RESUMO
Cancer cells induce a set of adaptive response pathways to survive in the face of stressors due to inadequate vascularization. One such adaptive pathway is the unfolded protein (UPR) or endoplasmic reticulum (ER) stress response mediated in part by the ER-localized transmembrane sensor IRE1 (ref. 2) and its substrate XBP1 (ref. 3). Previous studies report UPR activation in various human tumours, but the role of XBP1 in cancer progression in mammary epithelial cells is largely unknown. Triple-negative breast cancer (TNBC)--a form of breast cancer in which tumour cells do not express the genes for oestrogen receptor, progesterone receptor and HER2 (also called ERBB2 or NEU)--is a highly aggressive malignancy with limited treatment options. Here we report that XBP1 is activated in TNBC and has a pivotal role in the tumorigenicity and progression of this human breast cancer subtype. In breast cancer cell line models, depletion of XBP1 inhibited tumour growth and tumour relapse and reduced the CD44(high)CD24(low) population. Hypoxia-inducing factor 1α (HIF1α) is known to be hyperactivated in TNBCs. Genome-wide mapping of the XBP1 transcriptional regulatory network revealed that XBP1 drives TNBC tumorigenicity by assembling a transcriptional complex with HIF1α that regulates the expression of HIF1α targets via the recruitment of RNA polymerase II. Analysis of independent cohorts of patients with TNBC revealed a specific XBP1 gene expression signature that was highly correlated with HIF1α and hypoxia-driven signatures and that strongly associated with poor prognosis. Our findings reveal a key function for the XBP1 branch of the UPR in TNBC and indicate that targeting this pathway may offer alternative treatment strategies for this aggressive subtype of breast cancer.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fatores de Transcrição/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Animais , Antígeno CD24/metabolismo , Hipóxia Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Inativação Gênica , Humanos , Receptores de Hialuronatos/metabolismo , Camundongos , Invasividade Neoplásica , Recidiva Local de Neoplasia , Prognóstico , RNA Polimerase II/metabolismo , Fatores de Transcrição de Fator Regulador X , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Transcrição Gênica , Neoplasias de Mama Triplo Negativas/irrigação sanguínea , Neoplasias de Mama Triplo Negativas/genética , Resposta a Proteínas não Dobradas , Proteína 1 de Ligação a X-BoxRESUMO
Cell-based bone regeneration strategies offer promise for traumatic bone injuries, congenital defects, non-union fractures and other skeletal pathologies. Postnatal bone remodeling and fracture healing provide evidence that an osteochondroprogenitor cell is present in adult life that can differentiate to remodel or repair the fractured bone. However, cell-based skeletal repair in the clinic is still in its infancy, mostly due to poor characterization of progenitor cells and lack of knowledge about their in vivo behavior. Here, we took a combined approach of high-throughput screening, flow-based cell sorting and in vivo transplantation to isolate markers that identify osteochondroprogenitor cells. We show that the presence of tetraspanin CD9 enriches for osteochondroprogenitors within CD105(+) mesenchymal cells and that these cells readily form bone upon transplantation. In addition, we have used Thy1.2 and the ectonucleotidase CD73 to identify subsets within the CD9(+) population that lead to endochondral or intramembranous-like bone formation. Utilization of this unique cell surface phenotype to enrich for osteochondroprogenitor cells will allow for further characterization of the molecular mechanisms that regulate their osteogenic properties.
Assuntos
Biomarcadores/metabolismo , Regeneração Óssea/fisiologia , Condrócitos/fisiologia , Osteoblastos/fisiologia , Células-Tronco/fisiologia , 5'-Nucleotidase/metabolismo , Animais , Condrócitos/citologia , Condrócitos/metabolismo , Citometria de Fluxo , Ensaios de Triagem em Larga Escala , Processamento de Imagem Assistida por Computador , Rim/diagnóstico por imagem , Camundongos , Camundongos Endogâmicos C57BL , Análise em Microsséries , Osteoblastos/citologia , Osteoblastos/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Tetraspanina 29/metabolismo , Microtomografia por Raio-XRESUMO
Osteocytes within the mineralized bone matrix control bone remodeling by regulating osteoblast and osteoclast activity. Osteocytes express the aging suppressor Klotho, but the functional role of this protein in skeletal homeostasis is unknown. Here we identify Klotho expression in osteocytes as a potent regulator of bone formation and bone mass. Targeted deletion of Klotho from osteocytes led to a striking increase in bone formation and bone volume coupled with enhanced osteoblast activity, in sharp contrast to what is observed in Klotho hypomorphic (kl/kl) mice. Conversely, overexpression of Klotho in cultured osteoblastic cells inhibited mineralization and osteogenic activity during osteocyte differentiation. Further, the induction of chronic kidney disease with high-turnover renal osteodystrophy led to downregulation of Klotho in bone cells. This appeared to offset the skeletal impact of osteocyte-targeted Klotho deletion. Thus, our findings establish a key role of osteocyte-expressed Klotho in regulating bone metabolism and indicate a new mechanism by which osteocytes control bone formation.
Assuntos
Envelhecimento/metabolismo , Distúrbio Mineral e Ósseo na Doença Renal Crônica/patologia , Glucuronidase/metabolismo , Osteócitos/metabolismo , Osteogênese/fisiologia , Animais , Densidade Óssea , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Diferenciação Celular , Modelos Animais de Doenças , Regulação para Baixo , Feminino , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/metabolismo , Glucuronidase/genética , Humanos , Imuno-Histoquímica , Proteínas Klotho , Camundongos , Camundongos Knockout , Osteoblastos/fisiologia , Osteoclastos/fisiologia , Cultura Primária de Células , Transdução de SinaisRESUMO
The unfolded protein response (UPR) is a cellular homeostatic mechanism that is activated in many human cancers and plays pivotal roles in tumor progression and therapy resistance. However, the molecular mechanisms for UPR activation and regulation in cancer cells remain elusive. Here, we show that oncogenic MYC regulates the inositol-requiring enzyme 1 (IRE1)/X-box binding protein 1 (XBP1) branch of the UPR in breast cancer via multiple mechanisms. We found that MYC directly controls IRE1 transcription by binding to its promoter and enhancer. Furthermore, MYC forms a transcriptional complex with XBP1, a target of IRE1, and enhances its transcriptional activity. Importantly, we demonstrate that XBP1 is a synthetic lethal partner of MYC. Silencing of XBP1 selectively blocked the growth of MYC-hyperactivated cells. Pharmacological inhibition of IRE1 RNase activity with small molecule inhibitor 8866 selectively restrained the MYC-overexpressing tumor growth in vivo in a cohort of preclinical patient-derived xenograft models and genetically engineered mouse models. Strikingly, 8866 substantially enhanced the efficacy of docetaxel chemotherapy, resulting in rapid regression of MYC-overexpressing tumors. Collectively, these data establish the synthetic lethal interaction of the IRE1/XBP1 pathway with MYC hyperactivation and provide a potential therapy for MYC-driven human breast cancers.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Docetaxel/farmacologia , Sistemas de Liberação de Medicamentos , Endorribonucleases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Elementos de Resposta , Transdução de Sinais/efeitos dos fármacos , Proteína 1 de Ligação a X-Box/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Endorribonucleases/genética , Feminino , Humanos , Camundongos , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-myc/genética , Saccharomyces cerevisiae , Transdução de Sinais/genética , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Resposta a Proteínas não Dobradas/genética , Proteína 1 de Ligação a X-Box/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Sclerostin is a potent inhibitor of osteoblastogenesis. Interestingly, newly diagnosed multiple myeloma (MM) patients have high levels of circulating sclerostin that correlate with disease stage and fractures. However, the source and impact of sclerostin in MM remains to be defined. Our goal was to determine the role of sclerostin in the biology of MM and its bone microenvironment as well as investigate the effect of targeting sclerostin with a neutralizing antibody (scl-Ab) in MM bone disease. Here we confirm increased sclerostin levels in MM compared with precursor disease states like monoclonal gammopathy of undetermined significance (MGUS) and smoldering MM. Furthermore, we found that a humanized MM xenograft mouse model bearing human MM cells (NOD-SCID.CB17 male mice injected intravenously with 2.5 million of MM1.S-Luc-GFP cells) demonstrated significantly higher concentrations of mouse-derived sclerostin, suggesting a microenvironmental source of sclerostin. Associated with the increased sclerostin levels, activated ß-catenin expression levels were lower than normal in MM mouse bone marrow. Importantly, a high-affinity grade scl-Ab reversed osteolytic bone disease in this animal model. Because scl-Ab did not demonstrate significant in vitro anti-MM activity, we combined it with the proteasome inhibitor carfilzomib. Our data demonstrated that this combination therapy significantly inhibited tumor burden and improved bone disease in our in vivo MM mouse model. In agreement with our in vivo data, sclerostin expression was noted in marrow stromal cells and osteoblasts of MM patient bone marrow samples. Moreover, MM cells stimulated sclerostin expression in immature osteoblasts while inhibiting osteoblast differentiation in vitro. This was in part regulated by Dkk-1 secreted by MM cells and is a potential mechanism contributing to the osteoblast dysfunction noted in MM. Our data confirm the role of sclerostin as a potential therapeutic target in MM bone disease and provides the rationale for studying scl-Ab combined with proteasome inhibitors in MM. © 2016 American Society for Bone and Mineral Research.