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Purpose: Oestrogen (E2)-stimulated growth re-emerges after a c-Src inhibitor blocking E2-induced apoptosis. A resulting cell line, MCF-7:PF, is selected with features of functional oestrogen receptor (ER) and over-expression of insulin-like growth factor-1 receptor beta (IGF-1Rβ). We addressed the question of whether the selective ER modulator (SERM), 4-hydroxytamoxifen (4-OHT) or other SERMs could target ER to prevent E2-stimulated growth in MCF-7:PF cells., Methods: Protein levels of receptors and signalling pathways were examined by immunoblotting. Expression of mRNA was measured through real-time RT-PCR. Recruitment of ER or nuclear receptor coactivator 3 (SRC3) to the promoter of ER-target gene was detected by chromatin-immunoprecipitation (ChIP)., Results: 4-OHT and other SERMs stimulated cell growth in an ER-dependent manner. However, unlike E2, 4-OHT suppressed classical ER-target genes as does the pure antioestrogen ICI 182,780 (ICI). ChIP assay indicated that 4-OHT did not recruit ER or SRC3 to the promoter of ER-target gene, pS2. Paradoxically, 4-OHT reduced total IGF-1Rβ but increased phosphorylation of IGF-1Rβ. Mechanistic studies revealed that 4-OHT functioned as an agonist to enhance the non-genomic activity of ER and activate focal adhesion molecules to further increase phosphorylation of IGF-1Rβ. Disruption of membrane-associated signalling, IGF-1R and focal adhesion kinase (FAK), completely abolished 4-OHT-stimulated cell growth., Conclusions: This study is the first to recapitulate a cellular model in vitro of acquired tamoxifen resistance developed in athymic mice in vivo. Importantly, it provides a rationale that membrane-associated pathways may be valuable therapeutic targets for tamoxifen resistant patients in clinic., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Purpose: A c-Src inhibitor blocks oestrogen (E2)-induced stress and converts E2 responses from inducing apoptosis to growth stimulation in E2-deprived breast cancer cells. A reprogrammed cell line, MCF-7:PF, results in a functional oestrogen receptor (ER). We addressed the question of whether the selective ER modulator 4-hydroxytamoxifen (4-OHT) could target ER to prevent E2-stimulated growth in MCF-7:PF cells., Methods: Expression of mRNA was measured through real-time RT-PCR. Global gene expression profile was analysed through microarray. Transcriptome profiles were screened by RNA-sequencing., Results: Unexpectedly, both 4-OHT and E2 stimulated cell growth in a concentration-dependent manner. Expression profiling showed a remarkable overlap in genes regulated in the same direction by E2 and 4-OHT. Pathway enrichment analysis of the 280 genes commonly deregulated in MCF-7:PF cells by 4-OHT and E2 revealed functions mainly related to membrane, cytoplasm and metabolic processes. Further analysis of 98 genes up-regulated by both 4-OHT and E2 uncovered a significant enrichment in genes associated with membrane remodelling, cytoskeleton reorganisation, cytoplasmic adapter proteins, cytoplasm organelle proteins and related processes. 4-OHT was more potent than E2 in up-regulating some membrane remodelling molecules, such as EHD2, FHL2, HOMER3 and RHOF. In contrast, 4-OHT acted as an antagonist to inhibit expression of the majority of enriched membrane-associated genes in wild-type MCF-7 cells., Conclusions: Long-term selection pressure has changed the cell population responses to 4-OHT. Membrane-associated signalling is critical for 4-OHT-stimulated cell growth in MCF-7:PF cells. This study provides a rationale for the further investigation of target therapy for tamoxifen resistant patients., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Purpose: Our publications demonstrate that physiological concentrations of oestrogen (E2) induce endoplasmic reticulum and oxidative stress which finally result in apoptosis in E2-deprived breast cancer cells, MCF-7:5C. c-Src is involved in the process of E2-induced stress. To mimic the clinical administration of c-Src inhibitors, we treated cells with either E2, a c-Src inhibitor PP2, or the combination for 8 weeks to further explore the apoptotic potential of the c-Src inhibitor and E2 on MCF-7:5C cells., Methods: Protein levels of receptors and signalling pathways were examined by immunoblotting. Expression of mRNA was detected through real-time polymerase chain reaction (PCR). Cell cycles were analysed by flow cytometry., Results: Long-term treatment with PP2 alone or E2 alone decreased cell growth. In contrast, a combination of PP2 and E2 blocked apoptosis and the resulting cell line (MCF-7:PF) was unique, as they grew vigorously in culture with physiological levels of E2, which could be blocked by the pure antioestrogen ICI182,780. One major change was that PP2 collaborated with E2 to increase the level of insulin-like growth factor-1 receptor beta (IGF-1Rβ). Blockade of IGF-1Rβ completely abolished E2-stimulated growth in MCF-7:PF cells. Furthermore, combination treatment up-regulated transcription factors, Twist1 and Snail, and repressed E-cadherin expression which made MCF-7:PF cells display a characteristic phenotype of epithelial-mesenchymal transition (EMT)., Conclusions: These data illustrate the role of the c-Src inhibitor to block E2-induced apoptosis and enhance E2-stimulated growth. Caution must be exercised when considering c-Src inhibitors in clinical trials following the development of acquired resistance to aromatase inhibitors, especially in the presence of the patient's own oestrogen., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Purpose: c-Src is an important adapter protein with oestrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2), which validates it as an attractive target for the treatment of breast cancer. A specific c-Src inhibitor, 4-amino-5-(4-chlorophenyl)-7(t-butyl)pyrazolo[3,4-d]pyrinidine (PP2), was utilised to block c-Src activity to identify targeted vulnerabilities affected by ER and HER2 in a panel of breast cancer cell lines., Methods: ER, growth factor receptors and signalling pathways were detected by Western-blot. The DNA content of the cells was determined by using a DNA fluorescence quantitation kit. Cell cycles were analysed by flow cytometry., Results: The antiproliferative effect of PP2 closely correlated with the inhibition of c-Src mediated extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) and/or phosphoinositide 3-kinase (PI3K)/Akt growth pathways. Inhibition of c-Src tyrosine kinase predominantly blocked ER negative breast cancer cell growth, particularly the triple (i.e. ER, progesterone receptor (PR), and HER2) negative cells. In contrast, ER negative Sk-Br-3 cells with highest HER2 phosphorylation were resistant to PP2, in which hyper-activated HER2 directly regulated growth pathways. However, blocking c-Src recovered ER expression and down-regulated HER2 which made Sk-Br-3 cells regain responsiveness to 4-hydroxytamoxifen. The majority of ER positive cells were not sensitive to PP2 regardless of wild-type or endocrine resistant cell lines., Conclusions: c-Src mediates the essential role of growth pathways in ER negative breast cancer cells. The ER positive and HER2 over-activation are two important predictive biomarkers for the resistance to a c-Src inhibitor. These data provided an important therapeutic rationale for patient selection in clinical trials with c-Src inhibitors in breast cancer., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Purpose: Tamoxifen, a selective oestrogen receptor modulator (SERM), and brivanib alaninate, a vascular endothelial growth factor receptor 2 (VEGFR-2) inhibitor, are two target specific agents that result in a substantial decrease in tumour growth when given alone. Tamoxifen activates SERM stimulated breast and endometrial tumour growth. Tamoxifen and brivanib alaninate have side-effects that can affect therapeutic outcomes. The primary goal of the current study was to evaluate the therapeutic effects of lower doses of both agents when given in combination to mice with SERM sensitive, oestrogen stimulated tumour xenografts (MCF-7 E2 tumours). Experiments were conducted to evaluate the response of SERM stimulated breast (MCF-7 Tam, MCF-7 Ral) and endometrial tumours (EnCa 101) to demonstrate the activity of brivanib alaninate in SERM resistant models., Experimental Design: In the current study, tumour xenografts were minced and bi-transplanted into the mammary fat pads of athymic, ovariectomised mice. Preliminary experiments were conducted to determine an effective oral dose of tamoxifen and brivanib alaninate that had minimal effect on tumour growth. Doses of 125 microg of tamoxifen and 0.05 mg/g of brivanib alaninate were evaluated. An experiment was designed to evaluate the effect of the two agents together when started at the time of tumour implantation. An additional experiment was done in which tumours were already established and then treated, to obtain enough tumour tissue for molecular analysis., Results: Brivanib alaninate was effective at inhibiting tumour growth in SERM sensitive (MCF-7 E2) and SERM stimulated (EnCa 101, MCF-7 Ral, MCF-7 Tam) models. The effect of the low dose drug combination as an anti-tumour strategy for SERM sensitive (MCF-7 E2) in early treatment was as effective as higher doses of either drug used alone. In established tumours, the combination is successful at decreasing tumour growth, while neither agent alone is effective. Molecular analysis revealed a decreased phosphorylation of VEGFR-2 in tumours that were treated with brivanib alaninate and an increase in VEGFA transcription to compensate for the blockade of VEGFR-2 by increasing the transcription of VEGFA. Tamoxifen increases the phosphorylation of VEGFR-2 and this effect is abrogated by brivanib alaninate. There was also increased necrosis in tumours treated with brivanib alaninate., Conclusion: Historically, tamoxifen has a role in blocking angiogenesis as well as the blockade of the ER. Tamoxifen and a low dose of an angiogenesis inhibitor, brivanib alaninate, can potentially be combined not only to maximise therapeutic efficacy but also to retard SERM resistant tumour growth., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Tamoxifen is a standard endocrine therapy for the treatment of steroid receptor positive breast cancer. Tamoxifen efficacy depends on the formation of clinically active metabolites 4-hydroxytamoxifen and endoxifen which have a greater affinity to the oestrogen receptor and ability to control cell proliferation as compared to the parent drug. The cytochrome P450 2D6 enzyme plays a key role in this biotransformation and lack of tamoxifen efficacy has been linked to low activity. There is now considerable mechanistic, pharmacologic and clinical pharmacogenetic evidence in support of the notion that CYP2D6 genetic variants and phenocopying effects through drug interaction by CYP2D6 inhibitors influence plasma concentrations of active tamoxifen metabolites and negatively impact tamoxifen outcome. These interrelations are particularly critical for patients with non-functional (poor metaboliser) and severely impaired (intermediate metaboliser) CYP2D6 variants, and, moreover, for patients in need of co-medication such as serotonin re-uptake inhibitors to control adverse effects such as hot flashes and other menopausal symptoms. Therefore, in the future, a personalised approach for an optimal tamoxifen benefit should consider a CYP2D6 genotype guided adjuvant endocrine treatment strategy and avoid non-adherence as well as strong CYP2D6 inhibitors such as co-medications.

Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is an intercellular adhesion molecule that is overexpressed in a wide variety of human cancers, including colon, breast and lung and is associated with tumourigenesis, tumour cell adhesion, invasion and metastasis. In this study, we showed that CEACAM6 was overexpressed in a panel of oestrogen receptor (ERalpha)-positive human breast cancer cell lines (MCF-7:5C and MCF-7:2A) that have acquired resistance to oestrogen deprivation, and this overexpression was associated with a more aggressive invasive phenotype in vitro. Expression array analysis revealed that MCF-7:5C and MCF-7:2A cells overexpressed CEACAM6 mRNA by 27-fold and 12-fold, respectively, and were 6-15-times more invasive compared to non-invasive wild-type MCF-7 cells which expressed low levels of CEACAM6. Suppression of CEACAM6 expression using small interfering RNA (siRNA) completely reversed migration and invasion of MCF-7:5C and MCF-7:2A cells and it significantly reduced phosphorylated Akt and c-Src expression in these cells. In conclusion, our findings establish CEACAM6 as a unique mediator of migration and invasion of drug resistant oestrogen-deprived breast cancer cells and suggest that this protein could be an important biomarker of metastasis.

In the early 1970s, a failed post-coital contraceptive, ICI 46,474, was reinvented as tamoxifen, the first targeted therapy for breast cancer. A cluster of papers published in the European Journal of Cancer described the idea of targeting tamoxifen to patients with oestrogen receptor positive tumours, and proposed the strategic value of using long-term tamoxifen therapy in an adjuvant setting with a consideration of the antitumour properties of the hydroxylated metabolites of tamoxifen. At the time, these laboratory results were slow to be embraced by the clinical community. Today, it is estimated that hundreds of thousands of breast cancer patients are alive today because of targeted long-term adjuvant tamoxifen therapy. Additionally, the first laboratory studies for the use of tamoxifen as a chemopreventive were published. Eventually, the worth of tamoxifen was tested as a chemopreventive and the drug is now known to have an excellent risk benefit ratio in high risk pre-menopausal women. Overall, the rigorous investigation of the pharmacology of tamoxifen facilitated tamoxifen's ubiquitous use for the targeted treatment of breast cancer, chemoprevention and pioneered the exploration of selective oestrogen receptor modulators (SERMs). This new concept subsequently heralded the development of raloxifene, a failed breast cancer drug, for the prevention of osteoporosis and breast cancer without the troublesome side-effect of endometrial cancer noted in post-menopausal women who take tamoxifen. Currently, the pharmaceutical industry is exploiting the SERM concept for all members of the nuclear receptor superfamily so that medicines can now be developed for diseases once thought impossible.

The completion of the Study of Tamoxifen and Raloxifene (STAR) [Vogel VG, Costantino JP, Wickerham DL, et al. The Study of Tamoxifen and Raloxifene (STAR): Report of the National Surgical Adjuvant Breast and Bowel Project P-2 Trial. JAMA 2006;295:2727-2741.] and the ongoing studies with aromatase inhibitors [Goss PE. Breast cancer prevention-clinical trials strategies involving aromatase inhibitors. J Steroid Biochem Mol Biol 2003;86(3-5):487-93.] to assess their worth for the chemoprevention of breast cancer, creates an opportunity to consider the realistic future for chemoprevention as an option for women's healthcare and to identify strategies for future progress in an era of targeted therapeutics, managed healthcare and soaring costs.

The aim of this study was to determine the effects of low doses of tamoxifen (5 and 10mg/day) for 50 days compared with the standard dose (20 mg/day) on breast biomarkers measured in normal breast tissue from premenopausal patients. A randomised double-blind study was performed using tissue from 56 premenopausal women with a diagnosis of fibroadenoma of the breast. Excisional biopsy was performed on the 50th day of therapy. Normal breast tissue samples were collected during surgery. The patients were divided in groups: A (placebo, n=11); group B (5 mg, n=16), group C (10 mg, n=14) and group D (20 mg, n=15). In this cross-sectional study, differences in the expression of Oestrogen Receptor alpha (ERalpha), Progesterone Receptor (PR), Ki-67, apoptotic bodies and mitotic index between the different groups after treatment can be seen on the normal breast tissue. We believe that a lower dose of tamoxifen could reduce the side-effects associated with treatment without affecting its chemopreventive activity in the breast.

Tamoxifen has not only proved to be a valuable treatment for estrogen receptor (ER)-positive breast cancer, but is also a pioneering medicine for chemoprevention in high-risk pre- and postmenopausal women. Insights into the pharmacology and toxicology of tamoxifen have led to the recognition of selective ER modulators (SERMs) with estrogen-like actions in maintaining bone density and in lowering circulating cholesterol, but antiestrogenic actions in the breast. Raloxifene, a related SERM, is now available to treat osteoporosis and is also being tested as a preventive for breast cancer and coronary heart disease. Emerging knowledge about the action of SERMs will provide clues for the design of mechanism-based medicines.

Knowledge of the mechanism of action and pharmacology of tamoxifen and raloxifene, for the prevention of breast cancer and osteoporosis respectively, has opened the door for the discovery of multifunctional medicines. There is now the potential to prevent osteoporosis, coronary heart disease, breast and endometrial cancer in postmenopausal women with elevated risk factors.

Knowledge of the mechanism of action and pharmacology of tamoxifen and raloxifene, for the prevention of breast cancer and osteoporosis respectively, has opened the door for the discovery of multifunctional medicines. There is now the potential to prevent osteoporosis, coronary heart disease, breast and endometrial cancer in postmenopausal women with elevated risk factors.

Tamoxifen has been the endocrine treatment of choice for all stages of breast cancer for nearly a decade. Millions of women are currently receiving tamoxifen worldwide, while large-scale randomised trials have been launched aiming to investigate the drug's merit as a preventive agent. However, there are now concerns about tamoxifen's potential carcinogenicity. The goal of this review is to address these concerns, re-evaluate the available data from laboratory biological models and those from clinical reports and put the whole issue into perspective. Our focus is the association between tamoxifen and the increased frequency of endometrial tumours, while key issues, such as the role of duration of tamoxifen therapy, are also addressed. Finally, we discuss the various monitoring strategies for early detection of endometrial lesions and pertinent problems most likely to be encountered by clinicians taking care of patients who are receiving tamoxifen.

Growth stimulation by oestrogens in immunodeficient mice is characteristically restricted to tumours expressing oestrogen receptors (ER). We now describe oestrogen-stimulated growth of the ER-negative human breast cancer cell line MDA-MB-231, subclone 10A. Cell culture experiments confirmed that 10A cells are unresponsive to a wide concentration range of oestradiol (E2) in vitro. Analysis of growth curves in vivo revealed significantly longer tumour volume doubling times for the control group than for the E2-treated group. Cell cycle studies using in vivo labelling with bromodeoxyuridine (BrdU) and flow cytometric analysis showed essentially equal potential doubling times for controls and E2-treated animals. These results suggest that E2 reduces cell loss, rather than stimulating proliferation. E2-stimulated growth was seen in both natural killer (NK) cell producing athymic (nude) mice and congenitally NK cell deficient beige nude mice. We conclude that E2-induced natural killer cell suppression is an unlikely mechanism of action.

Serial IGF-1 levels in patients prior to and during adjuvant tamoxifen (TAM) treatment were followed in a retrospective study. Serum IGF-1 levels were determined by radioimmunoassay in 19 patients taking TAM and 19 controls, matched for age, body weight and other treatments. IGF-1 levels at 2 years were significantly lower in TAM patients (P < or = 0.05) compared to control patients. We observed a significant mean drop from pretreatment to treatment IGF-1 levels by 19.9% in the TAM group (P < or = 0.005), but also noted a mean 11.4% decline in the control group (P < or = 0.025). A subgroup analysis suggested that premenopausal were relatively resistant to the IGF-1 lowering effects of TAM as compared to postmenopausal women.

The anti-oestrogenic and anti-tumour properties of a sustained-release preparation of tamoxifen were evaluated in female C3H/OUJ mice. Tamoxifen decreased uterine weight compared with controls in intact mice but caused an initial uterotrophic response for 2 months in ovariectomized mice. Prolonged tamoxifen therapy in ovariectomized mice resulted in a uterine weight no different from controls, but these uteri were eventually (at 6 months) refractory to oestradiol. Spontaneous mammary tumours were detected in female mice between 6 months and 1 year of age during continuous cycles of pregnancy and weaning. A similar tumour frequency occurred after one cycle of pregnancy and weaning initiated at 31/2 months. Prolonged tamoxifen, started at 31/2 or 41/2 months of age (following pregnancy/weaning), reduced the appearance of tumours. Similarly ovariectomy at 31/2 months prevented mammary tumorigenesis and prolonged tamoxifen could not increase tumour incidence consistently in ovariectomized mice. Although tamoxifen is oestrogenic in short-term tests the compound has the properties of an anti-oestrogen during prolonged administration.

Serum concentrations of tamoxifen, 4-OH-tamoxifen, N-desmethyltamoxifen, and metabolites E and Y were assayed to assess the variation of tamoxifen-metabolism during short-term and long-term endocrine treatment for breast cancer. Once steady-state was achieved, serum levels of tamoxifen and its metabolites in individual patients were stable in the short (10 weeks) and long term (over 7 years) (coefficient of variation [CV], 10-15%), but the variation between individuals (CV 50-70%) was high. Serum tamoxifen and N-desmethyltamoxifen levels were not correlated with indices of obesity. Thus this does not explain the large variation between individuals. In addition to the metabolites that were measured, 4-hydroxy-N-desmethyltamoxifen was tentatively identified in patients' serum. Overall, this study demonstrated that the metabolites of tamoxifen are stable (i.e. no metabolic tolerance) for up to 10 years of drug administration.