RESUMO
Liposarcoma, the most common soft tissue sarcoma, is a group of fat cell mesenchymal tumors with different histological subtypes. The dysregulation of long non-coding RNAs (lncRNAs) has been observed in human cancers including a few studies in sarcoma. However, the global transcriptome analysis and potential role of lncRNAs remain unexplored in liposarcoma. The present investigation uncovers the transcriptomic profile of liposarcoma by RNA sequencing to gain insight into the global transcriptional changes in liposarcoma. Our RNA sequencing analysis has identified that many oncogenic lncRNAs are differentially expressed in different subtypes of liposarcoma including MALAT1, PVT1, SNHG15, LINC00152, and MIR210HG. Importantly, we identified a highly overexpressed, unannotated, and novel lncRNA in dedifferentiated liposarcomas. We have named it TODL, transcript overexpressed in dedifferentiated liposarcoma. TODL lncRNA displayed significantly higher expression in dedifferentiated liposarcoma cell lines and patient samples. Interestingly, functional studies revealed that TODL lncRNA has an oncogenic function in liposarcoma cells by regulating proliferation, cell cycle, apoptosis, differentiation, and tumorigenesis in the murine model. Silencing of TODL lncRNA highlighted the enrichment of several key oncogenic signaling pathways including cell cycle, transcriptional misregulation, FOXM1 network, p53 signaling, PLK1 signaling, FoxO, and signaling Aurora signaling pathways. RNA pull-down assay revealed the binding of TODL lncRNA with FOXM1, an oncogenic transcription factor, and the key regulator of the cell cycle. Silencing of TODL lncRNA also induces adipogenesis in dedifferentiated liposarcomas. Altogether, our finding indicates that TODL could be utilized as a novel, specific diagnostic biomarker, and a pharmacological target for therapeutic development in controlling aggressive and metastatic dedifferentiated liposarcomas.
Assuntos
Proteína Forkhead Box M1 , Lipossarcoma , RNA Longo não Codificante , Animais , Humanos , Camundongos , Carcinogênese/genética , Proliferação de Células , Proteína Forkhead Box M1/genética , Proteína Forkhead Box M1/metabolismo , Perfilação da Expressão Gênica , Lipossarcoma/genética , Lipossarcoma/metabolismo , Lipossarcoma/patologia , RNA Longo não Codificante/genética , TranscriptomaRESUMO
Proteasome inhibitors, such as bortezomib and carfilzomib, have shown efficacy in anti-cancer therapy in hematological diseases but not in solid cancers. Here, we found that liposarcomas (LPS) are susceptible to proteasome inhibition, and identified drugs that synergize with carfilzomib, such as selinexor, an inhibitor of XPO1-mediated nuclear export. Through quantitative nuclear protein profiling and phospho-kinase arrays, we identified potential mode of actions of this combination, including interference with ribosome biogenesis and inhibition of pro-survival kinase PRAS40. Furthermore, by assessing global protein levels changes, FADS2, a key enzyme regulating fatty acids synthesis, was found down-regulated after proteasome inhibition. Interestingly, SC26196, an inhibitor of FADS2, synergized with carfilzomib. Finally, to identify further combinational options, we performed high-throughput drug screening and uncovered novel drug interactions with carfilzomib. For instance, cyclosporin A, a known immunosuppressive agent, enhanced carfilzomib's efficacy in vitro and in vivo. Altogether, these results demonstrate that carfilzomib and its combinations could be repurposed for LPS clinical management.
Assuntos
Ácidos Graxos Dessaturases/genética , Carioferinas/genética , Lipossarcoma/tratamento farmacológico , Oligopeptídeos/farmacologia , Receptores Citoplasmáticos e Nucleares/genética , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Bortezomib/farmacologia , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Sinergismo Farmacológico , Ácidos Graxos Dessaturases/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Hidrazinas/farmacologia , Lipossarcoma/genética , Lipossarcoma/patologia , Piperazinas/farmacologia , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Inibidores de Proteassoma/farmacologia , Triazóis/farmacologia , Proteína Exportina 1RESUMO
Multiple myeloma (MM) is a hematological disease marked by abnormal growth of B cells in bone marrow. Inherent chromosomal instability and DNA damage are major hallmarks of MM, which implicates an aberrant DNA repair mechanism. Studies have implicated a role for CDK12 in the control of expression of DNA damage response genes. In this study, we examined the effect of a small molecule inhibitor of CDK12-THZ531 on MM cells. Treatment of MM cells with THZ531 led to heightened cell death accompanied by an extensive effect on gene expression changes. In particular, we observed downregulation of genes involved in DNA repair pathways. With this insight, we extended our study to identify synthetic lethal mechanisms that could be exploited for the treatment of MM cells. Combination of THZ531 with either DNA-PK inhibitor (KU-0060648) or PARP inhibitor (Olaparib) led to synergistic cell death. In addition, combination treatment of THZ531 with Olaparib significantly reduced tumor burden in animal models. Our findings suggest that using a CDK12 inhibitor in combination with other DNA repair inhibitors may establish an effective therapeutic regimen to benefit myeloma patients.
Assuntos
Anilidas/farmacologia , Biomarcadores Tumorais/genética , Reparo do DNA , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Mieloma Múltiplo/tratamento farmacológico , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Pirimidinas/farmacologia , Mutações Sintéticas Letais , Animais , Apoptose , Proteína BRCA1/genética , Proteína BRCA2/genética , Proliferação de Células , Quimioterapia Combinada , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Prognóstico , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND & AIMS: We investigated the transcriptome of esophageal squamous cell carcinoma (ESCC) cells, activity of gene regulatory (enhancer and promoter regions), and the effects of blocking epigenetic regulatory proteins. METHODS: We performed chromatin immunoprecipitation sequencing with antibodies against H3K4me1, H3K4me3, and H3K27ac and an assay for transposase-accessible chromatin to map the enhancer regions and accessible chromatin in 8 ESCC cell lines. We used the CRC_Mapper algorithm to identify core regulatory circuitry transcription factors in ESCC cell lines, and determined genome occupancy profiles for 3 of these factors. In ESCC cell lines, expression of transcription factors was knocked down with small hairpin RNAs, promoter and enhancer regions were disrupted by CRISPR/Cas9 genome editing, or bromodomains and extraterminal (BET) family proteins and histone deacetylases (HDACs) were inhibited with ARV-771 and romidepsin, respectively. ESCC cell lines were then analyzed by whole-transcriptome sequencing, immunoprecipitation, immunoblots, immunohistochemistry, and viability assays. Interactions between distal enhancers and promoters were identified and verified with circular chromosome conformation capture sequencing. NOD-SCID mice were given injections of modified ESCC cells, some mice where given injections of HDAC or BET inhibitors, and growth of xenograft tumors was measured. RESULTS: We identified super-enhancer-regulated circuits and transcription factors TP63, SOX2, and KLF5 as core regulatory factors in ESCC cells. Super-enhancer regulation of ALDH3A1 mediated by core regulatory factors was required for ESCC viability. We observed direct interactions between the promoter region of TP63 and functional enhancers, mediated by the core regulatory circuitry transcription factors. Deletion of enhancer regions from ESCC cells decreased expression of the core regulatory circuitry transcription factors and reduced cell viability; these same results were observed with knockdown of each core regulatory circuitry transcription factor. Incubation of ESCC cells with BET and HDAC disrupted the core regulatory circuitry program and the epigenetic modifications observed in these cells; mice given injections of HDAC or BET inhibitors developed smaller xenograft tumors from the ESCC cell lines. Xenograft tumors grew more slowly in mice given the combination of ARV-771 and romidepsin than mice given either agent alone. CONCLUSIONS: In epigenetic and transcriptional analyses of ESCC cell lines, we found the transcription factors TP63, SOX2, and KLF5 to be part of a core regulatory network that determines chromatin accessibility, epigenetic modifications, and gene expression patterns in these cells. A combination of epigenetic inhibitors slowed growth of xenograft tumors derived from ESCC cells in mice.
Assuntos
Epigênese Genética , Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas do Esôfago/genética , Regulação Neoplásica da Expressão Gênica , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição/genética , Transcrição Gênica , Proteínas Supressoras de Tumor/genética , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Montagem e Desmontagem da Cromatina , Epigênese Genética/efeitos dos fármacos , Neoplasias Esofágicas/tratamento farmacológico , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/tratamento farmacológico , Carcinoma de Células Escamosas do Esôfago/metabolismo , Carcinoma de Células Escamosas do Esôfago/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Humanos , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas/antagonistas & inibidores , Proteínas/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacos , Transcriptoma , Carga Tumoral , Proteínas Supressoras de Tumor/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
CCAAT/enhancer binding protein ε (CEBPE) is an essential transcription factor for granulocytic differentiation. Mutations of CEBPE occur in individuals with neutrophil-specific granule deficiency (SGD), which is characterized by defects in neutrophil maturation. Cebpe-knockout mice also exhibit defects in terminal differentiation of granulocytes, a phenotype reminiscent of SGD. Analysis of DNase I hypersensitive sites sequencing data revealed an open chromatin region 6 kb downstream of the transcriptional start site of Cebpe in murine myeloid cells. We identified an interaction between this +6-kb region and the core promoter of Cebpe using circular chromosome conformation capture sequencing (4C-seq). To understand the role of this putative enhancer in transcriptional regulation of Cebpe, we targeted it using catalytically inactive Cas9 fused to Krüppel-associated box (KRAB) domain and observed a significant downregulation of transcript and protein levels of CEBPE in cells expressing guide RNA targeting the +6-kb region. To further investigate the role of this novel enhancer further in myelopoiesis, we generated mice with deletion of this region using CRISPR/Cas9 technology. Germline deletion of the +6-kb enhancer resulted in reduced levels of CEBPE and its target genes and caused a severe block in granulocytic differentiation. We also identified binding of CEBPA and CEBPE to the +6-kb enhancer, which suggests their role in regulating the expression of Cebpe In summary, we have identified a novel enhancer crucial for regulating expression of Cebpe and required for normal granulocytic differentiation.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/biossíntese , Diferenciação Celular/genética , Regulação da Expressão Gênica/genética , Granulócitos/metabolismo , Mielopoese/genética , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
OBJECTIVES: Oesophageal squamous cell carcinoma (OSCC) is an aggressive malignancy and the major histological subtype of oesophageal cancer. Although recent large-scale genomic analysis has improved the description of the genetic abnormalities of OSCC, few targetable genomic lesions have been identified, and no molecular therapy is available. This study aims to identify druggable candidates in this tumour. DESIGN: High-throughput small-molecule inhibitor screening was performed to identify potent anti-OSCC compounds. Whole-transcriptome sequencing (RNA-Seq) and chromatin immunoprecipitation sequencing (ChIP-Seq) were conducted to decipher the mechanisms of action of CDK7 inhibition in OSCC. A variety of in vitro and in vivo cellular assays were performed to determine the effects of candidate genes on OSCC malignant phenotypes. RESULTS: The unbiased high-throughput small-molecule inhibitor screening led us to discover a highly potent anti-OSCC compound, THZ1, a specific CDK7 inhibitor. RNA-Seq revealed that low-dose THZ1 treatment caused selective inhibition of a number of oncogenic transcripts. Notably, further characterisation of the genomic features of these THZ1-sensitive transcripts demonstrated that they were frequently associated with super-enhancer (SE). Moreover, SE analysis alone uncovered many OSCC lineage-specific master regulators. Finally, integrative analysis of both THZ1-sensitive and SE-associated transcripts identified a number of novel OSCC oncogenes, including PAK4, RUNX1, DNAJB1, SREBF2 and YAP1, with PAK4 being a potential druggable kinase. CONCLUSIONS: Our integrative approaches led to a catalogue of SE-associated master regulators and oncogenic transcripts, which may significantly promote both the understanding of OSCC biology and the development of more innovative therapies.
Assuntos
Acrilamidas/farmacologia , Aminopiridinas/farmacologia , Antineoplásicos/farmacologia , Carcinoma de Células Escamosas/genética , Neoplasias Esofágicas/genética , Expressão Gênica/efeitos dos fármacos , Fenilenodiaminas/farmacologia , Pirimidinas/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Carcinoma de Células Escamosas/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Quinases Ciclina-Dependentes/antagonistas & inibidores , Ensaios de Seleção de Medicamentos Antitumorais , Neoplasias Esofágicas/tratamento farmacológico , Feminino , Perfilação da Expressão Gênica , Proteínas de Choque Térmico HSP40/genética , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Transplante de Neoplasias , Oncogenes/genética , Fosfoproteínas/genética , Análise de Sequência de RNA , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Fatores de Transcrição , Transcriptoma , Proteínas de Sinalização YAP , Quinases Ativadas por p21/genética , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
Acute myeloid leukemia (AML) with an FLT3 internal tandem duplication (FLT3-ITD) mutation is an aggressive hematologic malignancy with a grave prognosis. To identify the mutational spectrum associated with relapse, whole-exome sequencing was performed on 13 matched diagnosis, relapse, and remission trios followed by targeted sequencing of 299 genes in 67 FLT3-ITD patients. The FLT3-ITD genome has an average of 13 mutations per sample, similar to other AML subtypes, which is a low mutation rate compared with that in solid tumors. Recurrent mutations occur in genes related to DNA methylation, chromatin, histone methylation, myeloid transcription factors, signaling, adhesion, cohesin complex, and the spliceosome. Their pattern of mutual exclusivity and cooperation among mutated genes suggests that these genes have a strong biological relationship. In addition, we identified mutations in previously unappreciated genes such as MLL3, NSD1, FAT1, FAT4, and IDH3B. Mutations in 9 genes were observed in the relapse-specific phase. DNMT3A mutations are the most stable mutations, and this DNMT3A-transformed clone can be present even in morphologic complete remissions. Of note, all AML matched trio samples shared at least 1 genomic alteration at diagnosis and relapse, suggesting common ancestral clones. Two types of clonal evolution occur at relapse: either the founder clone recurs or a subclone of the founder clone escapes from induction chemotherapy and expands at relapse by acquiring new mutations. Relapse-specific mutations displayed an increase in transversions. Functional assays demonstrated that both MLL3 and FAT1 exert tumor-suppressor activity in the FLT3-ITD subtype. An inhibitor of XPO1 synergized with standard AML induction chemotherapy to inhibit FLT3-ITD growth. This study clearly shows that FLT3-ITD AML requires additional driver genetic alterations in addition to FLT3-ITD alone.
Assuntos
Exoma , Leucemia Mieloide Aguda , Mutação , Tirosina Quinase 3 Semelhante a fms/genética , Cromatina/genética , Cromatina/metabolismo , Metilação de DNA/genética , Feminino , Humanos , Quimioterapia de Indução , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidade , Masculino , Recidiva , Estudos RetrospectivosRESUMO
1,2,3-Triazole-based heterocycles have previously been shown to possess significant anticancer activity in various tumor models. In the present study, we attached a 1,2,3-triazole moiety to the third position of a 1,2-benzisoxazole heterocycle via copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) with various alkynes and established for the title compounds significant antiproliferative effect against human acute myeloid leukemia (AML) cells. Among the tested compounds, 3-(4-(4-phenoxyphenyl)-1H-1,2,3-triazol-1-yl)benzo[d]isoxazole (PTB) was found to be the most potent antiproliferative agent with an IC50 of 2 µM against MV4-11 cells using MTT assay. Notably, PTB induced cytotoxicity in MOLM13, MOLM14 and MV4-11 cells with selectivity over normal bone marrow cells (C57BL/6). Furthermore, PTB was found to induce cytotoxicity by increasing apoptosis of AML cells (MOLM13, MOLM14 and MV4-11) as well as sub-G1 cell population and apoptotic cells at submicromolar concentrations, as shown by flow cytometry and Annexin-V staining, respectively. On the protein level we suggested histone deacetylases (HDACs) as the potential protein target of those compounds in silico, and the predicted target was next experimentally validated by measuring the variations in the levels of p21, cyclin D and acetylation of histone H3 and tubulin. Molecular docking analysis of the title compounds with the second deacetylase domain of HDAC6 displayed high degree of shape complementarity to the binding site of the enzyme, forming multiple molecular interactions in the hydrophobic region as well as a hydrogen bond to the phenol side-chain of Tyr-782. Thus, 1,2,3-triazole derivatives appear to represent a class of novel, biologically active ligands against histone deacetylases which deserve to be further evaluated in their applications in the cancer field.
Assuntos
Antineoplásicos/síntese química , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidores de Histona Desacetilases/síntese química , Histona Desacetilases/química , Triazóis/química , Tubulina (Proteína)/metabolismo , Acetilação , Animais , Antineoplásicos/química , Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclina D/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/toxicidade , Histona Desacetilases/metabolismo , Histonas/metabolismo , Humanos , Isoxazóis/química , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos C57BL , Triazóis/síntese química , Triazóis/toxicidadeRESUMO
Colorectal cancer (CRC) is the second most common tumor in developed countries. The present study was undertaken to determine the expression of the sperm-associated antigen 9 gene (SPAG9) as a possible biomarker in CRC, to investigate its correlation with humoral immune response and different stages and grades in CRC patients, and to explore its possible role in colon tumorigenesis in vitro and in an in vivo mouse model. SPAG9 expression was determined by RT-PCR, in situ RNA hybridization, and immunohistochemistry. Humoral response against SPAG9 was detected by enzyme-linked immunosorbent assay and Western blotting. SPAG9 gene silencing was performed using plasmid-based small interfering RNA to study various malignant properties of colon cancer cells in vitro and in vivo. The majority of CRC patients showed SPAG9 expression and generated humoral response. There was a close relationship between SPAG9 protein expression and humoral immune response in the majority of early-stage CRC patients, indicating that anti-SPAG9 antibodies could be a novel serum biomarker for early diagnosis. The down-regulation of SPAG9 (mediated by small interfering RNA) inhibited malignant properties in in vitro and significantly suppressed tumor growth in vivo. These findings collectively suggest that SPAG9 may have a role in tumor development and early spread and thus could serve as a novel target for early detection and for cancer immunotherapy.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Neoplasias Colorretais/genética , Neoplasias Colorretais/imunologia , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Inativação Gênica , Humanos , Imunidade Humoral/imunologia , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Invasividade Neoplásica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Ensaio Tumoral de Célula-Tronco , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
To date, there have been no tumor biomarkers validated and incorporated into oncologic practice for the early diagnosis of breast cancer. Recently, we showed that sperm-associated antigen 9 (SPAG9), a member of cancer testis (CT) antigen family, is associated with ovarian carcinomas. In the present study, we investigated SPAG9 expression and humoral immune response in breast cancer. We further evaluated the diagnostic potential of autoantibodies to SPAG9 protein in various stages, grades, and histotypes of breast cancer. We analyzed the association of SPAG9 immunoreactivity score (IRS) with predicted risk of breast cancer recurrence over 10 years. Our reverse transcription-PCR and immunohistochemical analyses revealed SPAG9 expression in 88% breast cancer specimens independent of tumor stages and grades. Further, the humoral immune response against SPAG9 was detected in 80% breast cancer patients with SPAG9-expressing tumors. The linear regression modeling predicted a direct relationship between presence of lymphovascular invasion and high SPAG9 IRS, whereas the univariate and multivariate logistic regression models predicted a strong association of SPAG9 IRS with tumor grade. Further, our data indicated a significant higher trend of SPAG9 IRS with the predicted high risk of breast cancer recurrence. The present investigation reports for the first time SPAG9 expression and humoral immune response in early stages and low-grade breast cancer. Although our data indicated that autoantibodies against SPAG9 represent a promising approach for the development of biomarker, further large-scale validation studies are required to establish its potential use in early diagnosis and monitoring of breast cancer recurrence.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Distribuição de Qui-Quadrado , Diagnóstico Precoce , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Técnicas Imunoenzimáticas , Recidiva Local de Neoplasia/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estatísticas não ParamétricasRESUMO
Lipomas are benign fatty tumors with a high prevalence rate, mostly found in adults but have a good prognosis. Until now, reason for lipoma occurrence not been identified. We performed whole exome sequencing to define the mutational spectrum in ten lipoma patients along with their matching control samples. We presented genomic insight into the development of lipomas, the most common benign tumor of soft tissue. Our analysis identified 412 somatic variants including missense mutations, splice site variants, frameshift indels, and stop gain/lost. Copy number variation analysis highlighted minor aberrations in patients. Kinase genes and transcriptions factors were among the validated mutated genes critical for cell proliferation and survival. Pathway analysis revealed enrichment of calcium, Wnt and phospholipase D signaling in patients. In conclusion, whole exome sequencing in lipomas identified mutations in genes with a possible role in development and progression of lipomas.
Assuntos
Proliferação de Células/genética , Variações do Número de Cópias de DNA/genética , Sequenciamento do Exoma , Lipoma/genética , Adulto , Idoso , Códon sem Sentido/genética , Exoma/genética , Feminino , Mutação da Fase de Leitura/genética , Humanos , Mutação INDEL/genética , Lipoma/patologia , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto/genética , Fosfotransferases/genética , Fatores de Transcrição/genéticaRESUMO
Liposarcomas (LPSs) are a group of malignant mesenchymal tumors showing adipocytic differentiation. Here, to gain insight into the enhancer dysregulation and transcriptional addiction in this disease, we chart super-enhancer structures in both LPS tissues and cell lines. We identify a bromodomain and extraterminal (BET) protein-cooperated FUS-DDIT3 function in myxoid LPS and a BET protein-dependent core transcriptional regulatory circuitry consisting of FOSL2, MYC, and RUNX1 in de-differentiated LPS. Additionally, SNAI2 is identified as a crucial downstream target that enforces both proliferative and metastatic potentials to de-differentiated LPS cells. Genetic depletion of BET genes, core transcriptional factors, or SNAI2 mitigates consistently LPS malignancy. We also reveal a compelling susceptibility of LPS cells to BET protein degrader ARV-825. BET protein depletion confers additional advantages to circumvent acquired resistance to Trabectedin, a chemotherapy drug for LPS. Moreover, this study provides a framework for discovering and targeting of core oncogenic transcriptional programs in human cancers.
Assuntos
Lipossarcoma/genética , Proteínas de Neoplasias/metabolismo , Transcrição Gênica , Animais , Azepinas/farmacologia , Sequência de Bases , Carcinogênese/genética , Carcinogênese/patologia , Linhagem Celular Tumoral , Elementos Facilitadores Genéticos/genética , Genoma Humano , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas de Fusão Oncogênica/metabolismo , Talidomida/análogos & derivados , Talidomida/farmacologia , Transcrição Gênica/efeitos dos fármacosRESUMO
PURPOSE: Cancer testis antigens are a group of tumor antigens with gene expression restricted to male germ cells in the testis and in various cancerous tissues. Recently, we reported a novel testis-specific sperm-associated antigen 9 (SPAG9) gene, a new member of the c-Jun NH(2)-terminal kinase-interacting protein family, having functional role in sperm-egg fusion and mitogen-activated protein kinase signaling pathway. National Center for Biotechnology Information Blast searches revealed SPAG9 nucleotide sequence similarities with expressed sequence tags of various cancerous tissues. In an effort to examine the clinical utility of SPAG9, we investigated the SPAG9 mRNA and protein expression in epithelial ovarian cancer (EOC). Humoral immune response to SPAG9 was also evaluated in EOC patients. EXPERIMENTAL DESIGN: We determined the expression profile of SPAG9 transcript by reverse transcription-PCR and RNA in situ hybridization and SPAG9 protein expression by immunohistochemistry in EOC specimens and human ovarian cancer cell lines. Using ELISA and Western blotting, we analyzed specific antibodies for SPAG9 in sera from patients with EOC. RESULTS: SPAG9 mRNA and protein expression was detected in 90% of EOC tissues and in all three human ovarian cancer cell lines. Specific SPAG9 antibodies were detected in 67% of EOC patients and not in sera from healthy individuals. CONCLUSIONS: Our findings indicate that SPAG9 is highly expressed in EOC and immunogenic in patients. Humoral immune response against SPAG9 in early stages of EOC suggests its important role in early diagnostics. These results collectively suggest that SPAG9, a novel member of cancer testis antigen family, could be a potential target for the development of diagnostic and therapeutic methods in EOC.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antígenos de Neoplasias/metabolismo , Imunoterapia , Neoplasias Epiteliais e Glandulares/metabolismo , Neoplasias Ovarianas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Anticorpos Antineoplásicos/sangue , Antígenos de Neoplasias/imunologia , Biomarcadores Tumorais/análise , Western Blotting , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Hibridização In Situ , Neoplasias Epiteliais e Glandulares/sangue , Neoplasias Epiteliais e Glandulares/imunologia , Neoplasias Ovarianas/sangue , Neoplasias Ovarianas/imunologia , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Squamous cell carcinomas (SCCs) are aggressive malignancies. Previous report demonstrated that master transcription factors (TFs) TP63 and SOX2 exhibited overlapping genomic occupancy in SCCs. However, functional consequence of their frequent co-localization at super-enhancers remains incompletely understood. Here, epigenomic profilings of different types of SCCs reveal that TP63 and SOX2 cooperatively and lineage-specifically regulate long non-coding RNA (lncRNA) CCAT1 expression, through activation of its super-enhancers and promoter. Silencing of CCAT1 substantially reduces cellular growth both in vitro and in vivo, phenotyping the effect of inhibiting either TP63 or SOX2. ChIRP analysis shows that CCAT1 forms a complex with TP63 and SOX2, which regulates EGFR expression by binding to the super-enhancers of EGFR, thereby activating both MEK/ERK1/2 and PI3K/AKT signaling pathways. These results together identify a SCC-specific DNA/RNA/protein complex which activates TP63/SOX2-CCAT1-EGFR cascade and promotes SCC tumorigenesis, advancing our understanding of transcription dysregulation in cancer biology mediated by master TFs and super-enhancers.
Assuntos
Carcinoma de Células Escamosas/genética , Elementos Facilitadores Genéticos , RNA Longo não Codificante/genética , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Animais , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Receptores ErbB/genética , Receptores ErbB/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Camundongos Endogâmicos NOD , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Liposarcoma, the most common soft tissue tumor, is understudied cancer, and limited progress has been made in the treatment of metastatic disease. The Achilles heel of cancer often is their kinases that are excellent therapeutic targets. However, very limited knowledge exists of therapeutic critical kinase targets in liposarcoma that could be potentially used in disease management. METHODS: Large RNAi and small-molecule tyrosine kinase inhibitor screens were performed against the proliferative capacity of liposarcoma cell lines of different subtypes. Each small molecule inhibitor was either FDA approved or in a clinical trial. RESULTS: Screening assays identified several previously unrecognized targets including PTK2 and KIT in liposarcoma. We also observed that ponatinib, multi-targeted tyrosine kinase inhibitor, was the most effective drug with anti-growth effects against all cell lines. In vitro assays showed that ponatinib inhibited the clonogenic proliferation of liposarcoma, and this anti-growth effect was associated with apoptosis and cell cycle arrest at the G0/G1 phase as well as a decrease in the KIT signaling pathway. In addition, ponatinib inhibited in vivo growth of liposarcoma in a xenograft model. CONCLUSIONS: Two large-scale kinase screenings identified novel liposarcoma targets and a FDA-approved inhibitor, ponatinib with clear anti-liposarcoma activity highlighting its potential therapy for treatment of this deadly tumor.
Assuntos
Imidazóis/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Piridazinas/uso terapêutico , Animais , Proliferação de Células , Avaliação Pré-Clínica de Medicamentos , Humanos , Imidazóis/administração & dosagem , Imidazóis/farmacologia , Lipossarcoma , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Piridazinas/administração & dosagem , Piridazinas/farmacologia , Interferência de RNARESUMO
Exportin-1 mediates nuclear export of multiple tumor suppressor and growth regulatory proteins. Aberrant expression of exportin-1 is noted in human malignancies, resulting in cytoplasmic mislocalization of its target proteins. We investigated the efficacy of selinexor against liposarcoma cells both in vitro and in vivo. Exportin-1 was highly expressed in liposarcoma samples and cell lines as determined by immunohistochemistry, western blot, and immunofluorescence assay. Knockdown of endogenous exportin-1 inhibited proliferation of liposarcoma cells. Selinexor also significantly decreased cell proliferation as well as induced cell cycle arrest and apoptosis of liposarcoma cells. The drug also significantly decreased tumor volumes and weights of liposarcoma xenografts. Importantly, selinexor inhibited insulin-like growth factor 1 (IGF1) activation of IGF-1R/AKT pathway through upregulation of insulin-like growth factor binding protein 5 (IGFBP5). Further, overexpression and knockdown experiments showed that IGFBP5 acts as a tumor suppressor and its expression was restored upon selinexor treatment of liposarcoma cells. Selinexor decreased aurora kinase A and B levels in these cells and inhibitors of these kinases suppressed the growth of the liposarcoma cells. Overall, our study showed that selinexor treatment restored tumor suppressive function of IGFBP5 and inhibited aurora kinase A and B in liposarcoma cells supporting the usefulness of selinexor as a potential therapeutic strategy for the treatment of this cancer.
Assuntos
Antineoplásicos/farmacologia , Hidrazinas/farmacologia , Carioferinas/antagonistas & inibidores , Lipossarcoma/tratamento farmacológico , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Triazóis/farmacologia , Animais , Apoptose/efeitos dos fármacos , Aurora Quinase A/metabolismo , Aurora Quinase B/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação Neoplásica da Expressão Gênica , Humanos , Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Carioferinas/genética , Carioferinas/metabolismo , Lipossarcoma/genética , Lipossarcoma/metabolismo , Lipossarcoma/patologia , Masculino , Camundongos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Receptor IGF Tipo 1 , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Somatomedina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Transfecção , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína Exportina 1RESUMO
Anaplastic thyroid carcinoma (ATC) is one of the most lethal malignancies having no effective treatment. Exportin-1 (XPO1) is the key mediator of nuclear export of many tumor suppressor proteins and is overexpressed in human cancers. In this study, we examined the therapeutic potential of selinexor (XPO1 inhibitor) against human ATC cells both in vitro and in vivo. Here, we showed that XPO1 is robustly expressed in primary ATC samples and human ATC cell lines. Silencing of XPO1 by either shRNA or selinexor significantly reduced cellular growth and induced cell cycle arrest, apoptosis of ATC cells by altering the protein expression of cancer-related genes. Moreover, selinexor significantly inhibited tumor growth of ATC xenografts. Microarray analysis showed enrichment of DNA replication, cell cycle, cell cycle checkpoint and TNF pathways in selinexor treated ATC cells. Importantly, selinexor decreased AXL and GAS6 levels in CAL62 and HTH83 cells and suppressed the phosphorylation of downstream targets of AXL signaling such as AKT and P70S6K. Finally, a combination of selinexor with doxorubicin demonstrated a synergistic decrease in the cellular proliferation of several ATC cells. These results provide a rationale for investigating the efficacy of combining selinexor and doxorubicin therapy to improve the outcome of ATC patients.
Assuntos
Antineoplásicos/administração & dosagem , Doxorrubicina/administração & dosagem , Hidrazinas/administração & dosagem , Carcinoma Anaplásico da Tireoide/tratamento farmacológico , Neoplasias da Glândula Tireoide/tratamento farmacológico , Triazóis/administração & dosagem , Animais , Antineoplásicos/farmacologia , Apoptose , Pontos de Checagem do Ciclo Celular , Modelos Animais de Doenças , Doxorrubicina/farmacologia , Xenoenxertos , Humanos , Hidrazinas/farmacologia , Carioferinas/antagonistas & inibidores , Modelos Biológicos , Transplante de Neoplasias , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Resultado do Tratamento , Triazóis/farmacologia , Células Tumorais Cultivadas , Proteína Exportina 1RESUMO
Somatic mutations in the spliceosome gene ZRSR2-located on the X chromosome-are associated with myelodysplastic syndrome (MDS). ZRSR2 is involved in the recognition of 3'-splice site during the early stages of spliceosome assembly; however, its precise role in RNA splicing has remained unclear. Here we characterize ZRSR2 as an essential component of the minor spliceosome (U12 dependent) assembly. shRNA-mediated knockdown of ZRSR2 leads to impaired splicing of the U12-type introns and RNA-sequencing of MDS bone marrow reveals that loss of ZRSR2 activity causes increased mis-splicing. These splicing defects involve retention of the U12-type introns, while splicing of the U2-type introns remain mostly unaffected. ZRSR2-deficient cells also exhibit reduced proliferation potential and distinct alterations in myeloid and erythroid differentiation in vitro. These data identify a specific role for ZRSR2 in RNA splicing and highlight dysregulated splicing of U12-type introns as a characteristic feature of ZRSR2 mutations in MDS.
Assuntos
Processamento Alternativo , Mutação , Síndromes Mielodisplásicas/genética , Proteínas Nucleares/genética , RNA Nuclear Pequeno , Ribonucleoproteínas/genética , Animais , Antígenos CD34/metabolismo , Sequência de Bases , Células da Medula Óssea/citologia , Diferenciação Celular , Proliferação de Células , Éxons , Feminino , Genômica , Humanos , Íntrons , Células K562 , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Dados de Sequência Molecular , Transplante de Neoplasias , Proteínas Nucleares/metabolismo , Ribonucleoproteínas/metabolismo , SpliceossomosRESUMO
CONTEXT: Anaplastic thyroid cancer (ATC) has no effective treatment, resulting in a high rate of mortality. We established cell lines from a primary ATC and its lymph node metastasis, and investigated the molecular factors and genomic changes associated with tumor growth. OBJECTIVE: The aim of the study was to understand the molecular and genomic changes of highly aggressive ATC and its clonal evolution to develop rational therapies. DESIGN: We established unique cell lines from primary (OGK-P) and metastatic (OGK-M) ATC specimen, as well as primagraft from the metastatic ATC, which was serially xeno-transplanted for more than 1 year in NOD scid gamma mice were established. These cell lines and primagraft were used as tools to examine gene expression, copy number changes, and somatic mutations using RNA array, SNP Chip, and whole exome sequencing. RESULTS: Mice carrying sc (OGK-P and OGK-M) tumors developed splenomegaly and neutrophilia with high expression of cytokines including CSF1, CSF2, CSF3, IL-1ß, and IL-6. Levels of HIF-1α and its targeted genes were also elevated in these tumors. The treatment of tumor carrying mice with Bevacizumab effectively decreased tumor growth, macrophage infiltration, and peripheral WBCs. SNP chip analysis showed homozygous deletion of exons 3-22 of the PARD3 gene in the cells. Forced expression of PARD3 decreased cell proliferation, motility, and invasiveness, restores cell-cell contacts and enhanced cell adhesion. Next generation exome sequencing identified the somatic changes present in the primary, metastatic, and primagraft tumors demonstrating evolution of the mutational signature over the year of passage in vivo. CONCLUSION: To our knowledge, we established the first paired human primary and metastatic ATC cell lines offering unique possibilities for comparative functional investigations in vitro and in vivo. Our exome sequencing also identified novel mutations, as well as clonal evolution in both the metastasis and primagraft.
Assuntos
Linhagem Celular Tumoral/patologia , Carcinoma Anaplásico da Tireoide/patologia , Neoplasias da Glândula Tireoide/patologia , Idoso , Animais , Proliferação de Células , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Transplante de Neoplasias , Neutrófilos/patologia , Esplenomegalia/etiologia , Esplenomegalia/patologia , Carcinoma Anaplásico da Tireoide/complicações , Neoplasias da Glândula Tireoide/complicaçõesRESUMO
Liposarcoma (LPS) is the most common type of soft tissue sarcoma accounting for 20% of all adult sarcomas. Due to absence of clinically effective treatment options in inoperable situations and resistance to chemotherapeutics, a critical need exists to identify novel therapeutic targets. We analyzed LPS genomic landscape using SNP arrays, whole exome sequencing and targeted exome sequencing to uncover the genomic information for development of specific anti-cancer targets. SNP array analysis indicated known amplified genes (MDM2, CDK4, HMGA2) and important novel genes (UAP1, MIR557, LAMA4, CPM, IGF2, ERBB3, IGF1R). Carboxypeptidase M (CPM), recurrently amplified gene in well-differentiated/de-differentiated LPS was noted as a putative oncogene involved in the EGFR pathway. Notable deletions were found at chromosome 1p (RUNX3, ARID1A), chromosome 11q (ATM, CHEK1) and chromosome 13q14.2 (MIR15A, MIR16-1). Significantly and recurrently mutated genes (false discovery rate < 0.05) included PLEC (27%), MXRA5 (21%), FAT3 (24%), NF1 (20%), MDC1 (10%), TP53 (7%) and CHEK2 (6%). Further, in vitro and in vivo functional studies provided evidence for the tumor suppressor role for Neurofibromin 1 (NF1) gene in different subtypes of LPS. Pathway analysis of recurrent mutations demonstrated signaling through MAPK, JAK-STAT, Wnt, ErbB, axon guidance, apoptosis, DNA damage repair and cell cycle pathways were involved in liposarcomagenesis. Interestingly, we also found mutational and copy number heterogeneity within a primary LPS tumor signifying the importance of multi-region sequencing for cancer-genome guided therapy. In summary, these findings provide insight into the genomic complexity of LPS and highlight potential druggable pathways for targeted therapeutic approach.