignant (T)GCT. Moreover, miR-371a-3p might be an informative seminal biomarker for testicular germ cell composition.

BCAR1, a human homologue of the adapter protein p130Cas, and antiestrogen resistance in breast cancer cells.

BACKGROUND: Treatment of breast cancer with the antiestrogen tamoxifen is effective in approximately one half of the patients with estrogen receptor-positive disease, but tumors recur frequently because of the development of metastases that are resistant to tamoxifen. We have previously shown that mutagenesis of human estrogen-dependent ZR-75-1 breast cancer cells by insertion of a defective retrovirus genome caused the cells to become antiestrogen resistant. In this study, we isolated and characterized the crucial gene at the breast cancer antiestrogen resistance 1 (BCAR1) locus. METHODS/RESULTS: Transfer of the BCAR1 locus from retrovirus-mutated, antiestrogen-resistant cells to estrogen-dependent ZR-75-1 cells by cell fusion conferred an antiestrogen-resistant phenotype on the recipient cells. The complete coding sequence of BCAR1 was isolated by use of exon-trapping and complementary DNA (cDNA) library screening. Sequence analysis of human BCAR1 cDNA predicted a protein of 870 amino acids that was strongly homologous to rat p130Cas-adapter protein. Genomic analysis revealed that BCAR1 consists of seven exons and is located at chromosome 16q23.1. BCAR1 transcripts were detected in multiple human tissues and were similar in size to transcripts produced by retrovirus-mutated ZR-75-1 cells. Transfection of BCAR1 cDNA into ZR-75-1 cells again resulted in sustained cell proliferation in the presence of antiestrogens, confirming that BCAR1 was the responsible gene in the locus. CONCLUSIONS: Overexpression of the BCAR1 gene confers antiestrogen resistance on human ZR-75-1 breast cancer cells. Overexpression of BCAR1 in retrovirus-mutated cells appears to result from activation of the gene's promoter. The isolation and characterization of this gene open new avenues to elucidating mechanisms by which the growth of human breast cancer becomes independent of estrogen.

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 levels of Bcar1/p130Cas. In multivariate analysis for response, Bcar1/p130Cas was independent of classical predictive factors, such as estrogen receptor status, age/menopausal status, disease-free interval, and dominant site of relapse. CONCLUSION: Patients with primary breast tumors expressing a high level of Bcar1/p130Cas protein appear to experience more rapid disease recurrence and have a greater risk of (intrinsic) resistance to tamoxifen therapy. Thus, measurement of Bcar1/p130Cas may provide useful prognostic information for patients with primary or metastatic breast cancer.

Ectopic expression of epidermal growth factor receptors induces hormone independence in ZR-75-1 human breast cancer cells.

Epidermal growth factor (EGF) receptor is inversely related to expression of estrogen receptor (ER) and progesterone receptor in primary breast tumors and is a negative predictor for response to endocrine therapy. To investigate a possible causal role of EGF receptor expression in breast cancer progression to hormone independence, we have created an experimental cell system. Epidermal growth factor receptor complementary DNA was introduced in estrogen-dependent ZR-75-1 breast cancer cells, and the resulting ZR/HERc cells exhibited a mitogenic response to epidermal growth factor, thus bypassing estrogen dependence. This EGF-induced proliferation could not be inhibited by antiestrogens. In addition, we noted changes in cell morphology and keratin expression of EGF-stimulated ZR/HERc cells, suggestive of an altered differentiation state. Furthermore, intolerance of functional ER and EGF receptor signal transduction pathways in ZR/HERc cells was observed during simultaneous activation, which possibly explains the inverse relationship of ER and EGF receptor expression in primary tumors. In contrast to the parental cells, ZR/HERc cells rapidly progressed to a stable ER-negative phenotype when cultured in the presence of the antiestrogen hydroxy-tamoxifen. These results suggest a possible role for EGF receptor in progression of breast cancer to hormone independence.

Inhibition of breast cancer cell growth by combined treatment with vitamin D3 analogues and tamoxifen.

The steroid hormone 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] has potential to be used as an antitumor agent, but its clinical application is restricted by the strong calcemic activity. Therefore, new vitamin D3 analogues are developed with increased growth inhibitory and reduced calcemic activity. In the present study, we have examined the antiproliferative effects of four novel vitamin D3 analogues (CB966, EB1089, KH1060, and 22-oxa-calcitriol) on breast cancer cells, either alone or in combination with the antiestrogen tamoxifen. The estrogen-dependent ZR-75-1 and estrogen-responsive MCF-7 cell lines were used as a model. It was shown that, with EB1089 and KH1060, the same growth inhibitory effect as 1,25-(OH)2D3 could be reached at up to 100-fold lower concentrations, whereas CD966 and 22-oxa-calcitriol were nearly equipotent with 1,25-(OH)2D3. The growth inhibition by the vitamin D3 compounds could be augmented by combined treatment with tamoxifen. At the maximal effective concentrations of the vitamin D3 compounds, the effect of combined treatment was addictive (MCF-7 cells) or less than additive (ZR-75-1 cells). Tamoxifen increased the sensitivity of the cells to the vitamin D3 compounds 2- to 4000-fold, which was expressed by a shift to lower median effective concentration values. Thereby, the vitamin D3 compounds may be used at even lower dosages in combination therapy with tamoxifen. A major problem of tamoxifen therapy is the development of tamoxifen resistance. We have observed that tamoxifen-resistant clones of ZR-75-1 cells retain their response to the vitamin D3 compounds. Regulation of the growth-related oncogene c-myc (mRNA level) and the estrogen receptor (protein level) were studied but appeared not to be related to the antiproliferative action of the vitamin D3 compounds. Together, our data point to a potential benefit of combination therapy with 1,25-(OH)2D3 or vitamin D3 analogues and tamoxifen for the treatment of breast cancer.

Cloning and expression of interleukin-3 genes of chimpanzee and New World monkeys.

Interleukin-3 (IL-3) genes were cloned from chimpanzee (Pan troglodytes), tamarin (Saguinus oedipus) and marmoset (Callithrix jacchus) and expressed in COS cells. Although the IL-3 gene structure is well conserved in these primate species, sequence analysis revealed extensive base substitutions. The chimpanzee IL-3 protein, which is highly homologous (98.5% identity) to human IL-3, stimulated proliferation of human cells dependent on IL-3. In contrast, due to the numerous amino acid substitutions in the New World monkey IL-3 species, no stimulation of human cells was observed, illustrating the extensive evolutionary divergence of IL-3.

Clinical significance of t(14; 18)-positive cells in the circulation of patients with stage III or IV follicular non-Hodgkin's lymphoma during first remission.

PURPOSE: To evaluate polymerase chain reaction (PCR) analysis as a method for the detection of circulating lymphoma cells in patients with stage III and IV t(14; 18)-positive follicular Non-Hodgkin's lymphoma (NHL) in first remission in a longitudinal prospective study. PATIENTS AND METHODS: Peripheral blood or bone marrow from eight patients with stage III and IV t(14; 18)-positive NHL was studied using PCR to detect the presence of t(14; 18)-positive cells in the circulation at different times during first remission. RESULTS: In four of six patients with no clinical evidence of disease (NCED), t(14; 18)-positive cells were detectable in the circulation. In one of two patients with clinical evidence of disease (CED), no t(14; 18)-positive cells were found at the four different occasions tested during first remission. First-remission duration ranged from 17 to 81+ months. The duration from the first PCR determination in remission until first relapse or the end of the observation period ranged from 10 to 37+ months. CONCLUSION: In patients with t(14; 18)-positive follicular NHL stage III and IV, treated with conventional remission induction therapy, the presence or absence of t(14; 18)-positive cells in the circulation shows no obvious correlation with the clinical remission status and the remission duration.

Detection of residual disease in translocation (14;18) positive non-Hodgkin's lymphoma, using the polymerase chain reaction: a comparison with conventional staging methods.

This paper reports the detection of residual lymphoma cells in blood and bone marrow samples from patients with translocation (t) (14;18) positive non-Hodgkin's lymphoma by the polymerase chain reaction (PCR) compared with conventional staging techniques. In 15 of 22 samples, in which no lymphoma cells could be detected by morphological examination, t(14;18) positive cells were detected by PCR. In 13 of 21 samples, in which a monoclonal B-cell population was not detectable by immunological marker analysis, PCR was positive. The clinical status (physical examination, imaging techniques, leucocyte count, and occasionally morphology and immunological marker analysis) was documented in 30 patients at the time of PCR analysis. In three of 19 patients with clinical evidence of disease, circulating t(14;18) positive cells were not detectable by PCR. Five of 11 patients in clinical remission from 7 to 47 months, showed t(14;18) positive cells in the blood. Our data show that PCR analysis in t(14;18) positive non-Hodgkin's lymphoma offers a powerful tool in the study of residual disease.

Genomic organization of the translocations (8;14) and (14;18) in a new lymphoma cell line.

We generated a new lymphoma cell line carrying the translocations (8;14) and (14;18) and studied the genomic organization and expression of the BCL-2 and MYC genes. Polymerase chain reaction (PCR) and Southern analysis showed that the breakpoints of t(14;18) were located in the major breakpoint region (mbr) of the BCL-2 gene and just 5' of JH6 in the IgH locus. The breakpoints of the t(8;14) were located upstream of exon 2 in the non-coding region of the MYC gene and near the switch region of the IgH locus. Both IgH loci were involved in chromosomal translocations resulting in the absence of a functional B-cell receptor. Normal BCL-2 and truncated MYC transcripts were detected in these cells. The BCL-2 protein was expressed.

Rearranged immunoglobulin light chain genes as minimal residual disease markers in intermediate- and high-grade malignant B cell non-Hodgkin's lymphoma.

Minimal residual disease (MRD) detection in B cell non-Hodgkin's lymphoma (NHL) patients has been shown to be possible using the rearranged heavy (IgH) chain gene as a tumor marker. To explore a second independent tumor marker, we used specific PCR primer sets to identify tumor-specific rearranged Ig light chain (IgL) genes. Rearranged IgL genes were amplified from lymphoma DNA by multiplex PCR using separate primer sets for the Igkappa and the Iglambda genes. They were considered to be of tumor origin if they were monoclonal, and if the same rearrangement was isolated from at least two independent PCR products. From 12 out of 13 intermediate- and high-grade malignant NHL, PCR products could be obtained with IgL specific primers. PCR products from five NHL were studied in detail by cloning and sequencing. The rearranged IgL genes showed 85-100% homology with their closest germ line counterparts. Intraclonal IgL sequence heterogeneity was studied in five lymphomas and detected in only one. Minimal disease was studied in three patients by PCR, followed by Southern hybridization of the PCR product with a lymphoma-specific oligonucleotide probe, which allowed for detection of lymphoma DNA following 1000-fold dilution. Blood samples from one patient, who is in long-term clinical remission, were negative for the lymphoma-specific rearranged Igkappa gene. In the second patient the rearranged Iglambda gene was detected during the first clinical remission, that was followed by a nodal relapse, but not during the second remission, that has been stable for almost 3 years now. The third patient was negative for the rearranged Iglambda gene in blood samples up to 102 months after diagnosis. Circulating lymphoma cells were detected in blood and bone marrow samples which were negative by morphological and immunological criteria. Our studies show that the rearranged IgL gene can be used as a second independent tumor marker in intermediate- and high-grade malignant NHL.

Detection of minimal disease using rearranged immunoglobulin heavy chain genes from intermediate- and high-grade malignant B cell non-Hodgkins lymphoma.

Rearranged immunoglobulin heavy chain (IgH) genes provide unique clonal markers for B cells. Since amplification of the rearranged gene by polymerase chain reaction (PCR) and demonstrating that the amplified sequence is indeed derived from tumor cells is more problematic in non-Hodgkin's lymphoma (NHL) than in other B cell malignancies, we used a comprehensive PCR primer set and formulated stringent selection criteria to identify tumor-specific rearranged IgH genes. Rearranged IgH genes amplified from lymphoma DNA were considered to be of tumor origin if they were monoclonal, and if the same rearrangement was amplified with at least two independent VH-specific primers. From 11 of 13 (85%) intermediate- and high-grade malignant NHL, IgH rearrangements were isolated. Intraclonal IgH sequence heterogeneity was studied in four lymphomas, and detected in two of them. PCR using a lymphoma-specific primer followed by Southern hybridization of PCR product with a specific probe allowed detection of lymphoma DNA after 10,000-fold dilution. Circulating lymphoma cells were detected in patient blood and bone marrow samples which were negative by morphological and immunological criteria. Thus, also in intermediate- and high-grade malignant lymphoma, sensitive minimal disease detection using the rearranged IgH gene as a marker appears feasible.

Induction of antiestrogen resistance in human breast cancer cells by random insertional mutagenesis using defective retroviruses: identification of bcar-1, a common integration site.

Duration of response to antiestrogen therapy in metastatic breast cancer is limited due to the development of antiestrogen-resistant tumors. The mechanisms involved are not understood but could originate from (epi)genetic alterations within the tumor cells. We have applied in vitro random insertional mutagenesis with replication defective retroviruses to identify those genes playing a key role in development of antiestrogen resistance in human breast cancer cells. Eighty antiestrogen-resistant cell clones were isolated from 7 x 10(8) estrogen-dependent ZR-75-1 cells, mass-infected with defective retroviruses and subjected to 4-OH-tamoxifen selection. Integration site-specific DNA probes were made by inverse polymerase chain reaction techniques and used to search for common integration sites. Six cell clones were identified with retroviral genome integrations in the same orientation in a single locus, designated breast cancer antiestrogen resistance locus-1 (bcar-1). These bcar-1 cell clones had lost estrogen receptor expression and had become estrogen independent. Our results strongly suggest that alteration of the bcar-1 locus is responsible for development of antiestrogen resistance in human breast cancer cells in vitro. In addition, we have shown that in vitro insertional mutagenesis using defective retroviruses can be applied for gene tagging in human cells.

Induction of estrogen independence of ZR-75-1 human breast cancer cells by epigenetic alterations.

Antagonists of steroid hormones are clinically important in the management of breast cancer. However, the duration of response is limited due to the development of hormone-independent tumors in virtually all cases. In an attempt to obtain insight into the mechanisms underlying antiestrogen resistance, the consequences of epigenetic changes in gene expression were studied in vitro. Estrogen-dependent ZR-75-1 human breast cancer cells were treated with 5-azacytidine, an inhibitor of DNA methylation, and cultured in the absence of estradiol or in the presence of antiestrogens. Estrogen-independent cell colonies developed within 3 weeks at high frequency in 5-azacytidine-treated cultures (0.7 x 10(-3), in contrast to control cultures (< or = 10(-8). The derived cells (ZR/AZA) were resistant to 4-hydroxytamoxifen and ICI 164,384, independent of the selection protocol, but had lost the ability to grow anchorage-independent. Whereas expression of estrogen receptor, progesterone receptor, and pS2 were down-regulated, expression of epidermal growth factor (EGF) receptor and HER2/neu were increased in ZR/AZA cells. In contrast to the stable altered expression patterns of estrogen receptor and EGF receptor, transient keratin 7 expression was observed. Transforming growth factor-alpha mRNA was identified in ZR-75-1 cells and ZR/AZA cells and EGF-like peptides were secreted in the culture medium. Proliferation of ZR/AZA cells could be partially inhibited with an EGF receptor-blocking antibody. Presence of both growth factor receptors and possible ligands suggests the development of an autocrine growth mechanism. Our data show that epigenetic alterations of gene expression result in rapid progression of breast cancer cells to hormone independence.

Characterization of a human multilineage-colony-stimulating factor cDNA clone identified by a conserved noncoding sequence in mouse interleukin-3.

Blood-cell production is regulated by hemopoietic growth factors, which act at specific stages of hemopoietic cell differentiation. Murine interleukin-3 (mIL-3)/multilineage colony-stimulating factor (multi-CSF) has been shown to stimulate colony formation in vitro by multipotent hemopoietic cells and production of spleen colony-forming units (CFU-S) in suspension cultures. The molecular cloning of the human counterpart of mIL-3 is described here. Hybridization of radiolabeled mIL-3 cDNA with a cDNA library obtained from mRNA of stimulated human lymphocytes resulted in the identification of a human (h)multi-CSF cDNA clone. Sequence homology (73%) in the 3'-noncoding region of mIL-3 enabled the detection of the hmulti-CSF cDNA clone. Whereas only 45% sequence homology was found in the coding region, specific A + T-rich domains in the 3'-noncoding region were highly conserved (93%). As far as we know, this is the first example of gene identification by sequence homology occurring only within the 3'-noncoding region. The protein encoded by this hmulti-CSF cDNA stimulates in vitro colony formation by multipotent human hemopoietic stem cells. In addition, the growth factor strongly stimulates the in vitro proliferation of human leukemic blast cells.

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 resis- tance (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/pl30Cas 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.

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.

Genetic mechanisms of estrogen-independence in breast cancer.

Endocrine therapy is effective in the treatment of breast cancer. Adjuvant treatment with tamoxifen reduces tumor recurrence and achieves increased survival. In metastatic disease, tamoxifen treatment accomplishes objective responses in +/- 50% of the patients with estrogen receptor-positive primary tumors. However, the response duration is limited due to the inevitable development of metastases resistant to tamoxifen. The mechanisms leading to tamoxifen resistance are largely unknown. We have set out to identify genetic pathways in the tumor cells causing failure of tamoxifen therapy. We selected an estrogen-dependent human breast cancer cell line (ZR-75-1) and demonstrated that genetic and epigenetic alterations can change the hormone-response phenotype of these cells. Subsequently, we applied insertional mutagenesis with defective retroviruses to these ZR-75-1 breast cancer cells. Integration of a retrovirus in the cellular DNA alters the genome structure and may modify the expression of genes in its surroundings. As a result of the altered gene expression, the biological phenotype of the infected cell may be changed. The infected ZR-75-1 cells were subjected to tamoxifen selection and a panel of tamoxifen-resistant cell lines has been established. Screening for a common integration site for the retrovirus has provided, so far, compelling evidence for the involvement of at least one genetic locus (BCAR 1) in breast cancer antiestrogen resistance in vitro.