Estrogen-dependent Tumor Growth Research Articles

Estrogen Receptors-Mediated Apoptosis in Hormone-Dependent Cancers.

It is known that estrogen stimulates growth and inhibits apoptosis through estrogen receptor(ER)-mediated mechanisms in many cancer cell types. Interestingly, there is strong evidence that estrogens can also induce apoptosis, activating different ER isoforms in cancer cells. It has been observed that E2/ERα complex activates multiple pathways involved in both cell cycle progression and apoptotic cascade prevention, while E2/ERβ complex in many cases directs the cells to apoptosis. However, the exact mechanism of estrogen-induced tumor regression is not completely known. Nevertheless, ERs expression levels of specific splice variants and their cellular localization differentially affect outcome of estrogen-dependent tumors. The goal of this review is to provide a general overview of current knowledge on ERs-mediated apoptosis that occurs in main hormone dependent-cancers. Understanding the molecular mechanisms underlying the induction of ER-mediated cell death will be useful for the development of specific ligands capable of triggering apoptosis to counteract estrogen-dependent tumor growth.

RASSF1A-Mediated Suppression of Estrogen Receptor Alpha (ERα)-Driven Breast Cancer Cell Growth Depends on the Hippo-Kinases LATS1 and 2.

Around 70% of breast cancers express the estrogen receptor alpha (ERα). This receptor is of central importance for breast cancer development and estrogen-dependent tumor growth. However, the molecular mechanisms that are responsible for the control of ERα expression and function in the context of breast carcinogenesis are complex and not fully understood. In previous work, we have demonstrated that the tumor suppressor RASSF1A suppresses estrogen-dependent growth of breast cancer cells through a complex network that keeps ERα expression and function under control. We observed that RASSF1A mediates the suppression of ERα expression through modulation of the Hippo effector Yes-associated protein 1 (YAP1) activity. Here we report that RASSF1A-mediated alteration of YAP1 depends on the Hippo-kinases LATS1 and LATS2. Based on these results, we conclude that inactivation of RASSF1A causes changes in the function of the Hippo signaling pathway and altered activation of YAP1, and as a consequence, increased expression and function of ERα. Thus, the inactivation of RASSF1A might constitute a fundamental event that supports the initiation of ERα-dependent breast cancer. Furthermore, our results support the notion that the Hippo pathway is important for the suppression of luminal breast cancers, and that the tumor-suppressor function of RASSF1A depends on LATS1 and LATS2.

Synthesis of 16β-derivatives of 3-(2-bromoethyl)-estra-1,3,5(10)-trien-17β-ol as inhibitors of 17β-HSD1 and/or steroid sulfatase for the treatment of estrogen-dependent diseases

17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1) and steroid sulfatase (STS) are involved in the synthesis of the most potent estrogen in the human body, estradiol (E2). These enzymes are known to play a pivotal role in the progression of estrogen-dependent diseases, such as breast cancer and endometriosis. Therefore, the inhibition of 17β-HSD1 and/or STS represents a promising avenue to modulate the growth of estrogen-dependent tumors or lesions. We recently established the key role of a bromoethyl side chain added at the C3-position of a 16β-carbamoyl-benzyl-E2 nucleus to covalently inhibit 17β-HSD1. To extend the structure–activity relationship study to the C16β-position of this new selective irreversible inhibitor (PBRM), we synthesized a series of analog compounds by changing the nature of the C16β-side chain but keeping the 2-bromoethyl group at position C3. We determined their 17β-HSD1 inhibitions in T-47D cells (transformation of E1 into E2), but we did not obtain a stronger 17β-HSD1 inhibitor than PBRM. Compounds 16 and 17 were found to be more likely to bind to the catalytic site and showed a promising but moderate inhibitory activity with estimated IC50 values of 0.5 and 0.7 µM, respectively (about 10 times higher than PBRM). Interestingly, adding one or two sulfamate groups in the D-ring’s surroundings did not significantly decrease compounds’ potential to inhibit 17β-HSD1, but clearly improved their potential to inhibit STS. These results open the door to the development of a new family of steroid derivatives with dual (17β-HSD1 and STS) inhibiting actions.

SUN-743 Understanding the Role of Pancreas and Testis Specific lncRNA86 in Estrogen-Dependent Signaling in Breast Cancer

Long noncoding RNAs (lncRNAs) are emerging as key regulators of diverse cellular processes, but their roles in breast cancer biology are just beginning to be elucidated. In this study, integration of powerful techniques, RNA-seq data from subcellular fractionated RNA with GRO-seq data has yielded a comprehensive catalog of estrogen-regulated lncRNAs in MCF-7 cells. Analysis of RNA-seq data from samples representing molecular subtypes of breast cancer and normal tissue types, revealed that many lncRNAs (such as lincRNA86) show distinct expression patterns. LincRNA86 shows highest normal expression in pancreas followed by testis in normal human tissues. The hypothesis is lincRNA86 regulate estrogen-dependent signaling in breast cancer. In functional assays, knockdown of lncRNA86 inhibits the growth of ER-positive breast cancer cells. Amplified expression of lncRNA86 in breast cancer correlates with clinical outcome. LncRNA86 have now been fully annotated (transcription start and stop site, 5’ cap, polyA tail, and exon/intron structure), and cloned. We are now performing detailed molecular analyses to better understand the underlying mechanisms of action of the lncRNA. We are also currently have experiments underway to view cancer phenotypes: estrogen-dependent tumor growth. Collectively, our preliminary results suggest that lincRNA86 plays a critical role in ERα-dependent pathways.

Abstract P1-05-06: Functional characterization of lncRNA152 in ER+ and triple-negative breast cancers

Abstract Genomic analyses have revealed that many RNA species other than protein-coding mRNAs are transcribed from mammalian genomes and may have profound effects on cellular physiology and pathology. One such class of RNAs are the long noncoding RNAs (lncRNAs). In our previous work using Global Run-On and sequencing (GRO-seq), we identified and annotated ~1,900 lncRNAs in MCF-7 human breast cancer cells, which are differentially expressed across the distinct molecular subtypes of breast cancer. Further meta-analysis identified lncRNA152 as a predictor of increased metastasis-free survival. Thus, lncRNA152 levels may serve as a biomarker to track disease progression in breast cancer patients. However, the precise mechanisms by which lncRNA152 regulates these clinical outcomes remain largely unknown. Interestingly, lncRNA152 is highly expressed in the luminal subtypes of breast cancer, but downregulated in triple-negative breast cancers (TNBC), suggesting that lncRNA152 expression may be associated with more aggressive cancer features. Through multiple complementary experimental approaches, we found that (1) FoxA1 regulates lncRNA152 transcription, (2) knockdown of lncRNA152 inhibits the growth, but promotes cell migration and invasion, of luminal breast cancer cell subtypes, and (3) ectopic overexpression of lncRNA152 inhibits TNBC cell growth, migration, and invasion. Importantly, our xenograft studies provide evidence that lncRNA152 inhibits estrogen-dependent tumor growth of luminal and TNBC cells. In addition, transcriptome analysis of TNBC cell-derived xenografts indicates that lncRNA152 expression downregulates many genes that are involved in biological processes such as cell growth, migration, invasion, and angiogenesis. Collectively, these results suggest that lncRNA152 is a novel tumor suppressor in luminal and basal subtype of breast cancer. This work is supported by grants from the Cancer Prevention and Research Institute of Texas (CPRIT RP160318/RP190235) to W.L.K. Citation Format: Daeseok Kim. Functional characterization of lncRNA152 in ER+ and triple-negative breast cancers [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-05-06.

17β-Estradiol Activates HSF1 via MAPK Signaling in ERα-Positive Breast Cancer Cells.

Heat Shock Factor 1 (HSF1) is a key regulator of gene expression during acute environmental stress that enables the cell survival, which is also involved in different cancer-related processes. A high level of HSF1 in estrogen receptor (ER)-positive breast cancer patients correlated with a worse prognosis. Here we demonstrated that 17β-estradiol (E2), as well as xenoestrogen bisphenol A and ERα agonist propyl pyrazole triol, led to HSF1 phosphorylation on S326 in ERα positive but not in ERα-negative mammary breast cancer cells. Furthermore, we showed that MAPK signaling (via MEK1/2) but not mTOR signaling was involved in E2/ERα-dependent activation of HSF1. E2­activated HSF1 was transcriptionally potent and several genes essential for breast cancer cells growth and/or ERα action, including HSPB8, LHX4, PRKCE, WWC1, and GREB1, were activated by E2 in a HSF1-dependent manner. Our findings suggest a hypothetical positive feedback loop between E2/ERα and HSF1 signaling, which may support the growth of estrogen-dependent tumors.

Antisulfatase, Osteogenic, and Anticancer Activities of Steroid Sulfatase Inhibitor EO-33 in Mice.

Steroid sulfatase (STS) is a key enzyme involved in the biosynthesis of estrogens from inactive sulfated steroids. After we reported EO-33 as a potent in vitro STS inhibitor without undesirable estrogenic activity and with osteogenic properties, we are now interested in validating EO-33's in vivo potential to inhibit STS, to prevent bone deterioration, and to reduce estrogen-dependent tumor growth. A scale-up synthesis was first elaborated to prepare the multigram quantity of EO-33 needed to perform in vivo studies. EO-33 blocked the uterine weight stimulated by estrone sulfate in ovariectomized mice by 69% and the STS activity in the liver by 81%. It also produced a selective estrogen receptor modulator effect as assessed by measuring the tibia weight and calcium content. Using a human breast cancer (MCF-7 xenograft) model in nude mice, EO-33 blocked 90% of tumor growth induced by estradiol sulfate, and no toxic effect was observed by assessing the body and liver weights.

HMGA1a induces alternative splicing of estrogen receptor alpha in MCF-7 human breast cancer cells

The high-mobility group A protein 1a (HMGA1a) protein is known as an oncogene whose expression level in cancer tissue correlates with the malignant potential, and known as a component of senescence-related structures connecting it to tumor suppressor networks in fibroblasts. HMGA1 protein binds to DNA, but recent studies have shown it exerts novel functions through RNA-binding. Our previous studies have shown that sequence-specific RNA-binding of HMGA1a induces exon-skipping of Presenilin-2 exon 5 in sporadic Alzheimer disease. Here we show that HMGA1a induced exon-skipping of the estrogen receptor alpha (ERα) gene and increased ERα46 mRNA expression in MCF-7 breast cancer cells. An RNA-decoy of HMGA1a efficiently blocked this event and reduced ERα46 protein expression. Blockage of HMGA1a RNA-binding property consequently induced cell growth through reduced ERα46 expression in MCF-7 cells and increased sensitivity to tamoxifen in the tamoxifen-resistant cell line, MCF-7/TAMR1. Stable expression of an HMGA1a RNA-decoy in MCF-7 cells exhibited decreased ERα46 protein expression and increased estrogen-dependent tumor growth when these cells were implanted in nude mice. These results show HMGA1a is involved in alternative splicing of the ERα gene and related to estrogen-related growth as well as tamoxifen sensitivity in MCF-7 breast cancer cells.

Inhibitory effects of Ginkgo biloba extract on aromatase activity and tumor suppression in aromatase xenograft model

Aromatase is a responsible enzyme for the biosynthesis of estrogen, and Aromatase Inhibitor (AI) became a first line therapy in postmenopausal breast cancer patients. Ginkgo biloba extract (GBE) is the most popular natural extract, and widely used in various symptoms, such as cardiovascular, renal, respiratory and central nervous system disorders. In this study, we showed that GBE significantly inhibited aromatase activity in aromatase over-expressed MCF-7 (MCF-7 AROM) cells and reduced estrogen-dependent tumor growth in aromatase xenograft model. GBE (100 – 1,000 µg/ml) significantly inhibited aromatase activity in directly and indirectly and its inhibition was accompanied with reduced aromatase mRNA expression and E2 synthesis. 1,000 µg/ml of EGB 761 directly inhibited aromatase activity to 45.12 ± 0.57% and indirectly inhibited aromatase activity to 34.21 ± 5.16% compare to vehicle control group. Major components of GBE, kaempferol (10 – 100µM) and quercetin (10 – 100µM) were tested for aromatase inhibition, and kaempferol showed strong inhibitory effects on aromatase activity and mRNA expression. We examined the anti-tumor effects of GBE on aromatase expression using a xenograft model. Androstenedione pellet was inserted into ovariectomized BALB/c-Slc-nu mice and MCF-7 AROM cells were inoculated to establish the xenograft model. Mice were given oral administration of GBE (40 to 1,000 mg/kg) for 3 weeks after tumor size was measurable (200 – 300 mm3). 200 mg/kg, and 1,000 mg/kg of GBE strongly delayed tumor growth and suppressed tumor growth compared to vehicle treated group. After three weeks treatment, the % control changes in mean tumor volume of vehicle control group was 167.35 ± 14.42% and 200 mg/kg of GBE treated group was 89.09 ± 34.23%. This study showed that GBE can be potentially useful in the treatment of hormone dependent breast cancer through the inhibition of aromatase activity as well as tumor suppression.

Abstract 1402: A reporter mouse model reveals that human CYP19A1 (aromatase) gene expression is induced in breast cancer xenograft stroma and surrounding mammary gland by the cancer cells in vivo.

Abstract Aromatase enzyme coded by the CYP19A1 gene catalyzes the final step of estrogen biosynthesis and is of major significance in endocrine regulation of breast cancer (BCa). Human CYP19A1 gene expression is regulated through the utilization of various non-translated tissue selective first exons, each controlled by unique sets of e.g. hormones and cytokines. Extragonadal expression pattern and regulation of human CYP19A1 gene is significantly different from that of rodents, which has been a major obstacle in studies focusing on regulation of aromatase gene expression in vivo. To study the tissue-selective regulation of human CYP19A1 gene in vivo, we have generated a transgenic reporter mouse model, hARO-Luc mouse, with a construct composing of a 100-kbp-long 5’-regulatory region of the human CYP19A1 gene attached to a luciferase (Luc) reporter gene. The aim of this study was to investigate human CYP19A1 gene expression in human BCa xenografts, to determine how the BCa cells regulate CYP19A1 gene expression in tumor associated stroma in vivo. In vitro, BCa cells have been shown to induce CYP19A1 gene expression in human breast derived fibroblasts. Furthermore, in order to study the regulation of the human CYP19A1 gene expression in hARO-Luc derived undifferentiated fibroblasts, mesenchymal stromal cells were isolated from bone marrow of female reporter mice. In these cells, human CYP19A1 gene expression was induced by dexamethasone ± TNFα, and PGE2 that are among factors known to drive aromatase gene expression in human mammary fibroblasts through promoters I.4 and I.3/PII. In order to investigate human CYP19A1 expression in BCa xenografts and tumor surrounding mammary gland, the hARO-Luc reporter mice were cross-bred with athymic nude mice. Estrogen-responsive MCF-7 or T47D cells or non-estrogen-responsive MDA-MB-231 cells were injected into inguino-abdominal mammary fat pads of athymic female hARO-Luc mice, and mice that received MCF-7 or T47D cells were also implanted s.c. with estradiol releasing pellet to induce the estrogen-dependent tumor growth. The reporter gene activity was measured from tissue homogenates. MCF-7 and T47D tumors had 5-10 -fold higher Luc activity as compared with the intact mammary gland, indicating a strong up-regulation of the reporter gene in host derived tumor stroma. In contrast, in MDA-MB-231 tumor bearing mice the reporter activity in tumors was only slightly higher as compared with the intact mammary gland. Furthermore, reporter gene activity in mammary gland surrounding MDA-MB-231 tumors was 2-fold higher than in the tumor itself. In conclusion, the data indicate that human BCa cells up-regulate expression of human aromatase gene in the tumor associated stroma in vivo. Citation Format: Päivi Järvensivu, Leena Strauss, Sari Mäkelä, Matti Poutanen, Niina Saarinen. A reporter mouse model reveals that human CYP19A1 (aromatase) gene expression is induced in breast cancer xenograft stroma and surrounding mammary gland by the cancer cells in vivo. [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 1402. doi:10.1158/1538-7445.AM2013-1402

SIRT1 Positively Regulates Breast Cancer Associated Human Aromatase (CYP19A1) Expression

Breast cancer remains one of the leading causes of death in women diagnosed with cancer. In breast cancer, aberrant expression of the CYP19A1 gene, which encodes the aromatase enzyme, contributes to increased intratumoral levels of estradiol. Regardless of whether this estrogen is produced by peripheral tissues or within specific subpopulations of cells within the breast tumor, it is clear that the aromatase enzymatic activity is critical for the growth of estrogen-dependent tumors. Currently, aromatase inhibitors have proven to be highly effective in blocking the growth of estrogen-dependent forms of breast cancer. CYP19A1 transcription is tightly controlled by 10 tissue-specific promoters. In breast cancer, however, aromatase transcription is driven by multiple promoters that somehow override the tissue-specific regulation of normal tissue. Here, we explore the role that the deacetylase, sirtuin-1 (SIRT1), plays in positively regulating aromatase in breast cancer. We demonstrate that the use of cambinol and the SIRT1/2 inhibitor VII, 2 small molecule inhibitors of SIRT1 and SIRT2, as well as small molecule inhibitors and small interfering RNA specific to SIRT1, all reduce the levels of aromatase mRNA. We further demonstrate that pharmacologic inhibition causes a marked reduction in aromatase protein levels. Additionally, by chromatin immunoprecipitation, we demonstrate that SIRT1 occupies the promoter regions PI.3/PII and PI.4, and its inhibition leads to increased acetylation of estrogen-related receptorα, a transcription factor that positively regulates CYP19A1 transcription in epithelial cells. Finally, we demonstrate by immunohistochemistry that SIRT1 is significantly up-regulated in invasive ductal carcinoma relative to normal tissue adjacent to tumor, further suggesting a role of SIRT1 in breast cancer. This work uncovers a new mechanism for the regulation of aromatase and provides rationale for further investigation of how the inhibition of specific sirtuins may provide a unique strategy for inhibiting aromatase that may complement or synergize with existing therapies.

Inhibition of estrogen signaling activates the NRF2 pathway in breast cancer

Exposure to higher levels of estrogen produces genotoxic metabolites that can stimulate mammary tumorigenesis. Induction of NF-E2-related factor 2 (NRF2)-dependent detoxifying enzymes (e.g., NAD(P)H-quinone oxidoreductase 1 (NQO1)) is considered an important mechanism of protection against estrogen-associated carcinogenesis because they would facilitate removal of toxic estrogens. Here, we studied the impact of estrogen-receptor (ER) signaling on NRF2-dependent gene transcription. In luciferase assay experiments using the 5-flanking region of the human NQO1 gene promoter, we observe that ERα ligand-binding domain (LBD) is required for estrogen inhibition of NQO1 promoter activity in estrogen-dependent breast cancer cells. Chromatin immunoprecipitation (ChIP) assay shows that estrogen recruits ERα and a class III histone deacetylase SIRT1 at the NQO1 promoter, leading to inhibition of NQO1 transcription. Inhibition of ERα expression by the antiestrogen shikonin reverses the inhibitory effect of estrogen on NQO1 expression. As a consequence, a chemoprevention study was undertaken to monitor the impact of shikonin on DNA lesions and tumor growth. Treatment of MCF-7 breast cancer cells with shikonin inhibits estrogen-induced 8-hydroxy-2-deoxyguanosine (8-OHdG), a marker of DNA damage. NQO1 deficiency promotes estrogen-dependent tumor formation, and shikonin inhibits estrogen-dependent tumor growth in an NQO1-dependent manner in MCF-7 xenografts. These results suggest that estrogen-receptor signaling pathway has an inhibitory effect on NRF2-dependent enzymes. Moreover, shikonin reverses the inhibitory effects of estrogen on this pathway and may contribute to breast cancer prevention.

Abstract 715: Synthesis of potent anticancer genistein derivatives to cure estrogen receptor-related breast cancers

Abstract Abstract: Background: Estrogen Receptor (ER) is a major target for the treatment of breast cancer as estrogens act to stimulate the growth of estrogen-dependent tumors. Estrogens exert their physiological action through ER (ERα and ERβ). Both of them are expressed in normal breast tissue and several other tissues. In many breast cancers, an up-regulation of ERα expression as well as down-regulation of ERβ expression was observed by several groups. Studies also showed that high ERβ levels are associated with a reduced breast cancer risk. Genistein is a soy isoflavone with a structure similar to estrogen, can mimic and antagonize the estrogen effects in tissues, which could allow it to act as chemo-preventive agent in breast cancer. It has been noted that Genistein is an effective anticancer agent in breast cancer cell lines, but only at supraphysiological concentrations. We synthesized several structurally modified Genistein derivatives, to enhance its antiproliferative effects and to minimize its alpha-estrogenic effects. Methods: We first synthesized 7 Genistein derivatives (MA-6, MA-8, MA-11, MA-19, MA-20, MA-21 and MA-22) in which Genistein was linked to aliphatic side-chains or heterocyclic triazole moieties that precluded standard agonist binding-induced conformational changes in the ERα receptor pocket. Then we investigated the biochemical effects of these substituted genistein derivatives in the three ER positive breast cancer cell lines (MCF7, 21PT and T47D). Effects were compared to those seen with the parent compound genistein. Results: 1) Three derivatives (MA-6, MA-8 and MA-19) showed growth inhibitory activity at 5-15 times lower concentrations than the parent Genistein, as assessed by 50% inhibitory concentrations (IC50) in all the 3 breast cancer cell lines. They also induced apoptosis in the breast cancer cell lines as it was reported earlier by Genistein. 2) Treatment with these 3 genistein derivatives in the above low IC50 concentrations, resulted in a decrease of ERα levels and an increase in ERβ levels, at similar levels compared to Genistein itself. These effects were accompanied by the expected changes in gene expression of presenilin-2 and psoriasin, downstream genes of ERα and ERβ signaling respectively. 3) The observed increase of ER β /ER α ratio after MA8, MA6 and MA19 treatments also reached to similar levels achieved by Genistein, explain their high and better potency as anticancer drugs. Conclusion: Modified Genistein derivatives MA6, MA8 and MA19 reduce proliferation & ERα levels and induce ERβ levels in all the 3 estrogen positive breast cancer cell lines. These effects are seen at 5-15 times lower IC50 concentrations than the parent drug Genistein. ERβ level enhancement by the genistein derivatives may also be associated with a reduced breast cancer risk by the modulation of growth. These new drugs may therefore be much better chemopreventive agents than Genistein itself. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 715.

Estrogen-Induced Generation of Reactive Oxygen and Nitrogen Species, Gene Damage, and Estrogen-Dependent Cancers

In addition to the direct effect of estrogen on mitochondria and the redox cycling of catechol estrogen, estrogen-induced proinflammatory cytokines, such as interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α), also generate reactive oxygen and nitrogen species (RO/NS). Different cellular signaling pathways may operate in response to varying levels of estrogen-induced RO/NS, leading to genotoxic damage, cell apoptosis, or cell growth. At high levels of RO/NS, cells receiving genotoxic insults, if not repaired, may engage the apoptotic pathways. There is increasing evidence supporting that estrogen-induced alterations in the genome of cells is produced by oxidative attack. Furthermore, ROS generated by estrogen exposure and/or active metabolites of estrogen in combination with receptor-mediated proliferation of genetically damaged cells may be involved in tumor development. This view is supported by the findings of DNA modifications produced in vitro or in vivo by natural and synthetic estrogens in the target organs of cancer both in experimental models and in humans. Interaction of estrogen-induced oxidants and estrogen metabolites with DNA was shown to generate mutations in genes. Cotreatment with an inhibitor of IL-1β and TNF-α synthesis, pentoxifylline, decreased stilbene estrogen-induced levels of myeloperoxidase (MPO), 8-hydroxydeoxyguanosine formation, and gene mutations, and prevented stilbene estrogen-induced lesions. Stable MCF-7 clones overexpressing IL-1β resulted in a high level of IL-1β peptide secretion undergoing cell apoptosis, and an elevated level of p53 protein in response to high oxidative stress when compared to nontransfected cells, whereas MCF-7 clones overexpressing IL-1β that resulted in a moderate level of IL-1β secretion stimulated the clonal expansion of MCF-7 and TM3 cells. Estrogen-induced MCF-7 cell growth and cyclin D1 expression were suppressed by antioxidants and mitochondrial blockers. These studies support that in addition to ovarian estrogen-mediated ER signaling, mitogenic signals may also come from estrogen-induced RO/NS. Further validation of this concept that the concentration of the RO/NS within the cellular microenvironment determines its stimulatory or inhibitory growth signals as well as its genotoxic effects regulating the growth of estrogen-dependent tumors may result in novel preventive strategies.

Antiestrogenic Glyceollins Suppress Human Breast and Ovarian Carcinoma Tumorigenesis

We have identified the phytoalexin compounds glyceollins I, II, and III, which exhibit marked antiestrogenic effects on estrogen receptor function and estrogen-dependent tumor growth in vivo. The purpose of this study was to investigate the interactions among the induced soy phytoalexins glyceollins I, II, and III on the growth of estrogen-dependent MCF-7 breast cancer and BG-1 ovarian cancer cells implanted in ovariectomized athymic mice. Four treatment groups for each cell line were used: vehicle control, 20 mg/kg/mouse/d glyceollin mixture injection, 0.72 mg estradiol (E2) implant, and E2 implant + 20 mg/kg/mouse/d glyceollin injection. Treatment with glyceollin suppressed E2-stimulated tumor growth of MCF-7 cells (-53.4%) and BG-1 cells (-73.1%) in ovariectomized athymic mice. These tumor-inhibiting effects corresponded with significantly lower E2-induced progesterone receptor expression in the tumors. In contrast to tamoxifen, the glyceollins had no estrogen-agonist effects on uterine morphology and partially antagonized the uterotropic effects of estrogen. These findings identify glyceollins as antiestrogenic agents that may be useful in the prevention or treatment of breast and ovarian carcinoma.

Genistein stimulates growth of human breast cancer cells in a novel, postmenopausal animal model, with low plasma estradiol concentrations

We have demonstrated that genistein (GEN) stimulates growth of estrogen-dependent breast tumors in vivo. In this study, we evaluated whether dietary GEN can act in an additive manner with low circulating levels of 17beta-estradiol (E2). We developed an E2 delivery system using silastic implants that yield low circulating plasma E2 levels similar to those observed in postmenopausal women. We inserted various concentrations of E2 silastic implants (1:127, 1:63, 1:31, 1:15 and 1:7 = E2:cholesterol) and injected estrogen-dependent human breast cancer (MCF-7) cells into ovariectomized athymic mice. The E2 implants tested (1:127-1:7) generated 30.1-101.6 pM E2 in plasma, which is comparable to the E2 levels observed in postmenopausal women. The E2 implants stimulated MCF-7 tumor growth in a dose-dependent manner. We selected the 1:31 ratio of E2 implant to evaluate if dietary GEN acts in an additive manner with low E2 levels to influence the growth of MCF-7 tumors. Ovariectomized mice were divided into four groups: MCF-7 control, 500 ppm GEN, 1:31 E2, and 1:31 E2 + 500 ppm GEN. At week 17, the average tumor sizes were 7.6, 32.1, 67.4 and 106.8 mm2 for these groups, respectively (P < 0.05), demonstrating that 500 ppm GEN additively stimulated MCF-7 tumor growth in the presence of low levels of E2. In summary, we established a preclinical mouse model that results in E2 blood concentrations similar to those found in postmenopausal women. Further, we observed that these concentrations regulate the growth rate of MCF-7 breast tumors. Using this model, we demonstrated that dietary GEN in the presence of low levels of circulating E2 act in an additive manner to stimulate estrogen-dependent tumor growth in vivo. Results from this study suggest that consumption of products containing GEN may not be safe for postmenopausal women with estrogen-dependent breast cancer.

Nod1-dependent control of tumor growth

Nod1, a cytosolic protein that senses meso-diaminopimelic acid-containing ligands derived from peptidoglycan, plays a role in host responses to invasive bacteria. Here we describe a function for Nod1, whereby it controls tumor formation. Cell lines derived from the human breast cancer epithelial cell line MCF-7 were used in a severe combined immune deficiency (SCID) mouse xenograft model to characterize a pathway linking Nod1 to the growth of estrogen-sensitive tumors. In MCF-7 cells, the absence of Nod1 correlates with tumor growth, an increased sensitivity to estrogen-induced cell proliferation, and a failure to undergo Nod1-dependent apoptosis. Conversely, overexpression of Nod1 in MCF-7 cells results in inhibition of estrogen-dependent tumor growth and reduction of estrogen-induced proliferative responses in vitro.

Levels of IL-1 beta control stimulatory/inhibitory growth of cancer cells

Different cellular signaling pathways operate in response to varying levels of IL-1 beta leading to genotoxic damage, cell apoptosis or cell growth. At high levels of IL-1 beta, cells receiving genotoxic insults engage apoptotic pathways. The IL-1 beta over expressing stable MCF7 cell secreting high level of IL-1 beta peptides undergo cell apoptosis. Cotreatment with an inhibitor of IL-1 beta and TNF-alpha synthesis prevented stilbene estrogen-induced lesions. In addition to direct effect of 17 beta-estradiol (E2) on mitochondria and redox cycling of catechol estrogens, E2-induced overexpression of IL-1 beta can produce an increase in the level of ROS. Our recent data showed that MCF7 cell growth and cyclin D1 expression are suppressed by antioxidants and mitochondrial blockers. Stably IL-1 beta transfected cells secreting moderate level of IL-1 beta peptides stimulated the clonal expansion of MCF7 cells. These studies support that in addition to ovarian estrogens, mitogenic signals may also come from TNF-alpha and IL-1 beta-generated O2*- and hydrogen peroxide. Further validation of this concept that the concentrations of the peptide interleukin-1 beta within the cells determine its stimulatory or inhibitory signals regulating the growth of estrogen-dependent tumors might result in novel preventive strategies.

Progestins Initiate a Luminal to Myoepithelial Switch in Estrogen-Dependent Human Breast Tumors without Altering Growth

Although long-term clinical use of progestins is associated with an increased incidence of breast cancers, their role in established cancers is unclear. Estrogens are considered to be the main mitogens in the majority of breast cancers. Whether progesterone affects proliferation and/or differentiation is under debate. To assess the role of progesterone in established breast cancers, we used T47D human breast cancer cells that are estrogen receptor (ER) positive and either progesterone receptor (PR) negative or positive for PRA, PRB, or both. These cells were grown as strictly estrogen-dependent solid tumors in ovariectomized female nude mice. Progesterone or medroxyprogesterone acetate (MPA) alone did not support tumor growth, nor did progesterone or MPA given simultaneously with estrogen significantly alter estrogen-dependent tumor growth. However, treatment of mice bearing ER+PR+ but not ER+PR- tumors with either progesterone or MPA increased expression of the myoepithelial cytokeratins (CK) 5 and 6 in a subpopulation of tumor cells. These CK5+/CK6+ cells had decreased expression of luminal epithelial CK8, CK18, and CK19. We conclude that progestins exert differentiative effects on tumors characterized by transition of a cell subpopulation from luminal to myoepithelial. This may not be beneficial, however, because such a phenotype is associated with poor prognosis.

Arimidex inhibition on proliferation of human breast solid tumors measured by ATP bioluminescence

To determine the degree of Arimidex (Anastrozole) inhibition on proliferation of human breast solid tumors in vitro by ATP bioluminescence assay. Breast cancer solid tumors with different hormone receptors and grading were collected from 38 Chinese women with invasive breast cancers. Tumors were treated with three concentrations of Arimidex (1.5 mM, 15 mM and 150 mM). ATP bioluminescence assay was used to measure the metabolic rate in order to determine the degree of inhibition of Arimidex on the breast cancer tumors by comparing to the untreated tumors. 15 mM Arimidex shows greatest inhibitory effect on the proliferation of solid tumors with ER-postive/PR-positive. It can also inhibit the growth of metastatic tumors and tumors with HER-2/neu expression. It shows greater inhibitory effect in lower grading of tumors then higher. Arimidex may effectively inhibit the growth of breast tumors in in vitro system by inhibiting aromatase and block estrogen dependent tumor growth.