BRG1/SMARCA4 is essential for neuroblastoma cell viability through modulation of cell death and survival pathways.

Neuroblastoma (NB) is a neoplasm of the sympathetic nervous system, and is the most common solid tumor of infancy. NBs are very heterogeneous, with a clinical course ranging from spontaneous regression to resistance to all current forms of treatment. High-risk patients need intense chemotherapy, and only 30-40% will be cured. Relapsed or metastatic tumors acquire multi-drug resistance, raising the need for alternative treatments. Owing to the diverse mechanisms that are responsible of NB chemoresistance, we aimed to target epigenetic factors that control multiple pathways to bypass therapy resistance. We found that the SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 4 (SMARCA4/BRG1) was consistently upregulated in advanced stages of NB, with high BRG1 levels being indicative of poor outcome. Loss-of-function experiments in vitro and in vivo showed that BRG1 is essential for the proliferation of NB cells. Furthermore, whole-genome transcriptome analysis revealed that BRG1 controls the expression of key elements of oncogenic pathways such as PI3K/AKT and BCL2, which offers a promising new combination therapy for high-risk NB.

The Rho family GTPase Cdc42 regulates the activation of Ras/MAP kinase by the exchange factor Ras-GRF.

The Ras guanine-nucleotide exchange factor Ras-GRF/Cdc25(Mn) harbors a complex array of structural motifs that include a Dbl-homology (DH) domain, usually found in proteins that interact functionally with the Rho family GTPases, and the role of which is not yet fully understood. Here, we present evidence that Ras-GRF requires its DH domain to translocate to the membrane, to stimulate exchange on Ras, and to activate mitogen-activated protein kinase (MAPK). In an unprecedented fashion, we have found that these processes are regulated by the Rho family GTPase Cdc42. We show that GDP- but not GTP-bound Cdc42 prevents Ras-GRF recruitment to the membrane and activation of Ras/MAPK, although no direct association of Ras-GRF with Cdc42 was detected. We also demonstrate that catalyzing GDP/GTP exchange on Cdc42 facilitates Ras-GRF-induced MAPK activation. Moreover, we show that the potentiating effect of ionomycin on Ras-GRF-mediated MAPK stimulation is also regulated by Cdc42. These results provide the first evidence for the involvement of a Rho family G protein in the control of the activity of a Ras exchange factor.

Interaction of the corepressor Alien with DAX-1 is abrogated by mutations of DAX-1 involved in adrenal hypoplasia congenita.

DAX-1 is an unusual member of the nuclear hormone receptor (NHR) superfamily. Lack of DAX-1-mediated silencing leads to adrenal hypoplasia congenita and hypogonadotropic hypogonadism. Gene silencing through NHRs such as the thyroid hormone receptor (TR) is mediated by corepressors. We have previously characterized a novel corepressor, termed Alien, which interacts with TR and the ecdysone receptor but not with the retinoic acid receptors RAR or RXR. Here, we show that DAX-1 interacts with the corepressor Alien but not with the corepressor SMRT. This interaction is mediated by the DAX-1-silencing domain. Naturally occurring mutants of the DAX-1 gene fail to interact with Alien and have lost silencing function. Because the silencing domain of DAX-1 is unusual for NHRs, we mapped the interaction of Alien with DAX-1 and with TR. We show that Alien exhibits different binding characteristics to DAX-1 and TR. Furthermore, Northern experiments demonstrate that Alien is expressed in the adrenal gland and testis in tissues where DAX-1 is specifically expressed. Interestingly, a novel adrenal gland-specific mRNA of Alien was discovered. Thus, the impairment of Alien binding seems to play an important role in the pathogenesis mediated by DAX-1 mutants.

EcR interacts with corepressors and harbours an autonomous silencing domain functional in both Drosophila and vertebrate cells.

The ecdysone receptor (EcR) is a member of the large family of nuclear hormone receptors, which are ligand regulated transcription factors. In general, ligand converts these receptors into a transcriptional activator. Some vertebrate nuclear hormone receptors, such as the thyroid hormone and retinoic acid receptors, silence gene expression in the absence of ligand. EcR is involved in fly metamorphosis and is used in vertebrates as an inducible system for expression of transgenes. Here, we show that a Drosophila receptor, the EcR, harbours an autonomous silencing function in its carboxy-terminus. Interestingly, EcR mediates also silencing in vertebrate cells. In concordance with this EcR interacts with the corepressors SMRT and N-CoR, while addition of ligand reduces this interaction. Conversely, the v-erbA oncogene product, a thyroid hormone receptor derivative, mediates silencing in Drosophila cells. Thus, our data suggest the involvement of an evolutionarily conserved mechanism by which nuclear hormone receptors mediate gene silencing in multicellular organisms.

Alien, a highly conserved protein with characteristics of a corepressor for members of the nuclear hormone receptor superfamily.

Some members of nuclear hormone receptors, such as the thyroid hormone receptor (TR), silence gene expression in the absence of the hormone. Corepressors, which bind to the receptor's silencing domain, are involved in this repression. Hormone binding leads to dissociation of corepressors and binding of coactivators, which in turn mediate gene activation. Here, we describe the characteristics of Alien, a novel corepressor. Alien interacts with TR only in the absence of hormone. Addition of thyroid hormone leads to dissociation of Alien from the receptor, as shown by the yeast two-hybrid system, glutathione S-transferase pull-down, and coimmunoprecipitation experiments. Reporter assays indicate that Alien increases receptor-mediated silencing and that it harbors an autonomous silencing function. Immune staining shows that Alien is localized in the cell nucleus. Alien is a highly conserved protein showing 90% identity between human and Drosophila. Drosophila Alien shows similar activities in that it interacts in a hormone-sensitive manner with TR and harbors an autonomous silencing function. Specific interaction of Alien is seen with Drosophila nuclear hormone receptors, such as the ecdysone receptor and Seven-up, the Drosophila homologue of COUP-TF1, but not with retinoic acid receptor, RXR/USP, DHR 3, DHR 38, DHR 78, or DHR 96. These properties, taken together, show that Alien has the characteristics of a corepressor. Thus, Alien represents a member of a novel class of corepressors specific for selected members of the nuclear hormone receptor superfamily.