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
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
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
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
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
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
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
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
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
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.
Assuntos
Lipossarcoma/genética , Neoplasias de Tecidos Moles/genética , Animais , Análise Mutacional de DNA , Citometria de Fluxo , Técnicas de Silenciamento de Genes , Xenoenxertos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único , TranscriptomaRESUMO
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
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
The anti-apoptotic protein Bcl-2 is a well-known and attractive therapeutic target for cancer. In the present study the solution-phase T3P-DMSO mediated efficient synthesis of 2-amino-chromene-3-carbonitriles from alcohols, malanonitrile and phenols is reported. These novel 2-amino-chromene-3-carbonitriles showed cytotoxicity in human acute myeloid leukemia (AML) cell lines. Compound 4 g was found to be the most bioactive, decreasing growth and increasing apoptosis of AML cells. Moreover, compound 4 g (at a concentration of 5 µM) increased the G2/M and sub-G1 (apoptosis) phases of AML cells. The AML cells treated with compound 4 g exhibited decreased levels of Bcl-2 and increased levels of caspase-9. In silico molecular interaction analysis showed that compound 4 g shared a similar global binding motif with navitoclax (another small molecule that binds Bcl-2), however compound 4 g occupies a smaller volume within the P2 hot spot of Bcl-2. The intermolecular π-stacking interaction, direct electrostatic interactions, and docking energy predicted for 4 g in complex with Bcl-2 suggest a strong affinity of the complex, rendering 4 g as a promising Bcl-2 inhibitor for evaluation as a new anticancer agent.
Assuntos
Antineoplásicos/farmacologia , Benzopiranos/farmacologia , Nitrilas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Antineoplásicos/síntese química , Benzopiranos/síntese química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Concentração Inibidora 50 , Leucemia Mieloide Aguda/tratamento farmacológico , Simulação de Acoplamento Molecular , Terapia de Alvo Molecular , Nitrilas/síntese química , Proteínas Proto-Oncogênicas c-bcl-2/químicaRESUMO
CONTEXT: Anaplastic thyroid carcinoma (ATC) is an aggressive malignancy having no effective treatment. Laminin subunit-γ-2 (LAMC2) is an epithelial basement membrane protein involved in cell migration and tumor invasion and might represent an ideal target for the development of novel therapeutic approaches for ATC. OBJECTIVE: The objective of the investigation was to study the role of LAMC2 in ATC tumorigenesis. DESIGN: LAMC2 expression was evaluated by RT-PCR, Western blotting, and immunohistochemistry in tumor specimens, adjacent noncancerous tissues, and cell lines. The short hairpin RNA (shRNA) approach was used to investigate the effect of LAMC2 knockdown on the tumorigenesis of ATC. RESULTS: LAMC2 was highly expressed in ATC samples and cell lines compared with normal thyroid tissues. Silencing LAMC2 by shRNA in ATC cells moderately inhibited cell growth in liquid culture and dramatically decreased growth in soft agar and in xenografts growing in immunodeficient mice. Silencing LAMC2 caused cell cycle arrest and significantly suppressed the migration, invasion, and wound healing of ATC cells. Rescue experiments by overexpressing LAMC2 in LAMC2 knockdown cells reversed the inhibitory effects as shown by increased cell proliferation and colony formation. Microarray data demonstrated that LAMC2 shRNA significantly altered the expression of genes associated with migration, invasion, proliferation, and survival. Immunoprecipitation studies showed that LAMC2 bound to epidermal growth factor receptor (EGFR) in the ATC cells. Silencing LAMC2 partially blocked epidermal growth factor-mediated activation of EGFR and its downstream pathway. Interestingly, cetuximab (an EGFR blocking antibody) or EGFR small interfering RNA additively enhanced the antiproliferative activity of the LAMC2 knockdown ATC cells compared with the control cells. CONCLUSIONS: To our knowledge, this is the first report investigating the effect of LAMC2 on cell growth, cell cycle, migration, invasion, and EGFR signaling in ATC cells, suggesting that LAMC2 may be a potential therapeutic target for the treatment of ATC.
Assuntos
Movimento Celular/genética , Receptores ErbB/fisiologia , Laminina/genética , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Animais , Movimento Celular/efeitos dos fármacos , Progressão da Doença , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Invasividade Neoplásica , Metástase Neoplásica , RNA Interferente Pequeno/farmacologia , Transdução de Sinais/genética , Carcinoma Anaplásico da Tireoide , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Majority of bladder cancer deaths are caused due to transitional cell carcinoma (TCC) which is the most prevalent and chemoresistant malignancy of urinary bladder. Therefore, we analyzed the role of Sperm associated antigen 9 (SPAG9) in bladder TCC. METHODOLOGY AND FINDINGS: We examined SPAG9 expression and humoral response in 125 bladder TCC patients. Four bladder cancer cell lines were assessed for SPAG9 expression. In addition, we investigated the effect of SPAG9 ablation on cellular proliferation, cell cycle, migration and invasion in UM-UC-3 bladder cancer cells by employing gene silencing approach. Our SPAG9 gene and protein expression analysis revealed SPAG9 expression in 81% of bladder TCC tissue specimens. High SPAG9 expression (>60% SPAG9 positive cells) was found to be significantly associated with superficial non-muscle invasive stage (Pâ=â0.042) and low grade tumors (Pâ=â0.002) suggesting SPAG9 putative role in early spread and tumorigenesis. Humoral response against SPAG9 was observed in 95% of patients found positive for SPAG9 expression. All four bladder cancer cell lines revealed SPAG9 expression. In addition, SPAG9 gene silencing in UM-UC-3 cells resulted in induction of G0-G1 arrest characterized by up-regulation of p16 and p21 and consequent down-regulation of cyclin E, cyclin D and cyclin B, CDK4 and CDK1. Further, SPAG9 gene silencing also resulted in reduction in cellular growth, and migration and invasion ability of cancer cells in vitro. CONCLUSIONS: Collectively, our data in clinical specimens indicated that SPAG9 is potential biomarker and therapeutic target for bladder TCC.