Breast cancer. Cyr61 is overexpressed, estrogen-inducible, and associated with more advanced disease

To identify genes involved in breast cancer, polymerase chain reaction-selected cDNA subtraction was utilized to construct a breast cancer-subtracted library. Differential screening of the library isolated the growth factor-inducible immediate-early gene Cyr61, a secreted, cysteine-rich, heparin binding protein that promotes endothelial cell adhesion, migration, and neovascularization. Northern analysis revealed that Cyr61 was expressed highly in the invasive breast cancer cell lines MDA-MB-231, T47D, and MDA-MB-157; very low levels were found in the less tumorigenic MCF-7 and BT-20 breast cancer cells and barely detectable amounts were expressed in the normal breast cells, MCF-12A. Univariate analysis showed a significant or borderline significant association between Cyr61 expression and stage, tumor size, lymph node positivity, age, and estrogen receptor levels. Interestingly, expression of Cyr61 mRNA increased 8- to 12-fold in MCF-12A and 3- to 5-fold in MCF-7 cells after 24- and 48-h exposure to estrogen, respectively. Induction of Cyr61 mRNA was blocked by tamoxifen and ICI182,780, inhibitors of the estrogen receptor. Stable expression of Cyr61 cDNA under the regulation of a constitutive promoter in MCF-7 cells enhanced anchorage-independent cell growth in soft agar and significantly increased tumorigenicity and vascularization of these tumors in nude mice. Moreover, overexpression of Cyr61 in MCF-12A normal breast cells induced their tumor formation and vascularization in nude mice. In summary, these results suggest that Cyr61 may play a role in the progression of breast cancer and may be involved in estrogen-mediated tumor development.     

Endogenous aromatization of testosterone results in growth stimulation of the human MCF-7 breast cancer cell line

Estrogens produced within breast tumors may play a pivotal role in growth stimulation of the breast cancer cells. However, it is elusive whether the epithelial breast cancer cells themselves synthesize estrogens, or whether the surrounding tumor stromal cells synthesize and supply the cancer cells with estrogen. The aromatase enzyme catalyzes the estrogen production, aromatizing circulating androgens into estrogens. The aim of this study was to investigate aromatase expression and function in a model system of human breast cancer, using the estrogen responsive human MCF-7 breast cancer cell line. Cells were cultured in a low estrogen milieu and treated with estrogens, aromatizable androgens or non-aromatizable androgens. Cell proliferation, expression of estrogen-regulated proteins and aromatase activity were investigated. The MCF-7 cell line was observed to express sufficient aromatase enzyme activity in order to aromatize the androgen testosterone, resulting in a significant cell growth stimulation. The testosterone-mediated growth effect was completely inhibited by the aromatase inhibitors letrozole and 4-hydroxy-androstenedione. Expression studies of estrogen-regulated proteins confirmed that testosterone was aromatized to estrogen in the MCF-7 cells. Thus, the results indicate that epithelial breast cancer cells possess the ability to aromatize circulating androgens to estrogens.     

Letrozole-, anastrozole-, and tamoxifen-responsive genes in MCF-7aro cells: a microarray approach

Antiestrogens and aromatase inhibitors are important drugs in the treatment of estrogen-dependent breast cancer. To investigate the effects of these drugs on gene expression in breast cancer cells, we treated estrogen receptor-positive MCF-7 cells stably transfected with the aromatase gene (known as MCF-7aro cells) with testosterone, 17 beta-estradiol, two aromatase inhibitors (letrozole and anastrozole), and an antiestrogen (tamoxifen). We found that testosterone or 17 beta-estradiol induced the proliferation of MCF-7aro cells at a rate six times faster than the untreated cells. In addition, the testosterone-induced proliferation of MCF-7aro cells was effectively suppressed by letrozole, anastrozole, or tamoxifen. Microarray analyses on Affymetrix Human Genome U133A GeneChips (Affymetrix, Santa Clara, CA) were carried out using total RNA isolated from the control and treated cells. At the false discovery rate of 0.05 and a minimum fold-change criteria of 1.5, 104 genes were identified that were up-regulated and 109 genes were identified that were down-regulated by both androgen and estrogen. More than 50% of these hormone-regulated genes were counter-regulated by all three inhibitors and >90% were counter-regulated by at least one of the inhibitors. Comparing the effect of each inhibitor on gene expression, we observed that letrozole and anastrozole are more similar in terms of the genes they affect compared with treatment with tamoxifen. To validate the gene expression profiles identified from microarray analyses, the expression patterns of 13 representative genes were examined by Northern analysis. Finally, the genes identified as statistically significant were classified based on their expression patterns and biological function/pathways. The results of this study provide us with a better understanding of the actions of both aromatase inhibitors and antiestrogens at the molecular level. We believe that the results of this study serve as the first step in identifying unique expression patterns following drug treatment, and that this will ultimately be useful in customizing patient treatment strategies for hormone-dependent breast cancer.     

Nordihydroguaiaretic acid (NDGA), an inhibitor of the HER2 and IGF-1 receptor tyrosine kinases, blocks the growth of HER2-overexpressing human breast cancer cells

We have reported that nordihydroguaiaretic acid (NDGA) inhibits the tyrosine kinase activities of the IGF-1 receptor (IGF-1R) and the HER2 receptor in breast cancer cells. Herein, we studied the effects of NDGA on the growth of estrogen receptor (ER) positive MCF-7 cells engineered to overexpress HER2 (MCF-7/HER2-18). These cells are an in vitro model of HER2-driven, ER positive, tamoxifen resistant breast cancer. NDGA was equally effective at inhibiting the growth of both parental MCF-7 and MCF-7/HER2-18 cells. Half maximal effects for both cell lines were in the 10-15 microM range. The growth inhibitory effects of NDGA were associated with an S phase arrest in the cell cycle and the induction of apoptosis. NDGA inhibited both IGF-1R and HER2 kinase activities in these breast cancer cells. In contrast, Gefitinib, an epidermal growth factor receptor inhibitor but not an IGF-1R inhibitor, was more effective in MCF-7/HER2-18 cells than in the parental MCF-7 cells and IGF binding protein-3 (IGFBP-3) was more effective against MCF-7 cells compared to MCF-7/HER2-18. MCF-7/HER2-18 cells are known to be resistant to the effects of the estrogen receptor inhibitor, tamoxifen. Interestingly, NDGA not only inhibited the growth of MCF-7/HER2-18 on its own, but it also demonstrated additive growth inhibitory effects when combined with tamoxifen. These studies suggest that NDGA may have therapeutic benefits in HER2-positive, tamoxifen resistant, breast cancers in humans.     

Evaluation of exosomal miR-9 and miR-155 targeting PTEN and DUSP14 in highly metastatic breast cancer and their effect on low metastatic cells

Breast cancer is one of the most prevalent cancers in women. Triple-negative breast cancer consists 15% to 20% of breast cancer cases and has a poor prognosis. Cancerous transformation has several causes one of which is dysregulation of microRNAs (miRNAs) expression. Exosomes can transfer miRNAs to neighboring and distant cells. Thus, exosomal miRNAs can transfer cancerous phenotype to distant cells. We used gene expression omnibus (GEO) datasets and miRNA target prediction tools to find overexpressed miRNA in breast cancer cells and their target genes, respectively. Exosomes were extracted from MDA-MB-231 and MCF-7 cells and characterized. Overexpression of the miRNAs of MDA-MB-231 cells and their exosomes were analyzed using quantitative Real-time PCR. The target genes expression was also evaluated in the cell lines. Luciferase assay was performed to confirm the miRNAs: mRNAs interactions. Finally, MCF-7 cells were treated with MDA-MB-231 cells’ exosomes. The target genes expression was evaluated in the recipient cells. GSE60714 results indicated that miR-9 and miR-155 were among the overexpressed miRNAs in highly metastatic triple negative breast cancer cells and their exosomes. Bioinformatic studies showed that these two miRNAs target PTEN and DUSP14 tumor suppressor genes. Quantitative Real-time PCR confirmed the overexpression of the miRNAs and downregulation of their targets. Luciferase assay confirmed that the miRNAs target PTEN and DUSP14. Treatment of MCF-7 cells with MDA-MB-231 cells’ exosomes resulted in target genes downregulation in MCF-7 cells. We found that miR-9 and miR-155 were enriched in metastatic breast cancer exosomes. Therefore, exosomal miRNAs can transfer from cancer cells to other cells and can suppress their target genes in the recipient cells.     

Ribozyme targeting demonstrates that the nuclear receptor coactivator AIB1 is a rate-limiting factor for estrogen-dependent growth of human MCF-7 breast cancer cells

Human breast tumorigenesis is promoted by the estrogen receptor pathway, and nuclear receptor coactivators are thought to participate in this process. Here we studied whether one of these coactivators, AIB1 (amplified in breast cancer 1), was rate-limiting for hormone-dependent growth of human MCF-7 breast cancer cells. We developed MCF-7 breast cancer cell lines in which the expression of AIB1 can be modulated by regulatable ribozymes directed against AIB1 mRNA. We found that depletion of endogenous AIB1 levels reduced steroid hormone signaling via the estrogen receptor alpha or progesterone receptor beta on transiently transfected reporter templates. Down-regulation of AIB1 levels in MCF-7 cells did not affect estrogen-stimulated cell cycle progression but reduced estrogen-mediated inhibition of apoptosis and cell growth. Finally, upon reduction of endogenous AIB1 expression, estrogen-dependent colony formation in soft agar and tumor growth of MCF-7 cells in nude mice was decreased. From these findings we conclude that, despite the presence of different estrogen receptor coactivators in breast cancer cells, AIB1 exerts a rate-limiting role for hormone-dependent human breast tumor growth.     

Evaluation of synthetic isoflavones on cell proliferation, estrogen receptor binding affinity, and apoptosis in human breast cancer cells

Natural isoflavones have demonstrated numerous pharmacological activities in breast cancer cells, including antiproliferative activities and binding affinities for estrogen receptors (ERs). Chemical modifications on the isoflavone ring system have been prepared and explored for the development of new therapeutics for hormone-dependent breast cancer. The antiproliferative actions of the synthesized isoflavones on MCF-7 and MDA-MB-231 breast cancer cells were examined, as well as cytotoxicity, interaction with estrogen receptors, and proapoptotic activity. The compounds were screened in the absence and in the presence of estradiol to evaluate whether or not estradiol could rescue cell proliferation on MCF-7 cells. Several compounds were able to inhibit cell proliferation in a dose-dependent manner, and compounds containing the bulky 7-phenylmethoxy substituent resulted in cell toxicity not only in MCF-7 cells but also in MDA-MB-231 cells. Selected synthetic isoflavones were able to bind to estrogen receptor with low affinity. Apoptotic pathways were also activated by these compounds in breast cancer cells. The majority of the compounds can bind to both ERs with low affinity, and their effects on hormone-independent breast cancer cells suggest that their ability to inhibit cell growth in breast cancer cells is not exclusively mediated by ERs. Thus, the synthetic trisubstituted isoflavones act on multiple signaling pathways leading to activation of mechanisms of cell-death and ultimately affecting breast cancer cell survival.     

Estrogen withdrawal-induced human breast cancer tumour regression in nude mice is prevented by Bcl-2

We recently showed that estrogen induces expression of the anti-apoptotic protein, Bcl-2 in MCF-7 human breast cancer cells. Since estrogen-dependent breast tumours can regress following estrogen withdrawal, we hypothesized that stable Bcl-2 expression would prevent estrogen-withdrawal induced regression of MCF-7 tumours. We therefore established tumours in ovariectomized female nude mice implanted with an estrogen-release pellet using untransfected MCF-7 cells or MCF-7 cells stably transfected with a Bcl-2 cDNA sense or antisense expression vector. All tumours grew at similar rates indicating that Bcl-2 levels have no effect on tumour formation. After removal of the estrogen pellet, Bcl-2 antisense tumours and untransfected MCF-7 tumours regressed means of 49% and 52%, respectively, after estrogen pellet removal whereas Bcl-2 sense tumours were significantly stabilized. Regressing tumours displayed characteristics of apoptotic cells. These results show that Bcl-2 can prevent hormone-dependent breast tumour regression and are consistent with the notion that decreased Bcl-2 levels following estrogen withdrawal renders hormone-dependent breast tumour cells sensitive to apoptotic regression.     

Induction of Hsp22 (HspB8) by estrogen and the metalloestrogen cadmium in estrogen receptor-positive breast cancer cells

Estrogen (E2) plays a critical role in the etiology and progression of human breast cancer. The estrogenic response is complex and not completely understood, including in terms of the involved responsive genes. Here we show that Hsp22 (synonyms: HspB8, E2lG1, H11), a member of the small heat shock protein (sHSP) superfamily, was induced by E2 in estrogen receptor-positive MCF-7 breast cancer cells, resulting in an elevated Hsp22 protein level, whereas it was not induced in estrogen receptor-negative MDA-MB-231 cells. This induction was prevented by the pure anti-estrogen ICI182780 (faslodex, fulvestrant), whereas tamoxifen, a substance with mixed estrogenic and antiestrogenic properties, had no major inhibitory effect on this induction, nor did it induce Hsp22 on its own. Cadmium (Cd) is an environmental pollutant with estrogenic properties (metalloestrogen) that has been implicated in breast cancer. Treatment of MCF-7 cells with Cd also resulted in induction of Hsp22, and this induction was also inhibited by ICI182780. In live MCF-7 cells, Hsp22 interacted at the level of dimers with Hsp27, a related sHSP, as was shown by quantitative fluorescence resonance energy transfer measurements. In cytosolic extracts of MCF-7 cells, most of the E2- and Cd-induced Hsp22 was incorporated into high-molecular mass complexes. In part, Hsp22 and Hsp27 were components of distinct populations of these complexes. Finally, candidate elements in the Hsp22 promoter were identified by sequence analysis that could account for the induction of Hsp22 by E2 and Cd. Taken together, Hsp22 induction represents a new aspect of the estrogenic response with potential significance for the biology of estrogen receptor-positive breast cancer cells.