Selective estrogen-induced apoptosis in breast cancer

Steroids 90 (2014) 60–70

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Selective estrogen-induced apoptosis in breast cancer Ifeyinwa E. Obiorah, Ping Fan, Surojeet Sengupta, V. Craig Jordan ⇑ Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, United States

a r t i c l e

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Article history: Available online 11 June 2014 Keywords: Estradiol Apoptosis Inflammation Unfolded protein response Endoplasmicreticulum stress Breast cancer

a b s t r a c t Antihormone therapy remains the gold standard of care in the treatment of estrogen receptor (ER) positive breast cancer. However, development of acquired long term antihormone resistance exposes a vulnerability to estrogen that induces apoptosis. Laboratory and clinical studies indicate that successful therapy with estrogens is dependent on the duration of estrogen withdrawal and menopausal status of a woman. Interrogation of estradiol (E2) induced apoptosis using molecular studies indicate treatment of long term estrogen deprived MCF-7 breast cancer cells with estrogen causes an endoplasmic reticulum stress response that induces an unfolded protein response signal to inhibit protein translation. E2 binds to the ER and mediates apoptosis through the classical genomic pathway. Furthermore, the induction of apoptosis by estrogens is dependent on the conformation of the estrogen–ER complex. In this review, we explore the mechanism and the processes involved in the paradox of estrogen induced apoptosis and the new selectivity of estrogen action on different cell populations that is correctly been deciphered for clinical practice. Ó 2014 Published by Elsevier Inc.

1. Introduction Ovarian estrogen in premenopausal patients or estrogens produced by peripheral aromatization of adrenal androgenic precursors in postmenopausal patients support the growth of breast cancer. As a result of this knowledge, treatment practices evolved throughout the 20th century to either remove the source of estrogen synthesis by ablative surgery (oophorectomy, adrenalectomy or hypophysectomy) or block the actions of estrogen which stimulates tumor growth through the breast tumor estrogen receptor (ER)-signal transduction system [1]. Two clinical approaches to breast cancer therapy have proved to be successful [2]: either the development of nonsteroidal antiestrogens that block estrogen binding to the ER or the development of aromatase inhibitors which block the peripheral aromatase enzyme system that convert steroidal precursors from the adrenals to estrogens. Both therapeutic advances have resulted in dramatic increases in patient survival if the nonsteroidal antiestrogen tamoxifen or an aromatase inhibitor is given for extended periods (5–10 years) as an adjuvant therapy [3–5]. There is compelling support for the proposition that estrogen is an essential component for the development of breast cancer and is ⇑ Corresponding author. Address: Georgetown University Medical Center, 3970 Reservoir Rd NW. Research Building, Suite E501, Washington, DC 20057, United States. Tel.: +1 202 687 2897; fax: +1 202 687 6402. E-mail address: [email protected] (V.C. Jordan). http://dx.doi.org/10.1016/j.steroids.2014.06.003 0039-128X/Ó 2014 Published by Elsevier Inc.

essential for the promotion and replication of breast cancer cells. The first evidence that there was a link between estrogen and the development of breast cancer was presented at the annual meeting of the American Association for Cancer Research in Boston in 1936. Professor Antoine Lacassagne [6] presented his vision of the prevention of breast cancer in the future based on the results he had obtained in laboratory animals by either administering estrogens to develop mammary cancer [7] or removing estrogen through ovariectomy to prevent mammary cancer in high incident strains: ‘‘if one accepts the consideration of adenocarcinoma of the breast as the consequences of a special hereditary sensibility to the proliferative actions of oestrone, one is led to imagine a therapeutic preventive for subjects predisposed by their heredity to this cancer. It would consistin the near future when knowledge and use of hormones will be better understood- in the suitable use of a hormone antagonist to prevent the stagnation of estrone in the ducts of the breast.’’ This visionary strategy became a reality with the development of the nonsteroidal antiestrogen, tamoxifen for the treatment of breast cancer [8] and the successful testing of its worth in high risk women to reduce the incidence of breast cancer [9,10]. Thus, the use of an ‘‘antiestrogen’’ to prevent the development of breast cancer was further proof of the critical role of estrogen in the process of breast carcinogenesis. Today, two selective ER modulators (SERMs) tamoxifen and raloxifene are available for the chemoprevention of breast cancer in both the United States and United Kingdom [11,12]. The final proof of the direct role of estrogen to stimulate breast cancer cell proliferation came from the laboratory. Initially it was

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difficult to demonstrate that estrogen directly caused the replication of breast cancer cells in vitro but growth of ER positive tumors could be demonstrated in vivo if the same ER positive MCF7 breast cancer cells were inoculated into athymic (immune deficient mice) and treated with estrogen [13]. It was to remain a mystery why exogenous estrogen could not stimulate MCF-7 breast cancer cells to grow in vitro until Berthois and colleagues [14] made the landmark discovery that MCF-7 cells had been routinely maintained for more than a decade in media containing high concentrations of phenol red as a pH indicator. Phenol red or a contaminant was actually an estrogen, so cells were already growing maximally. Removal of the phenol red from the media stopped cell replication and the cells now had a robust proliferation in response to exogenous estrogen. The evidence of the relevance of the critical role of endogenous estrogen being necessary for breast cancer development and growth was overwhelming but there was an unexplained paradox lurking in the historical record of therapeutics. The first successful therapy used to treat any cancer was the use of high dose estrogen to treat metastatic breast cancer in postmenopausal patients [15]. The encouraging initial trial was the result of laboratory studies, so the treatment strategy was based on translational research. However, the clinical research went a step further. Haddow used this preliminary data [15] to conduct a multicentric clinical trial through the Royal Society of Medicine. He made a discovery: ‘‘When the various reports were assembled at the end of that time, it was fascinating to discover that rather general impression, not sufficiently strong from the relatively small numbers in any single group, became reinforced to the point of certainty; namely, the beneficial responses were three times more frequent in women over the age of 60 years than in those under that age; that estrogens may, on the contrary, accelerate the course of mammary cancer in younger women, and that their therapeutic use should be restricted to cases 5 years beyond the menopause. Here was an early and satisfying example of the advantages which may accrue from cooperative clinical trial’’ [16]. Dr. Basil Stoll [17] was able to quantify this finding within his own clinical practice and demonstrated that patients more than 5 years beyond menopause had a high probability of a response to high dose estrogen therapy but those less than five years from menopause were unlikely to have a responsive tumor (Table 1). High dose estrogen therapy for the treatment of metastatic breast cancer in postmenopausal women became the standard of care for 30 years [18–20] until the advent of tamoxifen. Response rates to tamoxifen for postmenopausal patients with metastatic breast cancer were similar at 30% [21,22], but side effects with tamoxifen were much less severe. This allowed tamoxifen to advance as long term adjuvant therapy and ultimately be shown to save lives. However, in 1970 at the dawn of interest ICI 46, 474 (to become tamoxifen) as an experimental antiestrogen for the treatment of breast cancer, Sir Alexander Haddow was selected to present the inaugural Karnofsky lecture at the American Society of Clinical Oncology [16]. His article paints a gloomy picture for the future of targeted cancer therapies and the remote prospects of success for anticancer agents as had been achieved with the selective toxicity of antibiotics for the cure of infectious disease. He did

Table 1 Objective response rates in postmenopausal women with metastatic breast cancer using high dose estrogen therapy. The 407 patients are divided in relation to menopausal status [17]. The objective remission rate of breast cancer tumors was higher in women more than 5 years postmenopausal. Reprinted with permission from Obiorah I and Jordan VC. Menopause 2013; 20: 372–382. Age since menopause

Patient number

% Regression

Postmenopausal 0–5 Years Postmenopausal >5 Years

63 344

9 35

61

however highlight a potential glimmer of hope with his statement that reflected upon the pioneering success he had achieved with his discovery of high dose estrogen treatment as the first ‘‘chemical therapy’’ in cancer. ‘‘The extraordinary extent of tumour regression observed in perhaps 1% of post-menopausal cases (with oestrogen) has always been regarded as of major theoretical importance, and it is a matter for some disappointment that so much of the underlying mechanism continues to elude us. . .. . .’’ [16]. However, resolution of the paradox of the antitumor actions of estrogen was to be discarded and dismissed with the refocusing on the accepted paradigm of the obvious understanding of the antitumor action of antiestrogen, tamoxifen. It is therefore ironic that through the clinical development of tamoxifen as a long term adjuvant therapy and the necessity to examine the evolution of acquired resistance to long term tamoxifen, that the veil should be lifted on Haddow’s paradox and the new biology of estrogeninduced apoptosis be discovered. 2. The evolution of acquired resistance to SERMs The first clinically relevant models of acquired resistance to tamoxifen were developed by inoculating MCF-7 cells into ovarectomized athymic mice and initially treating with estrogen for a short time to establish palpable tumors. Continuous tamoxifen treatment of the tumor bearing mice resulted in the growth of tumors despite tamoxifen treatment [23]. However, the finding through retransplantation studies that tumors grew because of tamoxifen treatment and also continued to respond to estrogen for growth [24,25], recapitulated acquired resistance to tamoxifen therapy in the treatment of metastatic breast cancer. Tamoxifen treatment fails in a year or two [21,22], tumors exhibit a ‘‘withdrawal’’ response from tamoxifen [26,27] so this is tamoxifen stimulated growth. Finally estrogen can still maintain tumor growth following the cessation of tamoxifen. Second line therapies with either an aromatase inhibitor to prevent estrogen synthesis or a pure antiestrogen fulvestrant to destroy ER, are effective second line therapies [28,29]. However, the characteristics of the model of acquired resistance did not explain why tamoxifen could be given effectively as a long term adjuvant therapy. If this model was true for all acquired resistance, micrometastatic disease would fail to be controlled for more than two years of adjuvant therapy. It was the retransplantation of tumors into new generations of athymic mice for at least 5 years that was to result in the discovery of a new biology of estrogen action: estrogen induced apoptosis. 3. The antitumor action of physiologic estrogen The finding that acquired resistance to tamoxifen evolves through phases also demonstrates that the cell selection pressure of tamoxifen and its metabolites exposes a vulnerability in cell populations that struggle to survive in a long term tamoxifen (antiestrogen environment) environment. Estrogen triggers apoptosis after 5 years of retransplantation in tamoxifen-treated mice [30,31]. Numerous publications in vivo with SERM (tamoxifen or raloxifene) stimulated tumors [32–35] were of value to document biological control mechanisms. The key to advancing the mechanistic understanding of estrogen induced apoptosis and the reasons for selectivity in breast cancer occurred with the development of long term estrogen deprived cell models to replicate resistance to aromatase inhibitors during adjuvant therapy. 4. Cellular models of estrogen deprivation in vitro To decipher the mechanism involved in antihormone resistance following long term treatment, antiestrogen resistant clonal

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variants of MCF-7 cells: MCF7:5C and MCF7:2A were established after long-term culture in estrogen free media [36,37] or long term estrogen deprived (LTED) MCF-7 cell populations examined [38]. The variant clone, MCF7:5Ccells express wild type ER but have drastically reduced levels of progesterone receptor (PR) when compared to the parent MCF-7 cells. Using DNA quantification assays 17b estradiol (E2) drastically reduced the growth of the MCF7:5C cells in a time dependent manner that resulted in 90% inhibition after six days of treatment [39]. The observed E2 induced inhibition in cell proliferation was confirmed to be apoptosis using annexin v-FITC and DNA binding dye, DAPI staining methods [39,40]. Although fulvestrant partially inhibited the growth of the MCF7:5C cells, this biological effect was not due to apoptosis. On the other hand, these cells are resistant to 4-hydroxytamoxifen (4OHT), while fulvestrant caused 40% growth inhibition. The induction of E2 induced apoptosis in vitro raised the question of its ability to induce tumor regression in vivo. MCF7:5C cells injected into athymic nude mice resulted in detectable spontaneously growing tumors within 4 weeks. Treatment of the MCF7:5C tumors with E2resulted in complete regression after 4 weeks of therapy. Involvement of apoptosis in the E2 induced tumor reduction was determined using TUNEL assay. Another clone, MCF7:2A was identified and characterized from long term estrogen deprived MCF-7 breast cancer cells [37]. Significant growth inhibition is observed in the second week of treatment with E2 [41]. Similar to the MCF7:5C cells, the MCF7:2A cells grow maximally in the absence of estrogens [37,41]. However, the MCF7:2A are inhibited by both antiestrogens 4OHT and fulvestrant and these cells are both ER/PR positive [37]. Because the MCF7:2A cells were initially resistant to E2 induced apoptosis with proapoptotic genes activated much later than in MCF7:5C cells [42], potential mechanisms of action for this resistance were explored. Glutathione (GSH), a tripeptide has been implicated in the tumorigenesis and progression of breast cancer [43]. Elevated levels of GSH were observed in MCF7:2A cells and microarray studies show high levels of glutathione synthetase and glutathione peroxidase 2 [41,42,44]. Both enzymes are involved in GSH synthesis. Depletion of the cells of GSH using L-buthionine sulfoximine (BSO), a GSH inhibitor, sensitized the MCF7:2A cells to E2 induced apoptosis [42,44,45]. Therefore, utilization of BSO with estrogen in patients with ER positive metastatic breast cancer in the context of a clinical trial could potentially inhibit disease progression in patients with exhaustive antihormone resistance. 5. Differential effects of estrogens in MCF7:5C cells Based on the fact that the ER is the major signaling pathway for breast cancer growth and apoptosis, a series of planar and angular estrogens (Fig. 1) were evaluated for their ability to trigger apoptosis in the MCF7:5C cells. Estrogens can be classified into class I (planar) and class II (angular) estrogens [46,47] based on the reported crystal structure of the ligand binding domain (LBD) ER with estrogens (E2, diethylstilbestrol) and antiestrogens (4OHT and raloxifene) [48,49]. The planar estrogens are sealed within the LBD by helix 12 to induce estrogenic action, whereas the bulky side chains of 4OHT and raloxifene prevent helix 12 from sealing the LBD resulting in antiestrogen action. We previously synthesized a range of estrogenic angular triphenylethylenes (TPEs) which are structurally similar to 4OHT. The TPEs (bisphenol (BP), ethoxytriphenylethylene (EtOX) and trihydroxytriphenylethylene (3OHTPE) cause proliferation of MCF-7 cells [50] at higher concentrations when compared to the planar estrogens. First, we compared the ability of bisphenol A (BPA), a planar estrogen to stimulate growth in MCF7 cells and induce apoptosis in the MCF7:5C cells to BP, an angular TPE [51]. The TPE, BP is a more potent estrogen than BPA in stimulating MCF-7 cell growth.

Despite the fact that BPA is only a weak estrogen, it inhibited the growth of MCF7:5C cells at high concentration at the end of a 7 day assay, whereas BP did not readily induce apoptosis at this time point but rather blocked E2 induced apoptosis. Planar estrogens, E2 and BPA induce similar apoptosis related genes by 48 h of treatment, whereas BP did not induce apoptosis genes but rather the pattern of genes down regulated by BP resembles the pattern observed with E2 and BPA. To further investigate the paradox of how BP which is fully estrogenic in MCF-7 cells but appeared to have antiestrogen-like in MCF7:5C cells [51], we examined the relationship whereby the structure of an estrogenic ligand can affect their ability to induce apoptosis by using a range of compounds [52]. Planar estrogens, which included E2, DES and constituents of conjugated equine estrogens (equilin, estrone and equilenin), prevented the growth and induced apoptosis of MCF7:5C cells even though they all caused cell proliferation of the MCF-7 cells [52,53]. This corresponds with what is observed in clinical practice. Clinical studies [54,55] have shown that thirty percent of postmenopausal patients with metastatic breast cancer show an objective response with estrogen therapy after undergoing exhaustive antihormone therapy. Similarly a persistent decrease was noted in the incidence and mortality of breast cancer in postmenopausal women from the Women Health Initiative trial, who were treated with conjugated equine estrogen (CEE) when compared to those on placebo [56,57]. In contrast, all TPEs, BP, EtOX and 3OHTPE did not readily induce apoptosis at the end of the first week but rather blocked E2 induced apoptosis in a similar manner as the selective estrogen receptor modulators (SERMS) [52]. However the TPEs were all able to induce apoptosis after 14 days of treatment, whereas the SERMS remain antiestrogenic in the MCF7:5C cells, and do not trigger apoptosis. We previously observed that E2 induced apoptosis is a slow process [41], therefore we sought to compare its antiproliferative effects to that of a classic cytotoxic chemotherapy [58]. Paclitaxel caused 50% growth reduction and almost 100% growth inhibition of the MCF7:5C cells by 24 h and 48 h respectively [58]. By contrast, inhibition of cell proliferation was not observed until after 72 h of E2 treatment and 80% growth inhibition was seen at 120 h [58]. Apoptosis was quantified using annexin v staining after 12 h of treatment with paclitaxel, while an apoptotic response was only detected with E2 treatment after 72 h. Interestingly in a related study, we found that BP was only able to significant prevent cell proliferation after 9 days of treatment, whereas apoptosis was determined to be starting after 6 days of treatment [59]. It is evident that the initial response of the MCF7:5C cells to E2 and BP is cell proliferation and this was confirmed using cell cycle flow cytometry studies. Both estrogens caused a persistent increase in S phase in the first 96 h following treatment. This contrasted with paclitaxel which causes a rapid G2/M blockade and apoptosis by 12 h. It became important to determine the point at which apoptosis is triggered for planar estrogen E2 [58] and angular TPE, BP [59] i.e. the time after which the cell is committed to apoptosis. The critical trigger point of apoptosis for E2 occurred between 24 h and 36 h, while that of BP occurred after 4 days of treatment. The delayed biological actions indicate that estrogen induced apoptosis involves a multidynamic process that is dramatically distinct from that of a chemotherapeutic drug such as paclitaxel. The conformation of the ER complex regulates the time at which the cell commits to apoptosis. 6. Importance of the conformation of the ligand-ER complex involved in estrogen induced apoptosis The angular TPEs possess short-term antiestrogenic properties in MCF7:5C cells similar to those of the 4OHT, which indicate that

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CLASS I (PLANAR) ESTROGENS

A

17β Estradiol (E2)

CLASS I I (ANGULAR)TRIPHENYLETHYLENE ESTROGENS

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Diethylslbestrol (DES) Bisphenol (BP) Trihydroxytriphenylethylene(3OHTPE)

Equilin

Equilenin

Ethoxytriphenylethylene(EtOX) Estrone

Bisphenol A (BPA) SELECTIVE ESTROGEN RECEPTOR MODULATORS (SERMS)

C

4-hydroxytamoxifen(4OHT)

Endoxifen (endox)

Raloxifene(ral)

Bazedoxifene(baze)

Fig. 1. Chemical structures of compounds.(A)Planar estrogens (B) Angular Triphenylethylenes(C) Selective estrogen receptor modulators.

they make an antiestrogenic like conformation with the ER. The recruitment of ER a or coactivator, SRC3 (AIB1) at the promoter of pS2 (TFF1) gene, an estrogen responsive gene, by the planar and angular estrogens was determined using chromatin immunoprecipitation assays in MCF-7 and MCF7:5C cells. Planar estrogens, E2 and BPA readily recruited high levels of ERa to the pS2 promoter [51,52]. On the other hand, TPEs were about 50% efficient as the planar estrogen in the recruitment of ERa, whereas 4OHT showed much lower levels of ERa recruitment to the promoter. SRC3 plays a key role in the transcriptional regulation of E2 induced growth [60–62] and apoptosis [63] in breast cancer cells. Recruitment of SRC3 shows a similar pattern as the ERa following treatment with the planar estrogens, TPEs and 4OHT. Planar estrogens show high levels of SRC recruitment, whereas 4OHT show no recruitment to the pS2 promoter and the TPEs show variable low levels of recruitment that lie between that of the planar estrogens and 4OHT. This indicates that the conformation of the TPE-ER complex results in a moderate reduction of ERa binding to the ERE region of the pS2 promoter and a severe inhibition of SCR3 binding when compared to the planar estrogens. The reduction of SCR3 recruitment observed with the TPEs correlates with another study, where Bourgoin-Voillard and colleagues [64] discovered that class II estrogens such as BP had a reduced tendency to induce recruitment of coactivators containing LxxLL motif, thus suggesting that the TPE:ER complex appears to be ‘‘antiestrogen-like’’ when compared to 4OHT. Furthermore molecular modelling data indicate that TPEs would bind to the ER in an antagonist conformation in a similar manner to that observed with 4OHT based on X-ray

crystallography [49]. These data suggest that the antiestrogenic conformation may be responsible for the initial retardation of TPE induced apoptosis. Nevertheless, the molecular dynamics of the TPE:ER complex must eventually create accumulated cellular damage to trigger apoptosis. 7. The modulation of c-Src on estrogen induced apoptosis c-Src is a non-receptor tyrosine kinase that plays a crucial role in signaling cascades that control cell growth, angiogenesis, invasion adhesion and metastasis and act as an adaptor protein in the crosstalk between the ER and growth factors such as the EGFR family [65,66]. Many of the proliferative actions of estrogen are dependent on c-Src [66]. The multiple involvement of c-Src in many intracellular signaling pathways, such as the mitogen- activated protein kinase (MAPK) and the phosphoinositide 3-kinase (PI3K) pathways makes it a potential therapeutic target in breast cancer cells. Elevated c-Src activity has been noted in tamoxifen resistant breast cancer cells and treatment of these cells with a c-Src inhibitor suppresses growth, invasion and motility of the endocrine resistant cells [67,68]. However, treatment with therapeutic c-Src inhibitors shows either modest or limited activity in patients with advanced breast cancer [69–71]. Because c-Src alone is not sufficient to cause oncogenic transformation, improvement in the value of a c-Src inhibitor could be achieved in combination with other targeted therapies. Due to the fact that we have previously shown that E2 induces apoptosis in the MCF7:5C cells, we reasoned that combination of PP2, an experimental c-Src inhibitor,

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+ -

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p-c-SrcTyr416 total-c-Src

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15 10 5 0

Fig. 2. The c-Src inhibitor blocked E2 activated nongenomic pathway. (A) E2 stimulated c-Src after 24 h treatment. (B) MCF-7:5C cells were treated with vehicle (0.1% DMSO), E2, 4-OHT, E2 plus 4-OHT, PP2, E2 plus PP2 respectively for 72 h and the cells were harvested for Annexin V binding assay through flow cytometry. The percentage of Annexin V binding was compared with control. P < 0.05, *compared with control. Reprinted with permission from Fan et al. Cancer Research 2013; 73: 4510–4520.

3

ER

2

Phosphorylaon Cascade

ER

ESTRADIOL 1

NON GENOMIC PATHWAY

GENOMIC PATHWAY

(APOPTOSIS)

Fig. 3. E2 induced estrogen receptor signaling. 1. The genomic mechanism of ER signaling is by estrogen binding to the nuclear ER and then binding to hormone response elements in the promoters of target genes (classic) or through protein–protein tethering with nuclear DNA-binding transcription factors (non-classic) to alter gene transcription. 2. E2 can act through nongenomic signaling by activating cell surface membrane localized extranuclear ER. 3. estrogen dendrimer conjugate(EDC) specifically activate the nongenomic signaling of ER action.

and E2 will potentiate the apoptotic effect of E2. Surprisingly, although PP2 was able to block E2 induction of c-Src (Fig. 2A), PP2 failed to induce apoptosis but rather blocked E2 apoptosis (Fig. 2B) [72], which was confirmed using siRNA to knockdown c-Src in MCF7:5C cells which resulted in a reduction of E2 induced apoptosis [73]. These data indicates that E2 may trigger apoptosis via the nongenomicc-Src signaling pathway. This hypothesis, though unlikely due to the long delay in estrogen induced apoptosis observed previously [58], was addressed using a novel reagent supplied by Dr. John Katzenellenbogen [74].

8. E2 induced apoptosis is through the genomic pathway in MCF7:5C cells Estrogens can exert their effects by either classically binding to the nuclear ER and hormone response elements to alter gene transcription (genomic pathway) or by acting through nongenomic signaling via cell surface membrane localized extranuclear ER (Fig. 3). The role of the nongenomic pathway in ER signaling was evaluated in E2 induced apoptosis. Estrogen dendrimer conjugate (EDC), a synthetic ligand that only interacts with the extra-nuclear ER to

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120 p-c-SrcTyr416

p-MAPK

total-c-Src

total-MAPK

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p-c-SrcTyr416 total-c-Src

total MAPK Fig. 4. Effect of estrogen dendrimer conjugate(EDC) on estrogen induced apoptosis. (A) E2,activates pS2, an estrogen responsive gene, but EDC and the empty dendrimer does not. (B) Cell growth was inhibited by E2 treatment whereas growth was enhanced by EDC and unaffected by the empty dendrimer. (C) The c-Src inhibitor blocked EDC activated nongenomic pathway via inhibition of EDC induced phosphorylation of MAPK. (D) EDC rapidly activated MAPK and c-Src in MCF-7:5C cells. (E) EDC activated signaling pathways after 24 h. Reprinted with permission from Fan et al. Cancer Research 2013; 73: 4510–4520.

induce nongenomic signaling was used to activate the nongenomic pathway [74]. EDC was neither able to activate the estrogen responsive gene, pS2 (Fig. 4A) nor induce apoptosis in the MCF7:5C cells (Fig. 4B). Nevertheless, EDC did activate the nongenomic pathway via induction of phosphorylated c-Src, MAPK and AKT in the MCF7:5C cells and PP2 blocked the EDC activated nongenomic pathway (Fig. 4C–E). This suggests that the nongenomic signaling pathway is not crucial for E2 induced apoptosis but that E2immediately activates the nongenomic pathway within minutes with subsequent activation of the genomic pathway. On the other hand, E2 induces ERE activity which can be blocked by 4OHT but not by PP2. Inhibition of c-Src increased expression of classic ER targeted genes such as pS2, by increasing the accumulated ER. C-Src is important for phosphorylation and degradation of ER [73]. Selective induction of AP-1 complexes, consisting of c-Fos, c-Jun and Jund, were activated by E2 in MCF7:5C cells suggesting that AP-1 may play an important role in E2 mediated apoptosis. 9. Estrogen induced endoplasmic reticulum stress and unfolded protein response Differential regulation of global gene expression and identification of genes and potential signaling pathways associated with E2 induced apoptosis was interrogated using Agilent microarray studies. The major groups of MCF7:5C specific genes overrepresented include estrogen signaling, endoplasmicreticulum stress (ERS)

and inflammatory response genes [41] and functional testing indicate that ERS and inflammatory stress response led to apoptosis. Endoplasmic reticulum is the key site for the synthesis and folding of proteins. Disturbances of the homeostasis within the endoplasmic reticulum can lead to accumulation of unfolded proteins that result in ERS. In order to overcome, a number of responses occur within the endoplasmic reticulum [75,76]. The first response is the synthesis of new proteins and prevention of accumulation of unfolded proteins [77]. Next chaperone proteins, such as BiP/GRP78, trigger an unfolded protein response (UPR) to relieve the ERS. Under normal conditions, BiP binds to unfolded proteins, PERK, ATF6 and IRE1 and maintains them in an inactive state. Under stress conditions, BiP dissociates from the UPR proteins and allows their oligomerization and autophosphorylation to initiate a UPR signal that serves to prevent protein synthesis and induce transportation of malfolded proteins to the cytosol for degradation. The UPR signal causes activation of PERK, ATF6 and IRE1. Activation of PERK induces phosphorylation of eIF2a resulting in inhibition of protein synthesis and translocation into the lumen of the endoplasmic reticulum [75,76]. Dissociation of BiP from ATF6 results in its transport to the golgi apparatus where it undergoes cleavage and translocate to the nucleus to induce transcription of UPR genes [75,78] such as XBP-1. Activated IRE1 induces splicing of XBP-1 which can now efficiently activate UPR [79,80]. Under severe or prolonged ERS, the UPR signal switches from cell survival to apoptosis. The regulation of UPR genes was evaluated

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in the MCF7:5C cells in response to E2. Significant induction of UPR sensors, IRE1a and PERK/eIF2a by E2 was detected after 24 h of treatment which further increased by prolongation of treatment to 72 h [73]. Treatment of the MCF7:5C cells with a PERK inhibitor blocked phosphorylation of eIF2a and blocked E2 induced apoptosis, thus confirming that ERS is necessary for E2 triggered apoptosis [73].

Apoptosis-related genes PPP1R15A HOMX1 SGK1 ZAK FOSL2 HIPK2 LGALS1 LGALS7B NUAK2

Stress-related genes

Indicator of Oxidative stress

10. Apoptotic pathways mediated by estrogens We previously reported that MCF7:5C cells respond to E2 by suppressing ERa signaling leading to activation of ERS and inflammatory stress [41]. One of the apoptosis pathways associated with ERS mediated apoptosis is via activation of DDIT3/CHOP [75,81]. Studies suggest that PERK/eIF2a and IRE1a/ATF6 signaling are necessary for maximum induction of DDIT3 [82–84]. Overexpression of DDIT3 leads to a decrease in bcl-2 protein and translocation of Bax protein from the cytosol to the mitochondria [85,86]. Puthalakath and colleagues [87] reported that ERS induced by diverse stimuli required Bim for initiating apoptosis in a variety of cell lines including MCF-7 cells. Knockdown of Bim expression resulted in protection from ERS induced apoptosis. Increased Bim levels noted with ERS induction was dependent on transcriptional activation of DDIT3 [87]. Therefore prolonged ERS potentially leads to activation of BCL2L11/Bim and Bax. The involvement of the intrinsic pathway in E2 induced apoptosis was first reported by

TP63

TP53-related genes

PMAIP1 CYFIP2 LTB FAS

Inflammation-related genes

TNFRSF21 TNFRSF11B

TNF family related genes

NGFR (TNFSF16) CXCR4 Fig. 5. Classification of E2-induced apoptosis-related genes selected by RNA-seq in MCF-7:5C cells. E2 induced stress related genes, TP53 related genes and inflammation related genes. Reprinted with permission from Fan et al. Cancer Research 2013; 73: 4510–4520.

Relative PPP1R15A levels

5

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Fig. 6. The c-Src inhibitor blocked apoptosis-related genes induced by E2. Apoptosis related genes: (A) PPP1R15A (GADD34) gene, (B) BCL2L11 (Bim) gene, (C) NUAK2 gene, (D) PMAIP1 (Noxa) gene, identified with RNA-seq analysis were confirmed using real-time PCR. P < 0.001, ** compared with control. Reprinted with permission from Fan et al. Cancer Research 2013; 73: 4510–4520.

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Fig. 7. The success of estrogen replacement therapy is dependent on menopausal status of a woman. A. Estrogen withdrawal in postmenopausal women causes ER positive dependent cells to die but some cells continue to grow independent of estrogen. B Treatment of women immediately after menopause with CEE results in sustained growth of nascent ER positive tumors, whereas treatment 5 years after menopause causes apoptotic cell death. Reprinted with permission from Obiorah I and Jordan VC. Menopause 2013; 20: 372–382.

Lewis and colleagues [40] who showed that E2 treatment increased expression of proapoptotic proteins including, Bax, Bak, Bim, Noxa, Puma and p53. Depletion of Bim and Bax using short interfering RNAs (siRNAs) reversed the apoptotic effect of E2. Furthermore mitochondrial pathway activity was determined by loss of mitochondrial potential, increase in cytochrome c release and cleavage of caspase 9 and poly ADP ribose polymerase (PARP) protein. The Fas/Fasl signaling (extrinsic) pathway has been implicated in E2 induced apoptosis. Song and colleagues [38] reported elevated levels of Fas in long term estrogen deprived (LTED)MCF-7 cells and a marked increase of FasL noted with E2 treatment. This correlated with the report from Osipo et al. [32] which showed that E2 induced reduction of tamoxifen resistant breast cancer tumors, by activating Fas expression and suppressing NF-kB and HER2/neu activity. A similar observation was noted in raloxifene resistant MCF7 cells [33]. The growth of cells in vitro and in vivo was repressed by E2 by increasing induction of Fas expression and reducing expression of NF-kB. Although there is obvious involvement of both intrinsic and extrinsic pathways in E2 induced apoptosis, none of the previous studies investigated a time course of the sequence of activation of the apoptotic pathways in estrogen induced apoptosis. Because the trigger of E2 induced apoptosis was observed to occur after 24 h [58], differential regulation of apoptotic gene expression was interrogated at 36 h and 48 h in response to E2using polymerase chain reaction (PCR) arrays. At 36 h, E2 induced ERS and proinflammatory related genes [58]. DDIT3 was one of the highest inducible genes during E2 mediated ERS. This is similar to what is observed in several microarray or PCR array studies [51,84] that analyzed differential gene expression associated with ERS. As expected, IRE1a was also upregulated at 36 h, giving further evidence of its involvement in the UPR induced by ERS. In addition, Bim expression was increased indicating an early involvement of the mitochondrial pathway possibly mediated by DDIT3. The gene expression expanded to involve the TNF

family of genes such as, Fas and TNF and continued increased expression of Bim, ERS and proinflammmatory genes at 48 h of E2 treatment [58]. However, a prolonged induction of ERS and inflammatory stress response was observed in the first week of treatment with BP and induction of caspase 4, an inflammatory caspase and downstream target of ERS, after 5 days of treatment [59]. Induction of both mitochondrial and extramitochondrial apoptotic related genes were activated after 7 days of treatment. The delay in induction of genes correlates with the more pronounced delayed apoptosis noted with BP when compared to that of E2 [59]. Furthermore, RNA-seq analysis of genes regulated by E2 [73] revealed a range of apoptosis-related genes functionally classified into: TP53-related genes, stress related genes and inflammatory response genes (Fig. 5). The majority of these apoptosisrelated genes observed with the RNA-seq experiments were confirmed with real-time-PCR (Fig. 6). 11. Conclusions Low physiologic concentrations of estrogen induce apoptosis in LTED MCF-7 cells. The laboratory phenomenon translates well with observation in clinical treatment of postmenopausal women with advanced antihormone resistant breast cancer. In postmenopausal women with metastatic breast cancer and acquired resistance to aromatase inhibitors, a daily dose of 6 mg of estradiol provided a similar clinical benefit rate (28% vs. 29%) as 30 mg [54,55]. Loning and colleagues [55] found an objective response in 30 percent of postmenopausal breast cancer patients with previous exhaustive antihormone therapy who received high dose diethylstilbestrol (15 mg). Estrogen deprivation is necessary to sensitize breast cancer tumor cell to estrogen treatment and subsequent tumor reduction. Estrogens induce cell proliferation of fully estrogenised MCF-7 cells after 3 days of culture in estrogen free medium because the cell population has adapted to an estrogen rich (phenol red) environment and will grow with a resupply of estrogens

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Recruited Response to Estrogen Inial Response to Estrogen

4

Intrinsic pathway Unfolded protein response

3

extrinsic pathway E2 Estrogen

TNF family

Ca 2+ Inflammaon

Bcl2 protein

FADD Mitochondria

Caspase 8 Endoplasmic reculum

Cytochrome C

5

Apaf1

2

Caspase 9 c-Fos/c-Jun Caspase 4 Caspase 12

Caspase 6 Caspase 7

1 E2

ER Apoptosis

Acvated receptor

ER Unliganded receptor

DEATH Fig. 8. The mechanism of E2 induced apoptosis. 1. Activation of ER by E2 induces activation AP-1 complex. 2. Endoplasmic reticulum stress caused accumulation of unfolded proteins that stimulates a UPR signal. 3. Failure to combat ERS induces apoptosis via induction of the mitochondrial pathway. 4. Subsequent activation of the extrinsic pathway of apoptosis occurs through the TNF family of proapoptotic genes. 5. Apoptosis can occur independent of the intrinsic and extrinsic pathway through activation of caspase 4.

(Fig. 7B). Therefore the ability of estrogens to induce apoptosis to treat or prevent breast tumors is dependent on the menopausal state of a woman and the duration of estrogen deprivation [53]. Beneficial responses with estrogen administration were noted in women over the age of 60 [15,57] and 35% remission rate of breast tumors was observed in women more than 5 years postmenopausal when compared to women who were less than 5 years postmenopausal (9%) [17]. The cells vulnerable to E2 mediated apoptosis have been selected because estrogen deprivation that occurs at menopause causes death of estrogen dependent nascent breast cancer cells (Fig. 7A). The surviving cells grow independent of estrogens and may induce breast cancer tumors unless exogenous estrogens induce apoptotic death. Therefore it may be best to use estrogens as a preventive therapy after 5 years of menopause. The mechanism of E2 induced apoptosis has been extensively investigated (Fig. 8). EDC was not able to activate estrogen targeted genes, nor was it able induces apoptosis in the MCF7:5C cells confirming that the nongenomic pathway was not critical for E2 triggered apoptosis. E2 appears to induce apoptosis through the genomic pathway. Treatment of the MCF7:5C cells with estrogens [58,59] induces an initial reaction of cell growth. However, there is a subsequent induction of ERS within 24 h of treatment with E2. In order to relieve ERS, UPR sensors PERK, ATF6 and IRE1a are activated. PERK serves to prevent protein translation via phosphorylation of eIF2a, whereas ATF6 and IRE1a act to upregulate UPR related genes. When UPR fails to contain ERS, apoptosis is activated in a number a ways (Fig. 8). Severe ERS induces apoptosis through activation of Bim which initiates the signaling cascade associated with the intrinsic mitochondrial pathway. Subsequent induction of the extrinsic pathway of apoptosis occurs through the TNF family of apoptosis related genes. ERS also induces apoptosis via activation of caspase 4. Inhibition of caspase 4 using a specific caspase 4 inhibitor abolished both E2 [41] and BP [59] induced apoptosis. Estrogens have been classified based on the conformation they create with the ER [46,47]. Planar estrogens are sealed within the LBD of the ER and coactivators are readily recruited resulting in a more rapid induction of apoptosis in the MCF7:5C cells. TPEs adopt an antiestrogen-like conformation with ER with reduced

coactivator recruitment which may be responsible to their ability to initially block E2 induced apoptosis. This correlates with the observed prolonged ERS with BP treatment and induction of apoptosis via caspase 4 after 5 days of treatment and subsequent induction of intrinsic and extrinsic pathway after 7 days of treatment. The fact that TPEs, though structurally similar to 4OHT, finally induce apoptosis is reassuring because TPEs were among the successful chemical therapies used by Haddow [15] to treat postmenopausal women with advanced breast cancer. In conclusion, estrogen induced apoptosis is a delayed process when compared to that of a classic cytotoxic chemotherapy. However, the multi-faceted but relentless process involved ultimately results in the paradoxical induction of cell death induced by estrogens. This mechanism plays a potential role in the chemoprevention of breast cancer with estrogen therapy alone leads to increased patient survival if used at least five years after menopause [53,57]. Here is the key to selective estrogen induced apoptosis in breast cancer. Occult breast cancer cells grow robustly in a replete environment of estrogen but die quickly at menopause when estrogen is decreased. Only cells that are adapted by trial and error will grow in an estrogen depleted environment (Fig. 7A). Estrogen given immediately after menopause (within 5 years) will maintain the cell replication of populations adapted to a replete environment but administration of estrogen to populations adapted to growing in an estrogen deprived environment will trigger estrogen induced apoptosis (Fig. 7B). Estrogen is selective in its action based on the environmental adaptation of the population of breast cancer cells. Acknowledgements This work (VCJ) was supported by the Department of Defense Breast Program under Award number W81XWH-06-1-0590 Center of Excellence; the Susan G. Komen for the Cure Foundation under Award number SAC100009, the Lombardi Comprehensive Cancer 1095 Center Support Grant (CCSG) Core Grant NIH P30 CA051008. The views and opinions of the author(s) do not reflect those of the US Army or the Department of Defense.

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