ABSTRACT
The anti-parasitic benzimidazole flubendazole has been used for many years to treat intestinal infections in humans and animals. Previous genotoxicity studies have shown that the compound is not a bacterial mutagen and a bone marrow micronucleus test, using a formulation that limited systemic absorption, was negative. The purpose of this study is to explore the genotoxicity of flubendazole and its main metabolites in in vitro micronucleus studies and to test a new oral formulation that improves systemic absorption in an in vivo micronucleus test. The isolated metabolites were also screened using the Ames test for bacterial mutagenicity. It was found that flubendazole, like other chemically related benzimidazoles used in anti-parasitic therapies, is a potent aneugen in vitro The hydrolysed metabolite of flubendazole is negative in these tests, but the reduced metabolite (R- and S-forms) shows both aneugenic and clastogenic activity. However, in vitro micronucleus tests of flubendazole in the presence of rat liver S9 gave almost identical signals for aneugenicity as they did in the absence of S9, suggesting that any clastogenicity from the reduced metabolite is not sufficient to change the overall profile. Like flubendazole itself, both metabolites are negative in the Ames test. Analysis of dose-response curves from the in vitro tests, using recently developed point of departure approaches, demonstrate that the aneugenic potency of flubendazole is very similar to related anti-parasitic benzimidazoles, including albendazole, which is used in mass drug administration programmes to combat endemic filarial diseases. The in vivo micronucleus test of the new formulation of flubendazole also showed evidence of induced aneugenicity. Analysis of the in vivo data allowed a reference dose for aneugenicity to be established which can be compared with therapeutic exposures of flubendazole when this has been established. Analysis of the plasma from the animals used in the in vivo micronucleus test showed that there is increased exposure to flubendazole compared with previously tested formulations, as well as significant formation of the non-genotoxic hydrolysed metabolite of flubendazole and small levels of the reduced metabolite. In conclusion, this study shows that flubendazole is a potent aneugen in vitro with similar potency to chemically related benzimidazoles currently used as anti-parasitic therapies. The reduced metabolite also has aneugenic properties as well as clastogenic properties. Treatment with a new formulation of flubendazole that allows increased systemic exposure, compared with previously used formulations, also results in detectable aneugenicity in vivo. Based on the lack of carcinogenicity of this class of benzimidazoles and the intended short-term dosing, it is unlikely that flubendazole treatment will pose a carcinogenic risk to patients.
Subject(s)
Aneugens/toxicity , Chromosome Aberrations , DNA Damage , Lymphocytes/drug effects , Mebendazole/analogs & derivatives , Activation, Metabolic , Aneugens/metabolism , Animals , Antinematodal Agents/metabolism , Antinematodal Agents/toxicity , Cells, Cultured , Chromosomes, Human/drug effects , DNA/drug effects , Humans , Lymphocytes/metabolism , Male , Mebendazole/metabolism , Mebendazole/toxicity , Micronucleus Tests , Mutagens/metabolism , Mutagens/toxicity , RatsABSTRACT
INTRODUCTION: Strategies to improve the efficacy of endocrine agents in breast cancer (BC) therapy and to delay the onset of resistance include concomitant targeting of the estrogen receptor alpha (ER) and the mammalian target of rapamycin complex 1 (mTORC1), which regulate cell-cycle progression and are supported by recent clinical results. METHODS: BC cell lines expressing aromatase (AROM) and modeling endocrine-sensitive (MCF7-AROM1) and human epidermal growth factor receptor 2 (HER2)-dependent de novo resistant disease (BT474-AROM3) and long-term estrogen-deprived (LTED) MCF7 cells that had acquired resistance associated with HER2 overexpression were treated in vitro and as subcutaneous xenografts with everolimus (RAD001-mTORC1 inhibitor), in combination with tamoxifen or letrozole. End points included proliferation, cell-cycle arrest, cell signaling, and effects on ER-mediated transactivation. RESULTS: Everolimus caused a concentration-dependent decrease in proliferation in all cell lines, which was associated with reductions in S6 phosphorylation. Everolimus plus letrozole or tamoxifen enhanced the antiproliferative effect and G1-accumulation compared with monotherapy, as well as increased phosphorylation (Ser10) and nuclear accumulation of p27 and pronounced dephosphorylation of Rb. Sensitivity was greatest to everolimus in the LTED cells but was reduced by added estrogen. Increased pAKT occurred in all circumstances with everolimus and, in the BT474 and LTED cells, was associated with increased pHER3. Decreased ER transactivation suggested that the effectiveness of everolimus might be partly related to interrupting cross-talk between growth-factor signaling and ER. In MCF7-AROM1 xenografts, letrozole plus everolimus showed a trend toward enhanced tumor regression, versus the single agents. In BT474-AROM3 xenografts, everolimus alone was equally effective at reducing tumor volume as were the combination therapies. CONCLUSIONS: The results provide mechanistic support for recent positive clinical data on the combination of everolimus and endocrine therapy, as well as data on potential routes of escape via enhanced HER2/3 signaling. This merits investigation for further improvements in treatment efficacy.
Subject(s)
Antineoplastic Agents, Hormonal/pharmacology , Nitriles/pharmacology , Protein Kinase Inhibitors/pharmacology , Sirolimus/analogs & derivatives , Tamoxifen/pharmacology , Triazoles/pharmacology , Animals , Antineoplastic Agents, Hormonal/administration & dosage , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Cycle/drug effects , Cell Line, Tumor , Cell Nucleolus/metabolism , Cell Proliferation/drug effects , Disease Models, Animal , Drug Resistance, Neoplasm , Everolimus , Female , Humans , Letrozole , Mechanistic Target of Rapamycin Complex 1 , Mice , Multiprotein Complexes/antagonists & inhibitors , Multiprotein Complexes/metabolism , Nitriles/administration & dosage , Phosphorylation , Protein Kinase Inhibitors/administration & dosage , Protein Transport , Proto-Oncogene Proteins c-akt/metabolism , Receptor, ErbB-3/metabolism , Receptors, Estrogen/metabolism , Signal Transduction/drug effects , Sirolimus/administration & dosage , Sirolimus/pharmacology , TOR Serine-Threonine Kinases/antagonists & inhibitors , TOR Serine-Threonine Kinases/metabolism , Tamoxifen/administration & dosage , Triazoles/administration & dosage , Xenograft Model Antitumor AssaysABSTRACT
IGF-IR-mediated signaling promotes survival, anchorage-independent growth, and oncogenic transformation, as well as tumor growth and metastasis formation in vivo. NVP-AEW541 is a pyrrolo[2,3-d]pyrimidine derivative small molecular weight kinase inhibitor of the IGF-IR, capable of distinguishing between the IGF-IR (IC50 = 0.086 microM) and the closely related InsR (IC50 = 2.3 microM) in cells. As expected for a specific IGF-IR kinase inhibitor, NVP-AEW541 abrogates IGF-I-mediated survival and colony formation in soft agar at concentrations that are consistent with inhibition of IGF-IR autophosphorylation. In vivo, this orally bioavailable compound inhibits IGF-IR signaling in tumor xenografts and significantly reduces the growth of IGF-IR-driven fibrosarcomas. Thus, NVP-AEW541 represents a class of selective, small molecule IGF-IR kinase inhibitors with proven in vivo antitumor activity and potential therapeutic application.
Subject(s)
Antineoplastic Agents/pharmacology , Receptor, IGF Type 1/metabolism , Signal Transduction/physiology , 3T3 Cells , Animals , Apoptosis/drug effects , Apoptosis/physiology , Cell Division , Enzyme Activation/drug effects , Enzyme Activation/physiology , Enzyme Inhibitors/pharmacology , Humans , MAP Kinase Signaling System/drug effects , MAP Kinase Signaling System/physiology , Mice , Phosphorylation/drug effects , Receptor, IGF Type 1/drug effects , Receptor, Insulin/drug effects , Receptor, Insulin/metabolism , Signal Transduction/drug effects , src-Family Kinases/drug effects , src-Family Kinases/metabolismABSTRACT
Implantation is initiated when the embryo attaches to the uterine luminal epithelium during early pregnancy. Following this event, uterine stromal cells undergo steroid hormone-dependent transformation into morphologically and functionally distinct decidual cells in a unique process known as decidualization. An angiogenic network is also formed in the uterine stromal bed, critically supporting the early development of the embryo. The steroid-induced mechanisms that promote stromal differentiation and endothelial proliferation during decidualization are not fully understood. Although the role of ovarian progesterone as a key regulator of decidualization is well established, the requirement of ovarian estrogen (E) during this process remains unresolved. Here we show that the expression of P450 aromatase, a key enzyme that converts androgens to E, is markedly induced in mouse uterine stromal cells undergoing decidualization. The aromatase then acts in conjunction with other steroid biosynthetic enzymes present in the decidual tissue to support de novo synthesis of E. This locally produced E is able to support the advancement of the stromal differentiation program even in the absence ovarian E in an ovariectomized, progesterone-supplemented pregnant mouse model. Administration of letrozole, a specific aromatase inhibitor, to these mice blocked the stromal differentiation process. Gene expression profiling further revealed that the intrauterine E induces the expression of several stromal factors that promote neovascularization in the decidual tissue. Collectively, these studies identified the decidual uterus as a novel site of E biosynthesis and uncovered E-regulated maternal signaling pathways that critically control uterine differentiation and angiogenesis during early pregnancy.
Subject(s)
Decidua/metabolism , Estrogens/biosynthesis , Neovascularization, Physiologic , Uterus/metabolism , Animals , Aromatase/genetics , Aromatase/metabolism , Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , Decidua/blood supply , Decidua/cytology , Embryo Implantation , Female , Gene Expression Regulation, Developmental/genetics , Immunohistochemistry , Male , Mice , Ovariectomy , Phosphoproteins/genetics , Phosphoproteins/metabolism , Pregnancy , Progesterone/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Stromal Cells/metabolism , Uterus/blood supply , Uterus/cytologyABSTRACT
Letrozole is a potent aromatase inhibitor and superior to other defined selective estrogen receptor modulators such as tamoxifen in treating hormone-responsive postmenopausal breast cancer patients. Patients who receive this drug may become insensitive to the effects of estrogen deprivation induced by letrozole. Letrozole has known side effects on bone metabolism due to systemic ablation of estrogen production. The purpose of this study was to examine the therapeutic efficacy of hyaluronic acid-bound letrozole nanoparticles (HA-Letr-NPs) in restoring sensitivity to letrozole-resistant (LTLT-Ca) cells. To target letrozole to LTLT-Ca cells, hyaluronic acid-bound letrozole nanoparticles were prepared by nanoprecipitation using biodegradable PLGA-PEG co-polymer. Binding specificity of HA to CD44 on the cell surface was analyzed in vitro using FITC-CD44 Ab and CD44 siRNA by flow cytometry. Effects on in vitro cytotoxicity and aromatase enzymatic activity of HA-Letr-NPs were performed in MCF-7 breast cancer cells, MCF-7 cells over-expressing aromatase (MCF-7/Aro), and LTLT-Ca cells resistant to letrozole. Preclinical efficacy of HA-Letr-NPs was examined in mice using LTLT-Ca xenograft tumors. HA-Letr-NPs were restricted to a maximum size of 100 nm. The in vitro drug release assay showed that the highest released concentration of letrozole occurred after 23 hours at 37 degrees C in phosphate-buffered saline. HA-Letr-NPs on MCF-7/Aro and LTLT-Ca cells showed an IC50 of 2 microM and 5 microM, respectively. HA-Letr-NPs were more efficacious in inhibiting tumor growth, reducing in vitro cellular and in vivo tumor aromatase enzyme activity more than the corresponding Letr-NPs or letrozole. HA-Letr-NPs restored and maintained a prolonged sensitivity and targeted delivery of letrozole in letrozole-resistant tumors in vivo.
Subject(s)
Antineoplastic Agents/pharmacology , Hyaluronic Acid/chemistry , Nanoparticles , Nitriles/pharmacology , Triazoles/pharmacology , Animals , Antineoplastic Agents/chemistry , Apoptosis/drug effects , Blotting, Western , Drug Resistance, Neoplasm , Flow Cytometry , Letrozole , Mice , Microscopy, Electron, Scanning , Nitriles/chemistry , RNA Interference , Triazoles/chemistry , Xenograft Model Antitumor AssaysABSTRACT
Estrogens induce breast tumor cell proliferation by directly regulating gene expression via the estrogen receptor (ER) transcriptional activity and by affecting growth factor signaling pathways such as mitogen-activated protein kinase (MAPK) and AKT/mammalian target of rapamycin Complex1 (mTORC1) cascades. In this study we demonstrated the preclinical therapeutic efficacy of combining the aromatase inhibitor letrozole with the multi-kinase inhibitor sorafenib in aromatase-expressing breast cancer cell lines. Treatment with letrozole reduced testosterone-driven cell proliferation, by inhibiting the synthesis of estrogens. Sorafenib inhibited cell proliferation in a concentration-dependent manner; this effect was not dependent on sorafenib-mediated inhibition of Raf1, but involved the down-regulation of mTORC1 and its targets p70S6K and 4E-binding protein 1 (4E-BP1). At concentrations of 5-10 µM the growth-inhibitory effect of sorafenib was associated with the induction of apoptosis, as indicated by release of cytochrome c and Apoptosis-Inducing Factor into the cytosol, activation of caspase-9 and caspase-7, and PARP-1 cleavage. Combination of letrozole and sorafenib produced a synergistic inhibition of cell proliferation associated with an enhanced accumulation of cells in the G(0)/G(1) phase of the cell cycle and with a down-regulation of the cell cycle regulatory proteins c-myc, cyclin D1, and phospho-Rb. In addition, longer experiments (12 weeks) demonstrated that sorafenib may be effective in preventing the acquisition of resistance towards letrozole. Together, these results indicate that combination of letrozole and sorafenib might constitute a promising approach to the treatment of hormone-dependent breast cancer.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Breast Neoplasms/pathology , Cell Proliferation/drug effects , Adaptor Proteins, Signal Transducing/metabolism , Apoptosis/drug effects , Apoptosis Inducing Factor/metabolism , Aromatase/genetics , Aromatase/metabolism , Aromatase Inhibitors/pharmacology , Benzenesulfonates/pharmacology , Breast Neoplasms/enzymology , Breast Neoplasms/genetics , Caspase 7/metabolism , Caspase 9/metabolism , Cell Cycle/drug effects , Cell Cycle Proteins , Cell Line, Tumor , Cyclin D1/metabolism , Cytochromes c/metabolism , Dose-Response Relationship, Drug , Drug Resistance, Neoplasm , Drug Synergism , Estradiol/metabolism , Female , Humans , Letrozole , Mechanistic Target of Rapamycin Complex 1 , Multiprotein Complexes , Niacinamide/analogs & derivatives , Nitriles/pharmacology , Phenylurea Compounds , Phosphoproteins/metabolism , Phosphorylation , Poly (ADP-Ribose) Polymerase-1 , Poly(ADP-ribose) Polymerases/metabolism , Protein Kinase Inhibitors/pharmacology , Proteins/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Pyridines/pharmacology , Retinoblastoma Protein/metabolism , Ribosomal Protein S6 Kinases, 70-kDa/metabolism , Sorafenib , TOR Serine-Threonine Kinases , Testosterone/metabolism , Time Factors , Transfection , Triazoles/pharmacologyABSTRACT
Mutations in the alpha catalytic subunit of phosphoinositol-3-kinase (PIK3CA) occur in approximately 30% of ER positive breast cancers. We therefore sought to determine the impact of PIK3CA mutation on response to neoadjuvant endocrine therapy. Exons 9 (helical domain) and 20 (kinase domain-KD) mutations in PIK3CA were determined samples from four neoadjuvant endocrine therapy trials.Interactions with clinical, pathological, and biomarker response parameters were examined. A weak negative interaction between PIK3CA mutation status and clinical response to neoadjuvant endocrine treatment was detected(N = 235 P < or = 0.05), but not with treatment-induced changes in Ki67-based proliferation index (N = 418). Despite these findings, PIK3CA KD mutation was a favorable prognostic factor for relapse-free survival (RFS log-rank P = 0.02) in the P024 trial (N = 153). The favorable prognostic effect was maintained in a multivariable analysis(N = 125) that included the preoperative endocrine prognostic index, an approach to predicting RFS based on post neoadjuvant endocrine therapy pathological stage, ER, and Ki67 levels (HR for no PIK3CA KD mutation, 14, CI 1.9-105 P = 0.01). PIK3CA mutation status did not strongly interact with neoadjuvant endocrine therapy responsiveness in estrogen receptor-positive breast cancer. Nonetheless, as with other recent studies, a favorable interaction between PIK3CA KD mutation and prognosis was detected. The mechanism for the favorable prognostic impact of PIK3CA mutation status therefore remains unexplained.
Subject(s)
Antineoplastic Agents, Hormonal/therapeutic use , Breast Neoplasms/drug therapy , Mutation , Phosphatidylinositol 3-Kinases/genetics , Receptors, Estrogen/analysis , Breast Neoplasms/chemistry , Breast Neoplasms/enzymology , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Proliferation/drug effects , Chemotherapy, Adjuvant , Class I Phosphatidylinositol 3-Kinases , Clinical Trials, Phase II as Topic , Disease-Free Survival , Exons , Female , Gene Expression Regulation, Neoplastic , Humans , Kaplan-Meier Estimate , Ki-67 Antigen/analysis , Neoadjuvant Therapy , Neoplasm Staging , Phosphorylation , Proportional Hazards Models , Proto-Oncogene Proteins c-akt/analysis , Randomized Controlled Trials as Topic , Receptor, ErbB-2/analysis , Receptors, Progesterone/analysis , Risk Assessment , Time Factors , Treatment OutcomeABSTRACT
Estrogen-secreting adrenal cancers are extremely rare, with feminizing symptoms attributed to aromatase expression in the adrenal tumor. We describe a case of hypogonadotropic hypogonadism as a consequence of aberrant aromatase expression in a patient with adrenocortical adenocarcinoma. A 54 year-old man presented with a two month history of gynecomastia and reduced libido. Endocrine biochemistry at presentation showed hypogonadotropic hypogonadism (LH 2.4 U/L, FSH <1.0 IU/L, testosterone 2.8 nmol/L) with increased serum estrone (E(1), 821 pmol/L) and estradiol (E(2), 797 pmol/L) and subclinical ACTH-independent hypercortisolism (serum cortisol post 1mg overnight dexamethasone suppression test, 291 nmol/L). A right adrenal mass was identified on CT scanning and the patient underwent an open adrenalectomy. Post-operative evaluation showed normalization of serum levels of E(1) (95 pmol/L), E(2 )(109 pmol/L), testosterone (11.4 nmol/L), LH (4.1 U/L) and FSH (5.9 IU/L), and of cortisol dynamics. Immunohistochemistry of the adrenal cancer confirmed aberrant expression of aromatase in most, although not all, carcinoma cells. Transcripts associated with utilization of promoters II, I.1 and I.3 were prominently represented in the tumor aromatase mRNA. This case highlights that clinical features of feminizing adrenocortical carcinomas can be secondary to estrogen production by aberrantly transcribed and translated aromatase within the tumor. Even in males, gonadotropin secretion is subject to predominantly estrogen-mediated feedback-inhibition. The diagnosis of adrenocortical adenocarcinoma should be considered in men presenting with low testosterone and gonadotropins, particularly in the presence of feminizing features.
Subject(s)
Adenocarcinoma/genetics , Adrenal Cortex Neoplasms/genetics , Aromatase/genetics , Hypogonadism/genetics , Adenocarcinoma/complications , Adenocarcinoma/metabolism , Adrenal Cortex Neoplasms/complications , Adrenal Cortex Neoplasms/metabolism , Adult , Age of Onset , Aromatase/metabolism , Gene Expression Regulation, Enzymologic/physiology , Gene Expression Regulation, Neoplastic/physiology , Genetic Predisposition to Disease , Humans , Hypogonadism/epidemiology , Hypogonadism/etiology , Hypogonadism/metabolism , Male , Middle AgedABSTRACT
PURPOSE: The aim of this study was to investigate the potential benefits of prolonged treatment with neoadjuvant letrozole. PATIENTS AND METHODS: About 182 consecutive patients have been treated in Edinburgh with neoadjuvant letrozole for 3 months or longer and 63 patients have continued on letrozole beyond 3 months. Outcomes are reported. RESULTS: Of the 63 patients who continued on letrozole, 38 patients took letrozole for more than 1 year and 23 took letrozole for more than 24 months. The median reduction in clinical volume in the first 3 months in these 63 patients was 52%. Similar reductions in median clinical volume were seen between three to 6 months (50%), 6-12 months and 12-24 months (medians 37 and 33%, respectively). At 3 months 69.8% of the 182 patients had a partial or complete response. The response rate increased to 83.5% with prolonged letrozole treatment. Continuing letrozole beyond 3 months increased the number of women who initially required mastectomy or had locally advanced breast cancer who were subsequently suitable for breast conserving surgery from 60% (81/134) at 3 months to 72% (96/134). Thirty-three women remain on letrozole alone (man age at diagnosis 83 years) and at 3 years the median time to treatment failure has not been reached. CONCLUSION: Continuing letrozole in responding patients beyond 3-4 months achieves further clinical reduction in tumour size. For elderly women with a short life expectancy letrozole alone may provide long-term disease control.
Subject(s)
Antineoplastic Agents/therapeutic use , Breast Neoplasms/drug therapy , Breast Neoplasms/surgery , Aged, 80 and over , Breast Neoplasms/pathology , Combined Modality Therapy , Disease Progression , Drug Administration Schedule , Female , Humans , Letrozole , Life Expectancy , Mastectomy , Mastectomy, Segmental , Neoadjuvant Therapy , Nitriles , Time Factors , Treatment Outcome , TriazolesABSTRACT
BACKGROUND: Expression of aromatase by malignant breast epithelial cells and/or the surrounding stroma implies local estrogen production that could influence the outcome of endocrine therapy for breast cancer. METHODS: A validated immunohistochemical assay for aromatase was applied to samples from the P024 neoadjuvant endocrine therapy trial that compared tamoxifen and letrozole. The presence of aromatase expression by tumor or stromal cells was correlated with tumor response, treatment induced changes in proliferation index (Ki67), relapse-free survival (RFS) and breast cancer-specific survival (BCSS). RESULTS: Tumor and stromal aromatase expression were highly correlated (P = 0.0001). Tumor cell aromatase, as a semi-continuous score, also correlated with smaller tumor size at presentation (P = 0.01) higher baseline ER Allred score (P = 0.006) and lower Ki67 levels (P = 0.003). There was no significant relationship with clinical response or treatment-induced changes in Ki67. However, in a Cox multivariable model that incorporated a post-treatment tumor profile (pathological T stage, N stage, Ki67 and ER status of the surgical specimen), the presence of tumor aromatase expression at baseline sample remained a favorable independent prognostic biomarker for both RFS (P = 0.01, HR 2.3, 95% CI 1.2-4.6 for absent expression) and BCSS (P = 0.008, HR 3.76, 95% CI 1.4-10.0). CONCLUSIONS: Autocrine estrogen synthesis may be most characteristic of smaller, more indolent and ER-rich breast cancers with lower baseline growth rates. However, response to endocrine treatment may not depend on whether the estrogenic stimulus has a local versus systemic source.
Subject(s)
Antineoplastic Agents, Hormonal/therapeutic use , Aromatase/biosynthesis , Biomarkers, Tumor/analysis , Breast Neoplasms/enzymology , Neoadjuvant Therapy/methods , Aged , Breast Neoplasms/drug therapy , Breast Neoplasms/mortality , Disease-Free Survival , Double-Blind Method , Female , Humans , Immunohistochemistry , Kaplan-Meier Estimate , Ki-67 Antigen/drug effects , Ki-67 Antigen/metabolism , Letrozole , Nitriles/therapeutic use , Prognosis , Receptors, Estrogen/metabolism , Tamoxifen/therapeutic use , Treatment Outcome , Triazoles/therapeutic useABSTRACT
Adrenocortical carcinoma is an uncommon malignancy and feminizing symptoms secondary to adrenal estrogen-secretion are extremely rare. The direct secretion of estradiol by adrenocortical tumors requires, in addition to the expression of aromatase (CYP19), the expression of one or more of the reductive 17beta-hydroxysteroid dehydrogenases. The expression of CYP19 transcripts and protein were markedly induced in the H295 adrenocortical carcinoma cell line after treatment with either forskolin or vasoactive intestinal peptide (VIP). Western immunoblotting demonstrated a marked induction of the CYP19 protein of characteristic size after only a short (6h) treatment period with VIP or forskolin. The CYP19 mRNA transcripts were derived from both promoters PII (Ic) and I.3 (Id) after treatment with both agents. The reductive type 5 17beta-hydroxysteroid dehydrogenase (AKR1C3) was also constitutively expressed in the H295 cells but neither its mRNA transcript nor protein levels were altered after forskolin or VIP treatment. Western immunoblotting of an estrogen-secreting adrenal carcinoma revealed notable levels of both aromatase and AKR1C3 expression while an aldosterone-producing adrenal adenoma lacked aromatase expression and showed a reduced level of AKR1C3 expression. Immunohistochemistry of the carcinoma-bearing adrenal revealed localization of AKR1C3 not only in the tumor but also principally in the zona reticularis of the normal adrenal tissue. Adrenal aromatase and AKR1C3 expression therefore appear to be features of adrenocortical malignancies that are associated with biosynthesis of active estrogen.
Subject(s)
Adrenal Cortex Neoplasms/metabolism , Cell Line, Tumor , Estrogens/biosynthesis , 17-Hydroxysteroid Dehydrogenases/genetics , 17-Hydroxysteroid Dehydrogenases/metabolism , 3-Hydroxysteroid Dehydrogenases , Adrenal Cortex Neoplasms/pathology , Adult , Aldo-Keto Reductase Family 1 Member C3 , Aromatase/genetics , Aromatase/metabolism , Colforsin/metabolism , Cytochrome P-450 CYP11B2/genetics , Cytochrome P-450 CYP11B2/metabolism , Female , Humans , Hydroxyprostaglandin Dehydrogenases , Progesterone Reductase/genetics , Progesterone Reductase/metabolism , Promoter Regions, Genetic , Steroid 11-beta-Hydroxylase/genetics , Steroid 11-beta-Hydroxylase/metabolism , Steroid 17-alpha-Hydroxylase/genetics , Steroid 17-alpha-Hydroxylase/metabolism , Vasoactive Intestinal Peptide/metabolismABSTRACT
BACKGROUND: New, third-generation aromatase inhibitors (AIs) have proven comparable or superior to the anti-estrogen tamoxifen for treatment of estrogen receptor (ER) and/or progesterone receptor (PR) positive breast cancer. AIs suppress total body and intratumoral estrogen levels. It is unclear whether in situ carcinoma cell aromatization is the primary source of estrogen production for tumor growth and whether the aromatase expression is predictive of response to endocrine therapy. Due to methodological difficulties in the determination of the aromatase protein, COX-2, an enzyme involved in the synthesis of aromatase, has been suggested as a surrogate marker for aromatase expression. METHODS: Primary tumor material was retrospectively collected from 88 patients who participated in a randomized clinical trial comparing the AI letrozole to the anti-estrogen tamoxifen for first-line treatment of advanced breast cancer. Semi-quantitative immunohistochemical (IHC) analysis was performed for ER, PR, COX-2 and aromatase using Tissue Microarrays (TMAs). Aromatase was also analyzed using whole sections (WS). Kappa analysis was applied to compare association of protein expression levels. Univariate Wilcoxon analysis and the Cox-analysis were performed to evaluate time to progression (TTP) in relation to marker expression. RESULTS: Aromatase expression was associated with ER, but not with PR or COX-2 expression in carcinoma cells. Measurements of aromatase in WS were not comparable to results from TMAs. Expression of COX-2 and aromatase did not predict response to endocrine therapy. Aromatase in combination with high PR expression may select letrozole treated patients with a longer TTP. CONCLUSION: TMAs are not suitable for IHC analysis of in situ aromatase expression and we did not find COX-2 expression in carcinoma cells to be a surrogate marker for aromatase. In situ aromatase expression in tumor cells is associated with ER expression and may thus point towards good prognosis. Aromatase expression in cancer cells is not predictive of response to endocrine therapy, indicating that in situ estrogen synthesis may not be the major source of intratumoral estrogen. However, aromatase expression in combination with high PR expression may select letrozole treated patients with longer TTP. TRIAL REGISTRATION: Sub-study of trial P025 for advanced breast cancer.
Subject(s)
Aromatase/biosynthesis , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Nitriles/therapeutic use , Receptors, Estrogen/biosynthesis , Tamoxifen/therapeutic use , Triazoles/therapeutic use , Antineoplastic Agents, Hormonal/therapeutic use , Aromatase Inhibitors/therapeutic use , Breast Neoplasms/enzymology , Carcinoma in Situ/drug therapy , Carcinoma in Situ/enzymology , Carcinoma in Situ/metabolism , Cyclooxygenase 2/biosynthesis , Female , Humans , Immunohistochemistry , Letrozole , Receptors, Progesterone/biosynthesis , Retrospective Studies , Tissue Array AnalysisABSTRACT
PURPOSE: To evaluate the influence of the third-generation aromatase inhibitor letrozole (Femara) on breast cancer tissue levels of estrone (E(1)), estradiol (E(2)), and estrone sulfate (E(1)S) in postmenopausal women undergoing primary treatment for locally advanced estrogen receptor/progesterone receptor-positive breast cancers. EXPERIMENTAL DESIGN: Breast cancer tissue samples were collected before and following 4 months of neoadjuvant therapy with letrozole (2.5 mg o.d.), and tissue estrogen levels measured using a highly sensitive RIA after high-pressure liquid chromatography purification. RESULTS: Letrozole suppressed pretreatment tumor levels of E(2), E(1), and E(1)S by 97.6%, 90.7%, and 90.1%, respectively. These data reveal that letrozole suppresses tissue estrogen levels significantly below what has previously been recorded with anastrozole (89.0%, 83.4%, and 72.9% suppression, respectively) using the same methods. To confirm the differential effect of letrozole and anastrozole on each plasma estrogen fraction, we re-analyzed plasma samples obtained from a previous intrapatient cross-over study comparing letrozole and anastrozole using an improved RIA (detection limits of 0.67, 1.14, and 0.55 pmol/L for E(2), E(1), and E(1)S, respectively). Letrozole consistently suppressed each plasma estrogen fraction below the levels recorded for anastrozole: E(2) (average suppression by 95.2% versus 92.8%; P = 0.018), E(1) (98.8% suppression versus 96.3%; P = 0.003), and E(1)S (98.9% suppression versus 95.3%; P = 0.003). CONCLUSION: Our data reveals that letrozole (2.5 mg o.d.) is more effective compared with anastrozole (1.0 mg o.d.) with respect to tissue as well as plasma estrogen suppression in patients with postmenopausal breast cancer.
Subject(s)
Antineoplastic Agents/therapeutic use , Breast Neoplasms/blood , Breast Neoplasms/drug therapy , Estrogens/blood , Nitriles/therapeutic use , Triazoles/therapeutic use , Aged , Aged, 80 and over , Anastrozole , Chromatography, High Pressure Liquid , Estradiol/metabolism , Estrone/analogs & derivatives , Estrone/metabolism , Female , Humans , Letrozole , Middle Aged , Postmenopause , RadioimmunoassayABSTRACT
PURPOSE: The possible involvement of gender-dependent factors has been suggested in human non-small cell lung carcinomas (NSCLC), but their precise roles remain largely unclear. Therefore, we examined intratumoral estradiol concentrations in NSCLC to examine local actions of estrogens in NSCLC. EXPERIMENTAL DESIGN: Fifty-nine frozen specimens of NSCLC were available for liquid chromatography/electrospray tandem mass spectrometry to study intratumoral estradiol concentrations. In addition, A549 NSCLC cells stably expressing estrogen receptor (ER) alpha (A549 + ERalpha) or ERbeta (A549 + ERbeta) were used in vitro studies. RESULTS: Forty-three (73%) of 59 NSCLC showed higher concentration of estradiol in carcinoma tissues than the corresponding nonneoplastic lung tissues from the same patient, and intratumoral estradiol concentrations were significantly (P = 0.0002 and 2.2-fold) higher than the corresponding nonneoplastic lungs. The intratumoral concentration of estradiol was positively correlated with aromatase expression, tumor size, and Ki-67 status in ERalpha- or ERbeta-positive cases. In in vitro studies, estradiol significantly increased cell proliferation of A549 + ERalpha or A549 + ERbeta, which was significantly suppressed by selective ER modulators, tamoxifen or raloxifene. Both A549 + ERalpha and A549 + ERbeta cells expressed aromatase. The cell proliferation level in these cells was significantly increased under treatment with testosterone, and it was inhibited by addition of the aromatase inhibitor letrozole. CONCLUSIONS: These results suggest that estradiol is locally produced in NSCLC mainly by aromatase and plays an important role in the growth of ERalpha- or ERbeta-positive NSCLC. Therefore, use of selective ER modulators and/or aromatase inhibitors may be clinically effective in NSCLC that are positive for both ER and aromatase.
Subject(s)
Carcinoma, Non-Small-Cell Lung/chemistry , Estrogens/analysis , Lung Neoplasms/metabolism , Receptors, Estrogen/metabolism , Aromatase/metabolism , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Cell Proliferation/drug effects , Chromatography, Liquid , Estrogen Receptor Modulators/pharmacology , Estrogens/metabolism , Estrogens/pharmacology , Humans , Ki-67 Antigen/metabolism , Lung Neoplasms/chemistry , Lung Neoplasms/pathology , Reverse Transcriptase Polymerase Chain Reaction , Selective Estrogen Receptor Modulators , Spectrometry, Mass, Electrospray IonizationABSTRACT
The prognostic value of aromatase, cyclooxygenase 2 (COX-2), HER-2/neu, and p53 expression was determined in endometrioid endometrial cancer. Tissue microarrays were constructed comprising samples from 315 endometrioid endometrial cancer patients. Expression of aromatase, COX-2, HER-2/neu, and p53 was determined by immunostaining and related to classical clinicohistopathologic parameters, in addition to recurrence of disease and survival. Median follow-up time for all patients was 5.0 years. Patients were classified as Fédération Internationale de Gynécologie Obstétrique stage I (59.0%), stage II (17.1%), stage III (19.4%), and stage IV (4.1%). Sixty-five patients (20.6%) developed recurrent disease, and 38 (12.1%) died because of endometrial cancer. Aromatase, COX-2, HER-2/neu, and p53 expression was observed in 133 (42.2%), 107 (34.0%), 17 (5.4%), and 21 (6.7%) tumor cases, respectively. Aromatase expression in tumor cells was related to aromatase expression in stromal cells (P < 0.0001) and to HER-2/neu expression in tumor cells (P = 0.019). Aromatase expression in tumor as well as stromal cells was related to a low stage of disease (P = 0.02 and P = 0.001, respectively), whereas aromatase expression in stromal cells was also related to a low tumor grade (P = 0.021). P53 expression was related to a high stage and a high grade (P = 0.006 and P < 0.0001, respectively). In multivariate analysis, p53 overexpression was independently related to death because of the disease (P = 0.043; odds ratio 3.0; 95% confidence interval, 1.0-8.7). For COX-2, HER-2/neu, and aromatase, no relation with any other histopathologic parameter or survival was found. In conclusion, aromatase and p53 expression are related to tumor grade and stage of disease, whereas p53 is an independent prognostic factor in endometrioid endometrial cancer.
Subject(s)
Aromatase/biosynthesis , Carcinoma, Endometrioid/metabolism , Cyclooxygenase 2/biosynthesis , Endometrial Neoplasms/metabolism , Receptor, ErbB-2/biosynthesis , Tumor Suppressor Protein p53/biosynthesis , Adult , Aged , Aged, 80 and over , Carcinoma, Endometrioid/enzymology , Carcinoma, Endometrioid/pathology , Carcinoma, Endometrioid/therapy , Disease-Free Survival , Endometrial Neoplasms/enzymology , Endometrial Neoplasms/pathology , Endometrial Neoplasms/therapy , Female , Humans , Middle AgedABSTRACT
Aromatase is a key enzyme in intratumoral estrogen production required for the production of estrogens through the conversion of serum androgens in postmenopausal breast cancer patients. There have been, however, controversies regarding the intratumoral localization of aromatase in human breast carcinoma tissues. Therefore, we have first examined the intratumoral localization of aromatase mRNA/protein in 19 breast carcinomas using laser capture microdissection/quantitative reverse transcription-PCR (RT-PCR) and immunohistochemistry. Aromatase mRNA and protein were detected in both intratumoral stromal and parenchymal cells in breast carcinoma tissues. Subsequent microarray expression profiling and clustering analyses, in addition to quantitative RT-PCR studies, showed a significant positive correlation between aromatase and estrogen-related receptor alpha mRNA expression in isolated carcinoma cells. We further examined an interaction between stromal cells isolated from human breast carcinoma tissues and breast carcinoma cell lines using a coculture system to study the biological characteristic of aromatase expression in carcinoma cells. Aromatase mRNA and enzyme activity and 17beta-hydroxysteroid dehydrogenase type 1 mRNA in breast carcinoma cell lines, including MCF-7 and SK-BR-3 cells, were up-regulated in the presence of patient-derived 32N or 74T intratumoral stromal cells. The results from steroid conversion assays were also consistent with the findings above. The results of our study also showed that aromatase inhibitors were more effective in inhibiting aromatization induced by coculture in MCF-7 than that in stromal 32N. The examination of the localization of aromatase and its regulation, including the interactions existing between different cell types in human breast carcinoma tissues, may provide important information as to achieving better clinical response to aromatase inhibitors in breast cancer patients.
Subject(s)
Aromatase/metabolism , Breast Neoplasms/enzymology , Carcinoma, Ductal, Breast/enzymology , 17-Hydroxysteroid Dehydrogenases/biosynthesis , 17-Hydroxysteroid Dehydrogenases/genetics , Adult , Aged , Aromatase/biosynthesis , Aromatase/genetics , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Carcinoma, Ductal, Breast/genetics , Carcinoma, Ductal, Breast/pathology , Cell Line, Tumor , Cluster Analysis , Estrogens/biosynthesis , Female , Humans , Middle Aged , Oligonucleotide Array Sequence Analysis , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Estrogen/biosynthesis , Receptors, Estrogen/genetics , Stromal Cells/enzymology , Stromal Cells/pathology , ERRalpha Estrogen-Related ReceptorABSTRACT
It is well known that sex steroids play important roles in the development of invasive ductal carcinoma (IDC) of the human breast. However, biological significance of sex steroids remains largely unclear in ductal carcinoma in situ (DCIS), regarded as a precursor lesion of IDC, which is partly due to the fact that the intratumoral concentration of sex steroids has not been examined in DCIS. Therefore, in this study, we first examined the intratumoral concentrations of estradiol and 5alpha-dihydrotestosterone (DHT) using liquid chromatography/electrospray tandem mass spectrometry in DCIS. Intratumoral concentrations of both estradiol and DHT were threefold higher in DCIS than non-neoplastic breast tissues and estrogen-producing enzymes (aromatase, steroid sulfatase, and 17beta-hydroxysteroid dehydrogenase type 1 (17betaHSD1)), and androgen-producing enzymes (17betaHSD5 and 5alpha-reductase type 1 (5alphaRed1)) were abundantly expressed in DCIS by real-time PCR and immunohistochemical analyses. The intratumoral concentration of DHT was significantly lower in IDC than DCIS, while the expression of aromatase mRNA in carcinoma cells and intratumoral stromal cells was significantly higher in IDC than those in DCIS. Immunohistochemistry for sex steroid-producing enzymes in DCIS demonstrated that 5alphaRed1 immunoreactivity was positively correlated with Ki-67 labeling index and histological grade and was also associated with an increased risk of recurrence in patients with DCIS examined. Results of our study suggest that intratumoral concentrations of estradiol and DHT are increased in DCIS, which is possibly due to intratumoral production of these steroids. Therefore, estradiol and DHT may play important roles in the development of DCIS of the human breast.
Subject(s)
17-Hydroxysteroid Dehydrogenases/metabolism , Aromatase/metabolism , Breast Neoplasms/metabolism , Carcinoma, Intraductal, Noninfiltrating/metabolism , Dihydrotestosterone/metabolism , Estradiol/metabolism , Steryl-Sulfatase/metabolism , 17-Hydroxysteroid Dehydrogenases/genetics , Adult , Aged , Aged, 80 and over , Aromatase/genetics , Breast Neoplasms/pathology , Carcinoma, Ductal, Breast/metabolism , Carcinoma, Ductal, Breast/pathology , Carcinoma, Intraductal, Noninfiltrating/pathology , Chromatography, Liquid , Female , Follow-Up Studies , Humans , Immunoenzyme Techniques , Middle Aged , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Spectrometry, Mass, Electrospray Ionization , Steryl-Sulfatase/genetics , Stromal Cells/metabolism , Stromal Cells/pathology , Survival RateABSTRACT
INTRODUCTION: Endocrine-dependent, estrogen receptor positive breast cancer cells proliferate in response to estrogens, synthesized by the cytochrome p450 aromatase enzyme. Letrozole is a potent nonsteroidal aromatase inhibitor that is registered for the treatment of postmenopausal women with advanced metastatic breast cancers and in the neoadjuvant, early, and extended adjuvant indications. Because crosstalk exists between estrogen receptor and insulin-like growth factor I receptor (IGF-IR), the effect of combining a selective IGF-IR inhibitor (NVP-AEW541) with letrozole was assessed in two independent in vitro models of estrogen-dependent breast cancer. METHODS: MCF7 and T47D cells stably expressing aromatase (MCF7/Aro and T47D/Aro) were used as in vitro models of aromatase-driven breast cancer. The role of the IGF-IR pathway in breast cancer cells stimulated only by 17beta-estradiol or androstenedione was assessed by proliferation assays. The combination of letrozole and NVP-AEW541 was assessed for synergy in inhibiting cell proliferation using Chou-Talalay derived equations. Finally, combination or single agent effects on proliferation and apoptosis were assessed using proliferation assays, flow cytometry, and immunoblotting. RESULTS: Both MCF7 and T47D cells, as well as MCF7/Aro and T47D/Aro, exhibited sensitivity to inhibition of 17beta-estradiol dependent proliferation by NVP-AEW541. Letrozole combined with NVP-AEW541 synergistically inhibited androstenedione-dependent proliferation in aromatase-expressing cells with combination index values of 0.6 or less. Synergistic combination effects correlated with higher levels of apoptosis as compared with cells treated with the single agent alone. Treatment with either agent also appeared to inhibit IGF-IR signalling via phosphoinositide 3-kinase. Notably, IGF-IR inhibition had limited effect on estrogen-dependent proliferation in the cell lines, but was clearly required for survival, suggesting that the combination of letrozole and IGF-IR inhibition sensitizes cells to apoptosis. CONCLUSION: Inhibition of the IGF-IR pathway and aromatase was synergistic in two independent estrogen-dependent in vitro models of breast cancer. Moreover, synergism of NVP-AEW541 and letrozole correlated with induction of apoptosis, but not cell cycle arrest, in the cell lines tested. Combination of IGF-IR inhibitors and letrozole may hold promise for the treatment of patients with estrogen-dependent breast cancers.
Subject(s)
Apoptosis , Aromatase Inhibitors/pharmacology , Breast Neoplasms/pathology , Estrogens/metabolism , Nitriles/pharmacology , Receptor, IGF Type 1/metabolism , Triazoles/pharmacology , Aromatase Inhibitors/chemistry , Breast Neoplasms/metabolism , Cell Line, Tumor , Cell Proliferation , Flow Cytometry , Humans , Letrozole , Neoplasm Metastasis , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Signal TransductionABSTRACT
Phosphorylation of the cytochrome P450 aromatase has been proposed as a switch to rapidly modulate enzymatic activity and estrogen biosynthesis. Herein, we demonstrate that aromatase serine-118 is a potential phosphorylation site in mammalian cells. The amino acid context surrounding S118 is highly conserved among diverse animal species and suggests that an AGC-like kinase may phosphorylate aromatase. Mutation of S118 to Ala blocked phosphorylation. Mutation of S118 to either Ala or Asp destabilized aromatase, indicating an important structural role for S118. The phosphomimetic S118D mutant showed decreased specific enzymatic activity, decreased Vmax, and increased Km, while the S118A phospho-inhibiting mutant showed opposite effects. Our findings suggest that phosphorylation of S118 may decrease aromatase activity, presenting a mechanism whereby kinase signaling may modulate estrogen production and hormone balance.