Helen R. Kim

Estrogen induces apoptosis in estrogen deprivation-resistant breast cancer through stress responses as identified by global gene expression across time

In laboratory studies, acquired resistance to long-term antihormonal therapy in breast cancer evolves through two phases over 5 y. Phase I develops within 1 y, and tumor growth occurs with either 17β-estradiol (E2) or tamoxifen. Phase II resistance develops after 5 y of therapy, and tamoxifen still stimulates growth; however, E2 paradoxically induces apoptosis. This finding is the basis for the clinical use of estrogen to treat advanced antihormone-resistant breast cancer. We interrogated E2-induced apoptosis by analysis of gene expression across time (2–96 h) in MCF-7 cell variants that were estrogen-dependent (WS8) or resistant to estrogen deprivation and refractory (2A) or sensitive (5C) to E2-induced apoptosis. We developed a method termed differential area under the curve analysis that identified genes uniquely regulated by E2 in 5C cells compared with both WS8 and 2A cells and hence, were associated with E2-induced apoptosis. Estrogen signaling, endoplasmic reticulum stress (ERS), and inflammatory response genes were overrepresented among the 5C-specific genes. The identified ERS genes indicated that E2 inhibited protein folding, translation, and fatty acid synthesis. Meanwhile, the ERS-associated apoptotic genes Bcl-2 interacting mediator of cell death (BIM; BCL2L11) and caspase-4 (CASP4), among others, were induced. Evaluation of a caspase peptide inhibitor panel showed that the CASP4 inhibitor z-LEVD-fmk was the most active at blocking E2-induced apoptosis. Furthermore, z-LEVD-fmk completely prevented poly (ADP-ribose) polymerase (PARP) cleavage, E2-inhibited growth, and apoptotic morphology. The up-regulated proinflammatory genes included IL, IFN, and arachidonic acid-related genes. Functional testing showed that arachidonic acid and E2 interacted to superadditively induce apoptosis. Therefore, these data indicate that E2 induced apoptosis through ERS and inflammatory responses in advanced antihormone-resistant breast cancer.

The Selective Estrogen Receptor Modulator Bazedoxifene Inhibits Hormone-Independent Breast Cancer Cell Growth and Down-Regulates Estrogen Receptor α and Cyclin D1

Bazedoxifene (BZA) is a third-generation selective estrogen receptor modulator (SERM) that has been approved for the prevention and treatment of postmenopausal osteoporosis. It has antitumor activity; however, its mechanism of action remains unclear. In the present study, we characterized the effects of BZA and several other SERMs on the proliferation of hormone-dependent MCF-7 and T47D breast cancer cells and hormone-independent MCF-7:5C and MCF-7:2A cells and examined its mechanism of action in these cells. We found that all of the SERMs inhibited the growth of MCF-7, T47D, and MCF-7:2A cells; however, only BZA and fulvestrant (FUL) inhibited the growth of hormone-independent MCF-7:5C cells. Cell cycle analysis revealed that BZA and FUL induced G1 blockade in MCF-7:5C cells; however, BZA down-regulated cyclin D1, which was constitutively overexpressed in these cells, whereas FUL suppressed cyclin A. Further analysis revealed that small interfering RNA knockdown of cyclin D1 reduced the basal growth of MCF-7:5C cells, and it blocked the ability of BZA to induce G1 arrest in these cells. BZA also down-regulated estrogen receptor-α (ERα) protein by increasing its degradation and suppressing cyclin D1 promoter activity in MCF-7:5C cells. Finally, molecular modeling studies demonstrated that BZA bound to ERα in an orientation similar to raloxifene; however, a number of residues adopted different conformations in the induced-fit docking poses compared with the experimental structure of ERα-raloxifene. Together, these findings indicate that BZA is distinct from other SERMs in its ability to inhibit hormone-independent breast cancer cell growth and to regulate ERα and cyclin D1 expression in resistant cells.

Buthionine sulfoximine sensitizes antihormone-resistant human breast cancer cells to estrogen-induced apoptosis.

IntroductionEstrogen deprivation using aromatase inhibitors is one of the standard treatments for postmenopausal women with estrogen receptor (ER)-positive breast cancer. However, one of the consequences of prolonged estrogen suppression is acquired drug resistance. Our group is interested in studying antihormone resistance and has previously reported the development of an estrogen deprived human breast cancer cell line, MCF-7:5C, which undergoes apoptosis in the presence of estradiol. In contrast, another estrogen deprived cell line, MCF-7:2A, appears to have elevated levels of glutathione (GSH) and is resistant to estradiol-induced apoptosis. In the present study, we evaluated whether buthionine sulfoximine (BSO), a potent inhibitor of glutathione (GSH) synthesis, is capable of sensitizing antihormone resistant MCF-7:2A cells to estradiol-induced apoptosis.MethodsEstrogen deprived MCF-7:2A cells were treated with 1 nM 17β-estradiol (E2), 100 μM BSO, or 1 nM E2 + 100 μM BSO combination in vitro, and the effects of these agents on cell growth and apoptosis were evaluated by DNA quantitation assay and annexin V and terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) staining. The in vitro results of the MCF-7:2A cell line were further confirmed in vivo in a mouse xenograft model.ResultsExposure of MCF-7:2A cells to 1 nM E2 plus 100 μM BSO combination for 48 to 96 h produced a sevenfold increase in apoptosis whereas the individual treatments had no significant effect on growth. Induction of apoptosis by the combination treatment of E2 plus BSO was evidenced by changes in Bcl-2 and Bax expression. The combination treatment also markedly increased phosphorylated c-Jun N-terminal kinase (JNK) levels in MCF-7:2A cells and blockade of the JNK pathway attenuated the apoptotic effect of E2 plus BSO. Our in vitro findings corroborated in vivo data from a mouse xenograft model in which daily administration of BSO either as a single agent or in combination with E2 significantly reduced tumor growth of MCF-7:2A cells.ConclusionsOur data indicates that GSH participates in retarding apoptosis in antihormone-resistant human breast cancer cells and that depletion of this molecule by BSO may be critical in predisposing resistant cells to E2-induced apoptotic cell death. We suggest that these data may form the basis of improving therapeutic strategies for the treatment of antihormone resistant ER-positive breast cancer.

Exemestane's 17-hydroxylated metabolite exerts biological effects as an androgen

Aromatase inhibitors (AI) are being evaluated as long-term adjuvant therapies and chemopreventives in breast cancer. However, there are concerns about bone mineral density loss in an estrogen-free environment. Unlike nonsteroidal AIs, the steroidal AI exemestane may exert beneficial effects on bone through its primary metabolite 17-hydroexemestane. We investigated 17-hydroexemestane and observed it bound estrogen receptor α (ERα) very weakly and androgen receptor (AR) strongly. Next, we evaluated 17-hydroexemestane in MCF-7 and T47D breast cancer cells and attributed dependency of its effects on ER or AR using the antiestrogen fulvestrant or the antiandrogen bicalutamide. 17-Hydroexemestane induced proliferation, stimulated cell cycle progression and regulated transcription at high sub-micromolar and micromolar concentrations through ER in both cell lines, but through AR at low nanomolar concentrations selectively in T47D cells. Responses of each cell type to high and low concentrations of the non-aromatizable synthetic androgen R1881 paralleled those of 17-hydroexemestane. 17-Hydroexemestane down-regulated ERα protein levels at high concentrations in a cell type–specific manner similarly as 17β-estradiol, and increased AR protein accumulation at low concentrations in both cell types similarly as R1881. Computer docking indicated that the 17β-OH group of 17-hydroexemestane relative to the 17-keto group of exemestane contributed significantly more intermolecular interaction energy toward binding AR than ERα. Molecular modeling also indicated that 17-hydroexemestane interacted with ERα and AR through selective recognition motifs employed by 17β-estradiol and R1881, respectively. We conclude that 17-hydroexemestane exerts biological effects as an androgen. These results may have important implications for long-term maintenance of patients with AIs. [Mol Cancer Ther 2007;6(11):2817–27]

Overexpression of CEACAM6 promotes migration and invasion of oestrogen-deprived breast cancer cells

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.

Experimental treatment of oestrogen receptor (ER) positive breast cancer with tamoxifen and brivanib alaninate, a VEGFR-2/FGFR-1 kinase inhibitor: A potential clinical application of angiogenesis inhibitors

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.

Modulating therapeutic effects of the c-Src inhibitor via oestrogen receptor and human epidermal growth factor receptor 2 in breast cancer cell lines

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.

New hypotheses and opportunities in endocrine therapy: amplification of oestrogen-induced apoptosis

AIMS To outline the progress being made in the understanding of acquired resistance to long term therapy with the selective oestrogen receptor modulators (SERMs, tamoxifen and raloxifene) and aromatase inhibitors. The question to be addressed is how we can amplify the new biology of oestrogen-induced apoptosis to create more complete responses in exhaustively antihormone treated metastatic breast cancer. METHODS AND RESULTS Three questions are posed and addressed. (1) Do we know how oestrogen works? (2) Can we improve adjuvant antihormonal therapy? (3) Can we enhance oestrogen-induced apoptosis? The new player in oestrogen action is GPR30 and there are new drugs specific for this target to trigger apoptosis. Similarly, anti-angiogenic drugs can be integrated into adjuvant antihormone therapy or to enhance oestrogen-induced apoptosis in Phase II antihormone resistant breast cancer. The goal is to reduce the development of acquired antihormone resistance or undermine the resistance of breast cancer cells to undergo apoptosis with oestrogen respectively. Finally, drugs to reduce the synthesis of glutathione, a subcellular molecule compound associated with drug resistance, can enhance oestradiol-induced apoptosis. CONCLUSIONS We propose an integrated approach for the rapid testing of agents to blunt survival pathways and amplify oestrogen-induced apoptosis and tumour regression in Phase II resistant metastatic breast cancer. This Pharma platform will provide rapid clinical results to predict efficacy in large scale clinical trials.

The Conformation of the Estrogen Receptor Directs Estrogen-Induced Apoptosis in Breast Cancer: A Hypothesis.

Abstract Background: Estrogens are classified as type I (planar) and type II (angular) based on their structures. In this study, we used triphenylethylenes (TPEs) compounds related to 4-hydroxytamoxifen 4OHT to address the hypothesis that the conformation of the liganded estrogen receptor (ERα) can dictate the E2-induced apoptosis of the ER+ breast cancer cells. Materials and methods: ERα positive MCF7:5C cells were used to study apoptosis induced by E2, 4OHT and TPEs. Growth and apoptosis assays were used to evaluate apoptosis and the ability to reverse E2-induced apoptosis. ERα protein was measured by Western blotting to investigate the destruction of ERα by TPEs in MCF7 cells. Chromatin immunoprecipitation (ChIP) assays were performed to study the in vivo recruitment of ERα and SRC3 at classical E2-responsive promoter TFF1 (PS2) by TPEs. Molecular modeling was used to predict the binding mode of the TPE to the ERα. Results: TPEs were not only unable to induce efficient apoptosis in MCF7:5C cells but also reversed the E2-induced apoptosis similar to 4OHT. Furthermore, the TPEs and 4OHT did not reduce the ERα protein levels unlike E2. ChIP assay confirmed very weak recruitment of SRC3 despite modest recruitment of ERα in the presence of TPEs. Mole-ular modeling suggests that TPE would bind in antagonistic mode with ERα. Conclusion: Our results advances the hypothesis that the TPE liganded ERα complex structurally resembles the 4OHT bound ERα and cannot efficiently recruit co-activator SRC3. As a result, the TPE complex cannot induce apoptosis of ER+ breast cancer cells, although it can cause growth of the breast cancer cells. The conformation of the estrogen-ER complex differentially controls growth and apoptosis.

Emerging principles for the development of resistance to antihormonal therapy: Implications for the clinical utility of fulvestrant

We seek to evaluate the clinical consequences of resistance to antihormonal therapy by studying analogous animal xenograft models. Two approaches were taken: (1) MCF-7 tumors were serially transplanted into selective estrogen receptor modulator (SERM)-treated immunocompromised mice to mimic 5 years of SERM treatment. The studies in vivo were designed to replicate the development of acquired resistance to SERMs over years of clinical exposure. (2) MCF-7 cells were cultured long-term under SERM-treated or estrogen withdrawn conditions (to mimic aromatase inhibitors), and then injected into mice to generate endocrine-resistant xenografts. These tumor models have allowed us to define Phase I and Phase II antihormonal resistance according to their responses to E(2) and fulvestrant. Phase I SERM-resistant tumors were growth stimulated in response to estradiol (E(2)), but paradoxically, Phase II SERM and estrogen withdrawn-resistant tumors were growth inhibited by E(2). Fulvestrant did not support growth of Phases I and II SERM-resistant tumors, but did allow growth of Phase II estrogen withdrawn-resistant tumors. Importantly, fulvestrant plus E(2) in Phase II antihormone-resistant tumors reversed the E(2)-induced inhibition and instead resulted in growth stimulation. These data have important clinical implications. Based on these and prior laboratory findings, we propose a clinical strategy for optimal third-line therapy: patients who have responded to and then failed at least two antihormonal treatments may respond favorably to short-term low-dose estrogen due to E(2)-induced apoptosis, followed by treatment with fulvestrant plus an aromatase inhibitor to maintain low tumor burden and avoid a negative interaction between physiologic E(2) and fulvestrant.