ABSTRACT
Epigenetic silencing of RASSF (Ras association domain family) genes RASSF1 and RASSF5 (also called NORE1) by CpG hypermethylation is found frequently in many cancers. Although the physiological roles of RASSF1 have been studied in some detail, the exact functions of RASSF5 are not well understood. Here, we show that RASSF5 plays an important role in mediating apoptosis in response to death receptor ligands, TNF-α and TNF-related apoptosis-inducing ligand. Depletion of RASSF5 by siRNA significantly reduced TNF-α-mediated apoptosis, likely through its interaction with proapoptotic kinase MST1, a mammalian homolog of Hippo. Consistent with this, siRNA knockdown of MST1 also resulted in resistance to TNF-α-induced apoptosis. To further study the role of Rassf5 in vivo, we generated Rassf5-deficient mouse. Inactivation of Rassf5 in mouse embryonic fibroblasts (MEFs) resulted in resistance to TNF-α- and TNF-related apoptosis-inducing ligand-mediated apoptosis. Importantly, Rassf5-null mice were significantly more resistant to TNF-α-induced apoptosis and failed to activate Mst1. Loss of Rassf5 also resulted in spontaneous immortalization of MEFs at earlier passages than the control MEFs, and Rassf5-null immortalized MEFs, but not the immortalized wild type MEFs, were fully transformed by K-RasG12V. Together, our results demonstrate a direct role for RASSF5 in death receptor ligand-mediated apoptosis and provide further evidence for RASSF5 as a tumor suppressor.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Apoptosis/physiology , Receptors, Death Domain/metabolism , Tumor Necrosis Factor-alpha/metabolism , Tumor Suppressor Proteins/metabolism , ras Proteins/metabolism , Adaptor Proteins, Signal Transducing/genetics , Animals , Apoptosis/drug effects , Apoptosis Regulatory Proteins , Cell Line , Embryo, Mammalian/cytology , Embryo, Mammalian/metabolism , Fibroblasts/cytology , Fibroblasts/metabolism , Gene Silencing/physiology , Mice , Mice, Knockout , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , RNA, Small Interfering/genetics , Receptors, Death Domain/genetics , Tumor Necrosis Factor-alpha/pharmacology , Tumor Suppressor Proteins/genetics , ras Proteins/geneticsABSTRACT
EWS/FLI1 is a fusion gene product generated by a chromosomal translocation t(11;22)(q24;q12) found in Ewing sarcoma. EWS/FLI1 encodes an aberrant transcription factor with oncogenic properties in vitro. Paradoxically, expression of EWS/FLI1 in nontransformed primary cells results in apoptosis, but the exact mechanism remains unclear. In primary mouse embryonic fibroblasts derived from conditional EWS/FLI1 knock-in embryos, expression of EWS/FLI1 resulted in apoptosis with concomitant increase in the endogenous Caspase 3 (Casp3) mRNA. EWS/FLI1 directly bound and activated the CASP3 promoter, whereas small interfering RNA-mediated knockdown of EWS/FLI1 led to a marked decrease in CASP3 transcripts in Ewing sarcoma cell lines. Ectopic expression of EWS/FLI1 resulted in an increased expression of CASP3 protein in heterologous cell lines. Importantly, expression of EWS/FLI1 in the mouse triggered an early onset of apoptosis in kidneys and acute lethality. These findings suggest that EWS/FLI1 induces apoptosis, at least partially, through the activation of CASP3 and show the cell context-dependent roles of EWS/FLI1 in apoptosis and tumorigenesis.
Subject(s)
Apoptosis , Caspase 3/metabolism , Oncogene Proteins, Fusion/metabolism , Proto-Oncogene Protein c-fli-1/metabolism , Animals , Antineoplastic Agents, Hormonal/pharmacology , Base Sequence , Binding Sites/genetics , Caspase 3/genetics , Cell Line, Tumor , Cells, Cultured , Embryo, Mammalian/cytology , Embryo, Mammalian/metabolism , Fibroblasts/cytology , Fibroblasts/metabolism , Gene Expression/drug effects , Heart/drug effects , Humans , Kidney/drug effects , Kidney/metabolism , Lung/drug effects , Lung/metabolism , Mice , Mice, Transgenic , Myocardium/metabolism , Oncogene Proteins, Fusion/genetics , Pancreas/drug effects , Pancreas/metabolism , Promoter Regions, Genetic/genetics , Protein Binding , Proto-Oncogene Protein c-fli-1/genetics , RNA Interference , RNA-Binding Protein EWS , Tamoxifen/pharmacology , Transcription, GeneticABSTRACT
BACKGROUND: Desmoplastic Small Round Cell Tumor (DSRCT) is a highly aggressive malignancy that affects mainly adolescents and young adults. A defining characteristic of DSRCT is a specific chromosomal translocation, t(11;22)(p13;q12), that fuses EWS with WT1, leading to a production of two isoforms of chimeric transcription factor, EWS/WT1(-KTS) and EWS/WT1(+KTS). The chimeric proteins are thought to play critical roles in various stages of oncogenesis through aberrant transcription of different genes, but only a few of these genes have been identified. METHODOLOGY/PRINCIPAL FINDINGS: We report the identification of a new target of EWS/WT1, ENT4 (equilibrative nucleoside transporter 4) which encodes a pH-dependent adenosine transporter. ENT4 is transcriptionally activated by both isoforms of EWS/WT1 as evidenced by promoter-reporter and chromatin immunoprecipitation (ChIP) analyses. Furthermore, ENT4 is highly and specifically expressed in primary tumors of DSRCT as well as in a DSRCT cell line, JN-DSRCT-1. Treatment of JN-DSRCT-1 cells with adenosine analogs, such as 2-chloro-2'-deoxyadenosine (2-CdA), resulted in an increased cytotoxic response in dose- and pH-dependent manner. CONCLUSIONS/SIGNIFICANCE: Our detailed analyses of a novel target of EWS/WT1 in DSRCT reveal an insight into the oncogenic mechanism of EWS-fusion chromosomal translocation gene products and provide a new marker for DSRCT. Furthermore, identification of ENT4 as a highly expressed transcript in DSRCT may represent an attractive pathway for targeting chemotherapeutic drugs into DSRCT.
