RESUMO
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.
Assuntos
Aneugênicos/toxicidade , Aberrações Cromossômicas , Dano ao DNA , Linfócitos/efeitos dos fármacos , Mebendazol/análogos & derivados , Ativação Metabólica , Aneugênicos/metabolismo , Animais , Antinematódeos/metabolismo , Antinematódeos/toxicidade , Células Cultivadas , Cromossomos Humanos/efeitos dos fármacos , DNA/efeitos dos fármacos , Humanos , Linfócitos/metabolismo , Masculino , Mebendazol/metabolismo , Mebendazol/toxicidade , Testes para Micronúcleos , Mutagênicos/metabolismo , Mutagênicos/toxicidade , RatosRESUMO
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.
Assuntos
Antineoplásicos Hormonais/farmacologia , Nitrilas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Sirolimo/análogos & derivados , Tamoxifeno/farmacologia , Triazóis/farmacologia , Animais , Antineoplásicos Hormonais/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Nucléolo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos , Everolimo , Feminino , Humanos , Letrozol , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Complexos Multiproteicos/antagonistas & inibidores , Complexos Multiproteicos/metabolismo , Nitrilas/administração & dosagem , Fosforilação , Inibidores de Proteínas Quinases/administração & dosagem , Transporte Proteico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor ErbB-3/metabolismo , Receptores de Estrogênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirolimo/administração & dosagem , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Tamoxifeno/administração & dosagem , Triazóis/administração & dosagem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We examined the efficacy of chemoendocrine therapy using capecitabine as a chemotherapeutic agent in premenopausal and postmenopausal models with estrogen receptor (ER)-positive human breast cancer xenografts. Tamoxifen and letrozole were used as endocrine therapeutic agents for premenopausal and postmenopausal models, respectively. The antitumor activity of capecitabine in combination was significantly superior to either monotherapy treatment in both premenopausal (p<0.01) and postmenopausal (p<0.05) models. No increase in toxicity in terms of body weight loss was observed during treatment in either of the xenograft models. In the premenopausal model, the level of thymidine phosphorylase (TP), a key enzyme generating 5-FU from capecitabine, was upregulated (p<0.05) in tumors by tamoxifen but not by letrozole treatment in the postmenopausal model. The combination of 5'-deoxy-5-fluorouridine (5'-DFUR; an intermediate of capecitabine) with 4-hydroxytamoxifen (4-OHT; an active form of tamoxifen) or letrozole was also evaluated in vitro by using estrogen-responsive element (ERE) reporter gene assays aimed to model premenopausal and postmenopausal breast cancer. Both combinations decreased the number of estrogen-responding cells in a concentration-dependent manner and further analysis by isobolograms revealed a synergistic effect of the combination of 5'-DFUR with 4-OHT, and at least an additive effect of the combination of 5'-DFUR with letrozole. These results suggest that chemoendocrine therapy using capecitabine may be a useful treatment modality for patients with hormone-receptor-positive breast cancer, regardless of the menopausal status and should be explored in clinical trials.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Desoxicitidina/análogos & derivados , Fluoruracila/análogos & derivados , Pós-Menopausa , Pré-Menopausa , Animais , Antineoplásicos Hormonais/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Aromatase/genética , Aromatase/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Capecitabina , Linhagem Celular Tumoral , Desoxicitidina/administração & dosagem , Desoxicitidina/uso terapêutico , Modelos Animais de Doenças , Feminino , Fluoruracila/administração & dosagem , Fluoruracila/uso terapêutico , Genes Reporter , Humanos , Letrozol , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Nitrilas/administração & dosagem , Tamoxifeno/administração & dosagem , Timidina Fosforilase/metabolismo , Triazóis/administração & dosagem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The gene expression grade index (GGI) is a 97-gene algorithm that measures proliferation and divides intermediate histological grade tumors into two distinct groups. We investigated the association between early changes in GGI and clinical response to neoadjuvant letrozole and compared this to Ki67 values. The paired gene expression data at the beginning and after 10-14 days of neoadjuvant letrozole treatment were available for 52 post-menopausal patients with estrogen receptor (ER)-positive breast cancer. Baseline values and changes in GGI, Ki67, and RNA expression modules representing oncogenic signaling pathways were compared to sonographic tumor volume changes after 3 months of treatment in the subsets of patients defined by high and low baseline GGI. The clinical response was observed in 80% genomic low-grade (24/30) and 59% genomic high-grade (13/22) tumors (P=0.10). Low residual proliferation after 10-14 days of neoadjuvant letrozole therapy, measured by either GGI or Ki67, was associated with sonographic response in genomic high-grade (GGI, P=0.003; Ki67, P=0.017) but not genomic low-grade (GGI, P=0.25; Ki67, P=1.0) tumors. The analysis of expression modules suggested that sonographic response to letrozole in genomic high-grade tumors was associated with an early reduction in IGF1 signaling (unadjusted P=0.018). The major conclusion of this study is that the early assessment of proliferation after short-term endocrine therapy may be useful to evaluate endocrine responsiveness, particularly in genomic high-grade ER-positive breast cancer.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Carcinoma/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Nitrilas/uso terapêutico , Triazóis/uso terapêutico , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Biomarcadores Farmacológicos/análise , Biomarcadores Farmacológicos/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/fisiologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinoma/genética , Carcinoma/metabolismo , Carcinoma/patologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Detecção Precoce de Câncer , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Letrozol , Análise em Microsséries , Pessoa de Meia-Idade , Neoplasia Residual , Nitrilas/farmacologia , Prognóstico , Receptores de Estrogênio/metabolismo , Fatores de Tempo , Triazóis/farmacologiaRESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Ácido Hialurônico/química , Nanopartículas , Nitrilas/farmacologia , Triazóis/farmacologia , Animais , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Western Blotting , Resistencia a Medicamentos Antineoplásicos , Citometria de Fluxo , Letrozol , Camundongos , Microscopia Eletrônica de Varredura , Nitrilas/química , Interferência de RNA , Triazóis/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
CONTEXT: Ovarian primordial follicle formation is critical for subsequent human female fertility. It is likely that steroid, and especially estrogen, signaling is required for this process, but details of the pathways involved are currently lacking. OBJECTIVE: The aim was to identify and characterize key members of the steroid-signaling pathway expressed in the second trimester human fetal ovary. DESIGN: We conducted an observational study of the female fetus, quantifying and localizing steroid-signaling pathway members. SETTING: The study was conducted at the Universities of Aberdeen, Edinburgh, and Glasgow. PATIENTS/PARTICIPANTS: Ovaries were collected from 43 morphologically normal human female fetuses from women undergoing elective termination of second trimester pregnancies. MAIN OUTCOME MEASURES: We measured mRNA transcript levels and immunolocalized key steroidogenic enzymes and steroid receptors, including those encoded by ESR2, AR, and CYP19A1. RESULTS: Levels of mRNA encoding the steroidogenic apparatus and steroid receptors increased across the second trimester. CYP19A1 transcript increased 4.7-fold during this period with intense immunostaining for CYP19A detected in pregranulosa cells around primordial follicles and somatic cells around oocyte nests. ESR2 was localized primarily to germ cells, but androgen receptor was exclusively expressed in somatic cells. CYP17A1 and HSD3B2 were also localized to oocytes, whereas CYP11A1 was detected in oocytes and some pregranulosa cells. CONCLUSIONS: The human fetal ovary expresses the machinery to produce and detect multiple steroid signaling pathways, including estrogenic signaling, with the oocyte acting as a key component. This study provides a step-change in our understanding of local dynamics of steroid hormone signaling during the key period of human primordial follicle formation.
Assuntos
Oócitos/metabolismo , Ovário/metabolismo , Transdução de Sinais/fisiologia , Análise de Variância , Aromatase/genética , Aromatase/metabolismo , Receptor beta de Estrogênio/genética , Receptor beta de Estrogênio/metabolismo , Feminino , Imunofluorescência , Humanos , Imuno-Histoquímica , Ovário/embriologia , Gravidez , Segundo Trimestre da Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Estrogens produced as a result of intratumoral aromatization has been recently shown to play important roles in proliferation of human non-small cell lung carcinomas (NSCLC), but the details have remained largely unknown. Therefore, in this study, we evaluated the possible roles of intratumoral aromatase in NSCLCs as follows: (a) evaluation of intratumoral localization of aromatase mRNA/protein in six lung adenocarcinoma cases using laser capture microdissection combined with quantitative reverse transcriptase-PCR and immunohistochemistry; (b) examination of the possible effects of isolated stromal cells from lung carcinoma tissues on aromatase mRNA transcript expression in lung carcinoma cell lines (A549 and LK87) through a coculture system; and (c) screening of cytokines derived from stromal LK001S and LK002S cells using cytokine antibody arrays and subsequent evaluation of effects of these cytokines on aromatase expression in A549 and LK87. Both aromatase mRNA and protein were mainly detected in intratumoral carcinoma cells but not in stromal cells. Aromatase expression of A549 and LK87 was upregulated in the presence of LK001S or LK002S cells. Several cytokines such as interleukin-6 (IL-6), oncostatin M, and tumor necrosis factor-alpha, all known as inducible factors of aromatase gene, were detected in conditioned media of LK001S and LK002S cells. Treatment of both oncostatin M and IL-6 induced aromatase gene expression in A549 an LK87, respectively. These results all indicated that intratumoral microenvironments, especially carcinoma-stromal cell interactions, play a pivotal role in the regulation of intratumoral estrogen synthesis through aromatase expression in human lung adenocarcinomas.
Assuntos
Aromatase/metabolismo , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Comunicação Celular/fisiologia , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologia , Idoso , Androgênios/biossíntese , Aromatase/biossíntese , Aromatase/genética , Linhagem Celular Tumoral , Técnicas de Cocultura , Estradiol/análogos & derivados , Estradiol/farmacologia , Estrogênios/biossíntese , Feminino , Fulvestranto , Humanos , Imuno-Histoquímica , Interleucina-6/biossíntese , Masculino , Pessoa de Meia-Idade , Oncostatina M/biossíntese , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Estromais/enzimologia , Células Estromais/patologia , Fator de Necrose Tumoral alfa/biossínteseRESUMO
PURPOSE: Targeting vascular endothelial growth factor (VEGF) and estrogen receptor signaling pathways concomitantly may enhance benefit in estrogen receptor-positive breast cancer. We had shown previously that the VEGF receptor tyrosine kinase inhibitor PTK787/ZK222584 (PTK/ZK) is a competitive aromatase inhibitor in vitro. Here we investigated (a) whether PTK/ZK shows both antiangiogenic and antiaromatase inhibitory properties in vivo, and (b) whether the combination of PTK/ZK and letrozole is superior to letrozole alone. EXPERIMENTAL DESIGN: Estrogen-dependent human breast cancer cells engineered to express aromatase (MCF7 AROM 1 and BT474 AROM) were used. Mice were treated with vehicle, PTK/ZK (25, 50, or 100 mg/kg), letrozole, or PTK/ZK in combination with letrozole. RESULTS: In MCF7 AROM 1 tumors, all treatments induced growth suppression and were associated with a reduction in cell turnover index, a composite measurement of both proliferation and apoptosis. PTK/ZK significantly reduced vessel density. Whereas letrozole caused tumor regression, PTK/ZK stabilized tumor volumes. The growth suppressive and antiangiogenic effects of PTK/ZK were confirmed in BT474 AROM xenografts. The addition of PTK/ZK did not enhance the growth-suppressive effects of letrozole. However, PTK/ZK decreased progesterone receptor (PgR) and TFF1 expression and uterine weight, indicating that PTK/ZK decreases 17beta-estradiol (E2) signaling in vivo. CONCLUSION: The VEGF receptor inhibitor PTK/ZK showed effects on E2-dependent gene expression consistent with aromatase inhibition as well as antiangiogenesis in xenograft models of breast cancer. The combination with letrozole was not superior to letrozole alone. Overall, these results provide further support for a potential therapeutic approach of dual inhibition of VEGF and E2 signaling using a single agent.
Assuntos
Inibidores da Angiogênese/farmacologia , Inibidores da Aromatase/farmacologia , Neoplasias Mamárias Experimentais/tratamento farmacológico , Ftalazinas/farmacologia , Piridinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Aromatase/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Imuno-Histoquímica , Letrozol , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Camundongos Nus , Neovascularização Patológica/tratamento farmacológico , Nitrilas/farmacologia , Receptores de Estrogênio/biossíntese , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Triazóis/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Adenocarcinoma/genética , Neoplasias do Córtex Suprarrenal/genética , Aromatase/genética , Hipogonadismo/genética , Adenocarcinoma/complicações , Adenocarcinoma/metabolismo , Neoplasias do Córtex Suprarrenal/complicações , Neoplasias do Córtex Suprarrenal/metabolismo , Adulto , Idade de Início , Aromatase/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Predisposição Genética para Doença , Humanos , Hipogonadismo/epidemiologia , Hipogonadismo/etiologia , Hipogonadismo/metabolismo , Masculino , Pessoa de Meia-IdadeRESUMO
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.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose/efeitos dos fármacos , Fator de Indução de Apoptose/metabolismo , Aromatase/genética , Aromatase/metabolismo , Inibidores da Aromatase/farmacologia , Benzenossulfonatos/farmacologia , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Caspase 7/metabolismo , Caspase 9/metabolismo , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Ciclina D1/metabolismo , Citocromos c/metabolismo , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Estradiol/metabolismo , Feminino , Humanos , Letrozol , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos , Niacinamida/análogos & derivados , Nitrilas/farmacologia , Compostos de Fenilureia , Fosfoproteínas/metabolismo , Fosforilação , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Piridinas/farmacologia , Proteína do Retinoblastoma/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Sorafenibe , Serina-Treonina Quinases TOR , Testosterona/metabolismo , Fatores de Tempo , Transfecção , Triazóis/farmacologiaRESUMO
Breast cancer tissue estrogen levels on an average exceed plasma as well as benign breast tissue levels. To evaluate the contribution of intra-tumor aromatization to individual tumor estrogen levels (estradiol, E2; estrone, E1; estrone sulfate, E1S), breast cancer tissue sections obtained during mastectomy in 28 postmenopausal breast cancer patients were stained for aromatase protein expression using the aromatase antibody 677. The findings were correlated to intra-tumor estrogen levels determined with a highly sensitive HPLC-RIA. Staining with 677 alone (irrespective of the hormone receptor status) revealed no difference in tumor E2 levels comparing 677+ versus 677- tumors, although a non-significant trend towards higher tumor E1 and E1S levels was observed in 677+ breast cancers. In contrast, tumor levels of E(2) were significantly higher in ER+ tumors compared to ER- tumors (P<0.001) and to benign breast tissue from the same breast (P<0.001). Analysing the additional effect of positive staining with the aromatase antibody 677 on tumor estrogen levels in the subgroup of ER+ tumors, revealed significantly higher tumor levels of E2 (mean level of 544.7 versus 197.1 fmol/g tissue) as well as a non-significant trend concerning tumor E1 (mean level of 296.9 versus 102.1 fmol/g tissue). The mean tumor tissue E1S level was observed somewhat lower in ER+677+ (103.5 fmol/g) versus ER+677- tumors (190.1 fmol/g). In the subgroup of ER+PgR+ tumors, tissue levels of E2 were also found to be significantly higher among 677+ compared to 677- tumors: 873.2 fmol/g (95% CI 395.9-1925.6) versus 217.9 fmol/g (95% CI 88.8-534.9) (P=0.015). In conclusion, our results indicate a moderate effect of aromatase enzyme expression evaluated by IHC using the antibody 677 on intra-tumor estrogen levels among ER+ breast cancers. A substantial interindividual variation in the ratios between the individual estrogen fractions suggests additional effects, like alterations in other enzymes to be involved in the intra-tumor estrogen homeostasis.
Assuntos
Aromatase/análise , Aromatase/metabolismo , Neoplasias da Mama/enzimologia , Carcinoma/enzimologia , Estrogênios/metabolismo , Receptores de Estrogênio/metabolismo , Anticorpos/imunologia , Aromatase/genética , Aromatase/imunologia , Neoplasias da Mama/patologia , Carcinoma/patologia , Estradiol/metabolismo , Estrogênios/análise , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Pós-Menopausa , Receptores de Estrogênio/análiseRESUMO
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.
Assuntos
Antineoplásicos Hormonais/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Mutação , Fosfatidilinositol 3-Quinases/genética , Receptores de Estrogênio/análise , Neoplasias da Mama/química , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Quimioterapia Adjuvante , Classe I de Fosfatidilinositol 3-Quinases , Ensaios Clínicos Fase II como Assunto , Intervalo Livre de Doença , Éxons , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Antígeno Ki-67/análise , Terapia Neoadjuvante , Estadiamento de Neoplasias , Fosforilação , Modelos de Riscos Proporcionais , Proteínas Proto-Oncogênicas c-akt/análise , Ensaios Clínicos Controlados Aleatórios como Assunto , Receptor ErbB-2/análise , Receptores de Progesterona/análise , Medição de Risco , Fatores de Tempo , Resultado do TratamentoRESUMO
Estradiol (E2), estrogen receptor (ER), ER-coregulators have been implicated in the development and progression of breast cancer. In situ E2 synthesis is implicated in tumor cell proliferation through autocrine or paracrine mechanisms, especially in post-menopausal women. Several recent studies demonstrated activity of aromatase P450 (Cyp19), a key enzyme that plays critical role in E2 synthesis in breast tumors. The mechanism by which tumors enhance aromatase expression is not completely understood. Recent studies from our laboratory suggested that PELP1 (Proline, Glutamic acid, Leucine rich Protein 1), a novel ER-coregulator, functions as a potential proto-oncogene and promotes tumor growth in nude mice models without exogenous E2 supplementation. In this study, we found that PELP1 deregulation contributes to increased expression of aromatase, local E2 synthesis and PELP1 cooperates with growth factor signaling components in the activation of aromatase. PELP1 deregulation uniquely up-regulated aromatase expression via activation of aromatase promoter I.3/II. Analysis of PELP1 driven mammary tumors in xenograft as well as in transgenic mouse models revealed increased aromatase expression. PELP1-mediated induction of aromatase requires functional Src and PI3K pathways. Chromatin immuno precipitation (ChIP) assays revealed that PELP1 is recruited to the Aro 1.3/II aromatase promoter. HER2 signaling enhances PELP1 recruitment to the aromatase promoter and PELP1 plays a critical role in HER2-mediated induction of aromatase expression. Mechanistic studies revealed that PELP1 interactions with orphan receptor ERRalpha, and histone demethylases play a role in the activation of aromatase promoter. Accordingly, ChIP analysis showed alterations in histone modifications at the aromatase promoter in the model cells that exhibit local E2 synthesis. Immunohistochemical analysis of breast tumor progression tissue arrays suggested that deregulation of aromatase expression occurs in advanced-stage and node-positive tumors, and that cooverexpression of PELP1 and aromatase occur in a sub set of tumors. Collectively, our results suggest that PELP1 regulation of aromatase represent a novel mechanism for in situ estrogen synthesis leading to tumor proliferation by autocrine loop and open a new avenue for ablating local aromatase activity in breast tumors.
Assuntos
Aromatase/genética , Neoplasias da Mama/enzimologia , Estrogênios/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Transativadores/metabolismo , Animais , Aromatase/metabolismo , Neoplasias da Mama/genética , Proteínas Correpressoras , Endometriose/enzimologia , Endometriose/genética , Epigênese Genética , Feminino , Humanos , Camundongos , Camundongos Transgênicos , Neoplasias Ovarianas/enzimologia , Neoplasias Ovarianas/genética , Proto-Oncogene Mas , Receptores de Estrogênio/metabolismo , Transativadores/genética , Fatores de Transcrição , Receptor ERRalfa Relacionado ao EstrogênioRESUMO
Estrogen receptor expression has been reported in non-small cell lung cancer. We examined the correlation between aromatase, a key enzyme in the synthesis of estrogen, and estrogen receptor expressions in 105 non-small cell lung cancer cases. All patients were older than 60 years, and all female patients were postmenopausal. Estrogen receptor alpha and progesterone receptor were detected in only 1 and 14 cases, respectively. Estrogen receptor beta and aromatase were positive in 75 and 89 cases respectively. Estrogen receptor beta expression in non-small cell lung cancer showed an inverse correlation with lymph node metastasis (P < .05). Only among females, both estrogen receptor beta and aromatase expressions were correlated with higher Ki-67 labeling index and younger age (P < .05). Among 89 aromatase-positive cases, 70 were positive for estrogen receptor beta, demonstrating a significant concordance (P < .05). Simultaneous immunohistochemical staining for aromatase and estrogen receptor beta showed a high rate of double positive association. Male non-small cell lung cancer cases with double positivity for aromatase and estrogen receptor beta demonstrated lower status in N factor by TNM classification (P < .05). In addition, among 89 aromatase-positive cases, a low-Allred total score of estrogen receptor beta showed a significant relationship with large tumor size and high T factor by TNM classification (P < .05). In conclusion, frequent coexpression of aromatase and estrogen receptor beta in non-small cell lung cancer might suggest some functional correlation between aromatase and estrogen receptor beta, whereas estrogen receptor beta negativity might be correlated with malignant progression of non-small cell lung cancer.
Assuntos
Aromatase/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Receptor beta de Estrogênio/metabolismo , Neoplasias Pulmonares/metabolismo , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/patologia , Distribuição de Qui-Quadrado , Feminino , Humanos , Imuno-Histoquímica , Antígeno Ki-67/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Receptores de Progesterona/metabolismo , Fatores SexuaisRESUMO
Immunohistochemistry is one of the most suitable methods for the detection of intratumoral aromatase in order to identify patients who may respond to aromatase inhibitor therapy in hormone-dependent breast cancer. Previous studies showed statistically significant correlation between results of immnuohistochemistry and biochemical analysis in carcinoma components stained by aromatase monoclonal antibody 677. In this study, determination of the antigenic peptides recognized by aromatase antibodies through epitope mapping, combined with the new knowledge on aromatase-reductase interaction, provide insights for understanding various immunostaining patterns using different aromatase antibodies. Our studies on aromatase-reductase interaction also provided critical information on how aromatase and reductase interact with each other on the endoplasmic reticulum membrane, and identified key residues, including K108 of aromatase, that are involved in the interaction with reductase. Through epitope mapping and taking into consideration the interference with aromatase immunohistochemical staining by NADPH-cytochrome P450 reductase, we demonstrated that monoclonal antibody 677 is a suitable antibody for an assessment of intratumoral aromatase activity in breast cancer patients for making clinical management decisions. These results also provide valuable information to identify new aromatase antibodies for immunohistochemical diagnosis of hormone-dependent breast cancer in future.
Assuntos
Anticorpos Monoclonais/química , Aromatase/química , Carcinoma/enzimologia , Neoplasias/enzimologia , Animais , Células CHO , Membrana Celular/enzimologia , Cricetinae , Cricetulus , Retículo Endoplasmático/metabolismo , Ensaio de Imunoadsorção Enzimática , Epitopos/química , Humanos , Imuno-Histoquímica/métodos , Cinética , Conformação Molecular , Peptídeos/química , Proteínas Recombinantes/química , Eletricidade Estática , Tripsina/químicaRESUMO
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.
Assuntos
Decídua/metabolismo , Estrogênios/biossíntese , Neovascularização Fisiológica , Útero/metabolismo , Animais , Aromatase/genética , Aromatase/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Decídua/irrigação sanguínea , Decídua/citologia , Implantação do Embrião , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Imuno-Histoquímica , Masculino , Camundongos , Ovariectomia , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Gravidez , Progesterona/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Estromais/metabolismo , Útero/irrigação sanguínea , Útero/citologiaRESUMO
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.
Assuntos
Aromatase/biossíntese , Carcinoma Endometrioide/metabolismo , Ciclo-Oxigenase 2/biossíntese , Neoplasias do Endométrio/metabolismo , Receptor ErbB-2/biossíntese , Proteína Supressora de Tumor p53/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Endometrioide/enzimologia , Carcinoma Endometrioide/patologia , Carcinoma Endometrioide/terapia , Intervalo Livre de Doença , Neoplasias do Endométrio/enzimologia , Neoplasias do Endométrio/patologia , Neoplasias do Endométrio/terapia , Feminino , Humanos , Pessoa de Meia-IdadeRESUMO
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.
Assuntos
Aromatase/biossíntese , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Nitrilas/uso terapêutico , Receptores de Estrogênio/biossíntese , Tamoxifeno/uso terapêutico , Triazóis/uso terapêutico , Antineoplásicos Hormonais/uso terapêutico , Inibidores da Aromatase/uso terapêutico , Neoplasias da Mama/enzimologia , Carcinoma in Situ/tratamento farmacológico , Carcinoma in Situ/enzimologia , Carcinoma in Situ/metabolismo , Ciclo-Oxigenase 2/biossíntese , Feminino , Humanos , Imuno-Histoquímica , Letrozol , Receptores de Progesterona/biossíntese , Estudos Retrospectivos , Análise Serial de TecidosRESUMO
Endocrine therapy is well established for the treatment of breast cancer, and antiangiogenic agents are showing considerable promise. Targeting the vascular endothelial growth factor (VEGF) and estrogen receptor (ER) signaling pathways concomitantly may provide enhanced therapeutic benefit in ER-positive breast cancer. Therefore, the effects of the VEGF receptor (VEGFR) tyrosine kinase inhibitor PTK787/ZK222584 (PTK/ZK) were investigated using human breast cancer cell lines engineered to express aromatase. As expected in this system, estrogen (E2) or androstenedione induced a proliferative response and increased ER-mediated transcription in ER-positive cell lines expressing aromatase. However, surprisingly, in the presence of androstenedione, PTK/ZK suppressed both the androstenedione-stimulated proliferation and ER-mediated transcription. PTK/ZK alone and in the presence of E2 had no observable effect on proliferation or ER-mediated transcription. These effects result from PTK/ZK having previously unrecognized antiaromatase activity and PTK/ZK being a competitive aromatase inhibitor. Computer-assisted molecular modeling showed that PTK/ZK could potentially bind directly to aromatase. The demonstration that PTK/ZK inhibits aromatase and VEGFR indicates that agents cross-inhibiting two important classes of targets in breast cancer could be developed.
Assuntos
Inibidores da Aromatase/farmacologia , Ftalazinas/farmacologia , Piridinas/farmacologia , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Inibidores da Angiogênese/farmacologia , Aromatase/metabolismo , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Estradiol/farmacologia , Humanos , Modelos Moleculares , Inibidores de Proteínas Quinases/farmacologia , Receptores de Estrogênio/fisiologia , Especificidade por Substrato , Transcrição Gênica/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
PURPOSE: Endocrine agents, such as letrozole, are established in the treatment of hormone-dependent breast cancer. However, response rates are only 50% to 70% in the neoadjuvant setting and lower in advanced disease. Thus there is a need to identify novel markers predicting for response and to understand molecular mechanisms of resistance. PATIENTS AND METHODS: Sequential tumor biopsies were taken before and after 10 to 14 days of neoadjuvant treatment with letrozole in patients with estrogen receptor-rich breast cancer. Expression profiles on high-density microarray chips were then related to clinical responses as assessed from tumor volume measurements after 3 months of treatment. RESULTS: Of 52 patients, 37 (71%) were classified as having a clinical response to letrozole and 15 being clinically resistant. Bioinformatic analysis identified 205 covariables (69 baseline expression, 45 day 14 expression, and 91 change in expression with treatment) which differentiated between clinical responders and nonresponders. Hierarchical clustering using the variables separated responders and nonresponders into two distinct groups. Ontological assessment indicated that discriminating genes were enriched toward cellular biosynthetic processes. In particular, functional gene assessment showed ribosomal protein probes to have higher baseline expression in tumors responsive to letrozole and increased expression with treatment in nonresponding cases. CONCLUSION: To our knowledge, this is the first study to describe genetic covariables and molecular processes discriminating between tumors clinically responsive and resistant to an aromatase inhibitor. The understanding of such molecular phenotypes will be important in optimizing the clinical use of aromatase inhibitors, both in terms of identifying responsive breast cancers and developing new agents to target resistance pathways.