Roles and Mechanisms of Estrogen and Estrogen Receptors in Breast Cancer Resistant to Chemotherapy

Breast cancer is the most common malignancy of women in many countries including United States and many European countries. Chemotherapy plays a major role in the treatment of advanced breast cancer, either as an adjuvant to primary therapy or as palliation therapy to improve symptoms and prolong survival. The previous 50 years have seen numerous advances in the properties of chemotherapeutic agents. However, a significant proportion of cancers are inherently unaffected by the administration of anticancer drugs. Furthermore, another considerable proportion of patients undergoing chemotherapy display an initial reduction in tumor size and then relapse with a marked insensitivity to a variety of drugs. Both phenomena are brought about by a resistant phenotype, which presents perhaps the single greatest barrier to successful chemotherapy. Biological mechanisms contributing to drug resistance may be present de novo or arise after exposure to anticancer drugs. At present, drug resistance is considered as a multifactorial phenomenon involving several major mechanisms (1, 2). In general, two main groups of factors contribute to the development of drug resistance. The first group includes pharmacological and physiological factors such as drug metabolism and excretion, inadequate access of the drug to the tumor, inadequate infusion rate and inadequate route of delivery. The second group includes cellor tissue-specific factors. For example, increased repair of DNA damage, reduced apoptotic cell death, altered metabolism of drugs, increased energy-dependent efflux (e.g. ATP-binding cassette transporters) of chemotherapeutic drugs and microRNAs are known factors correlated with the development of anticancer drug resistance (1). In recent years, both clinical observations and experimental studies suggested that steroid hormones and their receptors might also affect the therapeutic efficacy of antineoplastic drugs (3-8).


Introduction
Breast cancer is the most common malignancy of women in many countries including United States and many European countries.Chemotherapy plays a major role in the treatment of advanced breast cancer, either as an adjuvant to primary therapy or as palliation therapy to improve symptoms and prolong survival.The previous 50 years have seen numerous advances in the properties of chemotherapeutic agents.However, a significant proportion of cancers are inherently unaffected by the administration of anticancer drugs.Furthermore, another considerable proportion of patients undergoing chemotherapy display an initial reduction in tumor size and then relapse with a marked insensitivity to a variety of drugs.Both phenomena are brought about by a resistant phenotype, which presents perhaps the single greatest barrier to successful chemotherapy.Biological mechanisms contributing to drug resistance may be present de novo or arise after exposure to anticancer drugs.At present, drug resistance is considered as a multifactorial phenomenon involving several major mechanisms (1,2).In general, two main groups of factors contribute to the development of drug resistance.The first group includes pharmacological and physiological factors such as drug metabolism and excretion, inadequate access of the drug to the tumor, inadequate infusion rate and inadequate route of delivery.The second group includes cell-or tissue-specific factors.For example, increased repair of DNA damage, reduced apoptotic cell death, altered metabolism of drugs, increased energy-dependent efflux (e.g.ATP-binding cassette transporters) of chemotherapeutic drugs and microRNAs are known factors correlated with the development of anticancer drug resistance (1).In recent years, both clinical observations and experimental studies suggested that steroid hormones and their receptors might also affect the therapeutic efficacy of antineoplastic drugs (3)(4)(5)(6)(7)(8).

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Breast Cancer -Current and Alternative Therapeutic Modalities 506 Traditionally, steroid hormones can be grouped into five groups by the receptors to which they bind: glucocorticoids, mineralocorticoids, androgens, estrogens and progestagens (9)(10)(11).Previous studies from our laboratory showed that glucocorticoids, such as dexamethasone, could significantly interfere with the antitumor activities of paclitaxel in vitro and in vivo (3,4,12).Further studies suggest that paclitaxel may induce apoptotic cell death through activation of the NF-B/IB signaling pathway, whereas glucocorticoids inhibit paclitaxel-induced apoptosis through induction of IB synthesis, which antagonizes paclitaxel-mediated activation of NF-B and subsequently results in inhibition of paclitaxelinduced apoptosis (4，13, 14).Considering that cancer patients are routinely pretreated with glucocorticoids (such as dexamethasone) before receiving taxanes (e.g.paclitaxel, docetaxel) to prevent taxane-related hypersensitivity reactions or other adverse effects, the finding of glucocorticoid-mediated inhibition of paclitaxel-induced apoptosis raises a clinically relevant question as to whether pretreatment of glucocorticoids might actually interfere with the therapeutic efficacy of paclitaxel.We have recently reviewed the influence and impact of glucocorticoids on drug-induced apoptosis (4).The cuurent article is largely focused on the role of estrogen and estrogen receptors on the resistance to chemotherapy and the potential strategies to reverse the resistance or sensitize ER+ breast tumors to chemotherapy.

Estrogen and estrogen receptors in the development and treatment of breast cancer
Estrogens, such as 17- estradiol (E2) in human, are steroidal sex hormones that are synthesized from cholesterol and primarily secreted by the ovaries.They play a major role in the development and maintenance of the reproductive tract as well as in the development of the mammary glands.Estrogens also maintain bone density and reduce cardiovascular system by regulating cholesterol levels and influence some brain structures (15,16).However, besides their physiological functions, estrogens are also involved in the development and progression of breast and the uterus cancers and can maintain tumor cell proliferation (15,16).Estrogen action is primarily mediated by two types of estrogen receptors (ERs), i.e.ERand ER.ERs are members of the superfamily of nuclear receptors (17,18).ERs in the cell nucleus mediate the effects of the ligand E2 by functioning as transcriptional regulators that access various target gene promoters either by directly binding to specific estrogen response elements (EREs) within the promoter or indirectly by interacting with other transcriptional regulators bound to the promoter.Further, several cases of ligand-independent activation of ER mediated by its phosphorylation by various signaling pathways have been reported (19).In addition, ER localized in the extra-nuclear compartment (such as the plasma membrane or cytoplasm ER) of target cells, can also mediate several nongenomic effects of estrogen.These non-genomic actions are associated with the activation of a kinase cascade, such as growth factor receptor kinases (e.g.epidermal growth factor receptor).By these means, E2 and ERfacilitate pathways involved in the promotion of cell proliferation, inhibition of apoptosis, stimulation of metastasis, and angiogenesis.Although there is growing evidence that the ERmay inhibit the action of ERby heterodimerizing with it, the overall role of ER in breast cancer remains to be better clarified.A number of reviews have recently been published on the biological roles of estrogens and molecular activities of ERs (15)(16)(17)(18)(19)(20).Unless otherwise specified, ER refers to ERwithin this review.
Cumulative analysis of tumor biopsies has shown that ERs present in ~65% of human breast tumors (21,22).This is consistent with the crucial role of the ERsubtype in breast cancer etiology and progression, and with the role played by estrogens as tumor promoters.It has long been known that breast tumors that express the ER protein (ER+) behave in a fundamentally different fashion than ER-negative (ER-) tumors with regard to their response to hormonal therapies, given that outcomes are often favorable in ER-positive breast tumors treated by adjuvant endocrine therapy alone (23,24).Neoadjuvant chemotherapy has a well-established role in the management of early-stage, operable breast cancer, and remains the gold standard downstaging systemic therapy in many centers, regardless of ER status.However, the data from other clinical trials or retrospective analyses suggest that ER status might also affect the efficacy of chemotherapy (5)(6)(7)(8).Specifically, it has been observed that some chemotherapeutic agents may be less effective in patients with ER+ tumors than those with ER-tumors.Fig. 1.Hypothesized pathways of estrogen/ER-mediated chemoresistance using paclitaxel as an example.and represent inhibitory or antagonistic action.MDR, multidrug resistance.There are possible cross-talks between indicated pathways.

Current understnding of ER-mediated chemoresistance
More than one decade ago, Lippman ME et al first determined the relation between ER and the response rate to cytotoxic chemotherapy in 70 breast cancer patients (6).They found that 34 of 45 patients with low or absent ER values (<10 fmol/mg of cytoplasmic protein) had objective responses to chemotherapy, whereas only 3 of 25 patients with higher ER values (>10 fmol/mg of cytoplasmic protein) responded (p<0.0001).There were no statistically significant differences between the two groups in age, menopausal status, disease-free interval, Karnofsky index or prior therapy.Moreover, differences in sites of involvement or type of combination chemotherapy did not account for the increased response rate in ERpatients.This is the first report suggesting that ER status might be an important predictor of response to cytotoxic chemotherapy in breast tumors.Since then, evidence from clinical trials or retrospective analyses is accumulating that improvements in chemotherapy disproportionately benefit breast cancer patients with ER-tumors, in which multiple chemotherapeutic regimens have been tested in these studies (6,(25)(26)(27)(28)(29)(30)(31)(32)(33), such as taxanes-, anthracycline-and navelbine-containing regimens.More recently, in a retrospective clinical study conducted by us and our collaborators, we found that primary breast cancer patienhts with ER+ tumors achieved significant lower pathologic response than those with ER-breast tumors when treated with preoperative chemotherapeutic regimens including DEC (docetaxel+epirubicin+cyclophosphamide), VFC (vinorelbine/vincristine+5-fluorouracil+ cyclophosphamide) and EFC (epirubicin+5-fluorouracil+cyclophosphamide) (34).The involvement of ER in chemoresistance has also been confirmed in a number of in vitro studies (5,7,8,(35)(36)(37)(38)(39)(40).For example, ER-breast cancer tissue was found chemosensitive in vitro compared with ER+ tissue against six antitumor drugs including carboquone, adriamycin, mitomycin C, aclacinomycin A, cisplatin and 5-fluorouracil (5).When subjected ER+ human breast cancer MCF-7 and ZR-75-1 cells to paclitaxel or to UV irradiation, marked increases in cell apoptosis were induced.However, these responses were significantly reversed by incubation with E2, which was probably mediated through the plasma membrane estrogen receptor (40).Recently, we established several isogenic ER+ cell lines by stable transfection of ERexpression vectors into ER-breast cancer BCap37 cells to investigate the possible influence of ER on the therapeutic efficacy of paclitaxel and vinca alkaloids (7,8).We found that 17- estradiol significantly reduced the overall cytotoxicity of these antimicrotubule drugs in ER-expressing BCap37 but had no influence on the ERparental cells or ER-MDA-MB-468 cells.Further analyses indicate that expression of ERin BCap37 cells mainly interferes with the apoptotic cell death but not mitotic arrest induced by these drugs.Moreover, we found that the addition of ICI 182,780 (fulvestrant), a selective ER down-regulator, could completely reverse the above resistance observed in ER+ BCap37 cells, and sensitize MCF-7 and T47D cell lines to the treatment of the above drugs (see Fig. 2).These findings further confirmed the correlation between ER and drug resistance in ER+ tumor cells.

Possible mechanisms of ER-mediaed chemoresistance
Estrogens and ERs are well-known for their critical roles in the development and progression of breast tumors, through genomic or non-genomic pathways as described above.Plentiful data also indicate that estrogens and ER are involved in or interact with a number of apoptosis-or proliferation-related signal pathways existed in tumor cells.Therefore, it is believed that through interaction with and/or regulation on specific or various co-regulators or downstream molecules, estrogen/ER induce chemoresistance in tumor cells by promoting tumor growth and/or inhibiting the antitumor effect of chemotherapeutic drugs.Several mechanisms that may contribute to ER-mediated drug resistance are discussed below.It appears that the underlying mechanisms of ER-mediated chemoresistance are quite complicated and specifically related with the tumor models and chemotherapeutic drugs studied.

Role of apoptosis-related molecules in ER-mediated chemoresistance
Reduced apoptotic cell death or enhanced tumor cell proliferation are major factors involved in drug resistance.Whereas it is not completely understood how estrogen and ER regulate Fig. 2. ER expression attenuates the anticancer activity of paclitaxel (7).A, protein extracts of BCap37 cells transfected with empty vector (BC-V) or ERwere analyzed by Western blot.T47D and MCF-7 cells were used as positive controls of ER expression.B, cells were treated with 1 nmol/L 17- estradiol, 50 nmol/L paclitaxel, or the combination treatment in which cells were preincubated with 17- estradiol for 12 h before paclitaxel treatment.Cell viability was evaluated by MTT assays after both 48 and 72 h of paclitaxel treatment.BC-ER, pooled transfectants of BCap37 transfected with ER, BC-ER1-7, single clones 1 to 7 of BCap37 transfected with ER; CTL, control; EST, 17- estradiol; PTX, paclitaxel.#, P < 0.05, when compared with the group treated with paclitaxel alone in the same cell line; *, P < 0.001, when compared with the group treated with paclitaxel alone in the same cell line.C, cells treated with 1 nmol/L 17- estradiol, 50 nmol/L paclitaxel, or their combination for indicated time points were harvested, and DNA content stained with propidium iodide for flow cytometric analysis.Peaks corresponding to G1, G2-M, and S phases of the cell cycle and apoptotic cells (AP).
the growth of tumor cells, it is known that hormonal induction of growth factors/receptors such as transforming growth factor  epidermal growth factor, Her-2 contributes to the proliferative actions of E2 (41)(42)(43)(44).Recent studies indicate that several apoptosis-related moleculars or signal pathways, such as bcl-2 and p53, might be involved in E2/ER-mediated resistance to chemotherapy.Expression of the bcl-2 protein prevents apoptotic cell death induced by a variety of stimuli including most chemotherapeutic agents (45)(46)(47).Teixeira C et al demonstrated that depletion of estrogen from the medium results in loss of expression of the bcl-2 in MCF-7 cells, whereas reexposure to estrogen markedly induces the bcl-2 expression (48).Moreover, estrogen depletion, the simultaneous treatment of ICI 164,384, or the transfection of bcl-2 antisense significantly sensitized MCF-7 cells to adriamycin, consistent with a decrease in www.intechopen.comBreast Cancer -Current and Alternative Therapeutic Modalities 510 the bcl-2 levels.Their data suggest that estrogen can promote resistance of ER+ breast cancer cells to chemotherapeutic drugs through a mechanism that involves regulation of the bcl-2, which supports the recent report that bcl-2 expression usually occurs in ER+ breast tumors, whereas ER-breast cancer biopsies tend to lack this protein (48).Another study conducted by Razandi M et al showed that in ER+ human breast cancer cells, the apoptosis, activation of c-JNK, phosphorylation of Bcl-2 and Bcl-xl, activation of caspase induced by paclitaxel or UV radiation were significantly reversed by incubation with E2.E2 also independently activated extracellular signal-regulated protein kinase activity, which contributed to the antiapoptotic effects.In addition, our recent studies also demonstrated that E2 significantly inhibited paclitaxel or vinca alkaloids-induced phosphorylations of bcl-2 and c-raf-1, as well as the degradation of IB in BCap37 cells transfected with ER, which was accompanied with decreased sensitivity of BC-ER cells to the above anticancer drugs (7,8).In response to various extracellular and intracellular signals, p53 mediates cellular processes, such as apoptosis, cell cycle arrest, and senescence, depending on the signal and the cellular context (49)(50)(51).A body of accumulating evidence suggests the possibility of a cross-talk between pathways mediated by ER and p53.Das GM et al demonstrated the direct binding of ER to p53 both in vitro and in vivo to endogenous p53 target gene promoters, which subsequently resulted in inhibition of transcriptional activation by p53 (52).They further showed that ER bound to p53 on endogenous Survivin and MDR1 gene promoters, leading to inhibition of p53-mediated transcriptional repression of these genes.Further, alleviating p53-mediated transcriptional repression of Survivin contributes to the ability of ER to inhibit apoptosis in human breast cancer cells.RNA interference-mediated knockdown of ER resulted in reduced survivin expression and enhanced the propensity of MCF-7 cells to undergo apoptosis in response to staurosporine treatment.These data indicate that countering p53-mediated transcriptional repression of Survivin is at least one of the important mechanisms underlying the antiapoptotic function of ER.

Involvement of tumor growth rate in ER-mediated chemoresistance
Evidence has shown that ER-tumors have a higher growth rate as indicated by a higher labeling index and mitotic index (6).Since many agents used in chemotherapy for breast cancer have some degree of cell-cycle specificity, there might be a correlation between higher growth rate and chemotherapy response (6).Dougherty MK et al used three in vitro models (MCF-7, T47D and ZR-75) to examine and compare growth rates as well as paclitaxel-induced apoptosis in ER+ and ER-clones with the same originate (54).They found that in T47D and ZR-75 cell lines, loss of ER was associated with a decrease in doubling time and an increase in paclitaxel sensitivity.However, when cell culture conditions were altered to achieve equivalent cell proliferation rates, no difference in paclitaxel sensitivity was observed.Similarly, an ER-clone of MCF-7 cells that did not exhibit an enhanced growth rate compared to its ER+ counterpart also did not show increased paclitaxel sensitivity.In these in vitro models, the decreased sensitivity to paclitaxel appears to be correlated closely with the decreased growth rate observed in ER+ breast tumors (54).

ABC transportors and ER-mediated chemoresistance
The most widely studied phenomenon of drug resistance is multidrug resistance (MDR) that has been linked to overexpression of a membrane associated P-glycoprotein (1, 2), a member of ATP-binding cassette (ABC) transporter family that functions as an efflux pump for various structurally unrelated anticancer agents, such as the vinca alkaloids, anthracyclines and taxanes (1,2).Several studies have suggested that ABC transporters might be involved in E2/ER-induced drug resistance.For example, E2 increased the cytoplasmic concentration of P-gp in ER+ breast cancer cells that were resistant to doxorubicin treatment (55).In addition, ABCC11 (MRP8) expression is high in high-expressing ER breast cancers, supporting the notion that expression of ABCC11 in ER+ breast cancers may contribute to decreased sensitivity to chemotherapy combinations (56).Interestingly, Sugimoto Y et al recently reported that both estrogens (57) and antiestrogens (58) inhibit breast cancer resistance protein (BCRP)-mediated drug resistance.They also found that the physiological levels of E2 down-regulate both endogenous and exogenous BCRP expression in ER+ cells by post-transcriptional mechanisms (59).Moreover, they showed that estrogen decreases Pgp expression in MDR1-transduced, ER+ human breast cancer cells, and this E2-mediated Pgp down-regulation sensitizes tumor cells to vincristine.However, it is possible that the effects of estrogen on P-gp expression may differ in ER+ human breast cancer cells expressing endogenous and exogenous P-gp, which needs to be further assessed in appropriate models (60).

Enhanced 3-tubulin expression by E2/ER
It has been suggested that certain changes in cytoskeletons, such as tubulin mutations and isoforms, alterations in microtubule-bindings proteins (e.g.stathmin, tau), as well as enhanced 3-tubulin expression might be correlated with reduced response to antimicrotubule agentbased chemotherapy or worse outcome in a variety of tumor settings.In in vitro studies or in clinical investigations, enhanced expression of 3-tubulin has shown to play a crucial role in the development of chemoresistance to antimicrotubule agents in a variety of tumors such as lung, breast, prostate or orarian cancers (61-66), and has be considered as a predictive marker of paclitaxel resistance (25,(67)(68)(69)(70). Nevertheless, the mechanism underlying 3-tubulin expression still remains unclear.In Drosophila, 3-tubulin expression is enhanced by an exposure to ecdysone, a steroid hormone, through a transcriptional mechanism (71).Recently, Saussede-Aim J et al found that exposure of ER+ MCF-7 cells to estradiol induced 3-tubulin expression in both mRNA and protein levels, while estradiol had no effect on the expression of 3-tubulin in ER-MDA-MB-231 cells (72).They further showed that co-administration of antiestrogens including tamoxifen or fulvestrant, completely abolished the increase of 3tubulin mRNA levels due to estradiol in MCF-7 cells, implying that estradiol regulates 3tubulin expression, and thereby induces resistance of ER+ breast tumors to antimicrotubule drugs through an ER-dependent pathway.

Tumor-host interaction in ER-mediated chemoresistance
Estrogen regulates differentiation, maturation and function of many cell types in monocytemacrophage system directly or indirectly via other cells by autocrine/paracrine mechanisms (73).Estrogen effects on this system are primarily repressive, and mainly mediated by repression of expression of genes for cytokines or modulation of other inflammatory mediators by the ER-dependent or nongenomic pathways.The ER-dependent mechanisms mostly involve modulation of the NF-kappaB pathway for transcriptional regulation of cytokine or other mediator genes.In the context of hormone-regulated cancer, estrogen can influence production of cytokines or other inflammatory mediators by both tumor cells and tumor-invading macrophages (73).The interactions of breast cancer cells with tumor-associated macrophages, regulation of the monocyte-macrophage system by estrogen and cross-talk between the ER and cytokine-mediated pathways, may play an important role in tumor progression as well as the development of resistance to anticancer treatment (73)(74)(75).

Stratagies to sensitize ER+ breast tumors to chemotherapy
Considering that ERs are expressed in ~65% of human breast cancer, the ER-mediated resistance to chemotherapy has become a big challenge for clinical treatment of breast tumors.Unfortunately, despite the fact that the involvement of ER in drug resistance to www.intechopen.comchemotherapy has been observed for more than a decade, very few studies have investigated the potential strategies to reverse the ER-mediated chemoresistance or sensitize ER+ breast tumors to chemotherapy.Because the resistance of ER+ breast tumors to chemotherapy is mainly mediated by activation of estrogen/ER signal pathway, it is logical that agents targeting or inhibiting the ER signal pathway may have the potential to reverse the ER-mediated chemoresistance.Indeed, as described below, a number of studies have shown that antiestrogenic agents in combination with chemotherapeutic drugs are of significant therapeutic benefit in ER+ breast cancer over chemotherapy alone.Moreover, recent investigations indicate that the ER-derived peptide, MicroRNAs specifically targeting ER, as well as agents targeting estrogen-related receptors (ERRs) may hold great promise to sensitize ER+ breast tumors to chemotherapy.

Sensitization of ER+ breast tumors to chemotherapy by SERMs
Selective estrogen receptor modulators (or SERMs) bind ERs but have a mixed agonist/antagonist profile.Tamoxifen and raloxifene are well-known first and second generations of SERMs, respectively (76)(77)(78).New SERMs in clinical development include idoxifene, droloxifene, arzoxifene, acolbifene/EM-800, lasofoxifene, TAT-59, ERA-923, toremifene, GW5638/GW7604, etc (76-78).Kurebayashi J et al found that concurrent treatment of 5-FU and 4-hydroxytamoxifen (4OHT) additively inhibited the growth of ER+ ML-20 and KPL-1 breast cancer cells but not ER-MDA-MB-231 cells (79).They further demonstrated that 4OHT significantly decreased thymidilate synthase activity, which might increase the antitumor activity of 5-FU (79).However, conflicting observations on the interaction between tamoxifen and chemotherapeutic agents including 5-FU and doxorubicin in terms of antitumor activity have been reported by different laboratories (79)(80)(81)(82)(83).In addition, Wu L et al showed that arzoxifene and 4OHT can inhibit specifically the repopulation of ER+ MCF-7 and T47D breast cancer cells between courses of weekly treatment with 5-FU or methotrexate (84).Most recently, they further confirmed that combined treatment with arzoxifene given between cycles of 5-FU or paclitaxel can inhibit repopulation of MCF-7 breast cancer xenografts (85).They proposed that scheduling of short-acting antiestrogenic agents between courses of adjuvant chemotherapy for human breast cancer has potential to improve the outcome of treatment.Additionally, the increased etoposide cytotoxicity by tamoxifen as compared to cells treated with either drug alone was observed in brain tumor HTB-14 cells expressing ER, which was accompanied with enhanced inhibition of protein kinase C (PKC) and insulin-like growth factor II (IGF-II) (86).

Sensitization of ER+ breast tumors to chemotherapy by aromatase inhibitors
One strategy to inhibit the activation of estrogen/ER pathway is to block the conversion of estrogen precursors into estrogen by aromatase inhibitors (AIs) (87).Currently, thirdgeneration aromatase inhibitors, such as the non-steroidal agents anastrozole, letrozole and the steroidal agent exemestane, have been introduced into the market as endocrine therapy in postmenopausal patients, either alone or as part of multiple hormonal therapies (88).In addition to the above AIs, cyclooxygenase (COX) inhibitors also decrease aromatase mRNA expression and enzymatic activity (89).A recent study by Chen D et al showed that the combination of paclitaxel with exemestane produced additive antitumor effect in cultured human breast cancer cell lines.Interestingly, this additive effect was independent of ER expression, but dependent on the presence of androstenedione (90).The effects of AIs on sensitivity of ER+ breast tumors to chemotherapy remains unclear and need to be further investigated.

Sensitization of ER+ breast tumors to chemotherapy by SERDs
The pure antiestrogens, also called selective estrogen receptor downregulators (or SERDs), including fulvestrant (ICI 182, 780), ZK-703, ZK-253, RU58668 and TAS-108, act by decreasing the level of ERs through their ubiquitinylation and subsequent targeting to the proteasome (87).Unlike tamoxifen, fulvestrant is a pure antagonist of estrogen-regulated gene expression that could down-regulat ER expression without any concomitant rise in other growth signal pathways, e.g., EGFR or TGF-.Recently, our laboratory demonstrated that pretreatment with fulvestrant significantly prevented E2-induced resistance to paclitaxel and vinca alkaloids in human breast cancer BCap37 cells transfected with ER-expressing vector (BC-ER) while down-regulates the protein levels of ER in BC-ER cells (7,8).Similar sensitizing effect of fulvestrant was observed in MCF-7 and T47D breast cancer cells expressing endogenous ER (7,8)These results provided additional evidence for the correlation between ER and the resistance of breast tumors to chemotherapeutic drugs such as paclitaxel and vinca alkaloids.More recently, through implanted ER-and ER+ BCap37 cells into athymic nude mice, we established isogenic ER-and ER+ xenograft breast tumor models.Subsequently, we demonstrated that co-treatment of fulvestrant could significantly sensitize ER+ breast tumors to paclitaxel (unpublished data).Because fulvestrant has been successfully used in the treatment of ER+ advanced breast tumors, our experimental results may also suggest the clinical strategy to combine fulvestrant with certain chemotherapeutic drugs for the treatment of ER+ breast tumors.

Other strategies potentially useful for sensitizing ER+ breast tumors to chemotherapy
In addition to the well-known antiestrogens including the SERMs, AIs and SEDMs, studies have been conducted to explore new agents that may interfere the biological responses medicated by E2/ER.One example is the synthesis of ER-derived peptide.Two ER-derived peptides specifically targeting estradiol/ER action, pY-peptide (Ac-Leu-pTyr-Asp-Leu-Leu-Leu-NH2) and Tat-peptide (Ac-EFVCLKSIILLNS-AAA-RKKRRQRRR-NH2) have shown activity to inhibit the growth of ER+ breast tumors in vitro and in vivo (91,92).Moreover, accumulating evidence is revealing an important role of MicroRNAs in anticancer drug resistance (93).Adams et al reported that MicroRNA (miR)-206 could decrease endogenous ER in MCF-7 cells via two specific target sites within the 3'-untranslated region of the human ER transcript (22,94).They further found that miR-206 expression was markedly decreased in ER+ human breast cancer tissues, and that the introduction of miR-206 into estrogen-dependent MCF-7 cells led to the suppression of ERexpression and growth inhibition.These data suggest that miR-206 is a key factor for the regulation of ER expression in breast cancer, which could be a novel candidate for targeting ER (22,94).Nuclear receptor estrogen-related receptor (ERR) family, comprising ERR, ERR and ERR, are the closest relatives to ERafter ER(95).The ERRs share several biochemical activities with ERs, bind and regulate transcription via estrogen response elements (EREs) and extended ERE half-sites termed ERR response elements (ERREs), but do not bind endogenous estrogens.The ERRs act in an analogous fashion as ER, but the effect of ERR binding to an ERE or ERRE can be either negative or positive.ERRlikely plays a role in modulating estrogen responsiveness both by modulating levels of estrogens themselves and expression of estrogen-regulated genes in estrogen target-tissues such as breast cancer.The search for ligands of the ERRs is an active area of research.Targeting ERRs holds great promise and may open new opportunities for the management of breast cancers (95).As described above, the mechanisms underlying ER-mediated chemoresistance involve ERcoregulatory proteins and cross-talk between plasma membrane-localized ER, nuclearlocalized ER and other growth-factor signaling networks, such as EGFR, IGFR, VEGFR and HER2.As a consequence, targeting the ER-coregulators or "cross-talk" pathways may provide opportunities to overcome the ER-mediated chemoresistance, either alone or in combination with agents inhibiting E2/ER activation.However, the mechanisms of ERmediated chemoresistance need to be further clarified so that effective strategies could be developed to sensitize ER+ breast tumors to chemotherapy.Fig. 4. Possible strategies to reverse ER-mediated chemoresistance or sensitize ER positive tumors to chemotherapy.SERMs, selective estrogen receptor modulators; AIs, aromatase inhibitors; SERDs, selective estrogen receptor down-regulators; ERRs, estrogen-related receptors.

Future perspectives
Cumulative data from in vitro experiments and clinical investigations have demonstrated the association between ER expression and the resistance to chemotherapy in breast tumors.However, most of in vitro data were based on comparative studies between the tumor cell lines derived from different individuals.Although some paired cell lines were derived under the selective pressure of a low/no estrogen environments, these tumor cells are still not likely to be isogenic because many features, including their proliferative capacity, might have changed due to genetic alterations (54).Thus, it is difficult to elucidate the cellular and molecular mechanisms.The pairs of isogenic breast cell lines generated by stable transfection of ER or empty vector in our laboratory have provided a valuable model system to investigate the mechanism underlying ER-mediated breast tumor cell resistance to chemotherapeutic agents.Interestingly, we found that estrogen had marginal effect on microtubule dynamics in breast tumor cells expressing ER (BC-ER) treated with paclitaxel and vinca alkaloids, but may decrease the G2-M population through the increase of cells at the G1 phase.This phenomenon is similar to the finding previously reported by Zajchowski et al. (96,97).However, the question still remains whether G1 arrest and decreased G2-M population by estrogen may affect the above drug-induced apoptosis.Further studies are required to elucidate this issue, and it is important to integrate data obtained from breast tumors expressing endogenous ER with those expressing exogenous ER.

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Breast Cancer -Current and Alternative Therapeutic Modalities 516 Some previous reports about the association between ER status and response to chemotherapy can be confused by the use of chemo-endocrine therapy, where the ER+ population may have responded to the hormonal part of the treatment (98,99).Moreover, there are heterogeneous in design, in determination of marker and response evaluation, which could be partly responsible for conflicting results about the predictive and prognostic value of these markers (98)(99)(100)(101)(102)(103).Therefore, some cautions are required when interpret these results, considering that there are many factors need to be taken into account, such as differences in patient selection, whether the patients were previously untreated with chemotherapy or endocrine therapy, type of chemotherapy, size of the study, follow-up time, different evaluation methods, different cut-off value of ER or other related markers, interactions between combined chemotherapeutic drugs, etc.Compared to the available in vitro and clinical reports, very few animal studies have been conducted to determine the role and underlying mechanisms of estrogen and ER in development of chemoresistance, as well as to explorer the potential strategies to reverse the ER-mediated drug resistance.However, appropriate animal models may provide us with easily controlled ways to further evaluate various signal pathways/molecules in vivo, to determine the differences between in vitro and in vivo models, to test chemotherapeutic drugs that we have interests, to investigate agents that may hold promise to sensitize ER+ breast tumors to chemotherapy, either alone or in desirable combinations/sequences.There are less variables need to be taken into account when interpret or analyze the data obtained in animal models compared to clinical patients.The hope to overcome the ER-mediated chemoresistance relies on further clarification of specific pathways or molecules contributing most significantly to the resistance.More exhaustive and systematic attempts to provide this information are essential to reach deeper understandings on ER-mediated chemoresistance in breast tumors.Moreover, it is known that breast cancer patients show a wide range of ER expression levels, and the levels of ER expression in individual patients change during disease progression and/or in response to systemic therapies.Thus, the treatment plan for breast cancer patients might need to be optimized based on the most upto-date molecular characteristics and responses to therapy in individuals.

Fig. 3 .
Fig. 3. ICI 182, 780 abrogates the resistance of ER positive breast tumor cells to paclitaxel (7).BCap37 cells were treated with 1 nmol/L 17- estradiol, 50 nmol/L paclitaxel, 100 nmol/L ICI 182, 780 or their various combinations.MCF-7 and T47D cells were treated with 100 nmol/L 17- estradiol, 500 or 2000 nmol/L paclitaxel, 1mol/L ICI 182, 780 or their various combinations.A, determination of cell viability by MTT assays and apoptosis by DNA fragmentation assays after BC-V and BC-ER cells were exposed to paclitaxel for 48 h.B, effect of 17- estradiol, paclitaxel, ICI 182, 780 and their combinations on the expression of ER and IBin BC-ER cells.C, cell viability of MCF-7 and T47D cells after 72 h of paclitaxel treatment with MTT assays.D, effect of 17- estradiol, paclitaxel, ICI 182, 780 and their combinations on the expression of ERand IB in T47D cells.Proteins were extracted from cells after 24 h of paclitaxel treatment.