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Correctly identifying perturbed biological pathways is a critical step in uncovering basic disease mechanisms and developing much-needed therapeutic strategies. However, whether current tools are optimal for unbiased discovery of relevant pathways remains unclear. Here, we create "Benchmark" to critically evaluate existing tools and find that most function sub-optimally. We thus develop the "Pathway Ensemble Tool" (PET), which outperforms existing methods. Deploying PET, we identify prognostic pathways across 12 cancer types. PET-identified prognostic pathways offer additional insights, with genes within these pathways serving as reliable biomarkers for clinical outcomes. Additionally, normalizing these pathways using drug repurposing strategies represents therapeutic opportunities. For example, the top predicted repurposed drug for bladder cancer, a CDK2/9 inhibitor, represses cell growth in vitro and in vivo. We anticipate that using Benchmark and PET for unbiased pathway discovery will offer additional insights into disease mechanisms across a spectrum of diseases, enabling biomarker discovery and therapeutic strategies.
Assuntos
Benchmarking , Neoplasias , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/genética , Transdução de Sinais/efeitos dos fármacos , Biomarcadores Tumorais/metabolismo , Biomarcadores Tumorais/genética , Reposicionamento de Medicamentos , Animais , Prognóstico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Biologia Computacional/métodos , CamundongosRESUMO
Angiosarcoma is a vascular sarcoma that is highly aggressive and metastatic. Due to its rarity, treatment options for patients are limited, therefore more research is needed to identify possible therapeutic vulnerabilities. We previously found that conditional deletion of Dicer1 drives angiosarcoma development in mice. Given the role of DICER1 in canonical microRNA (miRNA) biogenesis, this suggests that miRNA loss is important in angiosarcoma development. After testing miRNAs previously suggested to have a tumor-suppressive role in angiosarcoma, microRNA-497-5p (miR-497) suppressed cell viability most significantly. We also found that miR-497 overexpression led to significantly reduced cell migration and tumor formation. To understand the mechanism of miR-497 in tumor suppression, we identified clinically relevant target genes using a combination of RNA-sequencing data in an angiosarcoma cell line, expression data from angiosarcoma patients, and target prediction algorithms. We validated miR-497 direct regulation of CCND2, CDK6, and VAT1. One of these genes, VAT1, is an understudied protein that has been suggested to promote cell migration and metastasis in other cancers. Indeed, we find that pharmacologic inhibition of VAT1 with the natural product Neocarzilin A reduces angiosarcoma migration. Implications: This work supports the potent tumor-suppressive abilities of miR-497 in angiosarcoma, providing evidence for its potential as a therapeutic, and provides insight into the mechanisms of tumor suppression through analysis of the target gene regulatory network of miR-497.
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Angiosarcoma (AS) is a vascular sarcoma that is highly aggressive and metastatic. Due to its rarity, treatment options for patients are limited, therefore more research is needed to identify possible therapeutic vulnerabilities. We previously found that conditional deletion of Dicer1 drives AS development in mice. Given the role of DICER1 in canonical microRNA (miRNA) biogenesis, this suggests that miRNA loss is important in AS development. After testing miRNAs previously suggested to have a tumor-suppressive role in AS, microRNA-497-5p (miR-497) suppressed cell viability most significantly. We also found that miR-497 overexpression led to significantly reduced cell migration and tumor formation. To understand the mechanism of miR-497 in tumor suppression, we identified clinically relevant target genes using a combination of RNA-sequencing data in an AS cell line, expression data from AS patients, and target prediction algorithms. We validated miR-497 direct regulation of CCND2, CDK6, and VAT1. One of these genes, VAT1, is an understudied protein that has been suggested to promote cell migration and metastasis in other cancers. Indeed, we find that pharmacologic inhibition of VAT1 with the natural product Neocarzilin A reduces AS migration. This work provides insight into the mechanisms of miR-497 and its target genes in AS pathogenesis.
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Angiosarcomas are aggressive vascular sarcomas that arise from endothelial cells and have an extremely poor prognosis. Because of the rarity of angiosarcomas, knowledge of molecular drivers and optimized treatment strategies is lacking, highlighting the need for in vivo models to study the disease. Previously, we generated genetically engineered mouse models of angiosarcoma driven by aP2-Cre-mediated biallelic loss of Dicer1 or conditional activation of KrasG12D with Cdkn2a loss that histologically and genetically resemble human tumors. In the present study, we found that DICER1 functions as a potent tumor suppressor and its deletion, in combination with either KRASG12D expression or Cdkn2a loss, is associated with angiosarcoma development. Independent of the genetic driver, the mTOR pathway was activated in all murine angiosarcoma models. Direct activation of the mTOR pathway by conditional deletion of Tsc1 with aP2-Cre resulted in tumors that resemble intermediate grade human kaposiform hemangioendotheliomas, indicating that mTOR activation was not sufficient to drive the malignant angiosarcoma phenotype. Genetic dissection of the spectrum of vascular tumors identified genes specifically regulated in the aggressive murine angiosarcomas that are also enriched in human angiosarcoma. The genetic dissection driving the transition across the malignant spectrum of endothelial sarcomas provides an opportunity to identify key determinants of the malignant phenotype, novel therapies for angiosarcoma, and novel in vivo models to further explore angiosarcoma pathogenesis. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
Assuntos
Hemangiossarcoma , Neoplasias de Tecidos Moles , Animais , Carcinogênese , Células Endoteliais/metabolismo , Hemangiossarcoma/genética , Hemangiossarcoma/patologia , Integrases , Camundongos , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
PTEN promoter hypermethylation is nearly universal and PTEN copy number loss occurs in ~25% of fusion-negative rhabdomyosarcoma (FN-RMS). Here we show Pten deletion in a mouse model of FN-RMS results in less differentiated tumors more closely resembling human embryonal RMS. PTEN loss activated the PI3K pathway but did not increase mTOR activity. In wild-type tumors, PTEN was expressed in the nucleus suggesting loss of nuclear PTEN functions could account for these phenotypes. Pten deleted tumors had increased expression of transcription factors important in neural and skeletal muscle development including Dbx1 and Pax7. Pax7 deletion completely rescued the effects of Pten loss. Strikingly, these Pten;Pax7 deleted tumors were no longer FN-RMS but displayed smooth muscle differentiation similar to leiomyosarcoma. These data highlight how Pten loss in FN-RMS is connected to a PAX7 lineage-specific transcriptional output that creates a dependency or synthetic essentiality on the transcription factor PAX7 to maintain tumor identity.
Assuntos
Fator de Transcrição PAX7/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Rabdomiossarcoma/metabolismo , Rabdomiossarcoma/patologia , Animais , Cruzamento , Diferenciação Celular , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Humanos , Integrases/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos Knockout , Desenvolvimento Muscular , PTEN Fosfo-Hidrolase/deficiência , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Rabdomiossarcoma/genéticaRESUMO
PURPOSE: Glioblastoma, the most common malignant brain tumor, was associated with a median survival of <1 year in the pre-temozolomide (TMZ) era. Despite advances in molecular and genetic profiling studies identifying several predictive biomarkers, none has been translated into routine clinical use. Our aim was to investigate the prognostic significance of a panel of diverse cellular molecular markers of tumor formation and growth in an annotated glioblastoma tissue microarray (TMA). METHODS AND MATERIALS: A TMA composed of archived glioblastoma tumors from patients treated with surgery, radiation, and non-TMZ chemother-apy, was provided by RTOG. RAD51, BRCA-1, phosphatase and tensin homolog tumor suppressor gene (PTEN), and miRNA-210 expression levels were assessed using quantitative in situ hybridization and automated quantitative protein analysis. The objectives of this analysis were to determine the association of each biomarker with overall survival (OS), using the Cox proportional hazard model. Event-time distributions were estimated using the Kaplan-Meier method and compared by the log-rank test. RESULTS: A cohort of 66 patients was included in this study. Among the 4 biomarkers assessed, only BRCA1 expression had a statistically significant correlation with survival. From univariate analysis, patients with low BRCA1 protein expression showed a favorable outcome for OS (p = 0.04; hazard ratio = 0.56) in comparison with high expressors, with a median survival time of 18.9 versus 4.8 months. CONCLUSIONS: BRCA1 protein expression was an important survival predictor in our cohort of glioblastoma patients. This result may imply that low BRCA1 in the tumor and the consequent low level of DNA repair cause vulnerability of the cancer cells to treatment.
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Proteína BRCA1/metabolismo , Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Adolescente , Adulto , Idoso , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/terapia , Estudos de Coortes , Terapia Combinada , Feminino , Glioblastoma/patologia , Glioblastoma/terapia , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Modelos de Riscos Proporcionais , Análise Serial de Tecidos , Adulto JovemRESUMO
Rhabdomyosarcoma is the most common soft-tissue sarcoma in childhood and histologically resembles developing skeletal muscle. Alveolar rhabdomyosarcoma (ARMS) is an aggressive subtype with a higher rate of metastasis and poorer prognosis. The majority of ARMS tumors (80%) harbor a PAX3-FOXO1 or less commonly a PAX7-FOXO1 fusion gene. The presence of either the PAX3-FOXO1 or PAX7-FOXO1 fusion gene foretells a poorer prognosis resulting in clinical re-classification as either fusion-positive (FP-RMS) or fusion-negative RMS (FN-RMS). The PAX3/7-FOXO1 fusion genes result in the production of a rogue transcription factors that drive FP-RMS pathogenesis and block myogenic differentiation. Despite knowing the molecular driver of FP-RMS, targeted therapies have yet to make an impact for patients, highlighting the need for a greater understanding of the molecular consequences of PAX3-FOXO1 and its target genes including microRNAs. Here we show FP-RMS patient-derived xenografts and cell lines display a distinct microRNA expression pattern. We utilized both loss- and gain-of function approaches in human cell lines with knockdown of PAX3-FOXO1 in FP-RMS cell lines and expression of PAX3-FOXO1 in human myoblasts and identified microRNAs both positively and negatively regulated by the PAX3-FOXO1 fusion protein. We demonstrate PAX3-FOXO1 represses miR-221/222 that functions as a tumor suppressing microRNA through the negative regulation of CCND2, CDK6, and ERBB3. In contrast, miR-486-5p is transcriptionally activated by PAX3-FOXO1 and promotes FP-RMS proliferation, invasion, and clonogenic growth. Inhibition of miR-486-5p in FP-RMS xenografts decreased tumor growth, illustrating a proof of principle for future therapeutic intervention. Therefore, PAX3-FOXO1 regulates key microRNAs that may represent novel therapeutic vulnerabilities in FP-RMS.
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MicroRNAs/genética , Neoplasias Musculares/genética , Proteínas de Fusão Oncogênica/fisiologia , Fatores de Transcrição Box Pareados/fisiologia , Rabdomiossarcoma Alveolar/genética , Animais , Proliferação de Células/genética , Transformação Celular Neoplásica/genética , Células Cultivadas , Criança , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Camundongos , Camundongos SCID , Análise em Microsséries , Neoplasias Musculares/patologia , Proteínas de Fusão Oncogênica/genética , Fatores de Transcrição Box Pareados/genética , Rabdomiossarcoma Alveolar/patologiaRESUMO
Rhabdomyosarcoma (RMS) is a pediatric soft tissue sarcoma that histologically resembles embryonic skeletal muscle. RMS occurs throughout the body and an exclusively myogenic origin does not account for RMS occurring in sites devoid of skeletal muscle. We previously described an RMS model activating a conditional constitutively active Smoothened mutant (SmoM2) with aP2-Cre. Using genetic fate mapping, we show SmoM2 expression in Cre-expressing endothelial progenitors results in myogenic transdifferentiation and RMS. We show that endothelium and skeletal muscle within the head and neck arise from Kdr-expressing progenitors, and that hedgehog pathway activation results in aberrant expression of myogenic specification factors as a potential mechanism driving RMS genesis. These findings suggest that RMS can originate from aberrant development of non-myogenic cells.
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Endotélio/metabolismo , Proteínas Hedgehog/metabolismo , Desenvolvimento Muscular/genética , Rabdomiossarcoma/metabolismo , Células-Tronco/metabolismo , Animais , Diferenciação Celular/fisiologia , Camundongos Transgênicos , Músculo Esquelético/metabolismo , Transdução de SinaisRESUMO
Angiosarcoma is an aggressive vascular sarcoma with an extremely poor prognosis. Because of the relative rarity of this disease, its molecular drivers and optimal treatment strategies are obscure. DICER1 is an RNase III endoribonuclease central to miRNA biogenesis, and germline DICER1 mutations result in a cancer predisposition syndrome, associated with an increased risk of many tumor types. Here, we show that biallelic Dicer1 deletion with aP2-Cre drives aggressive and metastatic angiosarcoma independent of other genetically engineered oncogenes or tumor suppressor loss. Angiosarcomas in aP2-Cre;Dicer1Flox/- mice histologically and genetically resemble human angiosarcoma. miR-23 target genes, including the oncogenes Ccnd1 as well as Adam19, Plau, and Wsb1 that promote invasiveness and metastasis, were enriched in mouse and human angiosarcoma. These studies illustrate that Dicer1 can function as a traditional loss-of-function tumor suppressor gene, and they provide a fully penetrant animal model for the study of angiosarcoma development and metastasis. Cancer Res; 77(22); 6109-18. ©2017 AACR.
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RNA Helicases DEAD-box/genética , Predisposição Genética para Doença/genética , Hemangiossarcoma/genética , Mutação , Ribonuclease III/genética , Animais , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Hemangiossarcoma/patologia , Homozigoto , Humanos , Estimativa de Kaplan-Meier , Camundongos Knockout , Camundongos Transgênicos , MicroRNAs/genéticaRESUMO
Oncogenic and tumor suppressing microRNAs (miRNAs) have emerged as key regulators of gene expression in many types of cancer including melanoma. We utilized quantitative in situ hybridization (qISH) to evaluate the tumor suppressing properties of miRNA, miR-205 in a population of human tumors. We hypothesize decreased miR-205 would be associated with more aggressive tumors. Multiplexing miR-205 qISH with immunofluorescent assessment of S100/GP100 allowed us to quantitatively evaluate miR-205 expression using the AQUA method of quantitative immunofluorescence. The specificity of the assay was validated using blocking oligos and transfected cell lines as controls. Outcomes were assessed on the Yale Melanoma Discovery Cohort consisting of 105 primary melanoma specimens and validated on an independent set of 206 primary melanomas (Yale Melanoma Validation Cohort). Measurement of melanoma cell miR-205 levels shows a significantly shorter melanoma-specific survival in patients with low expression. Multivariate analysis shows miR-205 levels are significantly independent of stage, age, gender, and Breslow depth. Low levels of melanoma cell miR-205 expression as quantified by ISH show worse outcome, supporting the role of miR-205 as a tumor suppressor miRNA. The quantification of miR-205 in situ suggests potential for the use of miRNAs in future prognostic or predictive models.
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Biomarcadores Tumorais/análise , Hibridização In Situ/métodos , Melanoma/diagnóstico , MicroRNAs/análise , RNA Neoplásico/análise , Neoplasias Cutâneas/diagnóstico , Idoso , Análise de Variância , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Melanoma/patologia , MicroRNAs/genética , Pessoa de Meia-Idade , Prognóstico , RNA Neoplásico/genética , Estudos Retrospectivos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas S100/imunologia , Proteínas S100/metabolismo , Neoplasias Cutâneas/patologia , Análise Serial de Tecidos/métodos , Antígeno gp100 de Melanoma/imunologia , Antígeno gp100 de Melanoma/metabolismoRESUMO
MicroRNAs (miRNAs) have emerged as key regulators in the pathogenesis of cancers where they can act as either oncogenes or tumor suppressors. Most miRNA measurement methods require total RNA extracts which lack critical spatial information and present challenges for standardization. We have developed and validated a method for the quantitative analysis of miRNA expression by in situ hybridization (ISH) allowing for the direct assessment of tumor epithelial expression of miRNAs. This co-localization based approach (called qISH) utilizes DAPI and cytokeratin immunofluorescence to establish subcellular compartments in the tumor epithelia, then multiplexed with the miRNA ISH, allows for quantitative measurement of miRNA expression within these compartments. We use this approach to assess miR-21, miR-92a, miR-34a, and miR-221 expression in 473 breast cancer specimens on tissue microarrays. We found that miR-221 levels are prognostic in breast cancer illustrating the high-throughput method and confirming that miRNAs can be valuable biomarkers in cancer. Furthermore, in applying this method we found that the inverse relationship between miRNAs and proposed target proteins is difficult to discern in large population cohorts. Our method demonstrates an approach for large cohort, tissue microarray-based assessment of miRNA expression.
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Hibridização In Situ/métodos , MicroRNAs/metabolismo , Análise Serial de Tecidos/métodos , Animais , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Camundongos Knockout , MicroRNAs/genética , Proteínas de Neoplasias/metabolismo , Prognóstico , Reprodutibilidade dos TestesRESUMO
BACKGROUND: Microtubule-associated proteins (MAPs) endogenously regulate microtubule stability. Here, the prognostic value of stathmin, a destabilizing protein, was assessed in combination with MAP-tau, a stabilizing protein, in order to evaluate microtubule stabilization as a potential biomarker. METHODS: Stathmin and MAP-tau expression levels were measured in a breast cancer cohort (n = 651) using the tissue microarray format and quantitative immunofluorescence (AQUA) technology, then correlated with clinical and pathological characteristics and disease-free survival. RESULTS: Univariate Cox proportional hazard models indicated that high stathmin expression predicts worse overall survival (hazard ratio [HR] = 1.48; 95% confidence interval [CI] = 1.119-1.966; P = .0061). Survival analysis showed 10-year survival of 53.1% for patients with high stathmin expression versus 67% for low expressers (log-rank, P < .003). Cox multivariate analysis showed high stathmin expression was independent of age, menopausal status, nodal status, nuclear grade, tumor size, and estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 expression (HR = 1.19; 95% CI = 1.03-1.37; P = .01). The ratio of MAP-tau to stathmin expression showed a positive correlation to disease-free survival (HR = 0.679; 95% CI = 0.517-0.891; P = .0053) with a 10-year survival of 65.4% for patients who had a high ratio of MAP-tau to stathmin versus 52.5% 10-year survival rate for those with a low ratio (log-rank, P = .0009). Cox multivariate analysis showed the ratio of MAP-tau to stathmin was an independent predictor of overall survival (HR = 0.609; 95% CI = 0.422-0.879; P = .008). CONCLUSIONS: Low stathmin and high MAP-tau are associated with increased microtubule stability and better prognosis in breast cancer.
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Biomarcadores Tumorais/análise , Neoplasias da Mama/química , Estatmina/análise , Proteínas tau/análise , Adulto , Idoso , Análise de Variância , Western Blotting , Mama/química , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Estudos de Coortes , Feminino , Imunofluorescência , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Metástase Linfática , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Razão de Chances , Valor Preditivo dos Testes , Prognóstico , Modelos de Riscos Proporcionais , RNA Interferente Pequeno/metabolismo , Medição de Risco , Fatores de Risco , Análise Serial de Tecidos , Resultado do TratamentoRESUMO
Clinical and genomic evidence suggests that the metastatic potential of a primary tumor may be dictated by prometastatic events that have additional oncogenic capability. To test this "deterministic" hypothesis, we adopted a comparative oncogenomics-guided function-based strategy involving: (1) comparison of global transcriptomes of two genetically engineered mouse models with contrasting metastatic potential, (2) genomic and transcriptomic profiles of human melanoma, (3) functional genetic screen for enhancers of cell invasion, and (4) evidence of expression selection in human melanoma tissues. This integrated effort identified six genes that are potently proinvasive and oncogenic. Furthermore, we show that one such gene, ACP5, confers spontaneous metastasis in vivo, engages a key pathway governing metastasis, and is prognostic in human primary melanomas.
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Melanoma/genética , Melanoma/patologia , Oncogenes/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Fosfatase Ácida/genética , Fosfatase Ácida/metabolismo , Animais , Linhagem da Célula/genética , Sequência Conservada/genética , Evolução Molecular , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genoma/genética , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Estimativa de Kaplan-Meier , Camundongos , Invasividade Neoplásica , Metástase Neoplásica , Estadiamento de Neoplasias , Fosforilação , Reprodutibilidade dos Testes , Fosfatase Ácida Resistente a Tartarato , Análise Serial de TecidosRESUMO
The hepatocyte growth factor (HGF) and its receptor, the Met receptor tyrosine kinase, form a signaling network promoting cell proliferation, invasion, and survival in normal and cancer cells. Improper regulation of this pathway is attributed to many cancer types through overexpression, activating mutations, or autocrine loop formation. Many studies describe the localization of Met as membranous/cytoplasmic, but some studies using antibodies targeted to the C-terminal domain of Met report nuclear localization. This chapter seeks to highlight the histopathology and expression of Met in cancer and its association with clinicopathological characteristics. We also discuss recent studies of the proteolytic processing of Met and effects of the processing on the subcellular localization of Met. Finally, we comment on Met as a therapeutic target for cancer treatment.