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.

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.

Differentially expressed genes in androgen-dependent and -independent prostate carcinomas.

Differential gene expression between androgen-dependent (LNCaP-FGC) and androgen-independent (LNCaP-LNO) prostate cancer cells has been investigated using RNA arbitrarily primed and differential display PCR of mRNA. Four differentially expressed cDNA transcripts were identified, of which differential expression was confirmed by Northern blot analysis. Sequence analysis revealed two unknown (JC19 and GC79) and two known genes [B-cell translocation gene 1 and UDP-glucuronosyltransferase 2B15 (UGT2B15)]. JC19, GC79, and B-cell translocation gene 1 were more highly expressed in LNCaP-FGC cells compared with LNCaP-LNO cells, whereas UGT2B15 was only expressed in LNCaP-LNO cells. Androgens and 1,25-dihydroxyvitamin D3 were able to down-regulate UGT2B15 mRNA in LNCaP-LNO cells. For GC79 mRNA, down-regulation was only observed with androgens in LNCaP-FGC cells. Expression of JC19 mRNA was studied using a panel of human prostate cancer xenografts. In androgen-dependent xenografts, expression of JC19 mRNA was much higher compared with androgen-independent xenografts, in which significant expression was hardly detectable. The mRNA expression pattern in the xenografts is in good agreement with that observed in the cell culture system. In conclusion, the differential display technique used in the present study allows analysis of gene expression in vitro and in vivo and can be used for the identification of important genes involved in androgen-independent prostate cancer development.

Identification of a novel breast-cancer-anti-estrogen-resistance (BCAR2) locus by cell-fusion-mediated gene transfer in human breast-cancer cells.

Development of anti-estrogen resistance limits the benefit of endocrine therapy of breast cancer. The mechanistic basis for resistance to the anti-estrogen tamoxifen may involve (epi)genetic alterations within tumor cells. We have initiated a random search for genes allowing estrogen-dependent ZR-75-1 human breast-cancer cells to proliferate in the presence of tamoxifen. The strategy was based on insertion mutagenesis of ZR-75-1 cells using defective retrovirus and subsequent identification of common integration sites. As an alternative approach to identify integration loci involved in anti-estrogen resistance, we have applied cell fusion. Integration regions from lethally irradiated, tamoxifen-resistant cells were transferred to hygromycin B-resistant ZR-75-1 cells. Somatic cell hybrids were established by selection for resistance to G418 (encoded by the integrated virus) and hygromycin B. Individual integration loci were thus separated among different cell hybrids and tested for their role in anti-estrogen resistance. Analysis of a panel of 29 somatic-cell hybrids revealed that tamoxifen resistance co-segregated with only 1 of the 2 integration loci present in the tamoxifen-resistant donor cell line. This locus was further identified as a common integration site in our panel of tamoxifen-resistant cell clones. Our results designate this integration site as the second breast-cancer-anti-estrogen-resistance locus (BCAR2), which most likely contains a gene responsible for the anti-estrogen-resistant phenotype in close proximity to the integrated virus. Our data also imply that individual genes can alter the estrogen dependency of human breast-cancer cells in a dominant manner in vitro.

Studies on the human prostatic cancer cell line LNCaP.

The effects of androgens, antiandrogens, and other steroid hormones on growth of the human prostate cancer cell line LNCaP were studied. Despite the absence of receptors for progesterone and estradiol, the growth rate of the androgen responsive LNCaP-FGC cells increased when cultured in the presence of either estrogens or progestagens. In addition, most antiandrogens were also growth stimulators. This aberrant response was due to a threonine to alanine substitution at amino acid position 868 in the steroid binding domain of the androgen receptor (AR). Only the antiandrogen ICI 176,334 could block transcription and cell growth by the mutant receptor. By immunoprecipitation of the AR from LNCaP cells with the specific antibody F39.4.1 and Western blotting, three types of heat-shock proteins co-precipitated: hsp90, hsp70 and hsp56. This co-isolation could be prevented by pre-incubating the cells with androgens or with the antiandrogen hydroxyflutamide. Only the antiandrogen ICI 176,334 could block the effect of androgens on complex dissociation and prevent tight nuclear binding of the AR. Hydroxyflutamide could only inhibit tight nuclear binding of the wild-type AR. Therefore, in LNCaP cells the mutation in the steroid binding domain of the AR prevents a blockade of receptor function by most antiandrogens, but not by ICI 176,334, probably because of a different mechanism by which this compound blocks receptor function.

Effects of antiandrogens on transformation and transcription activation of wild-type and mutated (LNCaP) androgen receptors.

LNCaP cells contain androgen receptors with a mutation in the steroid binding domain (Thr 868 changed to Ala) resulting in a changed hormone specificity. Both the wild-type and mutated androgen receptors were transfected into COS cells. Transcription activation was studied in cells co-transfected with an androgen sensitive reporter (CAT) gene. The wild-type androgen receptor was activated by the agonist R1881, but the antiandrogens did not enhance transcription apart from a partial agonistic effect at high concentrations of cyproterone acetate. The mutated androgen receptor was fully activated by R1881, cyproterone acetate and hydroxyflutamide, but not by ICI 176,334. Receptor transformation to a tight nuclear binding state was studied by preparation of detergent washed nuclei and Western blotting with a specific antibody against the androgen receptor. Nuclei of COS cells transfected with wild-type receptor retained the receptor when the cells had been treated with the agonist R1881, partially retained receptors when treated with antiandrogen cyproterone acetate, but did not retain receptor when treated with hydroxyflutamide or ICI 176,334. The cells transfected with the mutated receptor additionally retained nuclear receptors after treatment with hydroxyflutamide. We conclude that each one of the three antiandrogens tested displayed different characteristics with respect to its effect on transformation and transcription activation.

Hormone-induced dissociation of the androgen receptor-heat-shock protein complex: use of a new monoclonal antibody to distinguish transformed from nontransformed receptors.

The hormone-induced transformation process of the androgen receptor in the androgen-responsive human prostatic carcinoma cell line LNCaP was studied. Immunoprecipitation of the nontransformed cytosolic receptor (8S on sucrose gradients) with a specific monoclonal antibody (F39.4.1) resulted in coprecipitation of three heat-shock proteins (hsp90, hsp70, and hsp56). Upon incubation of the cells with the synthetic androgen R1881, the sedimentation value of the receptor complex decreased to an intermediate form of 6S, and an almost complete loss of coprecipitating heat-shock proteins was observed. After a 2-h incubation, the receptor was recovered in considerable part from the nuclear fraction (extraction with high salt; 4.6S form). By use of the bifunctional cross-linker dimethyl pimelimidate, dissociation of the 8S complex, but not of the 6S complex, was blocked. A newly developed monoclonal antibody (F52.24.4), directed against the C-terminal part of the DNA-binding domain of the androgen receptor, specifically recognized both the 4.6S and the 6S forms of the receptor but did not react with the nontransformed 8S form. It is concluded that the unoccupied androgen receptor is associated with several heat-shock proteins and that transformation of the receptor to the tight nuclear-binding form is a multistep process that involves the dissociation of heat-shock proteins from the receptor.

Anti-androgens and the mutated androgen receptor of LNCaP cells: differential effects on binding affinity, heat-shock protein interaction, and transcription activation.

Previous studies from this laboratory have described that LNCaP prostate tumor cells contain an androgen receptor (AR) with a point mutation in the steroid-binding domain (codon 868, Thr to Ala). This defect leads to a change in specificity of the AR. Estrogens, progestagens, and some anti-androgens (e.g., cyproterone acetate, hydroxyflutamide, nilutamide) stimulate LNCaP cell growth rate through the AR. The present studies indicate that not all anti-androgens showed agonistic effects with the mutated receptor. The growth rate of LNCaP cells did not increase with the anti-androgen ICI 176334, nor could this compound increase transcription activation of the reporter gene construct via the mutant receptor in a cotransfection system [HeLa cell cotransfection system with an androgen-regulated reporter gene construct (pG29G-tk-CAT) and the mutant receptor as trans-vector]. Interaction of the AR of LNCaP cells with heat-shock proteins was studied by isolation of the receptor with a specific monoclonal antibody and characterization of associated proteins. Hsp90, hsp70, and hsp56 were found to coprecipitate with the AR. Incubation of the cells at 37 degrees C with androgen (R1881, 10 nM) or the anti-androgen hydroxyflutamide, prior to receptor isolation, resulted in dissociation of the AR-heat-shock protein complex. This dissociation is paralleled by the transformation to a tight nuclear-binding form of the AR. In contrast, ICI 176334 could not induce a release of heat-shock proteins and did not increase nuclear binding, but inhibited the transformation process induced by R1881.(ABSTRACT TRUNCATED AT 250 WORDS)

The androgen receptor in LNCaP cells contains a mutation in the ligand binding domain which affects steroid binding characteristics and response to antiandrogens.

The human prostate tumor cell line LNCaP contains an abnormal androgen receptor system with broad steroid binding specificity. Progestagens, estradiol and several antiandrogens compete with androgens for binding to the androgen receptor in the cells to a higher extent than in other androgen sensitive systems. Optimal growth of LNCaP cells is observed after addition of the synthetic androgen R1881 (0.1 nM). In addition, estrogens, progestagens and several antiandrogens do not inhibit androgen responsive growth, but have striking growth stimulatory effects and increase EGF receptor level and acid phosphatase secretion. We have found that the androgen receptor in the LNCaP cells contains a single point mutation changing the sense of codon 868 (Thr to Ala) in the ligand binding domain. Expression vectors containing the normal or mutated androgen receptor sequence were transfected into COS or HeLa cells. Androgens, progestagens, estrogens and several antiandrogens bind the mutated androgen receptor protein and activate the expression of an androgen-regulated reporter gene (GRE-tk-CAT), indicating that the mutation directly affects both binding specificity and the induction of gene expression. Interestingly, the antiandrogen casodex showed antiandrogenic properties in growth studies of LNCaP cells and did not induce reporter gene activity in Hela cells transfected with the mutant receptor. The mutated androgen receptor of LNCaP cells is therefore a useful tool in the elucidation of different levels of action of steroids and antisteroids.

Regulation of growth of LNCaP human prostate tumor cells by growth factors and steroid hormones.

The mitogenic activity of several growth factors on androgen responsive LNCaP human prostate tumor cells was studied. A two-fold stimulation of cell proliferation was observed after a culture period of 6 days in 1 ng EGF/ml, 10 ng TGF-alpha/ml or 20 ng basic FGF/ml. TGF-beta (0.02 ng/ml), which did not affect cell proliferation when added alone to the culture medium, inhibited the EGF- and TGF-alpha-induced growth. The synthetic androgen R1881 (0.1 nM) stimulated cell proliferation three-fold and increased the number of EGF receptors from 11500 to 28500 sites/cell. One of the mechanisms involved in androgen action on these cells is therefore an increased EGF receptor expression and increased sensitivity to EGF. TGF-beta did not directly affect androgen-responsive growth but inhibited the synergistic effect of EGF. A considerable expression of TGF alpha (precursors) could be demonstrated on the cells by immunohistochemical staining. However the staining intensity was not affected by androgens. These results make it less likely that androgen-responsive growth is mediated by regulation of secretion of an EGF- or TGF alpha-like activity, which in turn acts in an autocrine manner to stimulate growth. Estrogens, progestagens and antiandrogens do not inhibit androgen responsive growth of LNCaP cells but have striking growth stimulatory effects, increase EGF receptor level and increase acid phosphatase secretion. LNCaP cells contain a modified androgen receptor system with respect to both steroid specificity and antiandrogen sensitivity. It has recently been shown that the stimulatory effects are due to a mutated amino acid in the steroid binding domain of the androgen receptor.

A mutation in the ligand binding domain of the androgen receptor of human LNCaP cells affects steroid binding characteristics and response to anti-androgens.

LNCaP prostate tumor cells contain an abnormal androgen receptor system. Progestagens, estradiol and anti-androgens can compete with androgens for binding to the androgen receptor and can stimulate both cell growth and excretion of prostate specific acid phosphatase. We have discovered in the LNCaP androgen receptor a single point mutation changing the sense of codon 868 (Thr to Ala) in the ligand binding domain. Expression vectors containing the normal or mutated androgen receptor sequence were transfected into COS or Hela cells. Androgens, progestagens, estrogens and anti-androgens bind the mutated androgen receptor protein and activate the expression of an androgen-regulated reporter gene construct (GRE-tk-CAT). The mutation therefore influences both binding and the induction of gene expression by different steroids and antisteroids.

Stimulatory effects of antiandrogens on LNCaP human prostate tumor cell growth, EGF-receptor level and acid phosphatase secretion.

LNCaP cells (derived from a lymph node carcinoma of the human prostate) show androgen responsive growth. Progestagens, estradiol and antiandrogens competed with androgens for binding to the androgen receptor in the cells to a higher extent than in other androgen-sensitive systems. Optimal growth (3-4 fold increase in DNA content of 6 day cell cultures vs controls) was observed after addition of the synthetic androgen R1881 (0.1 nM). Both steroidal and non-steroidal antiandrogens did not suppress the androgen responsive growth. At a concentration of 10 nM cyproterone acetate or 100 nM RU 23908, growth was even stimulated to an extent comparable to that observed after addition of androgen. Cyproterone acetate and RU 23908 also increased the number of epidermal growth factor receptors expressed at the cell surface to a comparable level as did the androgen. Like androgens, cyproterone acetate, RU 23908 or estradiol stimulated the secretion per cell of prostate specific acid phosphatase in the culture fluid. In conclusion, antiandrogens can exert striking stimulatory effects on the proliferation of LNCaP cells probably due to a defective androgen receptor system. It is discussed that comparable changes in the specificity of the androgen receptor in prostate cancer cells may give these cells an advantage in growth rate and may contribute to development of tumors characterized as hormone independent.

Unusual specificity of the androgen receptor in the human prostate tumor cell line LNCaP: high affinity for progestagenic and estrogenic steroids.

UNLABELLED: LNCaP tumor cells, derived from a metastatic lesion of a human prostatic carcinoma, are androgen-sensitive in cell culture. Although increase in growth rate is observed with low doses of progestagens or estradiol, these cells contain exclusively androgen receptors. In the present study the binding affinity of different ligands for both non-DNA- and DNA-binding (transformed) forms of the androgen receptor were analyzed. The cytosolic (non-transformed) form of the receptor displayed an abnormal high affinity for progestagens and estradiol when compared with the cytosolic androgen receptor from other sources. Subsequently the non-transformed forms of the androgen receptor obtained from LNCaP cell nuclei was studied. A high binding affinity was found not only for dihydrotestosterone, but also for progesterone and the synthetic progestagen R5020 (relative binding affinity 42% and 10% of dihydrotestosterone). The binding characteristics of the transformed androgen receptor were examined in intact cells at 37 degrees C. LNCaP cells were compared in this respect with COS cells containing the cloned human androgen receptor, normal human skin fibroblasts and PC3 (prostate) and NHIK (cervix) human tumor cell lines. The affinity of the transformed androgen receptors for the progestagen R5020 in LNCaP cells was significantly higher than in the other cell systems, although the differences were less pronounced than for the non-transformed receptor form. IN CONCLUSION: the LNCaP tumor cells contain an androgen receptor with an abnormal binding site. This might be due to a mutation and/or a post-transcriptional effect.

The release of iron by Sertoli cells in culture.

In seminiferous tubules, iron transport from the blood to the abluminal germinal cells must occur through the Sertoli cell cytoplasm. We investigated the release of previously accumulated iron by cultured Sertoli cells. We found that Sertoli cells contain easily releasable and less easily releasable iron pools. Iron is released in a low molecular weight form (molecular weight less than 30,000). A high concentration of this low molecular weight iron in the medium reduces further iron release by Sertoli cells, whereas the addition of more medium or fresh medium increases further iron release. Apotransferrin stimulates the release of iron in a dose-dependent manner by chelating the low molecular weight iron. Rat and human apotransferrin are completely competitive in this respect. Diethylenetriamine penta acetic acid (DTPA), an extracellular iron chelator, and apotransferrin compete for iron binding and stimulation of iron release, indicating that no binding or uptake of the chelator by the cells is required. Desferrioxamine (DFO), an intracellular iron chelator, on the other hand, increases iron release more drastically, and apotransferrin cannot compete with it for iron. The addition of extracellular iron also increases the amount of 59Fe in the medium, probably by reducing the re-uptake of 59Fe. This is also demonstrated with primaquine, which blocks endocytosis and increases the amount of 59Fe in the medium. The presence of germinal cells also stimulates the release of iron by Sertoli cells. When cocultured, the germinal cells internalize iron as it is release by Sertoli cells.