An Activin A/BMP2 chimera, AB215, blocks estrogen signaling via induction of ID proteins in breast cancer cells
© Jung et al.; licensee BioMed Central Ltd. 2014
Received: 25 April 2014
Accepted: 21 July 2014
Published: 29 July 2014
One in eight women will be affected by breast cancer in her lifetime. Approximately 75% of breast cancers express estrogen receptor alpha (ERα) and/or progesterone receptor and these receptors are markers for tumor dependence on estrogen. Anti-estrogenic drugs such as tamoxifen are commonly used to block estrogen-mediated signaling in breast cancer. However, many patients either do not respond to these therapies (de novo resistance) or develop resistance to them following prolonged treatment (acquired resistance). Therefore, it is imperative to continue efforts aimed at developing new efficient and safe methods of targeting ER activity in breast cancer.
AB215 is a chimeric ligand assembled from sections of Activin A and BMP2. BMP2’s and AB215’s inhibition of breast cancer cells growth was investigated. In vitro luciferase and MTT proliferation assays together with western blot, RT_PCR, and mRNA knockdown methods were used to determine the mechanism of inhibition of estrogen positive breast cancer cells growth by BMP2 and AB215. Additionally in vivo xenograft tumor model was used to investigate anticancer properties of AB215.
Here we report that AB215, a chimeric ligand assembled from sections of Activin A and BMP2 with BMP2-like signaling, possesses stronger anti-proliferative effects on ERα positive breast cancer cells than BMP2. We further show that AB215 inhibits estrogen signaling by inducing expression of inhibitor of DNA binding proteins (IDs). Specifically, we demonstrate that knockdown of ID proteins attenuates the anti-estrogen effects of AB215. Remarkably, we find that AB215 is more effective than tamoxifen in suppressing tumor growth in a xenograft model.
This study shows that IDs have profound role to inhibit estrogen signaling in ERα positive breast cancer cells, and that engineered TGF-beta ligands may have high therapeutic value.
KeywordsEstrogen receptor-positive breast cancer Transforming growth factor-β Bone morphogenetic protein Tamoxifen alternative Inhibitor of DNA binding proteins Bio-better AB215
Breast cancer is one of the leading causes of death for women worldwide, particularly in developed countries. During the early stage of breast cancer progression, estrogen plays a critical role by enhancing the tumor cell proliferation [1–4]. Estrogen’s pro-oncogenic effect is mediated via nuclear estrogen receptors (ER), ERα and ERβ, by forming steroid/receptor complexes, which in turn interact with DNA at estrogen response elements (EREs) in promoter regions of various genes [5, 6]. This binding of steroid/receptor complex at EREs, requires co-activators including nuclear receptor co-activator 1 (NCOA1), NCOA2, NCOA3 and aryl hydrocarbon receptor nuclear translocator (ARNT), which are all members of basic Helix-Loop-Helix (bHLH) family. Moreover, it was reported that over-expression of NCOAs in breast cancer cells significantly increased their survival .
Tamoxifen is an ER antagonist that is currently a major drug used in treatment of ERα-positive (ERα+)/pre-menopausal breast cancer patients. Tamoxifen is a competitive antagonist that predominantly blocks the binding of estrogen, 17-β-Estradiol (E2), to ERs. Tamoxifen treatment causes breast cancer cells to remain at the G0 and G1 phase of the cell cycle. Moreover, the ER/tamoxifen complex recruits co-repressors (e.g. Nuclear receptor co-repressor 1 and 2), which in turn stop the genes from being turned on by E2 . However, after prolonged tamoxifen usage, as many as ~30% of breast cancer patients who initially responded to tamoxifen develop resistance to this drug [9, 10]. The mechanism of tamoxifen resistance remains largely unclear and effective alternatives have yet to be discovered.
In addition to estrogen, growth factors including many Transforming Growth Factor-beta (TGF-β) superfamily ligands are also key regulators of ER+ breast tumor growth. Bone morphogenetic protein 2 (BMP2) is a TGF-β superfamily member that possesses high affinity for BMP type I receptors (e.g. Activin receptor like kinase 3 [ALK3]) [11, 12] and utilizes the SMAD1/5/8 signaling pathway to induce osteogenesis  and chondrogenesis . BMP2 is also reported to suppress the proliferation of MCF7 breast cancer cells by regulating the retinoblastoma  and the phosphatase and tensin homolog proteins . However, in contrast to this anti-oncogenic effect, BMP2 has also been reported as a pro-oncogene in breast cancer by promoting cancer cell invasion , increasing hormone-independent cancer growth , and angiogenesis in vitro. Interestingly, it has been reported that E2 treatment mitigated BMP2-induced gene transcription as well as osteoblast differentiation in 2T3 and C2C12 cell lines . Moreover, a BMP2-responsive reporter assay in breast cancer cells displayed a 50% decrease in BMP2 signaling when treated with E2 .
Because BMP2 suppresses estrogen-triggered breast cancer cell proliferation, we tested the anti-estrogenic effects of AB215, a chimeric ligand composed of approximately one third Activin A sequence and two thirds BMP2 sequence that possesses enhanced BMP2-like activity. We show that AB215 has stronger anti-estrogenic and anti-proliferative effects on breast cancer cells than BMP2. We further demonstrate that AB215 represses the proliferation of breast cancer cells by inhibiting E2/ERα-mediated signaling via a novel mechanism involving induction of ID proteins. Significantly, we demonstrate that AB215 suppresses ERα+ tumor growth and tumor cell proliferation more effectively than tamoxifen in a xenograft model in vivo.
AB215 was prepared as previously described . In brief, Activin A/BMP2 chimeras (AB2 library) have been engineered as a mix of six sequence segments originating from two parental molecules, Activin A and BMP2. AB215 is one such member of AB2 chimera library, which consists of two sequence segments from Activin A and four sequence segments from BMP2 in the order of BABBBA, where A and B denote corresponding segments of Activin A and BMP2, respectively. AB215 was expressed in Escherichia coli and chemically refolded . After the purification steps of heparin affinity and C4 reverse phase chromatography, the refolded protein was lyophilized for storage. BMP2 was purchased from joint Protein Central (http://jointproteincentral.com). Prior to use, the lyophilized proteins were reconstituted in 1 mM hydrochloric acid (HCl) in small volume before diluting by at least a factor of 100 in a relevant final buffer or media including phosphate buffered saline (PBS).
T47D and MCF7 cell lines were purchased from American Type Culture Collection (VA, USA) and SK-BR-3 cell lines from Korean Cell Line Bank (Seoul, Korea). Cells were grown at 37°C humidified atmosphere of 5% CO2 in RPMI-1640 medium (Invitrogen, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen). All assays were performed in RPMI-1640 without phenol red and supplemented with heat-inactivated and charcoal-stripped FBS (PAA Labs, Pasching, Austria), unless stated otherwise.
MTT proliferation assay
Cells were plated on a 96-well plate (BD, NJ, USA) at 4×103 cells/well with 2 ~ 5% heat-inactivated and charcoal-stripped FBS. After 24 hours, cells were treated with BMP2, or AB215, with or without 10nM E2 (Sigma) in ethanol. The final concentration of ethanol in all the condition was 0.001% (v/v). After desired period of treatment, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent (5 mg/ml in PBS, Sigma) was added and incubated at 37°C until purple precipitation was visible. MTT crystal was dissolved in 4 mM HCl, 0.1% NP-40 in isopropanol for 15 minutes and absorbance was measured at 590 nm and baseline corrected at 700 nm.
Cells were plated on a 96-well plate (BD) in Opti-MEM low serum medium (Invitrogen) at 2 × 104 cells/well and reverse co-transfected with ID1-Del2-Luc and β-Galactosidase (β-Gal) using FugeneHD (Roche). After 18 hours of transfection, cells were treated with BMP2 or AB215 with or without 10nM E2. After 24 hours of treatment, cells were lysed using Luciferase lysis buffer (Promega) and their luminescence was measured with plate luminometer (Berthhold, Bad Wildbad, Germany). Transfection variations were normalized by β-gal.
Cells were plated on a 6 or 12-well plate (BD) at 2 × 105 or 1 × 105 cells/well supplemented with 5% heat-inactivated and charcoal-stripped FBS. Cells were treated with 10nM E2, BMP2 or AB215, and exposed for 48 hours. Cells were lysed with cell lysis buffer (Cell Signaling, MA, USA) containing 1 mM PMSF and phosphatase inhibitor cocktail (Roche). Cell lysate’s total protein amount was quantified using Bradford assay. Proteins were separated on SDS-polyacrylamide gels transferred to nitrocellulose (GE healthcare, NJ, USA) or PVDF (Biorad, CA, USA) membrane and analyzed according to the manufacturer’s instruction. Trefoil factor 1 (TFF1) antibody was purchased from Santa Cruz Biotechnology (CA, USA), phosphorylated Extracellular signal-regulated kinases1/2 (ERK1/2), ERK1/2 from Cell Signaling Technology (MA, USA) and β-actin from Sigma.
List of RT-PCR primers
Human Cathepsin D
Genes of interest were knocked down using small interference RNA (siRNA) transfection. siRNA duplex was purchased/synthesized from Bioneer Inc (Korea). Cells were reverse transfected with siRNA duplex complexed with Lipofectamine RNAiMAX (Invitrogen) reagent in serum free RPMI1640 media without phenol red (Invitrogen) as specified by manufacturer’s instruction. Briefly, 15 pmol siRNA duplex was diluted in 200 ul serum free RPMI1640 without phenol red and complexed with Lipofectamine for15 ~ 20 minutes. 1×105 cells in RPMI1640 supplemented with10% heat-inactivated and charcoal-stripped FBS were added to the mixture in each well in a 12 well plate. Cells were treated with ligands after 24 ~ 48 hours of transfection. We tested 1 ~ 3 siRNAs from Bioneer to select the most efficient construct. The following sequences of siRNAs for particular gene knockdowns were used; ID1- FWD-5′-UCGCAUCUUGUGUCGCUGA, REV-5′-UCAGCGACACAAGAUGCGA; ID2- FWD-5′-CUUACUUGGACUGUGAUAU, REV-5′-AUAUCACAGUCCAAGUAAG; ID3- FWD-5′-CUGUAACAAUGCGAUGUAU, REV-5′-AUACAUCGCAUUGUUACAG; ID4- FWD-5′-GUGACAUUUCAUACCAUGU, REV-5′-ACAUGGUAUGAAAUGUCAC. Negative control was transfected with AccuTarget Negative control siRNA (Bioneer). Knockdown (KD) efficiency was determined by qRT-PCR.
In vivotumor xenograft model
Continuous E2-releasing pellets for 90 days (Innovative Research of America, FL, USA) were implanted subcutaneously into 4–6 weeks old KSN/Slc athymic mouse (n = 5) 3 days before xenograft. MCF7 breast cancer cells (5×106 cells) were subcutaneously xenografted in 50 μl RPMI1640 with 50 μl Matrigel Matrix (BD) using 21-gauge needle on the dorsal side. The ligand injection started when tumor was visible (after 17 days). Two doses (0.12 (low) or 0.4 (high) mg/kg of mice) of AB215 and 0.6 mg/kg dose of tamoxifen were subcutaneously injected, three times a week for 10 weeks (10 weeks total - low group- ~ 90 ug, high group- ~ 300 ug injected). After 70 days from injection started, mice were sacrificed, and tumor was surgically removed. Mice were also examined for tumors in other organs and the spleen size was measured to evaluate inflammation. All the in vivo experiments were done under the guideline of AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care International). All the procedures were performed at the Lee Gil Ya Cancer and Diabetes Institute and approved by Institutional Animal Care and Use Committee (IACUC No. 2011–0103) at Gachon University in South Korea.
Tumor tissues were fixed in formaldehyde, embedded in paraffin, sectioned, deparaffinized/hydrated and processed for antigen retrieval by microwaving 3 times for 5 minutes in 10 mM Tris–HCl/pH9.0 and 1 mM EDTA. The sections were then incubated with Ki67 antibody (Santa Cruz Biotechnology) at 4°C overnight and analyzed using ImmPress peroxidase polymer detection kit (Vector Labs, CA, USA). Harris Hematoxylin (BBC, WA, USA) was used for counter stain by following standard protocol.
Cell invasion assay
A fluorometric kit for cell invasion assay was purchased from Cell Biolabs (CA, USA). All the procedures followed the manufacturer’s protocol. Briefly, 2 × 106 cells were plated on upper chamber of transmembrane-welled plates in serum-free RPMI 1640 medium with or without ligands. Lower chamber contained 10% serum or 10nM E2. After 18 hours, penetrated cells were analyzed using CyQuant reagent and quantified by a multi-well fluorometer.
All the numerically quantifiable data have been statistically analyzed and graphically presented using Prism software (Graphpad, CA, USA). Column analysis was performed by one-way ANOVA with Dunnett’s post-hoc test adjustment.
AB215 strongly induces ID proteins
AB215 inhibits estrogen-induced growth of ERα+cells
One of the key mechanisms of estrogen-induced proliferation of breast cancer cells and tumor progression is the activation of mitogen activated protein kinase, by promoting phosphorylation of ERK1/2 . Consistent with its ability to block estrogen-induced proliferation, AB215 inhibits estrogen-induced phosphorylation of ERK1/2 in MCF7 cells and does so more strongly than BMP2 (Figure 2H).
AB215 blocks estrogen-induced ERK signaling by inducing ID proteins
AB215 inhibits expression of E2-induced genes
AB215 reduces in vivogrowth of breast cancer cells
We constructed the AB2 library of segmental chimeras between Activin A and BMP2  in order to create novel ligands with unique structural and functional properties and the potential to fulfill medical needs. The present study provides evidence that one of these, AB215 (BABBBA), can inhibit estrogen signaling and the growth of estrogen-fueled ER+ breast tumors. From the three-dimensional structure of the ternary complex of BMP2, Activin receptor Type II (ActRII)-Extracellular domain (ECD), and ALK3-ECD  it can be inferred that most of the type II receptor binding site of AB215 consists of Activin A sequence while almost all of its type I receptor binding site is derived from BMP2. Since both BMP2 and Activin A utilize the type II receptors ActRII and ActRIIb, we hypothesized that a chimeric ligand that possesses the type I receptor specificity of BMP2 together with the high affinity type II receptor binding properties of Activin A may have enhanced BMP2-like properties. Indeed, AB215 signals via the SMAD1/5/8 pathway but not the SMAD2/3 pathway and has increased potency relative to BMP2.
BMP2 can inhibit the progression of many different types of cancers but its role is also bi-directional since it is also implicated in tumor progression and angiogenesis in some cancers. Since BMP2 inhibits proliferation of ERα+ breast cancer cells, we hypothesized that the increased BMP2-like signaling activity of AB215 may augment AB215’s potency in anti-proliferation of ERα+ breast cancer cells. In the present study, we established that AB215 indeed inhibits E2-induced proliferation of ERα+ breast cancer cells to a greater extent than BMP2. Furthermore, like BMP2, AB215 has no proliferative effect on ERα− cells indicating that both ligands exert their anti-proliferative effects through effects on E2 signaling. Results led us to conclude that the anti-proliferative effects of AB215 are not only dependent on the ERα status, but also on the level of ERα expression since it had less of an effect on the proliferation and E2-induced gene expression in T47D cells which express ERα at lower levels than in MCF7 cells (Additional file 2: Figure S2a-e). The fact that T47D cells were less susceptible to AB215’s anti-proliferative effects than MCF7 cells (Figure 2B and 2D) strongly indicates that these effects are at least partially exerted via E2/ERα signaling.
E2-induced phosphorylation of ERK is thought to play essential role in mediating increases in cellular proliferation. Although the mechanism of E2-induced ERK phosphorylation remains unclear, epidermal growth factor receptor, protein kinase Cδ and HER-2/neu have each been shown to be involved . Here, we show that AB215 can inhibit E2-induced ERK phosphorylation and E2/ERα-induced gene expression. Consistent with our working hypothesis that AB215 blocks E2 signaling by inhibiting E2/ERα complex binding to EREs of various genes, we found that ID proteins are significantly up regulated downstream of AB215 signaling, and thus play a critical role in mediating inhibition of E2-induced ERK phosphorylation. We propose that ID proteins may interfere with the binding of E2/ERα to EREs by sequestering the E2/ER co-activator proteins such as NCOA and ARNT in nonproductive complexes. Intriguingly, our results also demonstrate that ID proteins act in a non-redundant and highly cooperative manner. Future studies will elucidate the precise mechanism through which ID proteins block E2-induced gene regulation.
Our in vivo studies demonstrate that the anti-tumorigenic effects of AB215 are similar to those of tamoxifen, not only in reducing tumor size, but also in improving tumor grade according to Ki67 expression level. It is important to note that prolonged injections of high concentration of AB215 had no apparent toxicity to mice and none of these mice developed abnormalities such as weight loss (Additional file 3: Figure S3), inflammation or tumorigenesis. Moreover, in vitro cell invasion assays of AB215-treated MCF7 cells did not show development of characteristic metastatic properties. (Additional file 4: Figure S4).
We show that the Activin A/BMP2 chimera AB215 strongly induces ID proteins and thereby interferes with the pro-proliferative and gene expression effects of E2/ERα signaling. Furthermore, our results suggest that this enhanced BMP2-like molecule is at least as efficient as tamoxifen in reducing the size of tumors resulting from breast cancer xenografts highlighting its potential effectiveness for the treatment of breast tumors, especially those resistant to tamoxifen. This discovery puts AB215 in a prime position as a novel endocrine therapeutic biologic and opens a new inroad to study the complex mechanisms regulating estrogen-driven cancer cell proliferation.
Estrogen responsive element
Aryl hydrocarbon receptor nuclear translocator
Transforming growth factor-beta
Bone morphogenetic protein
Activin receptor like kinase
Inhibitor of DNA binding protein
Phosphate buffered saline
Fetal bovine serum
Trefoil factor 1
Extracellular signal-regulated kinases1/2
Small interference RNA
Vascular Endothelial Growth Factor
Activin receptor Type II
We especially thank Byung Hak Yoon for the guidance of in vivo experiments. We thank Hye Jung Han and Jin Suk Lim for technical assistance, Somi Yoon and Yun Hui Jeon for scientific advices. We also thank members of CACU at LCDI of Gachon University for their assistance in making tissue sections and for conducting animal studies, and members of the CCMI of Salk institute for the guidance in molecular imaging. This work was supported by Incheon Free Economy Zone of Korea (jCB).
- Colditz GA: Relationship between estrogen levels, use of hormone replacement therapy, and breast cancer. J Natl Cancer Inst. 1998, 90: 814-823.View ArticlePubMedGoogle Scholar
- Helzlsouer KJ, Couzi R: Hormones and breast cancer. Cancer. 1995, 76 (10 Suppl): 2059-2063.View ArticlePubMedGoogle Scholar
- Henderson BE, Ross R, Bernstein L: Estrogens as a cause of human cancer: the Richard and Hinda Rosenthal Foundation award lecture. Cancer Res. 1988, 48: 246-253.PubMedGoogle Scholar
- Keen JC, Davidson NE: The biology of breast carcinoma. Cancer. 2003, 97 (3 Suppl): 825-833.View ArticlePubMedGoogle Scholar
- Cerillo G, Rees A, Manchanda N, Reilly C, Brogan I, White A, Needham M: The oestrogen receptor regulates NFkappaB and AP-1 activity in a cell-specific manner. J Steroid Biochem Mol Biol. 1998, 67: 79-88.View ArticlePubMedGoogle Scholar
- Khan S, Abdelrahim M, Samudio I, Safe S: Estrogen receptor/Sp1 complexes are required for induction of cad gene expression by 17beta-estradiol in breast cancer cells. Endocrinology. 2003, 144: 2325-2335.View ArticlePubMedGoogle Scholar
- Weldon CB, Elliott S, Zhu Y, Clayton JL, Curiel TJ, Jaffe BM, Burow ME: Regulation of estrogen-mediated cell survival and proliferation by p160 coactivators. Surgery. 2004, 136: 346-354.View ArticlePubMedGoogle Scholar
- Shang Y, Hu X, DiRenzo J, Lazar MA, Brown M: Cofactor dynamics and sufficiency in estrogen receptor-regulated transcription. Cell. 2000, 103: 843-852.View ArticlePubMedGoogle Scholar
- Brouillet JP, Spyratos F, Hacene K, Fauque J, Freiss G, Dupont F, Maudelonde T, Rochefort H: Immunoradiometric assay of pro-cathepsin D in breast cancer cytosol: relative prognostic value versus total cathepsin D. Eur J Cancer Oxf Engl 1990. 1993, 29A: 1248-1251.Google Scholar
- Stewart SS, Roldan JE, Lvov YM, Mills DK: Layer-by-Layer Adsorption of Biocompatible Polyelectrolytes onto Dexamethasone Aggregates. 28th Annu Int Conf IEEE Eng Med Biol Soc 2006 EMBS 06. 2006, 1474-1477. http://ieeexplore.ieee.org/xpls/abs_all.jsp?arnumber=4462041&tag=1,View ArticleGoogle Scholar
- Macías-Silva M, Hoodless PA, Tang SJ, Buchwald M, Wrana JL: Specific activation of Smad1 signaling pathways by the BMP7 type I receptor, ALK2. J Biol Chem. 1998, 273: 25628-25636.View ArticlePubMedGoogle Scholar
- Ten Dijke P, Yamashita H, Sampath TK, Reddi AH, Estevez M, Riddle DL, Ichijo H, Heldin CH, Miyazono K: Identification of type I receptors for osteogenic protein-1 and bone morphogenetic protein-4. J Biol Chem. 1994, 269: 16985-16988.PubMedGoogle Scholar
- Wozney JM, Rosen V, Celeste AJ, Mitsock LM, Whitters MJ, Kriz RW, Hewick RM, Wang EA: Novel regulators of bone formation: molecular clones and activities. Science. 1988, 242: 1528-1534.View ArticlePubMedGoogle Scholar
- Yoon BS, Lyons KM: Multiple functions of BMPs in chondrogenesis. J Cell Biochem. 2004, 93: 93-103.View ArticlePubMedGoogle Scholar
- Ghosh-Choudhury N, Ghosh-Choudhury G, Celeste A, Ghosh PM, Moyer M, Abboud SL, Kreisberg J: Bone morphogenetic protein-2 induces cyclin kinase inhibitor p21 and hypophosphorylation of retinoblastoma protein in estradiol-treated MCF-7 human breast cancer cells. Biochim Biophys Acta. 2000, 1497: 186-196.View ArticlePubMedGoogle Scholar
- Waite KA, Eng C: BMP2 exposure results in decreased PTEN protein degradation and increased PTEN levels. Hum Mol Genet. 2003, 12: 679-684.View ArticlePubMedGoogle Scholar
- Jin H, Pi J, Huang X, Huang F, Shao W, Li S, Chen Y, Cai J: BMP2 promotes migration and invasion of breast cancer cells via cytoskeletal reorganization and adhesion decrease: an AFM investigation. Appl Microbiol Biotechnol. 2012, 93: 1715-1723.View ArticlePubMedGoogle Scholar
- Clement JH, Raida M, Sänger J, Bicknell R, Liu J, Naumann A, Geyer A, Waldau A, Hortschansky P, Schmidt A, Höffken K, Wölft S, Harris AL: Bone morphogenetic protein 2 (BMP-2) induces in vitro invasion and in vivo hormone independent growth of breast carcinoma cells. Int J Oncol. 2005, 27: 401-407.PubMedGoogle Scholar
- Raida M, Clement JH, Leek RD, Ameri K, Bicknell R, Niederwieser D, Harris AL: Bone morphogenetic protein 2 (BMP-2) and induction of tumor angiogenesis. J Cancer Res Clin Oncol. 2005, 131: 741-750.View ArticlePubMedGoogle Scholar
- Roldán M, Oliva F, Gónzalez del Valle MA, Saiz-Jimenez C, Hernández-Mariné M: Does green light influence the fluorescence properties and structure of phototrophic biofilms?. Appl Environ Microbiol. 2006, 72: 3026-3031.View ArticlePubMedPubMed CentralGoogle Scholar
- Yamamoto T, Saatcioglu F, Matsuda T: Cross-talk between bone morphogenic proteins and estrogen receptor signaling. Endocrinology. 2002, 143: 2635-2642.View ArticlePubMedGoogle Scholar
- Allendorph GP, Read JD, Kawakami Y, Kelber JA, Isaacs MJ, Choe S: Designer TGFβ superfamily ligands with diversified functionality. PLoS One. 2011, 6: e26402-View ArticlePubMedPubMed CentralGoogle Scholar
- Venkateshwar GP, Padhye MN, Khosla AR, Kakkar ST: Complications of exodontia: a retrospective study. Indian J Dent Res Off Publ Indian Soc Dent Res. 2011, 22: 633-638.View ArticleGoogle Scholar
- Keshamouni VG, Mattingly RR, Reddy KB: Mechanism of 17-beta-estradiol-induced Erk1/2 activation in breast cancer cells. A role for HER2 AND PKC-delta. J Biol Chem. 2002, 277: 22558-22565.View ArticlePubMedGoogle Scholar
- Pinto A, Roldan R, Sollecito TP: Hypertension in children: an overview. J Dent Educ. 2006, 70: 434-440.PubMedGoogle Scholar
- Roldán G, Delgado L, Musé IM: Tumoral expression of BRCA1, estrogen receptor alpha and ID4 protein in patients with sporadic breast cancer. Cancer Biol Ther. 2006, 5: 505-510.View ArticlePubMedGoogle Scholar
- Roldan G, Ang RC: Overview of sleep disorders. Respir Care Clin N Am. 2006, 12: 31-54. viiiPubMedGoogle Scholar
- Brunnberg S, Pettersson K, Rydin E, Matthews J, Hanberg A, Pongratz I: The basic helix-loop-helix-PAS protein ARNT functions as a potent coactivator of estrogen receptor-dependent transcription. Proc Natl Acad Sci U S A. 2003, 100: 6517-6522.View ArticlePubMedPubMed CentralGoogle Scholar
- Pichon MF, Milgrom E: Clinical significance of the estrogen regulated pS2 protein in mammary tumors. Crit Rev Oncol Hematol. 1993, 15: 13-21.View ArticlePubMedGoogle Scholar
- Ravdin PM, De Moor CA, Hilsenbeck SG, Samoszuk MK, Vendely PM, Clark GM: Lack of prognostic value of cathepsin D levels for predicting short term outcomes of breast cancer patients. Cancer Lett. 1997, 116: 177-183.View ArticlePubMedGoogle Scholar
- Perillo B, Sasso A, Abbondanza C, Palumbo G: 17beta-estradiol inhibits apoptosis in MCF-7 cells, inducing bcl-2 expression via two estrogen-responsive elements present in the coding sequence. Mol Cell Biol. 2000, 20: 2890-2901.View ArticlePubMedPubMed CentralGoogle Scholar
- Shang Y, Brown M: Molecular determinants for the tissue specificity of SERMs. Science. 2002, 295: 2465-2468.View ArticlePubMedGoogle Scholar
- Stoner M, Wormke M, Saville B, Samudio I, Qin C, Abdelrahim M, Safe S: Estrogen regulation of vascular endothelial growth factor gene expression in ZR-75 breast cancer cells through interaction of estrogen receptor alpha and SP proteins. Oncogene. 2004, 23: 1052-1063.View ArticlePubMedGoogle Scholar
- Allendorph GP, Vale WW, Choe S: Structure of the ternary signaling complex of a TGF-β superfamily member. Proc Natl Acad Sci. 2006, 103: 7643-7648.View ArticlePubMedPubMed CentralGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/14/549/prepub
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.