Effects of a new clinically relevant antiestrogen (GW5638) related to tamoxifen on breast and endometrial cancer growth in vivo.

PURPOSE: Cross-resistance is an important issue for the evaluation of new antiestrogens to treat advanced breast cancer patients who have failed tamoxifen therapy. In addition, postmenopausal patients treated with long-term adjuvant tamoxifen show a 3-4-fold increase in the risk of developing endometrial cancer. Consequently, a new second line agent should be more antiestrogenic and less estrogen-like on the uterus, and be effective at controlling the growth of breast cancer after exposure to tamoxifen. The purpose was to evaluate the effects of the new tamoxifen analogue GW5638 on breast and endometrial cancer growth. EXPERIMENTAL DESIGN: Athymic mice were transplanted with an endometrial tumor model (ECC-1 E2) that is responsive to estrogen and has never been exposed to antiestrogen. In addition, we used three breast tumor models: a tamoxifen-na?ve tumor (T47D-E2) and two tamoxifen-stimulated tumors (MT2 TAM and MCF-7 TAM LT). The antiestrogen GW5638 (1.5 mg daily), tamoxifen (0.5 mg or 1.5 mg daily), and raloxifene (1.5 mg daily) were given p.o. The pure antiestrogen ICI182,780 (5 mg once a week) was given s.c. Western blots from MCF-7 TAM breast tumors were performed to demonstrate the regulation of estrogen receptor alpha expression by different ligands. RESULTS: Estradiol and GW5638 down-regulated the receptor compared with control. ICI182,780 completely degraded the receptor but tamoxifen had no effect. GW5638 did not promote tumor growth, and was effective in blocking the effects of postmenopausal estradiol on the growth of tamoxifen-na?ve breast and endometrial tumors. However, raloxifene did not completely block the effects of postmenopausal estradiol on the growth of tamoxifen-na?ve endometrial tumor after 14 weeks. GW5638 and ICI182,780 but not raloxifene were also effective in blocking the tamoxifen-stimulated breast tumor growth in athymic mice. CONCLUSIONS: GW5638 is more effective than raloxifene in blocking the effect of estrogen on tamoxifen-na?ve endometrial cancer. More importantly, GW5638, like the pure antiestrogen ICI182,780, is able to block the growth of breast cancer stimulated by tamoxifen differently from raloxifene. GW5638 down-regulates estrogen receptor but does not completely destroy the receptor. Therefore, based on our findings, GW5638 could be developed as a second line agent for advanced breast cancer patients and an important first line agent to evaluate as an adjuvant treatment or chemopreventive.     

Tamoxifen and ICI 182,780 increase Bcl-2 levels and inhibit growth of breast carcinoma cells by modulating PI3K/AKT, ERK and IGF-1R pathways independent of ERα

We recently showed that estrogen withdrawal from the ERα⁺, high Bcl-2-expressing breast carcinoma cells (MCF-7B) reduced Bcl-2 protein levels while increasing cell–cell adhesion, and junction formation. Here we compared these cells with the ERα⁺ and low Bcl-2-expressing MCF-7 cells and with the normal mammary epithelial cell line MCF-10-2A not expressing ERα or Bcl-2. All cell lines expressed normal HER2. Antiestrogen (Tamoxifen and ICI 182,780) treatment increased Bcl-2 levels in both MCF-7 and -7B cells and led to the formation of acinar structures. This treatment led to the dissociation of junctions and redistribution of junctional components to the cytoplasm in MCF-10-2A and -7 cells, while in MCF-7B cells junctional proteins redistributed to membranes. Antiestrogen treatment decreased PI3K/Akt activation and increased ERK activation regardless of ERα status. IGF-1R was inactivated in the antiestrogen-treated MCF-7 cells while it was activated in MCF-7B cells. Our data show that Tamoxifen and ICI 182,780 can induce growth inhibitory effects via the sustained activation/inactivation of signaling pathways that regulate cell survival, cell death and differentiation in the absence of ERα. Furthermore, Bcl-2 overexpression may alter the functional interactions among these pathways in response to antiestrogens, which also may provide a potential explanation for the observation that Bcl-2 overexpressing tumors have a better prognosis.     

Gene expression preferentially regulated by tamoxifen in breast cancer cells and correlations with clinical outcome

The beneficial effect of the selective estrogen receptor (ER) modulator tamoxifen in the treatment and prevention of breast cancer is assumed to be through its ability to antagonize the stimulatory actions of estrogen, although tamoxifen can also have some estrogen-like agonist effects. Here, we report that, in addition to these mixed agonist/antagonist actions, tamoxifen can also selectively regulate a unique set of >60 genes, which are minimally regulated by estradiol (E2) or raloxifene in ERalpha-positive MCF-7 human breast cancer cells. This gene regulation by tamoxifen is mediated by ERalpha and reversed by E2 or ICI 182,780. Introduction of ERbeta into MCF-7 cells reverses tamoxifen action on approximately 75% of these genes. To examine whether these genes might serve as markers of tamoxifen sensitivity and/or the development of resistance, their expression level was examined in breast cancers of women who had received adjuvant therapy with tamoxifen. High expression of two of the tamoxifen-stimulated genes, YWHAZ/14-3-3z and LOC441453, was found to correlate significantly with disease recurrence following tamoxifen treatment in women with ER-positive cancers and hence seem to be markers of a poor prognosis. Our data indicate a new dimension in tamoxifen action, involving gene expression regulation that is tamoxifen preferential, and identify genes that might serve as markers of tumor responsiveness or resistance to tamoxifen therapy. This may have a potential effect on the choice of tamoxifen versus aromatase inhibitors as adjuvant endocrine therapy.     

In vitro regulation of vascular endothelial growth factor by estrogens and antiestrogens in estrogen-receptor positive breast cancer

BACKGROUND: The effects of antiestrogens on angiogenesis in breast cancer are not fully defined. In this study we investigated the in vitro effects of antiestrogens at different concentrations on vascular endothelial growth factor (VEGF) production in estrogen receptor (ER)-positive breast cancer cells. METHODS: The dose-dependent effects of 17beta-estradiol (E2), 4-hydroxytamoxifen (4OHT), and ICI182,780 were analyzed both with reference to growth rates and VEGF protein production using enzyme-linked immunosorbent assay (ELISA) in MCF-7 cells. RESULTS: E2 stimulated both the growth rates and VEGF production of MCF-7 cells in the same manner. Although 4OHT stimulated the growth rates as an agonistic effect in an estrogen-free media at levels ranging from 1 nM to 1 micro M, it did not stimulate VEGF expression at the same levels except for at 1 micro M. Although 4OHT had a weak agonistic effect on VEGF production at 1 micro M in an estrogen-free media, it significantly inhibited E2-stimulated VEGF production at the same level. A cytotoxic effect was observed with 10 micro M 4OHT that paradoxically caused a prominent increase in VEGF production. ICI182,780 had no significant effects on the growth rates or VEGF production in this cell line. CONCLUSIONS: These results support the hypothesis that tamoxifen could inhibit angiogenesis induced by estrogens in ER-positive breast cancer cells.     

The effects of aromatase inhibitors and antiestrogens in the nude mouse model

The effects of antiestrogens, tamoxifen and ICI 182,780, and aromatase inhibitors, arimidex (anastrozole ZD1033) and letrozole (CGS 20,267), on the growth of tumors were studied in nude mice. In this model, estrogen dependent MCF-7 human breast cancer cells stably transfected with the aromatase gene were inoculated in four sites per mouse. Sufficient estrogen is produced from aromatization of androstenedione supplement (0.1 mg/mouse/day) by the cells to stimulate their proliferation, tumor formation, and maintain the uterus similar to that of the intact mouse. Once the tumors reached a measurable size, the mice were injected with antiestrogen or inhibitor for 35–56 days. Tumor volumes were measured weekly. At autopsy, the tumors were removed, cleaned, and weighed. Statistical data was determined from tumor weights. Both antiestrogens were effective in reducing tumor growth in these mice. Tamoxifen appears to be more effective than ICI 182,780, although the former stimulated the uterine weight whereas the pure antiestrogen did not. However, both aromatase inhibitors were more effective than the antiestrogens. Tumor regression was observed with letrozole. Thus, after-treatment tumor weights were less than those of a group of mice at the start of treatment. The aromatase inhibitors also reduced the weight of the uterus, suggesting that these compounds, as well as the pure antiestrogen, may not cause endometrial proliferation, unlike tamoxifen. These aromatase inhibitors may not only benefit patients who have relapsed from tamoxifen, but may be more effective in patients as first line agents for suppressing the effects of estrogen.     

Cooperative effects of Akt-1 and Raf-1 on the induction of cellular senescence in doxorubicin or tamoxifen treated breast cancer cells

Escape from cellular senescence induction is a potent mechanism for chemoresistance. Cellular senescence can be induced in breast cancer cell lines by the removal of estrogen signaling with tamoxifen or by the accumulation of DNA damage induced by the chemotherapeutic drug doxorubicin. Long term culturing of the hormone-sensitive breast cancer cell line MCF-7 in doxorubicin (MCF-7/DoxR) reduced the ability of doxorubicin, but not tamoxifen, to induce senescence. Two pathways that are often upregulated in chemo- and hormonal-resistance are the PI3K/PTEN/Akt/mTOR and Ras/Raf/MEK/ERK pathways. To determine if active Akt-1 and Raf-1 can influence drug-induced senescence, we stably introduced activated ΔAkt-1(CA) and ΔRaf-1(CA) into drug-sensitive and doxorubicin-resistant cells. Expression of a constitutively-active Raf-1 construct resulted in higher baseline senescence, indicating these cells possessed the ability to undergo oncogene-induced-senescence. Constitutive activation of the Akt pathway significantly decreased drug-induced senescence in response to doxorubicin but not tamoxifen in MCF-7 cells. However, constitutive Akt-1 activation in drug-resistant cells containing high levels of active ERK completely escaped cellular senescence induced by doxorubicin and tamoxifen. These results indicate that up regulation of the Ras/PI3K/PTEN/Akt/mTOR pathway in the presence of elevated Ras/Raf/MEK/ERK signaling together can contribute to drug-resistance by diminishing cell senescence in response to chemotherapy. Understanding how breast cancers containing certain oncogenic mutations escape cell senescence in response to chemotherapy and hormonal based therapies may provide insights into the design of more effective drug combinations for the treatment of breast cancer.     

A potent specific pure antiestrogen with clinical potential.

Previous studies from this laboratory have described a series of 7 alpha-alkylamide analogues of estradiol with pure antiestrogenic activity, exemplified by ICI 164,384. A new compound, 7 alpha-[9-(4,4,5,5,5-pentafluoropentylsulfinyl)nonyl]estra-1,3,5(10 )- triene-3,17 beta-diol (ICI 182,780) has now been identified which has significantly increased antiestrogenic potency and retains pure estrogen antagonist activity. The antiuterotrophic potency of ICI 182,780 in the immature rat was more than 10-fold greater than that of ICI 164,384 (50% effective doses of 0.06 and 0.9 mg/kg, respectively). This order of magnitude increase of in vivo potency was also reflected, in part, by intrinsic activity at the estrogen receptor. The relative binding affinities of ICI 182,780 and ICI 164,384 were 0.89 and 0.19, respectively, compared with that of estradiol (1.0). Similarly, the in vitro growth-inhibitory potency of ICI 182,780 exceeded that of ICI 164,384 in MCF-7 human breast cancer cells, where 50% inhibitory concentrations of 0.29 and 1.3 nM, respectively, were recorded. ICI 182,780 was a more effective inhibitor of MCF-7 growth than 4'-hydroxytamoxifen, producing an 80% reduction of cell number under conditions where 4'-hydroxytamoxifen achieved a maximum of 50% inhibition. This increased efficacy was reflected by a greater reduction of the proportion of cells engaged in DNA synthesis in ICI 182,780-treated cell cultures compared with tamoxifen-treated cells. Sustained antiestrogenic effects, following a single parenteral dose of ICI 182,780 in oil suspension, were apparent in both rats and pigtail monkeys. In vivo, antitumor activity of ICI 182,780 was demonstrated with xenografts of MCF-7 and Br10 human breast cancers in nude mice. A single injection of ICI 182,780 provided antitumor efficacy equivalent to that of daily tamoxifen treatment for at least 4 weeks. The properties of ICI 182,780 identify this pure antiestrogen as a prime candidate with which to evaluate the potential therapeutic benefits of complete estrogen withdrawal in endocrine-responsive human breast cancer.     

ICI 182,780 (Faslodex): development of a novel, "pure" antiestrogen

BACKGROUND: The nonsteroidal antiestrogen tamoxifen is well established as an effective treatment for patients with breast carcinoma, both for the treatment of metastatic disease and as an adjuvant to surgery for patients with primary breast carcinoma. In addition to exerting antagonistic effects on the estrogen receptor, tamoxifen and its derivatives act as partial agonists on certain tissues. These agonistic effects, for example, endometrial stimulation and stimulation of tumor growth after previous response to tamoxifen, may limit their clinical efficacy. ICI 182,780 (Faslodex) from AstraZeneca (Cheshire, United Kingdom) is a novel, steroidal estrogen antagonist that was designed to be devoid of estrogen agonist activity in preclinical models. METHODS: ICI 182,780 was tested in a large number of in vitro and in vivo preclinical models, and its value was assessed clinically when administered before surgery for breast carcinoma and hysterectomy for benign conditions and after failure of tamoxifen in patients with advanced breast carcinoma. RESULTS: All data indicated that ICI 182,780 is devoid of agonist activity in preclinical models and in clinical trials. It inhibits growth of the breast and endometrium. In animal models, it does not cross the blood-brain barrier and appears to be neutral with respect to lipids and bone. ICI 182,780 down-regulates the estrogen receptor and is active in tamoxifen-resistant breast carcinoma. In a small, Phase II study, durable responses were seen: Phase III clinical trials are in progress comparing ICI 182,780 with anastrozole and tamoxifen in the treatment of patients with advanced breast carcinoma. CONCLUSIONS: ICI 182,780 specifically down-regulates the estrogen receptor and, thus, represents the first of a new class of therapeutic agents. In this report, the authors present the current evidence that distinguishes ICI 182,780 from tamoxifen and related nonsteroidal compounds and establishes ICI 182,780 as the first in a new class of therapeutic agents.     

Antiestrogens modulate MT1 melatonin receptor expression in breast and ovarian cancer cell lines

An interaction between cellular estrogen response and melatonin signaling mediated by G-protein coupled receptors is present in breast cancer cells. In this study, the effect of antiestrogens on basal and melatonin-modulated expression of MT1 melatonin receptor in breast and ovarian cancer cells was examined. For this purpose, the effects of the selective estrogen receptor modulator tamoxifen and pure antiestrogen ICI 182,780 on MT1 expression in estrogen receptor (ER) alpha-positive and -negative breast and ovarian cancer cell lines cultured in medium supplemented with 1 nM 17-beta estradiol were assessed by Western blot analysis. We were able to detect expression of the MT1 receptor in SK-OV-3 and OVCAR-3 cells and report its up-regulation by melatonin in both ovarian cancer cell lines. MT1 expression was observed to be significantly weaker in ERalpha-positive MCF-7 and OVCAR-3 cells than in ERalpha-negative MDA-MB-231 and SK-OV-3 cells. Treatment with the pure antiestrogen ICI 182,780 increased MT1 receptor expression in OVCAR-3 ovarian cancer cells, but decreased MT1 expression in MCF-7 breast cancer cells. No effect of ICI 182,780 on MT1 expression was observed in the ERalpha-negative cell lines SK-OV-3 and MDA-MB-231. After treatment with 4-OH tamoxifen, down-regulation of basal MT1 receptor expression in ERalpha-positive MCF-7 cells and inhibition of melatonin-induced up-regulation of MT1 in OVCAR-3 ovarian cancer cells were observed. In contrast, treatment with 4-OH tamoxifen increased the MT1 receptor level in ERalpha-negative SK-OV-3 ovarian cancer cells. Our findings support the existence of close interaction between estrogen and melatonin signaling. Moreover, our data suggest that melatonin signaling is modulated by antiestrogens in breast and ovarian cancer cells.     

Low-dose dietary genistein negates the therapeutic effect of tamoxifen in athymic nude mice

The present study examined the effect of dietary genistein, a soy isoflavone, on breast cancer patients who take tamoxifen, an antiestrogen treatment, using a preclinical model. The interaction of various doses of genistein with tamoxifen on the growth of estrogen receptor-positive breast cancer MCF-7 cells was investigated by subcutaneously injecting MCF-7 cells into the flank of ovariectomized athymic mice. Animals were randomized into eight experimental groups with 10-13 mice per group: control (C), estrogen (E) (0.08 mg E implant), tamoxifen (T) (3 mg T implant), estrogen + tamoxifen (E + T), tamoxifen + 500 p.p.m. genistein (T + G500), estrogen + tamoxifen + 250 p.p.m. genistein (E + T + G250), estrogen + tamoxifen + 500 p.p.m. genistein (E + T + G500) and estrogen + tamoxifen + 1000 p.p.m. genistein (E + T + G1000). Treatment of tamoxifen significantly reduced the estrogen-induced MCF-7 tumor prevalence and tumor size. This inhibitory effect of tamoxifen was significantly negated by the low doses of dietary genistein (250 and 500 p.p.m.), whereas the 1000 p.p.m. genistein did not have the same effect. Cells harvested from tamoxifen-treated tumors retained estrogen responsiveness of their progenitor MCF-7 cells, indicating that the abrogating effect of genistein on tamoxifen-treated tumor growth was not caused by a diminished tamoxifen response but directly by genistein. The low doses of dietary genistein abrogated the inhibitory effect of tamoxifen potentially by acting on the tumor cell proliferation/apoptosis ratio and the messenger RNA (mRNA) expression of cyclin D1 in addition to regulating the mRNA expression of progesterone receptor. Therefore, data from the current study suggest that caution is warranted regarding the consumption of dietary genistein by breast cancer patients while on tamoxifen therapy.     

Human breast cancer cell lines resistant to pure anti-estrogens are sensitive to tamoxifen treatment

The pure steroidal anti-estrogens ICI 164,384 and ICI 182,780 are very potent growth inhibitors of the estrogen receptor-positive human breast cancer cell line MCF-7. However, long-term treatment of MCF-7 cells with 10^?7 M concentrations of these compounds results in selection of proliferating colonies of resistant cells. Our report describes 4 ICI 164,384- and 3 ICI 182,780-resistant MCF-7 sublines established after long-term treatment. Resistant sublines are estrogen receptor-positive, and all sublines have lost expression and estrogen inducibility of the progesterone receptor protein. Based on IC₅₀ concentrations, all tested resistant sublines had a reduced sensitivity to pure anti-estrogens on the order of 100- to 1000-fold compared with parent MCF-7 cells. All resistant cell lines have survived propagation for more than 15 subcultivations in the presence of 10^?7 M pure anti-estrogen. The MCF-7/182^R-6 subline has been tested for stability of resistance and appeared to be stably resistant after 13 weeks of propagation without the selective pressure of ICI 182,780. Cell lines resistant to the ICI 182,780 compound are cross-resistant to the ICI 164,384 compound and vice versa. However, the sublines resistant to pure anti-estrogens are sensitive to tamoxifen. Our results show that although the pure steroidal anti-estrogens are very potent growth inhibitors, they do not circumvent development of resistance. © 1995 Wiley-Liss, Inc.     

(-)-Epigallocatechin-3-gallate downregulates estrogen receptor alpha function in MCF-7 breast carcinoma cells

Background: (−)-Epigallocatechin-3-gallate (EGCG) is the most active catechin present in green tea, demonstrated to have chemopreventive action and to kill cancer cells selectively. As a previous study found that catechins could compete with 17-β-estradiol for binding to estrogen receptor alpha (ERα), we asked whether EGCG could regulate ERα action. Methods: We used MCF-7, a breast carcinoma cell line having a high level of ERα expression. The cells were treated with various EGCG concentrations and cell viability was evaluated by MTT assay. ERα and pS2 expression were analyzed by RT-PCR after RNA extraction. To better define EGCG action in relation to ERα, we studied EGCG cytotoxicity on MCF-7 resistant to tamoxifen (MCF-7tam), MCF-7 treated with 10⁻⁷ M ICI 182,780 for 8 days and on MDA-MB-231, a cell line that lacked ERα by flow cytometry (FCM). Results: Both ERα and pS2 mRNA were expressed in samples treated with low EGCG concentration (30 μg/ml). At this concentration, no cell change was detectable. In contrast, pS2 expression was lost in samples treated with 100 μg/ml EGCG for 24 h, indicating ERα alteration. EGCG cytotoxicity was lower when ERα was not present (MDA-MB-231) or inactivated (by tamoxifen or ICI 182,780). Conclusions: Functionally active ERα may have a role in EGCG cytotoxicity, increasing the sensitivity to the drug. As higher EGCG concentrations also killed cells resistant to tamoxifen or treated by 10⁻⁷ M ICI 182,780, EGCG ought to be better investigated in breast carcinoma cells treated with drugs targeted to steroid receptors, as a potential complement of therapy.     

Cross-resistance of triphenylethylene-type antiestrogens but not ICI 182,780 in tamoxifen-stimulated breast tumors grown in athymic mice.

The triphenylethylene antiestrogens, idoxifene (Idox) and toremifene (Tor), are structurally related analogues of tamoxifen (Tam) and were developed to improve the therapeutic index for advanced breast cancer patients. However, the issue of cross-resistance with Tam for these new agents is critical for clinical testing because the majority of breast cancer patients have already received or failed adjuvant Tam. The goal of this study was to determine the effectiveness of Idox as an antitumor agent in three models of Tam-stimulated breast cancer in athymic mice and compare the results with the actions of Tor and ICI 182,780 in a Tam-stimulated MCF-7 tumor model. We first compared the activities of Tam and Idox in the 17beta-estradiol (E2)-stimulated MCF-7 tumor line MT2:E2. Tam and Idox reduced E2-stimulated growth by 65-70% (week 9: P = 0.0009 for Tam, P = 0.0005 for Idox versus E2 alone). However, Tam (1.5 mg daily) and Idox (1.0 mg daily) both produced T47D breast tumors in athymic mice during 23 weeks of treatment (12 tumors/22 sites and 15 tumors/18 sites, respectively). Tam and Idox stimulated tumor growth equally in two different Tam-stimulated MCF-7 models and in a T47D model. Tor was completely cross-resistant with Tam in the MCF-7 tumor model, which implied that neither Idox nor Tor would be effective as a second-line endocrine therapy after Tam failure and may offer no therapeutic advantages over Tam as adjuvant therapies. In contrast, ICI 182,780, a pure antiestrogen currently being tested as a treatment for breast cancer after Tam failure, had no growth-stimulatory effect on the MCF-7 Tam-stimulated breast tumor line. This agent may provide an advantage as an adjuvant therapy and increase the time to treatment failure.     

Acquired antiestrogen resistance in MCF-7 human breast cancer sublines is not accomplished by altered expression of receptors in the ErbB-family

Development of acquired resistance against antiestrogen treatment is a serious problem in human breast cancer, and knowledge of alterations resulting in resistance is important for selection of further treatment. To mimic the clinical situation we have established a series of MCF-7 human breast cancer cell lines by long term treatment with the antiestrogens tamoxifen, ICI 164,384, and ICI 182,780. Common for these cell lines is a decreased expression of the estrogen receptor (ER). In human breast cancer, lack of response to endocrine therapy is often associated with decreased expression of the estrogen receptor and increased expression of epidermal growth factor receptor (EGFR) and/or HER-2/neu (ErbB-2). Our antiestrogen resistant cell lines did not express altered levels of EGFR, HER-2/neu, ErbB-3, or ErbB-4. Estrogen and antiestrogen regulation of HER-2/neu expression was essentially similar in parent and resistant MCF-7 cells. Treatment with antibodies to HER-2/neu (Herceptin^TM) did not affect growth of MCF-7 cells or resistant cells, indicating that in this in vitro model system, acquired antiestrogen resistance does not emerge from activation of the HER-2/neu signaling pathway. In MCF-7 cells transfected with HER-2/neu and/or ErbB-3, overexpression alone did not result in resistance. However, addition of heregulinl-1 abolished the inhibitory activity of ICI 182,780 on both vector and HER-2/neu/ErbB-3 transfected MCF-7 cells, demonstrating that activation of the HER-2/neu receptor signaling pathway can override the growth inhibitory effect of ICI 182,780.     

Upregulation of PKC-delta contributes to antiestrogen resistance in mammary tumor cells

Acquired resistance to tamoxifen (Tam) in breast cancer patients is a serious therapeutic problem. We have previously reported that protein kinase C-delta (PKC-delta) plays a major role in estrogen (E2)-mediated cell proliferation. To determine if PKC-delta is one of the major alternate signaling pathways that supports cell growth in the presence of Tam, we determined the levels of PKC isoforms in four different models of antiestrogen-resistant cells. Three out of four antiestrogen resistance cell lines (Tam/MCF-7, ICI/MCF-7 and HER-2/MCF-7) expressed significantly high levels of both total and activated PKC-delta levels compared to sensitive cells. Estrogen receptor (ER) alpha content and function are maintained in all the antiestrogen-resistant cell lines. Overexpressing active PKC-delta in Tam-sensitive MCF-7 cells (PKC-delta/MCF-7) led to Tam resistance both in vitro and in vivo. Inhibition of PKC-delta by rottlerin (a relatively specific inhibitor of PKC-delta) or siRNA significantly inhibited estrogen- and Tam-induced growth in antiestrogen-resistant cells. PKC-delta levels are significantly higher in Tam-resistant tumors compared to Tam-sensitive tumors in xenograft model (P<0.05). Taken together, these data suggest that PKC-delta plays a major role in antiestrogen resistance in breast tumor cells and thus provides a new target for treatment.     

Altered radiation responses of breast cancer cells resistant to hormonal therapy

Endocrine therapy agents (the selective estrogen receptor (ER) modulators such as tamoxifen or the selective ER down-regulators such as ICI 182,780) are key treatment regimens for hormone receptor-positive breast cancers. While these drugs are very effective in controlling ER-positive breast cancer, many tumors that initially respond well to treatment often acquire drug resistance, which is a major clinical problem. In clinical practice, hormonal therapy agents are commonly used in combination or sequence with radiation therapy. Tamoxifen treatment and radiotherapy improve both local tumor control and patient survival. However, tamoxifen treatment may render cancer cells less responsive to radiation therapy.Only a handful of data exist on the effects of radiation on cells resistant to hormonal therapy agents. These scarce data show that cells that were resistant to tamoxifen were also resistant to radiation. Yet, the existence and mechanisms of cross-resistance to endocrine therapy and radiation therapy need to be established.Here, we for the first time examined and compared radiation responses of MCF-7 breast adenocarcinoma cells (MCF-7/S0.5) and two antiestrogen resistant cell lines derived from MCF-7/S0.5: the tamoxifen resistant MCF-7/TAM^R-1 and ICI 182,780 resistant MCF-7/182^R-6 cell lines. Specifically, we analyzed the radiation-induced changes in the expression of genes involved in DNA damage, apoptosis, and cell cycle regulation. We found that the tamoxifen-resistant cell line in contrast to the parental and ICI 182,780-resistant cell lines displayed a significantly less radiation-induced decrease in the expression of genes involved in DNA repair. Furthermore, we show that MCF-7/TAM^R-1 and MCF-7/182^R-6 cells were less susceptible to radiation-induced apoptosis as compared to the parental line. These data indicate that tamoxifen-resistant breast cancer cells have a reduced sensitivity to radiation treatment. The current study may therefore serve as a roadmap to the future analysis of the mechanisms of cross-resistance between hormonal therapy and radiation.