Citations

Abstract Background The role of estrogen receptor alpha (ERα) in breast cancer has been studied extensively; yet, much less is known about full-length ERβ (ERβ1) and even less about its 4 variant forms (ERβ2-5). We have recently implicated a role for ERβ1 in sensitizing ERα expressing breast cancer cells to anti-estrogens. However, the ability of ERβ2-5 to modulate ERα and ERβ1 activity, and their association with cancer development, progression, and response to estradiol (E2) and anti-estrogens are not well understood. Here, we provide evidence that the presence of ERβ variants may be of diagnostic and clinical relevance for breast cancer patients and describe the development and characterization of a novel, highly specific monoclonal antibody (MC10) that is able to detect their expression in tumor biopsies. Methods: Transient transfection and luciferase assays were used to determine the transcriptional activity of ERβ2-5 in response to E2 and anti-estrogens alone or in combination with ERα and ERβ1. A novel monoclonal antibody targeting all ERβ variants (MC10) was developed and characterized. The sub-cellular localization of ERβ2-5 was determined via confocal microscopy. Finally, the MC10 antibody was used to assess ERβ positivity in breast tumors and was compared to that of another monoclonal antibody which only detects ERβ1. Results: Unlike ERβ1, ERβ2-5 do not activate an estrogen response element (ERE) in response to E2 and instead, slightly repress the activity of this reporter construct. Expression of ERβ2-5 does not significantly alter the transcriptional activity of ERβ1 following E2 treatment. However, ERβ2, 3 and 5, but not ERβ4, significantly enhance the E2-induced transcriptional activity of ERα. Interestingly, expression of ERβ3, 4 and 5, but not ERβ2, enhance the ability of anti-estrogens to block ERα mediated transcriptional activity. Confocal microscopy revealed that ERβ1 and 2 are almost exclusively localized to the cell nucleus. However, ERβ3-5 exhibit significant cytoplasmic and peri-nuclear localization. Immunohistochemistry of breast cancer biopsies using the MC10 antibody revealed multiple staining patterns including tumors which exhibit primarily nuclear staining and others primarily cytoplasmic, both in the presence and absence of ERα. These results are in contrast to the almost exclusive nuclear staining obtained on the same tumors with an ERβ1-specific antibody. Conclusions: ERβ variants exhibit variable sub-cellular localization patterns and can influence the function of ERα, both in response to E2 and anti-estrogens. Therefore, the differential expression of ERβ variants and their cellular localization may influence breast cancer progression and/or therapeutic responses. The use of ERβ antibodies which do not detect all ERβ variants, or the use of a single ERβ antibody which does not discriminate between ERβ1 and its variants, is unlikely to reveal the complete biological significance of total ERβ expression in breast cancer and may in part explain the conflicting studies which have been reported for ERβ in the field. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-02-03.

Abstract Background Commonly used endocrine therapies for breast cancer, such as aromatase inhibitors in postmenopausal women and tamoxifen in premenopausal women, have deleterious effects on bone mineral density. Therefore, the identification of novel cancer therapies which either maintain or improve bone mass are of clinical need. We have recently demonstrated that endoxifen is the most active tamoxifen metabolite with regard to inhibiting the growth of ERα+ breast cancer cells and these studies have led to the development of endoxifen as a novel anti-breast cancer drug for which first-in-human studies are now underway. At present, there are no data regarding endoxifen's effects on bone. Methods: The effects of endoxifen on osteoblast (OB) and osteoclast (OC) maturation and gene expression were monitored by cell differentiation assays and real-time PCR. Dual-energy X-ray absorptiometry (DXA), peripheral Quantitative Computed Tomography (pQCT) and micro-Computed Tomography (μCT) were used to determine changes in bone density, mass and architecture following 45 days of oral endoxifen administration (50mg/kg/day) to 3-month-old ovariectomized (OVX) C57BL/6 mice relative to vehicle control treated animals. Alterations in the numbers and activity of OBs and OCs were determined by histomorphometry and serum levels of P1NP and CTX-1 respectively. Results: Endoxifen treatment of mouse derived bone marrow stromal cells and human OBs led to significant increases in the expression of critical bone marker genes such as Runx2, osterix, osteocalcin, osteoprotegerin and alkaline phosphatase in a dose dependent manner. Daily administration of endoxifen to OVX mice led to significant increases in total body bone mineral density (BMD) (6%) and content (BMC) (9%), which was accompanied by a 50% decrease in fat tissue mass as determined by DXA. pQCT analysis of the tibial metaphysis revealed dramatic increases in BMD (35%) and BMC (20%), as well as trabecular density (52%), cortical content (62%), cortical area (60%) and cortical thickness (78%). μCT analysis of the femoral metaphysis revealed increases in bone volume/total volume (200%), trabecular number (38%) and trabecular thickness (18%), as well as decreased trabecular spacing (29%). Interestingly, there was nearly a 50% increase in the numbers of OCs derived from endoxifen treated mice which was associated with elevated expression of OC marker genes such as NFATcl, RANK, c-fms and cathepsin-K compared to control treated animals. Approximately 4 times as many OBs and OCs were observed on the bone surfaces of endoxifen treated mice which correlated with nearly 2-fold increases in serum levels of the bone formation (P1NP) and resorption (CTX-1) markers. Conclusions: These data are the first to demonstrate that endoxifen has anabolic effects on the mouse skeleton which are similar to that of estrogen. Additionally, these data reveal that endoxifen's mechanism of action in bone is different than that reported for tamoxifen and other selective estrogen receptor modulators in mice as it increases, rather than decreases, bone formation and remodeling. Therefore, the use of endoxifen for the treatment of endocrine responsive breast cancer may avoid the detrimental skeletal effects of many conventional endocrine therapies. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-16-09.

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 Background: Endoxifen, the active metabolite of tamoxifen (Tam), is currently being developed as a drug for the treatment of estrogen receptor (ER) + breast cancer (BC). HER2 expression in ER+ BC is associated with Tam resistance, and in vivo Tam administration to mice bearing ER+/HER2+ xenografts stimulates BC growth (Shou, JNCI 2004). In humans, endoxifen is the most important Tam metabolite responsible for inhibiting estrogen induced BC growth (Wu, Cancer Research 2009). CYP2D6 metabolism affects the concentrations (conc) of endoxifen (Stearns, JNCI, 2003) and associated with worse disease free survival (DFS) in Tam treated BC (Goetz, JCO, 2005). We sought to determine the activity of Tam and its metabolites in ER+/HER2+ BC cell lines and to evaluate the role of CYP2D6 metabolism in Tam-treated patients (pts) with ER+/HER2+ BC. Additionally, we sought to determine endoxifen conc in mice administered oral Tam.Methods: MCF7 (parental and HER2-expressing) and BT474 (ER+/HER2+) cells were used to compare the activity of Tam, 4HT, and endoxifen on estrogen- stimulated growth. Oral tam PK were characterized in mice treated with standard dose of Tam (4 mg/kg; 100 μg). Clinical data were obtained via a retrospective analysis of Tam-treated pts with ER+/ HER2+ BC randomized to receive 5 years of Tam (NCCTG 89-30-52). CYP2D6 metabolism (extensive or decreased) was based on CYP2D6 genotype (*3, 4, 6, 10, 17, 41) and co-administration of a CYP2D6 inhibitor (yes/no). HER2 was determined by immunohistochemistry (IHC) or FISH (tumors 0, 1, or 2+ by IHC). The association between CYP2D6 and DFS was assessed using the log-rank test and proportional hazards modeling.Results: Compared to Tam, endoxifen potently inhibited the growth of estrogen- stimulated BT474 cells. In MCF7 cells, expression of HER2 shifted the conc of endoxifen required for 50% inhibition of growth (IC50) from 54 nM (parental) to 131 nM (HER2 expressing). Using the range of conc of Tam and its metabolites observed in humans (Tam, 300-500 nM; 4HT, 5-10 nM; and endoxifen, 10-200 nM), only endoxifen potently inhibited estrogen- stimulated growth of MCF7HER2+ cells and only at conc achievable in CYP2D6 extensive metabolizers (>50nM). In mice, conc of 4HT and endoxifen were below 15 nM following an oral dose of 4 mg/kg. In NCCTG 89-30-52, both CYP2D6 phenotype and HER2 status was determined in 201/256 randomized pts. HER2 was expressed in 23/215 (11%) but not associated with DFS overall (p=0.62). In the HER2+ subset, pts with decreased CYP2D6 metabolism (n=10) had significantly shorter DFS compared to extensive metabolizers (n=9) (HR 9.5, p=0.03; 95% CI 1.16-76.9).Conclusions: Our in vitro and clinical data provide a simple pharmacological model for understanding HER2 resistance in Tam-treated breast cancer. Mice, which lack the CYP2D6 enzyme, may not be an appropriate model for understanding tam resistance given low conc of both 4HT and endoxifen. Given that the in vitro conc of endoxifen needed to inhibit the growth of ER+/HER-2+ BC are achievable in only a subset of humans (CYP2D6 extensive metabolizers), the primary administration of endoxifen could overcome de novo Tam resistance in ER+/HER2+ BC. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2006.

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.

Evidence has emerged that the clinical benefit of tamoxifen is related to the functional status of the hepatic metabolizing enzyme cytochrome P450 2D6 (CYP2D6). CYP2D6 is the key enzyme responsible for the generation of the potent tamoxifen metabolite, endoxifen. Multiple studies have examined the relationship of CYP2D6 status to breast cancer outcomes in tamoxifen-treated women; the majority of studies demonstrated that women with impaired CYP2D6 metabolism have lower endoxifen concentrations and a greater risk of breast cancer recurrence. As a result, practitioners must be aware that some of the most commonly prescribed medications coadministered with tamoxifen interfere with CYP2D6 function, thereby reducing endoxifen concentrations and potentially increasing the risk of breast cancer recurrence. After reviewing the published data regarding tamoxifen metabolism and the evidence relating CYP2D6 status to breast cancer outcomes in tamoxifen-treated patients, we are providing a guide for the use of medications that inhibit CYP2D6 in patients administered tamoxifen.

The purpose of this study was to characterize the effect of TIEG1 on the molecular structure of collagen within tail tendon fibers using 3-mo-old female C57BL/6 wild-type (WT) and TIEG1 KO mice. Synchrotron X-ray microdiffraction experiments were carried out on single tendon fibers extracted from the WT and TIEG1 KO dorsal tail tendon. The fibers were scanned in the radial direction, and X-ray patterns were obtained. From these patterns, the meridional direction was analyzed through X-ray intensity profile. In addition, collagen content was investigated using hydroxyproline assays, and qualitative real-time PCR experiments were performed on RNA isolated from fibroblasts to examine specific gene expression changes. The results showed different X-ray diffraction patterns between WT and TIEG1 KO tendon fibers, indicating a disorganization of the collagen structure for the TIEG1 KO compared with WT mice. Furthermore, the analyses of the X-ray intensity profiles exhibited a higher (23 Å) period of collagen for the TIEG1 KO compared with the WT mice. The results of the hydroxyproline assays revealed a significant decrease in the TIEG1 KO compared with WT mice, leading to a decrease in the total amount of collagen present within the TIEG1 KO tendons. Moreover, qualitative real-time PCR results showed differences in the expression profiles of specific genes known to play important roles in tendon fiber development. These data further elucidate the role of TIEG1 on tendon structure and could explain the previous defects in the structure-function relationship found for TIEG1 KO tendon fibers.