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BACKGROUND: Endocrine therapy is standard treatment for estrogen receptor (ER)-positive breast cancer. However, its efficacy is limited by intrinsic and acquired resistance. Here the potential of S100ß as a biomarker and inhibition of its signaling network as a therapeutic strategy in endocrine treated patients was investigated. METHODS: The expression of S100ß in tissue and serum was assessed by immunohistochemistry and an enzyme-linked immunosorbent assay, respectively. The S100ß signaling network was investigated in cell line models of endocrine resistance by western blot, PCR, immunoprecipitation, and chromatin-immunoprecipitation. Endocrine resistant xenografts and tumor explants from patients with resistant tumors were treated with endocrine therapy in the presence and absence of the p-Src kinase inhibitor, dasatinib. RESULTS: Tissue and serum levels of S100ß were found to predict poor disease-free survival in endocrine-treated patients (n = 509, HR 2.32, 95% CI is 1.58-3.40, p < 0.0001 and n = 187, HR 4.009, 95% CI is 1.66-9.68, p = 0.002, respectively). Moreover, elevated levels of serum S100ß detected during routine surveillance over the patient treatment period significantly associated with subsequent clinically confirmed disease recurrence (p = 0.019). In vivo studies demonstrated that endocrine treatment induced transcriptional regulation of S100ß which was successfully disrupted with tyrosine kinase inhibition. In endocrine resistant xenografts and tumor explants from patients with endocrine resistant breast cancer, combined endocrine and dasatinib treatment reduced tumor proliferation and down-regulated S100ß protein expression in comparison to endocrine treatment alone. CONCLUSIONS: S100ß has potential as a new surveillance tool for patients with ER-positive breast cancer to monitor ongoing response to endocrine therapy. Moreover, endocrine resistant breast cancer patients with elevated S100ß may benefit from combined endocrine and tyrosine-kinase inhibitor treatment. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01840293 ). Registered on 23 April 2013. Retrospectively registered.
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Antineoplásicos Hormonais/uso terapêutico , Biomarcadores/sangue , Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Subunidade beta da Proteína Ligante de Cálcio S100/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Biomarcadores/metabolismo , Neoplasias da Mama/sangue , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Intervalo Livre de Doença , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Células MCF-7 , Camundongos , Pessoa de Meia-Idade , Recidiva Local de Neoplasia , Subunidade beta da Proteína Ligante de Cálcio S100/genética , Transdução de Sinais/efeitos dos fármacos , Tamoxifeno/uso terapêutico , Análise Serial de Tecidos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
INTRODUCTION: HOX genes play vital roles in growth and development, however, atypical redeployment of these genes is often associated with steroidal adaptability in endocrine cancers. We previously identified HOXC11 to be an indicator of poor response to hormonal therapy in breast cancer. In this study we aimed to elucidate genes regulated by HOXC11 in the endocrine resistant setting. METHODS: RNA-sequencing paired with transcription factor motif-mapping was utilised to identify putative HOXC11 target genes in endocrine resistant breast cancer. Validation and functional evaluation of the target gene, prosaposin (PSAP), was performed in a panel of endocrine sensitive and resistant breast cancer cell lines. The clinical significance of this finding was explored in clinical cohorts at both mRNA and protein level. RESULTS: PSAP was shown to be regulated by HOXC11 in both tamoxifen and aromatase inhibitor (AI) resistant cell lines. Transcript levels of HOXC11 and PSAP correlated strongly in samples of primary breast tumours (r = 0.7692, n = 51). PSAP has previously been reported to activate androgen receptor (AR) in prostate cancer cells. In a panel of breast cancer cell lines it was shown that endocrine resistant cells exhibit innately elevated levels of AR compared to their endocrine sensitive counterparts. Here, we demonstrate that stimulation with PSAP can drive AR recruitment to a hormone response element (HRE) in AI resistant breast cancer cells. Functionally, PSAP promotes cell migration and invasion only in AI resistant cells and not in their endocrine sensitive counterparts. In a cohort of breast cancer patients (n = 34), elevated serum levels of PSAP were found to associate significantly with poor response to endocrine treatment (p = 0.04). Meta-analysis of combined PSAP and AR mRNA are indicative of poor disease-free survival in endocrine treated breast cancer patients (hazard ratio (HR): 2.2, P = 0.0003, n = 661). CONCLUSION: The HOXC11 target gene, PSAP, is an AR activator which facilitates adaptation to a more invasive phenotype in vitro. These findings have particular relevance to the development of resistance to AI therapy which is an emerging clinical issue. PSAP is a secreted biomarker which has potential in identifying patients failing to exhibit sustained response to hormonal treatment.
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Inibidores da Aromatase/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Resistencia a Medicamentos Antineoplásicos/genética , Receptores Androgênicos/genética , Saposinas/genética , Antineoplásicos Hormonais/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Intervalo Livre de Doença , Feminino , Proteínas de Homeodomínio/genética , Humanos , Células MCF-7 , Masculino , Neoplasias da Próstata/genética , RNA Mensageiro/genética , Tamoxifeno/farmacologiaRESUMO
Steroid hormone receptors are key mediators in the execution of hormone action through a combination of genomic and non-genomic action. Since their isolation and characterisation in the early 20th Century much of our understanding of the biological actions of steroid hormones are underpinned by their activated receptor activity. Over the past two decades there has been an acceleration of more omics-based research which has resulted in a major uptick in our comprehension of genomic steroid action. However, it is well understood that steroid hormones can induce very rapid signalling events in tandem with their genomic actions wherein they exert their influence through alterations in gene expression. Thus the totality of genomic and non-genomic steroid action occurs in a simultaneous and reciprocal manner and a greater appreciation of whole cell action is required to fully evaluate steroid hormone activity in vivo. In this mini-review we outline the most recent developments in non-genomic steroid action and cytoplasmic steroid hormone receptor biology in endocrine-related cancers with a focus on the 3-keto steroid receptors, in particular the androgen receptor.
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Polycystic ovary syndrome (PCOS) is the most common endocrine condition affecting women. It has traditionally been viewed as a primarily reproductive disorder; however, it is increasingly recognized as a lifelong metabolic disease. Women with PCOS are at increased risk of insulin resistance (IR), type 2 diabetes mellitus, non-alcoholic fatty liver disease and cardiovascular disease. Although not currently a diagnostic criterion, IR is a cardinal pathophysiological feature and highly prevalent in women with PCOS. Androgens play a bidirectional role in the pathogenesis of IR, and there is a complex interplay between IR and androgen excess in women with PCOS. Skeletal muscle has a key role in maintaining metabolic homeostasis and is also a metabolic target organ of androgen action. Skeletal muscle is the organ responsible for the majority of insulin-mediated glucose disposal. There is growing interest in the relationship between skeletal muscle, androgen excess and mitochondrial dysfunction in the pathogenesis of metabolic disease in PCOS. Molecular mechanisms underpinning defects in skeletal muscle dysfunction in PCOS remain to be elucidated, but may represent promising targets for future therapeutic intervention. In this review, we aim to explore the role of skeletal muscle in metabolism, focusing particularly on perturbations in skeletal muscle specific to PCOS as observed in recent molecular and in vivo human studies. We review the possible role of androgens in the pathophysiology of skeletal muscle abnormalities in PCOS, and identify knowledge gaps, areas for future research and potential therapeutic implications. Despite increasing interest in the area of skeletal muscle dysfunction in women with PCOS, significant challenges and unanswered questions remain, and going forward, novel innovative approaches will be required to dissect the underlying mechanisms.
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To date, almost all solid malignancies have implicated insulin-like growth factor (IGF) signalling as a driver of tumour growth. However, the remarkable level of crosstalk between sex hormones, the IGF-1 receptor (IGF-1R) and its ligands IGF-1 and 2 in endocrine driven cancers is incompletely understood. Similar to the sex steroids, IGF signalling is essential in normal development as well as growth and tissue homoeostasis, and undergoes a steady decline with advancing age and increasing visceral adiposity. Interestingly, IGF-1 has been found to play a compensatory role for both estrogen receptor (ER) and androgen receptor (AR) by augmenting hormonal responses in the absence of, or where low levels of ligand are present. Furthermore, experimental, and epidemiological evidence supports a role for dysregulated IGF signalling in breast and prostate cancers. Insulin-like growth factor binding protein (IGFBP) molecules can regulate the bioavailability of IGF-1 and are frequently expressed in these hormonally regulated tissues. The link between age-related disease and the role of IGF-1 in the process of ageing and longevity has gained much attention over the last few decades, spurring the development of numerous IGF targeted therapies that have, to date, failed to deliver on their therapeutic potential. This review will provide an overview of the sexually dimorphic nature of IGF signalling in humans and how this is impacted by the reduction in sex steroids in mid-life. It will also explore the latest links with metabolic syndromes, hormonal imbalances associated with ageing and targeting of IGF signalling in endocrine-related tumour growth with an emphasis on post-menopausal breast cancer and the impact of the steroidal milieu.
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PURPOSE: There is strong epidemiologic evidence indicating that estrogens may not be the sole steroid drivers of breast cancer. We hypothesize that abundant adrenal androgenic steroid precursors, acting via the androgen receptor (AR), promote an endocrine-resistant breast cancer phenotype. EXPERIMENTAL DESIGN: AR was evaluated in a primary breast cancer tissue microarray (n = 844). Androstenedione (4AD) levels were evaluated in serum samples (n = 42) from hormone receptor-positive, postmenopausal breast cancer. Levels of androgens, progesterone, and estradiol were quantified using LC/MS-MS in serum from age- and grade-matched recurrent and nonrecurrent patients (n = 6) before and after aromatase inhibitor (AI) therapy (>12 months). AR and estrogen receptor (ER) signaling pathway activities were analyzed in two independent AI-treated cohorts. RESULTS: AR protein expression was associated with favorable progression-free survival in the total population (Wilcoxon, P < 0.001). Pretherapy serum samples from breast cancer patients showed decreasing levels of 4AD with age only in the nonrecurrent group (P < 0.05). LC/MS-MS analysis of an AI-sensitive and AI-resistant cohort demonstrated the ability to detect altered levels of steroids in serum of patients before and after AI therapy. Transcriptional analysis showed an increased ratio of AR:ER signaling pathway activities in patients failing AI therapy (t test P < 0.05); furthermore, 4AD mediated gene changes associated with acquired AI resistance. CONCLUSIONS: This study highlights the importance of examining the therapeutic consequences of the steroid microenvironment and demonstrable receptor activation using indicative gene expression signatures.
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Androstenodiona/fisiologia , Inibidores da Aromatase/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/etiologia , Receptores Androgênicos/fisiologia , Androstenodiona/sangue , Neoplasias da Mama/sangue , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Ligantes , Transdução de Sinais , Células Tumorais CultivadasRESUMO
PURPOSE: This study investigates the role of the p160 coactivators AIB1 and SRC-1 independently, and their interactions with the estrogen receptor, in the development of resistance to endocrine treatments. EXPERIMENTAL DESIGN: The expression of the p160s and the estrogen receptor, and their interactions, was analyzed by immunohistochemistry and quantitative coassociation immunofluorescent microscopy, using cell lines, primary breast tumor cell cultures, and a tissue microarray with breast cancer samples from 560 patients. RESULTS: Coassociation of the p160s and estrogen receptor alpha was increased in the LY2 endocrine-resistant cell line following treatment with tamoxifen in comparison with endocrine-sensitive MCF-7 cells. In primary cultures, there was an increase in association of the coactivators with estrogen receptor alpha following estrogen treatment but dissociation was evident with tamoxifen. Immunohistochemical staining of the tissue microarray revealed that SRC-1 was a strong predictor of reduced disease-free survival (DFS), both in patients receiving adjuvant tamoxifen treatment and untreated patients (P < 0.0001 and P = 0.0111, respectively). SRC-1 was assigned a hazard ratio of 2.12 using a Cox proportional hazards model. Endocrine-treated patients who coexpressed AIB1 with human epidermal growth factor receptor 2 had a significantly shorter DFS compared with all other patients (P = 0.03). Quantitative coassociation analysis in the patient tissue microarray revealed significantly stronger colocalization of AIB1 and SRC-1 with estrogen receptor alpha in patients who have relapsed in comparison with those patients who did not recur (P = 0.026 and P = 0.00001, respectively). CONCLUSIONS: SRC-1 is a strong independent predictor of reduced DFS, whereas the interactions of the p160 proteins with estrogen receptor alpha can predict the response of patients to endocrine treatment.
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Neoplasias da Mama/tratamento farmacológico , Receptor alfa de Estrogênio/fisiologia , Histona Acetiltransferases/fisiologia , Recidiva Local de Neoplasia/etiologia , Proteínas Nucleares/fisiologia , Proteínas de Transporte Nucleocitoplasmático/fisiologia , Tamoxifeno/uso terapêutico , Fatores de Transcrição/fisiologia , Neoplasias da Mama/química , Neoplasias da Mama/mortalidade , Linhagem Celular Tumoral , Proteínas de Ligação a DNA , Intervalo Livre de Doença , Resistencia a Medicamentos Antineoplásicos , Receptor alfa de Estrogênio/análise , Feminino , Histona Acetiltransferases/análise , Humanos , Proteínas Nucleares/análise , Coativador 1 de Receptor Nuclear , Coativador 3 de Receptor Nuclear , Proteínas de Transporte Nucleocitoplasmático/análise , Prognóstico , Proteínas de Ligação a RNA , Análise Serial de Tecidos , Transativadores/análise , Transativadores/fisiologia , Fatores de Transcrição/análiseRESUMO
Cytochrome P450 1B1 (CYP1B1) mRNA is constitutively expressed in most normal extra-hepatic tissues; however the protein is not detectable in these tissues but is expressed in a wide variety of tumors. CYP1B1 is responsible for the activation of a number of carcinogens present in tobacco smoke and food. A surgical model of rat esophageal tumorigenesis, promoted by gastric or duodenal reflux was used to determine CYP1B1 expression in premalignant esophageal tissue. Immunohistochemistry was performed using a modified amplified fluorescein tyramide protocol. CYP1B1 was not observed in normal esophageal mucosa, submucosa, or muscularis mucosa. Animals exposed to gastric reflux developed mild hyperplasia. Varying degrees of hyperplasia were observed in the duodenal reflux group. All regions of hyperplasia showed moderate or strong CYP1B1 immunoreactivity. Duodenal reflux induced a small number of premalignant changes: immunoreactivity was absent from the epithelium of squamous dysplasia (0/10), Barrett's esophagus (0/7), and majority of dysplastic Barrett's esophagus (1/4). Moderate or strong immunoreactivity was observed in the majority (7/8) of squamous cell carcinomas (SCCs) in situ. Immunoreactivity was also observed in the lamina propria and submucosa in association with inflammation, regardless of the severity of inflammation. The expression of CYP1B1 in hyperplasia, SCCs in situ, or in association with inflammation may increase the production of carcinogenic metabolites, which may promote esophageal tumorigenesis.
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Hidrocarboneto de Aril Hidroxilases/genética , Refluxo Duodenogástrico/complicações , Neoplasias Esofágicas/enzimologia , Refluxo Gastroesofágico/complicações , Animais , Especificidade de Anticorpos , Hidrocarboneto de Aril Hidroxilases/imunologia , Western Blotting , Citocromo P-450 CYP1B1 , Refluxo Duodenogástrico/enzimologia , Neoplasias Esofágicas/etiologia , Feminino , Refluxo Gastroesofágico/enzimologia , Imuno-Histoquímica , Camundongos , Camundongos Knockout , RNA Mensageiro/genética , Ratos , Ratos Sprague-DawleyRESUMO
Divergent roles for androgen receptor (AR) in breast cancer have been reported. Following aromatase inhibitor (AI) treatment, the conversion of circulating androgens into estrogens can be diminished by >99%. We wished to establish whether the steroid environment can dictate the role of AR and the implications of this for subsequent therapy. This study utilizes models of AI resistance to explore responsiveness to PI3K/mTOR and anti-AR therapy when cells are exposed to unconverted weak androgens. Transcriptomic alterations driven by androstenedione (4AD) were assessed by RNA-sequencing. AR and estrogen receptor (ER) recruitment to target gene promoters was evaluated using ChIP, and relevance to patient profiles was performed using publicly available data sets. Although BEZ235 showed decreased viability across AI-sensitive and -resistant cell lines, anti-AR treatment elicited a decrease in cell viability only in the AI-resistant model. Serum and glucocorticoid-regulated kinase 3 (SGK3) and cAMP-dependent protein kinase inhibitor ß (PKIB) were confirmed to be regulated by 4AD and shown to be mediated by AR; crucially, reexposure to estradiol suppressed expression of these genes. Meta-analysis of transcript levels showed high expression of SGK3 and PKIB to be associated with poor response to endocrine therapy (HR = 2.551, P = 0.003). Furthermore, this study found levels of SGK3 to be sustained in patients who do not respond to AI therapy. This study highlights the importance of the tumor steroid environment. SGK3 and PKIB are associated with poor response to endocrine therapy and could have utility in tailoring therapeutic approaches.
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Inibidores da Aromatase/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Receptores Androgênicos/metabolismo , Esteroides/metabolismo , Adaptação Fisiológica/efeitos dos fármacos , Androstenodiona/farmacologia , Inibidores da Aromatase/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Intervalo Livre de Doença , Resistencia a Medicamentos Antineoplásicos/genética , Estradiol/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células MCF-7 , Fosfatidilinositol 3-Quinases/metabolismo , Pós-Menopausa/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinases/metabolismo , Quinolinas/farmacologia , Quinolinas/uso terapêutico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Estrogênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , Transcriptoma/genética , Regulação para Cima/efeitos dos fármacosRESUMO
Cyclooxygenase-2 (COX-2) is associated with breast tumour progression. Clinical and molecular studies implicate human epidermal growth factor receptor 2 (HER2) in the regulation of COX-2 expression. Recent reports raise the possibility that HER2 could mediate these effects through direct transcriptional mechanisms. The relationship between HER2 and COX-2 was investigated in a cohort of breast cancer patients with or without endocrine treatment. A tissue microarray comprising tumours from 560 patients with 10-year follow-up was analysed for HER2, ERK1/2, polyoma enhancer activator 3 (PEA3) and COX-2 expression. Subcellular localisation of HER2 was assessed by immunofluorescence and confocal microscopy. Expression of markers examined was analysed in relation to classic clinicopathological parameters and disease-free survival in the presence and absence of tamoxifen. COX-2 expression associated with both membranous and nuclear expression of HER2 (P=0.0033 and P<0.00001 respectively). No association was detected between COX-2 and either ERK1/2 or PEA3 (P=0.7 and P=0.3 respectively). None of the markers were found to be independently prognostic. Membrane HER2, nuclear HER2 and COX-2, however, were all found to predict poor disease-free survival in patients on endocrine treatment (P=0.0017, P=0.0003 and P=0.0202 respectively). Moreover, patients who were positive for COX-2 predicted adverse effects of tamoxifen (P=0.0427). These clinical ex vivo data are consistent with molecular observations that HER2 can regulate COX-2 expression through direct transcriptional mechanisms. COX-2 expression correlates with disease progression on endocrine treatment. This study supports a role for COX-2 as a predictor of adverse effects of tamoxifen in breast cancer patients.
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Neoplasias da Mama/diagnóstico , Carcinoma/diagnóstico , Núcleo Celular/metabolismo , Ciclo-Oxigenase 2/fisiologia , Receptor ErbB-2/fisiologia , Tamoxifeno/efeitos adversos , Antineoplásicos Hormonais/efeitos adversos , Antineoplásicos Hormonais/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Carcinoma/tratamento farmacológico , Carcinoma/metabolismo , Carcinoma/mortalidade , Núcleo Celular/efeitos dos fármacos , Estudos de Coortes , Progressão da Doença , Feminino , Humanos , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Prognóstico , Receptor ErbB-2/metabolismo , Análise de Sobrevida , Tamoxifeno/uso terapêutico , Distribuição Tecidual , Fatores de Transcrição/metabolismo , Resultado do TratamentoRESUMO
Androgen receptor (AR) is the most widely expressed steroid receptor protein in normal breast tissue and is detectable in approximately 90% of primary breast cancers and 75% of metastatic lesions. However, the role of AR in breast cancer development and progression is mired in controversy with evidence suggesting it can either inhibit or promote breast tumorigenesis. Studies have shown it to antagonize estrogen receptor alpha (ERα) DNA binding, thereby preventing pro-proliferative gene transcription; whilst others have demonstrated AR to take on the mantle of a pseudo ERα particularly in the setting of triple negative breast cancer. Evidence for a potentiating role of AR in the development of endocrine resistant breast cancer has also been mounting with reports associating high AR expression with poor response to endocrine treatment. The resurgence of interest into the function of AR in breast cancer has resulted in various emergent clinical trials evaluating anti-AR therapy and selective androgen receptor modulators in the treatment of advanced breast cancer. Trials have reported varied response rates dependent upon subtype with overall clinical benefit rates of ~19-29% for anti-androgen monotherapy, suggesting that with enhanced patient stratification AR could prove efficacious as a breast cancer therapy. Androgens and AR have been reported to facilitate tumor stemness in some cancers; a process which may be mediated through genomic or non-genomic actions of the AR, with the latter mechanism being relatively unexplored in breast cancer. Steroidogenic ligands of the AR are produced in females by the gonads and as sex-steroid precursors secreted from the adrenal glands. These androgens provide an abundant reservoir from which all estrogens are subsequently synthesized and their levels are undiminished in the event of standard hormonal therapeutic intervention in breast cancer. Steroid levels are known to be altered by lifestyle factors such as diet and exercise; understanding their potential role in dictating the function of AR in breast cancer development could therefore have wide-ranging effects in prevention and treatment of this disease. This review will outline the endogenous biochemical drivers of both genomic and non-genomic AR activation and how these may be modulated by current hormonal therapies.
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OBJECTIVE: Pericytes, located in close proximity to the underlying endothelium, form an integral component of the microvasculature. These cells are intimately involved in angiogenesis, which is of fundamental importance in many physiological and pathological processes. We evaluated the influence of pericyte-conditioned medium (PCM) on endothelial cell growth characteristics and modulation of endothelial gene expression. METHODS: Migration and tubule formation assays were performed in vitro to determine the effect of PCM on endothelial growth characteristics. cDNA microarray analysis was used to identify alterations in gene expression following exposure of human microvascular endothelial cells (HMEC-1) to PCM. Overexpression of PAI-1 using recombinant protein or transient transfection, and inhibition using an inhibitory antibody against PAI-1, were used to determine whether up- or down-regulation of this gene was responsible for the changes in endothelial cell characteristics observed in response to PCM exposure. RESULTS: We have shown that PCM exerts a dramatic inhibitory influence on endothelial cell migration in vitro. In addition, endothelial cells cultured on Matrigel and exposed to PCM were found to generate significantly fewer angiogenic branches. Microarray analysis of endothelial cells exposed to PCM identified PAI-1 as the gene showing the greatest level of differential expression (3.4-fold induction). Studies using an inhibitory antibody to PAI-1 suggest that induction of this protein by PCM is pivotal to the observed inhibitory influence on the migratory and angiogenic potential of HMEC-1. We further investigated this by overexpressing PAI-1, which was shown to have a potent inhibitory influence on EC migration and angiogenic branching, although the concentration of PAI-1 was clearly important. CONCLUSION: Collectively, these findings suggest that PCM contains a bioactive element(s) that controls both endothelial cell migration and tubule formation in vitro and that these responses may be partially controlled by increased endothelial cell expression of PAI-1.
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Células Endoteliais/metabolismo , Regulação da Expressão Gênica , Neovascularização Fisiológica , Pericitos/metabolismo , Inibidor 1 de Ativador de Plasminogênio/genética , Northern Blotting/métodos , Western Blotting/métodos , Movimento Celular , Proliferação de Células , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Células Endoteliais/citologia , Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Inibidor 1 de Ativador de Plasminogênio/metabolismoRESUMO
PURPOSE: Disease recurrence is a common problem in breast cancer and yet the mechanisms enabling tumor cells to evade therapy and colonize distant organs remain unclear. We sought to characterize global expression changes occurring with metastatic disease progression in the endocrine-resistant setting. EXPERIMENTAL DESIGN: Here, for the first time, RNAsequencing has been performed on matched primary, nodal, and liver metastatic tumors from tamoxifen-treated patients following disease progression. Expression of genes commonly elevated in the metastases of sequenced patients was subsequently examined in an extended matched patient cohort with metastatic disease from multiple sites. The impact of tamoxifen treatment on endocrine-resistant tumors in vivo was investigated in a xenograft model. RESULTS: The extent of patient heterogeneity at the gene level was striking. Less than 3% of the genes differentially expressed between sequential tumors were common to all patients. Larger divergence was observed between primary and liver tumors than between primary and nodal tumors, reflecting both the latency to disease progression and the genetic impact of intervening therapy. Furthermore, an endocrine-resistant in vivo mouse model demonstrated that tamoxifen treatment has the potential to drive disease progression and establish distant metastatic disease. Common functional pathways altered during metastatic, endocrine-resistant progression included extracellular matrix receptor interactions and focal adhesions. CONCLUSIONS: This novel global analysis highlights the influence of primary tumor biology in determining the transcriptomic profile of metastatic tumors, as well as the need for adaptations in cell-cell communications to facilitate successful tumor cell colonization of distant host organs.
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Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Transcriptoma , Adulto , Animais , Antineoplásicos Hormonais/farmacologia , Antineoplásicos Hormonais/uso terapêutico , Biomarcadores , Neoplasias da Mama/tratamento farmacológico , Comunicação Celular , Linhagem Celular Tumoral , Análise por Conglomerados , Terapia Combinada , Biologia Computacional/métodos , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/secundário , Camundongos , Pessoa de Meia-Idade , Gradação de Tumores , Metástase Neoplásica , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Transcriptional control is the major determinant of cell fate. The steroid receptor coactivator (SRC)-1 enhances the activity of the estrogen receptor in breast cancer cells, where it confers cell survival benefits. Here, we report that a global analysis of SRC-1 target genes suggested that SRC-1 also mediates transcriptional repression in breast cancer cells. Combined SRC-1 and HOXC11 ChIPseq analysis identified the differentiation marker, CD24, and the apoptotic protein, PAWR, as direct SRC-1/HOXC11 suppression targets. Reduced expression of both CD24 and PAWR was associated with disease progression in patients with breast cancer, and their expression was suppressed in metastatic tissues. Investigations in endocrine-resistant breast cancer cell lines and SRC-1(-/-)/PyMT mice confirmed a role for SRC-1 and HOXC11 in downregulation of CD24 and PAWR. Through bioinformatic analysis and liquid chromatography/mass spectrometry, we identified AP1 proteins and Jumonji domain containing 2C (JMD2C/KDM4C), respectively, as members of the SRC-1 interactome responsible for transcriptional repression. Our findings deepen the understanding of how SRC-1 controls transcription in breast cancers.
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Neoplasias da Mama/metabolismo , Carcinogênese/metabolismo , Inativação Gênica , Coativador 1 de Receptor Nuclear/fisiologia , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Sítios de Ligação , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Antígeno CD24/genética , Antígeno CD24/metabolismo , Carcinogênese/genética , Intervalo Livre de Doença , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/fisiologia , Humanos , Histona Desmetilases com o Domínio Jumonji/metabolismo , Estimativa de Kaplan-Meier , Células MCF-7 , Camundongos , Camundongos Knockout , Regiões Promotoras GenéticasRESUMO
Resistance to aromatase inhibitors (AIs) is a major clinical problem in the treatment of estrogen receptor (ER)-positive breast cancer. In two breast cancer cell line models of AI resistance, we identified widespread DNA hyper- and hypomethylation, with enrichment for promoter hypermethylation of developmental genes. For the homeobox gene HOXC10, methylation occurred in a CpG shore, which overlapped with a functional ER binding site, causing repression of HOXC10 expression. Although short-term blockade of ER signaling caused relief of HOXC10 repression in both cell lines and breast tumors, it also resulted in concurrent recruitment of EZH2 and increased H3K27me3, ultimately transitioning to increased DNA methylation and silencing of HOXC10. Reduced HOXC10 in vitro and in xenografts resulted in decreased apoptosis and caused antiestrogen resistance. Supporting this, we used paired primary and metastatic breast cancer specimens to show that HOXC10 was reduced in tumors that recurred during AI treatment. We propose a model in which estrogen represses apoptotic and growth-inhibitory genes such as HOXC10, contributing to tumor survival, whereas AIs induce these genes to cause apoptosis and therapeutic benefit, but long-term AI treatment results in permanent repression of these genes via methylation and confers resistance. Therapies aimed at inhibiting AI-induced histone and DNA methylation may be beneficial in blocking or delaying AI resistance.
Assuntos
Neoplasias da Mama/genética , Reprogramação Celular/genética , Resistencia a Medicamentos Antineoplásicos/genética , Epigênese Genética/genética , Estrogênios/farmacologia , Proteínas de Homeodomínio/genética , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Inibidores da Aromatase/farmacologia , Inibidores da Aromatase/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Reprogramação Celular/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Histonas/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Células MCF-7 , Camundongos , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Regiões Promotoras Genéticas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The development of breast cancer resistance to endocrine therapy results from an increase in cellular plasticity that permits the emergence of a hormone-independent tumor. The steroid coactivator protein SRC-1, through interactions with developmental proteins and other nonsteroidal transcription factors, drives this tumor adaptability. In this discovery study, we identified ADAM22, a non-protease member of the ADAM family of disintegrins, as a direct estrogen receptor (ER)-independent target of SRC-1. We confirmed SRC-1 as a regulator of ADAM22 by molecular, cellular, and in vivo studies. ADAM22 functioned in cellular migration and differentiation, and its levels were increased in endocrine resistant-tumors compared with endocrine-sensitive tumors in mouse xenograft models of human breast cancer. Clinically, ADAM22 was found to serve as an independent predictor of poor disease-free survival. Taken together, our findings suggest that SRC-1 switches steroid-responsive tumors to a steroid-resistant state in which the SRC-1 target gene ADAM22 has a critical role, suggesting this molecule as a prognostic and therapeutic drug target that could help improve the treatment of endocrine-resistant breast cancer.
Assuntos
Proteínas ADAM/fisiologia , Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/patologia , Proteínas do Tecido Nervoso/fisiologia , Coativador 1 de Receptor Nuclear/genética , Receptores de Estrogênio/metabolismo , Transcriptoma , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB CRESUMO
PURPOSE: The use of aromatase inhibitors (AI) in the treatment of estrogen receptor (ER)-positive, postmenopausal breast cancer has proven efficacy. However, inappropriate activation of ER target genes has been implicated in the development of resistant tumors. The ER coactivator protein AIB1 has previously been associated with initiation of breast cancer and resistance to endocrine therapy. EXPERIMENTAL DESIGN: Here, we investigated the role of AIB1 in the deregulation of ER target genes occurring as a consequence of AI resistance using tissue microarrays of patients with breast cancer and cell line models of resistance to the AI letrozole. RESULTS: Expression of AIB1 associated with disease recurrence (P = 0.025) and reduced disease-free survival time (P = 0.0471) in patients treated with an AI as first-line therapy. In a cell line model of resistance to letrozole (LetR), we found ERα/AIB1 promoter recruitment and subsequent expression of the classic ER target genes pS2 and Myc to be constitutively upregulated in the presence of both androstenedione and letrozole. In contrast, the recruitment of the ERα/AIB1 transcriptional complex to the nonclassic ER target cyclin D1 and its subsequent expression remained sensitive to steroid treatment and could be inhibited by treatment with letrozole. Molecular studies revealed that this may be due in part to direct steroid regulation of c-jun-NH(2)-kinase (JNK), signaling to Jun and Fos at the cyclin D1 promoter. CONCLUSION: This study establishes a role for AIB1 in AI-resistant breast cancer and describes a new mechanism of ERα/AIB1 gene regulation which could contribute to the development of an aggressive tumor phenotype.
Assuntos
Inibidores da Aromatase/uso terapêutico , Neoplasias da Mama/genética , Receptor alfa de Estrogênio/metabolismo , Regulação Neoplásica da Expressão Gênica , Coativador 3 de Receptor Nuclear/metabolismo , Transcrição Gênica , Androstenodiona/farmacologia , Inibidores da Aromatase/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Ciclina D1/genética , Ciclina D1/metabolismo , Intervalo Livre de Doença , Resistencia a Medicamentos Antineoplásicos , Receptor alfa de Estrogênio/genética , Feminino , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Letrozol , Sistema de Sinalização das MAP Quinases , Recidiva Local de Neoplasia , Nitrilas/farmacologia , Coativador 3 de Receptor Nuclear/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Proteínas Proto-Oncogênicas c-myc/biossíntese , Fator Trefoil-1 , Triazóis/farmacologia , Proteínas Supressoras de Tumor/biossíntese , Regulação para CimaRESUMO
Human cancers utilise telomerase to maintain telomeres and prohibit cell senescence. Human telomerase reverse transcriptase (hTERT), an essential component of this complex, is regulated at the level of gene transcription. Using SILAC-proteomic analysis and molecular studies, we identified the AAA+ ATPase, RuvBl2 as a transcriptional regulator of hTERT and established that this regulation is through cooperation with Ets-2. In colon cancer patients, nuclear expression of RuvBl2 associated with nuclear expression of hTERT, pEts2 and advanced nodal disease (P<0.01, P=0.05 and P=0.03 respectively, n=170). These data firmly implicate RuvBl2 in Ets2 mediated regulation of hTERT in colon cancer which has functional and clinical consequences.
Assuntos
Proteínas de Transporte/fisiologia , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , DNA Helicases/fisiologia , Regulação Neoplásica da Expressão Gênica , Proteína Proto-Oncogênica c-ets-2/fisiologia , Telomerase/genética , ATPases Associadas a Diversas Atividades Celulares , Neoplasias do Colo/química , Progressão da Doença , Humanos , Proteômica , Transcrição GênicaRESUMO
Epidemiological, clinical, and molecular studies suggest a role for oestrogen in thyroid cancer. How oestrogen mediates its effects and the consequence of it on clinical outcome has not been fully elucidated. The participation of coregulatory proteins in modulating oestrogen receptor (ER) function and input of crosstalk with the tyrosine kinase receptor HER2 was investigated. Oestrogen induced cell proliferation in the follicular thyroid cancer (FTC)-133 cells, but not in the anaplastic 8305C cell line. Knockdown of the coactivator steroid receptor coactivator (SRC)-1 inhibited FTC-133 basal, but not oestrogen induced, cell proliferation. Oestrogen also increased protein expression of SRC-1 and the ER target gene cyclin D1 in the FTC-133 cell line. ERalpha, ERbeta, the coregulatory proteins SRC-1 and nuclear corepressor (NCoR), and the tyrosine kinase receptor HER2 were localised by immunohistochemistry and immnofluorescence in paraffin-embedded tissue from thyroid tumour patients (n=111). ERalpha was colocalised with both SRC-1 and NCoR to the nuclei of the tumour epithelial cells. Expression of ERalpha and NCoR was found predominantly in non-anaplastic tumours and was significantly associated with well-differentiated tumours and reduced incidence of disease recurrence. In non-anaplastic tumours, HER2 was significantly associated with SRC-1, and these proteins were associated with poorly differentiated tumours, capsular invasion and disease recurrence. Totally, 87% of anaplastic tumours were positive for SRC-1. Kaplan-Meier estimates of disease-free survival indicated that in thyroid cancer, SRC-1 strongly correlates with reduced disease-free survival (P<0.001), whereas NCoR predicted increased survival (P<0.001). These data suggest opposing roles for the coregulators SRC-1 and NCoR in thyroid tumour progression.
Assuntos
Adenocarcinoma Folicular/etiologia , Receptor alfa de Estrogênio/fisiologia , Receptor ErbB-2/fisiologia , Neoplasias da Glândula Tireoide/etiologia , Fatores de Transcrição/fisiologia , Adenocarcinoma Folicular/metabolismo , Adenocarcinoma Folicular/mortalidade , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma/etiologia , Carcinoma/metabolismo , Carcinoma/mortalidade , Estudos de Casos e Controles , Criança , Pré-Escolar , Proteínas Correpressoras/metabolismo , Proteínas Correpressoras/fisiologia , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Proteína Tirosina Quinases/fisiologia , Receptor ErbB-2/metabolismo , Análise de Sobrevida , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/mortalidade , Transativadores/metabolismo , Transativadores/fisiologia , Fatores de Transcrição/metabolismo , Células Tumorais Cultivadas , Adulto JovemRESUMO
Mechanisms of acquired resistance to endocrine therapy in breast cancer, a major clinical challenge, are poorly understood. We have used a mass spectrometry-based screen to identify proteins that are associated with the endocrine-resistant phenotype. In this study, we report the identification of a novel pathway of resistance to endocrine therapy involving interactions of the developmental transcription HOXC11 with the steroid receptor coactivator protein SRC-1, which is a strong predictor of reduced disease-free survival in breast cancer patients. HOXC11 and SRC-1 cooperate to regulate expression of the calcium-binding protein S100beta in resistant breast cancer cells. Nuclear HOXC11 and S100beta were found to strongly predict poor disease-free survival in breast cancer patients (n = 560; hazard ratios: 5.79 and 5.82, respectively; P < 0.0001). Elevated serum levels of S100beta detected in patients also predicted reduced disease-free survival (n = 80; hazard ratio: 5.3; P = 0.004). Our findings define a biomolecular interaction network that drives an adaptive response to endocrine therapy with negative consequences for survival in breast cancer.