Estrogen-dependent Human Breast Cancer Cells Research Articles

Metabolites of n-Butylparaben and iso-Butylparaben Exhibit Estrogenic Properties in MCF-7 and T47D Human Breast Cancer Cell Lines.

Two oxidized metabolites of n-butylparaben (BuP) and iso-butylparaben (IsoBuP) discovered in human urine samples exhibit structural similarity to endogenous estrogens. We hypothesized that these metabolites bind to the human estrogen receptor (ER) and promote estrogen signaling. We tested this using models of ER-mediated cellular proliferation. The estrogenic properties of 3-hydroxy n-butyl 4-hydroxybenzoate (3OH) and 2-hydroxy iso-butyl 4-hydroxybenzoate (2OH) were determined using the ER-positive, estrogen-dependent human breast cancer cell lines MCF-7, and T47D. The 3OH metabolite induced cellular proliferation with EC50 of 8.2µM in MCF-7 cells. The EC50 for 3OH in T47D cells could not be reached. The 2OH metabolite induced proliferation with EC50 of 2.2µM and 43.0µM in MCF-7 and T47D cells, respectively. The EC50 for the parental IsoBuP and BuP was 0.30 and 1.2µM in MCF-7 cells, respectively. The expression of a pro-proliferative, estrogen-inducible gene (GREB1) was induced by these compounds and blocked by co-administration of an ER antagonist (ICI 182, 780), confirming the ER-dependence of these effects. The metabolites promoted significant ER-dependent transcriptional activity of an ERE-luciferase reporter construct at 10 and 20µM for 2OH and 10µM for 3OH. Computational docking studies showed that the paraben compounds exhibited the potential for favorable ligand-binding domain interactions with human ERα in a manner similar to known x-ray crystal structures of 17ß-estradiol in complex with ERα. We conclude that the hydroxylated metabolites of BuP and IsoBuP are weak estrogens and should be considered as additional components of potential endocrine disrupting effects upon paraben exposure.

Loss of estrogen-regulated microRNA expression increases HER2 signaling and is prognostic of poor outcome in luminal breast cancer.

Among the genes regulated by estrogen receptor (ER) are miRNAs that play a role in breast cancer signaling pathways. To determine whether miRNAs are involved in ER-positive breast cancer progression to hormone independence, we profiled the expression of 800 miRNAs in the estrogen-dependent human breast cancer cell line MCF7 and its estrogen-independent derivative MCF7:2A (MCF7:2A) using NanoString. We found 78 miRNAs differentially expressed between the two cell lines, including a cluster comprising let-7c, miR99a, and miR125b, which is encoded in an intron of the long noncoding RNA LINC00478. These miRNAs are ER targets in MCF7 cells, and nearby ER binding and their expression are significantly decreased in MCF7:2A cells. The expression of these miRNAs was interrogated in patient samples profiled in The Cancer Genome Atlas (TCGA). Among luminal tumors, these miRNAs are expressed at higher levels in luminal A versus B tumors. Although their expression is uniformly low in luminal B tumors, they are lost only in a subset of luminal A patients. Interestingly, this subset with low expression of these miRNAs had worse overall survival compared with luminal A patients with high expression. We confirmed that miR125b directly targets HER2 and that let-7c also regulates HER2 protein expression. In addition, HER2 protein expression and activity are negatively correlated with let-7c expression in TCGA. In summary, we identified an ER-regulated miRNA cluster that regulates HER2, is lost with progression to estrogen independence, and may serve as a biomarker of poor outcome in ER(+) luminal A breast cancer patients.

In vitro evaluation of a tetrahydroisoquinoline derivative as a steroid sulfatase inhibitor and a selective estrogen receptor modulator.

Selective estrogen receptor modulators (SERMs) are currently in use in the hormonal therapy of breast cancer. In that respect, a new hormone-related approach is the therapeutical inhibition of steroid sulfatase (STS), which converts inactive, sulfated steroids into active hormones. We investigated the potential of 6-EO-14, a non-steroidal STS inhibitor with SERM potential. The latter compound, which exhibits a sulfamate moiety, releases the phenol derivative 8-EO-14 after the irreversible inhibition of STS. STS was inhibited by 6-EO-14 (IC50 = 0.3 μM), but not 8-EO-14, in HEK-293 cells transfected with an STS expression vector. The SERM potential of 8-EO-14 was assessed in osteoblast-like Saos-2 cells by investigating its effect on cell proliferation and on the activity of alkaline phosphatase (ALP), a specific differentiation marker. Saos-2 cell proliferation was increased by 21 % following 8-EO-14 addition (1 μM), and 8-EO-14 induced ALP activity (31 % increase at 0.1 nM) via estrogen receptor alpha (ERα) similarly to the SERM raloxifene. As compared to estradiol (E2) (100 %), the relative binding affinity of 6-EO-14 and 8-EO-14) for ERα was found to be weak (0.09 and 0.01 %, respectively). When assessed in two estrogen-dependent human breast cancer cell lines (MCF-7 and T-47D), 8-EO-14 did not support MCF-7 cell proliferation, whereas both 8-EO-14 and 6-EO-14 exhibited estrogen-like growth stimulation in T-47D cells. These two compounds were also unable to block E2-induced cell proliferation, suggesting their lack of antiestrogenic activity. Despite the known potency of 6-EO-14 as an STS inhibitor, the observed trophic activity of this new scaffold towards ERα-positive cells needs to be carefully considered prior to its potential utilization as a therapeutic agent.

Icariin promote maturation of osteoblasts in vitro by an estrogen-independent mechanism

To investigate the estrogenic activity of icariin and genistein with estrogen-dependent human breast cancer (MCF-7) cells. MCF-7 cells were incubated with media containing 5% charcoal dextran-treated FBS in phenol red-free media for 48 h. CCK-8 kit was used to study the impact of defferent concentration of icariin and genistein on MCF-7 proliferation in vitro. Optimal concentration icariin and genistein were added into medium and total RNA was isolated after 12, 24, 36, 48 h. The gene expression of ERalpha, ERbeta, PS2, and PR were investigated by Real-time RT-PCR Total protein was also isolated and secretion of ERalpha, ERbeta, PS2, and PR were examined by Western blot. 10 micromol x L(-1) icariin and genistein could promote the proliferation of MCF-7 evidently. However, the ability of genistein to promote the proliferation was better than icariin. With the concentration of 10 micromol x L(-1), genistein group had a stronger expression of ERa, PS2 and PR mRNA levels than icariin while ERbetaexpression had no significant difference in two group. The same effects were detected by western blotting. Both genistein and icariin have a strong estrogen-like effect, but the estrogenic activity of genistein is stronger than icariin. It showed that the activity of icariin is stron-ger than genistein to promote ROB maturation. So it must be that icariin promotes the maturation of osteoblasts in vitro by a estogen-independent mechanism.

Abstract P5-04-15: Investigating the role of the AMPK signaling cascade in breast cancer tumor dormancy

Abstract Breast cancer can recur in patients long after treatment for initial disease. The mechanisms underlying recurrence as well as the characteristics of these dormant tumor cells remain poorly understood. AMPK (AMP-activated protein kinase) is the central metabolic regulator of the cell, and has two catalytic isoforms, a1 and α2. The loss of AMPK, more specifically AMPKα2, is observed in primary breast cancer and its expression could function to limit tumor expansion in vivo. We hypothesize that the expression of AMPKα2 will confer a survival advantage in dormant breast cancer cells by upregulating the p38MAPK signaling axis. In order to investigate breast cancer dormancy mechanisms in vitro, we generated mammospheres using the estrogen-dependent MCF-7 human breast cancer cell line, which has low expression of AMPKα2. Control MCF-7 cells expressing GFP and MCF-7 cells overexpressing the AMPKα1 or AMPKα2 isoforms were cultured as mammospheres and exposed to various metabolic stressors including estrogen, glucose, and serum deprivation. Spheroids were evaluated for size, viability, gene expression at the RNA level by qRT-PCR, and intracellular signaling by immunoblot as well as immunohistochemistry. AMPKα2 expressing mammospheres were larger and more viable than either the GFP or AMPKα1 expressing spheres. In addition, these cells were resistant to serum deprivation, and upregulated members of the p38MAPK signaling axis, a key player in tumor dormancy. To evaluate estrogen deprivation-induced dormancy in vivo, MCF-7 cell lines expressing either GFP or AMPKα2 embedded in a matrigel matrix were implanted in athymic nude mice containing supplemental slow-release estradiol pellets. After one week, estradiol pellets were removed to induce a dormant period for roughly thirty days. Evaluation of matrigel plugs at this time indicated that AMPKα2 expressing cells demonstrated significant survival whereas GFP expressing cells were significantly depleted. Estadiol pellets were then re-implanted for five weeks to determine if the AMPKα2 expressing cells still responded to estradiol supplementation.This resulted in the AMPKα2 expressing tumors rapidly expanding to a maximal size as compared to the GFP expressing tumors. Consistent with our findings in vitro using mammosphere cultures, the AMPKα2 expressing cells had higher levels of phospho-p38MAPK than the GFP expressing cells, as seen by immunofluorescence. We conclude that the expression of AMPKα2 promotes both survival and dormancy in estrogen-sensitive breast tumor cells, thus implying a significant role in breast cancer recurrence. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-04-15.

Proapoptotic and Antimetastatic Properties of Supercritical CO2 Extract of Nigella sativa Linn. Against Breast Cancer Cells

Nigella sativa, commonly referred as black cumin, is a popular spice that has been used since the ancient Egyptians. It has traditionally been used for treatment of various human ailments ranging from fever to intestinal disturbances to cancer. This study investigated the apoptotic, antimetastatic, and anticancer activities of supercritical carbon dioxide (SC-CO2) extracts of the seeds of N. sativa Linn. against estrogen-dependent human breast cancer cells (MCF-7). Twelve extracts were prepared from N. sativa seeds using the SC-CO2 extraction method by varying pressure and temperature. Extracts were analyzed using FTIR and UV-Vis spectrometry. Cytotoxicity of the extracts was evaluated on various human cancer and normal cell lines. Of the 12 extracts, 1 extract (A3) that was prepared at 60°C and 2500 psi (~17.24 MPa) showed selective antiproliferative activity against MCF-7 cells with an IC50 of 53.34±2.15 μg/mL. Induction of apoptosis was confirmed by evaluating caspases activities and observing the cells under a scanning electron microscope. In vitro antimetastatic properties of A3 were investigated by colony formation, cell migration, and cell invasion assays. The elevated levels of caspases in A3 treated MCF-7 cells suggest that A3 is proapoptotic. Further nuclear condensation and fragmentation studies confirmed that A3 induces cytotoxicity through the apoptosis pathway. A3 also demonstrated remarkable inhibition in migration and invasion assays of MCF-7 cells at subcytotoxic concentrations. Thus, this study highlights the therapeutic potentials of SC-CO2 extract of N. sativa in targeting breast cancer.

Abstract 1092: AMPK alpha 2 promotes tumor cell survival in models of breast cancer dormancy.

Abstract Breast cancer can recur after standard of care treatment, even after many years. As a result, there is a need to identify new ways to treat metastatic recurrence, and to understand the mechanisms behind long-term cancer cell dormancy. AMP-dependent kinase (AMPK) has been implicated in tumor cell survival pathways and is under investigation as a potential target for breast cancer treatment. A defining characteristic of dormant tumor cells has been shown to be a reduction in mitogenic ERK1/2 signaling with a concomitant activation of stress-induced p38MAPK signaling. This study aimed to investigate the role of AMPKα2 in establishing and/or maintaining a dormant state. Two estrogen-dependent human breast cancer cell lines that express different levels of AMPKα2 were used. MCF-7 cells, with little to no AMPKα2 expression, were transfected to express GFP control or AMPKα2, while ZR-75-1 cells, with high AMPKα2 expression, were transduced with lentiviral shAMPKα2 or shGFP control particles. Stable cell lines were plated into mammospheres and were exposed to stress conditions such as hypoxia, depletion of essential growth factors, or low glucose. These mammospheres were evaluated by direct cell count, size, and immunofluoresence. In addition, single cell suspensions were mixed with matrigel and injected into athymic nude mice containing slow-release estradiol cholesterol pellets. Estradiol pellets were removed one week after implantation, and re-introduced six weeks later. Matrigel plugs and tumors were harvested and analyzed for cell survival by histological analysis, as well as immunofluorescent analysis of the tumor cells for ERK/p38 activation, epithelial cadherin, differentiation proteins, and autophagy markers. In order to investigate the survival of MCF-7 cells with and without AMPKα2, the amount of cells remaining after prolonged estrogen deprivation was evaluated. There were twice as many viable AMPKα2 expressing cells as GFP cells. Re-exposure of these animals to estradiol resulted in a rapid expansion of the remaining GFP and AMPKα2 tumor cells. Notably, AMPKα2 tumors maintained a significant growth advantage and were double the size of the GFP control tumors at harvest. Evaluation of the p38MAPK pathway by immunoblot revealed that those cells expressing increased amounts of AMPKα2 exhibited high levels of phospho-p38MAPK. Furthermore, those cells that expressed AMPKα2 also expressed increased amounts of LC3-II and autophagosome formation, standard autophagy markers. In conclusion, AMPK promotes breast cancer cell survival under stress in models of tumor dormancy potentially through promotion of the p38MAPK pathway with concomitant reduction in MAPK signaling, and/or autophagy pathways. This study suggests that AMPKα2 contributes to breast cancer recurrence by promotion of tumor cell dormancy and may be a novel therapeutic target. Citation Format: Katie L. Sullivan, Kathryn N. Phoenix, Melissa M. Fox, Stavros Kopsiaftis, Kevin P. Claffey. AMPK alpha 2 promotes tumor cell survival in models of breast cancer dormancy. [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 1092. doi:10.1158/1538-7445.AM2013-1092

Indole-3-Carbinol disrupts Estrogen Receptor-alpha dependent expression of Insulin-like Growth Factor-1 Receptor and Insulin Receptor Substrate-1 and proliferation of human breast cancer cells

We previously established that Indole-3-Carbinol (I3C), a natural hydrolysis product of glucobrassicin in cruciferous vegetables, arrests the proliferation of estrogen-dependent human breast cancer cells and induces protein degradation of Estrogen Receptor-alpha (ERα). We demonstrate in human MCF-7 breast cancer cells that I3C ablates expression of Insulin-like Growth Factor Receptor-1 (IGF1R) and Insulin Receptor Substrate-1 (IRS1), downstream effectors of the IGF1 signaling pathway. Exogenous ERα reversed the I3C mediated loss of IGF1R and IRS1 gene expression demonstrating that down-regulation of ERα is functionally linked to I3C control of IGF1R and IRS1 expression. I3C disrupted binding of endogenous ERα, but not Sp1, to ERE-Sp1 composite elements within the IGF1R/IRS1 promoters. Exogenous ERα abrogated, and combined expression of IGF1R and IRS1 attenuated, the I3C mediated cell cycle arrest. Therefore, I3C inhibits proliferation of estrogen-sensitive breast cancer cells through disruption of ERα-mediated transcription of cell signaling components within the IGF1 cascade.

Abstract 258: Development of death receptor agonists - A novel therapy for breast cancer

Abstract Binding of TRAIL to death receptors (TRAIL-R1/DR4 and TRAIL-R2/DR5) induces apoptosis in cancer cells without affecting normal cells. The purpose of this study was to identify and characterize TRAIL-death receptor agonist (DRA) that induces apoptosis in breast cancer cells. DRA is a small organic molecule. The pharmacokinetics of the DRA with a single oral dose was also characterized in nude mice. Blood was collected over 72-hour period after DRA administration, and analyzed by HPLC-MS/MS for plasma drug concentration. The Cmax, tmax, and AUC of DRA were measured. DRA inhibited cell viability and colony formation, and induced apoptosis in estrogen-dependent (MCF-7) and independent (MDA-MB-231 and MDA-MB-468) human breast cancer cell lines, but had no effect on human normal mammary epithelial cells and hepatocytes. The combination of DRA with paclitaxel or doxorubicin had synergistic effects on apoptosis in breast cancer cell lines. DRA formed active TRAIL-DISC (death-inducing signaling complex) similar to that of TRAIL. DRA inhibited the growth of orthotopically implanted MDA-MB-231 and MDA-MB-468 tumors in nude mice. Histological examination of tumor tissues derived from DRA-treated nude mice revealed massive caspase-3 activation and apoptosis. Pharmacokinetic data demonstrate that DRA possesses a long terminal half-life, which is acceptable for further clinical development. In conclusion, we have identified DRA that can be used either alone or in combination with chemotherapeutic drugs for the treatment of human breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 258. doi:1538-7445.AM2012-258

Soy isoflavone extracts stimulate the growth of nude mouse xenografts bearing estrogen-dependent human breast cancer cells (MCF-7)

We explored the effects of different lifetime exposures to soy isoflavone extracts on the growth of estrogen-dependent human breast cancer cells (MCF-7) implanted into athymic mice of different ovarian statuses. The athymic mice, ovariectomized or not, were implanted with MCF-7 cells. Mice were fed with low, moderate and high doses of soy isoflavone extract, at dietary concentrations of 6.25, 12.5 and 25 g/kg, in different reproductive models, respectively. The expression of ki-67 was detected by immunohistochemistry. pS2 expression in tumors was analyzed by real time PCR. Estrogen level in the serum was measured by chemiluminescence enzyme immunoassay. Total genistein and daidzein levels in serum and urine were determined by liquid chromatography-electrospray tandem mass spectrometry (LC-ES/MS/MS). In Group A, on week 4, nude mice were exposed to different doses of soy iosflavone extracts. In Group B, the experimental diets were given to the nude mice following ovariectomy and tumor implantation. In both groups, 6.25 and 12.5 g/kg soy isoflavone extracts stimulated the growth of MCF-7 xenografts, increased pS2 expression, proliferation and estrogen level in serum. In both Group B (postmenopausal mouse model) and Group C (premenopausal mouse model), soy isoflavone extracts at doses of 6.25 and 12.5 g/kg showed stimulatory effects on the growth of MCF-7 tumors. In conclusion, administration of soy isoflavone extracts at doses of 6.25 and 12.5 g/kg during adolescence or later in life stimulated tumor growth in both menopausal and postmenopausal mouse models.

Antioxidant, Cytotoxic Activities, and Structure–Activity Relationship of Gallic Acid‐based Indole Derivatives

A new series of gallic hydrazones containing an indole moiety was synthesized through the reaction of gallic hydrazide and different indole carboxaldehydes. Their antioxidant activities were determined on DPPH radical scavenging and inhibition of lipid peroxidation. The in-vitro cytotoxic activities of the compounds were evaluated against HCT-116 (human colon cancer cell line) and MCF-7 (estrogen-dependent human breast cancer cell line) by the MTT method. An attempt to correlate the biological results with their structural characteristics has been done. A limited positive structure activity relationship was found between cytotoxic and antioxidant activities.

Effects of chlorpyrifos on growth of estrogen-dependent human breast cancer cell line

Effects of chlorpyrifos on growth of estrogen-dependent human breast cancer cell line

Abstract S3-8: RNAi Screening Identifies the Insulin/Insulin-Like Growth Factor-I Receptor Pathway as a Mechanism of Escape from Hormone Dependence in Estrogen Receptor-Positive Breast Cancer

Abstract Estrogen receptor-positive (ER+) breast cancers are treated with strategies aimed at inducing estrogen deprivation such as aromatase inhibitors (AIs). However, many ER+ tumors adapt to hormone deprivation and acquire resistance to AIs. The purpose of this study was to identify kinases essential for the growth of endocrine-resistant ER+ breast cancer cells. To model acquired resistance to AIs, we maintained four ER+, estrogen-dependent human breast cancer cell lines (MCF-7, MDA-361, HCC-1428, and ZR75- 1) under hormone-depleted conditions for several months until hormone-independent populations emerged (termed long-term estrogen-deprived, LTED). We then performed an RNAi screen targeting 779 protein kinases to identify molecules required for growth of MCF-7/LTED cells in hormonefree conditions. Individual knockdown of 42 kinases significantly inhibited MCF-7/LTED cell growth ≥33% (p≥0.05) in at least 3/4 independent experiments. Of these 42 hits, knockdown of the insulin receptor (InsR) inhibited MCF-7/LTED growth (n=4 experiments) compared to control siRNA. Knockdown of InsR expression using independent siRNA oligonucleotides significantly inhibited growth of MCF-7/LTED, ZR75-1/LTED, and HCC-1428/LTED cells. The InsR can heterodimerize with the highly homologous insulin-like growth factor-I receptor (IGF-IR) to potently activate the phosphatidylinositol-3 kinase (PI3K)/AKT signaling pathway. Immunoblot analysis revealed that siRNA knockdown of InsR or IGF-IR caused a compensatory upregulation of IGF-IR and InsR expression, respectively. Knockdown of InsR or IGF-IR inhibited growth of 3/4 LTED lines, but knockdown of both InsR and IGF-IR was required for maximal inhibition of PI3K/AKT. Inhibition of these receptors by treatment with the InsR/IGF-IR tyrosine kinase inhibitor (TKI) OSI-906 decreased PI3K signaling and disrupted the basal association between p85 and the IGF-IR/InsR effectors IRS-1 and IRS-2 in MCF-7/LTED cells. Further, OSI-906 inhibited the hormone-independent growth of all four LTED lines and prevented the emergence of hormone-independent ER+ parental cells following estrogen deprivation. Treatment with the TKI or the neutralizing IGF-IR monoclonal antibody MK-0646 induced feedback upregulation and phosphorylation of the ErbB3/HER3 receptor tyrosine kinase (RTK). Induction of P-HER3 was blocked by the EGFR/HER2 TKI lapatinib. Combined treatment of lapatinib and OSI-906 prevented the emergence of hormone-independent (HER2+/ER+) MDA-361 cells more effectively than either drug alone. Finally, treatment of ovariectomized nude female mice with OSI-906 suppressed the growth of established MCF-7 xenografts. In addition, treatment with OSI-906 decreased PI3K signaling as measured by immunoblot analysis of tumor extracts using P-AKT antibodies. These results suggest that 1) InsR/IGF-IR signaling is required for PI3K/AKT signaling and hormone-independent growth of ER+ breast cancer cells; 2) these cells compensate for the inhibition of InsR/IGF-IR by upregulating alternative RTK pathways such as HER3; 3) combined targeting of InsR/IGF-IR and ER signaling may be effective for the treatment of endocrine-resistant breast cancer. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr S3-8.

Biological Evidence for Dual Antiangiogenic-Antiaromatase Activity of the VEGFR Inhibitor PTK787/ZK222584In vivo

Targeting vascular endothelial growth factor (VEGF) and estrogen receptor signaling pathways concomitantly may enhance benefit in estrogen receptor-positive breast cancer. We had shown previously that the VEGF receptor tyrosine kinase inhibitor PTK787/ZK222584 (PTK/ZK) is a competitive aromatase inhibitor in vitro. Here we investigated (a) whether PTK/ZK shows both antiangiogenic and antiaromatase inhibitory properties in vivo, and (b) whether the combination of PTK/ZK and letrozole is superior to letrozole alone. Estrogen-dependent human breast cancer cells engineered to express aromatase (MCF7 AROM 1 and BT474 AROM) were used. Mice were treated with vehicle, PTK/ZK (25, 50, or 100 mg/kg), letrozole, or PTK/ZK in combination with letrozole. In MCF7 AROM 1 tumors, all treatments induced growth suppression and were associated with a reduction in cell turnover index, a composite measurement of both proliferation and apoptosis. PTK/ZK significantly reduced vessel density. Whereas letrozole caused tumor regression, PTK/ZK stabilized tumor volumes. The growth suppressive and antiangiogenic effects of PTK/ZK were confirmed in BT474 AROM xenografts. The addition of PTK/ZK did not enhance the growth-suppressive effects of letrozole. However, PTK/ZK decreased progesterone receptor (PgR) and TFF1 expression and uterine weight, indicating that PTK/ZK decreases 17beta-estradiol (E2) signaling in vivo. The VEGF receptor inhibitor PTK/ZK showed effects on E2-dependent gene expression consistent with aromatase inhibition as well as antiangiogenesis in xenograft models of breast cancer. The combination with letrozole was not superior to letrozole alone. Overall, these results provide further support for a potential therapeutic approach of dual inhibition of VEGF and E2 signaling using a single agent.

Abstract 754: Development of death receptor agonists for breast cancer therapy

Abstract TRAIL and death receptor (TRAIL-R1 and TRAIL-R2) agonistic antibodies have been shown to induce apoptosis in cancer cells and their clinical trials have produced positive results. However, there are no small organic molecular weight compounds that bind to TRAIL-death receptors and induce apoptosis specifically in cancer cells. The purpose of this study was to identify and characterize death receptor agonists (DRAs)/ small molecular weight organic compounds that induce apoptosis in breast cancer cells. The pharmacokinetics of the DRA with a single oral dose was characterized in nude mice. Blood was collected over 72 hour period after drug administration, and analyzed by HPLC-MS/MS for plasma drug concentration. The Cmax, tmax, and AUC of DRA was measured. DRAs inhibited cell viability and colony formation, and induced apoptosis in estrogen-dependent (MCF-7) and independent (MDA-MB-231 and MDA-MB-468) human breast cancer cell lines, but had no effect on human normal mammary epithelial cells and hepatocytes. The ability of DRAs to induce apoptosis was further enhanced in the presence of TRAIL. DRAs formed active TRAIL-DISC (death-inducing signaling complex) similar to that of TRAIL. DRAs inhibited the growth of orthotopically implanted MDA-MB-231 and MDA-MB-468 tumors in nude mice. Histological examination of tumor tissues derived from DRAs-treated nude mice revealed massive caspase-3 activation and apoptosis. Pharmacokinetic data demonstrate that DRA possesses a long terminal half-life, which is acceptable for further clinical development. In conclusion, we have identified, for the first time, DRAs that can be used in the clinic for the treatment of human breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 754.

Selective recruitment of breast cancer anti-estrogen resistance genes and relevance for breast cancer progression and tamoxifen therapy response

Although endocrine treatment of breast cancer is effective and common practice, in advanced disease the development of resistance is nearly inevitable. To get more insight into individual genes that account for resistance against hormonal agents, we have executed functional genetic screens and subsequently evaluated the clinical relevance of several identified genes with respect to tumor aggressiveness and tamoxifen resistance in estrogen receptor-positive patients. Estrogen-dependent human breast cancer cells were transduced with different retroviral cDNA expression libraries and subjected to selective cultures with various anti-estrogens. From a total of 264 resistant cell clones, 132 different genes were recovered by PCR. By applying stringent selection criteria, we identified 15 breast cancer anti-estrogen resistance (BCAR) genes individually yielding resistance. BCAR genes were recovered with differential frequencies for the diverse culture conditions and anti-estrogen drugs. Analysis of the relation of BCAR genes (EIF1, FBXL10, HRAS, NRG1, PDGFRA, PDGFRB, RAD21, and RAF1) with tamoxifen treatment in patients with advanced disease showed significant association with clinical benefit and progression-free survival for EIF1 and PDGFRA mRNA levels. Furthermore, PDGFRA and HRAS mRNA levels were significantly associated with tumor aggressiveness in lymph node-negative patients who had not received adjuvant systemic therapy. In conclusion, our functional genetic screens showed that BCAR genes differ in their ability to confer resistance towards distinct anti-estrogens. Based on the clinical relevance of several BCAR genes, further studies are warranted to characterize the underlying mechanisms, which may ultimately lead to the development of novel treatments and more individualized management of breast cancer patients.

Trigonelline Is a Novel Phytoestrogen in Coffee Beans ,

Drinking coffee has been associated with the development of several endocrine-related cancers. The interpretation of these data has often been limited to the role that caffeine plays. Trigonelline (Trig), a niacin-related compound, is a natural constituent of coffee accounting for ∼1% dry matter in roasted beans. Studies exploring the effects of this bioactive compound on mammalian cells are limited. The initial purpose of our studies was to determine whether Trig alters the actions of estradiol (E2), using proliferation of estrogen-dependent human breast cancer (MCF-7) cells as a model system. When cells were cotreated with suboptimal doses of E2 (10 pmol/L) and Trig (100 pmol/L), an additive enhancement of MCF-7 growth was observed. In the absence of E2, Trig stimulated MCF-7 cell proliferation in a dose-responsive manner and significantly enhanced cell growth at concentrations as low as 100 pmol/L. Cotreatment of MCF-7 cells with Trig and ICI 182,780, an estrogen receptor (ER) antagonist, inhibited Trig-induced cell proliferation. Trig treatment also induced activation of estrogen response element reporter assays in MCF-7 cells and increased expression of ER target genes (pS2, progesterone receptor, and cyclin D1) similar to E2. While our data demonstrate that Trig activates the ER, competitive binding assays showed that Trig does not compete E2 off of the ER at any concentration. This suggests that Trig is activating the ER through a separate mechanism. Collectively, these data demonstrate that Trig even at low concentrations stimulates MCF-7 cell growth and that this effect is mediated through ER, clearly identifying Trig as a novel phytoestrogen.

Dietary genistein negates the inhibitory effect of letrozole on the growth of aromatase-expressing estrogen-dependent human breast cancer cells (MCF-7Ca) in vivo

Genistein (GEN), a soy isoflavone, stimulates growth of estrogen-dependent human tumor cells (MCF-7) in a preclinical mouse model for postmenopausal breast cancer. Antiestrogens and aromatase inhibitors are frontline therapies for estrogen-dependent breast cancer. We have demonstrated that dietary GEN can negate the inhibitory effect of tamoxifen. In this study, we evaluated the interaction of dietary GEN (at 250-1000 p.p.m. in the American Institute of Nutrition 93 growth diet) and an aromatase inhibitor, letrozole (LET), on the growth of tumors in an aromatase-expressing breast cancer xenograft model (MCF-7Ca) in the presence and absence of the substrate androstenedione (AD). Dietary GEN (250 and 500 p.p.m.) or implanted AD stimulated MCF-7Ca tumor growth. Implanted LET inhibited AD-stimulated MCF-7Ca tumor growth. In the presence of AD and LET, dietary GEN (250, 500 and 1000 p.p.m.) reversed the inhibitory effect of LET in a dose-dependent manner. Uterine wet weight, plasma estradiol (E(2)) levels (enzyme-linked immunosorbent assay) and total plasma GEN and LET levels (liquid chromatography-electrospray/tandem mass spectrometry) were measured. Ki-67 (cellular proliferation), aromatase and pS2 protein expression in tumors were evaluated using immunohistochemical (IHC) analysis. In conclusion, dietary GEN increased the growth of MCF-7Ca tumors implanted in ovariectomized mice and could also negate the inhibitory effect of LET on MCF-7Ca tumor growth. These findings are significant because tumors, which express aromatase and synthesize estrogen, are good candidates for aromatase therapy dietary and GEN can reverse the inhibitory effect of LET on tumor growth and adversely impact breast cancer therapy. Caution is warranted for consumption of dietary GEN by postmenopausal women with estrogen-dependent breast cancer taking LET treatment.

New steroidal aromatase inhibitors: suppression of estrogen-dependent breast cancer cell proliferation and induction of cell death.

BackgroundAromatase, the cytochrome P-450 enzyme (CYP19) responsible for estrogen biosynthesis, is an important target for the treatment of estrogen-dependent breast cancer. In fact, the use of synthetic aromatase inhibitors (AI), which induce suppression of estrogen synthesis, has shown to be an effective alternative to the classical tamoxifen for the treatment of postmenopausal patients with ER-positive breast cancer. New AIs obtained, in our laboratory, by modification of the A and D-rings of the natural substrate of aromatase, compounds 3a and 4a, showed previously to efficiently suppress aromatase activity in placental microsomes. In the present study we have investigated the effects of these compounds on cell proliferation, cell cycle progression and induction of cell death using the estrogen-dependent human breast cancer cell line stably transfected with the aromatase gene, MCF-7 aro cells.ResultsThe new steroids inhibit hormone-dependent proliferation of MCF-7aro cells in a time and dose-dependent manner, causing cell cycle arrest in G0/G1 phase and inducing cell death with features of apoptosis and autophagic cell death.ConclusionOur in vitro studies showed that the two steroidal AIs, 3a and 4a, are potent inhibitors of breast cancer cell proliferation. Moreover, it was also shown that the antiproliferative effects of these two steroids on MCF-7aro cells are mediated by disrupting cell cycle progression, through cell cycle arrest in G0/G1 phase and induction of cell death, being the dominant mechanism autophagic cell death. Our results are important for the elucidation of the cellular effects of steroidal AIs on breast cancer.

Role of manganese superoxide dismutase on growth and invasive properties of human estrogen-independent breast cancer cells

Manganese superoxide dismutase (MnSOD) is known to play a role in cancer. MnSOD exerts a tumor suppressive effect in estrogen-dependent human breast cancer cells. In the present study we investigated the in vitro role of MnSOD in the growth of some aggressive and highly metastatic estrogen-independent breast cancer cells, i.e., MDA-MB231 and SKBR3 cells. We show that estrogen-independent cells expressed a significantly higher basal MnSOD level compared to estrogen-dependent human breast cancer cell lines (MCF-7 and T47D). For MDA-MB231 cells, the high-MnSOD level was accompanied by an overproduction of intracellular hydrogen peroxide (H2O2) and by a low expression of the major H2O2-detoxifying enzymes, catalase, and peroxiredoxin 3, compared to MCF-7 cells. Suppression of MnSOD expression by antisense RNA was associated with a decrease of H2O2 content and caused a stimulation of growth with a reduced cell doubling time but induced a decrease of colony formation. Furthermore, treatment of MDA-MB231 cells with H2O2 scavengers markedly reduced tumor cell growth and colony formation. In addition, MnSOD suppression or treatment with H2O2 scavengers reduced the invasive properties of MDA-MB231 cells up to 43%, with a concomitant decrease of metalloproteinase-9 activity. We conclude that MnSOD plays a role in regulating tumor cell growth and invasive properties of estrogen-independent metastatic breast cancer cells. These action are mediated by MnSOD-dependent H2O2 production. In addition, these results suggest that MnSOD up-regulation may be one mechanism that contributes to the development of metastatic breast cancers.