RESUMO
Accumulation of amyloid ß (Aß) induces neuronal, synaptic, and cognitive deficits in patients and animal models of Alzheimer's disease (AD). The underlying mechanisms, however, remain to be fully elucidated. In the present study, we found that Aß interacted with ErbB4, a member of the receptor tyrosine kinase family and mainly expressed in GABAergic interneurons. Deleting ErbB4 in parvalbumin-expressing neurons (PV neurons) significantly attenuated oligomeric Aß-induced suppression of long term potentiation (LTP). Furthermore, specific ablation of ErbB4 in PV neurons via Cre/loxP system greatly improved spatial memory and synaptic plasticity in the hippocampus of hAPP-J20 mice. The deposition of Aß detected by 3D6 and Thioflavin S staining and the proteolytic processing of hAPP analyzed by western blotting were not affected in the hippocampus of hAPP-J20 mice by deleting ErbB4 in PV neurons. Our data suggested that ErbB4 in PV neurons mediated Aß-induced synaptic and cognitive dysfunctions without affecting Aß levels.
Assuntos
Doença de Alzheimer/metabolismo , Cognição/fisiologia , Potenciação de Longa Duração/fisiologia , Neurônios/metabolismo , Parvalbuminas/metabolismo , Receptor ErbB-4/metabolismo , Doença de Alzheimer/patologia , Doença de Alzheimer/psicologia , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Modelos Animais de Doenças , Células HEK293 , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Aprendizagem em Labirinto/fisiologia , Camundongos Transgênicos , Neurônios/patologia , Fragmentos de Peptídeos/metabolismo , Placa Amiloide/metabolismo , Placa Amiloide/patologia , Receptor ErbB-4/genética , Memória Espacial/fisiologia , Técnicas de Cultura de TecidosRESUMO
Breast cancer is a complex disease with at least five different molecular subtypes identified. The breast tumor molecular subtypes guide stratification of patients for specific targeted therapy regimens and each subtype is associated with significantly different patient outcomes. For example, patients with the HER2 positive molecular subtype benefit from the HER2 targeted therapy trastuzumab. Unfortunately, women with the HER2 positive molecular subtype have the worst overall prognosis and nearly 70% of women with HER2 positive breast cancer exhibit de novo or acquired resistance to trastuzumab. Identification of tumor markers predicting trastuzumab response can be used to further stratify patients for life-saving personalized therapeutic options. The aim of this study was to identify clinically useful tumor markers predicting de novo tumor cell resistance to trastuzumab treatment. To identify oncogenic signaling pathways activated in response to trastuzumab treatment, we performed a Human Phospho-Kinase Proteome Profiler Array analysis comparing trastuzumab sensitive MCF-7/HER2.2 and trastuzumab resistant MCF-7/HER2Δ16H cells following acute treatment with 20 µg/ml of trastuzumab for 2 h. We found that of the 43 phosphorylation activated human kinases represented on the array, S6K1 was the only kinase altered greater than 1.5-fold in response to trastuzumab treatment of the trastuzumab resistant MCF-7/HER2Δ16H cells. Trastuzumab activation of S6K1 was confirmed in the two trastuzumab resistant SUM190 and SUM225 cell lines. Significantly, trastuzumab failed to stimulate S6K1 activation in the trastuzumab sensitive MCF-7/HER2.2, BT474, and SKBR3 cell lines suggesting that trastuzumab activation of S6K1 is a tumor cell marker for trastuzumab resistance. Consistent with a role for mTORC1/S6K1 signaling promoting trastuzumab resistance, all cell lines were sensitive to S6K1 inactivation with significant growth inhibition following treatment with the mTORC1 inhibitor rapamycin. In conclusion, characterizing rapid trastuzumab induced molecular alterations resulted in the identification of activated S6K1 as an early breast tumor cell marker for trastuzumab resistance. Our results further suggest that trastuzumab resistant breast tumor cells are addicted to mTORC1/S6K1 oncogenic signaling and targeting mTORC1 with rapamycin reverses trastuzumab resistance.
Assuntos
Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Trastuzumab/uso terapêutico , Neoplasias da Mama/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Células MCF-7 , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos/antagonistas & inibidores , Complexos Multiproteicos/metabolismo , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismoRESUMO
KEY POINTS: Synaptic excitation and inhibition must be properly balanced in individual neurons and neuronal networks to allow proper brain function. Disrupting this balance may lead to autism spectral disorders and epilepsy. We show the basic helix-loop-helix transcription factor NeuroD2 promotes inhibitory synaptic drive but also decreases cell-intrinsic neuronal excitability of cortical pyramidal neurons both in vitro and in vivo. We identify two genes potentially downstream of NeuroD2-mediated transcription that regulate these parameters: gastrin-releasing peptide and the small conductance, calcium-activated potassium channel, SK2. Our results reveal an important function for NeuroD2 in balancing synaptic neurotransmission and intrinsic excitability. Our results offer insight into how synaptic innervation and intrinsic excitability are coordinated during cortical development. ABSTRACT: Synaptic excitation and inhibition must be properly balanced in individual neurons and neuronal networks for proper brain function. Disruption of this balance during development may lead to autism spectral disorders and epilepsy. Synaptic excitation is counterbalanced by synaptic inhibition but also by attenuation of cell-intrinsic neuronal excitability. To maintain proper excitation levels during development, neurons must sense activity over time and regulate the expression of genes that control these parameters. While this is a critical process, little is known about the transcription factors involved in coordinating gene expression to control excitatory/inhibitory synaptic balance. We show here that the basic helix-loop-helix transcription factor NeuroD2 promotes inhibitory synaptic drive but also decreases cell-intrinsic neuronal excitability of cortical pyramidal neurons both in vitro and in vivo as shown by ex vivo analysis of a NeuroD2 knockout mouse. Using microarray analysis and comparing wild-type and NeuroD2 knockout cortical networks, we identified two potential gene targets of NeuroD2 that contribute to these processes: gastrin-releasing peptide (GRP) and the small conductance, calcium-activated potassium channel, SK2. We found that the GRP receptor antagonist RC-3059 and the SK2 specific blocker apamin partially reversed the effects of increased NeuroD2 expression on inhibitory synaptic drive and action potential repolarization, respectively. Our results reveal an important function for NeuroD2 in balancing synaptic neurotransmission and intrinsic excitability and offer insight into how these processes are coordinated during cortical development.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Neuropeptídeos/fisiologia , Células Piramidais/fisiologia , Córtex Somatossensorial/fisiologia , Sinapses/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Células Cultivadas , Peptídeo Liberador de Gastrina/genética , Potenciais Pós-Sinápticos Inibidores , Camundongos Knockout , Neuropeptídeos/genética , Ratos , Canais de Potássio Ativados por Cálcio de Condutância Baixa/genéticaRESUMO
Conditional knock-out of Hif1a in the mouse mammary gland impairs lobuloalveolar differentiation during lactation. Here, we demonstrate that expression of ErbB4 was reduced in the lobulalveoli of mice with mammary gland-specific deletion of Hif1a. Erbb4 was not, however, a direct target gene for transcriptional regulation by HIF-1α in vitro. HIF-1α overexpression or HIF accumulating prolyl hydroxylase inhibitors reduced ErbB4 endocytosis, promoted transcriptional co-regulatory activity of ErbB4, and stimulated ErbB4-induced differentiation of mammary carcinoma cells. Consistently, RNA interference-mediated down-regulation of HIF-1α resulted in reduced ErbB4 protein amount and reduced mammary carcinoma cell differentiation. These findings indicate that HIF-1α is a physiologically relevant regulator of ErbB4 and that ErbB4 is involved in HIF-regulated differentiation of the mammary gland.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Glândulas Mamárias Animais/crescimento & desenvolvimento , Glândulas Mamárias Animais/metabolismo , Receptor ErbB-4/metabolismo , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Endocitose , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/deficiência , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Lactação/genética , Lactação/metabolismo , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Humanas/citologia , Glândulas Mamárias Humanas/crescimento & desenvolvimento , Glândulas Mamárias Humanas/metabolismo , Camundongos , Camundongos Knockout , Fragmentos de Peptídeos/metabolismo , Gravidez , Transdução de SinaisRESUMO
The EGFR-family member HER4 undergoes regulated intramembrane proteolysis (RIP) to generate an intracellular domain (4ICD) that functions as a transcriptional coactivator. Accordingly, 4ICD coactivates the estrogen receptor (ER) and associates with ER at target gene promoters in breast tumor cells. However, the extent of 4ICD coactivation of ER and the functional significance of the 4ICD/ER transcriptional complex is unclear. To identify 4ICD coactivated genes we performed a microarray gene expression analysis of ß-estradiol treated cells comparing control MCF-7 breast cancer cells to MCF-7 cells where HER4 expression was stably suppressed using a shRNA. In the MCF-7 cell line, ß-estradiol significantly stimulated or repressed by 2-fold or more 726 or 53 genes, respectively. Significantly, HER4/4ICD was an obligate coactivator for 277 or 38% of the ß-estradiol stimulated genes. Ingenuity Pathway Analysis of ß-estradiol regulated genes identified significant associations with multiple cellular functions regulating cellular growth and proliferation, cell cycle progression, cancer metastasis, decreased hypoplasia, tumor cell migration, apoptotic resistance of tumor cells, and increased transcription. Genes coactivated by 4ICD displayed functional specificity by only significantly contributing to cellular growth and proliferation, cell cycle progression, and decreased hypoplasia. In direct concordance with these in situ results we show that HER4 knockdown in MCF-7 cells results in a loss of estrogen stimulated tumor cell proliferation and cell cycle progression, whereas, estrogen stimulated tumor cell migration was unaffected by loss of HER4 expression. In summary, we demonstrate for the first time that a cell surface receptor functions as an obligate ER coactivator with functional specificity associated with breast tumor cell proliferation and cell cycle progression. Nearly 90% of ER positive tumors coexpress HER4, therefore we predict that the majority of breast cancer patients would benefit from a strategy to therapeutic disengage ER/4ICD coregulated tumor cell proliferation.
Assuntos
Neoplasias da Mama/metabolismo , Receptores ErbB/metabolismo , Estrogênios/metabolismo , Regulação Neoplásica da Expressão Gênica , Proliferação de Células , Humanos , Células MCF-7 , Receptor ErbB-4RESUMO
HER2 overexpression is associated with aggressive breast cancer with high recurrence rate and poor patient prognosis. Treatment of HER2 overexpressing patients with the HER2 targeting therapy trastuzumab results in acquired resistance within a year. The HER2/EGFR dual kinase inhibitor lapatinib was shown to inhibit some trastuzumab resistant breast cancer cell lines and is currently in clinical trials. Our group has found two new quinone compounds that show excellent inhibition of breast tumor cells expressing HER2 or the trastuzumab resistant HER2 oncogenic isoform, HER2Δ16. Compound 4 ((1R,2S,3S)-1,2,3,5,8-pentahydroxy-1,2,3,4-tetrahydroanthracene-9,10-dione) and compound 5 (5,8-dihydroxy-2,3-bis(hydroxymethyl)naphthalene-1,4-dione) showed sub-micromolar inhibition potency against these cell lines. These compounds also inhibit auto-phosphorylation of the Y1248 and Y1068 residues of HER2 and EGFR, respectively.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Quinonas/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Neoplasias da Mama/metabolismo , Relação Dose-Resposta a Droga , Feminino , Ensaios de Triagem em Larga Escala , Humanos , Modelos Moleculares , Estrutura Molecular , Quinonas/química , Receptor ErbB-2/metabolismo , Relação Estrutura-Atividade , TrastuzumabRESUMO
The receptor-tyrosine kinase ErbB4 was identified as a direct regulator of hypoxia-inducible factor-1α (HIF-1α) signaling. Cleaved intracellular domain of ErbB4 directly interacted with HIF-1α in the nucleus, and stabilized HIF-1α protein in both normoxic and hypoxic conditions by blocking its proteasomal degradation. The mechanism of HIF stabilization was independent of VHL and proline hydroxylation but dependent on RACK1. ErbB4 activity was necessary for efficient HRE-driven promoter activity, transcription of known HIF-1α target genes, and survival of mammary carcinoma cells in vitro. In addition, mammary epithelial specific targeting of Erbb4 in the mouse significantly reduced the amount of HIF-1α protein in vivo. ERBB4 expression also correlated with the expression of HIF-regulated genes in a series of 4552 human normal and cancer tissue samples. These data demonstrate that soluble ErbB4 intracellular domain promotes HIF-1α stability and signaling via a novel mechanism.
Assuntos
Núcleo Celular/metabolismo , Receptores ErbB/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteólise , Transdução de Sinais/fisiologia , Animais , Linhagem Celular Tumoral , Núcleo Celular/genética , Receptores ErbB/genética , Feminino , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Humanos , Hidroxilação , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Masculino , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Estabilidade Proteica , Estrutura Terciária de Proteína , Receptor ErbB-4 , Receptores de Quinase C Ativada , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismoRESUMO
MicroRNAs (miRs) function as tumor suppressors or oncogenes in multiple tumor types. Although miR expression is tightly regulated, the molecular basis of miR regulation is poorly understood. Here, we investigated the influence of the histone demethylase Jumonji/ARID1 B (JARID1B) on miR regulation in breast tumor cells. In MCF-7 cells with stable RNAi-mediated suppression of JARID1B expression we identified altered regulation of multiple miRs including let-7e, a member of the let-7 family of tumor suppressor miRs. Chromatin immunoprecipitation analysis demonstrated JARID1B binding to the let-7e promoter region as well as removal of the of H3K4me3 histone mark associated with active gene expression. These results suggest that JARID1B epigenetically represses let-7e expression. JARID1B stimulates tumor cell proliferation by promoting the G(1) to S transition. As predicted, suppression of JARID1B resulted in an accumulation of MCF-7 cells in G(1). We confirmed that cyclin D1, which also promotes G(1) progression, is a direct target of let-7e, and we show that cyclin D1 expression is suppressed in JARID1B knockdown cells. Cyclin D1 expression and cell cycle progression were restored following inhibition of let-7e, suggesting that JARID1B repression of let-7e contributes to cyclin D1 expression and JARID1B-mediated cell cycle progression. Our results indicate that the JARID1B demethylase contributes to tumor cell proliferation through the epigenetic repression of a tumor suppressor miR.
Assuntos
Neoplasias da Mama/patologia , Ciclo Celular/genética , Epigênese Genética/genética , Inativação Gênica , Histona Desmetilases com o Domínio Jumonji/metabolismo , MicroRNAs/genética , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Linhagem Celular Tumoral , Ciclina D1/genética , Feminino , Histonas/química , Histonas/metabolismo , Humanos , Lisina , MetilaçãoRESUMO
Sufficient pulmonary surfactant production is required for the fetal-neonatal transition, especially in preterm infants. Neuregulin (NRG) and its transmembrane receptor ErbB4 positively regulate the onset of fetal surfactant synthesis. Details of this signaling process remain to be elucidated. ErbB4 is known to regulate gene expression in the mammary gland, where the receptor associates with the signal transducer and activator of transcription Stat5a to transactivate the ß-casein gene promoter. We hypothesized that in the fetal lung, ErbB4 functions as a transcriptional regulator for surfactant protein B (Sftpb), the most critical surfactant protein gene. Re-expressing full-length ErbB4 in primary fetal ErbB4-depleted Type II epithelial cells led to an increased expression of Sftpb mRNA. This stimulatory effect required the nuclear translocation of ErbB4 and association with Stat5a, with the resultant binding to and activation of the Sftpb promoter. We conclude that ErbB4 directly regulates important aspects of fetal lung maturation that help prepare for the fetal-neonatal transition.
Assuntos
Células Epiteliais Alveolares/metabolismo , Receptores ErbB/metabolismo , Pulmão/metabolismo , Neuregulina-1/metabolismo , Proteína B Associada a Surfactante Pulmonar/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Sítios de Ligação , Células Cultivadas , Receptores ErbB/deficiência , Receptores ErbB/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Idade Gestacional , Humanos , Pulmão/embriologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Neuregulina-1/genética , Regiões Promotoras Genéticas , Proteína B Associada a Surfactante Pulmonar/genética , RNA Mensageiro/metabolismo , Receptor ErbB-4 , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismo , Fatores de Tempo , Transcrição Gênica , TransfecçãoRESUMO
Tamoxifen is the most commonly prescribed therapy for patients with estrogen receptor (ER)α-positive breast tumors. Tumor resistance to tamoxifen remains a serious clinical problem especially in patients with tumors that also overexpress human epidermal growth factor receptor 2 (HER2). Current preclinical models of HER2 overexpression fail to recapitulate the clinical spectrum of endocrine resistance associated with HER2/ER-positive tumors. Here, we show that ectopic expression of a clinically important oncogenic isoform of HER2, HER2Δ16, which is expressed in >30% of ER-positive breast tumors, promotes tamoxifen resistance and estrogen independence of MCF-7 xenografts. MCF-7/HER2Δ16 cells evade tamoxifen through upregulation of BCL-2, whereas mediated suppression of BCL-2 expression or treatment of MCF-7/HER2Δ16 cells with the BCL-2 family pharmacological inhibitor ABT-737 restores tamoxifen sensitivity. Tamoxifen-resistant MCF-7/HER2Δ16 cells upregulate BCL-2 protein levels in response to suppressed ERα signaling mediated by estrogen withdrawal, tamoxifen treatment or fulvestrant treatment. In addition, HER2Δ16 expression results in suppression of BCL-2-targeting microRNAs miR-15a and miR-16. Reintroduction of miR-15a/16 reduced tamoxifen-induced BCL-2 expression and sensitized MCF-7/HER2Δ16 to tamoxifen. Conversely, inhibition of miR-15a/16 in tamoxifen-sensitive cells activated BCL-2 expression and promoted tamoxifen resistance. Our results suggest that HER2Δ16 expression promotes endocrine-resistant HER2/ERα-positive breast tumors and in contrast to wild-type HER2, preclinical models of HER2Δ16 overexpression recapitulate multiple phenotypes of endocrine-resistant human breast tumors. The mechanism of HER2Δ16 therapeutic evasion, involving tamoxifen-induced upregulation of BCL-2 and suppression of miR-15a/16, provides a template for unique therapeutic interventions combining tamoxifen with modulation of microRNAs and/or ABT-737-mediated BCL-2 inhibition and apoptosis.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Antagonistas de Estrogênios/uso terapêutico , Genes bcl-2 , MicroRNAs/antagonistas & inibidores , Receptor ErbB-2/fisiologia , Tamoxifeno/uso terapêutico , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Nus , MicroRNAs/fisiologiaRESUMO
INTRODUCTION: The pineal gland hormone, melatonin, has been shown by numerous studies to inhibit the proliferation of estrogen receptor α (ERα)-positive breast cancer cell lines. Here, we investigated the role of melatonin in the regulation of breast cancer cell invasion. METHODS: Three invasive MCF-7 breast cancer cell clones - MCF-7/6, MCF-7/Her2.1, and MCF-7/CXCR4 cells - were employed in these studies. All three cell lines exhibited elevated phosphorylation of the ERK1/2 and p38 mitogen-activated protein kinase (MAPK) as determined by Western blot analysis. The effect of melatonin on the invasive potential of these human breast cancer cells was examined by matrigel invasion chamber assays. The expression and proteinase activity of two matrix metalloproteinases (MMPs), MMP-2 and MMP-9, were analyzed by Western blot analysis and gelatin zymography, respectively. RESULTS: Melatonin (10-9 M) significantly suppressed the invasive potential of MCF-7/6 and MCF-7/Her2.1 cells as measured by matrigel invasion chamber assays, and significantly repressed the proteinase activity of MMP-2 and MMP-9. In MCF-7/CXCR4 cells, melatonin significantly inhibited stromal-derived factor-1 (SDF-1/CXCL12) induced cell invasion and activity of MMP-9. Elevated expression of the MT1 melatonin receptor further enhanced, while luzindole, an MT1/MT2 antagonist, abrogated melatonin's anti-invasive effect, suggesting that melatonin's effect on invasion is mediated, principally, through the MT1 receptor. Furthermore, melatonin repressed the phosphorylation of p38 MAPK in MCF-7/Her2.1 cells and blocked stromal-derived factor-1 (SDF-1) induced p38 phosphorylation in MCF-7/CXCR4 cells. SB230580, a p38 inhibitor, was able to mimic, while transfection of the cells with a constitutively-active MKK6b construct blocked melatonin's effect on cell invasion, suggesting that the anti-invasive action of melatonin is mediated through the p38 pathway. CONCLUSIONS: Melatonin exerts an inhibitory effect on breast cancer cell invasion through down-regulation of the p38 pathway, and inhibition of MMP-2 and MMP-9 expression and activity.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Melatonina/metabolismo , Melatonina/farmacologia , Receptor MT1 de Melatonina/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Western Blotting , Linhagem Celular Tumoral , Quimiocina CXCL12/antagonistas & inibidores , Feminino , Expressão Gênica , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz/metabolismo , Invasividade Neoplásica/prevenção & controle , Fosforilação , Receptor MT1 de Melatonina/antagonistas & inibidores , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Triptaminas/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
The HER4 intracellular domain (4ICD) is a potent estrogen receptor (ERalpha) coactivator with activities in breast cancer and the developing mammary gland that appear to overlap with progesterone receptor (PgR). In fact, 4ICD has recently emerged as an important regulator and predictor of tamoxifen response, a role previously thought to be fulfilled by PgR. Here we investigated the possibility that the 4ICD coactivator regulates PgR expression thereby providing a mechanistic explanation for their partially overlapping activities in breast cancer. We show that 4ICD is both sufficient and necessary to potentiate estrogen stimulation of gene expression. Suppression of HER4/4ICD expression in the MCF-7 breast tumor cell line completely eliminated estrogen stimulated expression of PgR. In addition, the HER4/4ICD negative MCF-7 variant, TamR, failed to express PgR in response to estrogen. Reintroduction of wild-type HER4 but not the gamma-secretase processing mutant HER4V673I into the TamR cell line restored PgR expression indicating that 4ICD is an essential PgR coactivator in breast tumor cells. These results were substantiated in vivo using two different physiologically relevant experimental systems. In the mouse mammary gland estrogen regulates expression of PgR-A whereas expression of PgR-B is estrogen independent. Consistent with a role for 4ICD in estrogen regulated PgR expression in vivo, PgR-A, but not PgR-B, expression was abolished in HER4-null mouse mammary glands during pregnancy. Coexpression of PgR and 4ICD is also commonly observed in ERalpha positive breast carcinomas. Using quantitative AQUA IHC technology we found that 4ICD potentiated PgR expression in primary breast tumors and the highest levels of PgR expression required coexpression of ERalpha and the 4ICD coactivator. In summary, our results provide compelling evidence that 4ICD is a physiologically important ERalpha coactivator and 4ICD cooperates with ERalpha to potentiate PgR expression in the normal and malignant breast. We propose that direct coupling of these signaling pathways may have important implications for mammary development, breast carcinogenesis, and patient response to endocrine therapy.
Assuntos
Neoplasias da Mama/genética , Receptores ErbB/genética , Receptor alfa de Estrogênio/genética , Regulação da Expressão Gênica/genética , Glândulas Mamárias Humanas/metabolismo , Receptores de Progesterona/genética , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Receptores ErbB/metabolismo , Receptor alfa de Estrogênio/metabolismo , Feminino , Expressão Gênica , Humanos , Glândulas Mamárias Animais/metabolismo , Camundongos , Camundongos Transgênicos , Gravidez , Receptor ErbB-4 , Receptores de Progesterona/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genéticaRESUMO
BACKGROUND: Tumor resistance to the selective estrogen receptor modulator tamoxifen remains a serious clinical problem especially in patients with tumors that also overexpress HER2. We have recently demonstrated that the clinically important isoform of HER2, HERΔ16, promotes therapeutically refractory breast cancer including resistance to endocrine therapy. Likewise additional breast tumor cell models of tamoxifen resistance have been developed that do not involve HER2 overexpression. However, a unifying molecular mechanism of tamoxifen resistance has remained elusive. RESULTS: Here we analyzed multiple cell models of tamoxifen resistance derived from MCF-7 cells to examine the influence of microRNAs (miRNAs) on tamoxifen resistance. We compared miRNA expression profiles of tamoxifen sensitive MCF-7 cells and tamoxifen resistant MCF-7/HER2Δ16 cells. We observed significant and dramatic downregulation of miR-342 in the MCF-7/HER2Δ16 cell line as well as the HER2 negative but tamoxifen resistant MCF-7 variants TAMR1 and LCC2. Restoring miR-342 expression in the MCF-7/HER2Δ16 and TAMR1 cell lines sensitized these cells to tamoxifen-induced apoptosis with a dramatic reduction in cell growth. Expression of miR-342 was also reduced in a panel of tamoxifen refractory human breast tumors, underscoring the potential clinical importance of miR-342 downregulation. Towards the goal of identifying direct and indirect targets of miR-342 we restored miR-342 expression in MCF-7/HER2Δ16 cells and analyzed changes in global gene expression by microarray. The impact of miR-342 on gene expression in MCF-7/HER2Δ16 cells was not limited to miR-342 in silica predicted targets. Ingenuity Pathways Analysis of the dataset revealed a significant influence of miR-342 on multiple tumor cell cycle regulators. CONCLUSIONS: Our findings suggest that miR-342 regulates tamoxifen response in breast tumor cell lines and our clinical data indicates a trend towards reduced miR-342 expression and tamoxifen resistance. In addition, our results suggest that miR-342 regulates expression of genes involved in tamoxifen mediated tumor cell apoptosis and cell cycle progression. Restoring miR-342 expression may represent a novel therapeutic approach to sensitizing and suppressing the growth of tamoxifen refractory breast tumors.
Assuntos
Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/genética , Resistencia a Medicamentos Antineoplásicos/genética , MicroRNAs/metabolismo , Tamoxifeno/farmacologia , Regiões 3' não Traduzidas/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Northern Blotting , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Hibridização In Situ , MicroRNAs/genética , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Conflicting reports of the prognostic value of HER4 in breast cancer may be explained by distinct activities of the HER4 intracellular domain, 4ICD. Here, immunohistochemical 4ICD staining of archival invasive breast cancers (n = 923) was scored separately for nuclear and cytosolic expression, and these data were tested for associations with clinicopathological markers, disease-free survival, and disease-specific survival. By univariate analysis, cytosolic 4ICD expression was independently associated with estrogen receptor and progesterone receptor expression and tumor cell apoptosis. Nuclear 4ICD inversely correlated with tumor grade and tumor mitosis. In multivariate analyses cytosolic, but not nuclear 4ICD, significantly correlated with disease-free survival (P = 0.035) and disease-specific survival (P < 0.004) in lymph node-negative patients. Our results demonstrate for the first time that cytosolic 4ICD has significant positive prognostic value in node-negative breast cancer patients. At present, tumor grade and size are the primary clinicopathological parameters commonly used to guide decision making in these patients. Our results suggest that cytosolic 4ICD has important pathological functions and may be used to identify node-negative breast cancer patients at low risk of relapse and an improved survival, thereby avoiding systemic overtreatment of these patients. Our results also suggest that pan-receptor tyrosine kinase inhibitors, currently in clinical trials, or HER4 antagonists, which disengage 4ICD signaling, may have untoward activity in patients whose tumors express cytosolic 4ICD.
Assuntos
Neoplasias da Mama , Receptores ErbB , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Intervalo Livre de Doença , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Prognóstico , Estrutura Terciária de Proteína , Receptor ErbB-4 , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismoRESUMO
In the lactating breast, ERBB4 localizes to the nuclei of secretory epithelium while regulating activities of the signal transducer and activator of transcription (STAT) 5A transcription factor essential for milk-gene expression. We have identified an intrinsic ERBB4 NLS (residues 676-684) within the ERBB4 intracellular domain (4ICD) that is essential for nuclear accumulation of 4ICD. To determine the functional significance of 4ICD nuclear translocation in a physiologically relevant system, we have demonstrated that cotransfection of ERBB4 and STAT5A in a human breast cancer cell line stimulates beta-casein promoter activity. Significantly, nuclear localization of STAT5A and subsequent stimulation of the beta-casein promoter requires nuclear translocation of 4ICD. Moreover, 4ICD and STAT5A colocalize within nuclei of heregulin beta 1 (HRG)-stimulated cells and both proteins bind to the endogenous beta-casein promoter in T47D breast cancer cells. Together, our results establish a novel molecular mechanism of transmembrane receptor signal transduction involving nuclear cotranslocation of the receptor intracellular domain and associated transcription factor. Subsequent binding of the two proteins at transcription factor target promoters results in activation of gene expression.
Assuntos
Transporte Ativo do Núcleo Celular , Proteínas de Ligação a DNA/metabolismo , Receptores ErbB/fisiologia , Regulação da Expressão Gênica , Proteínas do Leite/metabolismo , Chaperonas Moleculares/fisiologia , Transativadores/metabolismo , Sequência de Aminoácidos , Neoplasias da Mama/patologia , Caseínas/genética , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Humanos , Proteínas do Leite/genética , Neuregulina-1/metabolismo , Sinais de Localização Nuclear , Regiões Promotoras Genéticas , Ligação Proteica , Receptor ErbB-4 , Fator de Transcrição STAT5 , Transativadores/genética , Transfecção , Proteínas Supressoras de TumorRESUMO
Human epidermal growth factor receptor 2 (HER2) is overexpressed in nearly 20-30% of breast cancers and is associated with metastasis resulting in poor patient survival and high recurrence. The dual EGFR/HER2 kinase inhibitor lapatinib has shown promising clinical results, but its limitations have also led to the resistance and activation of tumor survival pathways. Following our previous investigation of quinones as HER2 kinase inhibitors, we synthesized several naphthoquinone derivatives that significantly inhibited breast tumor cells expressing HER2 and trastuzumab-resistant HER2 oncogenic isoform, HER2Δ16. Two of these compounds were shown to be more effective than lapatinib at the inhibition of HER2 autophosphorylation of Y1248. Compounds 7 (5,8-dihydroxy-2-methylnaphthalene-1,4-dione) and 9 (2-(bromomethyl)-5,8-dihydroxynaphthalene-1,4-dione) inhibited HER2-expressing MCF-7 cells (IC50 0.29 and 1.76 µM, respectively) and HER2Δ16-expressing MCF-7 cells (IC50 0.51 and 1.76 µM, respectively). Compound 7 was also shown to promote cell death in multiple refractory breast cancer cell lines with IC50 values ranging from 0.12 to 2.92 µM. These compounds can function as lead compounds for the design of a new series of nonquinonoid structural compounds that can maintain a similar inhibition profile.
RESUMO
Tumor necrosis factor alpha converting enzyme (TACE) mediates shedding of human epidermal growth factor receptor-4 (HER4). Recent data suggest that released HER4 intracellular domain (4ICD) induces apoptosis in breast cancer. TACE expression, as measured by immunohistochemical analysis, was observed in 183 of 383 breast carcinomas, 39 of 217 ovarian carcinomas, and 16 of 24 and 17 of 24 hormonesensitive and hormone-insensitive prostate carcinomas, respectively. HER4 expression was detected in breast carcinomas by using 2 antibodies recognizing an extracellular or intracellular epitope. TACE expression was predominantly seen in tumors with high levels of 4ICD and membranous HER4. Apoptotic activity was measured by the terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) assay and cleaved caspase-3 staining in breast carcinomas. There was no significant association between cleaved caspase-3 or TUNEL positivity and 4ICD, whereas TUNEL positivity was seen predominantly in tumors with high levels of internalized HER4. The data presented herein show TACE expression in endocrine cancers and further support a role for TACE in breast cancer apoptosis.
Assuntos
Proteínas ADAM/biossíntese , Neoplasias da Mama/enzimologia , Neoplasias das Glândulas Endócrinas/enzimologia , Neoplasias Ovarianas/enzimologia , Neoplasias da Próstata/enzimologia , Proteína ADAM17 , Apoptose/fisiologia , Neoplasias da Mama/patologia , Neoplasias das Glândulas Endócrinas/patologia , Receptores ErbB/biossíntese , Feminino , Expressão Gênica , Humanos , Marcação In Situ das Extremidades Cortadas , Masculino , Neoplasias Ovarianas/patologia , Neoplasias da Próstata/patologia , Receptor ErbB-4 , Receptores de Estrogênio/biossínteseRESUMO
ERBB4/HER4 (referred to here as ERBB4) is a unique member of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases. In contrast to the other three members of the EGFR family (i.e., EGFR, ERBB2/HER2/NEU, and ERBB3), which are associated with aggressive forms of human cancers, ERBB4 expression seems to be selectively lost in tumors with aggressive phenotypes. Consistent with this observation, we show that ERBB4 induces apoptosis when reintroduced into breast cancer cell lines or when endogenous ERBB4 is activated by a ligand. We further show that ligand activation and subsequent proteolytic processing of endogenous ERBB4 results in mitochondrial accumulation of the ERBB4 intracellular domain (4ICD) and cytochrome c efflux, the essential and committed step of mitochondrial regulated apoptosis. Our results indicate that 4ICD is functionally similar to BH3-only proteins, proapoptotic members of the BCL-2 family required for initiation of mitochondrial dysfunction through activation of the proapoptotic multi-BH domain proteins BAX/BAK. Similar to other BH3-only proteins, 4ICD cell-killing activity requires an intact BH3 domain and 4ICD interaction with the antiapoptotic protein BCL-2, suppressed 4ICD-induced apoptosis. Unique among BH3-only proteins, however, is the essential requirement of BAK but not BAX to transmit the 4ICD apoptotic signal. Clinically, cytosolic but not membrane ERBB4/4ICD expression in primary human breast tumors was associated with tumor apoptosis, providing a mechanistic explanation for the loss of ERBB4 expression during tumor progression. Thus, we propose that ligand-induced mitochondrial accumulation of 4ICD represents a unique mechanism of action for transmembrane receptors, directly coupling a cell surface signal to the tumor cell mitochondrial apoptotic pathway.
Assuntos
Apoptose/fisiologia , Neoplasias da Mama/patologia , Receptores ErbB/metabolismo , Sequência de Aminoácidos , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Citocromos c/metabolismo , Receptores ErbB/biossíntese , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Neuregulina-1/farmacologia , Fragmentos de Peptídeos/metabolismo , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptor ErbB-4 , Homologia de Sequência de Aminoácidos , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismoRESUMO
Tamoxifen, a selective estrogen receptor (ER) modulator, is the most widely prescribed hormonal therapy treatment for breast cancer. Despite the benefits of tamoxifen therapy, almost all tamoxifen-responsive breast cancer patients develop resistance to therapy. In addition, tamoxifen displays estrogen-like effects in the endometrium increasing the incidence of endometrial cancer. New therapeutic strategies are needed to circumvent tamoxifen resistance in breast cancer as well as tamoxifen toxicity in endometrium. Organic selenium compounds are highly effective chemopreventive agents with well-documented benefits in reducing total cancer incidence and mortality rates for a number of cancers. The present study shows that the organic selenium compound methylseleninic acid (MSA, 2.5 micromol/L) can potentiate growth inhibition of 4-hydroxytamoxifen (10(-7) mol/L) in tamoxifen-sensitive MCF-7 and T47D breast cancer cell lines. Remarkably, in tamoxifen-resistant MCF-7-LCC2 and MCF7-H2Delta16 breast cancer cell lines and endometrial-derived HEC1A and Ishikawa cells, coincubation of 4-hydroxytamoxifen with MSA resulted in a marked growth inhibition that was substantially greater than MSA alone. Growth inhibition by MSA and MSA + 4-hydroxytamoxifen in all cell lines was preceded by a specific decrease in ER(alpha) mRNA and protein without an effect on ER(beta) levels. Estradiol and 4-hydroxytamoxifen induction of endogenous ER-dependent gene expression (pS2 and c-myc) as well as ER-dependent reporter gene expression (ERE(2)e1b-luciferase) was also attenuated by MSA in all cell lines before effect on growth inhibition. Taken together, these data strongly suggest that specific decrease in ER(alpha) levels by MSA is required for both MSA potentiation of the growth inhibitory effects of 4-hydroxytamoxifen and resensitization of tamoxifen-resistant cell lines.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias do Endométrio/tratamento farmacológico , Antagonistas de Estrogênios/uso terapêutico , Receptor alfa de Estrogênio/antagonistas & inibidores , Neoplasias Hormônio-Dependentes/tratamento farmacológico , Compostos Organosselênicos/uso terapêutico , Tamoxifeno/análogos & derivados , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias do Endométrio/metabolismo , Antagonistas de Estrogênios/farmacologia , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/antagonistas & inibidores , Receptor beta de Estrogênio/genética , Receptor beta de Estrogênio/metabolismo , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Compostos Organosselênicos/farmacologia , Proteínas Proto-Oncogênicas c-myc/genética , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Transdução de Sinais , Tamoxifeno/farmacologia , Tamoxifeno/uso terapêutico , Fator Trefoil-1 , Proteínas Supressoras de Tumor/genéticaRESUMO
The HER4 receptor tyrosine kinase and STAT5A cooperate to promote mammary luminal progenitor cell maturation and mammary epithelial cell differentiation. Coupled HER4 and STAT5A signaling is mediated, in part, through association of the HER4 intracellular domain (4ICD) with STAT5A at STAT5A target gene promoters where 4ICD functions as a STAT5A transcriptional coactivator. Despite an essential role for coupled 4ICD and STAT5A signaling in mammary gland development, the mechanistic basis of 4ICD and STAT5A cooperative signaling remains unexplored. Here we show for the first time that 4ICD and STAT5A directly interact through STAT5A recruitment and binding to HER4/4ICD residue Y984. Accordingly, altering the 4ICD Y984 to phenylalanine results in a dramatic reduction of STAT5A and 4ICD-Y984F interacting complexes coimmunoprecipitated with HER4 or STAT5A specific antibodies. We further show that disrupting the 4ICD and STAT5A interaction has an important physiological impact on mammary epithelial cell differentiation. HC11 mammary epithelial cells with stable expression of 4ICD undergo differentiation with significantly increased expression of the STAT5A target genes and differentiation markers ß-casein and WAP. In contrast, HC11 cells stably expressing 4ICD-Y984F failed to undergo differentiation with basal expression levels of ß-casein and WAP. Differentiation in this cell system was induced in the absence of exogenous prolactin indicating that 4ICD activity is sufficient to induce mammary epithelial cell differentiation. Finally, we show that suppression of STAT5A expression abolishes the ability of 4ICD to induce HC11 differentiation and activate ß-casein or WAP expression. Taken together our results demonstrate for the first time that direct coupling of 4ICD and STAT5A is both necessary and sufficient to drive mammary epithelial differentiation. In conclusion, our findings that 4ICD and STAT5A directly interact to form a physiologically important transcriptional activation complex, provide a mechanistic basis for the in vivo observations that HER4/4ICD and STAT5A cooperate to promote mammary gland progenitor cell maturation and initiate lactation at parturition.