Species-specific pharmacology of antiestrogens: role of metabolism

The nonsteroidal antiestrogen tamoxifen exhibits a paradoxical species-specific pharmacology. The drug is a full estrogen in the mouse, a partial estrogen/antiestrogen in humans and the rat, and an antiestrogen in the chick oviduct. Inasmuch as tamoxifen has antiestrogenic effects in vitro, differential metabolism of tamoxifen to estrogens might occur in the species in which it has an estrogenic pharmacology. Tamoxifen or its metabolite 4-hydroxytamoxifen could lose the alkylaminoethane side chain to form the estrogenic compound metabolite E or bisphenol. Sensitive metabolic studies with [3H]tamoxifen in chicks, rats, and mice identified 4-hydroxytamoxifen as the major metabolite, but no potentially estrogenic metabolites were observed. Athymic mice with transplanted human breast tumors can be used to study the ability of tamoxifen to stimulate target tissue or tumor growth. Estradiol caused the growth of transplanted MCF-7 breast cancer cells into solid tumors and a uterotrophic response. However, tamoxifen does not support tumor growth when administered alone, although it stimulates uterine growth. Since a similar profile of metabolites is sequestered in human and mouse tissues, these studies strongly support the concept that the drug can selectively stimulate or inhibit events in the target tissues of different species without metabolic intervention.     

Estrogen induces death of tamoxifen-resistant MCF-7 cells: contrasting effect of the estrogen receptor downregulator fulvestrant

A common problem in breast cancer therapy is resistance to the antiestrogen tamoxifen. However, tamoxifen-resistant breast tumors can still respond to other hormonal therapies. In animal models of tamoxifen-resistant breast cancer cells, physiological levels of estrogen can induce tumor regression. Recently, the estrogen receptor downregulator fulvestrant was shown to promote tumor growth of tamoxifen-resistant cells when added in combination with physiological levels of estrogen. Here, we show, using a cell culture model, that continuous exposure of tamoxifen-resistant cells to physiological levels of estrogen leads to cell death. Addition of the estrogen receptor downregulator fulvestrant prevents estrogen-induced death in a dose-dependent manner. Our data indicate that endogenous levels of estrogen affect the response of tamoxifen-resistant cells to fulvestrant. These results suggest that failure of fulvestrant to inhibit tumor growth in some tamoxifen-resistant patients may be due to endogenous estrogen levels. Moreover, these studies support short-term treatment with estrogen as a second-line hormonal therapy for tamoxifen-resistant breast cancer.     

Partial inhibition of estrogen-induced mammary carcinogenesis in rats by tamoxifen: balance between oxidant stress and estrogen responsiveness

Epidemiological and experimental evidences strongly support the role of estrogens in breast tumor development. Both estrogen receptor (ER)-dependent and ER-independent mechanisms are implicated in estrogen-induced breast carcinogenesis. Tamoxifen, a selective estrogen receptor modulator is widely used as chemoprotectant in human breast cancer. It binds to ERs and interferes with normal binding of estrogen to ERs. In the present study, we examined the effect of long-term tamoxifen treatment in the prevention of estrogen-induced breast cancer. Female ACI rats were treated with 17β-estradiol (E2), tamoxifen or with a combination of E2 and tamoxifen for eight months. Tissue levels of oxidative stress markers 8-iso-Prostane F(2α) (8-isoPGF(2α)), superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase, and oxidative DNA damage marker 8-hydroxydeoxyguanosine (8-OHdG) were quantified in the mammary tissues of all the treatment groups and compared with age-matched controls. Levels of tamoxifen metabolizing enzymes cytochrome P450s as well as estrogen responsive genes were also quantified. At necropsy, breast tumors were detected in 44% of rats co-treated with tamoxifen+E2. No tumors were detected in the sham or tamoxifen only treatment groups whereas in the E2 only treatment group, the tumor incidence was 82%. Co-treatment with tamoxifen decreased GPx and catalase levels; did not completely inhibit E2-mediated oxidative DNA damage and estrogen-responsive genes monoamine oxygenase B1 (MaoB1) and cell death inducing DFF45 like effector C (Cidec) but differentially affected the levels of tamoxifen metabolizing enzymes. In summary, our studies suggest that although tamoxifen treatment inhibits estrogen-induced breast tumor development and increases the latency of tumor development, it does not completely abrogate breast tumor development in a rat model of estrogen-induced breast cancer. The inability of tamoxifen to completely inhibit E2-induced breast carcinogenesis may be because of increased estrogen-mediated oxidant burden.     

Src Drives Growth of Antiestrogen Resistant Breast Cancer Cell Lines and Is a Marker for Reduced Benefit of Tamoxifen Treatment

The underlying mechanisms leading to antiestrogen resistance in estrogen-receptor α (ER)-positive breast cancer is still poorly understood. The aim of this study was therefore to identify biomarkers and novel treatments for antiestrogen resistant breast cancer. We performed a kinase inhibitor screen on antiestrogen responsive T47D breast cancer cells and T47D-derived tamoxifen and fulvestrant resistant cell lines. We found that dasatinib, a broad-spectrum kinase inhibitor, inhibited growth of the antiestrogen resistant cells compared to parental T47D cells. Furthermore western blot analysis showed increased expression and phosphorylation of Src in the resistant cells and that dasatinib inhibited phosphorylation of Src and also signaling via Akt and Erk in all cell lines. Immunoprecipitation revealed Src: ER complexes only in the parental T47D cells. In fulvestrant resistant cells, Src formed complexes with the Human Epidermal growth factor Receptor (HER)1 and HER2. Neither HER receptors nor ER were co-precipitated with Src in the tamoxifen resistant cell lines. Compared to treatment with dasatinib alone, combined treatment with dasatinib and fulvestrant had a stronger inhibitory effect on tamoxifen resistant cell growth, whereas dasatinib in combination with tamoxifen had no additive inhibitory effect on fulvestrant resistant growth. When performing immunohistochemical staining on 268 primary tumors from breast cancer patients who had received tamoxifen as first line endocrine treatment, we found that membrane expression of Src in the tumor cells was significant associated with reduced disease-free and overall survival. In conclusion, Src was identified as target for treatment of antiestrogen resistant T47D breast cancer cells. For tamoxifen resistant T47D cells, combined treatment with dasatinib and fulvestrant was superior to treatment with dasatinib alone. Src located at the membrane has potential as a new biomarker for reduced benefit of tamoxifen.     

MGMT Inhibition Restores ERα Functional Sensitivity to Antiestrogen Therapy

Antiestrogen therapy resistance remains a huge stumbling block in the treatment of breast cancer. We have found significant elevation of O(6) methylguanine DNA methyl transferase (MGMT) expression in a small sample of consecutive patients who have failed tamoxifen treatment. Here, we show that tamoxifen resistance is accompanied by upregulation of MGMT. Further we show that administration of the MGMT inhibitor, O(6)-benzylguanine (BG), at nontoxic doses, leads to restoration of a favorable estrogen receptor alpha (ERα) phosphorylation phenotype (high p-ERα Ser167/low p-ERα Ser118), which has been reported to correlate with sensitivity to endocrine therapy and improved survival. We also show BG to be a dual inhibitor of MGMT and ERα. In tamoxifen-resistant breast cancer cells, BG alone or in combination with antiestrogen (tamoxifen [TAM]/ICI 182,780 [fulvestrant, Faslodex]) therapy enhances p53 upregulated modulator of apoptosis (PUMA) expression, cytochrome C release and poly (ADP-ribose) polymerase (PARP) cleavage, all indicative of apoptosis. In addition, BG increases the expression of p21(cip1/waf1). We also show that BG, alone or in combination therapy, curtails the growth of tamoxifen-resistant breast cancer in vitro and in vivo. In tamoxifen-resistant MCF7 breast cancer xenografts, BG alone or in combination treatment causes significant delay in tumor growth. Immunohistochemistry confirms that BG increases p21(cip1/waf1) and p-ERα Ser167 expression and inhibits MGMT, ERα, p-ERα Ser118 and ki-67 expression. Collectively, our results suggest that MGMT inhibition leads to growth inhibition of tamoxifen-resistant breast cancer in vitro and in vivo and resensitizes tamoxifen-resistant breast cancer cells to antiestrogen therapy. These findings suggest that MGMT inhibition may provide a novel therapeutic strategy for overcoming antiestrogen resistance.     

Treatment of breast cancer with different antiprogestins: Preclinical and clinical studies

Treatment with antiprogestins in a new treatment modality for breast cancer. Previously, in rats with DMBA-induced mammary tumors we observed significant growth inhibitory effects of chronic treatment with the antiprogestin mifepristone (RU486). In addition, in 11 postmenopausal breast cancer patients, we observed one objective response, six instances of short-term stable disease, and four instances of progressive disease. Side-effects appeared mainly due to antiglucocorticoid properties of the drug. Increased plasma estradiol levels were observed which probably resulted from ovarian (rat) and adrenal (patients) steroidogenesis.

Combined treatment with an antiestrogen in the rat model caused additive growth inhibitory effects. Tumor inhibition after single treatment with mifepristone or tamoxifen was 90 and 75%, respectively. In contrast, when combined, tumor remission similar to that caused by LHRH-agonist treatment (50%) was observed. Even higher tumor remission was found after combined treatment with mifepristone plus LHRH-agonist (75%). In first studies in the rat model we observed significant tumor growth inhibitory effects with two new antiprogestins of seemingly greater potency which cause less unfavorable endocrine side-effects.

In conclusion: combined treatment (antiprogestin plus antiestrogen or LHRH-agonist) may be of value in endocrine therapy of breast cancer.     

Sensitivity and insensitivity of breast cancer to tamoxifen

Tamoxifen is the endocrine treatment of choice for breast cancer. In several laboratory models in vivo tamoxifen is a tumoristatic agent. When MCF-7 breast cancer cells are inoculated into athymic mice, palpable tumors do not grow unless the animals are treated with estrogen, and tamoxifen inhibits estrogen-stimulated growth. If tamoxifen is stopped, tumors regrow. These results suggest that adjuvant tamoxifen therapy should involve long treatment periods (even lifetime) to prevent tumor recurrence. Unfortunately resistance to therapy and patient relapse inevitably occur, and such disease recurrence involving tamoxifen resistance is difficult to treat successfully. A laboratory model of endocrine therapy failure has been developed. When athymic mice with MCF-7 tumors are treated for 6–8 months with tamoxifen, several tumors grew and continued to grow in tamoxifen-treated mice. These estrogen receptor-positive tumors grow with either tamoxifen or estradiol. Tamoxifen-stimulated tumor growth has been observed in human endometrial tumors implanted into athymic animals. Growth of these tamoxifen-stimulated tumors can be inhibited with the pure antiestrogen ICI 164,384 upon withdrawal of tamoxifen. These data are discussed in terms of treatment strategies for tamoxifen-failed patients.     

Effect of antiestrogens and aromatase inhibitor on basal growth of the human breast cancer cell line MCF-7 in serum-free medium

Antiestrogens are efficient inhibitors of estrogen-mediated growth of human breast cancer. Besides inhibiting estradiol-stimulated growth, antiestrogens may have a direct growth-inhibitory effect on estrogen receptor (ER) positive cells and thus be more efficient than aromatase inhibitors, which will only abrogate estrogen-dependent tumor growth. To address this issue, we have used the human breast cancer cell line MCF-7/S9 as a model system which is maintained in a chemically defined medium without serum and estrogen. The addition of estradiol results in an increase in cell growth rate. Thus, the MCF-7/S9 cell line is estrogen-responsive but not estrogen-dependent. Three different types of antiestrogens, namely tamoxifen, ICI 182,780 and EM-652 were found to exert a significant and dose-dependent inhibition of basal growth of MCF-7/S9 cells. The growth-inhibitory effect of the three antiestrogens was prevented by simultaneous estradiol treatment. Antiestrogen treatment also reduced the basal pS2 mRNA expression level, thus indicating spontaneous estrogenic activity in the cells. However, treatment with the aromatase inhibitor had no effect on basal cell growth, excluding that endogenous estrogen synthesis is involved in basal growth. These data demonstrate that in addition to their estrogen antagonistic effect, antiestrogens have a direct growth-inhibitory effect which is ER-mediated. Consequently, in the subset of ER positive breast cancer patients with estrogen-independent tumor growth, antiestrogen therapy may be superior to treatment with aromatase inhibitors which only inhibit estrogen formation but do not affect cancer cell growth in the absence of estrogens.     

MGMT Inhibition Restores ERα Functional Sensitivity to Antiestrogen Therapy

Antiestrogen therapy resistance remains a huge stumbling block in the treatment of breast cancer. We have found significant elevation of O⁶ methylguanine DNA methyl transferase (MGMT) expression in a small sample of consecutive patients who have failed tamoxifen treatment. Here, we show that tamoxifen resistance is accompanied by upregulation of MGMT. Further we show that administration of the MGMT inhibitor, O⁶-benzylguanine (BG), at nontoxic doses, leads to restoration of a favorable estrogen receptor alpha (ERα) phosphorylation phenotype (high p-ERα Ser167/low p-ERα Ser118), which has been reported to correlate with sensitivity to endocrine therapy and improved survival. We also show BG to be a dual inhibitor of MGMT and ERα. In tamoxifen-resistant breast cancer cells, BG alone or in combination with antiestrogen (tamoxifen [TAM]/ICI 182,780 [fulvestrant, Faslodex]) therapy enhances p53 upregulated modulator of apoptosis (PUMA) expression, cytochrome C release and poly (ADP-ribose) polymerase (PARP) cleavage, all indicative of apoptosis. In addition, BG increases the expression of p21^cip1/waf1. We also show that BG, alone or in combination therapy, curtails the growth of tamoxifen-resistant breast cancer in vitro and in vivo. In tamoxifen-resistant MCF7 breast cancer xenografts, BG alone or in combination treatment causes significant delay in tumor growth. Immunohistochemistry confirms that BG increases p21^cip1/waf1 and p-ERα Ser167 expression and inhibits MGMT, ERα, p-ERα Ser118 and ki-67 expression. Collectively, our results suggest that MGMT inhibition leads to growth inhibition of tamoxifen-resistant breast cancer in vitro and in vivo and resensitizes tamoxifen-resistant breast cancer cells to antiestrogen therapy. These findings suggest that MGMT inhibition may provide a novel therapeutic strategy for overcoming antiestrogen resistance.     

Tamoxifen induces TGF-β1 activity and apoptosis of human MCF-7 breast cancer cells in vitro

We report here that the antiestrogen tamoxifen (TAM) induces cell death in human breast cancer cell line MCF-7. We assessed the type of cell death induced by TAM in this breast cancer cell line on the basis of morphological and biochemical characteristics. Dying cells showed morphological characteristics of apoptosis, such as chromatin condensation and nuclear disintegration. DNA isolated from these cells revealed a pattern of distinctive DNA bands on agarose gel. The DNA fragmentation in MCF-7 cells induced by TAM could also be detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling. Northern blot hybridization revealed a substantial increase in the amounts of TRPM-2 and TGF-β1 mRNAs in MCF-7 cells after treatment with TAM. In contrast, the mRNA level of the estrogen-induced pS2 gene was strongly suppressed. The biological activity of TGF-β was increased at least fourfold in the media from MCF-7 cells treated with TAM. The results presented in this study suggest that TAM induces apoptosis of MCF-7 cells and it may be mediated by the secretion of active TGF-β. © 1996 Wiley-Liss, Inc.     

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.     

Oldenlandia diffusa extracts exert antiproliferative and apoptotic effects on human breast cancer cells through ERα/Sp1-mediated p53 activation

Breast cancer is the most frequent tumor and a major cause of death among women. Estrogens play a crucial role in breast tumor growth, which is the rationale for the use of hormonal antiestrogen therapies. Unfortunately, not all therapeutic modalities are efficacious and it is imperative to develop new effective antitumoral drugs. Oldenlandia diffusa (OD) is a well-known medicinal plant used to prevent and treat many disorders, especially cancers. The aim of this study was to investigate the effects of OD extracts on breast cancer cell proliferation. We observed that OD extracts strongly inhibited anchorage-dependent and -independent cell growth and induced apoptosis in estrogen receptor alpha (ERα)-positive breast cancer cells, whereas proliferation and apoptotic responses of MCF-10A normal breast epithelial cells were unaffected. Mechanistically, OD extracts enhance the tumor suppressor p53 expression as a result of an increased binding of ERα/Sp1 complex to the p53 promoter region. Finally, we isolated ursolic and oleanolic acids as the bioactive compounds able to upregulate p53 expression and inhibit breast cancer cell growth. These acids were greatly effective in reducing tamoxifen-resistant growth of a derivative MCF-7 breast cancer cell line resistant to the antiestrogen treatment. Our results evidence how OD, and its bioactive compounds, exert antiproliferative and apoptotic effects selectively in ERα-positive breast cancer cells, highlighting the potential use of these herbal extracts as breast cancer preventive and/or therapeutic agents.     

Autophagy facilitates the progression of ERα-positive breast cancer cells to antiestrogen resistance

A major impediment to the successful treatment of estrogen receptor α (ERα)-positive breast cancer is the development of antiestrogen resistance. Tamoxifen, the most commonly used antiestrogen, exerts its pharmacological action by binding to ERα and blocking the growth- promoting action of estrogen-bound ERα in breast cancer cells. Tamoxifen treatment primarily induces cytostasis (growth arrest) and the surviving breast cancer cells commonly acquire tamoxifen resistance. Numerous clinically-relevant mechanisms of acquired antiestrogen resistance have been identified by in vitro studies. Our recent studies (Mol Cancer Ther 2008: 7:2977-87) now demonstrate that autophagy (also referred to as macroautophagy) is critical to the development of antiestrogen resistance. Under conditions of compromised autophagy, including treatments with pharmacological inhibitors and RNAi targeting of the beclin 1 gene, the cytotoxicity (death-inducing effects) of the antiestrogen 4-hydroxytamoxifen (4-OHT) was significantly increased. 4-OHT is an active metabolite of tamoxifen commonly used for in vitro studies. A step-wise drug selection protocol, using 4-OHT as the selecting drug, established antiestrogen-resistant breast cancer cell lines. Analysis of a representative resistant cell line showed an increased ability of the cells to sustain high levels of antiestrogen-induced autophagy without progression to death. Importantly, blockade of autophagosome function in the 4-OHT-treated, antiestrogen-resistant cells induced a robust death response. These data provide strong evidence that autophagy is a key mechanism of cell survival during antiestrogen challenge and progression to antiestrogen resistance. We discuss the potential benefit of blocking autophagosome function to significantly reduce the emergence of antiestrogen-resistant breast cancer cells.     

Antiproliferation and apoptosis induced by tamoxifen in human bile duct carcinoma QBC939 cells via upregulated p53 expression

Tamoxifen (TAM) is a nonsteroidal antiestrogen that has been used in the treatment of breast cancer for over 30 years. Recently, it was shown that TAM also has efficacy on gastrointestinal neoplasms such as hepatocarcinoma and pancreatic carcinoma, and that the chemopreventive activities of TAM might be due to its abilities to inhibit cell growth and induce apoptosis. In the present study, we investigated the effects of tamoxifen on growth and apoptosis in the human bile duct carcinoma (BDC) cell line QBC939 using MTT assay, inverted microscopy, fluorescence microscopy, transmission electron microscopy, classic DNA fragmentation agarose gel electrophoresis assay, PI single- and FITC/PI double-staining flow cytometry, and Western blotting. Our data revealed that TAM could significantly inhibit growth and induce apoptosis in QBC939 cells. Increased expression of p53 was observed in TAM-treated cells, indicating that p53 might play an important role in TAM-induced apoptosis in QBC939 cells. These results provide significant insight into the anticarcinogenic action of TAM on BDC.