Estrogen Receptor-negative Human Breast Cancer Research Articles

Strong Protection by 4-Hydroxyestrone against Erastin-Induced Ferroptotic Cell Death in Estrogen Receptor-Negative Human Breast Cancer Cells: Evidence for Protein Disulfide Isomerase as a Mechanistic Target for Protection.

Ferroptosis is a recently identified form of regulated cell death, characterized by excessive iron-dependent lipid peroxidation. Recent studies have demonstrated that protein disulfide isomerase (PDI) is an important mediator of chemically induced ferroptosis and also a new target for protection against ferroptosis-associated cell death. In the present study, we identified that 4-hydroxyestrone (4-OH-E1), a metabolic derivative of endogenous estrogen, is a potent small-molecule inhibitor of PDI, and can strongly protect against chemically induced ferroptotic cell death in the estrogen receptor-negative MDA-MB-231 human breast cancer cells. Pull-down and CETSA assays demonstrated that 4-OH-E1 can directly bind to PDI both in vitro and in intact cells. Computational modeling analysis revealed that 4-OH-E1 forms two hydrogen bonds with PDI His256, which is essential for its binding interaction and thus inhibition of PDI's catalytic activity. Additionally, PDI knockdown attenuates the protective effect of 4-OH-E1 as well as cystamine (a known PDI inhibitor) against chemically induced ferroptosis in human breast cancer cells. Importantly, inhibition of PDI by 4-OH-E1 and cystamine or PDI knockdown by siRNAs each markedly reduces iNOS activity and NO accumulation, which has recently been demonstrated to play an important role in erastin-induced ferroptosis. In conclusion, this study demonstrates that 4-OH-E1 is a novel inhibitor of PDI and can strongly inhibit ferroptosis in human breast cancer cells in an estrogen receptor-independent manner. The mechanistic understanding gained from the present study may also aid in understanding the estrogen receptor-independent cytoprotective actions of endogenous estrogen metabolites in many noncancer cell types.

Sevoflurane modulates breast cancer cell survival via modulation of intracellular calcium homeostasis

BackgroundSome retrospective and in vitro studies suggest that general anesthetics influence breast cancer recurrence and metastasis. We compared the effects of general anesthetics sevoflurane versus propofol on breast cancer cell survival, proliferation and invasion in vitro. The investigation focused on effects in intracellular Ca2+ homeostasis as a mechanism for general anesthetic-mediated effects on breast cancer cell survival and metastasis.MethodsEstrogen receptor-positive (MCF7) and estrogen receptor-negative (MDA-MB-436) human breast cancer cell lines along with normal breast tissue (MCF10A) were used. Cells were exposed to sevoflurane or propofol at clinically relevant and extreme doses and durations for dose- and time-dependence studies. Cell survival, proliferation and migration following anesthetic exposure were assessed. Intracellular and extracellular Ca2+ concentrations were modulated using Ca2+ chelation and a TRPV1 Ca2+ channel antagonist to examine the role of Ca2+ in mediating anesthetic effects.ResultsSevoflurane affected breast cancer cell survival in dose-, time- and cell type-dependent manners. Sevoflurane, but not propofol, at equipotent and clinically relevant doses (2% vs. 2 μM) for 6 h significantly promoted breast cell survival in all three types of cells. Paradoxically, extreme exposure to sevoflurane (4%, 24 h) decreased survival in all three cell lines. Chelation of cytosolic Ca2+ dramatically decreased cell survival in both breast cancer lines but not control cells. Inhibition of TRPV1 receptors significantly reduced cell survival in all cell types, an effect that was partially reversed by equipotent sevoflurane but not propofol. Six-hour exposure to sevoflurane or propofol did not affect cell proliferation, metastasis or TRPV1 protein expression in any type of cell.ConclusionSevoflurane, but not propofol, at clinically relevant concentrations and durations, increased survival of breast cancer cells in vitro but had no effect on cell proliferation, migration or TRPV1 expression. Breast cancer cells require higher cytoplasmic Ca2+ levels for survival than normal breast tissue. Sevoflurane affects breast cancer cell survival via modulation of intracellular Ca2+ homeostasis.

Granulocyte-colony stimulating factor-associated aortitis in a woman with advanced breast cancer: a case report and review of the literature

BackgroundGranulocyte-colony stimulating factor (G-CSF) is increasingly been used to prevent febrile neutropenia (FN) associated with the administration of chemotherapy for various cancers. The most common adverse effects of G-CSF are bone pain and injection-site reactions and aortitis has rarely been reported. We report herein a rare case of G-CSF associated with aortitis in a woman with advanced breast cancer.Case presentationA 72-year-old woman with estrogen receptor-negative human epidermal growth factor 2-positive breast cancer with distant metastases in the lung was admitted. Her treatment was initiated with docetaxel in combination with trastuzumab and pertuzumab followed by the supportive use of a long-acting G-CSF, pegfilgrastim. After administration of pegfilgrastim on day 5, the patient had an intermittent fever (body temperature up to 39.6 °C) on day 9 which continued irrespective of taking levofloxacin. She visited our outpatient clinic on day 13 with no objective symptoms other than fever. Laboratory tests revealed a high neutrophil count (15,000/μl) and a high C-reactive protein (CRP) level (46.35 mg/dl) without any other abnormalities. There was no response upon administration of antimicrobial agents. An 18F-fluorodeoxyglucose-positron emission tomography/computed tomography (FDG-PET/CT) revealed thickening of the wall of the descending thoracic aorta and left pleural effusion. Therefore, thoracic aortitis induced by pegfilgrastim was suspected. On day 19, the fever resolved spontaneously followed by a gradual reduction in the neutrophil count and CRP level. In the follow-up CT, the aortic wall thickness and pleural effusion had disappeared.ConclusionsG-CSF may cause aortitis due to stimulation of the production of inflammatory cytokines. In case of high continuous fever after administration of pegfilgrastim, aortitis should be suspected unless there are other infectious findings.

Profiling of gene expression regulated by 17β-estradiol and tamoxifen in estrogen receptor-positive and estrogen receptor-negative human breast cancer cell lines.

One area of great importance in breast cancer (BC) research is the study of gene expression regulated by both estrogenic and antiestrogenic agents. Although many studies have been performed in this area, most of them have only addressed the effects of 17β-estradiol (E2) and tamoxifen (TAM) on MCF7 cells. This study aimed to determine the effect of low doses of E2 and TAM on the expression levels of 84 key genes, which are commonly involved in breast carcinogenesis, in four BC cell lines differentially expressing estrogen receptor (ER) α and HER2 (MCF7, T47D, BT474, and SKBR3). The results allowed us to determine the expression patterns modulated by E2 and TAM in ERα+ and ERα− cell lines, as well as to identify differences in expression patterns. Although the MCF7 cell line is the most frequently used model to determine gene expression profiles in response to E2 and TAM, the changes in gene expression patterns identified in ERα+ and ERα− cell lines could reflect distinctive properties of these cells. Our results could provide important markers to be validated in BC patient samples, and subsequently used for predicting the outcome in ERα+ and ERα− tumors after TAM or hormonal therapy. Considering that BC is a molecularly heterogeneous disease, it is important to understand how well, and which cell lines, best model that diversity.

Cdx2 polymorphism affects the activities of vitamin D receptor in human breast cancer cell lines and human breast carcinomas.

Vitamin D plays a role in cancer development and acts through the vitamin D receptor (VDR). It regulates the action of hormone responsive genes and is involved in cell cycle regulation, differentiation and apoptosis. VDR is a critical component of the vitamin D pathway and different common single nucleotide polymorphisms have been identified. Cdx2 VDR polymorphism can play an important role in breast cancer, modulating the activity of VDR. The objective of this study is to assess the relationship between the Cdx2 VDR polymorphism and the activities of VDR in human breast cancer cell lines and carcinomas breast patients. Cdx2 VDR polymorphism and antiproliferative effects of vitamin D treatment were investigated in a panel of estrogen receptor-positive (MCF7 and T-47D) and estrogen receptor-negative (MDA-MB-231, SUM 159PT, SK-BR-3, BT549, MDA-MB-468, HCC1143, BT20 and HCC1954) human breast cancer cell lines. Furthermore, the potential relationship among Cdx2 VDR polymorphism and a number of biomarkers used in clinical management of breast cancer was assessed in an ad hoc set of breast cancer cases. Vitamin D treatment efficacy was found to be strongly dependent on the Cdx2 VDR status in ER-negative breast cancer cell lines tested. In our series of breast cancer cases, the results indicated that patients with variant homozygote AA were associated with bio-pathological characteristics typical of more aggressive tumours, such as ER negative, HER2 positive and G3. Our results may suggest a potential effect of Cdx2 VDR polymorphism on the efficacy of vitamin D treatment in aggressive breast cancer cells (estrogen receptor negative). These results suggest that Cdx2 polymorphism may be a potential biomarker for vitamin D treatment in breast cancer, independently of the VDR receptor expression.

Tamoxifen induces apoptosis through cancerous inhibitor of protein phosphatase 2A-dependent phospho-Akt inactivation in estrogen receptor-negative human breast cancer cells.

IntroductionTamoxifen, a selective estrogen receptor (ER) modulator, may affect cancer cell survival through mechanisms other than ER antagonism. In the present study, we tested the efficacy of tamoxifen in a panel of ER-negative breast cancer cell lines and examined the drug mechanism.MethodsIn total, five ER-negative breast cancer cell lines (HCC-1937, MDA-MB-231, MDA-MB-468, MDA-MB-453 and SK-BR-3) were used for in vitro studies. Cellular apoptosis was examined by flow cytometry and Western blot analysis. Signal transduction pathways in cells were assessed by Western blot analysis. The in vivo efficacy of tamoxifen was tested in xenograft nude mice.ResultsTamoxifen induced significant apoptosis in MDA-MB-231, MDA-MB-468, MDA-MB-453 and SK-BR-3 cells, but not in HCC-1937 cells. Tamoxifen-induced apoptosis was associated with inhibition of cancerous inhibitor of protein phosphatase 2A (CIP2A) and phospho-Akt (p-Akt) in a dose-dependent manner. Ectopic expression of either CIP2A or Akt protected MDA-MB-231 cells from tamoxifen-induced apoptosis. In addition, tamoxifen increased protein phosphatase 2A (PP2A) activity, and tamoxifen-induced apoptosis was attenuated by the PP2A antagonist okadaic acid in the sensitive cell lines, but not in resistant HCC-1937 cells. Moreover, silencing CIP2A by small interfering RNA sensitized HCC-1937 cells to tamoxifen-induced apoptosis. Furthermore, tamoxifen regulated CIP2A protein expression by downregulating CIP2A mRNA. Importantly, tamoxifen inhibited the in vivo growth of MDA-MB-468 xenograft tumors in association with CIP2A downregulation, whereas tamoxifen had no significant effect on CIP2A expression and anti-tumor growth in HCC-1937 tumors.ConclusionsInhibition of CIP2A determines the effects of tamoxifen-induced apoptosis in ER-negative breast cancer cells. Our data suggest a novel “off-target“ mechanism of tamoxifen and suggest that CIP2A/PP2A/p-Akt signaling may be a feasible anti-cancer pathway.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0431-9) contains supplementary material, which is available to authorized users.

Gene expression profiles of NO- and HNO-donor treated breast cancer cells: insights into tumor response and resistance pathways

Nitric oxide (NO) synthase 2 (NOS2), a major inflammatory protein, modulates disease progression via NO in a number of pathologies, including cancer. The role of NOS2-derived NO is not only flux-dependent, which is higher in mouse vs human cells, but also varies based on spatial and temporal distribution both within tumor cells and in the tumor microenvironment. NO donors have been utilized to mimic NO flux conditions and to investigate the effects of varied NO concentrations. As a wide range of effects mediated by NO and other nitrogen oxides such as nitroxyl (HNO) have been elucidated, multiple NO- and HNO-releasing compounds have been developed as potential therapeutics, including as tumor modulators. One of the challenges is to determine differences in biomarker expression from extracellular vs intracellular generation of NO or HNO. Taking advantage of new NO and HNO releasing agents, we have characterized the gene expression profile of estrogen receptor-negative human breast cancer (MDA-MB-231) cells following exposure to aspirin, the NO donor DEA/NO, the HNO donor IPA/NO andtheir intracellularly-activated prodrug conjugates DEA/NO-aspirin and IPA/NO-aspirin. Comparison of the gene expression profiles demonstrated that several genes were uniquely expressed with respect to NO or HNO, such as miR-21, HSP70, cystathionine γ-lyase and IL24. These findings provide insight into targets and pathways that could be therapeutically exploited by the redox related species NO and HNO.

Dinuclear berenil-platinum (II) complexes as modulators of apoptosis in human MCF-7 and MDA-MB231 breast cancer cells.

The metabolism of alkylating agents is accompanied by the generation of reactive oxygen species. The aim of this study was to treat estrogen receptor-positive and estrogen receptor-negative human breast cancer cells, MCF-7 and MDA-MB-231, respectively, with cisplatin and five different berenil-platinum (II) complexes, and then to investigate the oxidative modifications of DNA, lipid and protein, and to compare them with the profile of various pro- and antiapoptotic proteins. Changes in the levels of 8-hydroxy-2'-deoxyguanosine, 4-hydroxynonenal, carbonyl groups, dityrosine, active caspases 3, 8 and 9, as well as the expression of Bcl-2, Bax, cytochrome c, and p53 were subsequently examined. Activities of superoxide dismutase, catalase and glutathione, vitamin C levels were also investigated. Cellular reactions to cisplatin and the berenil-platinum (II) derivatives were more pronounced in MCF-7 cells as compared with the MDA-MB231 cells. Furthermore, the berenil-platinum (II) derivatives were found to be more effective than cisplatin. All of the complexes reduced the activity of superoxide dismutase and catalase, and also lowered the levels of non-enzymatic antioxidants. Increased level of lipid, protein as well as DNA damage markers was also observed after berenil-platinum (II) derivatives treatment. Similarly, the increase in the levels of the proapoptotic factors, were detected in MCF-7 and MDA-MB231 cells. Incubation of examined cells with the berenil-platinum (II) complexes also led to the increase in the levels of active caspases 3, 8 and 9. In conclusion, the results of the present study demonstrated that berenil-platinum (II) complexes more efficiently mediate cellular oxidative modifications and proapoptotic metabolism, particularly in MCF-7 cells, compared to cisplatin. Pt2(isopropylamine)4berenil2 and Pt2(piperidine)4(berenil)2 notably affected the cellular metabolism of estrogen-positive breast cancer cells. Thus, these complexes may be valuable for the design of additional anticancer drugs.

Sensitivity Evaluation of Two Human Breast Cancer Cell Lines to Tamoxifen through Apoptosis Induction

Tamoxifen citrate (TAM) has been used to treat breast cancer in women for many years. The com-parative effects of TAM in inducing apoptosis were evaluated in estrogen receptor-positive (ER- positive MCF-7) and estrogen receptor-negative (ER-negative MDA-MB-231) human breast cancer cell lines in vitro in order to determine if these two cell lines differ in their sensitivity to TAM. Mi-tochondrial membrane permeability potential disruption was assessed in both cell lines by a lip-ophilic cationic dye (DePsipher assay, Trevigen, Inc.) utilizing fluorescence microscopy. Using this specific fluorochrome, we were able to associate mitochondrial membrane disruption to early, mid-, and late apoptotic cells. TAM induced cell death via apoptosis in both ER-positive and ER- negative cells, however, apoptosis induction was more pronounced in ER-positive MCF-7 compared to ER-negative MDA-MB-231 breast cancer cells. These findings may have some therapeutic use in the treatment of estrogen dependent and estrogen independent breast cancer.

Arsenic Induces Functional Re-Expression of Estrogen Receptor α by Demethylation of DNA in Estrogen Receptor-Negative Human Breast Cancer

Estrogen receptor α (ERα) is a marker predictive for response of breast cancers to endocrine therapy. About 30% of breast cancers, however, are hormone- independent because of lack of ERα expression. New strategies are needed for re-expression of ERα and sensitization of ER-negative breast cancer cells to selective ER modulators. The present report shows that arsenic trioxide induces reactivated ERα, providing a target for therapy with ER antagonists. Exposure of ER-negative breast cancer cells to arsenic trioxide leads to re-expression of ERα mRNA and functional ERα protein in in vitro and in vivo. Luciferase reporter gene assays and 3-(4,5-dimethylthiazol-2-yl)- 5-(3-carboxymethoxyphenyl)- 2-(4-sulfophenyl)- 2H-tetrazolium (MTS) assays show that, upon exposure to arsenic trioxide, formerly unresponsive, ER-negative MDA-MB-231 breast cancer cells become responsive to ER antagonists, 4-hydroxytamoxifen and ICI 182,780. Furthermore, methylation- specific PCR and bisulfite-sequencing PCR assays show that arsenic trioxide induces partial demethylation of the ERα promoter. A methyl donor, S-adenosylmethionine (SAM), reduces the degree of arsenic trioxide-induced re-expression of ERα and demethylation. Moreover, Western blot and ChIP assays show that arsenic trioxide represses expression of DNMT1 and DNMT3a along with partial dissociation of DNMT1 from the ERα promoter. Thus, arsenic trioxide exhibits a previously undefined function which induces re-expression ERα in ER-negative breast cancer cells through demethylation of the ERα promoter. These findings could provide important information regarding the application of therapeutic agents targeting epigenetic changes in breast cancers and potential implication of arsenic trioxide as a new drug for the treatment of ER–negative human breast cancer.

Abstract 865: Fat-1 gene suppression of breast cancer growth in mice

Abstract The high ratio of n-6/n-3 polyunsaturated fatty acids (PUFAs) content in Western diets (approximately 10 to 30) may be related to the high breast cancer rate in the United States compared to Japan (approximately 4). Numerous studies in literature report about the tumor suppression effect of n-3 PUFAs, a main component of fish or fish oil. A recent study on B16 melanoma on C57BL/6 mice carrying the fat-1 gene, suppressed the melanoma by changing the n-6/n-3 ratio in tumors from 27:1 in wild type mice to 4.6:1 in fat-1 mice. In this project, a fat-1 gene, which encodes a fatty acyl desaturase that acts on substrates of 16-20 carbons and converts n-6 to n-3 fatty acids, was transfected into the human breast cancer cells MDA-MB-231, parental MCF-7 and tamoxifen-resistant MCF-7/TAM-R cells, Sk-BR3 and murine EMT6 mammary tumor cells. Both fat-1 transgenic mice and fat-1 gene treated breast cancer cells were employed to test our hypothesis that fat-1 suppresses the growth of both estrogen receptor (ER) positive and ER negative breast cancer cells that respond poorly to endocrine therapies. We have also treated the breast cancer cell lines with docosahexaenoic acid (DHA, n-3 fatty acid), arachidonic acid (AA, n-6 fatty acid) with a vehicle control. We found that for the ER negative human breast cancer cells, MDA- MB-231 and SK-BR-3 cells, the cell proliferation rate of DHA treated cells decreased 50%, compared to the corresponding control cells treated with AA and vehicle control. The fatty acid analysis of the fat-1 gene expressing breast cancer cells MDA-MB-231 and MCF-7 showed 30% and 46% reduction of n-6/n-3 ratio, respectively, and ∼30% reduction of cell proliferation rate. The fat-1 transfected MDA-MB-231 cells have ∼4-fold increase of cell apoptosis by Tunel assay. Fat-1 gene expression also suppresses the growth of tamoxifen resistant human breast cancer cells MCF-7F/Tam-R. These results indicate that fat-1 or n-3 fatty acid suppresses breast cancer cells in an ER independent way. Normal human mammary epithelial cells treated with DHA, AA and BSA showed no significant difference of growth rates, suggesting that the n-3 suppression effect is breast cancer selective. In a preliminary study, the growth rate of the EMT6 mammary tumors were significantly reduced when the cell line was treated with fat-1 gene before inoculation into the FVB/NJ mice. The transgenic mice expressing fat-1 gene are also used to investigate the breast cancer suppression effect of fat-1 gene in vivo. The underlying mechanism of the breast cancer suppression effect by fat-1 gene is currently under investigation. This study may provide a potential adjuvant therapeutic method for hormonal resistant human breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 865. doi:10.1158/1538-7445.AM2011-865

Preparation and Evaluation of Carborane Analogues of Tamoxifen

A stereoselective synthesis of closo carborane analogues of tamoxifen was developed where the products represent a new approach to developing metabolically robust SERMs. The A-ring found in the backbone of tamoxifen was replaced with an ortho carborane cluster; the product was determined to be the desired Z isomer, which showed superior chemical stability to tamoxifen both in solution and in the solid state. By use of microwave heating, it was possible to convert some of the Z carborane tamoxifen analogue to the corresponding E isomer. Cell growth assays using both isomers and a carborane that is known to target the ER were conducted using estrogen receptor (ER) positive and ER negative human breast cancer cells with and without the presence of estradiol (E2). The Z carborane isomer was able to inhibit cell proliferation better than tamoxifen in an E2 free environment, while the E isomer inhibited cell growth better than tamoxifen when E2 was present.

Genistein inhibits osteolytic bone metastasis and enhances bone mineral in nude mice

In this study, the effective activity of genistein on osteolytic bone metastasis and bone mineral was investigated. Female BALB/c-nu/nu mice were injected with estrogen receptor-negative human breast cancer cells, MDA-MB-231, into left cardiac ventricle to form osteolytic bone metastases, and administered genistein subcutaneously after radiologically small but defined osteolytic metastases had been observed (protocol 1), simultaneously with cancer cells inoculation (protocol 2) and prophylactically 7 days before inoculation of cancer cells (protocol 3). In all protocols, genistein (10 mg/kg/day) markedly reduced the number and volume of osteolytic bone metastases assessed by radiography and the number of osteoclasts. Furthermore, histomorphometrical analysis revealed that genistein markedly increased trabecular area (Tb.Ar%), trabecular thickness (Tb.Th) and trabecular number (Tb.N), and decreased trabecular separation (Tb.Sp). These results thus demonstrate that genistein could inhibit osteolytic bone metastases, suppress bone resorption, increase bone mass and improve bone microstructure in bone metastases of breast cancer.

Modification of survival pathway gene expression in human breast cancer cells by tetraiodothyroacetic acid (tetrac)

Tetraiodothyroacetic acid (tetrac) inhibits the cellular actions of thyroid hormone initiated at the hormone receptor on plasma membrane integrin αvβ3. Via interaction with the integrin, tetrac is also capable of inhibiting the angiogenic effects of vascular endothelial growth factor and basic fibroblast growth factor. MDA-MB-231 cells are estrogen receptor-negative human breast cancer cells shown to be responsive to tetrac in terms of decreased cell proliferation. Here we describe actions initiated at the cell surface receptor by unmodified tetrac and nanoparticulate tetrac on a panel of survival pathway genes in estrogen receptor-negative human breast cancer (MDA-MB-231) cells. Nanoparticulate tetrac is excluded from the cell interior. Expression of apoptosis inhibitors XIAP (X-linked inhibitor of apoptosis) and MCL1 (myeloid cell leukemia sequence 1) was downregulated by nanoparticulate tetrac in these breast cancer cells whereas apoptosis-promoting CASP2 and BCL2L14 were upregulated by the nanoparticulate formulation. Unmodified tetrac affected only XIAP expression. Expression of the angiogenesis inhibitor thrombospondin 1 (THBS1) gene was increased by both formulations of tetrac, as was the expression of CBY1, a nuclear inhibitor of catenin activity. The majority of differentially regulated Ras-oncogene family members were downregulated by nanoparticulate tetrac. The latter downregulated expression of epidermal growth factor receptor gene and unmodified tetrac did not. Nanoparticulate tetrac has coherent anti-cancer actions on expression of differentially-regulated genes important to survival of MDA-MB-231 cells.

Isolation and characterization of a highly metastatic hybrid cell line generated between estrogen receptor (ER) negative and ER positive breast cancer cells in mouse bone marrow.

Abstract Abstract #2053 Background: Breast cancer cells in a patient are heterogeneous. How the presence of these heterogeneous cancer cells may contribute to cancer progression is not clear. Experimental evidence indicates that soluble factors produced by aggressive cancer cells may generate metastasis niches for less aggressive cancer cells. In this study, we sought to determine how a growing orthotopic tumor formed by an aggressive ER negative breast cancer cell line might affect the metastatic potential of a less aggressive ER positive breast cancer cell line.
 Methods and Results: Five-week-old female nude mice were injected orthotopically with metastasis-competent ER negative human breast cancer MDA-MB-231/GFP/Neo cells. After three weeks, metastasis-incompetent human breast cancer ER positive ZR-75-1/GFP/puro cells were inoculated into these mice via intra-cardiac (IC) route and puromycin resistant metastatic ZR-75-1/GFP/puro cells were flushed out of the bone marrow of one mouse and established as a variant cell line called B6. B6 cells have both estrogen-dependent and -independent cells when they were orthotopically injected into female nude mice with or without estrogen supplementation. The cells isolated from an estrogen-dependent tumor were called B6TE and the cells isolated from an estrogen-independent tumor were called B6TC. Further characterization revealed that B6TC cell was more tumorigenic without estrogen supplementation and has a unique metastatic property. More interestingly, B6TC cell was resistant to both puromycin and G418 suggesting it was derived from the fusion of MDA-MB-231/GFP/Neo and ZR-75-1/GFP/puro in the mouse bone marrow. When compared with parental MDA-MB-231/GFP/Neo, the B6TC cells were found to be more metastatic to lung and bone after IC inoculation as detected by GFP imaging, with significant development of paraplegia in the hind limbs after four weeks. More significantly, B6TC mice also developed metastases to brain, which was not observed in the MDA-MB-231/GFP/Neo group. B6TC cell has a low expression of ER alpha and CD24, and high expression of p-ERK, CD44, ALDH. It also expresses vimentin, CXCR4 and Integrin-β1. Its growth is refractory to the anti-estrogens, raloxifene and tamoxifen.
 Conclusion: Our study indicates that B6 cells generated in the bone marrow were a heterogeneous population of ER positive and ER negative cells with marked differences in their proliferative potential and malignancy. The low ER-expressing B6TC is a novel hybrid cell line generated spontaneously in a metastatic site, which has propensity to metastasize to brain in addition to lung and bone. The expression of CD44 and ALDH indicates that it has stem cell-like features. Further characterization and molecular profiling is underway to reveal the novel biomarkers. This cell line should be a useful model for the investigation of the molecular mechanism of brain metastasis and the therapeutic strategies for the treatment of metastatic breast cancer resistant to anti-estrogens. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2053.

Cadmium regulation of apoptotic and stress response genes in tumoral and immortalized epithelial cells of the human breast

Cadmium (Cd) is a widely-disseminated metal which can be imported and accumulated in living cells thereby drastically interfering with their biological mechanisms. Increasing interest has been recently focused on the elucidation of the cellular and molecular aspects of Cd-dependent regulation of gene expression and signal transduction pathways in different model system. Concerning breast cancer, very limited studies have been produced so far on the role played by Cd on estrogen receptor-negative human breast cancer cells, that are expected to be insensitive to the already-proven metallo-estrogenic effect exerted by Cd on the estrogen receptor-positive cell counterparts. Here, we have examined the effects of long-term (96 h) exposure of estrogen receptor-negative MDA-MB231 malignant adenocarcinoma cells to CdCl 2 at 5 μM concentration, corresponding to the IC 50 for this time of incubation, by evaluating the expression levels of genes coding for stress response factors (e.g. heat shock proteins and metallothioneins), and for apoptosis-related factors and enzymes. In parallel, we tested the gene expression pattern of immortalized HB2 breast epithelial cells, taken as non-tumoral counterpart, after the same exposure to the metal which instead did not exert any change in their cell number with respect to controls. Our cumulative results indicate that, whilst HB2 cells appear to activate defense mechanisms against metal stress principally via metallothionein massive up-regulation and appearance of the spliced form of XBP-1 message, MDA-MB231 cells seem to couple the onset of a protective reaction (e.g. up-regulation of hsp27 and metallothioneins) to the switching-on of new intracellular pathways directing cells to a kind of death which shares several aspects with the apoptotic program, such as down-regulation of Bcl-2 and over-expression of Dap kinase and several caspases.

Estrogen Receptor-Positive and Estrogen Receptor-Negative Human Breast Cancer Cells: Regulation of Expression of Cancer-Related Genes by Estradiol and Tamoxifen

Breast cancer is one of the most prevalent forms of carcinomas among women world-wide. The oncogenes, tumor suppressor genes and antioxidant enzyme genes are cancer-related genes which play an important role in the initiation, metastasis and malignancy of many cancers, including breast cancer. Estrogen which is a key steroid hormone in the regulation and differentiation of the normal breast also appears to be involved in the carcinogenesis of this tissue. Antiestrogen therapy, such as the use of tamoxifen, targets the estrogen receptors (ER) through which estrogen exerts both its normal biological function as well as the molecular processes leading to cancer formation. The objectives of the present study were to characterize and quantify the expression of genes related to human breast carcinogenesis, using ER- positive (ZR-75-1) and ER-negative (MDA-MB-231) cells as in vitro cellular models of breast cancer. We have examined the expression of a number of genes which have an important clinical significance in human breast cancer, when expressed at levels below or above the normal range. Among these are the receptor oncogenes (EGFR, c-erbB2), other oncogenes (pS2, hMAM, MUC1 and CK-19), the tumor suppressor gene, p53, as well as four of the major antioxidant enzyme genes. These are superoxide dismutase-1 (SOD1), superoxide dismutase-2 (SOD2), Se-glutathione peroxidase (GPx1) and catalase (CAT), all of which have been well characterized in human cells, and whose activities have been localized to different cellular compartments, such as the cytoplasm, mitochondria and peroxisomes. Gene expressions were determined using real-time PCR, in control untreated cells as well as when these two cell types were exposed to 17β-estradiol (E), or tamoxifen (TAM) or a combination of E and TAM. Our results show that basal level expressions of receptor oncogenes (EGFR and c-erbB2) as well as two of the four antioxidant enzyme genes (SOD2 and GPx1) were several folds lower in ER-positive breast cancer cells (ZR-75-1) as compared to ER-negative cells (MDAMB- 231). However, treatment with E and TAM, either individually or in combination, produced much greater effects on the ER-positive cells than on the ER-negative cells, with the result that these genes were greatly overexpressed in the ER-positive cells as compared to the ER-negative cells. Of these, the receptor oncogene, c-erbB2, and the antioxidant enzyme gene, SOD2, were the most affected, resulting in striking upregulations ranging over several hundred folds. This, and other observations, would suggest that the carcinogenic effects of estradiol and the anticarcinogenic effects of tamoxifen are not only mediated by the presence of estrogen receptors, but may also be regulated, either directly or indirectly, through other mechanisms, including the mitochondrial antioxidant system. Keywords: Breast cancer, estrogen and antiestrogens, tamoxifen, oncogenes, tumor suppressor genes, antioxidant enzyme genes, oxidative stress and antioxidants, quantitative real-time PCR

Aberrant Expression of Fra-1 in Estrogen Receptor-negative Breast Cancers and Suppression of their Propagation In Vivo by Ascochlorin, an Antibiotic that Inhibits Cellular Activator Protein-1 Activity

Estrogen receptor-negative breast cancers generally are highly malignant, resistant to chemotherapy and poorly prognostic. Here we demonstrate that estrogen receptor-negative human breast cancer cell lines highly express Fra-1, c-Fos and c-Jun, components of the transcription factor, activator protein-1 (AP-1). Retrospective observation of breast cancer tissues obtained by core needle biopsy before surgery from stages II and III patients demonstrates that Fra-1 expression is high in estrogen receptor-negative human breast cancers, and negatively correlated to paclitaxel sensitivity. Ascochlorin, which suppresses cellular AP-1 activity, selectively kills estrogen receptor-negative human and mouse breast cancer cell lines, and prolongs the survival time of mice implanted with an estrogen receptor-negative mammary carcinoma. These results suggest that chemotherapy targeting AP-1 activity is a potent strategy for estrogen receptor-negative human breast cancers.

Estrogenic Regulation of Host Immunity against an Estrogen Receptor–Negative Human Breast Cancer

The risk of developing breast cancer is positively correlated with exposure to increased levels of estrogen and/or an increased duration of estrogen exposure. Many different mechanisms have been proposed to explain the association of estrogens with breast cancer risk; however, the well-documented immune modulatory properties of estrogen have received little attention. In part, this is due to a lack of suitable models for studying this relationship. We have developed an animal model using estrogen receptor (ER)-negative human breast cancer cell line, MDA-MB-468, xenografted into severe combined immunodeficient (SCID) mice. We also generated the ER-alpha knockout (ER-alphaKO) mice on the SCID background and then tested the ability of 17beta-estradiol to stimulate growth of xenografted ER-negative human breast cancer tumors in wild-type and ER-alphaKO SCID mice. We quantified vascularization of tumors, macrophage recruitment to the tumor site by immunocytochemistry, and inflammatory cytokine production. We show that estrogen treatment of C57BL/6/SCID mice promotes the growth of xenografted ER-negative tumors in wild-type mice and this estrogen-induced tumor growth is abrogated in ER-alphaKO mice. Tumor neovascularization of estrogen-treated mice was unchanged versus control; however, estrogen treatment of the C57BL/6/SCID host suppressed macrophage recruitment to and inflammatory cytokine production at the tumor site. These data are consistent with estrogen modulation of the inflammatory response as a contributing factor in estrogen-stimulated growth of an ER-negative tumor. This effect on the host innate immune response was mediated by ER-alpha.

Selective cytotoxicity of ascochlorin in ER-negative human breast cancer cell lines

While agents targeting estrogen receptors are most effective in adjuvant therapy for human breast cancers expressing estrogen receptors after surgery, breast cancers lacking estrogen receptor are clinically serious, because they are highly malignant and exhibit resistance to the usual anti-cancer drugs, including estrogen receptor-antagonists and DNA breaking agents. Here, we found that MX-1, a human breast cancer cell line lacking estrogen receptors, exhibited higher AP-1 activity and expressed higher levels of c-Jun, c-Fos, and Fra-1 when compared with conventional estrogen receptor-positive human breast cancer cell lines. The prenylphenol antibiotic ascochlorin suppressed the AP-1 activity of MX-1 cells, and selectively killed MX-1 cells, partly due to induction of apoptosis. Our results suggest that AP-1 is an effective clinical target molecule for the treatment of estrogen receptor-negative human breast cancer.