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Dysregulation of RNA splicing by spliceosome mutations or in cancer genes is increasingly recognized as a hallmark of cancer. Small molecule splicing modulators have been introduced into clinical trials to treat solid tumors or leukemia bearing recurrent spliceosome mutations. Nevertheless, further investigation of the molecular mechanisms that may enlighten therapeutic strategies for splicing modulators is highly desired. Here, using unbiased functional approaches, we report that the sensitivity to splicing modulation of the anti-apoptotic BCL2 family genes is a key mechanism underlying preferential cytotoxicity induced by the SF3b-targeting splicing modulator E7107. While BCL2A1, BCL2L2 and MCL1 are prone to splicing perturbation, BCL2L1 exhibits resistance to E7107-induced splicing modulation. Consequently, E7107 selectively induces apoptosis in BCL2A1-dependent melanoma cells and MCL1-dependent NSCLC cells. Furthermore, combination of BCLxL (BCL2L1-encoded) inhibitors and E7107 remarkably enhances cytotoxicity in cancer cells. These findings inform mechanism-based approaches to the future clinical development of splicing modulators in cancer treatment.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Melanoma/tratamento farmacológico , Antígenos de Histocompatibilidade Menor/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Splicing de RNA/efeitos dos fármacos , Proteína bcl-X/genética , Células A549 , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Doxiciclina/farmacologia , Sinergismo Farmacológico , Compostos de Epóxi/farmacologia , Feminino , Humanos , Neoplasias Pulmonares/genética , Macrolídeos/farmacologia , Melanoma/genética , Camundongos , Camundongos Nus , Interferência de RNA , Splicing de RNA/genética , RNA Interferente Pequeno/genética , Spliceossomos/efeitos dos fármacos , Spliceossomos/genética , Sequenciamento do Exoma , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Pladienolide, herboxidiene and spliceostatin have been identified as splicing modulators that target SF3B1 in the SF3b subcomplex. Here we report that PHF5A, another component of this subcomplex, is also targeted by these compounds. Mutations in PHF5A-Y36, SF3B1-K1071, SF3B1-R1074 and SF3B1-V1078 confer resistance to these modulators, suggesting a common interaction site. RNA-seq analysis reveals that PHF5A-Y36C has minimal effect on basal splicing but inhibits the global action of splicing modulators. Moreover, PHF5A-Y36C alters splicing modulator-induced intron-retention/exon-skipping profile, which correlates with the differential GC content between adjacent introns and exons. We determine the crystal structure of human PHF5A demonstrating that Y36 is located on a highly conserved surface. Analysis of the cryo-EM spliceosome Bact complex shows that the resistance mutations cluster in a pocket surrounding the branch point adenosine, suggesting a competitive mode of action. Collectively, we propose that PHF5A-SF3B1 forms a central node for binding to these splicing modulators.
Assuntos
Adenosina/química , Processamento Alternativo , Proteínas de Transporte/química , Fosfoproteínas/química , Fatores de Processamento de RNA/química , Proliferação de Células , Sobrevivência Celular , Microscopia Crioeletrônica , Cristalografia por Raios X , Compostos de Epóxi/química , Éxons , Álcoois Graxos/química , Células HCT116 , Humanos , Íntrons , Macrolídeos/química , Espectrometria de Massas , Mutagênese Sítio-Dirigida , Mutação , Proteína de Sequência 1 de Leucemia de Células Mieloides/química , Fosfoproteínas/metabolismo , Ligação Proteica , Conformação Proteica , Piranos/química , Interferência de RNA , Fatores de Processamento de RNA/metabolismo , Proteínas de Ligação a RNA , Proteínas Recombinantes/química , Análise de Sequência de RNA , Compostos de Espiro/química , Spliceossomos/metabolismo , TransativadoresRESUMO
The epithelial-mesenchymal transition program becomes activated during malignant progression and can enrich for cancer stem cells (CSCs). We report that inhibition of protein kinase C α (PKCα) specifically targets CSCs but has little effect on non-CSCs. The formation of CSCs from non-stem cells involves a shift from EGFR to PDGFR signaling and results in the PKCα-dependent activation of FRA1. We identified an AP-1 molecular switch in which c-FOS and FRA1 are preferentially utilized in non-CSCs and CSCs, respectively. PKCα and FRA1 expression is associated with the aggressive triple-negative breast cancers, and the depletion of FRA1 results in a mesenchymal-epithelial transition. Hence, identifying molecular features that shift between cell states can be exploited to target signaling components critical to CSCs.
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Neoplasias da Mama/metabolismo , Células-Tronco Neoplásicas/metabolismo , Proteína Quinase C-alfa/metabolismo , Transdução de Sinais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Análise por Conglomerados , Transição Epitelial-Mesenquimal/genética , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Proteína Quinase C-alfa/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição da Família Snail , Fatores de Transcrição/metabolismo , Proteína 1 Relacionada a Twist/metabolismoRESUMO
Epigenetic regulators represent a promising new class of therapeutic targets for cancer. Enhancer of zeste homolog 2 (EZH2), a subunit of Polycomb repressive complex 2 (PRC2), silences gene expression via its histone methyltransferase activity. We found that the oncogenic function of EZH2 in cells of castration-resistant prostate cancer is independent of its role as a transcriptional repressor. Instead, it involves the ability of EZH2 to act as a coactivator for critical transcription factors including the androgen receptor. This functional switch is dependent on phosphorylation of EZH2 and requires an intact methyltransferase domain. Hence, targeting the non-PRC2 function of EZH2 may have therapeutic efficacy for treating metastatic, hormone-refractory prostate cancer.
Assuntos
Proteínas Oncogênicas/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Neoplasias da Próstata/metabolismo , Animais , Castração , Linhagem Celular Tumoral , Estudos de Coortes , Proteína Potenciadora do Homólogo 2 de Zeste , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Histona Desmetilases com o Domínio Jumonji/metabolismo , Masculino , Metiltransferases/química , Metiltransferases/genética , Metiltransferases/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Camundongos SCID , Proteínas Oncogênicas/genética , Complexo Repressor Polycomb 2/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/mortalidade , Estrutura Terciária de Proteína , Receptores Androgênicos/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We present a powerful application of ultra high-throughput sequencing, SAGE-Seq, for the accurate quantification of normal and neoplastic mammary epithelial cell transcriptomes. We develop data analysis pipelines that allow the mapping of sense and antisense strands of mitochondrial and RefSeq genes, the normalization between libraries, and the identification of differentially expressed genes. We find that the diversity of cancer transcriptomes is significantly higher than that of normal cells. Our analysis indicates that transcript discovery plateaus at 10 million reads/sample, and suggests a minimum desired sequencing depth around five million reads. Comparison of SAGE-Seq and traditional SAGE on normal and cancerous breast tissues reveals higher sensitivity of SAGE-Seq to detect less-abundant genes, including those encoding for known breast cancer-related transcription factors and G protein-coupled receptors (GPCRs). SAGE-Seq is able to identify genes and pathways abnormally activated in breast cancer that traditional SAGE failed to call. SAGE-Seq is a powerful method for the identification of biomarkers and therapeutic targets in human disease.
