ERα-positive Breast Cancer Cells Research Articles

Xanthohumol suppresses oestrogen-signalling in breast cancer through the inhibition of BIG3-PHB2 interactions.

Xanthohumol (XN) is a natural anticancer compound that inhibits the proliferation of oestrogen receptor-α (ERα)-positive breast cancer cells. However, the precise mechanism of the antitumour effects of XN on oestrogen (E2)-dependent cell growth, and especially its direct target molecule(s), remain(s) largely unknown. Here, we focus on whether XN directly binds to the tumour suppressor protein prohibitin 2 (PHB2), forming a novel natural antitumour compound targeting the BIG3-PHB2 complex and acting as a pivotal modulator of E2/ERα signalling in breast cancer cells. XN treatment effectively prevented the BIG3-PHB2 interaction, thereby releasing PHB2 to directly bind to both nuclear- and cytoplasmic ERα. This event led to the complete suppression of the E2-signalling pathways and ERα-positive breast cancer cell growth both in vitro and in vivo, but did not suppress the growth of normal mammary epithelial cells. Our findings suggest that XN may be a promising natural compound to suppress the growth of luminal-type breast cancer.

Highlights of This Issue

Although SMAD4 has been suggested to inhibit the activity of the WNT/β-catenin signaling cascade in cancer, the mechanism of this inhibition remains largely unknown. Jia and colleagues have discovered a novel interaction between SMAD4 and Aurora A kinase (AURKA) in cancer cells that facilitates AURKA degradation and suppresses WNT/β-catenin signaling. Furthermore, manipulation of TGFβ did not alter the ability of SMAD4 to repress AURKA and β-catenin signaling. Elucidation of this unprecedented molecular mechanism demonstrates how the balance between suppression and activation of the WNT/β-catenin signaling pathway in dysplastic precancerous cells could be broken to promote carcinogenesis in SMAD4-deleted advanced cancerous cells.The ETV6–RUNX1 genomic fusion is a frequent initiating event in childhood leukemia; however, its role in leukemogenesis is only partly understood. Here, Palmi and colleagues show defective adhesion and migration properties in ETV6–RUNX1-positive B-cell progenitors (BCP). In particular, alterations in the expression of adhesion molecules impaired chemotactic response to the chemokine SDF1 (CXCL12) and a block in the GTPase CDC42 signaling pathway are described. The molecular details revealed in this study posit an altered interaction of ETV6–RUNX1-positive BCP with the microenvironment and represent initiating events in the evolution of the disease, from its preleukemic phase to clinical onset, with potential implications to develop strategies for effective leukemia eradication.Most breast cancers are estrogen receptor (ER) positive and many exhibit intrinsic or acquired resistance to commonly used endocrine therapies. Wright and colleagues demonstrate that the proto-oncogene AGR2 is required for the viability of tamoxifen-sensitive and -resistant ERα-positive breast cancer cells and highlight the therapeutic utility, in vitro and in vivo, of interfering with the production and/or activity of this secreted protein. Further, a new regulatory pathway is defined wherein ligand activated ERα facilitates AGR2 expression, which is required for the maintenance of ERα expression. Surprisingly, AGR2 expression was shown to occur in an ERα-independent manner in tamoxifen-resistant breast cancer cells, an activity that was attributed to dysregulated signaling by the transcription factor FOXA1.Extracellular adenosine mediates diverse anti-inflammatory, angiogenic, tumor-promoting, and other signaling effects through activation of G-protein–coupled adenosine receptors. Along with “classical” receptor-mediated pathways, adenosine also exerts cytotoxic and other biologic effects through intrinsic mechanisms. Virtanen and colleagues establish that pretreatment of either human prostate or breast cancer cells with low concentrations of adenosine markedly inhibits cell invasion and migration capacity. Molecularly, these inhibitory effects involve cellular uptake of nucleosides, their intracellular interconversion into ADP/ATP, and eventually, the inhibition of phospho-AMPK1α and other signaling pathways. This study provides novel insight into the ability of adenosine to prevent tumor invasion via two different adenosine receptor-dependent and -independent mechanisms.

ERβ1: characterization, prognosis, and evaluation of treatment strategies in ERα-positive and -negative breast cancer.

BackgroundThe role and clinical value of ERβ1 expression is controversial and recent data demonstrates that many ERβ antibodies are insensitive and/or non-specific. Therefore, we sought to comprehensively characterize ERβ1 expression across all sub-types of breast cancer using a validated antibody and determine the roles of this receptor in mediating response to multiple forms of endocrine therapy both in the presence and absence of ERα expression.MethodsNuclear and cytoplasmic expression patterns of ERβ1 were analyzed in three patient cohorts, including a retrospective analysis of a prospective adjuvant tamoxifen study and a triple negative breast cancer cohort. To investigate the utility of therapeutically targeting ERβ1, we generated multiple ERβ1 expressing cell model systems and determined their proliferative responses following anti-estrogenic or ERβ-specific agonist exposure.ResultsNuclear ERβ1 was shown to be expressed across all major sub-types of breast cancer, including 25% of triple negative breast cancers and 33% of ER-positive tumors, and was associated with significantly improved outcomes in ERα-positive tamoxifen-treated patients. In agreement with these observations, ERβ1 expression sensitized ERα-positive breast cancer cells to the anti-cancer effects of selective estrogen receptor modulators (SERMs). However, in the absence of ERα expression, ERβ-specific agonists potently inhibited cell proliferation rates while anti-estrogenic therapies were ineffective.ConclusionsUsing a validated antibody, we have confirmed that nuclear ERβ1 expression is commonly present in breast cancer and is prognostic in tamoxifen-treated patients. Using multiple breast cancer cell lines, ERβ appears to be a novel therapeutic target. However, the efficacy of SERMs and ERβ-specific agonists differ as a function of ERα expression.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-749) contains supplementary material, which is available to authorized users.

Abstract LB-183: Y-box binding protein-1 YB-1 negatively regulates ERα expression accompanying by enhanced HER2/ErbB2 expression in breast cancer

Abstract [Purpose] YB-1 containing a cold-shock domain is a potent oncogene for breast cancer (Bergmann et al., Cancer Res, 2005). Our previous studies have demonstrated that YB-1 plays pivotal roles not only in acquisition of global drug resistance and cell proliferation (Kuwano et al., Mol. Cancer Ther., 2004), but also in expression of HER2/ErbB2 in breast cancer cells (Fujii et al., Cancer Res., 2008) and stomach cancer cells (Shibata et al., Mol. Cancer Ther., 2013) in culture and clinical samples. In our present study, we first asked whether ERα affect YB-1-induced HER2 expression in breast cancer cells, and then asked whether YB-1 activation could be biostatistically linked to HER2 and/or ER expression in breast cancer patients. [Results] Is this study, we first examined the role of YB-1 in expression of HER2 and ERα by using ERα-positive and -negative breast cancer cell lines in culture, and we next examined whether YB-1 expression in nucleus could be biostatistically associated with HER2 or ERα expression by using clinical specimens of breast cancer. We observed novel findings as follows. [1] Knockdown of YB-1 markedly reduced HER2/ErbB2 expression in ERα positive human breast cancer cells, but only slightly reduced HER2/ErbB2 expression in ERα-negative breast cancer cells. [2] Nuclear YB-1 was forced expression by the tetracycline-induced YB-1/Tet-On system. Nuclear YB-1 expression enhanced HER2 expression and decreased ERα expression in ERα-positive cancer cells. By contrast, in ERα-negative breast cancer cells, there was no apparent increase in HER2 expression by YB-1. [3] Exogenous introduction of ERα cDNA resulted in suppression of HER2 expression dependent on YB-1 in ERα negative cells. Furthermore, treatment with tamoxifen stimulated YB-1-dependent HER2 expression in ERα-positive cells. [4] We further examined expression of YB-1, ERα, PgR and HER2 by immunohistochemical (IHC) analysis in clinical samples of breast cancer patients who had received no treatments by hormonal and chemical therapeutics. Based on expression levels of various factors, nuclear YB-1 expression was significantly correlated with HER2 expression, but negatively correlated with ERα expression in tumors of postmenopausal patients (n=114). By contrast, such significant association of YB-1 expression with ERα or HER2 expression was not seen in tumors of premenopausal patients (n=57). [Conclusions] YB-1 positively regulated HER2 expression and negatively ERα expression in ERα positive breast cancer cells, and the presence of ERα seems to be required for YB-1-dependent HER2 expression. Furthermore, in breast tumors of post-menopausal patients, nuclear localization of YB-1 was positively correlated with HER2 expression and negatively correlated with ERα expression. This study may contribute to further development of optimized endocrine- and HER2- targeted therapeutics against breast cancer. Citation Format: Tomohiro Shibata, Hiroto Izumi, Akihiko Kawahara, Satoshi Hattori, Chihiro Fukumitsu, Ryuji Takahashi, Kosuke Watari, Yuichi Murakami, Kimitoshi Kohno, Ken-ichi Ito, Masayoshi Kage, Michihiko Kuwano, Mayumi Ono. Y-box binding protein-1 YB-1 negatively regulates ERα expression accompanying by enhanced HER2/ErbB2 expression in breast cancer. [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 LB-183. doi:10.1158/1538-7445.AM2014-LB-183

Abstract 623: BIG3-PHB2 interaction is a key therapeutic target in luminal-type of breast cancer

Abstract More than 70% of primary breast tumors are estrogen receptor alpha (ERα)-positive, and the interactions between estrogen (E2) and ERα dramatically enhance the proliferative and metastatic activity of breast tumor cells. The selective estrogen receptor modulator tamoxifen directly inhibits E2 and ERα interactions and is a standard treatment offered to patients with ERα-positive breast cancer. The acquisition of endocrine resistance is a common obstacle in endocrine therapy for ERα-positive breast tumors. Therefore, identifying the factors and pathways responsible for resistance and defining ways to overcome it represent important therapeutic challenges in breast cancer research. Prohibitin 2 (PHB2) is known to function as a co-repressor of ERα because it regulates ERα transcriptional activity via its direct binding to ERα. However, it remains unclear how endogenous PHB2 is inactivated in ERα-positive breast cancer cells, despite its abundant expression and the lack of genomic mutations or methylations in breast cancer clinical specimens or cell lines. Here we report that brefeldin A-inhibited guanine nucleotide-exchange protein 3 (BIG3) protein interacts PP1α, and dephosphorylates the E2-dependent phosphorylation of PHB2, which is indispensable for its tumor suppressive ability, thereby causing an apparent “loss-of-function” of PHB2 protein, resulting in the constitutive activation of ERα signaling pathways. These findings demonstrate for the first time the novel mechanism for the inactivation of tumor suppressor protein. From a therapeutic perspective, intrinsic PHB2 released from BIG3 by the cell-permeable peptide inhibitor directly binds to both nuclear- and membrane-associated ERα, which leads to the inhibition of multiple ERα-signaling pathways, and the growth of ERα-positive breast cancer cells both in vitro and in vivo. More importantly, this peptide inhibitor treatment suppressed tamoxifen resistance and enhanced tamoxifen responsiveness in ERα-positive breast cancer cells. These findings mark the first step toward uncovering a new E2/ ERα signaling network in breast cancer and shed light on novel therapeutic strategies utilizing PHB2 protein functions for E2/ ERα-positive breast cancer. Citation Format: Toyomasa Katagiri, Tesuro Yoshimaru, Masato Komatsu, Taisuke Matsuo. BIG3-PHB2 interaction is a key therapeutic target in luminal-type of breast cancer. [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 623. doi:10.1158/1538-7445.AM2014-623

Abstract 1987: The role of lipolysis-stimulated lipoprotein receptor in directing breast cancer cell behavior and subtype

Abstract The claudin-low molecular subtype of breast cancer is of particular interest for clinically the majority of these tumors are poor prognosis, triple negative, invasive ductal carcinomas. Claudin-low tumors are characterized by low gene expression of cell junction and adhesion proteins, and de-regulation of tight junction proteins is often been implicated in tumorigenesis. Herein, we sought to define the role of tricellular tight junction (tTJ) proteins in breast cancer and cancer cell behavior with a focus on lipolysis-stimulated lipoprotein receptor (LSR). LSR was expressed in epithelium, endothelium, adipose and stromal cells within the healthy breast tissue, as well as in tumor epithelium. LSR expression was significantly correlated with invasive ductal carcinomas compared to invasive lobular carcinomas, and ERα positive tumors and breast cancer cell lines. Consistent with the role of LSR in tTJs, LSR levels were significantly reduced in claudin-low breast cancer cell lines. Functional studies illustrated that that re-introduction of LSR into a claudin-low cell line suppressed the EMT phenotype and reduced individual cell migration. However, our data suggest that LSR may direct collective cell migration. Moreover, re-introduction of LSR in claudin-low breast cancer cell lines reestablished a family of TJ expression, thereby reverting claudin-low lines to other molecular subtypes. Overexpression of LSR enhanced proliferation and survival in anchorage independent conditions, suggesting that reestablishment of tight junctions is not sufficient to inhibit aggressive cell behaviors in vitro. Collectively, these data highlight an unanticipated role for the tTJ protein, LSR in directing aggressive breast cancer behavior. Citation Format: Jodie M. Fleming, Denise K. Reaves, Katerina D. Fagan-Solis, Karen Dunphy, Shannon D. Oliver. The role of lipolysis-stimulated lipoprotein receptor in directing breast cancer cell behavior and subtype. [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 1987. doi:10.1158/1538-7445.AM2014-1987

Abstract 3341: Cross-talk between PI3K/AKT and ERα signaling leads to STAT3/6-modulated epigenetic transcription in aggressive breast cancer

Abstract The phosphatidylinositol 3-kinase (PI3K)/AKT pathway is a critical survival cascade in tumor progression. Our previous study demonstrated that combinational treatment of a potent PI3K inhibitor, LY294002 (LY), and DNA methyltransferase inhibitor, 5’-Aza-deoxycytidine (DAC), compounds effectively reduce tumor formation in the immunodeficiency mouse model, suggesting that PI3K/AKT signaling suppresses tumorigenesis through the epigenetic modulation. While tumor size of the LY-treated xenografts is dramatically decreased compared to the control group, the tumor growth cannot be completely attenuated by either LY or LY/DAC treatment, indicating that there is an alternative route for tumor survival. Here, we performed a microarray analysis to identify LY-mediated expression profiling on an ERα-positive breast cancer cell line, T-47D, the cell line applied in the previous xenograft experiments. A subset of 24 LY-activated genes associated with better clinical outcome are found to be not only direct targets of PI3K/AKT but involved in estrogen (E2)/ERα signaling by ingenuity pathway analysis, suggesting the involvement of ERα signaling in PI3K/AKT-mediated tumor growth. To clarify their relationship, pharmacological experiments using an ERα antagonist, ICI 182, 780 (ICI) and LY coupled with E2 was conducted. We observed that E2 stimulation induces both AKT and ERα activation in a time-dependent manner. Either ICI or LY treatment cannot repress the activation levels of AKT or ERα, respectively. Though, either single or combined inhibitor treatment can effectively restore E2-suppressed gene expression. These findings indicate that cross-talk between PI3K/AKT and ERα signaling cascades may cooperatively regulate cell growth and associated gene expression. In addition, transcription factor motif analysis showed that signal transducer and activator of transcription 3 and 6 (STAT3/6) binding motifs are enriched with highest prediction score on the promoter regions of the examined AKT-directed targets. Inhibitors prohibit E2-mediated assembly of STAT3/6 with ERα and STAT3/6 recruitment onto the promoter regions of target loci using co-immunoprecipitation (co-IP) and chromatin IP-qPCR. Moreover, repressive histone marks, H3K27me3 and H3K9me3, are found to occupy on the same regions as STAT3/6 upon E2 treatment. DAC treatment can effectively restore E2-induced suppression of AKT-directed target genes. Further association studies are ongoing to interrogate whether STAT3/6 is part of repressive transcription complex with H3K27me3 and H3K9me3. In summary, we propose that cross-talk of ERα with PI3K/AKT signaling represents the potential function switch of ERα from genomic binding to no-genomic binding. Combined treatment of PI3K/AKT inhibitors with hormone therapy may shed a light of therapeutic strategy to patients with aggressive or metastatic breast tumors. Citation Format: Hang-Kai Hsu, Pei-Yin Hsu, Yao Wang, Zelton D. Sharp, Tim H.-M. Huang, Victor X. Jin. Cross-talk between PI3K/AKT and ERα signaling leads to STAT3/6-modulated epigenetic transcription in aggressive breast cancer. [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 3341. doi:10.1158/1538-7445.AM2014-3341

Abstract 622: S-equol, an estrogen receptor β agonist, inhibits tumor growth and progression of breast cancer

Abstract Breast cancer is the primary cause of cancer-associated mortality in women worldwide. Estrogen and the Estrogen Receptors (ER) play a significant role in breast cancer, with over two-thirds of breast cancers expressing ERα. Current endocrine therapy, such as aromatase inhibitors, target estrogen biosynthesis and anti-estrogens target ERα. However, therapeutic resistance frequently arises. In addition to the importance of ERα, ERβ has also been shown to play a critical, but opposing role in breast cancer. ERβ has been shown to inhibit the growth of ERα-positive breast cancer cells. The ratio of ERα to ERβ, in addition to the cross talk between ER's and growth factor signaling, has been implicated in the development of therapeutic resistance. Recently, several plant-derived compounds that exhibit ERβ agonist activity have been identified. S-equol is a potent ERβ agonist and a metabolite from the soy isoflavone daidzein, and has been previously shown to alleviate menopausal symptoms in a clinical trial. Activation of ERβ, or its over expression, shifts the balance of ER's from the oncogenic action of ERα to the tumor suppressor activity of ERβ, and therefore may be a valuable therapeutic approach in the treatment of breast cancer. In this study we sought out to determine the efficacy of the ERβ agonist, S-equol, in inhibiting the growth and progression of breast tumors using a syngeneic mouse model. We used mouse mammary tumor cells expressing endogenous ERβ, and to determine the contribution of ERβ, cells with knockdown of ERβ were generated using shRNA. ERβ mRNA and protein expression was analyzed using qRT-PCR and western blot respectively. Syngeneic tumors were established and mice were treated with either a vehicle control or S-equol. Treatment with S-equol reduces tumor volume and inhibits the progression of mouse mammary tumor cells in tumors expressing ERβ. Our mechanistic studies show that S-equol reduces the expression of ERα, and increases the expression of p53 and p27 in an ERβ dependent manner. Additionally, S-equol modulates the expression of inflammatory molecules involved in aromatase expression and promotes the differentiation of cancer stem cells. In conclusion, this study suggests that targeting ERβ may be a valuable strategy in treatment of breast cancer. Citation Format: Cathy Samayoa, Naveen K. Krishnegowda, Samaya R. Krishnan, Ratna K. Vadlamudi, Rajeshwar R. Tekmal. S-equol, an estrogen receptor β agonist, inhibits tumor growth and progression of breast cancer. [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 622. doi:10.1158/1538-7445.AM2014-622

Human glutathione S-transferase P1-1 functions as an estrogen receptor α signaling modulator

Estrogen receptor α (ERα) plays a crucial role in estrogen-mediated signaling pathways and exerts its action as a nuclear transcription factor. Binding of the ligand-activated ERα to the estrogen response element (ERE) is a central part of ERα-associated signal transduction pathways and its aberrant modulation is associated with many disease conditions. Human glutathione S-transferase P1-1 (GSTP) functions as an enzyme in conjugation reactions in drug metabolism and as a regulator of kinase signaling pathways. It is overexpressed in tumors following chemotherapy and has been associated with a poor prognosis in breast cancer. In this study, a novel regulatory function of GSTP has been proposed in which GSTP modulates ERE-mediated ERα signaling events. Ectopic expression of GSTP was able to induce the ERα and ERE-mediated transcriptional activities in ERα-positive but GSTP-negative MCF7 human breast cancer cells. This inductive effect of GSTP on the ERE-transcription activity was diminished when the cells express a mutated form of the enzyme or are treated with a GSTP-specific chemical inhibitor. It was found that GSTP inhibited the expression of the receptor interacting protein 140 (RIP140), a negative regulator of ERα transcription, at both mRNA and protein levels. Our study suggests a novel non-enzymatic role of GSTP which plays a significant role in regulating the classical ERα signaling pathways via modification of transcription cofactors such as RIP140.

Indole-3-carbinol and its N-alkoxy derivatives preferentially target ERα-positive breast cancer cells

Indole-3-carbinol (I3C) is a natural anti-carcinogenic compound found at high concentrations in Brassica vegetables. I3C was recently reported to inhibit neutrophil elastase (NE) activity, while consequently limiting the proteolytic processing of full length cyclin E into pro-tumorigenic low molecular weight cyclin E (LMW-E). In this study, we hypothesized that inhibition of NE activity and resultant LMW-E generation is critical to the anti-tumor effects of I3C. LMW-E was predominately expressed by ERα-negative breast cancer cell lines. However, ERα-positive cell lines demonstrated the greatest sensitivity to the anti-tumor effects of I3C and its more potent N-alkoxy derivatives. We found that I3C was incapable of inhibiting NE activity or the generation of LMW-E. Therefore, this pathway did not contribute to the anti-tumor activity of I3C. Gene expression analyzes identified ligand-activated aryl hydrocarbon receptor (AhR), which mediated sensitivity to the anti-tumor effects of I3C in ERα-positive MCF-7 cells. In this model system, the reactive oxygen species (ROS)-induced upregulation of ATF-3 and pro-apoptotic BH3-only proteins (e.g. NOXA) contributed to the sensitivity of ERα-positive breast cancer cells to the anti-tumor effects of I3C. Overexpression of ERα in MDA-MB-231 cells, which normally lack ERα expression, increased sensitivity to the anti-tumor effects of I3C, demonstrating a direct role for ERα in mediating the sensitivity of breast cancer cell lines to I3C. Our results suggest that ERα signaling amplified the pro-apoptotic effect of I3C-induced AhR signaling in luminal breast cancer cell lines, which was mediated in part through oxidative stress induced upregulation of ATF-3 and downstream BH3-only proteins.

CDK11p58 inhibits ERα-positive breast cancer invasion by targeting integrin β3 via the repression of ERα signaling.

BackgroundCDK11p58, a Ser/Thr kinase that belongs to the cell division cycle 2-like 1 (CDC2L1) subfamily, is associated with cell cycle progression, tumorigenesis and apoptotic signaling. CDK11p58 is also involved in the regulation of steroid receptors, such as androgen and estrogen receptors. We previously found that CDK11p58 was abnormally expressed in prostate cancer. However, its role in breast cancer remains unclear.MethodsCDK11p58 expression was evaluated by immunohistochemical staining in a tissue array. A Transwell assay was used to detect invasion and metastasis in breast cancer cells. The TaqMan® Metastasis Gene Expression Assay was used to search for potential downstream factors in the CDK11p58 signaling pathway. qRT-PCR was used to evaluate mRNA levels, and the dual luciferase array was used to analyze promoter activity. Western blotting was used to detect the protein level.ResultsCDK11p58 expression was negatively correlated with node status (P = 0.012), relapse status (P = 0.002) and metastasis status (P = 0.023). Kaplan-Meier survival curves indicated that the disease-free survival (DFS) was significantly poor in breast cancer patients with low CDK11 expression. Interestingly, using the breast cancer cell lines ZR-75-30 and MDA-MB-231, we found that CDK11p58 was capable of repressing the migration and invasion of ERα-positive breast cancer cells, but not ERα-negative breast cancer cells, in a kinase-dependent manner. Gene expression assays demonstrated that integrin β3 mRNA was dramatically repressed by CDK11p58, and luciferase results confirmed that the integrin β3 promoter was inhibited by CDK11p58 through ERα repression. The expression of integrin β3 was highly related to ERα signaling; ERα overexpression stimulated integrin β3 expression, whereas siRNA-mediated knockdown of ERα attenuated integrin β3 expression.ConclusionsThese data indicate that CDK11p58 is an anti-metastatic gene in ERα-positive breast cancer and that the regulation of integrin β3 by CDK11p58 via the repression of ERα signaling may constitute part of a signaling pathway underlying breast cancer invasion.

Tongshu Capsule Down-Regulates the Expression of Estrogen Receptor α and Suppresses Human Breast Cancer Cell Proliferation

The Tongshu Capsule (TSC) is a prevalent form of traditional Chinese medicine widely used for its purported effects in treating mammary gland hyperplasia and inflammation. Though successful in several clinical studies, there is no clear evidence as to why TSC has a positive treatment effect, and little known about underlying mechanism that may account for it. In this study, we examined the effects of TSC and found that it has a comparatively strong growth inhibition on ERα positive breast cancer cells. TSC seems to cause G1 cell cycle arrest instead of apoptosis. Interestingly, TSC also down-regulated the expression of ERα and Cyclin D1. Consistently, TSC suppressed E2 mediated ERα downstream gene expression and cell proliferation in ERα positive breast cancer cell lines MCF7 and T47D. Depletion of ERα partially abolished the effects of TSC on the decrease of Cyclin D1 and cell viability. Our findings suggest that TSC may have therapeutic effects on ERα positive breast cancers and moreover that TSC may suppress breast epithelial cell proliferation by inhibiting the estrogen pathway.

An Activin A/BMP2 chimera, AB215, blocks estrogen signaling via induction of ID proteins in breast cancer cells.

BackgroundOne 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.MethodsAB215 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.ResultsHere 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.ConclusionThis 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.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-549) contains supplementary material, which is available to authorized users.

Promoting E2F1-mediated apoptosis in oestrogen receptor-α-negative breast cancer cells

BackgroundBecause oestrogen receptor α (ERα) regulates E2F1 expression to mediate tamoxifen resistance in ERα-positive breast cancer cells, we aimed to define the possible roles of ERα and E2F1 in promoting the resistance of ERα-negative breast cancer cells to 4-hydroxy-tamoxifen (4OHT).MethodsThis study utilised conventional techniques to demonstrate the effects of 4OHT on the expression of ERα and E2F1 and also examined the individual and combined effects of 4OHT with dipyridamole (DIPY) and 3-O-(3,4,5-trimethoxybenzoyl)-(-)-catechin (TMCG) on the oestrogen-negative MDA-MB-231 breast cancer cell line using viability assays, Hoechst staining, MALDI-TOF mass spectroscopy, and confocal microscopy.ResultsDespite the ERα-negative status of the MDA-MB-231 cells, we observed that 4OHT efficiently up-regulated ERα in these cells and that this upregulation promoted E2F1-mediated cell growth. Because E2F1 plays a dual role in cell growth/apoptosis, we designed a therapy incorporating TMCG/DIPY to take advantage of the elevated E2F1 expression in these 4OHT-treated cells. 4OHT enhances the toxicity of TMCG/DIPY in these ERα-negative breast cancer cells.ConclusionsBecause TMCG/DIPY treatment modulates the methylation status/stability of E2F1, the results demonstrate that therapies targeting the epigenetic machinery of cancer cells in the presence of overexpressed E2F1 may result in efficient E2F1-mediated cell death.

Non-tumorigenic epithelial cells secrete MCP-1 and other cytokines that promote cell division in breast cancer cells by activating ERα via PI3K/Akt/mTOR signaling

Efforts in understanding the role of the microenvironment in the development of breast cancer have focused on tumor-stroma cross-talk, but the possibility that normal epithelial cells might also play a role in tumor progression has received little attention. Here, we show that non-tumorigenic human mammary epithelial cells (MCF10A and HMEC) secrete factors able to enhance the proliferation of estrogen receptor α (ERα) positive breast cancer cells (MCF7 and T47D) and suppress their ability to undergo apoptosis. Conditioned medium (CM) derived from MCF10A and HMEC cells was capable of activating ERα in a hormone-independent way, by phosphorylating ERα on Ser167. Co-exposure with PI3K and mTORC1 inhibitors significantly reduced the ERα Ser167 phosphorylation and suppressed the proliferation-enhancing effects of both 10A-CM and HMEC-CM on MCF7 cells. We show that MCF10A and HMEC secrete numerous cytokines, among them MCP-1, which was one of the most prevalent. MCP-1 was shown to have a role in the effects elicited by the 10A-CM. It activated the ERα by phosphorylating Ser167 via the PI3K/Akt/mTORC1 signaling pathway, an effect which was further confirmed by silencing the MCP-1 receptors, CCR2 and CCR4. To our knowledge, this is the first time MCP-1 has been shown to contribute to ERα signaling activation.These data suggest that normal mammary cells could have the capability of supporting the proliferation of breast cancer cells via paracrine interactions. A better understanding of the role of these cells may be useful for designing strategies for the prevention of tumor progression at early stages.

Estrogen receptor α and aryl hydrocarbon receptor independent growth inhibitory effects of aminoflavone in breast cancer cells.

BackgroundNumerous studies have implicated the aryl hydrocarbon receptor (AhR) as a potential therapeutic target for several human diseases, including estrogen receptor alpha (ERα) positive breast cancer. Aminoflavone (AF), an activator of AhR signaling, is currently undergoing clinical evaluation for the treatment of solid tumors. Of particular interest is the potential treatment of triple negative breast cancers (TNBC), which are typically more aggressive and characterized by poorer outcomes. Here, we examined AF’s effects on two TNBC cell lines and the role of AhR signaling in AF sensitivity in these model cell lines.MethodsAF sensitivity in MDA-MB-468 and Cal51 was examined using cell counting assays to determine growth inhibition (GI50) values. Luciferase assays and qPCR of AhR target genes cytochrome P450 (CYP) 1A1 and 1B1 were used to confirm AF-mediated AhR signaling. The requirement of endogenous levels of AhR and AhR signaling for AF sensitivity was examined in MDA-MB-468 and Cal51 cells stably harboring inducible shRNA for AhR. The mechanism of AF-mediated growth inhibition was explored using flow cytometry for markers of DNA damage and apoptosis, cell cycle analysis, and β-galactosidase staining for senescence. Luciferase data was analyzed using Student’s T test. Three-parameter nonlinear regression was performed for cell counting assays.ResultsHere, we report that ERα-negative TNBC cell lines MDA-MB-468 and Cal51 are sensitive to AF. Further, we presented evidence suggesting that neither endogenous AhR expression levels nor downstream induction of AhR target genes CYP1A1 and CYP1B1 is required for AF-mediated growth inhibition in these cells. Between these two ERα negative cell lines, we showed that the mechanism of AF action differs slightly. Low dose AF mediated DNA damage, S-phase arrest and apoptosis in MDA-MB-468 cells, while it resulted in DNA damage, S-phase arrest and cellular senescence in Cal51 cells.ConclusionsOverall, this work provides evidence against the simplified view of AF sensitivity, and suggests that AF could mediate growth inhibitory effects in ERα-positive and negative breast cancer cells, as well as cells with impaired AhR expression and signaling. While AF could have therapeutic effects on broader subtypes of breast cancer, the mechanism of cytotoxicity is complex, and likely, cell line- and tumor-specific.

Induction of the CLOCK gene by E2-ERα signaling promotes the proliferation of breast cancer cells.

Growing genetic and epidemiological evidence suggests a direct connection between the disruption of circadian rhythm and breast cancer. Moreover, the expression of several molecular components constituting the circadian clock machinery has been found to be modulated by estrogen-estrogen receptor α (E2-ERα) signaling in ERα-positive breast cancer cells. In this study, we investigated the regulation of CLOCK expression by ERα and its roles in cell proliferation. Immunohistochemical analysis of human breast tumor samples revealed high expression of CLOCK in ERα-positive breast tumor samples. Subsequent experiments using ERα-positive human breast cancer cell lines showed that both protein and mRNA levels of CLOCK were up-regulated by E2 and ERα. In these cells, E2 promoted the binding of ERα to the EREs (estrogen-response elements) of CLOCK promoter, thereby up-regulating the transcription of CLOCK. Knockdown of CLOCK attenuated cell proliferation in ERα-positive breast cancer cells. Taken together, these results demonstrated that CLOCK could be an important gene that mediates cell proliferation in breast cancer cells.

Chaperonin CCT-mediated AIB1 folding promotes the growth of ERα-positive breast cancer cells on hard substrates.

Clinical observations have revealed a strong association between estrogen receptor alpha (ERα)-positive tumors and the development of bone metastases, however, the mechanism underlying this association remains unknown. We cultured MCF-7 (ERα-positive) on different rigidity substrates. Compared with cells grown on more rigid substrates (100 kPa), cells grown on soft substrates (10 kPa) exhibited reduced spreading ability, a lower ratio of cells in the S and G2/M cell cycle phases, and a decreased proliferation rate. Using stable isotope labeling by amino acids (SILAC), we further compared the whole proteome of MCF-7 cells grown on substrates of different rigidity (10 and 100 kPa), and found that the expression of eight members of chaperonin CCT increased by at least 2-fold in the harder substrate. CCT folding activity was increased in the hard substrate compared with the soft substrates. Amplified in breast cancer 1 (AIB1), was identified in CCT immunoprecipitates. CCT folding ability of AIB1 increased on 100-kPa substrate compared with 10- and 30-kPa substrates. Moreover, using mammalian two-hybrid protein-protein interaction assays, we found that the polyglutamine repeat sequence of the AIB1 protein was essential for interaction between CCTζ and AIB1. CCTζ-mediated AIB1 folding affects the cell area spreading, growth rate, and cell cycle. The expressions of the c-myc, cyclin D1, and PgR genes were higher on hard substrates than on soft substrate in both MCF-7 and T47D cells. ERα and AIB1 could up-regulate the mRNA and protein expression levels of the c-myc, cyclin D1, and PgR genes, and that 17 β-estradiol could enhance this effects. Conversely, 4-hydroxytamoxifen, could inhibit these effects. Taken together, our studies demonstrate that some ERα-positive breast cancer cells preferentially grow on more rigid substrates. CCT-mediated AIB1 folding appears to be involved in the rigidity response of breast cancer cells, which provides novel insight into the mechanisms of bone metastasis.

ERα directly activated the MDR1 transcription to increase paclitaxel-resistance of ERα-positive breast cancer cells in vitro and in vivo

Chemotherapy is commonly used to treat early-stage invasive and advanced-stage breast cancer either before or after surgery. Increasing evidence from clinical analysis and in vitro studies has shown that ER-positive breast cancer cells are insensitive to chemotherapy. Complete understanding of how ERα mediates drug resistance is prerequisite to improvement of the chemotherapeutic efficacy. Over-expression of P-glycoprotein (P-gp) encoded by MDR1 gene is one of the major causes of drug resistance. The association between ERα and MDR1 in breast cancer is still unclear and the limited reports are conflict. This study systematically explored intrinsic link between ERα and the P-gp over-expression in paclitaxel-resistant ERα(+) breast cancer cell lines and mouse model in molecular details. Our data showed that ERα activated the MDR1 transcription in MCF-7/PTX breast cancer cells by binding to ERE1/2 and interacting with Sp1 that bridged to the downstream CG-rich element within the MDR1 promoter. Knockdown of MDR1 restrained the effect of ERα in MCF-7 cells and sensitized the cells to paclitaxel. Treatment of ICI 182,780 that selectively suppressed ERα significantly decreased the MDR1 expression and increased the sensitivity of drug resistant breast cancer cells and xenograft tumors to paclitaxel. Our data strongly demonstrated that ERα was able to increase drug resistance of breast cancer cells through activating MDR1 transcription. This novel mechanism provides new insight to how the ERα signaling regulates response of ERα(+) breast tumors to chemotherapy, which may be exploited for developing novel therapeutic strategies for breast cancer in the future.

IL-13Rα2 mediates PNR-induced migration and metastasis in ERα-negative breast cancer.

Emerging evidence has linked photoreceptor cell-specific nuclearreceptor (PNR/NR2E3), an orphan nuclear hormone receptor, to human breast cancer. PNR was shown to be a transcriptional activator of estrogen receptor-α (ERα) in ERα-positive breast cancer cell linesand high-level expression of PNR correlates with favorable response of ERα-positive breast cancer patients to tamoxifen. Interestingly, gene expression microarray study shows that PNR regulates distinct genes from those regulated by ERα, suggesting that PNR could have ERα-independent functions. Herein, we investigated the function of PNR in ERα-negative breast cancer cells. Our results showed that PNR-induced cell migration and metastasis of ERα-negative breast cancer cells both in vitro and in vivo, and the effect was attributed to the upregulation of interleukin (IL)-13Rα2, a high-affinity receptor for IL-13 that regulates tumor growth, invasion and metastasis of various human cancers. Mechanistically, PNR activated transcription of IL-13Rα2 through direct recruitment to IL-13Rα2 promoter. Upon stimulation with IL-13, IL-13Rα2 increased the extracellular signal-regulated kinases 1 and 2phosphorylation, which led to breast cancer migration and metastasis. The IL-13 triggered signal cascade was specific to IL-13Rα2, as the closely related IL-13Rα1 was not regulated by PNR. IL-13Rα2 is a novel tumor antigen that is overexpressed in a variety of solid tumor types. This study presents the first evidence that PNR could promote ERα-negative breast cancer metastasis through activation of IL-13Rα2-mediated signaling pathway.