RESUMO
Brain metastasis (br-met) develops in an immunologically unique br-met niche. Central nervous system-native myeloid cells (CNS-myeloids) and bone-marrow-derived myeloid cells (BMDMs) cooperatively regulate brain immunity. The phenotypic heterogeneity and specific roles of these myeloid subsets in shaping the br-met niche to regulate br-met outgrowth have not been fully revealed. Applying multimodal single-cell analyses, we elucidated a heterogeneous but spatially defined CNS-myeloid response during br-met outgrowth. We found Ccr2+ BMDMs minimally influenced br-met while CNS-myeloid promoted br-met outgrowth. Additionally, br-met-associated CNS-myeloid exhibited downregulation of Cx3cr1. Cx3cr1 knockout in CNS-myeloid increased br-met incidence, leading to an enriched interferon response signature and Cxcl10 upregulation. Significantly, neutralization of Cxcl10 reduced br-met, while rCxcl10 increased br-met and recruited VISTAHi PD-L1+ CNS-myeloid to br-met lesions. Inhibiting VISTA- and PD-L1-signaling relieved immune suppression and reduced br-met burden. Our results demonstrate that loss of Cx3cr1 in CNS-myeloid triggers a Cxcl10-mediated vicious cycle, cultivating a br-met-promoting, immune-suppressive niche.
Assuntos
Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/secundário , Quimiocina CXCL10/metabolismo , Terapia de Imunossupressão , Células Mieloides/metabolismo , Animais , Células da Medula Óssea/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Receptor 1 de Quimiocina CX3C/metabolismo , Sistema Nervoso Central/patologia , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Interferons/metabolismo , Macrófagos/metabolismo , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Testes de Neutralização , Fenótipo , Linfócitos T/imunologia , Transcriptoma/genéticaRESUMO
INTRODUCTION: The androgen receptor (AR) is widely expressed in breast cancers and has been proposed as a therapeutic target in estrogen receptor alpha (ER) negative breast cancers that retain AR. However, controversy exists regarding the role of AR, particularly in ER + tumors. Enzalutamide, an AR inhibitor that impairs nuclear localization of AR, was used to elucidate the role of AR in preclinical models of ER positive and negative breast cancer. METHODS: We examined nuclear AR to ER protein ratios in primary breast cancers in relation to response to endocrine therapy. The effects of AR inhibition with enzalutamide were examined in vitro and in preclinical models of ER positive and negative breast cancer that express AR. RESULTS: In a cohort of 192 women with ER + breast cancers, a high ratio of AR:ER (≥2.0) indicated an over four fold increased risk for failure while on tamoxifen (HR = 4.43). The AR:ER ratio had an independent effect on risk for failure above ER % staining alone. AR:ER ratio is also an independent predictor of disease-free survival (HR = 4.04, 95% CI: 1.68, 9.69; p = 0.002) and disease specific survival (HR = 2.75, 95% CI: 1.11, 6.86; p = 0.03). Both enzalutamide and bicalutamide inhibited 5-alpha-dihydrotestosterone (DHT)-mediated proliferation of breast cancer lines in vitro; however, enzalutamide uniquely inhibited estradiol (E2)-mediated proliferation of ER+/AR + breast cancer cells. In MCF7 xenografts (ER+/AR+) enzalutamide inhibited E2-driven tumor growth as effectively as tamoxifen by decreasing proliferation. Enzalutamide also inhibited DHT- driven tumor growth in both ER positive (MCF7) and negative (MDA-MB-453) xenografts, but did so by increasing apoptosis. CONCLUSIONS: AR to ER ratio may influence breast cancer response to traditional endocrine therapy. Enzalutamide elicits different effects on E2-mediated breast cancer cell proliferation than bicalutamide. This preclinical study supports the initiation of clinical studies evaluating enzalutamide for treatment of AR+ tumors regardless of ER status, since it blocks both androgen- and estrogen- mediated tumor growth.
Assuntos
Antagonistas de Androgênios/uso terapêutico , Antagonistas de Receptores de Andrógenos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Receptor alfa de Estrogênio/metabolismo , Feniltioidantoína/análogos & derivados , Anilidas/uso terapêutico , Animais , Antineoplásicos Hormonais/uso terapêutico , Apoptose/efeitos dos fármacos , Benzamidas , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Intervalo Livre de Doença , Feminino , Humanos , Células MCF-7 , Camundongos , Pessoa de Meia-Idade , Transplante de Neoplasias , Nitrilas/uso terapêutico , Feniltioidantoína/uso terapêutico , Receptores Androgênicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tamoxifeno/uso terapêutico , Compostos de Tosil/uso terapêutico , Transplante HeterólogoRESUMO
The purpose of this pilot study was to explore the impact of a certified nurse's aide (CNA)-led interdisciplinary teamwork and communication intervention on perceived quality of work environment and six-month job intentions. CNAs are frequently excluded from team communication and decision-making, which often leads to job dissatisfaction with high levels of staff turnover. Using a mixed quantitative and qualitative approach with pre- post-program design, the intervention utilized the strategy of debriefing from the national patient safety initiative, TeamSTEPPS. Inherent in the program design, entitled Long Term Care (LTC) Team Talk, was the involvement of the CNAs in the development of the intervention as an empowering process on two wings of a transitional care unit in a long-term care facility in upstate NY. CNAs' perceptions of work environment quality were measured using a Quality of Work Life (QWL) instrument. Additionally, job turnover intent within six months was assessed. Results indicated improved scores on nearly all QWL subscales anticipated to be impacted, and enhanced perceived empowerment of the CNAs on each wing albeit through somewhat different experiential processes. The program is highly portable and can potentially be implemented in a variety of long-term care settings.
Assuntos
Satisfação no Emprego , Assistentes de Enfermagem , Equipe de Assistência ao Paciente , Poder Psicológico , Qualidade de Vida , Certificação , Comunicação , HumanosRESUMO
Carcinogenesis is a complex process during which cells undergo genetic and epigenetic alterations. These changes can lead tumor cells to acquire characteristics that enable movement from the primary site of origin when conditions become unfavorable. Such characteristics include gain of front-rear polarity, increased migration/invasion, and resistance to anoikis, which facilitate tumor survival during metastasis. An epithelial to mesenchymal transition (EMT) constitutes one way that cancer cells can gain traits that promote tumor progression and metastasis. Two microRNA (miRNA) families, the miR-200 and miR-221 families, play crucial opposing roles that affect the differentiation state of breast cancers. These two families are differentially expressed between the luminal A subtype of breast cancer as compared to the less well-differentiated triple negative breast cancers (TNBCs) that exhibit markers indicative of an EMT. The miR-200 family promotes a well-differentiated epithelial phenotype, while high miR-221/222 results in a poorly differentiated, mesenchymal-like phenotype. This review focuses on the mechanisms (specific proven targets) by which these two miRNA families exert opposing effects on cellular plasticity during breast tumorigenesis and metastasis.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Transição Epitelial-Mesenquimal/genética , MicroRNAs/genética , Animais , Feminino , HumanosRESUMO
Chemotherapy prior to immune checkpoint blockade (ICB) treatment appears to improve ICB efficacy but resistance to ICB remains a clinical challenge and is attributed to highly plastic myeloid cells associating with the tumor immune microenvironment (TIME). Here we show by CITE-seq single-cell transcriptomic and trajectory analyses that neoadjuvant low-dose metronomic chemotherapy (MCT) leads to a characteristic co-evolution of divergent myeloid cell subsets in female triple-negative breast cancer (TNBC). Specifically, we identify that the proportion of CXCL16 + myeloid cells increase and a high STAT1 regulon activity distinguishes Programmed Death Ligand 1 (PD-L1) expressing immature myeloid cells. Chemical inhibition of STAT1 signaling in MCT-primed breast cancer sensitizes TNBC to ICB treatment, which underscores the STAT1's role in modulating TIME. In summary, we leverage single-cell analyses to dissect the cellular dynamics in the tumor microenvironment (TME) following neoadjuvant chemotherapy and provide a pre-clinical rationale for modulating STAT1 in combination with anti-PD-1 for TNBC patients.
Assuntos
Neoplasias de Mama Triplo Negativas , Humanos , Feminino , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Radioimunoterapia , Células Mieloides , Quimiocina CXCL16 , Microambiente Tumoral , Fator de Transcrição STAT1/genéticaRESUMO
INTRODUCTION: miR-200c and other members of the miR-200 family promote epithelial identity by directly targeting ZEB1 and ZEB2, which repress E-cadherin and other genes involved in polarity. Loss of miR-200c is often observed in carcinoma cells that have undergone epithelial to mesenchymal transition (EMT). Restoration of miR-200c to such cells leads to a reduction in stem cell-like characteristics, reduced migration and invasion, and increased sensitivity to taxanes. Here we investigate the functional role of novel targets of miR-200c in the aggressive behavior of breast and endometrial cancer cells. METHODS: Putative target genes of miR-200c identified by microarray profiling were validated as direct targets using dual luciferase reporter assays. Following restoration of miR-200c to triple negative breast cancer and type 2 endometrial cancer cell lines that had undergone EMT, levels of endogenous target mRNA and respective protein products were measured. Migration and sensitivity to anoikis were determined using wound healing assays or cell-death ELISAs and viability assays respectively. RESULTS: We found that restoration of miR-200c suppresses anoikis resistance, a novel function for this influential miRNA. We identified novel targets of miR-200c, including genes encoding fibronectin 1 (FN1), moesin (MSN), neurotrophic tyrosine receptor kinase type 2 (NTRK2 or TrkB), leptin receptor (LEPR), and Rho GTPase activating protein 19 (ARHGAP19). These targets all encode proteins normally expressed in cells of mesenchymal or neuronal origin; however, in carcinoma cells that lack miR-200c they become aberrantly expressed and contribute to the EMT phenotype and aggressive behavior. We showed that these targets are inhibited upon restoration of miR-200c to aggressive breast and endometrial cancer cells. We demonstrated that inhibition of MSN and/or FN1 is sufficient to mediate the ability of miR-200c to suppress cell migration. Lastly, we showed that targeting of TrkB mediates the ability of miR-200c to restore anoikis sensitivity. CONCLUSIONS: miR-200c maintains the epithelial phenotype not only by targeting ZEB1/2, which usually facilitates restoration of E-cadherin expression, but also by actively repressing a program of mesenchymal and neuronal genes involved in cell motility and anoikis resistance.
Assuntos
Anoikis , Neoplasias da Mama/metabolismo , Movimento Celular , Neoplasias do Endométrio/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal , Feminino , Fibronectinas/genética , Proteínas Ativadoras de GTPase/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Proteínas dos Microfilamentos/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptor trkB/genética , Receptores para Leptina/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Homeobox 2 de Ligação a E-box com Dedos de Zinco , Homeobox 1 de Ligação a E-box em Dedo de ZincoRESUMO
Age is a major risk factor for cancer. While the importance of age related genetic alterations in cells on cancer progression is well documented, the effect of aging extracellular matrix (ECM) has been overlooked. This study shows that the aging breast ECM alone is sufficient to drive normal human mammary epithelial cells (KTB21) to a more invasive and cancer-like phenotype, while promoting motility and invasiveness in MDA-MB-231 cells. Decellularized breast matrix from aged mice leads to loss of E-cadherin membrane localization in KTB21 cells, increased cell motility and invasion, and increased production of inflammatory cytokines and cancer-related proteins. The aged matrix upregulates cancer-related genes in KTB21 cells and enriches a cell subpopulation highly expressing epithelial-mesenchymal transition-related genes. Lysyl oxidase knockdown reverts the aged matrix-induced changes to the young levels; it relocalizes E-cadherin to cell membrane, and reduces cell motility, invasion, and cytokine production. These results show for the first time that the aging ECM harbors key biochemical, physical, and mechanical cues contributing to invasive and cancer-like behavior in healthy and cancer mammary cells. Differential response of cells to young and aged ECMs can lead to identification of new targets for cancer treatment and prevention.
Assuntos
Envelhecimento/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Matriz Extracelular/metabolismo , Animais , Mama/metabolismo , Mama/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Invasividade Neoplásica , FenótipoRESUMO
Breast cancer brain metastases (BCBM) have a 5-20 year latency and account for 30% of mortality; however, mechanisms governing adaptation to the brain microenvironment remain poorly defined. We combine time-course RNA-sequencing of BCBM development with a Drosophila melanogaster genetic screen, and identify Rab11b as a functional mediator of metastatic adaptation. Proteomic analysis reveals that Rab11b controls the cell surface proteome, recycling proteins required for successful interaction with the microenvironment, including integrin ß1. Rab11b-mediated control of integrin ß1 surface expression allows efficient engagement with the brain ECM, activating mechanotransduction signaling to promote survival. Lipophilic statins prevent membrane association and activity of Rab11b, and we provide proof-of principle that these drugs prevent breast cancer adaptation to the brain microenvironment. Our results identify Rab11b-mediated recycling of integrin ß1 as regulating BCBM, and suggest that the recycleome, recycling-based control of the cell surface proteome, is a previously unknown driver of metastatic adaptation and outgrowth.
Assuntos
Neoplasias Encefálicas/metabolismo , Neoplasias da Mama/patologia , Integrina beta1/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/fisiopatologia , Neoplasias Encefálicas/secundário , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Proliferação de Células , Modelos Animais de Doenças , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Feminino , Humanos , Integrina beta1/genética , Camundongos , Camundongos Endogâmicos C57BL , Metástase Neoplásica , Transporte Proteico , Transdução de Sinais , Microambiente Tumoral , Proteínas rab de Ligação ao GTP/genéticaRESUMO
With evolutionary drug resistance impacting efforts to treat disease, the need for small molecules that exhibit novel molecular mechanisms of action is paramount. In this study, we combined scaffold-directed synthesis with a hybrid experimental and transcriptome analysis to identify bis-spirooxindole cyclopropanes that inhibit cancer cell proliferation through disruption of ribosomal function. These findings demonstrate the value of an integrated, biologically inspired synthesis and assay strategy for the accelerated identification of first-in-class cancer therapeutic candidates.
Assuntos
Antineoplásicos/farmacologia , Ciclopropanos/farmacologia , Oxindóis/farmacologia , RNA Neoplásico/efeitos dos fármacos , Ribossomos/efeitos dos fármacos , Compostos de Espiro/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Ciclopropanos/síntese química , Ciclopropanos/química , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Oxindóis/síntese química , Oxindóis/química , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Compostos de Espiro/síntese química , Compostos de Espiro/química , Relação Estrutura-Atividade , Transcriptoma , Células Tumorais CultivadasRESUMO
Lacking targetable molecular drivers, triple-negative breast cancer (TNBC) is the most clinically challenging subtype of breast cancer. In this study, we reveal that Death Effector Domain-containing DNA-binding protein (DEDD), which is overexpressed in > 60% of TNBCs, drives a mitogen-independent G1/S cell cycle transition through cytoplasm localization. The gain of cytosolic DEDD enhances cyclin D1 expression by interacting with heat shock 71 kDa protein 8 (HSC70). Concurrently, DEDD interacts with Rb family proteins and promotes their proteasome-mediated degradation. DEDD overexpression renders TNBCs vulnerable to cell cycle inhibition. Patients with TNBC have been excluded from CDK 4/6 inhibitor clinical trials due to the perceived high frequency of Rb-loss in TNBCs. Interestingly, our study demonstrated that, irrespective of Rb status, TNBCs with DEDD overexpression exhibit a DEDD-dependent vulnerability to combinatorial treatment with CDK4/6 inhibitor and EGFR inhibitor in vitro and in vivo. Thus, our study provided a rationale for the clinical application of CDK4/6 inhibitor combinatorial regimens for patients with TNBC.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Lapatinib/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Antineoplásicos/farmacologia , Ciclo Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/genética , Receptores ErbB/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Receptor ErbB-2/antagonistas & inibidores , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismoRESUMO
Zinc-finger E-box-binding homeobox 1 (ZEB1) is a transcription factor containing two clusters of Kruppel-type zinc-fingers, by which it binds E-box-like sequences on target DNAs. A role for ZEB1 in tumor progression, specifically, epithelial to mesenchymal transitions, has recently been revealed. ZEB1 acts as a master repressor of E-cadherin and other epithelial markers. We previously demonstrated that ZEB1 is confined to the stromal compartment in normal endometrium and low-grade endometrial cancers. Here, we quantify ZEB1 protein expression in endometrial samples from 88 patients and confirm that it is expressed at significantly higher levels in the tumor-associated stroma of low-grade endometrioid adenocarcinomas (type I endometrial cancers) compared to hyperplastic or normal endometrium. In addition, as we previously reported, ZEB1 is aberrantly expressed in the epithelial-derived tumor cells of highly aggressive endometrial cancers, such as FIGO grade 3 endometrioid adenocarcinomas, uterine serous carcinomas, and malignant mixed Müllerian tumors (classified as type II endometrial cancers). We now demonstrate, in both human endometrial cancer specimens and cell lines, that when ZEB1 is inappropriately expressed in epithelial-derived tumor cells, E-cadherin expression is repressed, and that this inverse relationship correlates with increased migratory and invasive potential. Forced expression of ZEB1 in the nonmigratory, low-grade, relatively differentiated Ishikawa cell line renders them migratory. Conversely, reduction of ZEB1 in a highly migratory and aggressive type II cell line, Hec50co, results in reduced migratory capacity. Thus, ZEB1 may be a biomarker of aggressive endometrial cancers at high risk of recurrence. It may help identify women who would most benefit from chemotherapy. Furthermore, if expression of ZEB1 in type II endometrial cancers could be reversed, it might be exploited as therapy for these highly aggressive tumors.
Assuntos
Carcinoma Endometrioide/metabolismo , Neoplasias do Endométrio/metabolismo , Endométrio/metabolismo , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Caderinas/metabolismo , Carcinoma Endometrioide/classificação , Carcinoma Endometrioide/patologia , Linhagem Celular Tumoral , Progressão da Doença , Hiperplasia Endometrial/metabolismo , Hiperplasia Endometrial/patologia , Neoplasias do Endométrio/classificação , Neoplasias do Endométrio/patologia , Endométrio/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Proteínas de Homeodomínio/genética , Humanos , Histerectomia , Imuno-Histoquímica , Pessoa de Meia-Idade , Recidiva Local de Neoplasia , Estadiamento de Neoplasias , RNA Mensageiro/metabolismo , Análise Serial de Tecidos , Fatores de Transcrição/genética , Homeobox 1 de Ligação a E-box em Dedo de ZincoRESUMO
The tumor microenvironment (TME), the dynamic tissue space in which the tumor exists, plays a significant role in tumor initiation, and is a key contributor in cancer progression; however, little is known about tumor-induced changes in the adjacent tissue stroma. Herein, tumor-induced changes in the TME were explored at the morphologic and molecular level to further understand cancer progression. Tumor-adjacent mammary glands (TAG) displayed altered branching morphology, expansion of myofibroblasts, and increased mammosphere formation, broadly suggesting a tumor-induced field effect. FACS analysis of TAGs demonstrated an increased number of Lin-CD24+/CD49+ enriched mammary gland stem cells (MaSC), suggesting deregulated tissue homeostasis in TAGs. Comparative transcriptome analysis of TAGs and contralateral control glands coupled with meta-analysis on differentially expressed genes with two breast cancer stromal patient microarray datasets identified shared upregulation of STAT1. Knockdown of STAT1 in cancer-associated fibroblast (CAF) cocultured with human breast cancer cells altered cancer cell proliferation, indicating a role for STAT1 as a stromal contributor of tumorigenesis. Furthermore, depletion of STAT1 in CAFs significantly reduced periductal reactive fibrosis and delayed early breast cancer progression in vivo Finally, cotreatment with fludarabine, a FDA-approved STAT1 activation inhibitor and DNA synthesis inhibitor, in combination with doxorubicin, showed enhanced therapeutic efficacy in treating mouse mammary gland tumors. Taken together, these results demonstrate that stromal STAT1 expression promotes tumor progression and is a potential therapeutic target for breast cancer.Implications: Tumors induce stromal STAT1-dependent cytokine secretion that promotes tumor cell proliferation and can be targeted using clinically-approved inhibitors of STAT1. Mol Cancer Res; 15(5); 585-97. ©2017 AACR.
Assuntos
Neoplasias da Mama/patologia , Fibroblastos Associados a Câncer/citologia , Carcinoma Intraductal não Infiltrante/metabolismo , Fator de Transcrição STAT1/genética , Animais , Neoplasias da Mama/genética , Fibroblastos Associados a Câncer/metabolismo , Carcinoma Intraductal não Infiltrante/genética , Linhagem Celular Tumoral , Proliferação de Células , Técnicas de Cocultura , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Microambiente Tumoral , Regulação para CimaRESUMO
The impact of altered amino acid metabolism on cancer progression is not fully understood. We hypothesized that a metabolic transcriptome shift during metastatic evolution is crucial for brain metastasis. Here, we report a powerful impact in this setting caused by epigenetic upregulation of glutamate decarboxylase 1 (GAD1), a regulator of the GABA neurotransmitter metabolic pathway. In cell-based culture and brain metastasis models, we found that downregulation of the DNA methyltransferase DNMT1 induced by the brain microenvironment-derived clusterin resulted in decreased GAD1 promoter methylation and subsequent upregulation of GAD1 expression in brain metastatic tumor cells. In a system to dynamically visualize cellular metabolic responses mediated by GAD1, we monitored the cytosolic NADH:NAD+ equilibrium in tumor cells. Reducing GAD1 in metastatic cells by primary glia cell coculture abolished the capacity of metastatic cells to utilize extracellular glutamine, leading to cytosolic accumulation of NADH and increased oxidative status. Similarly, genetic or pharmacologic disruption of the GABA metabolic pathway decreased the incidence of brain metastasis in vivo Taken together, our results show how epigenetic changes in GAD1 expression alter local glutamate metabolism in the brain metastatic microenvironment, contributing to a metabolic adaption that facilitates metastasis outgrowth in that setting. Cancer Res; 77(11); 2844-56. ©2017 AACR.
Assuntos
Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/secundário , Metilação de DNA , Glutamato Descarboxilase/metabolismo , Animais , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Biologia Computacional , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Metástase Neoplásica , Transfecção , Microambiente Tumoral , Regulação para CimaRESUMO
The anti-diabetic drug metformin (1,1-dimethylbiguanide hydrochloride) reduces both the incidence and mortality of several types of cancer. Metformin has been shown to selectively kill cancer stem cells, and triple-negative breast cancer (TNBC) cell lines are more sensitive to the effects of metformin as compared to luminal breast cancer. However, the mechanism underlying the enhanced susceptibility of TNBC to metformin has not been elucidated. Expression profiling of metformin-treated TNBC lines revealed fatty acid synthase (FASN) as one of the genes most significantly downregulated following 24 h of treatment, and a decrease in FASN protein was also observed. Since FASN is critical for de novo fatty acid synthesis and is important for the survival of TNBC, we hypothesized that FASN downregulation facilitates metformin-induced apoptosis. Profiling studies also exposed a rapid metformin-induced increase in miR-193 family members, and miR-193b directly targets the FASN 3'UTR. Addition of exogenous miR-193b mimic to untreated TNBC cells decreased FASN protein expression and increased apoptosis of TNBC cells, while spontaneously immortalized, non-transformed breast epithelial cells remained unaffected. Conversely, antagonizing miR-193 activity impaired the ability of metformin to decrease FASN and cause cell death. Further, the metformin-stimulated increase in miR-193 resulted in reduced mammosphere formation by TNBC lines. These studies provide mechanistic insight into metformin-induced killing of TNBC.
Assuntos
Ácido Graxo Sintase Tipo I/metabolismo , Metformina/uso terapêutico , MicroRNAs/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Regiões 3' não Traduzidas/genética , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Ácido Graxo Sintase Tipo I/genética , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Glândulas Mamárias Humanas/efeitos dos fármacos , MicroRNAs/genética , Células-Tronco Neoplásicas/patologia , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
MicroRNAs (miRNAs) are small non-coding RNA molecules capable of regulating multiple target genes through repression of mRNA stability or translation. miRNAs regulate a wide variety of developmental and pathological processes, and many have oncogenic or tumor suppressor activity. Carcinoma cells can undergo changes resembling a partial or transient epithelial-mesenchymal transition (EMT) as part of the metastatic cascade. Numerous miRNAs regulate EMT by targeting genes that control epithelial or mesenchymal characteristics. The involvement of miRNAs in feedback loops with EMT-regulating transcription factors reveals a critical role in governing epithelial plasticity. In this review we summarize miRNAs involved in carcinoma cell plasticity.
Assuntos
Células Epiteliais/patologia , Regulação Neoplásica da Expressão Gênica , MicroRNAs/fisiologia , Neoplasias/genética , Neoplasias/patologia , Caderinas/genética , Caderinas/metabolismo , Retroalimentação Fisiológica , Humanos , Transdução de Sinais/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Anoikis is apoptosis initiated upon cell detachment from the native extracellular matrix. Since survival upon detachment from basement membrane is required for metastasis, the ability to resist anoikis contributes to the metastatic potential of breast tumors. miR-200c, a potent repressor of epithelial to mesenchymal transition, is expressed in luminal breast cancers, but is lost in more aggressive basal-like, or triple negative breast cancers (TNBC). We previously demonstrated that miR-200c restores anoikis sensitivity to TNBC cells by directly targeting the neurotrophic receptor tyrosine kinase, TrkB. In this study, we identify a TrkB ligand, neurotrophin 3 (NTF3), as capable of activating TrkB to induce anoikis resistance, and show that NTF3 is also a direct target of miR-200c. We present the first evidence that anoikis resistant TNBC cells up-regulate both TrkB and NTF3 when suspended, and show that this up-regulation is necessary for survival in suspension. We further demonstrate that NF-κB activity increases 6 fold in suspended TNBC cells, and identify RelA and NF-κB1 as the transcription factors responsible for suspension-induced up-regulation of TrkB and NTF3. Consequently, inhibition of NF-κB activity represses anoikis resistance. Taken together, our findings define a critical mechanism for transcriptional and post-transcriptional control of suspension-induced up-regulation of TrkB and NTF3 in anoikis resistant breast cancer cells.
Assuntos
Anoikis/genética , Neoplasias da Mama , MicroRNAs , Receptor trkB , Apoptose/genética , Comunicação Autócrina , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Neurotrofina 3/genética , Neurotrofina 3/metabolismo , Receptor trkB/genética , Receptor trkB/metabolismo , Transdução de Sinais/genéticaRESUMO
Progesterone receptors (PRs) mediate response to progestins in the normal breast and breast cancer. To determine if liganded PR regulate microRNAs (miRNAs) as a component of their action, we profiled mature miRNA levels following progestin treatment. Indeed, 28 miRNAs are significantly altered by 6h of progestin treatment. Many progestin-responsive genes are putative targets of progestin-regulated miRNAs; for example, progestin treatment decreases miR-29, thereby relieving repression of one of its direct targets, the gene encoding ATPase, Na(+)/K(+) transporting, beta 1 polypeptide (ATP1B1). Thus, liganded PR regulates ATP1B1 through sites in the promoter and the 3'UTR, to achieve maximal tight hormonal regulation of ATP1B1 protein via both transcriptional and translational control. We find that ATP1B1 serves to limit migration and invasion in breast cancer cells. Lastly, we demonstrate that PR itself is regulated by a progestin-upregulated miRNA, miR-513a-5p, providing a novel mechanism for tight control of PR protein expression.
Assuntos
Neoplasias da Mama/metabolismo , MicroRNAs/genética , Progestinas/farmacologia , Receptores de Progesterona/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Regiões 3' não Traduzidas , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular , Feminino , Genes Reporter , Humanos , Luciferases , MicroRNAs/metabolismo , Análise em Microsséries , Progestinas/metabolismo , Regiões Promotoras Genéticas , Biossíntese de Proteínas/efeitos dos fármacos , ATPase Trocadora de Sódio-Potássio/genética , Transcrição Gênica/efeitos dos fármacos , TransfecçãoRESUMO
A therapeutic intervention that could decrease tumor burden and increase sensitivity to chemotherapy would have a significant impact on the high morbidity rate associated with ovarian cancer. miRNAs have emerged as potential therapeutic candidates due to their ability to downregulate multiple targets involved in tumor progression and chemoresistance. miRNA-200c (miR-200c) is downregulated in ovarian cancer cell lines and stage III ovarian tumors, and low miR-200c correlates with poor prognosis. miR-200c increases sensitivity to taxanes in vitro by targeting class III ß-tubulin gene (TUBB3), a tubulin known to mediate chemoresistance. Indeed, we find that patients with tumors having low TUBB3 had significantly prolonged survival (average survival 52.73 ± 4.08 months) as compared with those having high TUBB3 (average survival 42.56 ± 3.19 months). miR-200c also targets TrkB, a mediator of resistance to anoikis. We show that restoration of miR-200c to ovarian cancer cells results in increased anoikis sensitivity and reduced adherence to biologic substrates in vitro. Because both chemo- and anoikis-resistance are critical steps in the progression of ovarian cancer, we sought to determine how restoration of miR-200c affects tumor burden and chemosensitivity in an in vivo preclinical model of ovarian cancer. Restoration of miR-200c in an intraperitoneal xenograft model of human ovarian cancer results in decreased tumor formation and tumor burden. Furthermore, even in established tumors, restoration of miR-200c, alone or in combination with paclitaxel, results in significantly decreased tumor burden. Our study suggests that restoration of miR-200c immediately before cytotoxic chemotherapy may allow for a better response or lower effective dose.
Assuntos
MicroRNAs/administração & dosagem , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/terapia , Paclitaxel/farmacologia , Animais , Anoikis/efeitos dos fármacos , Anoikis/genética , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , MicroRNAs/biossíntese , MicroRNAs/genética , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Transdução Genética/métodos , Transfecção , Tubulina (Proteína)/biossíntese , Carga Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We focus on unique roles of miR-200c in breast, ovarian, and endometrial cancers. Members of the miR-200 family target ZEB1, a transcription factor which represses E-cadherin and other genes involved in polarity. We demonstrate that the double negative feedback loop between miR-200c and ZEB1 is functional in some, but not all cell lines. Restoration of miR-200c to aggressive cancer cells causes a decrease in migration and invasion. These effects are independent of E-cadherin status. Additionally, we observe that restoration of miR-200c to ovarian cancer cells causes a decrease in adhesion to laminin. We have previously reported that reintroduction of miR-200c to aggressive cells that lack miR-200c expression restores sensitivity to paclitaxel. We now prove that this ability is a result of direct targeting of class III beta-tubulin (TUBB3). Introduction of a TUBB3 expression construct lacking the miR-200c target site into cells transfected with miR-200c mimic results in no change in sensitivity to paclitaxel. Lastly, we observe a decrease in proliferation in cells transfected with miR-200c mimic, and cells where ZEB1 is knocked down stably, demonstrating that the ability of miR-200c to enhance sensitivity to paclitaxel is not due to an increased proliferation rate.
RESUMO
To identify microRNAs (miRNAs) associated with estrogen receptor (ESR1) status, we profiled luminal A, ESR1+ breast cancer cell lines versus triple negative (TN), which lack ERα, progesterone receptor and Her2/neu. Although two thirds of the differentially expressed miRNAs are higher in ESR1+ breast cancer cells, some miRNAs, such as miR-222/221 and miR-29a, are dramatically higher in ESR1- cells (â¼100- and 16-fold higher, respectively). MiR-222/221 (which target ESR1 itself) and miR-29a are predicted to target the 3' UTR of Dicer1. Addition of these miRNAs to ESR1+ cells reduces Dicer protein, whereas antagonizing miR-222 in ESR1- cells increases Dicer protein. We demonstrate via luciferase reporter assays that these miRNAs directly target the Dicer1 3' UTR. In contrast, miR-200c, which promotes an epithelial phenotype, is 58-fold higher in the more well-differentiated ERα+ cells, and restoration of miR-200c to ERα- cells causes increased Dicer protein, resulting in increased levels of other mature miRNAs typically low in ESR1- cells. Together, our findings explain why Dicer is low in ERα negative breast cancers, since such cells express high miR-221/222 and miR-29a levels (which repress Dicer) and low miR-200c (which positively affect Dicer levels). Furthermore, we find that miR-7, which is more abundant in ERα+ cells and is estrogen regulated, targets growth factor receptors and signaling intermediates such as EGFR, IGF1R, and IRS-2. In summary, miRNAs differentially expressed in ERα+ versus ERα- breast cancers actively control some of the most distinguishing characteristics of the luminal A and TN subtypes, such as ERα itself, Dicer, and growth factor receptor levels.