ZR-75 Cells Research Articles

Abstract A39: A role for src kinase in estrogen receptor-positive breast cancer

Abstract Estrogen (E2) stimulation promotes proliferation in estrogen receptor-positive (ER+) breast cancer, which accounts for 60-75% of all breast cancer. E2 signals through the ER via three pathways: the classical genomic pathway involving transcriptional activation of the ER, crosstalk between ER and other transcription factors, and “non-genomic” signaling cascades, which do not involve the transcriptional activity of the ER. This “non-genomic” pathway likely involves Src family kinases (SFKs) and has been implicated in cell cycle progression in fibroblasts engineered to express the ER (Castoria 1999). While not typically amplified or mutationally activated in ER+ breast cancer, Src is hyperactive in most breast cancers. To test whether SFK activity is required for G1/S cell cycle progression in breast cancer cells, we pretreated quiescent ER+ MCF7 or ZR75-1 cells with a selective SFK inhibitor (SU11333) prior to stimulation with E2. Inhibition of SFK activity blocked progression from G1 to S phase. Previous data from our laboratory has demonstrated that G1/S progression in response to peptide growth factor stimulation of quiescent fibroblasts is dependent upon SFK-mediated stabilization of myc mRNA (Bromann 2005). We therefore tested whether SFK activity also regulates myc expression in breast cancer cells. Our data suggest that SFK activity is required for accumulation of myc mRNA in quiescent MCF7s after E2 stimulation, and that E2 stimulated stabilization of myc mRNA is dependent on SFK activity. We are currently investigating the mechanism involved in stabilizing myc mRNA levels.

Bergapten induces metabolic reprogramming in breast cancer cells.

Alterations in cellular metabolism are among the most consistent hallmarks of cancer. Herein, after a comprehensive metabolic phenotype characterization of MCF7 and ZR75 breast cancer cells, we investigated the activity of bergapten (Bg), a plant-derived compound, against breast cancer. The study of different biochemical pathways involved in cell metabolism revealed that the two cell lines have different bioenergetic phenotypes: MCF7 cells express a glycolytic phenotype only partially oxidative, while ZR75 cells mainly have an oxidative phenotype. In both cell lines, Bg blocked glycolysis and significantly decreased glucose-6-phosphate dehydrogenase (G6PDH) activity promoting glucose accumulation; modulated bioenergetic requirements altering the expression of oxidative phosphorylation (OXPHOS) complexes and ATP production; and induced a lipid-lowering effect since an increased lipase activity concomitantly to a reduction in triglyceride levels was observed. Quantitative data of different metabolites and enzymatic activities were presented. Treatment with Bg resulted in an alteration in different metabolic pathways inducing death in the cells. We report a novel action of the natural product Bg on breast cancer, since it induced metabolic reprogramming by disrupting the interconnected network of different metabolic mechanisms. Bg can be used in combination with other forms of targeted chemotherapy to improve cancer treatment outcomes.

Abstract 5109: The CARMA3-Bcl10-MALT1 signalosome mediates NF-κB activation and cellular invasion in AGTR1-positive breast cancer

Abstract Angiotensin II (Ang II) is a potent vasoconstrictor and vascular pro-inflammatory mediator, known classically for its role in promoting arterial dysfunction. Many of these pathogenic effects result from activation of NF-κB in response to stimulation of the type-1 Ang II receptor (AGTR1), a G protein-coupled receptor. We previously discovered that a complex composed of three proteins, CARMA3, Bcl10 and MALT1 (CBM signalosome) mediates AGTR1-dependent activation of NF-κB in vascular cells. Despite its principal role in vascular pathobiology, we recently found that AGTR1 is aberrantly overexpressed in 10-20% of breast cancers (characterized as Luminal, ER+, PR+, Her2-). In this context, AGTR1 promotes cellular invasion and in vivo tumorigenesis, but the mechanisms driving this effect of AGTR1 have remained unclear. We hypothesized that stimulation of AGTR1 in this subset of breast tumors induces CBM-dependent NF-κB activation, thus contributing to tumorigenesis. To develop experimental models for studying the impact of AGTR1 in breast cancer (BC), we screened a panel of BC cell lines and found overexpression of endogenous AGTR1 in BT549 cells, among a few others, but no expression in most lines. To complement the BT549 line, we stably over-expressed exogenous AGTR1 in ZR75 cells (an AGTR1-, luminal line) to produce the ZR75-AGTR1 line. All BC lines tested, irrespective of AGTR1 status, expressed CARMA3, Bcl10 and MALT1. We found that Ang II treatment induces time-dependent phosphorylation of IκB, indicating NF-κB activation, in both BT549 and ZR75-AGTR1 cells, but not in control ZR75 cells. We then employed an NF-κB reporter assay to generate further evidence of NF-κB activation in ZR75-AGTR1 cells. We also demonstrated induction of several endogenous NF-κB target genes using real-time Q-PCR. Importantly, Ang II-dependent NF-κB activation is effectively blocked by pretreatment with losartan (AGTR1 antagonist) or with IKK-VI (IKKβ inhibitor), as well as by siRNA-mediated knockdown of Bcl10 or MALT1. These data indicate that Ang II dependent NF-κB activation in these cells is dependent on activation of AGTR1, proceeds through the canonical NF-κB machinery, and requires the CBM signalosome. Moreover, we showed that AGTR1 activation triggers a dramatic increase in migration and invasion of both BT549 and ZR75-AGTR1 cells, and that this response is completely blocked by siRNA-mediated Bcl10 knockdown. These results demonstrate for the first time that AGTR1 stimulation leads to NF-κB activation in a subset of breast cancer cells, and link this response to aggressive phenotypic behavior. These studies have significant clinical implications since AGTR1 antagonists, currently used for treatment of hypertension, might be effectively re-deployed for treatment of breast cancers that harbor AGTR1 overexpression. Inhibition of CBM-dependent signaling may also represent a novel treatment approach. Citation Format: Prasanna Ekambaram, Nathaniel Hubel, Jia-Ying Lee, Linda Klei, Vincent Concel, Philip Delekta, Scott Tomlins, Linda McAllister-Lucas, Peter Lucas. The CARMA3-Bcl10-MALT1 signalosome mediates NF-κB activation and cellular invasion in AGTR1-positive 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 5109. doi:10.1158/1538-7445.AM2015-5109

Sclerotium rolfsii lectin induces stronger inhibition of proliferation in human breast cancer cells than normal human mammary epithelial cells by induction of cell apoptosis.

Sclerotium rolfsii lectin (SRL) isolated from the phytopathogenic fungus Sclerotium rolfsii has exquisite binding specificity towards O-linked, Thomsen-Freidenreich (Galβ1-3GalNAcα1-Ser/Thr, TF) associated glycans. This study investigated the influence of SRL on proliferation of human breast cancer cells (MCF-7 and ZR-75), non-tumorigenic breast epithelial cells (MCF-10A) and normal mammary epithelial cells (HMECs). SRL caused marked, dose-dependent, inhibition of proliferation of MCF-7 and ZR-75 cells but only weak inhibition of proliferation of non-tumorigenic MCF-10A and HMEC cells. The inhibitory effect of SRL on cancer cell proliferation was shown to be a consequence of SRL cell surface binding and subsequent induction of cellular apoptosis, an effect that was largely prevented by the presence of inhibitors against caspases -3, -8, or -9. Lectin histochemistry using biotin-labelled SRL showed little binding of SRL to normal human breast tissue but intense binding to cancerous tissues. In conclusion, SRL inhibits the growth of human breast cancer cells via induction of cell apoptosis but has substantially less effect on normal epithelial cells. As a lectin that binds specifically to a cancer-associated glycan, has potential to be developed as an anti-cancer agent.

Abstract LB-18: Bcl2-associated athanogene 3 and major vault protein regulate cell survival and resistance to apoptosis in senescent cells by regulating ERK activation

Abstract Senescence is a prominent solid tumor response to therapy in which cells avoid apoptosis and instead enter into prolonged cell cycle arrest. We applied a quantitative proteomics screen to identify signals that lead to therapy-induced senescence (TIS) and discovered that Bcl2-associated athanogene 3 (Bag3) is upregulated after adriamycin treatment in MCF7 cells. Bag3 protein is a member of the BAG family of co-chaperones that interacts with Hsp70. Bag3 also regulates major signaling pathways. Mass Spectrometry analysis of the Bag3 Complex revealed a novel interaction between Bag3 and Major Vault Protein (MVP). Silencing of Bag3 or MVP shifts the cellular response to adriamycin in favor apoptosis. We demonstrate that Bag3 and MVP contribute to apoptosis resistance in TIS by activation of extracellular signal-regulated kinase1/2 (ERK1/2). Bag3 and MVP were required for ERK1/2 activation in response to adriamycin and silencing either gene product decreased cell survival in response to the drug. An increase in nuclear accumulation of MVP is observed during TIS and the shift in MVP subcellular localization is Bag3-dependent. We propose a model in which Bag3 binds to MVP and facilitates MVP accumulation in the nucleus, which sustains ERK1/2 activation. We demonstrate that Bag3 and MVP are expressed in a panel of diverse breast cancer cell lines and human tumor samples from patients. Silencing of Bag3 and MVP shifts the response to apoptosis and regulates ERK1/2 signaling in ER positive MCF7, ZR751 cell lines and triple negative MDA-MB-231 and MDA-MB-468 cells. Overall this study highlights Bag3-MVP as an important complex that regulates a potent pro-survival signaling pathway in breast cancer. Citation Format: Martina Pasillas, Sarah Shields, Rebecca Reilly, Laoighse Mulrane, Jan Strnadnel, Karin Jirstrom, Darran O Connor, William Gallagher, Steven Gonias, Judith Coppinger. Bcl2-associated athanogene 3 and major vault protein regulate cell survival and resistance to apoptosis in senescent cells by regulating ERK activation. [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-18. doi:10.1158/1538-7445.AM2014-LB-18

Abstract 1034: ZEB2 promotes cell motility and metastasis in ER+ breast cancer cells

Abstract While the precise molecular mechanisms underlying metastasis remain unclear, epithelial-to-mesenchymal transition (EMT), the loss of an epithelial cell phenotype and acquisition of a mesenchymal cell phenotype, has been implicated in cancer cell invasion and dissemination. The ZEB family of transcription factors, which includes ZEB1 and ZEB2, has been demonstrated to mediate this transition by downregulating the expression of genes associated with an epithelial phenotype. We sought to investigate the effects of direct ZEB family overexpression on EMT in estrogen receptor-positive (ER+) breast cancer cell systems. We overexpressed ZEB1 or ZEB2 in the epithelial, ER+, luminal A breast cancer cell lines MCF-7 and ZR75. Overexpression of individual ZEB1 and ZEB2 levels were confirmed and localization of the ZEB factors to the nucleus was confirmed by confocal microscopy in both cell lines. ZEB2 overexpressing cells, but not ZEB1 overexpressing cells, showed increased migration and invasion in vitro compared to the vector control in both MCF-7 and ZR75 cell lines, suggesting differential function of the two ZEB family members. Additionally, MCF-7-ZEB2 xenografts exhibited increased lung metastasis compared to MCF-7-vector cells. To elucidate the effects of ZEB on our ER+ cell line we performed next generation deep sequencing on MCF-7 -vector, ZEB1 and ZEB2 overexpressing cells. Analysis of total gene regulation using the NCI Pathway Interaction Database demonstrated an increase in genes associated with RhoA activity, an important mediator of cell motility, in ZEB2 overexpressing cells. However, the ZEB overexpressing cells show no change in morphology and canonical EMT markers remained unchanged between cell lines, suggesting a potential post-translational modification affecting ZEB1 and ZEB2 function. Together these results indicate that ZEB factors drive motility in breast cancer cells but are incapable of promoting a complete EMT in ER+ cells, warranting further investigation into the mechanisms involved in ZEB action. Elucidating the pathways involved in ZEB family function is an important step in understanding the mechanisms underlying metastasis and has the potential to yield new therapeutic targets. Citation Format: Hope E. Burks, Lyndsay Rhodes, Elizabeth Martin, Theresa Phamduy, Steven Elliot, Van Hoang, Henry Segar, Aaron Buechlein, Douglas Rusch, Dave Miller, Melody Baddoo, Erik Flemington, Kenneth Nephew, Douglas Chrisey, Bridgette Collins-Burow, Matthew Burow. ZEB2 promotes cell motility and metastasis in ER+ breast cancer cells. [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 1034. doi:10.1158/1538-7445.AM2014-1034

Cephalotaxus griffithii Hook.f. needle extract induces cell cycle arrest, apoptosis and suppression of hTERT and hTR expression on human breast cancer cells.

BackgroundCephalotaxus spp. are known to possess anticancer potential. In this present work, for the first time the effects of C. griffithii needle (CGN) extracts on human cancer cells were examined.MethodsThe CGN was successively extracted with petroleum ether (PE), acetone and methanol. The extracts were tested for its effect on proliferation of cancer cells (MTT assay on HeLa, ZR751 and HepG2). Extract that showed the maximum growth inhibitory effect was subjected for mechanism of action study. These included apoptosis (morphological and DNA fragmentation assay), cell cycle (flow cytometry), caspase expression (Western blot) and activity (assay kit), p53 (western blot and TP53 siRNA interference) and telomerase expression (reverse transcriptase PCR) analysis.ResultsAmong the extracts, PE extract induced maximum cytotoxicity, with highest death occurred in ZR751 cells. Since, PE extract induced cell death was highest among the CGN extracts, with maximum cancer cell death occurred in ZR751 cells; we carried out mechanism study of PE extract induced ZR751 cell death. It was observed that PE extract induced ZR751 cell death was associated with cell cycle arrest and apoptosis by activating both intrinsic and extrinsic apoptotic pathways. Knock down study revealed that p53 is essential for loss of ZR751 cell viability induced by PE extract. Further, PE extract down-regulated hTERT, hTR, and c-Myc expression. Thin layer chromatography analysis indicated the presence of unique phytochemicals in PE extract.ConclusionBased on the observations, we concluded that PE extract of C. griffithii needle contains important phyto-components with multiple cellular targets for control of breast cancer and is worthy of future studies.Electronic supplementary materialThe online version of this article (doi:10.1186/1472-6882-14-305) contains supplementary material, which is available to authorized users.

Progesterone regulation of tissue factor depends on MEK1/2 activation and requires the proline-rich site on progesterone receptor.

To characterize the molecular mechanism and map the response element used by progesterone (P) to upregulate tissue factor (TF) in breast cancer cells. TF expression and mRNA levels were analyzed in breast cancer ZR-75 and T47D cells, using Western blot and real-time PCR, respectively. Mapping of the TF promoter was performed using luciferase vectors. Progesterone receptor (PR) and specificity protein 1 (Sp1) binding to the TF promoter were analyzed by chromatin immuno precipitation assay. Specific or selective inhibitors were used for the MEK1/2 and the c-Src pathways (UO126 and PP2, respectively). TF mRNA increase peaks at 18h following P treatment in ZR-75 and T47D cells. P upregulation occurs via a transcriptional mechanism that depends on PR and MEK1/2 activation, PR and Sp1 transcription factors bind to a region in the TF promoter that contains three Sp1 sites. TF mRNA upregulation requires an intact PR proline-rich site (mPRO), but it is independent from c-Src. TF upregulation by P is mediated by Sp1 sites in the TF promoter region. Transcriptional upregulation in breast cancer cells occurs via a new mechanism that requires MEK1/2 activation and the mPRO site but independent of c-Src activity. PR Phosphorylation at serine 294 and 345 is not essential.

Abstract P5-05-08: Obesity suppresses estrogen receptor beta expression in breast cancer cells via a HER2-mediated pathway

Abstract Purpose: Increased levels of estrogen receptor beta (ERb) are correlated with an improved breast cancer prognosis. Our preliminary data suggests that obesity may decrease ERβ expression in certain breast cancer subtypes and may contribute to the more aggressive disease associated with obesity. This study aims to elucidate a potential mechanism(s) mediating this suppression. Experimental Design: We utilized an in vitro model of obesity in which breast cancer cells were exposed to sera collected from postmenopausal breast cancer patients at the Cancer Therapy and Research Center at the University of Texas Health Science Center at San Antonio (UTHSCSA) and pooled by BMI category (obese (OB): ≥30 kg/m2; normal weight (NW): 18.5-24.9 kg/m2). Four human mammary tumor cell lines were used: SKBR3 (HER2 over-expressing, ERα negative), MCF-7 and ZR75 (ERα positive, HER2 negative) and MDA-MB-231 (triple negative), in addition to the mouse mammary tumor cell line, MMTV/neu (HER2 over-expressing). The effects of the sera on ERβ expression were assessed by qPCR. Protein expression levels were determined using Western blot analyses. Herceptin, a HER2 antagonist, was used to evaluate the role of HER2 in OB sera-induced ERβ modulation. Results: OB sera exposure led to lower ERβ expression in SKBR3 and MMTV/neu cells in comparison to NW. Treatment of the SKBR3 cells with Herceptin at the time of OB sera exposure resulted in a three-fold increase in ERβ expression. In contrast, there was only a small increase in SKBR3 cell ERβ expression when Herceptin was added to NW sera, suggesting that the effects of the OB sera may be mediated by HER2 activity. In support of this hypothesis, exposure to OB versus NW sera did not modulate ERβ expression in MDA-MB-231, MCF-7 and ZR75 cells, which have very low levels of HER2. Lapatinib, another HER2 antagonist, will be used to inhibit HER2 signaling in MMTV/neu cells. On-going studies utilizing siRNA to the HER2 receptor aim to elucidate the HER2-regulated pathways contributing to the ERβ suppressing effects of OB sera in SKBR3 cells. Conclusion: Based on our preliminary data, we hypothesize that obesity suppresses ERβ expression in breast cancer cells via a HER2-mediated pathway. Elucidation of the mechanism(s) mediating this effect could provide important insight into both the regulation of ERβ expression as well as how obesity promotes a more aggressive disease. Increased knowledge regarding the regulation of this effect may result in the identification of a new treatment target for the obese breast cancer patient population. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-05-08.

Abstract P5-07-05: Oncogene PELP1 regulates alternative splicing in breast cancer through PRMT6

Abstract Background: Alternative splicing (AS) is essential in regulating gene expression and protein diversity; however, dysregulation leads to cancer development with tumor-specific splice variants that act by disrupting a tumor suppressor function. Protein arginine methylation is a modification involved in signal transduction, pre-RNA metabolism and transcriptional activation and deregulation of arginine methylase expression or functions can contribute to cancer progression via alternate splicing. PELP1 is a proto-oncogene, whose expression is associated with higher histological grade and shorter breast cancer specific survival, and PELP1 localization is a determinant of hormone sensitivity. This study focuses on examining the role of PELP1 in regulating gene expression and its possible role in alternative splicing. Methods: ZR75 and MCF7 cells stably expressing PELP1-shRNA or overexpressing PELP1 were used in RNASeq and ChipSeq analysis. Gene differential expression lists were generated using DEseq and PELP1 regulated pathways were analyzed using Ingenuity Pathway Analysis (IPA). Cell proliferation, migration and ERE reporter gene assays were used to test the role of PRMT6 in PELP1's oncogenic functions. Immunoprecipitation, confocal microscopy, ChIP, and GST pull down assays were used to demonstrate PELP1-PRMT6 interactions. Exon specific RTqPCR assays and CD44 minigene reporter assays were used to demonstrate PELP1's role in splicing. Results: RNA- and ChIP-sequencing results showed that the ERa coregulator PELP1 is a novel regulator of alternative splicing in breast cancer through the modulation of several genes involved in splicing including protein arginine methyltransferase 6 (PRMT6). PRMT6 plays a key role in coupling of transcription and alternative splicing by functioning as a transcriptional coactivator that can also regulate alternative splicing in a hormone independent manner. We discovered the novel complex of PELP1 and PRMT6 by co-immunoprecipitation. Biochemical assays revealed that PELP1 binds RNA, colocalizes with the splicing factor SC35 and promotes alternative splicing in reporter gene assays. PELP1 regulates the exon inclusion:skipping ratio of alternatively spliced exons in endogenous vascular endothelial growth factor (VEGF). PELP1's regulation of VEGF splicing is affected by the status of PRMT6. Interestingly, PRMT6 is needed for optimal oncogenic functions of PELP1. Further PELP1 has the potential to regulate the histone methyltransferase activity of PRMT6 and chromatin immunoprecipitation showed that PELP1 affects the enzyme recruitment and enrichment of the histone modification H3R2me2 at estrogen responsive genes. Conclusions: Our findings show a role of PELP1-PRMT6 axis in ERα-mediated RNA splicing and their deregulation has implications for targeted therapeutics in ERα-driven breast cancer. This work was supported by the NIH grant CA095681 and NIH F31 Fellowship 1F31CA173909-01A1. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-07-05.

λ Phage Nanobioparticle Expressing Apoptin Efficiently Suppress Human Breast Carcinoma Tumor Growth In Vivo

Using phages is a novel field of cancer therapy and phage nanobioparticles (NBPs) such as λ phage could be modified to deliver and express genetic cassettes into eukaryotic cells safely in contrast with animal viruses. Apoptin, a protein from chicken anemia virus (CAV) has the ability to specifically induce apoptosis only in carcinoma cells. We presented a safe method of breast tumor therapy via the apoptin expressing λ NBPs. Here, we constructed a λ ZAP-CMV-apoptin recombinant NBP and investigated the effectiveness of its apoptotic activity on BT-474, MDA-MB-361, SKBR-3, UACC-812 and ZR-75 cell lines that over-expressing her-2 marker. Apoptosis was evaluated via annexin-V fluorescent iso-thiocyanate/propidium iodide staining, flow-cytometric method and TUNEL assay. Transfection with NBPs carrying λ ZAP-CMV-apoptin significantly inhibited growth of all the breast carcinoma cell lines in vitro. Also nude mice model implanted BT-474 human breast tumor was successfully responded to the systemic and local injection of untargeted recombinant λ NBPs. The results presented here reveal important features of recombinant λ nanobioparticles to serve as safe delivery and expression platform for human cancer therapy.

Synthesis, characterization and anticancer screening of some novel piperonyl–tetrazole derivatives

A series of new 1,2-substituted tetrazole derivatives were synthesized and evaluated on MCF-7 (ER positive), MDA-MB-231 and ZR-75 (ER negative) breast cancer cell lines. Out of the fourteen compounds, three compounds 10, 12 and 14 showed higher inhibitory effects on MCF-7 cells. Whereas, compound 8 exhibited higher inhibition in MDA-MB-231 and ZR-75 cells at 10−5 M concentration. Total RNA was extracted and effect of compounds on different marker genes was studied. The gene expression of CD44, BRAC and BAX were significantly affected. The compounds were screened against the HepG2 cell line, to know if they are selectively targeting specific cancers and only 1–10 percent inhibition was found at 10−5 M concentration.

Abstract C283: Non-malignant cells from tissues targeted by metastatic breast cancer induce tumor cell resistance to antiestrogens and conventional chemotherapeutic agents.

Abstract The role of the microenvironment of the metastatic niche on breast cancer (BrCa) de novo or secondary resistance to currently available treatments is poorly understood. To study the tumor-stroma interactions and explore potential therapeutic targets in the context of the local metastatic microenvironment of disseminated BrCa we assembled heterotypic in vitro three-dimensional (3D) tissue cultures comprised of estrogen receptor-positive (ER+) BrCa cells and non-malignant accessory cells from organs frequently targeted by metastatic disease, which model the composition and architecture of metastatic lesions. MCF7, T47D and ZR75-1 cells expressing luciferase were immersed in extracellular matrices (collagen type I and Matrigel) alone or with accessory cells, and exposed to clinically achievable concentrations of FDA-approved antineoplastic agents widely used for the treatment of BrCa. The presence of HS-5 bone marrow stromal cells (BMSCs) induced BrCa cell resistance to vinca alkaloids (vincristine, vinblastin and vinorelbin) and taxanes (docetaxel, paclitaxel, cabazitaxel) both in 2D and 3D co-cultures. BMSCs sensitized the MCF7 to antifolates (methotrexate, pralatrexate) in 2D (but not in 3D) cultures. Importantly, BrCa spheroid response to antiestrogens (hydroxytamoxifen (4-OHT), fulvestrant or raloxifene) in 3D conditions was marked by acinar differentiation of tumor spheroids that resembles normal breast epithelium. Co-culture of BrCa cell and immortalized accessory cells from the bone (BMSCs; HOBIT and hFOB osteoblasts), brain (SVGp12 astrocytes), liver (THLE3 hepatocytes), and lung (MRC9 fibroblasts) in 3D conditions, conferred resistance to 4-OHT, raloxifene, and fulvestrant at clinically relevant doses. MCF7 cells injected s.c. in nude mice together with BMSCs (heterotypic xenografts) had reduced response to tamoxifen compared with monotypic xenografts (MCF7 alone). BMSCs induced in MCF7 cells downregulation of TGFβ2; upregulation of a transcriptional signature enriched for genes associated with high-grade tumors, including members of the JAK/STAT pathway, interferon signal gene signature, signaling through EGFR superfamily members, NFkB and other antiapoptotic pathways; and elevated c-Myc protein levels. The growth of MCF7 cells in co-culture with BMSCs or hepatocytes was inhibited by exogenous TGFβ1/2 or neutralizing antibodies against IL-6, INF-gamma, and TNF-alpha. Inhibition of JAK1/2 using ruxolitinib also inhibited MCF7 cell growth in co-culture conditions. Our results indicate that accessory cells representing the microenvironment of metastatic sites confer BrCa cell resistance to antiestrogens and chemotherapeutic agents in conventional and/or 3D tissue cultures and xenograft models. The mechanisms of stroma-induced drug resistance reveal candidate therapeutic targets for refractory BrCa patients with metastatic disease. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C283. Citation Format: Eugen Dhimolea, Richard W.J. Groen, Kornelia Polyak, Constantine S. Mitsiades. Non-malignant cells from tissues targeted by metastatic breast cancer induce tumor cell resistance to antiestrogens and conventional chemotherapeutic agents. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C283.

Abstract B071: Targeting the antiapoptotic protein Mcl-1 in luminal B breast cancers

Abstract In an effort to improve therapeutic tumor cell killing in luminal B breast cancers, we are investigating the combination of endocrine therapies with targeted inhibition of pro-survival proteins of the Bcl2 family, including Bcl2, Bcl-xL, and Mcl-1. Bcl2-family proteins are often overexpressed in breast cancers and are associated with tumor progression, therapeutic resistance, and poor patient survival. These proteins function by sequestering pro-apoptotic BH3-only proteins (BIM, BID, Noxa and Puma), thus inhibiting caspase-dependent apoptosis. We found Mcl1 gene amplification in 15/133 luminal B breast tumors (11%) curated through the Cancer Genome Atlas (TCGA), and Bcl2l1 (encoding Bcl-xL) gene amplification in 1/133 luminal B breast tumors, although Bcl2l1 mRNA was upregulated in 19/133 samples. Bcl2 was not amplified or overexpressed in TCGA-curated luminal B breast tumors. The BH3 mimetic ABT-263, which specifically binds Bcl2 and Bcl-xL, increased tumor cell killing in 3 out of 4 estrogen-deprived luminal B human breast cancer cell lines. However, ZR75-1 cells, which are Mcl1 gene amplified, were resistant to ABT-263. Further, Mcl-1 protein upregulation was induced in HCC-1428 and T47D cells treated with ABT-263, suggesting that cells may use Mcl-1 upregulation to partially mitigate the effects of Bcl-xL targeting with ABT-263. Mcl-1 knockdown using an shRNA approach, decreased cell growth and increased caspase-dependent apoptosis, and increased sensitivity to ABT-263 in 4 of the 4 cell lines tested, including ZR75-1 cells. We used long-term estrogen withdrawal (LTED) to model the sustained response of breast cancer cells to aromatase inhibitors (e.g., exemestane). As compared to parental breast cancer cell lines under estrogen withdraw conditions, LTED populations drastically increased protein levels of Mcl-1. Future experiments will be aimed at assessing the impact of Mcl-1 depletion on ABT-263-treated LTED populations. We predict that combined inhibition of Bcl-2, Bcl-xL and Mcl-1 will improve response of cells to estrogen deprivation. In summary, these preliminary studies suggest that BH3 mimetics targeting Bcl-2 family proteins may be therapeutically efficacious in endocrine-refractory luminal B breast tumors, and support future endeavors to generate BH3-mimetics specifically targeting Mcl-1. Citation Format: Michelle M. Williams, Steve Fesik, Donna Hicks, Rebecca S. Cook. Targeting the antiapoptotic protein Mcl-1 in luminal B breast cancers. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B071.

Synthesis and characterization of quinazolinone derivatives against mammary carcinoma

Abstract Aim The present study was aimed to synthesis series of quinazolinone derivatives against mammary carcinoma. Methods The compounds were synthesized by reaction of nucleophilic attack, Wohler's classical synthesis followed by condensation reaction. The synthesized quinazolinone derivatives were characterized by physico chemical analysis like melting point, TLC, IR, UV and NMR. Compounds have been evaluated for their in-vitro antioxidant methods like DPPH method, H2O2 method, nitrous oxide scavenging method, super oxide scavenging method, ABTS radical scavenging, lipid peroxidation and also in-vitro cytotoxicity study against MCF-7, BT-549, ZR-75 (human breast cancer) cell lines by MTT assay method. Results From physico chemical data the structure of synthesized compounds was confirmed. Antioxidant activity was found from the IC50 value. Qc showed lowest IC50 value in DPPH radical scavenging assay and nitric oxide scavenging assay and was observed to be 27.20 μg/ml and 26.3 μg/ml respectively. In super oxide scavenging activity and lipid peroxidation and H2O2 assays showed a good activity it was observed 26.3 μg/ml, 18.10 μg/ml and 21.4 μg/ml respectively. Qb showed lowest IC50 value in ABTS radical scavenging assay and was observed to be 26.9 μg/ml. Qe showed lowest IC50 value in lipid peroxidation method and was observed to be 149 μg/ml. QN-D showed a moderate antioxidant. In cytotoxic activity, Qc showed lowest IC50 value, i.e. 21.77, 23.03 and 21.14 μg/ml and Qb showed moderate values like 23.25 μg/ml, 27.39 μg/ml and 27.86 μg/ml in MCF-7, BT-549 and ZT-75 respectively. Conclusion All the synthesized compounds showed moderate to good antioxidant and potent cytotoxic activity.

Autocrine IGF-I/insulin receptor axis compensates for inhibition of AKT in ER-positive breast cancer cells with resistance to estrogen deprivation

IntroductionEstrogen receptor α-positive (ER+) breast cancers adapt to hormone deprivation and acquire resistance to antiestrogen therapies. Upon acquisition of hormone independence, ER+ breast cancer cells increase their dependence on the phosphatidylinositol-3 kinase (PI3K)/AKT pathway. We examined the effects of AKT inhibition and its compensatory upregulation of insulin-like growth factor (IGF)-I/InsR signaling in ER+ breast cancer cells with acquired resistance to estrogen deprivation.MethodsInhibition of AKT using the catalytic inhibitor AZD5363 was examined in four ER+ breast cancer cell lines resistant to long-term estrogen deprivation (LTED) by western blotting and proliferation assays. Feedback upregulation and activation of receptor tyrosine kinases (RTKs) was examined by western blotting, real-time qPCR, ELISAs, membrane localization of AKT PH-GFP by immunofluorescence and phospho-RTK arrays. For studies in vivo, athymic mice with MCF-7 xenografts were treated with AZD5363 and fulvestrant with either the ATP-competitive IGF-IR/InsR inhibitor AZD9362 or the fibroblast growth factor receptor (FGFR) inhibitor AZD4547.ResultsTreatment with AZD5363 reduced phosphorylation of the AKT/mTOR substrates PRAS40, GSK3α/β and S6K while inducing hyperphosphorylation of AKT at T308 and S473. Inhibition of AKT with AZD5363 suppressed growth of three of four ER+ LTED lines and prevented emergence of hormone-independent MCF-7, ZR75-1 and MDA-361 cells. AZD5363 suppressed growth of MCF-7 xenografts in ovariectomized mice and a patient-derived luminal B xenograft unresponsive to tamoxifen or fulvestrant. Combined treatment with AZD5363 and fulvestrant suppressed MCF-7 xenograft growth better than either drug alone. Inhibition of AKT with AZD5363 resulted in upregulation and activation of RTKs, including IGF-IR and InsR, upregulation of FoxO3a and ERα mRNAs as well as FoxO- and ER-dependent transcription of IGF-I and IGF-II ligands. Inhibition of IGF-IR/InsR or PI3K abrogated AKT PH-GFP membrane localization and T308 P-AKT following treatment with AZD5363. Treatment with IGFBP-3 blocked AZD5363-induced P-IGF-IR/InsR and T308 P-AKT, suggesting that receptor phosphorylation was dependent on increased autocrine ligands. Finally, treatment with the dual IGF-IR/InsR inhibitor AZD9362 enhanced the anti-tumor effect of AZD5363 in MCF-7/LTED cells and MCF-7 xenografts in ovariectomized mice devoid of estrogen supplementation.ConclusionsThese data suggest combinations of AKT and IGF-IR/InsR inhibitors would be an effective treatment strategy against hormone-independent ER+ breast cancer.

Sox2 suppresses the invasiveness of breast cancer cells via a mechanism that is dependent on Twist1 and the status of Sox2 transcription activity

BackgroundSox2, an embryonic stem cell marker, is aberrantly expressed in a subset of breast cancer (BC). While the aberrant expression of Sox2 has been shown to significantly correlate with a number of clinicopathologic parameters in BC, its biological significance in BC is incompletely understood.MethodsIn-vitro invasion assay was used to evaluate whether the expression of Sox2 is linked to the invasiveness of MCF7 and ZR751 cells. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and/or Western blots were used to assess if Sox2 modulates the expression of factors known to regulate epithelial mesenchymal transition (EMT), such as Twist1. Chromatin immunoprecipitation (ChIP) was used to assess the binding of Sox2 to the promoter region of Twist1.ResultsWe found that siRNA knockdown of Sox2 expression significantly increased the invasiveness of MCF7 and ZR751 cells. However, when MCF7 cells were separated into two distinct subsets based on their differential responsiveness to the Sox2 reporter, the Sox2-mediated effects on invasiveness was observed only in ‘reporter un-responsive’ cells (RU cells) but not ‘reporter responsive’ cells (RR cells). Correlating with these findings, siRNA knockdown of Sox2 in RU cells, but not RR cells, dramatically increased the expression of Twist1. Accordingly, using ChIP, we found evidence that Sox2 binds to the promoter region of Twist1 in RU cells only. Lastly, siRNA knockdown of Twist1 largely abrogated the regulatory effect of Sox2 on the invasiveness in RU cells, suggesting that the observed Sox2-mediated effects are Twist1-dependent.ConclusionSox2 regulates the invasiveness of BC cells via a mechanism that is dependent on Twist1 and the transcriptional status of Sox2. Our results have further highlighted a new level of biological complexity and heterogeneity of BC cells that may carry significant clinical implications.

Abstract 3567: Estrogen signaling promotes effective progesterone receptor engagement with DNA in breast cancer cells.

Abstract Estrogens and progesterone (PROG) are key mediators of breast cancer aetiology and progression. The predominant actions of these hormones in the breast are mediated via their respective cognate steroid receptors, estrogen receptor alpha (ERα) and progesterone receptor (PGR). As ERα upregulates PGR expression, both are currently measured in clinical breast cancer samples to determine the utility of anti-estrogen therapy. Although the molecular actions of estrogens have been extensively studied, the role of PROG and PGR need further elucidation, particularly in the context of activated ERα signaling. Here, we treated ZR75-1 cells with 17β-estradiol (E2) alone or in combination with PROG and assessed global transcriptional responses by microarray expression profiling and receptor binding profiles by chromatin immunoprecipitation-sequencing (ChIP-seq). PROG treatment alone significantly altered the regulation of only seven genes, whereas long term E2 treatment increased the PROG transcriptional response altering 785 genes (p value ≤ 0.05), and regulated pathways involved in ErbB signalling and cell proliferation. Consistent with these findings, ChIP-seq identified 10 times more PGR binding sites following long term E2 treatment (4566 at equivalent peak threshold) compared with PROG treatment alone. Candidate PGR ChIP validated these findings at 100% (12/12) sites tested. The long term E2-treated PGR cistrome was highly conserved amongst species, and enriched for progesterone receptor response elements (PREs) and FOXA1 binding sites. E2 enhancement of PGR binding could be abrogated by long term (72h) but not short term (4h) tamoxifen co-treatment. In T-47D cells, long term E2 treatment was not required for efficient interaction of PGR with DNA and FOXA1 silencing resulted in decreased PGR DNA occupancy at several sites. These data suggests that the combination of E2 and PROG regulated a distinct transcriptional response in breast cancer cells, perhaps due to the dependence of PGR on E2 signaling for maintenance of cellular PGR levels, and via the receptors sharing mutual collaborators and coregulators, such as FOXA1. ERα and PGR collectively shape a mitogenic response in breast cancer cells that is dependent on the combined presence of E2 and PROG. Citation Format: Erin E. Swinstead, Andrew P. Trotta, Eric Smith, Phulwinder K. Grover, Eleanor F. Need, Grant Buchanan. Estrogen signaling promotes effective progesterone receptor engagement with DNA in breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3567. doi:10.1158/1538-7445.AM2013-3567

Abstract 1816: SULT1A1 gene expression level is correlated with gene copy number in different human epithelial and breast cancer cell lines.

Abstract Sulfotransferase isoform 1A1 (SULT1A1) is a member of a subfamily of cytosolic enzymes which catalyze the transfer of a sulfonate group from 3’-phosphoadenosine 5’ -phosphosulfate (PAPS) to a range of xenobiotic and endogenous compounds such as drugs, carcinogens, and steroid hormones. SULT1A1 plays a role in detoxification of tamoxifen (TAM), a drug for estrogen receptor (ER) positive breast cancer treatment and chemoprevention. Besides its involvement in drug metabolism, SULT1A1 also contributes to increased cancer risk. SULT1A1 activity varies several-fold among individuals. Earlier studies have shown that individuals can possess one to five copies of the SULT1A1 gene. SULT1A1 gene expression is higher in individuals with more than one copy number, although there is no significant difference among individuals who have more than 3 copy numbers. We determined the copy number of SULT1A1 in cell lines ZR75, MCF7, T47D, MDA-MB-231, and MCF10A using a pre-designed Custom Plus TaqMan® Copy Number kit from Life Technology. Similar to the human population, the human cell lines tested also contain one to five copies of SULT1A1. Genomic DNA of MCF7, MDA-MB-231, and MCF10A cells contains only one copy of SULT1A1, while ZR75 DNA contains 3 copies and T47D DNA contains 5 copies of SULT1A1. To determine if copy number correlates with gene expression level, we compared the amount of SULT1A1 mRNA by RT-PCR in each cell line with corresponding copy numbers. Cell lines with one copy of SULT1A1 (MCF7 and MCF10A) had lower gene expression; while cell lines with more than one copy number (ZR75 and T47D) had higher gene expression. ZR75 cells expressed significantly more SULT1A1 mRNA than that of T47D, even though ZR75 DNA has two less copies than T47D cells. Although MDA-MB-231 DNA contains one copy of SULT1A1, no SULT1A1 gene expression was detected, indicating that other mechanisms exist in regulating SULT1A1 gene expression. In summary, the current data suggest that differential SULT1A1 gene expression levels in human breast cancer cells could be partially regulated by its copy numbers. Furthermore, the cell lines tested here could be used as in vitro models to study the mechanisms of SULT1A1 gene expression regulations since the copy number differences in those cell lines represented the copy number differences in human population. Citation Format: Aiwei Yao-Borengasser, Lora J. Rogers, Rosalind B. Penny, Vineetha K. Edvana, Susan A. Kadlubar. SULT1A1 gene expression level is correlated with gene copy number in different human epithelial and breast cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1816. doi:10.1158/1538-7445.AM2013-1816

Regulation of human Cripto‐1 expression by nuclear receptors and DNA promoter methylation in human embryonal and breast cancer cells

Human Cripto-1 (CR-1) plays an important role in regulating embryonic development while also regulating various stages of tumor progression. However, mechanisms that regulate CR-1 expression during embryogenesis and tumorigenesis are still not well defined. In the present study, we investigated the effects of two nuclear receptors, liver receptor homolog (LRH)-1 and germ cell nuclear factor receptor (GCNF) and epigenetic modifications on CR-1 gene expression in NTERA-2 human embryonal carcinoma cells and in breast cancer cells. CR-1 expression in NTERA-2 cells was positively regulated by LRH-1 through direct binding to a DR0 element within the CR-1 promoter, while GCNF strongly suppressed CR-1 expression in these cells. In addition, the CR-1 promoter was unmethylated in NTERA-2 cells, while T47D, ZR75-1, and MCF7 breast cancer cells showed high levels of CR-1 promoter methylation and low CR-1 mRNA and protein expression. Treatment of breast cancer cells with a demethylating agent and histone deacetylase inhibitors reduced methylation of the CR-1 promoter and reactivated CR-1 mRNA and protein expression in these cells, promoting migration and invasion of breast cancer cells. Analysis of a breast cancer tissue array revealed that CR-1 was highly expressed in the majority of human breast tumors, suggesting that CR-1 expression in breast cancer cell lines might not be representative of in vivo expression. Collectively, these findings offer some insight into the transcriptional regulation of CR-1 gene expression and its critical role in the pathogenesis of human cancer.