A Target for Breast Cancer Prevention and Treatment

Steroids, 1999

It is known that steroids can induce cell surface receptor aggregation followed by activation of receptor and nonreceptor tyrosine kinases. It has been shown recently that 17␤-estradiol (E 2) can stimulate the Src/p21 ras /mitogen-activated protein kinase pathway in breast cancer cells, and this effect is supposed to mediate the E 2-induced stimulation of breast cancer cell proliferation, possibly via activation of the c-fos and c-jun early genes or of genes involved in cell cycle control. Here we demonstrate the existence of an alternative mechanism of the cancer-promoting effect of E 2. Human breast cancer cells (MCF-7) were exposed to the known proapoptotic agent vitamin E succinate (VES), added alone or together with different concentrations of E 2. E 2 conjugated with bovine serum albumin (E 2-BSA), which cannot cross the plasma membrane of living cells, was also used in some experiments to assess whether E 2 acted on the cell surface or at intracellular receptors. Apoptosis was analyzed by fluorescence-activated cell sorting after cell staining with propidium iodide and FITC-labeled annexin V. E 2 showed a concentration-dependent stimulatory effect on spontaneous apoptosis but inhibited the VES-induced apoptosis. However, effects produced by the same molar concentrations of E 2 were different when the hormone was free and when it was used in the form of the E 2-BSA conjugate. The effects of E 2 and E 2-BSA were sensitive to genistein, a tyrosine kinase inhibitor. These data show that E 2 modulates apoptosis of breast cancer cells, probably acting both at the cell surface and inside the cells. Tyrosine phosphorylation is involved in the signaling pathways mediating this E 2 effect.

2006

Steroid and xenobiotic receptor (SXR) or human pregnane X receptor (hPXR) has been shown to play an important role in the regulation of genes related to xenobiotic detoxification, such as cytochrome P450 3A4 and multidrug resistance gene 1. Cytochrome P450 enzymes, conjugation enzymes, and transporters are all considered to be involved in the resistance of breast carcinoma to chemotherapeutic or

Japanese Journal of Cancer Research, 2002

Overexpression of the oncoprotein MDM2, an important regulator of the p53 tumor suppressor protein, is often observed in breast cancer tissues and cell lines, particularly in those which express estrogen receptor a (ERa). In MCF–7 breast cancer cell line possessing wild‐type p53, ERa, and overexpressing MDM2, p53 accumulation was stimulated by 17β‐estradiol (E2) in a concentrationdependent manner. On the other hand, E2 caused no change of the expression of p53 mRNA, indicating that E2 affects p53 at the post‐transcriptional level. To analyze the mechanism of p53 accumulation by E2, the stability of p53, ERa and MDM2 proteins was analyzed in the presence of cycloheximide under an E2–supplemented or‐depleted condition. E2 significantly extended the half‐life of p53 protein, but shortened that of ERa in MCF–7 cells. E2 significantly decreased the stability of p90(MDM2) and p60(MDM2) in MCF–7. Interestingly, E2 increased the ratio p60(MDM2)/p90(MDM2) inversely proportionally to the degr...

Breast Cancer Research, 2008

Introduction Estrogen deprivation using aromatase inhibitors is one of the standard treatments for postmenopausal women with estrogen receptor (ER)-positive breast cancer. However, one of the consequences of prolonged estrogen suppression is acquired drug resistance. Our group is interested in studying antihormone resistance and has previously reported the development of an estrogen deprived human breast cancer cell line, MCF-7:5C, which undergoes apoptosis in the presence of estradiol. In contrast, another estrogen deprived cell line, MCF-7:2A, appears to have elevated levels of glutathione (GSH) and is resistant to estradiol-induced apoptosis. In the present study, we evaluated whether buthionine sulfoximine (BSO), a potent inhibitor of glutathione (GSH) synthesis, is capable of sensitizing antihormone resistant MCF-7:2A cells to estradiolinduced apoptosis. Methods Estrogen deprived MCF-7:2A cells were treated with 1 nM 17β-estradiol (E 2), 100 μM BSO, or 1 nM E 2 + 100 μM BSO combination in vitro, and the effects of these agents on cell growth and apoptosis were evaluated by DNA quantitation assay and annexin V and terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) staining. The in vitro results of the MCF-7:2A cell line were further confirmed in vivo in a mouse xenograft model. Results Exposure of MCF-7:2A cells to 1 nM E 2 plus 100 μM BSO combination for 48 to 96 h produced a sevenfold increase in apoptosis whereas the individual treatments had no significant effect on growth. Induction of apoptosis by the combination treatment of E 2 plus BSO was evidenced by changes in Bcl-2 and Bax expression. The combination treatment also markedly increased phosphorylated c-Jun Nterminal kinase (JNK) levels in MCF-7:2A cells and blockade of the JNK pathway attenuated the apoptotic effect of E 2 plus BSO. Our in vitro findings corroborated in vivo data from a mouse xenograft model in which daily administration of BSO either as a single agent or in combination with E 2 significantly reduced tumor growth of MCF-7:2A cells. Conclusions Our data indicates that GSH participates in retarding apoptosis in antihormone-resistant human breast cancer cells and that depletion of this molecule by BSO may be critical in predisposing resistant cells to E 2-induced apoptotic cell death. We suggest that these data may form the basis of improving therapeutic strategies for the treatment of antihormone resistant ER-positive breast cancer.

2000

Carcinogenesis, 2005

Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a mammary gland carcinogen present in the human diet. Herein, the expression of estrogen receptor alpha (ERa), estrogen receptor beta (ERb) and progesterone receptor (PR) was examined in mammary gland carcinomas induced by PhIP in female Sprague-Dawley rats. Quantitative real-time polymerase chain reaction demonstrated that ERa, ERb and PR were statistically elevated by 3-, 4-and 8-fold in carcinomas compared with normal mammary glands. By immunohistochemistry, carcinomas showed statistically higher nuclear expression of all three steroid receptors with the majority of carcinomas showing at least 10% of epithelial cells stained for ERa (49/55, 89%), ERb (41/55, 75%) and PR (48/55, 87%). Furthermore, the level of expression of the three steroid hormone receptors was positively correlated with each other across the bank of carcinomas (Spearman analysis, P 5 0.05). The expression of ERa in carcinomas was associated with tumor grade, extent of nuclear pleomorphism and cellular proliferation as measured by proliferating cell nuclear antigen (PCNA) and phospho-Rb immunostaining (Spearman analysis, P 5 0.05). Confocal microscopy was used to measure the percentage of epithelial cells showing nuclear colocalization of receptors, PCNA, and cyclin D1. Colocalization of the receptors, and the colocalization of the receptors with PCNA and cyclin D1 was strikingly higher in carcinomas than in the normal mammary gland. In carcinoma cells, 37% of ERa positive epithelial cells were colocalized with PCNA in contrast to just 0.25% of cells in the normal mammary gland. The findings from this study indicate that ERa, ERb and PR were coupregulated and nuclear localized in epithelial cells from rat mammary carcinomas compared with normal mammary glands, and that the co-upregulation was positively correlated with proliferation and cell cycle progression in carcinomas.

Cancer Research Journal, 2014

A commercial food supplement called Citozym (CIZ), was tested for antiproliferative activity against estrogen receptor (ER)-positive MCF-7 and (ER)-negative BT-20 human breast cancer cells, performing two independent experiments using SRB assay. A CIZ concentration of 100mg/mL, showed the most potent antiproliferative activity for cells in a potential range for further investigation on estrogen-insensitive breast cancer therapy.

Proceedings of the National Academy of Sciences, 2012

More than two-thirds of breast cancers express the estrogen receptor (ER) and depend on estrogen for growth and survival. Therapies targeting ER function, including aromatase inhibitors that block the production of estrogens and ER antagonists that alter ER transcriptional activity, play a central role in the treatment of ER+ breast cancers of all stages. In contrast to ER− breast cancers, which frequently harbor mutations in the p53 tumor suppressor, ER+ breast cancers are predominantly wild type for p53. Despite harboring wild-type p53, ER+ breast cancer cells are resistant to chemotherapy-induced apoptosis in the presence of estrogen. Using genomewide approaches, we have addressed the mechanism by which ER antagonizes the proapoptotic function of p53. Interestingly, both ER agonists such as estradiol and the selective ER modulator (SERM) tamoxifen promote p53 antagonism. In contrast, the full ER antagonist fulvestrant blocks the ability of ER to inhibit p53-mediated cell death. This inhibition works through a mechanism involving the modulation of a subset of p53 and ER target genes that can predict the relapse-free survival of patients with ER+ breast cancer. These findings suggest an improved strategy for the treatment of ER+ breast cancer using antagonists that completely block ER action together with drugs that activate p53-mediated cell death. cistrome | DNA damage | nuclear receptor | nutlin | doxorubicin

Scientific Reports, 2016

Tamoxifen is the accepted therapy for patients with estrogen receptor-α (ERα)-positive breast cancer. However, clinical resistance to tamoxifen, as demonstrated by recurrence or progression on therapy, is frequent and precedes death from metastases. To improve breast cancer treatment it is vital to understand the mechanisms that result in tamoxifen resistance. This study shows that concentrations of tamoxifen and its metabolites, which accumulate in tumors of patients, killed both ERα-positive and ERα-negative breast cancer cells. This depended on oxidative damage and anti-oxidants rescued the cancer cells from tamoxifen-induced apoptosis. Breast cancer cells responded to tamoxifen-induced oxidation by increasing Nrf2 expression and subsequent activation of the anti-oxidant response element (ARE). This increased the transcription of anti-oxidant genes and multidrug resistance transporters. As a result, breast cancer cells are able to destroy or export toxic oxidation products leading to increased survival from tamoxifen-induced oxidative damage. These responses in cancer cells also occur in breast tumors of tamoxifen-treated mice. Additionally, high levels of expression of Nrf2, ABCC1, ABCC3 plus NAD(P)H dehydrogenase quinone-1 in breast tumors of patients at the time of diagnosis were prognostic of poor survival after tamoxifen therapy. Therefore, overcoming tamoxifeninduced activation of the ARE could increase the efficacy of tamoxifen in treating breast cancer. Breast cancer is the most common malignancy among women in western societies 1. After being diagnosed, primary breast tumors are surgically resected and those patients with estrogen receptor-α (ERα)-positive tumors are typically prescribed tamoxifen as an adjuvant treatment 2-4. Tamoxifen is composed of triphenylethylene backbone structure and works by blocking the actions of ERα 4. Α bout 75% of all breast tumors are ERα positive and thus tamoxifen is the most widely used therapy for breast cancer leading to tumor stabilization in about 50% of previously untreated patients with metastatic breast cancer 3,5. Tamoxifen has been credited with much of the decrease in breast cancer mortality over the last decade. Nevertheless, nearly one-third of patients receiving adjuvant tamoxifen eventually experience disease relapse and almost all patients with metastatic tumors treated with tamoxifen will have progression and die from their disease 3,6. The purpose of the present study was to elucidate the mechanism that leads to a decreased responsiveness to tamoxifen therapy. Binding of the primary human estrogen, 17β-estradiol, to ERα seals the hydrophobic pocket by the helix-12 domain 7,8. ERα then translocates to the nucleus, where it activates the estrogen response element and drives the transcription of estrogen-dependent genes 9. Tamoxifen is metabolized to 4-hydroxytamoxifen (4HT) 10,11 , which also binds to ERα. However, this leads to a different conformational change as compared to 17β-estradiol, and the hydrophobic pocket is not sealed by helix-12 8. Consequently, 4HT blocks ERα activation.

Forum on Immunopathological Diseases and Therapeutics, 2012

Chronic inflammation within the tumor microenvironment is a major driver of tumor progression and poor prognosis. Inducible nitric oxide synthase (NOS2) is present in numerous solid tumors. Estrogen receptor-negative (ER−) patients with high expression of tumor NOS2 have a poorer outcome than patients with low expression of NOS2. Furthermore, expression of NOS2 is associated with the basal-like breast cancer phenotype. Using an in vitro model, we have found that nitrosation of critical thiols and nitration of tyrosines lead to the activation of membrane receptors such as epithelial growth factor receptor, Src, Ras, and CD63. These nitric oxidemediated events in itiate oncogenic signaling pathways such as PI3K/Akt, Ras/ERK, β-catenin, nuclear factor-κB, and AP-1. These data suggest that NOS2 can serve as a major "nonmutatational driver" of ER− breast cancer.