RESUMO
The epithelial splicing regulatory proteins 1 and 2 (ESRP1 and ESRP2) control the epithelial-to-mesenchymal transition (EMT) splicing program in cancer. However, their role in breast cancer recurrence is unclear. In this study, we report that high levels of ESRP1, but not ESRP2, are associated with poor prognosis in estrogen receptor positive (ER+) breast tumors. Knockdown of ESRP1 in endocrine-resistant breast cancer models decreases growth significantly and alters the EMT splicing signature, which we confirm using TCGA SpliceSeq data of ER+ BRCA tumors. However, these changes are not accompanied by the development of a mesenchymal phenotype or a change in key EMT-transcription factors. In tamoxifen-resistant cells, knockdown of ESRP1 affects lipid metabolism and oxidoreductase processes, resulting in the decreased expression of fatty acid synthase (FASN), stearoyl-CoA desaturase 1 (SCD1), and phosphoglycerate dehydrogenase (PHGDH) at both the mRNA and protein levels. Furthermore, ESRP1 knockdown increases the basal respiration and spare respiration capacity. This study reports a novel role for ESRP1 that could form the basis for the prevention of tamoxifen resistance in ER+ breast cancer.
Assuntos
Neoplasias da Mama/metabolismo , Metabolismo Energético , Redes e Vias Metabólicas , Proteínas de Ligação a RNA/metabolismo , Receptores de Estrogênio/metabolismo , Processamento Alternativo , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Modelos de Riscos Proporcionais , Fatores de Processamento de RNA/metabolismo , Proteínas de Ligação a RNA/genéticaRESUMO
The αvß3 integrin has been shown to promote aggressive phenotypes in many types of cancers, including prostate cancer. We show that GFP-labeled αvß3 derived from cancer cells circulates in the blood and is detected in distant lesions in NOD scid gamma (NSG) mice. We, therefore, hypothesized that αvß3 travels through exosomes and tested its levels in pools of vesicles, which we designate extracellular vesicles highly enriched in exosomes (ExVs), and in exosomes isolated from the plasma of prostate cancer patients. Here, we show that the αvß3 integrin is found in patient blood exosomes purified by sucrose or iodixanol density gradients. In addition, we provide evidence that the αvß3 integrin is transferred through ExVs isolated from prostate cancer patient plasma to ß3-negative recipient cells. We also demonstrate the intracellular localization of ß3-GFP transferred via cancer cell-derived ExVs. We show that the ExVs present in plasma from prostate cancer patients contain higher levels of αvß3 and CD9 as compared to plasma ExVs from age-matched subjects who are not affected by cancer. Furthermore, using PSMA antibody-bead mediated immunocapture, we show that the αvß3 integrin is expressed in a subset of exosomes characterized by PSMA, CD9, CD63, and an epithelial-specific marker, Trop-2. Finally, we present evidence that the levels of αvß3, CD63, and CD9 remain unaltered in ExVs isolated from the blood of prostate cancer patients treated with enzalutamide. Our results suggest that detecting exosomal αvß3 integrin in prostate cancer patients could be a clinically useful and non-invasive biomarker to follow prostate cancer progression. Moreover, the ability of αvß3 integrin to be transferred from ExVs to recipient cells provides a strong rationale for further investigating the role of αvß3 integrin in the pathogenesis of prostate cancer and as a potential therapeutic target.
Assuntos
Adenocarcinoma/genética , Biomarcadores Tumorais/genética , Exossomos/metabolismo , Integrina alfaVbeta3/genética , Neoplasias da Próstata/genética , Adenocarcinoma/sangue , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antineoplásicos/farmacologia , Benzamidas , Biomarcadores Tumorais/sangue , Exossomos/química , Expressão Gênica , Humanos , Integrina alfaVbeta3/sangue , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Nitrilas , Células PC-3 , Feniltioidantoína/análogos & derivados , Feniltioidantoína/farmacologia , Neoplasias da Próstata/sangue , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Tetraspanina 29/sangue , Tetraspanina 29/genética , Tetraspanina 30/sangue , Tetraspanina 30/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Co-occurrence of Flt3ITD and TET2 mutations provoke an animal model of AML by epigenetic repression of Wnt pathway antagonists, including RUNX3, and by hyperexpression of ID1, encoding Wnt agonist. These affect HOXA over-expression and treatment resistance. A comparable epigenetic phenotype was identified among adult AML patients needing novel intervention. We chose combinations of targeted agents acting on distinct effectors, at the levels of both signal transduction and chromatin remodeling, in relapsed/refractory AML's, including Flt3ITD+ve, described with a signature of repressed tumor suppressor genes, involving Wnt antagonist RUNX3, occurring along with ID1 and HOXA over-expressions. We tracked patient response to combination of Flt3/Raf inhibitor, Sorafenib, and Vorinostat, pan-histone deacetylase inhibitor, without or with added Bortezomib, in consecutive phase I trials. A striking association of rapid objective remissions (near-complete, complete responses) was noted to accompany induced early pharmacodynamic changes within patient blasts in situ, involving these effectors, significantly linking RUNX3/Wnt antagonist de-repression (80%) and ID1 downregulation (85%), to a response, also preceded by profound HOXA9 repression. Response occurred in context of concurrent TET2 mutation/hypomorphy and Flt3ITD+ve mutation (83% of complete responses). Addition of Bortezomib to the combination was vital to attainment of complete response in Flt3ITD+ve cases exhibiting such Wnt pathway dysregulation.
RESUMO
Telomere dysfunction resulting from telomere shortening and deregulation of shelterin components has been linked to the pathogenesis of age-related disorders, including cancer. Recent evidence suggests that BRCA1/2 (BRCA1 and BRCA2) tumor suppressor gene products play an important role in telomere maintenance. Although telomere shortening has been reported in BRCA1/2 carriers, the direct effects of BRCA1/2 haploinsufficiency on telomere maintenance and predisposition to cancer development are not completely understood. In this study, we assessed the telomere-associated and telomere-proximal gene expression profiles in peripheral blood leukocytes from patients with a BRCA1 or BRCA2 mutation, compared to samples from sporadic and familial breast cancer individuals. We found that 25 genes, including TINF2 gene (a negative regulator of telomere length), were significantly differentially expressed in BRCA1 carriers. Leukocyte telomere length analysis revealed that BRCA1/2 carriers had relatively shorter telomeres than healthy controls. Further, affected BRCA1/2 carriers were well differentiated from unaffected BRCA1/2 carriers by the expression of telomere-proximal genes. Our results link BRCA1/2 haploinsufficiency to changes in telomere length, telomere-associated as well as telomere-proximal gene expression. Thus, this work supports the effect of BRCA1/2 haploinsufficiency in the biology underlying telomere dysfunction in cancer development. Future studies evaluating these findings will require a large study population.
Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Perfilação da Expressão Gênica , Leucócitos/metabolismo , Homeostase do Telômero/genética , Adulto , Idoso , Neoplasias da Mama/genética , Feminino , Predisposição Genética para Doença/genética , Heterozigoto , Humanos , Pessoa de Meia-Idade , MutaçãoRESUMO
Adipose tissue is a rich source of multipotent mesenchymal stem-like cells, located in the perivascular niche. Based on their surface markers, these have been assigned to two main categories: CD31- /CD45- /CD34+ /CD146- cells (adventitial stromal/stem cells [ASCs]) and CD31- /CD45- /CD34- /CD146+ cells (pericytes [PCs]). These populations display heterogeneity of unknown significance. We hypothesized that aldehyde dehydrogenase (ALDH) activity, a functional marker of primitivity, could help to better define ASC and PC subclasses. To this end, the stromal vascular fraction from a human lipoaspirate was simultaneously stained with fluorescent antibodies to CD31, CD45, CD34, and CD146 antigens and the ALDH substrate Aldefluor, then sorted by fluorescence-activated cell sorting. Individual ASCs (n = 67) and PCs (n = 73) selected from the extremities of the ALDH-staining spectrum were transcriptionally profiled by Fluidigm single-cell quantitative polymerase chain reaction for a predefined set (n = 429) of marker genes. To these single-cell data, we applied differential expression and principal component and clustering analysis, as well as an original gene coexpression network reconstruction algorithm. Despite the stochasticity at the single-cell level, covariation of gene expression analysis yielded multiple network connectivity parameters suggesting that these perivascular progenitor cell subclasses possess the following order of maturity: (a) ALDHbr ASC (most primitive); (b) ALDHdim ASC; (c) ALDHbr PC; (d) ALDHdim PC (least primitive). This order was independently supported by specific combinations of class-specific expressed genes and further confirmed by the analysis of associated signaling pathways. In conclusion, single-cell transcriptional analysis of four populations isolated from fat by surface markers and enzyme activity suggests a developmental hierarchy among perivascular mesenchymal stem cells supported by markers and coexpression networks. Stem Cells 2017;35:1273-1289.
Assuntos
Tecido Adiposo/citologia , Linhagem da Célula , Redes Reguladoras de Genes , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Aldeído Desidrogenase/metabolismo , Diferenciação Celular/genética , Feminino , Citometria de Fluxo , Regulação da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Pericitos/citologia , Análise de Célula ÚnicaRESUMO
Molecular mechanisms underlying development of acute pneumonitis and/or late fibrosis following thoracic irradiation remain poorly understood. Here, we hypothesize that heterogeneity in disease progression and phenotypic expression of radiation-induced lung disease (RILD) across murine strains presents an opportunity to better elucidate mechanisms driving tissue response toward pneumonitis and/or fibrosis. Distinct differences in disease progression were observed in age- and sex-matched CBA/J, C57L/J and C57BL/6J mice over 1â year after graded doses of whole-thorax lung irradiation (WTLI). Separately, comparison of gene expression profiles in lung tissue 24â h post-exposure demonstrated >5000 genes to be differentially expressed (P<0.01; >twofold change) between strains with early versus late onset of disease. An immediate divergence in early tissue response between radiation-sensitive and -resistant strains was observed. In pneumonitis-prone C57L/J mice, differentially expressed genes were enriched in proinflammatory pathways, whereas in fibrosis-prone C57BL/6J mice, genes were enriched in pathways involved in purine and pyrimidine synthesis, DNA replication and cell division. At 24â h post-WTLI, different patterns of cellular damage were observed at the ultrastructural level among strains but microscopic damage was not yet evident under light microscopy. These data point toward a fundamental difference in patterns of early pulmonary tissue response to WTLI, consistent with the macroscopic expression of injury manifesting weeks to months after exposure. Understanding the mechanisms underlying development of RILD might lead to more rational selection of therapeutic interventions to mitigate healthy tissue damage.
Assuntos
Progressão da Doença , Perfilação da Expressão Gênica , Pneumopatias/genética , Pneumopatias/patologia , Lesões por Radiação/genética , Lesões por Radiação/patologia , Proteínas de Fase Aguda/genética , Proteínas de Fase Aguda/metabolismo , Animais , Relação Dose-Resposta à Radiação , Feminino , Regulação da Expressão Gênica , Estimativa de Kaplan-Meier , Pulmão/patologia , Pulmão/efeitos da radiação , Pneumopatias/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Lesões por Radiação/fisiopatologia , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Risco , Tórax/patologia , Tórax/efeitos da radiaçãoRESUMO
The PI3K pathway is activated in approximately 70% of breast cancers. PIK3CA gene mutations or amplifications that affect the PI3K p110α subunit account for activation of this pathway in 20% to 40% of cases, particularly in estrogen receptor alpha (ERα)-positive breast cancers. AKT family of kinases, AKT1-3, are the downstream targets of PI3K and these kinases activate ERα. Although several inhibitors of PI3K have been developed, none has proven effective in the clinic, partly due to an incomplete understanding of the selective routing of PI3K signaling to specific AKT isoforms. Accordingly, we investigated in this study the contribution of specific AKT isoforms in connecting PI3K activation to ERα signaling, and we also assessed the utility of using the components of PI3K-AKT isoform-ERα signaling axis as predictive biomarkers of response to PI3K inhibitors. Using a variety of physiologically relevant model systems with defined natural or knock-in PIK3CA mutations and/or PI3K hyperactivation, we show that PIK3CA-E545K mutations (found in â¼20% of PIK3CA-mutant breast cancers), but not PIK3CA-H1047R mutations (found in 55% of PIK3CA-mutant breast cancers), preferentially activate AKT1. Our findings argue that AKT1 signaling is needed to respond to estrogen and PI3K inhibitors in breast cancer cells with PIK3CA-E545K mutation, but not in breast cancer cells with other PIK3CA mutations. This study offers evidence that personalizing treatment of ER-positive breast cancers to PI3K inhibitor therapy may benefit from an analysis of PIK3CA-E545K-AKT1-estrogen signaling pathways. Cancer Res; 76(13); 3989-4001. ©2016 AACR.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Receptor alfa de Estrogênio/genética , Mutação/genética , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais , Neoplasias da Mama/patologia , Classe I de Fosfatidilinositol 3-Quinases , Feminino , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
UNLABELLED: Why breast cancers become resistant to tamoxifen despite continued expression of the estrogen receptor-α (ERα) and what factors are responsible for high HER2 expression in these tumors remains an enigma. HOXB7 chromatin immunoprecipitation analysis followed by validation showed that HOXB7 physically interacts with ERα, and that the HOXB7-ERα complex enhances transcription of many ERα target genes, including HER2. Investigating strategies for controlling HOXB7, our studies revealed that MYC, stabilized via phosphorylation mediated by EGFR-HER2 signaling, inhibits transcription of miR-196a, a HOXB7 repressor. This leads to increased expression of HOXB7, ER target genes, and HER2. Repressing MYC using small-molecule inhibitors reverses these events and causes regression of breast cancer xenografts. The MYC-HOXB7-HER2 signaling pathway is eminently targetable in endocrine-resistant breast cancer. SIGNIFICANCE: HOXB7 acts as an ERα cofactor regulating a myriad of ER target genes, including HER2, in tamoxifen-resistant breast cancer. HOXB7 expression is controlled by MYC via transcriptional regulation of the HOXB7 repressor miR-196a; consequently, antagonists of MYC cause reversal of selective ER modulator resistance both in vitro and in vivo.
Assuntos
Antineoplásicos Hormonais/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Receptor alfa de Estrogênio/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Receptor ErbB-2/metabolismo , Animais , Antineoplásicos Hormonais/uso terapêutico , Sítios de Ligação , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Análise por Conglomerados , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Proteínas de Homeodomínio/genética , Humanos , Camundongos , MicroRNAs/genética , Prognóstico , Ligação Proteica , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Interferência de RNA , Receptor ErbB-2/genética , Transdução de Sinais , Tamoxifeno/farmacologia , Tamoxifeno/uso terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Breast cancer metastasizes to bone, visceral organs, and/or brain depending on the subtype, which may involve activation of a host organ-specific signaling network in metastatic cells. To test this possibility, we determined gene expression patterns in MDA-MB-231 cells and its mammary fat pad tumor (TMD-231), lung-metastasis (LMD-231), bone-metastasis (BMD-231), adrenal-metastasis (ADMD-231) and brain-metastasis (231-BR) variants. When gene expression between metastases was compared, 231-BR cells showed the highest gene expression difference followed by ADMD-231, LMD-231, and BMD-231 cells. Neuronal transmembrane proteins SLITRK2, TMEM47, and LYPD1 were specifically overexpressed in 231-BR cells. Pathway-analyses revealed activation of signaling networks that would enable cancer cells to adapt to organs of metastasis such as drug detoxification/oxidative stress response/semaphorin neuronal pathway in 231-BR, Notch/orphan nuclear receptor signals involved in steroidogenesis in ADMD-231, acute phase response in LMD-231, and cytokine/hematopoietic stem cell signaling in BMD-231 cells. Only NF-κB signaling pathway activation was common to all except BMD-231 cells. We confirmed NF-κB activation in 231-BR and in a brain metastatic variant of 4T1 cells (4T1-BR). Dimethylaminoparthenolide inhibited NF-κB activity, LYPD1 expression, and proliferation of 231-BR and 4T1-BR cells. Thus, transcriptome change enabling adaptation to host organs is likely one of the mechanisms associated with organ-specific metastasis and could potentially be targeted therapeutically.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Metástase Neoplásica/genética , Transdução de Sinais/fisiologia , Transcriptoma , Linhagem Celular Tumoral , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The overexpression or amplification of the human epidermal growth factor receptor 2 gene (HER2/neu) is associated with high risk of brain metastasis (BM). The identification of patients at highest immediate risk of BM could optimize screening and facilitate interventional trials. We performed gene expression analysis using complementary deoxyribonucleic acid-mediated annealing, selection, extension and ligation and real-time quantitative reverse transcription PCR (qRT-PCR) in primary tumor samples from two independent cohorts of advanced HER2 positive breast cancer patients. Additionally, we analyzed predictive relevance of clinicopathological factors in this series. Study group included discovery Cohort A (84 patients) and validation Cohort B (75 patients). The only independent variables associated with the development of early BM in both cohorts were the visceral location of first distant relapse [Cohort A: hazard ratio (HR) 7.4, 95 % CI 2.4-22.3; p < 0.001; Cohort B: HR 6.1, 95 % CI 1.5-25.6; p = 0.01] and the lack of trastuzumab administration in the metastatic setting (Cohort A: HR 5.0, 95 % CI 1.4-10.0; p = 0.009; Cohort B: HR 10.0, 95 % CI 2.0-100.0; p = 0.008). A profile including 13 genes was associated with early (≤36 months) symptomatic BM in the discovery cohort. This was refined by qRT-PCR to a 3-gene classifier (RAD51, HDGF, TPR) highly predictive of early BM (HR 5.3, 95 % CI 1.6-16.7; p = 0.005; multivariate analysis). However, predictive value of the classifier was not confirmed in the independent validation Cohort B. The presence of visceral metastases and the lack of trastuzumab administration in the metastatic setting apparently increase the likelihood of early BM in advanced HER2-positive breast cancer.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/secundário , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Receptor ErbB-2/genética , Adulto , Idoso , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/terapia , Neoplasias da Mama/mortalidade , Neoplasias da Mama/terapia , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/mortalidade , Carcinoma Ductal de Mama/secundário , Carcinoma Ductal de Mama/terapia , Carcinoma Lobular/genética , Carcinoma Lobular/mortalidade , Carcinoma Lobular/secundário , Carcinoma Lobular/terapia , Estudos de Coortes , Terapia Combinada , Feminino , Seguimentos , Perfilação da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de SobrevidaRESUMO
CONTEXT: Next-generation sequencing allows for high-throughput processing and sensitive variant detection in multiple genes from small samples. For many diseases, including cancer, a comprehensive mutational profile of a targeted list of genes can be used to simultaneously inform patient care, establish eligibility for ongoing clinical trials, and further research. OBJECTIVE: To validate a pan-cancer, next-generation-sequencing assay for use in the clinical laboratory. DESIGN: DNA was extracted from 68 clinical specimens (formalin-fixed, paraffin-embedded; fine-needle aspirates; peripheral blood; or bone marrow) and 5 normal controls. Sixty-four DNA samples (94%; 64 of 68) were successfully processed with the TruSeq Amplicon Cancer Panel (Illumina Inc, San Diego, California) and sequenced in 4 sequencing runs. The data were analyzed at 4 different filter settings for sequencing coverage and variant frequency cutoff. RESULTS: Libraries created from 40 specimens could be successfully sequenced in a single run and still yield sufficient coverage for robust data analysis of individual samples. Sensitivity for mutation detection down to 5% was demonstrated using dilutions of clinical specimens and control samples. The test was highly repeatable and reproducible and showed 100% concordance with clinically validated Sanger sequencing results. Comparison to an alternate next-generation sequencing technology was performed by also processing 9 of the specimens with the AmpliSeq Cancer Hotspot Panel (version 2; Life Technologies, Grand Island, New York). Thirty of the 31 (97%) TruSeq-detected variants covered by the designs of both panels were confirmed. CONCLUSIONS: A sensitive, high-throughput, pan-cancer mutation panel for sequencing of cancer hot-spot mutations in 42 genes was validated for routine use in clinical testing.
Assuntos
Técnicas de Laboratório Clínico/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mutação , Neoplasias/genética , DNA de Neoplasias/química , DNA de Neoplasias/genética , DNA de Neoplasias/isolamento & purificação , Predisposição Genética para Doença/genética , Testes Genéticos/métodos , Humanos , Neoplasias/diagnóstico , Reação em Cadeia da Polimerase , Reprodutibilidade dos TestesRESUMO
De novo or acquired resistance to endocrine therapy limits its utility in a significant number of estrogen receptor-positive (ER-positive) breast cancers. It is crucial to identify novel targets for therapeutic intervention and improve the success of endocrine therapies. Splicing factor 3b, subunit 1 (SF3B1) mutations are described in luminal breast cancer albeit in low frequency. In this study, we evaluated the role of SF3B1 and SF3B3, critical parts of the SF3b splicing complex, in ER-positive endocrine resistance. To ascertain the role of SF3B1/SF3B3 in endocrine resistance, their expression levels were evaluated in ER-positive/endocrine-resistant cell lines (MCF-7/LCC2 and MCF-7/LCC9) using a real-time quantitative reverse transcription PCR (qRT-PCR). To further determine their clinical relevance, expression analysis was performed in a cohort of 60 paraffin-embedded ER-positive, node-negative breast carcinomas with low, intermediate, and high Oncotype DX recurrence scores. Expression levels of SF3B1 and SF3B3 and their prognostic value were validated in large cohorts using publicly available gene expression data sets including The Cancer Genome Atlas. SF3B1 and SF3B3 levels were significantly increased in ERα-positive cells with acquired tamoxifen (MCF-7/LCC2; both P<0.0002) and fulvestrant/tamoxifen resistance (MCF-7/LCC9; P=0.008 for SF3B1 and P=0.0006 for SF3B3). Expression levels of both MCF-7/LCC2 and MCF-7/LCC9 were not affected by additional treatments with E2 and/or tamoxifen. Furthermore, qRT-PCR analysis confirmed that SF3B3 expression is significantly upregulated in Oncotype DX high-risk groups when compared with low risk (P=0.019). Similarly, in publicly available breast cancer gene expression data sets, overexpression of SF3B3, but not SF3B1, was significantly correlated with overall survival. Furthermore, the correlation was significant in ER-positive, but not in ER-negative tumors.This is the first study to document the role of SF3B3 in endocrine resistance and prognosis in ER-positive breast cancer. Potential strategies for therapeutic targeting of the splicing mechanism(s) need to be evaluated.
Assuntos
Biomarcadores Tumorais/análise , Neoplasias da Mama/patologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Proteínas de Ligação a RNA/biossíntese , Antineoplásicos Hormonais/uso terapêutico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Receptor alfa de Estrogênio/biossíntese , Feminino , Humanos , Estimativa de Kaplan-Meier , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/mortalidade , Recidiva Local de Neoplasia/patologia , Reação em Cadeia da Polimerase , Prognóstico , Fatores de Processamento de RNA , Transcriptoma , Regulação para CimaRESUMO
BACKGROUND: We recently published PROGgene, a tool that can be used to study prognostic implications of genes in various cancers. The first version of the tool had several areas for improvement. In this paper we present some major enhancements we have made on the existing tool in the new version, PROGgeneV2. RESULTS: In PROGgeneV2, we have made several modifications to enhance survival analysis capability of the tool. First, we have increased the repository of public studies catalogued in our tool by almost two folds. We have also added additional functionalities to perform survival analysis in a variety of new ways. Survival analysis can now be performed on a) single genes b) multiple genes as a signature, c) ratio of expression of two genes, and d) curated/published gene signatures in new version. Users can now also adjust the survival analysis models for available covariates. Users can study prognostic implications of entire gene signatures in different cancer types, which are searchable by keywords. Also, unique to our tool, in the new version, users will be able to upload and use their own datasets to perform survival analysis on genes of interest. CONCLUSIONS: We believe, like its predecessor, PROGGeneV2 will continue to be useful for the scientific community for formulating research hypotheses and designing mechanistic studies. With added datasets PROGgeneV2 is the most comprehensive survival analysis tool available. PROGgeneV2 is available at http://www.compbio.iupui.edu/proggene.
Assuntos
Bases de Dados Genéticas , Neoplasias/genética , Neoplasias/mortalidade , Software , Conjuntos de Dados como Assunto , Humanos , Prognóstico , Navegador , Fluxo de TrabalhoRESUMO
Children with neurofibromatosis type 1 (NF1) are increasingly recognized as having a high prevalence of social difficulties and autism spectrum disorders (ASDs). We demonstrated a selective social learning deficit in mice with deletion of a single Nf1 allele (Nf1(+/-)), along with greater activation of the mitogen-activated protein kinase pathway in neurons from the amygdala and frontal cortex, structures that are relevant to social behaviors. The Nf1(+/-) mice showed aberrant amygdala glutamate and GABA neurotransmission, deficits in long-term potentiation and specific disruptions in the expression of two proteins that are associated with glutamate and GABA neurotransmission: a disintegrin and metalloprotease domain 22 (Adam22) and heat shock protein 70 (Hsp70), respectively. All of these amygdala disruptions were normalized by the additional deletion of the p21 protein-activated kinase (Pak1) gene. We also rescued the social behavior deficits in Nf1(+/-) mice with pharmacological blockade of Pak1 directly in the amygdala. These findings provide insights and therapeutic targets for patients with NF1 and ASDs.
Assuntos
Tonsila do Cerebelo/metabolismo , Hipocampo/fisiologia , Aprendizagem/fisiologia , Neurofibromina 1/metabolismo , Comportamento Social , Quinases Ativadas por p21/metabolismo , Animais , Comportamento Animal , Transtornos Globais do Desenvolvimento Infantil/metabolismo , Transtornos Globais do Desenvolvimento Infantil/fisiopatologia , Modelos Animais de Doenças , Potenciação de Longa Duração/fisiologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação/genética , Neurofibromina 1/deficiênciaRESUMO
Metastasis suppressor genes (MSGs) have contributed to an understanding of regulatory pathways unique to the lethal metastatic process. When re-expressed in experimental models, MSGs block cancer spread to, and colonization of distant sites without affecting primary tumor formation. Genes have been identified with expression patterns inverse to a single MSG, and found to encode functional, druggable signaling pathways. We now hypothesize that common signaling pathways mediate the effects of multiple MSGs. By gene expression profiling of human MCF7 breast carcinoma cells expressing a scrambled siRNA, or siRNAs to each of 19 validated MSGs (NME1, BRMS1, CD82, CDH1, CDH2, CDH11, CASP8, MAP2K4, MAP2K6, MAP2K7, MAPK14, GSN, ARHGDIB, AKAP12, DRG1, CD44, PEBP1, RRM1, KISS1), we identified genes whose expression was significantly opposite to at least five MSGs. Five genes were selected for further analysis: PDE5A, UGT1A, IL11RA, DNM3 and OAS1. After stable downregulation of each candidate gene in the aggressive human breast cancer cell line MDA-MB-231T, in vitro motility was significantly inhibited. Two stable clones downregulating PDE5A (phosphodiesterase 5A), an enzyme involved in the regulation of cGMP-specific signaling, exhibited no difference in cell proliferation, but reduced motility by 47 and 66 % compared to the empty vector-expressing cells (p = 0.01 and p = 0.005). In an experimental metastasis assay, two shPDE5A-MDA-MB-231T clones produced 47-62 % fewer lung metastases than shRNA-scramble expressing cells (p = 0.045 and p = 0.009 respectively). This study demonstrates that previously unrecognized genes are inversely related to the expression of multiple MSGs, contribute to aspects of metastasis, and may stand as novel therapeutic targets.
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Neoplasias da Mama/patologia , Perfilação da Expressão Gênica , Metástase Neoplásica/genética , Animais , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/genética , Regulação para Baixo , Feminino , Humanos , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Bevacizumab, the recombinant antibody targeting vascular endothelial growth factor (VEGF), improves progression-free but not overall survival in metastatic breast cancer. To seek further insights in resistance mechanisms to bevacizumab at the molecular level, we developed VEGF and non-VEGF-driven ER-positive MCF7-derived xenograft models allowing comparison of tumor response at different timepoints. VEGF gene (MV165) overexpressing xenografts were initially sensitive to bevacizumab, but eventually acquired resistance. In contrast, parental MCF7 cells derived tumors were de novo insensitive to bevacizumab. Microarray analysis with qRT-PCR validation revealed that Follistatin (FST) and NOTCH were the top signaling pathways associated with resistance in VEGF-driven tumors (P<0.05). Based on the presence of VEGF, treatment with bevacizumab resulted in altered patterns of metagenes and PAM50 gene expression. In VEGF-driven model after short and long-term bevacizumab treatments, a change in the intrinsic subtype (luminal to myoepithelial/basal-like) was observed in association with increased expression of genes implicated with cancer stem cell phenotype (P<0.05). Our results show that the presence or absence of VEGF expression affects the response to bevacizumab therapy and gene pathways. In particular, long-term bevacizumab treatment shifts the cancer cells to a more aggressive myoepithelial/basal subtype in VEGF-expressing model, but not in non-VEGF model. These findings could shed light on variable results to anti-VEGF therapy in patients and emphasize the importance of patient stratification based on the VEGF expression. Our data strongly suggest consideration of patient subgroups for treatment and designing novel combinatory therapies in the clinical setting.
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Circulating microRNAs (miRNA) are emerging as important biomarkers of various diseases, including cancer. Intriguingly, circulating levels of several miRNAs are lower in patients with cancer compared with healthy individuals. In this study, we tested the hypothesis that a circulating miRNA might serve as a surrogate of the effects of cancer on miRNA expression or release in distant organs. Here we report that circulating levels of the muscle-enriched miR486 is lower in patients with breast cancer compared with healthy individuals and that this difference is replicated faithfully in MMTV-PyMT and MMTV-Her2 transgenic mouse models of breast cancer. In tumor-bearing mice, levels of miR486 were relatively reduced in muscle, where there was elevated expression of the miR486 target genes PTEN and FOXO1A and dampened signaling through the PI3K/AKT pathway. Skeletal muscle expressed lower levels of the transcription factor MyoD, which controls miR486 expression. Conditioned media (CM) obtained from MMTV-PyMT and MMTV-Her2/Neu tumor cells cultured in vitro were sufficient to elicit reduced levels of miR486 and increased PTEN and FOXO1A expression in C2C12 murine myoblasts. Cytokine analysis implicated tumor necrosis factor α (TNFα) and four additional cytokines as mediators of miR486 expression in CM-treated cells. Because miR486 is a potent modulator of PI3K/AKT signaling and the muscle-enriched transcription factor network in cardiac/skeletal muscle, our findings implicated TNFα-dependent miRNA circuitry in muscle differentiation and survival pathways in cancer.
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Neoplasias da Mama/fisiopatologia , Coração/fisiopatologia , MicroRNAs/metabolismo , Músculo Esquelético/fisiopatologia , Animais , Neoplasias da Mama/sangue , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Diferenciação Celular/fisiologia , Feminino , Humanos , Masculino , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/fisiopatologia , Camundongos , Camundongos Transgênicos , MicroRNAs/biossíntese , MicroRNAs/sangue , MicroRNAs/genética , Músculo Esquelético/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: Identification of prognostic mRNA biomarkers has been done for various cancer types. The data that are published from such studies are archived in public repositories. There are hundreds of such datasets available for multiple cancer types in public repositories. Wealth of such data can be utilized to study prognostic implications of mRNA in different cancers as well as in different populations or subtypes of same cancer. DESCRIPTION: We have created a web application that can be used for studying prognostic implications of mRNA biomarkers in a variety of cancers. We have compiled data from public repositories such as GEO, EBI Array Express and The Cancer Genome Atlas for creating this tool. With 64 patient series from 18 cancer types in our database, this tool provides the most comprehensive resource available for survival analysis to date. The tool is called PROGgene and it is available at http://www.compbio.iupui.edu/proggene. CONCLUSIONS: We present this tool as a hypothesis generation tool for researchers to identify potential prognostic mRNA biomarkers to follow up with further research. For this reason, we have kept the web application very simple and straightforward. We believe this tool will be useful in accelerating biomarker discovery in cancer and quickly providing results that may indicate disease-specific prognostic value of specific biomarkers.
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Recently developed genomics-based tools are allowing repositioning of Food and Drug Administration (FDA)-approved drugs as cancer treatments, which were employed to identify drugs that target cancer stem cells (CSCs) of breast cancer. Gene expression datasets of CSCs from six studies were subjected to connectivity map to identify drugs that may ameliorate gene expression patterns unique to CSCs. All-trans retinoic acid (ATRA) was negatively connected with gene expression in CSCs. ATRA reduced mammosphere-forming ability of a subset of breast cancer cells, which correlated with induction of apoptosis, reduced expression of SOX2 but elevated expression of its antagonist CDX2. SOX2/CDX2 ratio had prognostic relevance in CSC-enriched breast cancers. K-ras mutant breast cancer cell line enriched for CSCs was resistant to ATRA, which was reversed by MAP kinase inhibitors. Thus, ATRA alone or in combination can be tested for efficacy using SOX2, CDX2, and K-ras mutation/MAPK activation status as biomarkers of response.
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Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Aprovação de Drogas/métodos , Perfilação da Expressão Gênica/métodos , Células-Tronco Neoplásicas/metabolismo , Tretinoína/uso terapêutico , Antineoplásicos/uso terapêutico , Neoplasias da Mama/diagnóstico , Humanos , Células-Tronco Neoplásicas/efeitos dos fármacos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Resultado do Tratamento , Estados Unidos , United States Food and Drug AdministrationRESUMO
PURPOSE: Thymoma represents one of the rarest of all malignancies. Stage and completeness of resection have been used to ascertain postoperative therapeutic strategies albeit with limited prognostic accuracy. A molecular classifier would be useful to improve the assessment of metastatic behaviour and optimize patient management. METHODS: qRT-PCR assay for 23 genes (19 test and four reference genes) was performed on multi-institutional archival primary thymomas (nâ=â36). Gene expression levels were used to compute a signature, classifying tumors into classes 1 and 2, corresponding to low or high likelihood for metastases. The signature was validated in an independent multi-institutional cohort of patients (nâ=â75). RESULTS: A nine-gene signature that can predict metastatic behavior of thymomas was developed and validated. Using radial basis machine modeling in the training set, 5-year and 10-year metastasis-free survival rates were 77% and 26% for predicted low (class 1) and high (class 2) risk of metastasis (Pâ=â0.0047, log-rank), respectively. For the validation set, 5-year metastasis-free survival rates were 97% and 30% for predicted low- and high-risk patients (Pâ=â0.0004, log-rank), respectively. The 5-year metastasis-free survival rates for the validation set were 49% and 41% for Masaoka stages I/II and III/IV (Pâ=â0.0537, log-rank), respectively. In univariate and multivariate Cox models evaluating common prognostic factors for thymoma metastasis, the nine-gene signature was the only independent indicator of metastases (Pâ=â0.036). CONCLUSION: A nine-gene signature was established and validated which predicts the likelihood of metastasis more accurately than traditional staging. This further underscores the biologic determinants of the clinical course of thymoma and may improve patient management.