RESUMO
Ewing sarcoma cells depend on the EWS-FLI1 fusion transcription factor for cell survival. Using an assay of EWS-FLI1 activity and genome-wide RNAi screening, we have identified proteins required for the processing of the EWS-FLI1 pre-mRNA. We show that Ewing sarcoma cells harboring a genomic breakpoint that retains exon 8 of EWSR1 require the RNA-binding protein HNRNPH1 to express in-frame EWS-FLI1. We also demonstrate the sensitivity of EWS-FLI1 fusion transcripts to the loss of function of the U2 snRNP component, SF3B1. Disrupted splicing of the EWS-FLI1 transcript alters EWS-FLI1 protein expression and EWS-FLI1-driven expression. Our results show that the processing of the EWS-FLI1 fusion RNA is a potentially targetable vulnerability in Ewing sarcoma cells.
Assuntos
Proteínas de Fusão Oncogênica/metabolismo , Proteína Proto-Oncogênica c-fli-1/metabolismo , Proteína EWS de Ligação a RNA/metabolismo , Sequência de Bases , Sítios de Ligação , Proteínas de Ligação a Calmodulina/antagonistas & inibidores , Proteínas de Ligação a Calmodulina/genética , Proteínas de Ligação a Calmodulina/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Éxons , Regulação Neoplásica da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/antagonistas & inibidores , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/metabolismo , Humanos , Proteínas dos Microfilamentos/antagonistas & inibidores , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Proteínas de Fusão Oncogênica/antagonistas & inibidores , Proteínas de Fusão Oncogênica/genética , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteína Proto-Oncogênica c-fli-1/antagonistas & inibidores , Proteína Proto-Oncogênica c-fli-1/genética , Interferência de RNA , Precursores de RNA/metabolismo , Splicing de RNA , Fatores de Processamento de RNA , RNA Interferente Pequeno/metabolismo , Proteína EWS de Ligação a RNA/antagonistas & inibidores , Proteína EWS de Ligação a RNA/genética , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Ribonucleoproteína Nuclear Pequena U2/antagonistas & inibidores , Ribonucleoproteína Nuclear Pequena U2/genética , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Sarcoma de Ewing/patologia , Transativadores , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
INTRODUCTION: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) binds to its receptors, TRAIL-receptor 1 (TRAIL-R1) and TRAIL-receptor 2 (TRAIL-R2), leading to apoptosis by activation of caspase-8 and the downstream executioner caspases, caspase-3 and caspase-7 (caspase-3/7). Triple-negative breast cancer (TNBC) cell lines with a mesenchymal phenotype are sensitive to TRAIL, whereas other breast cancer cell lines are resistant. The underlying mechanisms that control TRAIL sensitivity in breast cancer cells are not well understood. Here, we performed small interfering RNA (siRNA) screens to identify molecular regulators of the TRAIL pathway in breast cancer cells. METHODS: We conducted siRNA screens of the human kinome (691 genes), phosphatome (320 genes), and about 300 additional genes in the mesenchymal TNBC cell line MB231. Forty-eight hours after transfection of siRNA, parallel screens measuring caspase-8 activity, caspase-3/7 activity, or cell viability were conducted in the absence or presence of TRAIL for each siRNA, relative to a negative control siRNA (siNeg). A subset of genes was screened in cell lines representing epithelial TNBC (MB468), HER2-amplified breast cancer (SKBR3), and estrogen receptor-positive breast cancer (T47D). Selected putative negative regulators of the TRAIL pathway were studied by using small-molecule inhibitors. RESULTS: The primary screens in MB231 identified 150 genes, including 83 kinases, 4 phosphatases, and 63 nonkinases, as potential negative regulators of TRAIL. The identified genes are involved in many critical cell processes, including apoptosis, growth factor-receptor signaling, cell-cycle regulation, transcriptional regulation, and DNA repair. Gene-network analysis identified four genes (PDPK1, IKBKB, SRC, and BCL2L1) that formed key nodes within the interaction network of negative regulators. A secondary screen of a subset of the genes identified in additional cell lines representing different breast cancer subtypes and sensitivities to TRAIL validated and extended these findings. Further, we confirmed that small-molecule inhibition of SRC or BCL2L1, in combination with TRAIL, sensitizes breast cancer cells to TRAIL-induced apoptosis, including cell lines resistant to TRAIL-induced cytotoxicity. CONCLUSIONS: These data identify novel molecular regulators of TRAIL-induced apoptosis in breast cancer cells and suggest strategies for the enhanced application of TRAIL as a therapy for breast cancer.
