Hawse, JR

Abstract Background: A major issue surrounding the use of therapeutic drugs for the treatment of breast cancer is the eventual development of resistance. Endoxifen, the most potent tamoxifen metabolite, is being developed as a novel endocrine therapy for the treatment of endocrine responsive breast cancer patients. While numerous studies have investigated the process of tamoxifen resistance, no such data exist regarding the mechanism by which cells develop resistance to endoxifen. Here, we describe the development and characterization of a novel endoxifen resistant MCF7 breast cancer cell line and the identification of a specific treatment to effectively target these resistant cells. Methods: MCF7 cells were chronically exposed to concentrations of endoxifen previously demonstrated to be associated with the greatest reductions in estrogen stimulated proliferation and transcription (1000 nM). Changes in the physiological and molecular properties of these cells were monitored during the course of resistance using a wide range of techniques. Results: Following 15 months of endoxifen exposure, an epithelial to mesenchymal transition (EMT) was induced in MCF7 cells, characterized by loss of E-cadherin expression, up-regulation of fibronectin and vimentin expression and increased responsiveness to TGFβ. Resistant cells exhibit a 7-fold increase in their proliferation rates relative to parental cells and display basal like properties (triple negative) due to silencing of ERα , progesterone receptor and CD24 expression. Resistant cells were confirmed to be estrogen insensitive through the use of cell proliferation and gene expression studies. Wound healing and cell migration assays revealed that resistant cells are highly aggressive with a significant level of metastatic potential. Microarray analysis revealed over 7500 genes to be differentially expressed in the resistant cell line relative to parental cells using a 2-fold cutoff, of which only 52 genes (0.7%) were determined to be up-regulated. Interestingly, a number of the most highly altered genes have previously been implicated in the development of EMT and/or resistance including Twist2, IGF binding protein 5, GATA and β-tubulin. Based on the identification of P-tubulin as the most up-regulated gene in resistant cells, 2-methoxyestradiol (2ME2) was recognized as a candidate drug to specifically target this resistant cell line due to it known roles in blocking tubulin polymerization. Indeed, 2ME2 exposure resulted in the induction of apoptosis and significant cell death in vitro. Conclusions: Chronic exposure of MCF7 cells to high concentrations of endoxifen led to induction of EMT with cells that are basal like, estrogen and SERM insensitive, highly TGFP responsive with significantly increased proliferation rates and metastatic potential relative to parental cells. These findings suggest that the mechanisms of resistance to endoxifen may differ from those observed with long term exposure to tamoxifen and have identified 2ME2 as a potentially successful alternative therapy for endoxifen resistant breast cancer cells. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD05-11.

Abstract Background: The role of estrogen receptor alpha (ERα) in breast cancer has been studied extensively, and its protein expression is prognostic and a primary determinant of endocrine sensitivity; however, much less is known about the role of ERß. In vitro studies demonstrate a tumor suppressive function for ERß, and we have recently implicated a role for ERα in sensitizing ERα expressing breast cancer cells to the anti-estrogenic effects of endoxifen. However, the in vivo relevance of ERα remains unclear due to conflicting reports. Here, we provide evidence that some of this controversy may be explained by variability in antibody specificity. In addition, we describe the development and characterization of a novel, highly specific monoclonal antibody and provide data regarding ERα expression in human breast cancers. Methods: Five commercially available ERα antibodies were screened for their sensitivity and specificity using western blotting, immunoprecipitation, immunofluorescence and immunohistochemistry in known ERα negative and positive cell lines as well as in normal human tissue samples. A novel monoclonal ERα antibody (C10) was developed and characterized in the same manner. Following identification of two specific antibodies, ERα expression was assessed in 66 breast tumors collected prior to adjuvant therapy. Samples were scored separately for nuclear and cytoplasmic staining. Results: In depth analysis of commercially available ERα antibodies reveled that the majority were non-specific with substantial cross-reactivity to ERα . Only one commercial antibody (PPG5/10), which solely recognizes full-length ERß, and our newly developed monoclonal antibody, which recognizes full-length and all 4 ERα variants, were determined to be sensitive and specific for ERα expression. These same two antibodies resulted in strong staining for endogenous levels of ERα protein in normal prostate tissue by immunohistochemistry. We further assessed these two antibodies in a set of breast tumors. Preliminary analysis revealed significant differences for ERα positivity between these two antibodies. Based on nuclear staining, 92% of tumors were ERα positive using the PPG5/10 antibody while only 34% were positive with C10. Approximately 50% of all tumors exhibited cytoplasmic staining with both antibodies. Conclusions: Our studies demonstrate that the majority of commercially available ERα antibodies are either non-specific or insensitive for the detection of ERα via immunohistochemistry. The present data call into question the relevance of prior studies which tested the association between clinical outcome and ERα expression and demonstrate the need to further analyze the role of ERα in breast cancer using highly specific and validated antibodies. While both the PPG5/10 and C10 antibodies are highly specific for ERß, the significant discrepancy in nuclear staining between them in breast tumors may be due to changes in epitope availability as a result of post-translational processing. Our newly developed C10 antibody could provide additional discriminatory features which may be useful in predicting response to therapy and/or associations with other clinicopathological factors and such studies are currently underway. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-09-25.

Abstract Background: We have previously demonstrated that endoxifen is the most potent tamoxifen metabolite responsible for eliciting the anti-estrogenic effects of this drug in breast cancer cells expressing ERα (Wu, Hawse et al. Cancer Res 2009;69:1722). These studies support the clinical findings which demonstrate that women with genetically impaired CYP2D6 metabolism have significantly reduced endoxifen levels and a higher risk of breast cancer recurrence. More recently, data has emerged from several laboratories suggesting that expression of ERβ in breast tumors leads to increased benefits from tamoxifen therapy. However, the role of ERβ in mediating endoxifen action has yet to be explored.Methods: Using U2OS osteosarcoma cells, as well as Hs578T and MCF7 breast cancer cells, stably expressing ERβ, we examined the effects of endoxifen on ERβ protein levels by western blotting, ERβ transcriptional activity by real-time RT-PCR, and on ERβ inhibition of breast cancer cell growth through the use of proliferation assays.Results: We have discovered that endoxifen stabilizes ERβ protein, unlike its targeted degradation of ERα, and induces ERα/ERβ heterodimerization in a concentration dependent manner. We have also found that endoxifen is a more potent inhibitor of estrogen induced gene expression in MCF7 cells stably expressing ERβ. Concentrations of endoxifen that mimic patients with impaired CYP2D6 activity (20-40 nM) do not significantly alter E2-induced cell proliferation in MCF7 cells expressing only ERα. However, these concentrations of endoxifen significantly reduce the estrogen induced proliferation rate of MCF7 cells expressing ERβ.Conclusions: Our data demonstrate that endoxifen stabilizes ERβ protein, induces ER heterodimerization and more potently inhibits the actions of estrogen in breast cancer cells expressing ERβ. The presence of ERβ sensitizes cells to the anti-estrogenic effects of endoxifen and growth inhibition occurs at substantially lower endoxifen concentrations (20-40 nM) than in cells that express only ERα (100 nm). Our data suggest that tamoxifen therapy may be beneficial even in patients with reduced CYP2D6 metabolism when ERβ is expressed. These studies also suggest that the increased effectiveness of tamoxifen therapy in patients whose tumors express ERβ may be due to the effects of endoxifen. Finally, these findings underscore the need to further elucidate the role of ERβ in the biology and treatment of breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 405.

Abstract Abstract #19 Background: Tamoxifen (TAM) is a standard endocrine therapy for the treatment of women with estrogen receptor (ER) positive breast cancer. TAM is activated by the cytochrome P450 2D6 enzyme system into two potent and active metabolites, 4-hydroxytamoxifen (4HT) and 4-hydroxy-N-desmethyl-tamoxifen (endoxifen). While human concentrations of 4HT are negligible and vary little in plasma (5-10 nM), endoxifen concentrations vary widely (10-180 nm), and women with genetically impaired CYP2D6 metabolism have significantly reduced endoxifen levels and a higher risk of breast cancer recurrence. Despite these observations, endoxifen's contribution to tamoxifen's overall drug effectiveness is uncertain.
 Methods: Using cells endogenously expressing ERa (MCF7, T47D) and cells stably transfected with ERa (Hs578T and U2OS), we examined the relative effects of TAM and its primary metabolites on ERa protein levels by western blotting, ERa transcriptional activity by luciferase reporter assays and real-time RT-PCR, and on ER positive breast cancer cell growth through the use of proliferation assays.
 Results: We have discovered that endoxifen induces ERa protein turnover through proteasomal degradation similar to that of ICI in a concentration and time-dependent manner. These findings are in stark contrast to TAM, N-desmethyl-tamoxifen (NDT) and 4HT, which stabilize the ER. Optimal degradation occurs only at endoxifen concentrations observed in human CYP2D6 extensive metabolizers (> 40 nM) and persists even in the presence of TAM (300 nM), 4HT (7 nM), and NDT (700 nM) at concentrations observed in patients receiving tamoxifen therapy. In contrast, reducing endoxifen concentrations to those observed in a CYP2D6 poor metabolizer (20 nM), without altering TAM, 4HT, and NDT, results in ER stabilization. High endoxifen concentrations (100-1000 nM) completely block estrogen (E2)-induced ER transcriptional activity even in the presence of TAM, 4HT, and NDT, while low endoxifen concentrations (20-40 nM) do not. Further, low concentrations of endoxifen (20 nM) do not significantly alter E2-induced cell proliferation; however, high concentrations of endoxifen (100-1000 nM) completely block this process. Discussion: Our data demonstrate that endoxifen is a potent anti-estrogen that targets ERa for proteasomal degradation, blocks ERa transcriptional activity and inhibits E2-induced breast cancer cell proliferation. Importantly, these effects of endoxifen are observed at concentrations found in CYP2D6 extensive metabolizers and are maintained even in the presence of TAM, 4HT and NDT. These studies suggest that endoxifen may be the primary metabolite responsible for tamoxifen's effectiveness in the treatment of ER positive breast cancer and provide the impetus to evaluate whether TAM-related side-effects, such as thrombo-embolism and endometrial hyperplasia/carcinoma, are inversely associated with a patient's ability to metabolically activate tamoxifen. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 19.