Development and characterization of a tamoxifen-resistant breast carcinoma xenograft

A human tamoxifen-resistant mammary carcinoma, MaCa 3366/TAM, originating from a sensitive parental xenograft 3366 was successfully established by treatment of tumour-bearing nude mice with 1-50 mg kg(-1) tamoxifen for 3 years during routine passaging. Both tumours did not differ significantly in OR- and PR-positivity, however, when compared with the sensitive tumour line, the mean OR content of the TAM-resistant subline is slightly lower. An OR-upregulation following withdrawal of oestradiol treatment was observed in the parental tumours but not in the resistant xenografts. Following long-term treatment with tamoxifen, the histological pattern of the breast carcinoma changed. The more differentiated structures being apparent after treatment with 17beta-oestradiol in the original 3366 tumour were not induced in the resistant line. Tamoxifen failed to induce a tumour growth inhibition in comparison to the tamoxifen-sensitive line. The pure anti-oestrogen, ICI 182 780, revealed cross-resistance. Sequence analysis of the hormone-binding domain of the OR of both lines showed no differences, suggesting that either mutations in other regions of the OR are involved in the TAM-resistance phenotype or that mechanisms outside of this protein induced this phenotype. Oestrogen and anti-oestrogen regulate pS2 and cathepsin D expression in 3366 tumours as in the human breast cancer cell line MCF-7. The resistant 3366/TAM tumours have lost this regulation. The established breast cancer xenografts 3366 and 3366/TAM offer the possibility of investigating mechanisms of anti-oestrogen resistance in an in vivo situation. They can be used to test novel approaches to prevent, or to overcome, this resistance in a clinically related manner.     

The importance of tamoxifen metabolism in tamoxifen-stimulated breast tumor growth

The acquired ability of tamoxifen to stimulate tumor growth has been suggested as one mechanism for the development of treatment failure in breast cancer. We have reported that tamoxifen-stimulated MCF-7 breast tumors in nude mice display reduced tamoxifen levels as compared with tamoxifen-inhibited tumors and an altered metabolite profile with isomerization oftrans-4-hydroxytamoxifen to a weak antiestrogen and the production of metabolite E, an estrogenic metabolite. To investigate further the importance of tamoxifen metabolism in this model, we quantified levels of tamoxifen and major metabolites in tamoxifen-stimulated as compared with tamoxifen-inhibited MCF-7 tumors growing in nude mice and employed tamoxifen analogs resistant to metabolism. Tamoxifen-stimulated tumors have a relative abundance ofcis-4-hydroxytamoxifen and metabolite E. However, in vivo treatment of mice carrying tamoxifen-stimulated tumors with fixed-ring nonisomerizable tamoxifen analogs or with nafoxidine, a nonsteroidal antiestrogen with a different structure, nonetheless resulted in tumor growth stimulation. Tumors were also stimulated by a deoxytamoxifen analog resistant to conversion to metabolite E. Growth of tamoxifen-stimulated tumors was inhibited by a pure steroidal antiestrogen, ICI 182, 780, suggesting the need for clinical trials of this drug in patients with tamoxifen resistance. Growth of tamoxifen-stimulated tumors was further stimulated by estrogen replenishment, and this estrogen stimulation could be blocked by tamoxifen indicating that tamoxifen has both agonist and antagonist properties in these tumors. This study suggests that tamoxifen-stimulated tumor growth in this model is not due to isomerization or metabolism of tamoxifen to less antiestrogenic or more estrogenic metabolities. The mechanisms by which tamoxifen acquires more potent in vivo agonist properties, resulting in tumor growth stimulation over time, remain to be defined.This work was supported in part by Cancer Center Support grants P30 CA 54174, P50 CA58183, and RO1 CA 30251 from the National Cancer Institute     

Measuring IGF-1, ER-α and EGFR expression can predict tamoxifen-resistance in ER-positive breast cancer

In vitro studies have suggested that tamoxifen resistance may be due to altered expression and downstream signalling of insulin-like growth factor-1 (IGF-1) receptor (IGF-1l), oestrogen receptor-alpha (ERα), epidermal growth factor receptor (EGFR) and HER-2. We investigated which gene expressions could predict tamoxifen resistant breast cancer. Expression of IGF-1R, IGF-1 ligand (IGF-1), ERα, EGFR and HER-2 in 91 ER-positive breast cancer tumours were measured using real-time PCR and correlated with clinical outcome. The tamoxifen resistant group (n=20) consisted of: i) tumours which were resistant to neoadjuvant tamoxifen treatment and ii) tumours which were excised from patients who later developed recurrence or metastasis during adjuvant tamoxifen treatment. These were compared with tamoxifen sensitive tumours which were surgical excision specimens from patients who did not develop recurrence/metastasis during adjuvant tamoxifen treatment. Tumours with higher IGF-1 ligand and ERα expression took longer to develop tamoxifen resistance. Tamoxifen resistant tumours had lower IGF-1 and ERα expression compared to tamoxifen-sensitive tumours. IGF-1 expression strongly correlated with ERα expression in the tamoxifen sensitive group only. ERα inversely correlated with EGFR expression in the tamoxifen resistant group only. We conclude that IGF-1 ligand and ERα expression in breast carcinomas can be measured to predict tamoxifen resistance. Measuring ERα expression using RT-PCR may be more sensitive than immunohistochemistry in determining anti-oestrogen sensitivity.     

Changes in the expression and binding properties of the estrogen receptor in MCF-7 breast cancer cells during growth inhibition by tamoxifen and cisplatin

Most mammary carcinomas contain estrogen receptors (ER), which are an important factor in diagnosis and prognosis, and in deciding on the type of therapy. ER-positive tumors are most commonly treated with the antiestrogen tamoxifen or with a combination of chemotherapeutic drugs. An important aspect for further treatment and anticipating possible side effects is the fate of the ER during the course of therapy. To study the effect of drug-induced growth inhibition on ER expression and binding properties. human breast cancer MCF-7 cells were treated with tamoxifen and cisplatin. and also estradiol (E2) for 5 days. Following this incubation, intact cells were incubated with [3H]E2 to determine the dissociation constant (KD) and maximal number of binding sites (Bmax) of the ER. The amount of ER protein per cell was quantified using anti-ER antibodies. For analysis of ER mRNA, total cellular RNA was subjected to Northern blotting. The 5-day treatment with E2 reduced Bmax and the amount of ER protein by about 70%, while the cellular level of ER mRNA was reduced by 40%. Treatment with E2 did not affect the subsequent growth inhibitory response to tamoxifen or cisplatin. In contrast, tamoxifen reduced the capacity for E2 binding; it caused about a 30-fold increase in the KD value. At the same time, Bmax and ER protein content were increased (about 3.5- and 2-fold, respectively), but the cellular level of ER mRNA was again reduced by 40%. The growth of tamoxifen-treated cells remained sensitive to subsequent treatment with estradiol, tamoxifen or cisplatin. Treatment of MCF-7 cells with cisplatin likewise reduced E2 binding due to a 20-fold increase in KD value. In this case, both Bmax and the amount of ER protein were decreased when calculated per milligram of protein, but were increased on a cellular basis due to an increase in cell size. The ER mRNA content was not altered in cisplatin-treated cells. Growth of these cells also remained sensitive to tamoxifen and cisplatin. In conclusion, drug-induced changes in ER expression and binding capacity do not necessarily indicate a loss of sensitivity of breast cancer cells to a subsequent chemotherapeutic treatment.     

Changes in Radiation Sensitivity and Steroid Receptor Content Induced by Hormonal Agents and Ionizing Radiation in Breast Cancer Cells in Vitro

Possible influences of tamoxifen and estradiol on in vitro radiation sensitivity and cellular receptor content after irradiation and/or tamoxifen treatment were studied in breast cancer cell lines; estrogen receptor (ER) and progesterone receptor (PgR) positive cell lines MCF-7 and MCF-7/TAM^R-1 and the ER and PgR negative cell line MDA-MB-231. The tamoxifen resistant MCF-7/TAM^R-1 cells were more resistant to ionizing radiation than the MCF-7 and MDA-MB-231 cells. Exposure to tamoxifen made the MCF-7 cells more radiation resistant, while estradiol made the MDA-MB-231 cells more radiation sensitive. A radiation dose of 6 Gy reduced the ER content in cytosol in both MCF-7 and MCF-7/TAM^R-1 cells, but brought no alterations to the PgR content. In MCF-7/TAM^R-1 cells tamoxifen exposure significantly increased the ER and reduced the PgR content, an effect not observed in the MCF-7 cells. To conclude, the present study indicates that irradiation and tamoxifen may modify the ER and PgR content in cytosol in breast cancer cells. Hormonal treatment may alter the radiation sensitivity, even in ER negative cells, suggesting that hormonal agents may act both via receptor and non-receptor binding mechanisms.     

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.     

Tamoxifen resistance in breast tumors is driven by growth factor receptor signaling with repression of classic estrogen receptor genomic function

Not all breast cancers respond to tamoxifen, and many develop resistance despite initial benefit. We used an in vivo model of estrogen receptor (ER)-positive breast cancer (MCF-7 xenografts) to investigate mechanisms of this resistance and develop strategies to circumvent it. Epidermal growth factor receptor (EGFR) and HER2, which were barely detected in control estrogen-treated tumors, increased slightly with tamoxifen and were markedly increased when tumors became resistant. Gefitinib, which inhibits EGFR/HER2, improved the antitumor effect of tamoxifen and delayed acquired resistance, but had no effect on estrogen-stimulated growth. Phosphorylated levels of p42/44 and p38 mitogen-activated protein kinases (both downstream of EGFR/HER2) were increased in the tamoxifen-resistant tumors and were suppressed by gefitinib. There was no apparent increase in phosphorylated AKT (also downstream of EGFR/HER2) in resistant tumors, but it was nonetheless suppressed by gefitinib. Phosphorylated insulin-like growth factor-IR (IGF-IR), which can interact with both EGFR and membrane ER, was elevated in the tamoxifen-resistant tumors compared with the sensitive group. However, ER-regulated gene products, including total IGF-IR itself and progesterone receptor, remained suppressed even at the time of acquired resistance. Tamoxifen's antagonism of classic ER genomic function was retained in these resistant tumors and even in tumors that overexpress HER2 (MCF-7 HER2/18) and are de novo tamoxifen-resistant. In conclusion, EGFR/HER2 may mediate tamoxifen resistance in ER-positive breast cancer despite continued suppression of ER genomic function by tamoxifen. IGF-IR expression remains dependent on ER but is activated in the tamoxifen-resistant tumors. This study provides a rationale to combine HER inhibitors with tamoxifen in clinical studies, even in tumors that do not initially overexpress EGFR/HER2.     

The mTOR pathway inhibitor RAD001 (everolimus) is highly efficacious in tamoxifen-sensitive and -resistant breast cancer xenografts

The rapamycin derivative RAD001 (everolimus) is presently in clinical trials. Preclinical studies have suggested preferential activity in antiestrogen resistant breast cancer cells. We investigated the response of breast cancer xenografts with different tamoxifen (TAM) sensitivity towards RAD001 and analyzed the regulatory machinery as well as the cross-talk between different signaling pathways. The ERα-positive and TAM-sensitive patient-derived breast carcinoma model 3366, its TAM-resistant counterpart 3366/TAM and 4049, a breast cancer with inherent TAM-resistance, were transplanted to immunodeficient nude mice and treated with RAD001 or TAM or the combination of both compounds. Shock frozen tumors were prepared for Western Blot and immunohistochemical analysis to semi-quantitatively evaluate the expression of the ERα and the ERα regulated IGF-IR as well as PTEN, pAkt, mTOR, (phospho)-p70^S6K, (phospho)-4E-BP1 and cyclin D1. RAD001 significantly inhibited the growth of tamoxifen responding and non-responding xenografts. The highest efficacy was found for the combined treatment with TAM and RAD001. RAD001 modified the protein expression of mTOR and its downstream molecule 4E-BP1 as well as the level of PTEN and ERα, but independent of the tumors sensitivity towards TAM. The protein kinase Akt was found in the active phosphorylated form (pAkt) only in TAM-resistant xenografts, but not detectable in the TAM-responding 3366 line. All treatment modalities down-regulated pAkt expression in the TAM-resistant tumors. p70^S6K and IGF-IR proteins were not significantly influenced by RAD001 treatment. Our findings document the linkage between different growth-controlling pathways. Due to its capability to be active in a TAM-resistant in vivo model, RAD001 could potentially serve as a promising second-line therapy in breast cancer.     

Effects of tamoxifen and interferon-β or the combination on tumor-induced angiogenesis

Inhibition of angiogenesis by anti-tumor agents may play a role in tumor growth arrest. Tamoxifen and interferon-α/β (IFN-α/β) exhibit potentiated anti-proliferative activity against tumor cells. However, additional host-mediated effects such as modulation of angiogenesis may also inhibit tumor growth in vivo. The effect of tamoxifen and IFN-β on angiogenesis induced by 2 human tumors, MCF-7 breast carcinoma (estradiol dependent) and NIH-OVCAR-3 ovarian carcinoma (estradiol independent), was assessed. Treatment of nude mice bearing MCF-7 tumors with tamoxifen resulted in a 68% decrease in the number of vessels at the tumor periphery. Treatment with IFN-β yielded a 33% reduction. Treatment of nude mice bearing NIH-OVCAR-3 tumors with tamoxifen resulted in a 73% decrease in the number of vessels. Treatment with IFN-β yielded a 57% reduction. Combination treatment resulted in augmented anti-angiogenic effects. As single agents, both tamoxifen and IFN-β inhibited xenograft tumor growth. Ten weeks of tamoxifen treatment resulted in growth inhibition of MCF-7 and NIH-OVCAR-3 carcinomas by 85% and 66%, respectively. Ten weeks of IFN-β treatment resulted in inhibition of growth of MCF-7 and NIH-OVCAR-3 carcinomas by 67% and 88%, respectively. The combination of tamoxifen and IFN-β completely prevented growth of MCF-7 and NIH-OVCAR-3 carcinomas. The anti-angiogenic effects of tamoxifen and IFN-β were additive. Inhibition of angiogenesis was detectable before measurable effects on tumor volume in both MCF-7 and NIH-OVCAR-3 tumors. Potentiation of anti-angiogenic effects by tamoxifen and IFN-β, possibly resulting from enhanced IFN-induced gene expression, may contribute to anti-tumor activity in both estradiol-dependent and estradiol-independent tumors in vivo. Int. J. Cancer 71:456-461, 1997. © 1997 Wiley-Liss Inc.     

Identification of estrogenic tamoxifen metabolite(s) in tamoxifen-resistant human breast tumors.

PURPOSE: We have shown previously that acquired tamoxifen resistance in an in vivo experimental model is associated with reduced tamoxifen accumulation, isomerization of trans-4-hydroxytamoxifen, and tamoxifen-stimulated tumor growth. The purpose of this study is to isolate and verify the presence of estrogenic tamoxifen metabolites in human breast tumors using high-performance liquid chromatography (HPLC) and mass-spectrometry (MS) techniques. PATIENTS AND METHODS: In the present study, we used HPLC and MS to identify the presence of estrogenic metabolites in tumor samples excised from athymic nude mice and in human breast tumors isolated from patients receiving adjuvant tamoxifen therapy. RESULTS: We identified the presence of metabolite E, a known estrogenic metabolite of tamoxifen, in tamoxifen-resistant MCF-7 human breast tumors implanted in athymic nude mice, as well as in tumors from patients with clinical resistance. Additionally, we separated another estrogenic metabolite, bisphenol, by HPLC, and this was also tentatively confirmed by MS analysis. CONCLUSION: These data suggest that cellular tamoxifen metabolism to estrogenic metabolites may in part contribute to stimulating the growth of hormone-responsive breast tumors following prolonged exposure to tamoxifen. Further evaluation of the relationship between cellular metabolism and acquired tamoxifen resistance is warranted.     

PC cell-derived growth factor mediates tamoxifen resistance and promotes tumor growth of human breast cancer cells

PC cell-derived growth factor, also known as progranulin, is an M(r) 88,000 growth factor (referred as PCDGF/GP88) overexpressed in human breast cancer. Antisense inhibition of PCDGF/GP88 expression in MDA-MB-468 cells inhibited tumor formation in nude mice. In estrogen receptor-positive cells, PCDGF/GP88 was expressed in response to estradiol and shown to mediate its mitogenic effect. Pathologic studies indicated that PCDGF/GP88 was expressed in 80% of invasive ductal carcinomas in correlation with parameters of poor prognosis. In the present article, the relationship between PCDGF/GP88 expression and tamoxifen resistance was examined in MCF-7 cells. PCDGF/GP88 overexpression rendered MCF-7 cells able to proliferate in the absence of estrogen and in the presence of tamoxifen. The PCDGF/GP88-overexpressing cells formed tumors in ovariectomized nude mice in the absence of estradiol and in its presence, in contrast to MCF-7 cells. Tumor growth of the overexpressing cells was increased significantly when the mice were treated with tamoxifen. PCDGF/GP88 blocked tamoxifen-induced apoptosis by preventing down-regulation of bcl-2 expression and poly(ADP-ribose) polymerase cleavage. In addition, PCDGF/GP88-overexpressing cells presented higher level of the angiogenic factors vascular endothelial growth factor and angiopoietin-1 than MCF-7 control cells. Tamoxifen treatment additionally increased the level of vascular endothelial growth factor. These studies suggest that PCDGF/GP88 plays a critical role in breast cancer tumorigenesis and in the transition to estrogen independence and tamoxifen resistance, a hallmark of poor prognosis. On the basis of the in vivo studies, it is postulated that tamoxifen treatment of patients with estrogen receptor-positive breast tumors overexpressing PCDGF/GP88 could have adverse clinical consequences.     

A new antiestrogen, 2-(4-hydroxy-phenyl)-3-methyl-1-[4-(2-piperidin-1-yl-ethoxy)-benzyl]-1H-indol-5-ol hydrochloride (ERA-923), inhibits the growth of tamoxifen-sensitive and -resistant tumors and is devoid of uterotropic effects in mice and rats.

PURPOSE: Tamoxifen is an antiestrogen used in women who have estrogen receptor (ER)-alpha-positive breast cancer. Unfortunately, resistance to tamoxifen is common in women with metastatic disease and side effects, including increased risk of endometrial cancer, exist. Here we describe the activity of a new selective ER modulator, ERA-923, in preclinical models focused on these limitations. EXPERIMENTAL DESIGN: The ability of ERA-923, 4-OH tamoxifen, or raloxifene to inhibit estrogen-stimulated growth was evaluated in cell-based and xenograft assays with tumor cells that are sensitive or resistant to tamoxifen. Uterine effects of selective ER modulators were compared in rodents. RESULTS: ERA-923 potently inhibits estrogen binding to ER-alpha (IC(50), 14 nM). In ER-alpha-positive human MCF-7 breast carcinoma cells, ERA-923 inhibits estrogen-stimulated growth (IC(50), 0.2 nM) associated with cytostasis. In vitro, a MCF-7 variant with inherent resistance to tamoxifen (10-fold) or 4-OH tamoxifen (>1000-fold) retains complete sensitivity to ERA-923. Partial sensitivity to ERA-923 exists in MCF-7 variants that have acquired profound tamoxifen resistance. In tumor-bearing animals, ERA-923 (10 mg/kg/day given p.o.) inhibits 17beta-estradiol-stimulated growth in human tumors derived from MCF-7, EnCa-101 endometrial, or BG-1 ovarian carcinoma cells, including a MCF-7-variant that is inherently resistant to tamoxifen. Raloxifene is inactive in the MCF-7 xenograft model. Unlike tamoxifen, droloxifene, or raloxifene, ERA-923 is not uterotropic in immature rats or ovariectomized mice. Consistent with this, tamoxifen, but not ERA-923, stimulates the growth of EnCa-101 tumors. CONCLUSIONS: In preclinical models, ERA-923 has an improved efficacy and safety compared with tamoxifen. Clinical trials with ERA-923 are in progress.     

Acquired tamoxifen resistance: correlation with reduced breast tumor levels of tamoxifen and isomerization of trans-4-hydroxytamoxifen

Acquired tamoxifen resistance represents a major cause of treatment failure in breast cancer. We implanted estrogen receptor-positive MCF-7 human breast cancer cells in athymic nude BALB/c mice as a model to study in vivo acquired tamoxifen resistance. After 4-6 months of tumor growth suppression by trans-tamoxifen, tumor progression was observed despite continued tamoxifen administration. Acquired resistance was not due to loss of estrogen receptors, to alterations in serum or tumor estrogen levels, or to changes in tamoxifen or its major metabolites in serum. Tamoxifen-resistant tumors remained estrogen dependent in vivo. However, resistance was also associated with the ability of tamoxifen to stimulate tumor growth. Resistant tumors were characterized by markedly lower intracellular tamoxifen levels and by isomerization of the potent antiestrogenic metabolite trans-4-hydroxy-tamoxifen to the less potent cis isomer. Metabolic tolerance, as manifested by alterations in cellular concentrations of tamoxifen and its metabolites, may thus be one mechanism for acquired tamoxifen resistance in breast cancer.     

Tamoxifen and the isomers of 4-hydroxytamoxifen in tamoxifen-resistant tumors from breast cancer patients.

PURPOSE: The antiestrogen tamoxifen is effective in therapy for breast cancer. However, its use is limited by the eventual development of acquired tamoxifen resistance in many patients. The mechanisms responsible for tamoxifen resistance remain unknown; loss of estrogen receptor (ER), selection of hormone-independent breast cancer clones, or alterations in serum tamoxifen levels after long-term use do not explain acquired resistance in most patients. Using an experimental model in which human breast cancer cells develop resistance in athymic mice treated with tamoxifen, we have recently shown that acquired resistance is associated with markedly reduced cellular concentrations of tamoxifen and by isomerization of the trans-4-hydroxy metabolite to the less potent cis isomer. MATERIALS AND METHODS: Using a sensitive high-performance liquid chromatography (HPLC) assay, we have now measured levels of tamoxifen and its major metabolites in a series of 14 tumors from patients treated with tamoxifen. The duration of therapy ranged from 1 month to 6 years. RESULTS: Tumor tamoxifen levels varied over a wide range. Low concentrations were observed in tumors from eight patients, all demonstrating progressive disease at the time of biopsy after a minimum duration of treatment of 6 months. Six tumors had moderate to high tamoxifen levels, two from patients responding to tamoxifen, one from a patient with stable disease, and three from patients with disease progression. Both the cis and trans isomers of the potent antiestrogenic metabolite 4-hydroxy-tamoxifen were detected in 11 tumors. Six tumors had high ratios of the cis to trans isomer (1.10:2.06), all from patients not responding to tamoxifen. The five tumors with low cis:trans ratios included the two tumors from responding patients and three from patients with progression. All but one of the 11 nonresponding patients had either a low tumor tamoxifen level, a high cis:trans ratio, or both. CONCLUSION: This study clearly demonstrates a wide range of tumor tamoxifen levels and accumulation of the less antiestrogenic cis isomer of 4-hydroxytamoxifen in some patients on tamoxifen therapy. Additional study is necessary to determine if these metabolic profiles are related to the development of tamoxifen resistance.     

Clinical impact of assay of estrogen receptor beta cx in breast cancer

Since 1996 when estrogen receptor beta(ER beta) was discovered, much effort has been devoted to the question of the value of ER beta as a prognostic and/or predictive factor in breast cancer and its potential as a novel target for pharmacological intervention. When estrogen receptors are applied on sucrose gradients and quantified by ligand binding, we found that in contrast to ER alpha, which has a narrow tissue distribution, ER beta is expressed in many tissues including both normal and malignant breast tissue. Receptor protein levels in tissues can also be measured from the intensities of bands after Western blotting and can be quantified when purified and quantified receptor is used as a standard. With this technique, we found that there were some tumors which had over 600 fmol/mg of ER beta protein but no detectable estradiol binding. In such tumors, RT-PCR analysis revealed that ER beta cx is the only ER beta isoform present. ER beta cx is a splice variant which utilizes an alternative exon 8. This change in the C-terminus results in very poor binding to estradiol (E2) and has a dominant negative effect on ER alpha function. Immunohistochemical analysis with an ER beta cx specific antibody in 115 ER alpha-positive breast cancers revealed that about half of the samples expressed ER beta cx protein. Initial analysis of samples from patients with preoperative tamoxifen treatment revealed that ER alpha-positive tumors expressing ER beta cx and lacking PR seemed to be resistant to the anti-estrogen. We conclude that, in order to better characterize breast cancers and design appropriate therapy for individual patients, assays for ER beta cx must be made available to clinicians.     

Effect on growth and cell cycle kinetics of estradiol and tamoxifen on MCF-7 human breast cancer cells grown in vitro and in nude mice

The effects of estradiol and tamoxifen (TAM) on the estrogen-dependent human breast cancer cell line MCF-7 grown in vitro and in nude mice were compared. The effect on growth was determined by cell number in vitro and by tumor growth curves in nude mice. The effects on the cell cycle kinetics were determined by repeated flow cytometric DNA analyses in vitro and in vivo and by the technique of labeled mitosis in nude mouse-grown tumors. Under in vitro conditions, estradiol induced a pronounced increase in S-phase fraction and cell number. TAM inhibited growth of MCF-7 cells with a concomitant increase in the G1 phase from 60% to 75%. In nude mice, MCF-7 only formed tumors in estradiol-supplemented mice. No differences were observed in growth and cell kinetics between 0.1 and 1.0 mg of estradiol. Daily i.p. injections of TAM resulted in tumor growth inhibition with shrinkage of tumors. The flow cytometric DNA analysis and percentage of labeled mitosis investigations revealed no significant differences in the proliferation kinetics of TAM-treated and control tumors. Calculating the cell loss factor demonstrated an increase from 69% in control tumors to 107% in TAM-treated tumors. These experiments have shown that the cell kinetic effect of TAM is different when MCF-7 cells are grown in vitro versus in vivo. In contrast to the in vitro data, the in vivo data indicate that the growth-inhibitory effect of TAM is not mediated through a perturbation of the cell cycle.     

Decreased secretion of Cathepsin D in breast cancer in vivo by tamoxifen: Mediated by the mannose-6-phosphate/IGF-II receptor?

The lysosomal protease Cathepsin D (Cath D) is associated with increased invasiveness and metastasis in breast cancer. Both estrogen and tamoxifen have been reported to increase Cath D, which seems to contradict the efficacy of tamoxifen as an adjuvant for estrogen dependent breast cancer. Cath D is bioactive in the extracellular space but very little is known about hormonal regulation of secreted Cath D in vivo. In this study we used microdialysis to sample the extracellular fluid in estrogen receptor positive MCF-7 tumors in nude mice. We show that tamoxifen in combination with estradiol decreased secreted Cath D compared with estradiol treatment only in solid tumors in situ. Cell culture of MCF-7 cells revealed that estradiol and tamoxifen increased intracellular proteolytic activity of Cath D in a similar fashion whereas secretion of Cath D was increased by estradiol and inhibited by tamoxifen. Immunofluorescence showed that estradiol located Cath D to the cell surface, while tamoxifen accumulated Cath D to dense lysosomes in perinuclear regions. Moreover, tamoxifen increased the intracellular transporter of Cath D, the mannose 6-phosphate/IGF-II receptor (M6P/IGF2R). In contrast, estradiol decreased the levels of this receptor. Thus, secretion of Cath D is hormone dependent and may be mediated by altered expression of the M6P/IGF2R. Our results highlight the importance of measurements of proteins in all compartments where they are biological active and show that microdialysis is a viable technique for sampling of Cath D in vivo.     

Intratumoral aromatase model: The effects of letrozole (CGS 20267)

An intratumoral aromatase model in the ovariectomized nude mouse was developed which simulated the hormone responsive postmenopausal breast cancer patient. MCF-7, human breast cancer cells transfected with the aromatase gene, inoculated into ovariectomized nude mice are able to synthesize sufficient estrogens to enhance cell proliferation and the development of tumors. These tumors are responsive to both antiestrogens and aromatase inhibitors. However, letrozole was found to be more effective than tamoxifen and caused tumor regression, a result not previously noted in nude mice with endocrine treatments. When the aromatase inhibitors were combined with tamoxifen, tumor growth was suppressed to about the same extent as treatment with the aromatase inhibitors alone. Thus, there was no additive or synergistic effects of combining tamoxifen with aromatase inhibitors. These results suggest that letrozole has the potential to be more effective than tamoxifen for achieving greater reduction in estrogenic effects on tumors and uterus in postmenopausal breast cancer patients. In addition, sequential treatment with these agents is likely to be more beneficial to the patient in terms of longer response to treatment.     

Increased endostatin generation and decreased angiogenesis via MMP-9 by tamoxifen in hormone dependent ovarian cancer

There are several similarities between breast and ovarian cancer but anti-estrogen treatment is rarely used in ovarian cancer. We have previously shown that the most widely used anti-estrogen tamoxifen increased MMP-9 activity and endostatin generation in breast cancer. Here, we show that tamoxifen exposure of highly hormone responsive ovarian cancer cells decreased proliferation, and increased MMP-9 activity leading to increased levels of endostatin both in cell culture in vitro and in solid tumors of nude mice. Tamoxifen exposed tumors also exhibited significantly decreased tumor growth and vascularisation. Moreover, in ascites from ovarian cancer patients, MMP-9 was undetectable in majority of cases but a significant correlation of MMP-2 and endostatin was found. The effects on MMPs and endostatin generation are previously unknown mechanisms of estradiol and tamoxifen in ovarian cancer, which may have therapeutic implications in future anti-cancer options of hormone dependent ovarian cancer.     

人结肠癌裸鼠移植瘤免疫组织化学与生物学特性初步观察

目的为实验药理学开展内分泌治疗建立人结肠癌裸鼠移植瘤。方法将雌孕激素受体阳性人结肠癌移植于裸鼠并传５代，连续观察各代肿瘤病理特征、免疫组织化学、细胞动力学、ＤＮＡ倍体及受体表达规律，并与原人体结肠癌比较。部分移植瘤进行超微结构透射电镜观察。结果人结肠癌裸鼠异种移植建株成功，保持原人体结肠癌相同高分化管状腺癌，持续阳性雌孕激素受体表达（生化ＤＣＣ法），稳定细胞动力学特征与ＤＮＡ倍体（异倍体），原代和第１、３、５代荷瘤分别显示出大肠癌最具有特征的超微结构如微绒毛和小根等，特异性大肠癌标记物癌胚抗原与花生凝集素受体表达阳性。结论性激素受体作用失常参与大肠癌发病，为内分泌治疗提供基础。