Activation of mitogen-activated protein kinase in estrogen receptor alpha-positive breast cancer cells in vitro induces an in vivo molecular phenotype of estrogen receptor alpha-negative human breast tumors

Breast cancer presents as either estrogen receptor alpha (ERalpha) positive or negative, with ERalpha+ tumors responding to antiestrogen therapy and having a better prognosis. By themselves, mRNA expression signatures of estrogen regulation in ERalpha+ breast cancer cells do not account for the vast molecular differences observed between ERalpha+ and ERalpha- cancers. In ERalpha- tumors, overexpression of epidermal growth factor receptor (EGFR) or c-erbB-2, leading to increased growth factor signaling, is observed such that mitogen-activated protein (MAP) kinase (MAPK) is significantly hyperactivated compared with ERalpha+ breast cancer. In ERalpha+/progesterone receptor-positive, estrogen-dependent MCF-7 breast cancer cells, we stably overexpressed EGFR or constitutively active erbB-2, Raf, or MAP/extracellular signal-regulated kinase kinase, resulting in cell lines exhibiting hyperactivation of MAPK, estrogen-independent growth, and the reversible down-regulation of ERalpha expression. By global mRNA profiling, we found a "MAPK signature" of approximately 400 genes consistently up-regulated or down-regulated in each of the MAPK+ cell lines. In several independent profile data sets of human breast tumors, the in vitro MAPK signature was able to accurately distinguish ER+ from ER- tumors. In addition, our in vitro mRNA profile data revealed distinct mRNA signatures specific to either erbB-2 or EGFR activation. A subset of breast tumor profiles was found to share extensive similarities with either the erbB-2-specific or the EGFR-specific signatures. Our results confirm that increased MAPK activation causes loss of ERalpha expression and suggest that hyperactivation of MAPK plays a role in the generation of the ERalpha- phenotype in breast cancer. These MAPK+ cell lines are excellent models for investigating the underlying mechanisms behind the ERalpha- phenotype.     

MEK ablation in MCF-7 cells blocks DNA synthesis induced by serum, but not by estradiol or growth factors

The role of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) signaling in estrogen receptor positive (ER(+)) MCF-7 breast carcinoma cells is not well understood. We depleted MEK by cotransfection of MEK1 and MEK2 siRNA duplexes in a MCF-7 derived line (MCF-7/ lacZ, ML-20) and determined its effect on serum, 17beta-estradiol (E(2)), and growth factor induced DNA synthesis. MEK knockdown did not decrease fetal bovine serum-induced DNA synthesis in ML-20 cells although it did inhibit DNA synthesis induced by estrogen-stripped calf serum (CCS) suggesting that MEK activation plays an important role in growth signaling induced by serum components other than estrogen. Consistent with this notion, MEK knockdown only modestly decreased DNA synthesis induced by E(2)-supplemented CCS medium in ML-20 cells. Similarly, MEK knockdown only caused moderate decreases in DNA synthesis induced by fibroblast growth factor-1 (FGF-1) or heregulin-beta1 (HRGbeta1) in this media. Also, there were only minimal effects of MEK knockdown in cells treated with growth factor-supplemented serum-free medium. Although MEK depletion inhibited ERK1/2 phosphorylation induced by CCS in these cells, that induced by growth factor supplemented CCS media was relatively unaffected. Similarly, ERK1/2 phosphorylation induced by growth factor-supplemented serum-free media was also relatively unaffected by MEK depletion. These results suggest that pathways regulating DNA synthesis induced by serum in MCF-7 cells are significantly more dependent on constitutive MEK levels than that induced by E(2) or growth factors.     

Stopping treatment can reverse acquired resistance to letrozole

Using the intratumoral aromatase xenograft model, we have observed that despite long-lasting growth inhibition, tumors eventually begin to grow during continued letrozole treatment. In cells isolated from these long-term letrozole-treated tumors (LTLT-Ca), estrogen receptor-alpha (ERalpha) levels were decreased, whereas signaling proteins in the mitogen-activated protein kinase cascade were up-regulated along with human epidermal growth factor receptor 2 (Her-2). In the current study, we evaluated the effect of discontinuing letrozole treatment on the growth of letrozole-resistant cells and tumors. The cells formed tumors equally well in the absence or presence of letrozole and had similar growth rates. After treatment was discontinued for 6 weeks, letrozole was administered again. Marked tumor regression was observed with this second course of letrozole treatment. Similarly, in MCF-7Ca xenografts, a 6-week break in letrozole treatment prolonged the responsiveness of the tumors to letrozole. To understand the mechanisms of this effect, LTLT-Ca cells were cultured in the absence of letrozole for 16 weeks. The resulting cell line (RLT-Ca) exhibited properties similar to MCF-7Ca cells. The cell growth was inhibited by letrozole and stimulated by estradiol. The expression of phosphorylated mitogen-activated protein kinase (MAPK) was reduced and ERalpha and aromatase levels increased compared with LTLT-Ca cells and were similar to levels in MCF-7Ca cells. These results indicate that discontinuing treatment can reverse letrozole resistance. This could be a beneficial strategy to prolong responsiveness to aromatase inhibitors for patients with breast cancer.     

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.     

Effects of novel retinoic acid metabolism blocking agent (VN/14-1) on letrozole-insensitive breast cancer cells

Aromatase inhibitors are proving to be more effective than tamoxifen for postmenopausal estrogen receptor (ER)-positive breast cancer. However, the inevitable development of resistance to treatment is a concern. We investigated the effects of novel retinoic acid metabolism blocking agent, VN/14-1, in overcoming letrozole resistance in long-term letrozole cultured (LTLC) cells. Compared with MCF-7 cells stably transfected with aromatase (MCF-7Ca), LTLC cells were no longer sensitive to growth inhibition by aromatase inhibitors. The HER-2/phosphorylated mitogen-activated protein kinase (pMAPK) growth factor signaling pathways were activated, and ERalpha and coactivator amplified in breast cancer 1 (AIB1) were up-regulated approximately 3-fold in LTLC cells. VN/14-1 inhibited aromatase activity and growth values of in MCF-7Ca cells with IC(50) of 8.5 and 10.5 nmol/L, respectively. In human placental microsomes, aromatase activity was inhibited with IC(50) of 8.0 pmol/L. The IC(50) in LTLC cells was 0.83 nmol/L, similar to letrozole (IC(50), 0.3 nmol/L) in MCF-7Ca cells. LTLC cells were 10-fold more sensitive to growth inhibition by VN/14-1 than MCF-7Ca cells. VN/14-1 treatment effectively down-regulated ERalpha, AIB1, pMAPK, HER-2, cyclin D1, cyclin-dependent kinase 4 (CDK4), and Bcl2 and up-regulated cytokeratins 8/18, Bad, and Bax. Tumor growth of LTLC cells in ovariectomized nude mice was independent of estrogens but was inhibited by VN/14-1 (20 mg/kg/d; P < 0.002). Decreases in ERalpha, cyclin D1, CDK4, and pMAPK and up-regulation of cytokeratins, Bad, and Bax with VN/14-1 in tumor samples may be responsible for the efficacy of this compound in inhibiting LTLC cell growth in vitro and in vivo.     

Bidirectional cross talk between ERα and EGFR signalling pathways regulates tamoxifen-resistant growth

Summary We have previously demonstrated that oestrogen receptor α (ERα) modulates epidermal growth factor receptor (EGFR)/mitogen-activated protein kinase (MAPK) signalling efficiency in a tamoxifen-resistant MCF-7 breast cancer cell line (Tam-R). In the present study we have investigated whether this cross-talk between EGFR/MAPK and ERα signalling pathways is bidirectional by examining the effects of EGFR/MAPK activity on ER functionality in the same cell line. Elevated expression levels of phosphorylated serine 118 (S118) ERα were observed in the Tam-R compared to the parental wild type MCF-7 cell line (WT-MCF-7) under basal growth conditions. Phosphorylation of ERα at S118 was regulated by the EGFR/MAPK pathway in Tam-R cells being increased in response to amphiregulin (AR) and inhibited by the selective EGFR tyrosine kinase inhibitor, gefitinib and the MEK1/2 inhibitor, PD184352. Recruitment of the co-activators p68 RNA helicase and SRC1 to ERα, oestrogen response element (ERE) activity and Tam-R cell growth were similarly EGFR/MAPK-regulated. Chromatin immunoprecipitation (ChIP) studies revealed that in Tam-R cells the ERα assembled on the AR gene promoter and this was associated with elevated basal expression of AR mRNA. Furthermore, AR mRNA expression was under the regulation of the EGFR/MAPK and ERα signalling pathways. Neutralising antibodies to AR inhibited EGFR/ERK1/2 activity, reduced S118 ERα phosphorylation and reduced AR mRNA expression in TAM-R cells. These findings suggest that ERα function in Tam-R cells is maintained as a consequence of EGFR/MAPK-mediated phosphorylation at serine residue 118 resulting in the generation of a self-propogating autocrine growth-regulatory loop through the ERα-mediated production of AR.     

Functional implications of altered subcellular localization of PELP1 in breast cancer cells

It is increasingly accepted that steroidal receptor co-regulators may also function in the cytoplasmic compartment. Proline-, glutamic acid-, and leucine-rich protein-1 (PELP1) is a novel coregulator that plays a role in both the genomic and extranuclear actions of estrogen receptors (ER) in hormonally responsive tissues. In this study using breast tumor arrays, we found that PELP1 was localized only in the cytoplasm in 58% of the PELP1-positive breast tumors. To help explain the significance of the cytoplasmic localization of PELP1 in human breast tumors, we created a mutant protein that was expressed only in the cytoplasm (PELP1-cyto) and then generated a model system wherein MCF-7 breast cancer cells were engineered to specifically express this mutant. We found that PELP1-cyto cells were hypersensitive to estrogen but resistant to tamoxifen. PELP1-cyto cells, but not parental MCF-7 cells, formed xenograft tumors in nude mice. In addition, PELP1-cyto cells exhibited increased association of PELP1 with Src, enhanced mitogen-activated protein kinase (MAPK) activation, and constitutive activation of AKT. The altered localization of PELP1 was sufficient to trigger the interaction of PELP1 with the p85 subunit of phosphatidylinositol-3-kinase (PI3K), leading to PI3K activation. In addition, PELP1 interacted with epidermal growth factor receptors and participated in growth factor-mediated ER transactivation functions. Our results suggest that the altered localization of PELP1 modulates sensitivity to antiestrogens, potentiates tumorigenicity, presumably via the stimulation of extranuclear estrogen responses, such as the activation of MAPK and AKT, and also enhance cross-regulation of ER transactivation activity by growth factors.     

A novel taspine derivative, HMQ1611, inhibits breast cancer cell growth via estrogen receptor α and EGF receptor signaling pathways

Breast cancer is a common cancer with a leading cause of cancer mortality in women. Currently, the chemotherapy for breast cancer is underdeveloped. Here, we report a novel taspine derivative, HMQ1611, which has anticancer effects using in vitro and in vivo breast cancer models. HMQ1611 reduced cancer cell proliferation in four human breast cancer cell lines including MDA-MB-231, SK-BR-3, ZR-75-30, and MCF-7. HMQ1611 more potently reduced growth of estrogen receptor α (ERα)-positive breast cancer cells (ZR-75-30 and MCF-7) than ERα-negative cells (MDA-MB-231 and SK-BR-3). Moreover, HMQ1611 arrested breast cancer cell cycle at S-phase. In vivo tumor xenograft model, treatment of HMQ1611 significantly reduced tumor size and weight compared with vehicles. We also found that HMQ1611 reduced ERα expression and inhibited membrane ERα-mediated mitogen-activated protein kinase (MAPK) signaling following the stimulation of cells with estrogen. Knockdown of ERα by siRNA transfection in ZR-75-30 cells attenuated HMQ1611 effects. In contrast, overexpression of ERα in MDA-MB-231 cells enhanced HMQ1611 effects, suggesting that ERα pathway mediated HMQ1611's inhibition of breast cancer cell growth in ERα-positive breast cancer. HMQ1611 also reduced phosphorylation of EGF receptor (EGFR) and its downstream signaling players extracellular signal-regulated kinase (ERK)1/2 and AKT activation both in ZR-75-30 and MDA-MB-231 cells. These results showed that the novel compound HMQ1611 had anticancer effects, and partially via ERα and/or EGFR signaling pathways, suggesting that HMQ1611 may be a potential novel candidate for human breast cancer intervention.     

Potential of endogenous estrogen receptor beta to influence the selective ER modulator ERbeta complex

The ratio of estrogen receptor beta (ERbeta) to ERalpha can alter the estrogen-like properties of tamoxifen. Transient transfection of ERbeta cDNA into cells can decrease the estrogen-like properties of the ERalpha:tamoxifen complex, whereas an increase in the amount of ERbeta is associated with tamoxifen-resistant breast cancer. We have addressed each of these hypotheses by examining well characterized laboratory models. We determined whether changes in endogenous ERbeta are responsible for the estrogen-like or antiestrogenic properties of tamoxifen or raloxifene in MDA-MB-231 cells transfected with cDNAs for ERalpha or mutants D351G, D351Y. We found that the amount of ERbeta mRNA in separate, stable transfectants of mutant ERalpha cDNA was always < 2% of ERalpha. Since at least a 50:50 mixture of ERalpha:ERbeta is needed to silence the tamoxifen:ERalpha complex, we conclude that insufficient ERbeta mRNA is available for selective ER modulation in stable transfectants of D351G and D351Y ERalpha. Similarly, to test the hypothesis that ERbeta is up-regulated and plays an important role during the development of tamoxifen-stimulated tumor growth, we quantitatively analyzed ERbeta and ERalpha mRNA in tamoxifen-na?ve (MCF-7:E2, ECC1:E2) and tamoxifen-stimulated tumors (MCF-7:TAM, EnCa 101:TAM). We found that ERbeta mRNA levels were not significantly elevated in tamoxifen-stimulated tumors and the ERalpha mRNA remained over 99% out of all ER species for all the tumors tested. The same results were also obtained when mRNA levels of ERbeta and ERalpha in a series of tamoxifen-na?ve and tamoxifen-resistant breast cancer was analyzed. We conclude that endogenous ERbeta may not play a dominant role in the modulation of the tamoxifen ERalpha complex, or in the development of tamoxifen-stimulated resistant tumor growth.     

Inhibition of the MAP kinase activity suppresses estrogen-induced breast tumor growth both in vitro and in vivo

Elevated expression of mitogen-activated protein kinase (Erk/MAPK) has been noted in a significant percentage of primary human breast cancers. To directly assess the importance of Erk/MAPK activation in estrogen (E2)-induced tumor progression, we blocked E2-signaling with MEK-inhibitor CI-1040 and/or tamoxifen (Tam). Our data show that both MEK-inhibitor CI-1040 and Tam blocked E2-induced MAPK phosphorylation and cell proliferation in MCF-7 breast cancer cells in vitro. However, in vivo studies show that anti-tumor efficacy of combining the CI-1040 and Tam was similar to single agent(s). Furthermore, sequential treatment with Tam followed by CI-1040 or CI-1040 followed by Tam did not significantly reduce E2-induced tumor growth. This suggests that the combination of CI-1040 and Tam may not be synergistic in inhibiting E2-induced tumor growth. However, these findings also indicate that MAPK plays a critical role in E2-induced tumor growth, and that this could be a potential therapeutic target to combat hormonally regulated growth in ER-positive tumors.     

Cytotoxicity induced by manipulation of signal transduction pathways is associated with down-regulation of Bcl-2 but not Mcl-1 in MCF-7 human breast cancer

We examined the role of Mcl-1 and Bcl-2 expression in the induction of apoptosis, through blocking protein tyrosine kinase (PTK), protein kinase C (PKC), phosphatidylinositol 3-kinase (PI3-K) and mitogen-activated protein kinase (MAPK)/Erk kinase (MEK) signaling pathways by various kinase inhibitors in MCF-7 breast cancer cells. The PTK inhibitor genistein (GEN) and PKC inhibitor staurosporine (STP) down-regulated Mcl-1 and Bcl-2 expression, and induced growth inhibition by blocking at the G₂/M phase of cell cycle, followed by apoptosis, leading to chromatin condensation and DNA fragmentation. LY294002 (LY)-mediated inhibition of PI3-K activity down-regulated Bcl-2 but not Mcl-1 expression, triggered growth arrest at the G₁/G₀ phase of cell cycle and also led to apoptosis marked with chromatin condensation and DNA fragmentation. The MEK inhibitor U0126 (U0) decreased Bcl-2 expression but not Mcl-1 expression, inhibited cells growth and induced G₁/G₀ arrest, but in this case cell death occurred without significant apoptotic features. The kinase inhibitor concentration dependence of cytotoxicity correlated well with down-regulation of Bcl-2 but not with changes in Mcl-1 levels. This suggests that Bcl-2 plays a predominant role in the regulation of cell death induced by cell signaling alterations whereas Mcl-1 does not appear to control cell survival under these conditions in MCF-7 cells. Further studies showed that the combination of GEN, STP and LY with U0 can produce synergetic cytotoxic effects on MCF-7 cells. Our results suggest that PTK, PKC, PI3-K and MEK signaling pathways can regulate Bcl-2 expression and form an integrated network that plays a critical role in cell survival.     

抑制雌激素调控的靶基因LRP 16表达能削弱ERα介导的转录激活活性

目的探讨抑制雌激素(E2)调控的靶基因LRP16表达对雌激素受体α(ERα)介导转录激活活性的影响.方法将针对LRP16合成的小干扰RNA,与ERα表达载体、雌激素反应性元件荧光素酶报道载体(pGL-3-S10或3×ERE-Luc)共转染乳腺癌细胞MCF-7,双荧光素酶检测方法测定pGL-3-S10或3×ERE-Luc报道子的相对荧光素酶活性;E2刺激培养已建立的稳定抑制LRP16表达的MCF-7细胞,Northern bl0t方法检测比较具有不同LRP16表达水平的MCF-7细胞中ERα下游靶基因对E2的反应性变化.结果抑制LRP16表达降低了ERα对其反应性报道子(pGL-3-S10和3×ERE-Luc)的激活活性;同时也降低了ERα下游靶基因对E2的反应性上调效应,其中包括E2F1、维甲酸受体α(RARα)、c-fos、cyclin D1和MTA3,但对cathepsin D的表达无影响.结论抑制ERα靶基因LRP16的表达可以下调ERα介导的转录激活活性,表明LRP16对ERα信号转导通路具有反馈增强效应,提示LRP16在ERα阳性的乳腺癌发生与进展中应有重要作用.     

TAS-108, a novel oral steroidal antiestrogenic agent, is a pure antagonist on estrogen receptor alpha and a partial agonist on estrogen receptor beta with low uterotrophic effect.

PURPOSE: Investigators are currently conducting phase II trials on TAS-108, a novel oral steroidal antiestrogenic agent. The purpose of this study is to investigate the molecular and pharmacologic properties of TAS-108 compared with other antiestrogenic agents such as tamoxifen,raloxifene, and fulvestrant. EXPERIMENTAL DESIGN: The antagonistic or agonistic activities of these agents against both estrogen receptors (ER) alpha and beta were compared in the reporter assay systems. Their effects on the uterus were evaluated in ovariectomized rat models. The antitumor activity of TAS-108 given p.o. was evaluated in both dimethylbenzanthracene-induced mammary tumor model and human breast cancer MCF-7 cell line xenografts. RESULTS: TAS-108 inhibited the transactivation of ERalpha under the presence of 17beta-estradiol (E2) and did not induce the transactivation of ERalpha in the absence of E2, unlike the agonistic activity of tamoxifen. On the other hand, it exhibited the most agonistic activity on ERbeta among the antiestrogenic agents tested. When given p.o. in the ovariectomized rat, TAS-108 showed a much weaker estrogenic effect on utterine weight compared to tamoxifen, or with similar levels of raloxifene, a selective estrogen receptor modulator. Also, TAS-108 strongly inhibited tumor growth in dimethylbenzanthracene-induced mammary carcinomain the rat, the endogenous E2 model, at a dosage of 1 to 3 mg/kg/day. It also inhibited high exogenous E2, inducing tumor growth against MCF-7 xenografts at a dosage of 1 mg/kg/day without any toxic manifestation. CONCLUSIONS: Taken together, p.o. treatment with TAS-108 has a novel mode of action on ERs and inhibits E2-dependent tumor growth with little uterotrophic effect.