RESUMEN
Bone metastasis affects >70% of patients with advanced breast cancer. However, the molecular mechanisms underlying this process remain unclear. On the basis of analysis of clinical datasets, and in vitro and in vivo experiments, we report that the ZNF217 oncogene is a crucial mediator and indicator of bone metastasis. Patients with high ZNF217 mRNA expression levels in primary breast tumours had a higher risk of developing bone metastases. MDA-MB-231 breast cancer cells stably transfected with ZNF217 (MDA-MB-231-ZNF217) showed the dysregulated expression of a set of genes with bone-homing and metastasis characteristics, which overlapped with two previously described 'osteolytic bone metastasis' gene signatures, while also highlighting the bone morphogenetic protein (BMP) pathway. The latter was activated in MDA-MB-231-ZNF217 cells, and its silencing by inhibitors (Noggin and LDN-193189) was sufficient to rescue ZNF217-dependent cell migration, invasion or chemotaxis towards the bone environment. Finally, by using non-invasive multimodal in vivo imaging, we found that ZNF217 increases the metastatic growth rate in the bone and accelerates the development of severe osteolytic lesions. Altogether, the findings of this study highlight ZNF217 as an indicator of the emergence of breast cancer bone metastasis; future therapies targeting ZNF217 and/or the BMP signalling pathway may be beneficial by preventing the development of bone metastases. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Asunto(s)
Neoplasias Óseas/genética , Neoplasias Óseas/secundario , Neoplasias de la Mama/genética , Transactivadores/genética , Animales , Proteínas Morfogenéticas Óseas/metabolismo , Neoplasias Óseas/metabolismo , Remodelación Ósea/genética , Neoplasias de la Mama/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Predisposición Genética a la Enfermedad , Xenoinjertos , Humanos , Estimación de Kaplan-Meier , Ratones Desnudos , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Trasplante de Neoplasias , ARN Mensajero/genética , ARN Neoplásico/genética , Transducción de Señal/genética , Transactivadores/biosíntesis , Células Tumorales CultivadasRESUMEN
INTRODUCTION: Increasing evidence indicates that microRNAs (miRNAs) are important players in oncogenesis. Considering the widespread use of aromatase inhibitors (AIs) in endocrine therapy as a first-line treatment for postmenopausal estrogen receptor α-positive breast cancer patients, identifying deregulated expression levels of miRNAs in association with AI resistance is of utmost importance. METHODS: To gain further insight into the molecular mechanisms underlying the AI resistance, we performed miRNA microarray experiments using a new model of acquired resistance to letrozole (Res-Let cells), obtained by long-term exposure of aromatase-overexpressing MCF-7 cells (MCF-7aro cells) to letrozole, and a model of acquired anastrozole resistance (Res-Ana cells). Three miRNAs (miR-125b, miR-205 and miR-424) similarly deregulated in both AI-resistant cell lines were then investigated in terms of their functional role in AI resistance development and breast cancer cell aggressiveness and their clinical relevance using a cohort of 65 primary breast tumor samples. RESULTS: We identified the deregulated expression of 33 miRNAs in Res-Let cells and of 18 miRNAs in Res-Ana cells compared with the sensitive MCF-7aro cell line. The top-ranked Kyoto Encyclopedia of Genes and Genomes pathways delineated by both miRNA signatures converged on the AKT/mTOR pathway, which was found to be constitutively activated in both AI-resistant cell lines. We report for the first time, to our knowledge, that ectopic overexpression of either miR-125b or miR-205, or the silencing of miR-424 expression, in the sensitive MCF-7aro cell line was sufficient to confer resistance to letrozole and anastrozole, to target and activate the AKT/mTOR pathway and to increase the formation capacity of stem-like and tumor-initiating cells possessing self-renewing properties. Increasing miR-125b expression levels was also sufficient to confer estrogen-independent growth properties to the sensitive MCF-7aro cell line. We also found that elevated miR-125b expression levels were a novel marker for poor prognosis in breast cancer and that targeting miR-125b in Res-Let cells overcame letrozole resistance. CONCLUSION: This study highlights that acquisition of specific deregulated miRNAs is a newly discovered alternative mechanism developed by AI-resistant breast cancer cells to achieve constitutive activation of the AKT/mTOR pathway and to develop AI resistance. It also highlights that miR-125b is a new biomarker of poor prognosis and a candidate therapeutic target in AI-resistant breast cancers.
Asunto(s)
Inhibidores de la Aromatasa/farmacología , Neoplasias de la Mama/genética , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Antineoplásicos/farmacología , Antineoplásicos Hormonales , Inhibidores de la Aromatasa/uso terapéutico , Biomarcadores de Tumor , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Análisis por Conglomerados , Estudios de Cohortes , Resistencia a Antineoplásicos/genética , Femenino , Perfilación de la Expresión Génica , Humanos , Letrozol , Células MCF-7 , Recurrencia Local de Neoplasia , Nitrilos/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Pronóstico , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Estrógenos , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Triazoles/farmacología , Células Tumorales Cultivadas , Regulación hacia ArribaRESUMEN
Acquisition of resistance to aromatase inhibitors (AIs) remains a major drawback in the treatment of estrogen receptor alpha (ERα)-positive breast cancers. The Res-Ana cells, a new model of acquired resistance to anastrozole, were established by long-term exposure of aromatase-overexpressing MCF-7 cells to this drug. These resistant cells developed ER-independent mechanisms of resistance and decreased sensitivity to the AI letrozole or to ERα antagonists. They also displayed a constitutive activation of the PI3K/Akt/mTOR pathway and a deregulated expression of several ErbB receptors. An observed increase in the phospho-Akt/Akt ratio between primary and matched recurrent breast tumors of patients who relapsed under anastrozole adjuvant therapy also argued for a pivotal role of the Akt pathway in acquired resistance to anastrozole. Ectopic overexpression of constitutively active Akt1 in control cells was sufficient to induce de novo resistance to anastrozole. Strikingly, combining anastrozole with the highly selective and allosteric Akt inhibitor MK-2206 or with the mTOR inhibitor rapamycin increased sensitivity to this AI in the control cells and was sufficient to overcome resistance and restore sensitivity to endocrine therapy in the resistant cells. Our findings lead to us proposing a model of anastrozole-acquired resistance based on the selection of cancer-initiating-like cells possessing self-renewing properties, intrinsic resistance to anastrozole and sensitivity to MK-2206. Altogether, our work demonstrated that the Akt/mTOR pathway plays a key role in resistance to anastrozole and that combining anastrozole with Akt/mTOR pathway inhibitors represents a promising strategy in the clinical management of hormone-dependent breast cancer patients.
Asunto(s)
Inhibidores de la Aromatasa/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Compuestos Heterocíclicos con 3 Anillos/farmacología , Nitrilos/farmacología , Triazoles/farmacología , Anastrozol , Antineoplásicos Hormonales/farmacología , Antineoplásicos Hormonales/uso terapéutico , Inhibidores de la Aromatasa/uso terapéutico , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Resistencia a Antineoplásicos , Receptores ErbB/biosíntesis , Receptor alfa de Estrógeno/metabolismo , Femenino , Humanos , Células MCF-7 , Recurrencia Local de Neoplasia/metabolismo , Nitrilos/uso terapéutico , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor ErbB-2/biosíntesis , Receptor ErbB-3/biosíntesis , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo , Triazoles/uso terapéuticoRESUMEN
BACKGROUND: ZNF217 is a candidate oncogene located at 20q13, a chromosomal region frequently amplified in breast cancers. The precise mechanisms involved in ZNF217 pro-survival function are currently unknown, and utmost importance is given to deciphering the role of ZNF217 in cancer therapy response. RESULTS: We provide evidence that stable overexpression of ZNF217 in MDA-MB-231 breast cancer cells conferred resistance to paclitaxel, stimulated cell proliferation in vitro associated with aberrant expression of several cyclins, and increased tumor growth in mouse xenograft models. Conversely, siRNA-mediated silencing of ZNF217 expression in MCF7 breast cancer cells, which possess high endogenous levels of ZNF217, led to decreased cell proliferation and increased sensitivity to paclitaxel. The paclitaxel resistance developed by ZNF217-overexpressing MDA-MB-231 cells was not mediated by the ABCB1/PgP transporter. However, ZNF217 was able to counteract the apoptotic signals mediated by paclitaxel as a consequence of alterations in the intrinsic apoptotic pathway through constitutive deregulation of the balance of Bcl-2 family proteins. Interestingly, ZNF217 expression levels were correlated with the oncogenic kinase Aurora-A expression levels, as ZNF217 overexpression led to increased expression of the Aurora-A protein, whereas ZNF217 silencing was associated with low Aurora-A expression levels. We showed that a potent Aurora-A kinase inhibitor was able to reverse paclitaxel resistance in the ZNF217-overexpressing cells. CONCLUSION: Altogether, these data suggest that ZNF217 might play an important role in breast neoplastic progression and chemoresistance, and that Aurora-A might be involved in ZNF217-mediated effects.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Neoplasias de la Mama/metabolismo , Regulación Neoplásica de la Expresión Génica , Paclitaxel/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Transactivadores/metabolismo , Animales , Antineoplásicos Fitogénicos/uso terapéutico , Aurora Quinasa A , Aurora Quinasas , Western Blotting , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/terapia , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Citometría de Flujo , Humanos , Ratones , Ratones Desnudos , Paclitaxel/uso terapéutico , Proteínas Serina-Treonina Quinasas/genética , ARN Interferente Pequeño , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transactivadores/genéticaRESUMEN
The Krüppel-like zinc finger protein ZNF217 is a candidate oncogene in breast cancer. In this study, we showed that high levels of expression of ZNF217 mRNA are associated with poor prognosis and the development of metastases in breast cancer. Overexpression of ZNF217 in breast cancer cells stimulated migration and invasion in vitro and promoted the development of spontaneous lung or node metastases in mice in vivo. ZNF217 also promoted epithelial-mesenchymal transition (EMT) in human mammary epithelial cells, and the TGF-ß-activated Smad signaling pathway was identified as a major driver of ZNF217-induced EMT. In addition, a TGF-ß autocrine loop sustained activation of the TGF-ß pathway in ZNF217-overexpressing mammary epithelial cells, most likely because of ZNF217-mediated direct upregulation of TGFB2 or TGFB3. Inhibition of the TGF-ß pathway led to the reversal of ZNF217-mediated EMT. Together, our findings indicate that ZNF217 mRNA expression may represent a novel prognostic biomarker in breast cancer. Therapeutic targeting of ZNF217 of the TGF-ß signaling pathway may benefit the subset of patients whose tumors express high levels of ZNF217.