ERalpha-positive Breast Cancer Cells Research Articles

Abstract 5511: Endoxifen inhibits polyamine biosynthetic enzyme activity and up-regulates metabolizing enzymes, spermine oxidase (SMO) and acetyl spermine oxidase (APAO)in breast cancer

Abstract Tamoxifen is the most widely used selective estrogen receptor modulator for anti-estrogen therapy for women with estrogen receptor alpha (ERalpha)-positive breast cancer. Clinical trials recognized that tamoxifen was a pro-drug and got converted to active metabolites in the human body. Earlier studies recognized 4-hydroxytamoxifen as the active metabolite of tamoxifen, although recent studies showed that the concentration of another metabolite, endoxifen was up to 10-fold higher than that of 4-hydorxytamoxifen in patients treated with tamoxifen. These metabolites are formed by the action of cytochrome P450 2D6 (CYP2D6). To elucidate the mechanism of action of endoxifen, we determined its effects on cell growth, activity of polyamine biosynthetic and metabolizing enzymes, and oncogene expression in ERalpha-positive MCF-7 breast cancer cells. Polyamines (putrescine, spermidine and spermine) are ubiquitous cellular components and play important roles in cell proliferation. Estradiol (4 nM) increased the proliferation of MCF-7 cells by 2- to 3-fold by 3 days of treatment. Endoxifen significantly suppressed the effects of estradiol at 100 nM and higher concentrations (P < 0.01; n = 3). Estradiol increased the activity of the polyamine biosynthetic enzymes, ornithine decarboxlase (ODC) and S-adenosylmethionine decarboxlase (AdoMetDC), whereas endoxifen suppressed ODC and AdoMetDC activities in a concentration- and time-dependent manner. The change in biosynthetic enzyme activities was reflected in polyamine pools, as estradiol increased and endoxifen suppressed the levels of putrescine and spermidine in MCF-7 cells. There was no significant change in spermine level. Endoxifen had a significant effect on increasing the activities of two polyamine metabolizing enzymes, SMO and APAO. Estradiol suppressed the activities of SMO and APAO by 40% compared to control group at 24 hours of treatment. Endoxifen increased SMO and APAO activities by 2- to 4-fold compared to the activities of these enzymes in estradiol treated group. The expression of c-myc, c-fos and Tiff1 genes was significantly increased by 4 nM estradiol, while the addition of endoxifen suppressed estradiol-mediated increase in gene expression, as measured by quantitative real-time PCR analysis. These results indicate that the polyamine pathway plays an important role in the growth inhibitory and anti-estrogenic effects of endoxifen in breast cancer cells. Upregulation of SMO and APAO might be useful biomarkers for the action of endoxifen. Citation Format: T. J. Thomas, Hui-Chen Hsu, Mervi Hyvonen, Tuomo Keinanen. Endoxifen inhibits polyamine biosynthetic enzyme activity and up-regulates metabolizing enzymes, spermine oxidase (SMO) and acetyl spermine oxidase (APAO)in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5511. doi:10.1158/1538-7445.AM2015-5511

Abstract 5578: Breast cancer cell growth regulatory mechanisms of tamoxifen metabolites

Abstract The proliferation of estrogen receptor alpha (ERalpha)-positive breast cancer cells is stimulated by estradiol. Tamoxifen [(Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethylethanamine] is the most commonly used endocrine treatment for ERalpha-positive breast cancer in pre- and post-menopausal women. Tamoxifen is metabolized to 4-hydroxytamoxifen and endoxifen. ICI 182780 (Faslodex or Fulvestrant) is a pure antiestrogen targeted to the ERalpha. We investigated the effects of endoxifen and 4-hydroxytamoxifen on ERalpha-positive MCF-7 breast cancer cells using cell growth assay, oncogene expression and polyamine regulatory pathways. Polyamines are ubiquitous cellular components and are essential for cell growth and differentiation. Treatment of cells for 1-5 days with 4 nM estradiol increased cells number, whereas the addition of endoxifen, 4-hydroxytamoxifen or ICI 182780 in combination with estradiol suppressed cell growth in a concentration- and time-dependent manner. Real time quantitative polymerase chain reaction (RT-qPCR) experiments showed that the addition of 4 nM estradiol to MCF-7 cells increased the expression of c-myc, c-fos and Tiff1 genes significantly, while the addition endoxifen, 4-hydroxytamoxifen, or ICI 182780 suppressed estradiol-mediated increase in gene expression. Polyamine biosynthetic enzymes were also affected by tamoxifen metabolites. Estradiol increased the activities of the polyamine biosynthetic enzyme, ornithine decarboxlase (ODC) and S-adenosylmethionine decarboxlase (AdoMetDC), whereas the antiestrogens suppressed ODC and AdoMetDC activities in a concentration- and time-dependent manner. However, these agents had little effect on the activity of the polyamine catabolic enzyme, spermidine/spermine N1-acetyl transferase (SSAT). The change in biosynthetic enzyme activities was reflected in polyamine pools, as decreased putrescine and spermidine levels. There was no significant change in spermine level. These results suggest that the mechanism of action of tamoxifen metabolites involves multiple cell growth regulatory pathways. In addition, endoxifen can act as an antiestrogen and suppress cell growth stimulatory effects of estradiol on ERalpha-positive breast cancer cells in a manner similar to that of ICI 182780. Note: This abstract was not presented at the meeting. Citation Format: T. Thomas, Huichen Hsu, PingAr Yang, Mervi T. Hyvonen, Tuomo A. Keinanen. Breast cancer cell growth regulatory mechanisms of tamoxifen metabolites. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5578. doi:10.1158/1538-7445.AM2014-5578

Abstract SY36-03: Development of small-molecule inhibitors of autophagy as anticancer therapy

Abstract Autophagy is an intracellular catabolic mechanism that sequesters selected cytoplasmic components and organelles into double-membrane vesicles, called autophagosomes; the content of autophagosomes is degraded by lysosomal hydrolases after fusing with lysosomes. Autophagy is an important homeostatic mechanism that regulates intracellular recycling and supports cellular survival under stressful conditions (Moreau et al., 2010). Activation of autophagy confers stress resistance and sustains cell survival under adverse conditions. Consistently, autophagy has been found to play an important role in promoting cancer cell survival in the tumor micro-environment in vivo and contribute to metabolic changes to promote oncogenic transformation (Lock et al., 2010). Furthermore, activation of autophagy has been implicated in mediating resistance to existing anticancer therapy. Multiple anti-cancer treatments, including conventional chemotherapy, radiation, as well as targeted therapies such as histone deacetylase inhibitors and imatinib, have been found to activate autophagy as a pro-survival mechanism in cancer cells (Amaravadi and Thompson, 2007; Amaravadi et al., 2007; Kondo et al., 2005; Schoenlein et al., 2009). However, there is no pharmacological method currently available that can selectively inhibit autophagy. We have developed a series of small molecule inhibitors of autophagy, termed “spautins” for specific and potent autophagy inhibitors (Liu et al., 2011). Spautins target USP10 and USP13, two ubiquitin specific peptidases that regulate the ubiquitination and stability of Vps34 complexes, which are critical for autophagy initiation. Vps34 is the class III PI3 kinase that phosphorylates PI (phosphatidylinositol) to produce PI3P (phosphatidylinositol 3-phosphate). Treatment with spautin effectively reduces the levels of Vps34 complexes and PI3P, a key lipid signaling molecule essential for autopaghy, and consequently, leads to inhibition of autophagy. Since USP10 also mediates the deubiquitination of p53 (Yuan et al., 2010), spautins promote the degradation of p53 (Liu et al., 2011) and mutant p53. Our studies demonstrated that spautins show selective cytotoxicity to a subset of cancer cells while normal cells are relatively resistant to spautins (Liu et al., 2011). We will discuss our new data on the ability of spautin to induce the death of cancer cells in vitro and in vivo. Our study demonstrates a proof-of-principle for developing inhibitors of autophagy by targeting protein deubiquitination. Spautin family of autophagy inhibitors represent promising lead candidates for developing novel anti-cancer drugs. Reference: Amaravadi, R.K., and Thompson, C.B. (2007). The roles of therapy-induced autophagy and necrosis in cancer treatment. Clin Cancer Res 13, 7271-7279. Amaravadi, R.K., Yu, D., Lum, J.J., Bui, T., Christophorou, M.A., Evan, G.I., Thomas-Tikhonenko, A., and Thompson, C.B. (2007). Autophagy inhibition enhances therapy-induced apoptosis in a Myc-induced model of lymphoma. J Clin Invest 117, 326-336. Hoyer-Hansen, M., and Jaattela, M. (2008). Autophagy: an emerging target for cancer therapy. Autophagy 4, 574-580. Kondo, Y., Kanzawa, T., Sawaya, R., and Kondo, S. (2005). The role of autophagy in cancer development and response to therapy. Nat Rev Cancer 5, 726-734. Liu, J., Xia, H.G., Kim, M., Xu, L., Li, Y., Zhang, L., Yu, C., Norberg-Vakifahmetoglu, H., Zhang, T., Furuya, T., et al. (2011). Beclin1 controls the levels of p53 by regulating the deubiquitination activity of USP10 and USP13. Cell 147, 223-234. Lock, R., Roy, S., Kenific, C.M., Su, J.S., Salas, E., Ronen, S.M., and Debnath, J. (2010). Autophagy Facilitates Glycolysis During Ras Mediated Oncogenic Transformation. Mol Biol Cell 22, 165-178. Moreau, K., Luo, S., and Rubinsztein, D.C. (2010). Cytoprotective roles for autophagy. Curr Opin Cell Biol 22, 206-211. Schoenlein, P.V., Periyasamy-Thandavan, S., Samaddar, J.S., Jackson, W.H., and Barrett, J.T. (2009). Autophagy facilitates the progression of ERalpha-positive breast cancer cells to antiestrogen resistance. Autophagy 5, 400-403. Yuan, J., Luo, K., Zhang, L., Cheville, J.C., and Lou, Z. (2010). USP10 regulates p53 localization and stability by deubiquitinating p53. Cell 140, 384-396. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY36-03. doi:1538-7445.AM2012-SY36-03

Targeting the receptor tyrosine kinase RET sensitizes breast cancer cells to tamoxifen treatment and reveals a role for RET in endocrine resistance.

Endocrine therapy is the main therapeutic option for patients with estrogen receptor (ERalpha)-positive breast cancer. Resistance to this treatment is often associated with estrogen-independent activation of ERalpha. In this study, we show that in ERalpha-positive breast cancer cells, activation of the receptor tyrosine kinase RET (REarranged during Transfection) by its ligand GDNF results in increased ERalpha phosphorylation on Ser118 and Ser167 and estrogen-independent activation of ERalpha transcriptional activity. Further, we identify mTOR as a key component in this downstream signaling pathway. In tamoxifen response experiments, RET downregulation resulted in 6.2-fold increase in sensitivity of MCF7 cells to antiproliferative effects of tamoxifen, whereas GDNF stimulation had a protective effect against the drug. In tamoxifen-resistant (TAM(R)-1) MCF7 cells, targeting RET restored tamoxifen sensitivity. Finally, examination of two independent tissue microarrays of primary human breast cancers revealed that expression of RET protein was significantly associated with ERalpha-positive tumors and that in primary tumors from patients who subsequently developed invasive recurrence after adjuvant tamoxifen treatment, there was a twofold increase in the number of RET-positive tumors. Together these findings identify RET as a potentially important therapeutic target in ERalpha-positive breast cancers and in particular in tamoxifen-resistant tumors.

Adenosine A1 receptor, a target and regulator of estrogen receptorα action, mediates the proliferative effects of estradiol in breast cancer

Estrogen receptor-α (ERα) and its ligand estradiol (E2) play critical roles in breast cancer growth and are key therapeutic targets. Here, we report a novel dual role of the adenosine A1 receptor (Adora1) as an E2/ERα target and a regulator of ERα transcriptional activity. In ERα-positive breast cancer cells, E2 up-regulated Adora1 mRNA and protein levels, an effect that was reversed by the E2 antagonist ICI 182,780. siRNA ablation of Adora1 in ERα-positive cells reduced basal and E2-dependent proliferation, whereas Adora1 over-expression in an ERα-negative cell line induced proliferation. The selective Adora1 antagonist, DPCPX, reduced proliferation, establishing Adora1 as a mediator of E2/ERα-dependent breast cancer growth. Intriguingly, Adora1 ablation decreased both mRNA and protein levels of ERα and, consequently, estrogen responsive element-dependent ERα transcriptional activity. Moreover, Adora1 ablation decreased binding activity of ERα to the promoter of its target gene TFF1 and led to reduced TFF1 promoter activity and mRNA levels, suggesting that Adora1 is required for full transcriptional activity of ERα upon E2 stimulation. Taken together, we demonstrated a short feed-forward loop involving E2, ERα, and Adora1 that favors breast cancer growth. These data suggest that Adora1 may represent an important target for therapeutic intervention in hormone-dependent breast cancer.

Notch-1 activates estrogen receptor-α-dependent transcription via IKKα in breast cancer cells

Approximately 80% of breast cancers express the estrogen receptor-alpha (ERalpha) and are treated with anti-estrogens. Resistance to these agents is a major cause of mortality. We have shown that estrogen inhibits Notch, whereas anti-estrogens or estrogen withdrawal activate Notch signaling. Combined inhibition of Notch and estrogen signaling has synergistic effects in ERalpha-positive breast cancer models. However, the mechanisms whereby Notch-1 promotes the growth of ERalpha-positive breast cancer cells are unknown. Here, we demonstrate that Notch-1 increases the transcription of ERalpha-responsive genes in the presence or absence of estrogen via a novel chromatin crosstalk mechanism. Our data support a model in which Notch-1 can activate the transcription of ERalpha-target genes via IKKalpha-dependent cooperative chromatin recruitment of Notch-CSL-MAML1 transcriptional complexes (NTC) and ERalpha, which promotes the recruitment of p300. CSL binding elements frequently occur in close proximity to estrogen-responsive elements (EREs) in the human and mouse genomes. Our observations suggest that a hitherto unknown Notch-1/ERalpha chromatin crosstalk mediates Notch signaling effects in ERalpha-positive breast cancer cells and contributes to regulate the transcriptional functions of ERalpha itself.

A novel antiestrogen agent Shikonin inhibits estrogen-dependent gene transcription in human breast cancer cells

Shikonin (SK) has been isolated and identified as a key bioactive component in an herbal plant, Shikon (gromwell). In this study, we investigated antiestrogen activity of SK in breast cancer cells. In human breast cancer cells, we observed that treatment with SK inhibits tumor cell growth in estrogen receptor alpha (ERalpha)-positive, but not ERalpha-negative breast cancer cells. Estrogen-dependent cell growth was inhibited by co-treatment with SK. A potential molecular mechanism by which SK inhibits estrogen action was explored. We found that SK has no effect on ERalpha mRNA expression, but decreases its protein level. This effect is associated with an increase in ubiquitinated ERalpha for degradation. Our results suggest that SK downregulates ERalpha protein through a proteasome-mediated pathway. We also found that the treatment with SK inhibits estrogen-induced estrogen response elements reporter gene activity. Furthermore, SK inhibits recruitment of ERalpha at the estrogen-dependent gene promoters, and subsequently suppresses gene expression. Finally, co-treatment with SK enhanced sensitivity of breast cancer cells to endocrine therapy. Collectively, our studies suggested that SK has a potential for antihormone therapy in ERalpha-positive breast cancer cells, and should serve as a target for new drug developments.

KLF6 inhibits estrogen receptor-mediated cell growth in breast cancer via a c-Src-mediated pathway

Estrogen receptors play a key role in breast cancer development and progression. Kruppel-like factor 6 (KLF6) is a tumour-suppressing protein. The aim of this study was to identify the role of KLF6 inhibition in estrogen receptor(alpha) (ERalpha)-elicited breast cancer development. Protein expression levels were examined by western blot analysis and immunoprecipitation was used to analyse interactions between proteins. An MTT assay was used to study cell proliferation. We found that KLF6 mediates cell growth in ERalpha-positive breast cancer cells through interaction with the c-Src protein. This interaction causes inactivation of the Erk and Akt proteins. These pathways are critical for the proliferation and survival of breast cancer cells. We also established that KLF6 could not mediate cell growth in ERalpha-negative cells. We conclude that KLF6 can modulate ERalpha-mediated cell growth in breast cancer cells. The unique role of KLF6 in mediating cell growth in breast cancer cells opens up the possibility of a new therapeutic strategy for treating breast cancer.

PKA-mediated stabilization of FoxH1 negatively regulates ERalpha activity.

Estrogen receptor alpha (ERalpha) mediates the mitogenic effects of estrogen. ERalpha signaling regulates the normal growth and differentiation of mammary tissue, but uncontrolled ERalpha activation increases the risk to breast cancer. Estrogen binding induces ligand-dependent ERalpha activation, thereby facilitating ERalpha dimerization, promoter binding and coactivator recruitment. ERalpha can also be activated in a ligand-independent manner by many signaling pathways, including protein kinase A (PKA) signaling. However, in several ERalpha-positive breast cancer cells, PKA inhibits estrogen-dependent cell growth. FoxH1 represses the transcriptional activities of estrogen receptors and androgen receptors (AR). Interestingly, FoxH1 has been found to inhibit the PKA-induced and ligand-induced activation of AR. In the present study, we examined the effects of PKA activation on the ability of FoxH1 to represses ERalpha transcriptional activity. We found that PKA increases the protein stability of FoxH1, and that FoxH1 inhibits PKA-induced and estradiol-induced activation of an estrogen response element (ERE). Furthermore, in MCF7 cells, FoxH1 knockdown increased the PKA-induced and estradiol-induced activation of the ERE. These results suggest that PKA can negatively regulate ERalpha, at least in part, through FoxH1.

Resistance to Antiestrogen Arzoxifene Is Mediated by Overexpression of Cyclin D1

Resistance to tamoxifen treatment occurs in approximately 50% of the estrogen receptor (ER)alpha-positive breast cancer patients. Resistant patients would benefit from treatment with other available antiestrogens. Arzoxifene is an effective growth inhibitor of ERalpha-positive breast cancer cells, including tamoxifen-resistant tumors. In this study, we show that overexpression of a regular component of the ERalpha transcription factor complex, cyclin D1, which occurs in approximately 40% of breast cancer patients, renders cells resistant to the new promising antiestrogen, arzoxifene. Overexpression of cyclin D1 alters the conformation of ERalpha in the presence of arzoxifene. In this altered conformation, ERalpha still recruits RNA polymerase II to an estrogen response element-containing promoter, inducing transcription of an ERalpha-dependent reporter gene and of endogenous pS2, and promoting arzoxifene-stimulated growth of MCF-7 cells. Arzoxifene is then converted from an ERalpha antagonist into an agonist. This can be explained by a stabilization of the ERalpha/steroid receptor coactivator-1 complex in the presence of arzoxifene, only when cyclin D1 is overexpressed. These results indicate that subtle changes in the conformation of ERalpha upon binding to antiestrogen are at the basis of resistance to antiestrogens.

Interleukin-8 Expression Is Regulated by Histone Deacetylases through the Nuclear Factor-κB Pathway in Breast Cancer

We have reported recently that the chemokine interleukin 8 (IL-8)/CXCL8 was overexpressed in invasive estrogen receptor (ERalpha)-negative breast cancer cells compared with ERalpha-positive breast cancer cells. We now demonstrate that histone deacetylases (HDACs) play an essential role in the regulation of IL-8 gene expression in ERalpha-positive MCF-7 breast cancer cells. Treatment of MCF-7 cells with the HDAC inhibitor trichostatin A (TSA) led to a strong up-regulation of IL-8 protein and RNA levels in MCF-7 cells. The up-regulation of IL-8 in MCF-7 cells was time- and concentration-dependent. Moreover, run-on and transfection experiments demonstrated that IL-8 induction by HDAC inhibitors was transcriptional and involved mainly the nuclear factor-kappaB (NF-kappaB) site of the IL-8 promoter. These observations are corroborated by an up-regulation of NF-kappaB activity in MCF-7 cells in the presence of TSA. In addition, blocking NF-kappaB pathway by adenoviral delivery of a dominant-negative IkappaBorIkappaB kinase complex 2 (IKK2) mutant abolished IL-8 gene induction by histone deacetylase inhibitors. HDAC inhibitors triggered IKK phosphorylation and up-regulated p65 nuclear translocation, although they decreased the protein levels of IkappaBalpha, which accounts for NF-kappaB activation. TSA increased binding of acetylated histone 3 to the IL-8 gene promoter. In summary, our results demonstrate that NF-kappaB pathway repression by HDAC is responsible for the low expression of IL-8 in ERalpha-positive breast cancer cells.

Epigenetic repression of the estrogen-regulated Homeobox B13 gene in breast cancer

Several studies have reported that a high expression ratio of HOXB13 to IL17BR predicts tumor recurrence in node-negative, estrogen receptor (ER) alpha-positive breast cancer patients treated with tamoxifen. The molecular mechanisms underlying this dysregulation of gene expression remain to be explored. Our epigenetic analysis has found that increased promoter methylation of one of these genes, HOXB13, correlate with the decreased expression of its transcript in breast cancer cell lines (P < 0.005). Transcriptional silencing of this gene can be reversed by a demethylation treatment. HOXB13 is suppressed by the activation of estrogen signaling in ERalpha-positive breast cancer cells. However, treatment with 4-hydroxytamoxifen (4-OHT), an antiestrogen, abrogates the ERalpha-mediated suppression in cancer cells. The notion that this transcriptional induction of HOXB13 occurs in vitro with simultaneous exposure to both estrogen and 4-OHT may provide a biological explanation for its aberrant expression in many node-negative patients undergoing tamoxifen therapy. Interestingly, promoter hypermethylation of HOXB13 is more frequently observed in ERalpha-positive patients with increased lymph node metastasis (P = 0.031) and large tumor sizes (>5 cm) (P = 0.008). In addition, this aberrant epigenetic event is associated with shorter disease-free survival (P = 0.029) in cancer patients. These results suggest that hypermethylation of HOXB13 is a late event of breast tumorigenesis and a poor prognostic indicator of node-positive cancer patients.

Glycogen Synthase Kinase-3 Protects Estrogen Receptor α from Proteasomal Degradation and Is Required for Full Transcriptional Activity of the Receptor

Glycogen synthase kinase-3 (GSK-3) plays a key role in the regulation of transcription factors including steroid receptors. Having identified estrogen receptor-alpha (ERalpha) as substrate for GSK-3, the impact of GSK-3 on ERalpha function and activity upon 17beta-estradiol (E2)-dependent activation remains to be clarified. Here we show by using small interfering technology in combination with immunoblot, gene expression analysis, and luciferase reporter assays that silencing of GSK-3alpha or GSK-3beta results in the reduction of ERalpha levels and transcriptional activity in ERalpha-positive breast cancer cells. Using MCF-7 cells we demonstrate that reduction of ERalpha levels upon GSK-3 silencing was due to increased proteasomal degradation of ERalpha rather than inhibition of ERalpha protein synthesis. Indeed, under this condition, ERalpha protein was rescued using the proteasome inhibitor MG132 in presence of the protein synthesis inhibitor cycloheximide. In addition, strong accumulation of ubiquitinated ERalpha was obtained after GSK-3 silencing in the presence of MG132. We conclude that GSK-3 protects ERalpha from proteasomal degradation and plays a crucial role in ERalpha protein stabilization and turnover. Furthermore, in vitro kinase assay depicted that GSK-3beta phosphorylates ERalpha at Ser-118. GSK-3 silencing resulted in decrease of E2-induced nuclear ERalpha phosphorylation at Ser-118 and E2-induced estrogen response element-dependent luciferase reporter gene expression. Neither Ser-118 phosphorylation nor luciferase activity was restored by use of MG132. Moreover, the expression of estrogen-responsive genes (pS2 and progesterone receptor) was decreased upon GSK-3 silencing. These findings demonstrated that GSK-3 is required for E2-induced ERalpha phosphorylation at Ser-118 and full transcriptional activity of the receptor upon E2 stimulation.

Expression of ZER6 in ERα-Positive Breast Cancer

ZER6 is a C2H2 zinc finger transcription factor with two isoforms (p52-ZER6 and p71-ZER6), which are differentially repressed by a ligand-dependent interaction with estrogen receptor-alpha (ERalpha). We sought to determine if ZER6 proteins are expressed in ERalpha-positive breast cancer cells and if ZER6 is expressed in association with ERalpha in breast cancers. The expression of ZER6 protein was examined by Western blot and the pattern of ZER6 expression was examined in a panel of ERalpha-positive and ERalpha-negative breast cancers using RT-PCR. COS-1 cells transfected with expression vectors for p52-ZER6 express a major protein of 52 kDa and a minor protein of 75 kDa, whereas cells transfected with the p71-ZER6 expression vector express a major protein of 77 kDa and a minor protein of 100 kDa. Breast carcinoma cells express ZER6-specific proteins of similar size, and expression of the p52-ZER6 isoform was only detected in the ERalpha-positive cell lines. In primary breast cancer tissue, 8/16 (50%) of the ERalpha-positive tumors had high ZER6 expression, whereas only 1/12 (8%) of the ERalpha-negative tumors had a high ZER6 level of expression. The relative abundance of ZER6 mRNA in the ERalpha-positive group was statistically greater than the ERalpha-negative group (188 versus 106, P < 0.05). We have confirmed that breast carcinoma cells express ZER6 proteins and identified an association between the level of ZER6 expression and ERalpha expression in primary breast cancers. These data support a role for the ZER6 transcription factors in regulating the expression of genes in hormone-responsive breast cancer.

Selective Estrogen Receptor Modulators in the Ruthenocene Series. Synthesis and Biological Behavior

A series of ruthenocene derivatives, 1-[4-(O(CH(2))(n)()N(CH(3))(2))phenyl]-1-(4-hydroxyphenyl)-2-ruthenocenylbut-1-ene, with n = 2-5, based on the structure of the breast cancer drug tamoxifen has been prepared. These compounds were obtained, via a McMurry cross-coupling reaction, as a mixture of Z and E isomers that could not be separated by HPLC. The relative binding affinity values for estrogen receptor alpha (ERalpha) for n = 2 and 3 were very high (85 and 53%) and surpassed even that of hydroxytamoxifen (38.5%), the active metabolite of tamoxifen. Ruthenocene derivatives act as anti-estrogens as effective (n = 2) or slightly more effective (n = 3-5) than hydroxytamoxifen on ERalpha-positive breast cancer cell lines but, unlike ferrocifens, do not show antiproliferative effects on ERalpha-negative breast cancer cell lines. Electrochemical studies showed that the ruthenocifen radical cations are unstable, which may account for this behavior. Some of these compounds could be useful as radiopharmaceuticals for ERalpha-positive breast cancer tumors.

Identifying estrogen receptor target genes using integrated computational genomics and chromatin immunoprecipitation microarray

The estrogen receptor alpha (ERalpha) regulates gene expression by either direct binding to estrogen response elements or indirect tethering to other transcription factors on promoter targets. To identify these promoter sequences, we conducted a genome-wide screening with a novel microarray technique called ChIP-on-chip. A set of 70 candidate ERalpha loci were identified and the corresponding promoter sequences were analyzed by statistical pattern recognition and comparative genomics approaches. We found mouse counterparts for 63 of these loci and classified 42 (67%) as direct ERalpha targets using classification and regression tree (CART) statistical model, which involves position weight matrix and human-mouse sequence similarity scores as model parameters. The remaining genes were considered to be indirect targets. To validate this computational prediction, we conducted an additional ChIP-on-chip assay that identified acetylated chromatin components in active ERalpha promoters. Of the 27 loci upregulated in an ERalpha-positive breast cancer cell line, 20 having mouse counterparts were correctly predicted by CART. This integrated approach, therefore, sets a paradigm in which the iterative process of model refinement and experimental verification will continue until an accurate prediction of promoter target sequences is derived.

Valproic acid increases the stimulatory effect of estrogens on proliferation of human endometrial adenocarcinoma cells.

Long-term use of valproic acid (VA), a well-tolerated anticonvulsant agent widely used for treating epilepsia, has been recently shown to inhibit histone deacetylases, which in turn are involved in the regulation of the expression of estrogen receptor alpha (ERalpha) by suppressing gene transcription. Because estrogens are known to increase cell proliferation of human endometrial tumors, in this study we investigated whether treatment with VA may increase the proliferative response of human endometrial adenocarcinoma cells to 17-beta-estradiol through induction of ERalpha. The results clearly show that VA, at concentrations of clinical interest, significantly enhanced the proliferative activity exerted by 17-beta-estradiol in the endometrial adenocarcinoma Ishikawa cell line. Moreover, in these cells treatment with VA resulted in increased ERalpha gene expression. Similar effects of VA on cell proliferation were also observed in an ERalpha-positive breast cancer cell line (MCF-7). These findings indicate that VA might favor proliferation of estrogen-dependent human tumors.

The NEDD8 pathway is required for proteasome-mediated degradation of human estrogen receptor (ER)-alpha and essential for the antiproliferative activity of ICI 182,780 in ERalpha-positive breast cancer cells.

Steroid hormone receptors, including estrogen receptor-alpha (ERalpha), are ligand-activated transcription factors, and hormone binding leads to depletion of receptor levels via preteasome-mediated degradation. NEDD8 (neural precursor cell-expressed developmentally down-regulated) is an ubiquitin-like protein essential for protein processing and cell cycle progression. We recently demonstrated that ubiquitin-activating enzyme (Uba)3, the catalytic subunit of the NEDD8-activating enzyme, inhibits ERalpha transcriptional activity. Here we report that Uba3-mediated inhibition of ERalpha transactivation function is due to increased receptor protein turnover. Coexpression of Uba3 with ERalpha increased receptor degradation by the 26S proteasome. Inhibition of NEDD8 activation and conjugation diminished polyubiquitination of ERalpha and blocked proteasome-mediated degradation of receptor protein. The antiestrogen ICI 182,780 is known to induce ER degradation. In human MCF7 breast cancer cells modified to contain a disrupted NEDD8 pathway, ICI 182,780 degradation of ERalpha was impaired, and the antiestrogen was ineffective at inhibiting cell proliferation. This study provides the first evidence linking nuclear receptor degradation with the NEDD8 pathway and the ubiquitin-proteasome system, suggesting that the two pathways can act together to modulate ERalpha turnover and cellular responses to estrogens. Based on our observation that an intact NEDD8 pathway is essential for the antiproliferation activity of the ICI 182,780 in ERalpha positive breast cancer cells, we propose that disruptions in the NEDD8 pathway provide a mechanism by which breast cancer cells acquire antiestrogen resistance while retaining expression of ERalpha.

Antitumor and toxic effects of two derivatives of podophyliotoxin (GP 4 and GP 7) as compared with etopostde (VP 16)

Furanodiene is a natural product isolated from Rhizoma curcumae, and exhibits broad-spectrum anticancer activities in vitro and in vivo. Our previous study proved that furanodiene could increase growth inhibition of steroidal agent in ER alpha-positive breast cancer cells, but whether furanodiene can influence ER status is not clear. In this study, we confirmed that furanodiene down-regulated the ER alpha protein expression level and inhibited E2-induced estrogen response element (ERE)-driven reporter plasmid activity in ER alpha-positive MCF-7 cells. Actually, ERoc-knockdown cells were more sensitive than ER alpha positive cells to furanodiene on the cytotoxicity effect. So the anti-cancer effects of furanodiene and non steroidal agent in breast cancer cells still requires further investigation. Our results showed that furanodiene exposure could enhance growth inhibitory effects of doxorubicin in ERoc-negative MDA-MB-231 cells and ER alpha-low expression 4T1 cells. However, furanodiene did not increase the cytotoxicity of doxorubicin in ER alpha-positive breast cancer cells, non-tumorigenic breast epithelial cells, macrophage cells, hepatocytes cells, pheochromocytoma cells and cardiac myoblasts cells. Furanodiene enhances the anti-cancer effects of doxorubicin in ER alpha-negative breast cancer cells through suppressing cell viability via inducing apoptosis in mitochondria-caspases-dependent and reactive oxygen species-independent manners. These results indicate that furanodiene may be a promising and safety natural agent for cancer adjuvant therapy in the future. (C) 2016 Published by Elsevier B.V.