RESUMO
MYC family oncoproteins regulate the expression of a large number of genes and broadly stimulate elongation by RNA polymerase II (RNAPII). While the factors that control the chromatin association of MYC proteins are well understood, much less is known about how interacting proteins mediate MYC's effects on transcription. Here, we show that TFIIIC, an architectural protein complex that controls the three-dimensional chromatin organisation at its target sites, binds directly to the amino-terminal transcriptional regulatory domain of MYCN. Surprisingly, TFIIIC has no discernible role in MYCN-dependent gene expression and transcription elongation. Instead, MYCN and TFIIIC preferentially bind to promoters with paused RNAPII and globally limit the accumulation of non-phosphorylated RNAPII at promoters. Consistent with its ubiquitous role in transcription, MYCN broadly participates in hubs of active promoters. Depletion of TFIIIC further increases MYCN localisation to these hubs. This increase correlates with a failure of the nuclear exosome and BRCA1, both of which are involved in nascent RNA degradation, to localise to active promoters. Our data suggest that MYCN and TFIIIC exert an censoring function in early transcription that limits promoter accumulation of inactive RNAPII and facilitates promoter-proximal degradation of nascent RNA.
Assuntos
Cromatina , Proteína Proto-Oncogênica N-Myc , Regiões Promotoras Genéticas , RNA Polimerase II , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Proteína Proto-Oncogênica N-Myc/metabolismo , Proteína Proto-Oncogênica N-Myc/genética , Humanos , Cromatina/metabolismo , Ligação Proteica , Fatores de Transcrição TFII/metabolismo , Fatores de Transcrição TFII/genética , Transcrição Gênica , Linhagem Celular TumoralRESUMO
The MYCN oncoprotein binds active promoters in a heterodimer with its partner protein MAX. MYCN also interacts with the nuclear exosome, a 3'-5' exoribonuclease complex, suggesting a function in RNA metabolism. Here, we show that MYCN forms stable high-molecular-weight complexes with the exosome and multiple RNA-binding proteins. MYCN binds RNA in vitro and in cells via a conserved sequence termed MYCBoxI. In cells, MYCN associates with thousands of intronic transcripts together with the ZCCHC8 subunit of the nuclear exosome targeting complex and enhances their processing. Perturbing exosome function results in global re-localization of MYCN from promoters to intronic RNAs. On chromatin, MYCN is then replaced by the MNT(MXD6) repressor protein, inhibiting MYCN-dependent transcription. RNA-binding-deficient alleles show that RNA-binding limits MYCN's ability to activate cell growth-related genes but is required for MYCN's ability to promote progression through S phase and enhance the stress resilience of neuroblastoma cells.
Assuntos
Proteína Proto-Oncogênica N-Myc , Proteínas Nucleares , Proteínas Oncogênicas , Proteínas de Ligação a RNA , Proteína Proto-Oncogênica N-Myc/metabolismo , Proteína Proto-Oncogênica N-Myc/genética , Humanos , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas Oncogênicas/metabolismo , Proteínas Oncogênicas/genética , Regiões Promotoras Genéticas , Linhagem Celular Tumoral , Neuroblastoma/metabolismo , Neuroblastoma/genética , Neuroblastoma/patologia , Exossomos/metabolismo , Exossomos/genética , Íntrons , Ligação Proteica , Núcleo Celular/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Regulação Neoplásica da Expressão Gênica , RNA/metabolismo , RNA/genética , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Proliferação de CélulasRESUMO
Ferroptosis has emerged as an attractive strategy in cancer therapy. Understanding the operational networks regulating ferroptosis may unravel vulnerabilities that could be harnessed for therapeutic benefit. Using CRISPR-activation screens in ferroptosis hypersensitive cells, we identify the selenoprotein P (SELENOP) receptor, LRP8, as a key determinant protecting MYCN-amplified neuroblastoma cells from ferroptosis. Genetic deletion of LRP8 leads to ferroptosis as a result of an insufficient supply of selenocysteine, which is required for the translation of the antiferroptotic selenoprotein GPX4. This dependency is caused by low expression of alternative selenium uptake pathways such as system Xc- . The identification of LRP8 as a specific vulnerability of MYCN-amplified neuroblastoma cells was confirmed in constitutive and inducible LRP8 knockout orthotopic xenografts. These findings disclose a yet-unaccounted mechanism of selective ferroptosis induction that might be explored as a therapeutic strategy for high-risk neuroblastoma and potentially other MYCN-amplified entities.
Assuntos
Ferroptose , Neuroblastoma , Humanos , Linhagem Celular Tumoral , Proteína Proto-Oncogênica N-Myc/genética , Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/genética , Neuroblastoma/tratamento farmacológico , Selenocisteína/uso terapêutico , AnimaisRESUMO
Amplification of MYCN is the driving oncogene in a subset of high-risk neuroblastoma. The MYCN protein and the Aurora-A kinase form a complex during S phase that stabilizes MYCN. Here we show that MYCN activates Aurora-A on chromatin, which phosphorylates histone H3 at serine 10 in S phase, promotes the deposition of histone H3.3 and suppresses R-loop formation. Inhibition of Aurora-A induces transcription-replication conflicts and activates the Ataxia telangiectasia and Rad3 related (ATR) kinase, which limits double-strand break accumulation upon Aurora-A inhibition. Combined inhibition of Aurora-A and ATR induces rampant tumor-specific apoptosis and tumor regression in mouse models of neuroblastoma, leading to permanent eradication in a subset of mice. The therapeutic efficacy is due to both tumor cell-intrinsic and immune cell-mediated mechanisms. We propose that targeting the ability of Aurora-A to resolve transcription-replication conflicts is an effective therapy for MYCN-driven neuroblastoma (141 words).
Assuntos
Aurora Quinase A , Neuroblastoma , Animais , Apoptose/genética , Aurora Quinase A/genética , Linhagem Celular Tumoral , Camundongos , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/tratamento farmacológicoRESUMO
Effective treatment of pediatric solid tumors has been hampered by the predominance of currently "undruggable" driver transcription factors. Improving outcomes while decreasing the toxicity of treatment necessitates the development of novel agents that can directly inhibit or degrade these elusive targets. MYCN in pediatric neural-derived tumors, including neuroblastoma and medulloblastoma, is a paradigmatic example of this problem. Attempts to directly and specifically target MYCN have failed due to its similarity to MYC, the unstructured nature of MYC family proteins in their monomeric form, the lack of an understanding of MYCN-interacting proteins and ability to test their relevance in vivo, the inability to obtain structural information on MYCN protein complexes, and the challenges of using traditional small molecules to inhibit protein-protein or protein-DNA interactions. However, there is now promise for directly targeting MYCN based on scientific and technological advances on all of these fronts. Here, we discuss prior challenges and the reasons for renewed optimism in directly targeting this "undruggable" transcription factor, which we hope will lead to improved outcomes for patients with pediatric cancer and create a framework for targeting driver oncoproteins regulating gene transcription.
Assuntos
Antineoplásicos/isolamento & purificação , Resistencia a Medicamentos Antineoplásicos , Proteína Proto-Oncogênica N-Myc/fisiologia , Neoplasias/tratamento farmacológico , Terapias em Estudo , Idade de Início , Antineoplásicos/história , Antineoplásicos/uso terapêutico , Criança , Descoberta de Drogas/história , Descoberta de Drogas/métodos , Descoberta de Drogas/tendências , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Ensaios de Seleção de Medicamentos Antitumorais/história , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Ensaios de Seleção de Medicamentos Antitumorais/tendências , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , História do Século XX , História do Século XXI , Humanos , Proteína Proto-Oncogênica N-Myc/antagonistas & inibidores , Proteína Proto-Oncogênica N-Myc/genética , Neoplasias/epidemiologia , Neoplasias/genética , Terapias em Estudo/história , Terapias em Estudo/métodos , Terapias em Estudo/tendênciasRESUMO
MYC is an oncogenic transcription factor that binds globally to active promoters and promotes transcriptional elongation by RNA polymerase II (RNAPII)1,2. Deregulated expression of the paralogous protein MYCN drives the development of neuronal and neuroendocrine tumours and is often associated with a particularly poor prognosis3. Here we show that, similar to MYC, activation of MYCN in human neuroblastoma cells induces escape of RNAPII from promoters. If the release of RNAPII from transcriptional pause sites (pause release) fails, MYCN recruits BRCA1 to promoter-proximal regions. Recruitment of BRCA1 prevents MYCN-dependent accumulation of stalled RNAPII and enhances transcriptional activation by MYCN. Mechanistically, BRCA1 stabilizes mRNA decapping complexes and enables MYCN to suppress R-loop formation in promoter-proximal regions. Recruitment of BRCA1 requires the ubiquitin-specific protease USP11, which binds specifically to MYCN when MYCN is dephosphorylated at Thr58. USP11, BRCA1 and MYCN stabilize each other on chromatin, preventing proteasomal turnover of MYCN. Because BRCA1 is highly expressed in neuronal progenitor cells during early development4 and MYC is less efficient than MYCN in recruiting BRCA1, our findings indicate that a cell-lineage-specific stress response enables MYCN-driven tumours to cope with deregulated RNAPII function.
Assuntos
Proteína BRCA1/metabolismo , Proteína Proto-Oncogênica N-Myc/metabolismo , RNA Polimerase II/metabolismo , Elongação da Transcrição Genética , Linhagem Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Regulação da Expressão Gênica , Humanos , Neuroblastoma/genética , Neuroblastoma/patologia , Estabilidade Proteica , Tioléster Hidrolases/metabolismoRESUMO
Galectin-1 (Gal-1) has been described to promote tumour growth by inducing angiogenesis and to contribute to the tumour immune escape. We had previously identified up-regulation of Gal-1 in preclinical models of aggressive neuroblastoma (NB), the most common extracranial tumour of childhood. While Gal-1 did not confer a survival advantage in the absence of exogenous stressors, Gal-1 contributed to enhanced cell migratory and invasive properties. Here, we review these findings and extend them by analyzing Gal-1 mediated effects on immune cell regulation and radiation resistance. In line with previous results, cell autonomous effects as well as paracrine functions contribute to Gal-1 mediated pro-tumourigenic functions. Interfering with Gal-1 functions in vivo will add to a better understanding of the role of the Gal-1 axis in the complex tumour-host interaction during immune-, chemo- and radiotherapy of neuroblastoma.
Assuntos
Galectina 1/metabolismo , Imunidade , Neuroblastoma/etiologia , Neuroblastoma/metabolismo , Tolerância a Radiação , Receptor trkB/metabolismo , Animais , Modelos Animais de Doenças , Galectina 1/genética , Dosagem de Genes , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Imunidade/genética , Neovascularização Patológica/genética , Neovascularização Patológica/imunologia , Neovascularização Patológica/metabolismo , Neuroblastoma/radioterapia , Fenótipo , Tolerância a Radiação/genética , Receptor trkB/genética , Transdução de Sinais/efeitos da radiaçãoRESUMO
MYC proteins bind globally to active promoters and promote transcriptional elongation by RNA polymerase II (Pol II). To identify effector proteins that mediate this function, we performed mass spectrometry on N-MYC complexes in neuroblastoma cells. The analysis shows that N-MYC forms complexes with TFIIIC, TOP2A, and RAD21, a subunit of cohesin. N-MYC and TFIIIC bind to overlapping sites in thousands of Pol II promoters and intergenic regions. TFIIIC promotes association of RAD21 with N-MYC target sites and is required for N-MYC-dependent promoter escape and pause release of Pol II. Aurora-A competes with binding of TFIIIC and RAD21 to N-MYC in vitro and antagonizes association of TOP2A, TFIIIC, and RAD21 with N-MYC during S phase, blocking N-MYC-dependent release of Pol II from the promoter. Inhibition of Aurora-A in S phase restores RAD21 and TFIIIC binding to chromatin and partially restores N-MYC-dependent transcriptional elongation. We propose that complex formation with Aurora-A controls N-MYC function during the cell cycle.
Assuntos
Aurora Quinase A/metabolismo , Proteína Proto-Oncogênica N-Myc/metabolismo , RNA Polimerase II/metabolismo , Fase S , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , DNA Topoisomerases Tipo II/metabolismo , DNA Intergênico/metabolismo , Proteínas de Ligação a DNA , Humanos , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , RNA Polimerase II/genética , Elongação da Transcrição Genética , Fatores de Transcrição TFIII/metabolismoRESUMO
Galectin-1 (Gal-1) has been described to promote tumor growth by inducing angiogenesis and to contribute to tumor immune escape by promoting apoptosis of activated T cells. We had previously identified upregulation of Gal-1 in preclinical models of aggressive neuroblastoma (NB), a solid tumor of childhood. However, the clinical and biological relevance of Gal-1 in this tumor entity is unclear. Here, the effect of Gal-1 on the immune system and tumorigenesis was assessed using modulation of Gal-1 expression in immune effector cells and in a transgenic NB model, designated TH-MYCN. The fraction of CD4(+) T cells was decreased in tumor-bearing TH-MYCN mice compared to tumor-free littermates, while both CD4(+) T cells as well as CD8(+) T cells were less activated, compatible with a reduced immune response in tumor-bearing mice. Tumor incidence was not significantly altered by decreasing Gal-1/LGALS1 gene dosage in TH-MYCN mice, but TH-MYCN/Gal-1(-/-) double transgenic mice displayed impaired tumor angiogenesis, splenomegaly, and impaired T cell tumor-infiltration with no differences in T cell activation and apoptosis rate. Additionally, a lower migratory capacity of Gal-1 deficient CD4(+) T cells toward tumor cells was observed in vitro. Transplantation of TH-MYCN-derived tumor cells into syngeneic mice resulted in significantly reduced tumor growth and elevated immune cell infiltration when Gal-1 was downregulated by shRNA. We therefore conclude that T cell-derived Gal-1 mediates T cell tumor-infiltration, whereas NB-derived Gal-1 promotes tumor growth. This opposing effect of Gal-1 in NB should be considered in therapeutic targeting strategies, as currently being developed for other tumor entities.
RESUMO
In many cancer types, MYC proteins are known to be master regulators of the RNA-producing machinery. Neuroblastoma is a tumor of early childhood characterized by heterogeneous clinical courses. Amplification of the MYCN oncogene is a marker of poor patient outcome in this disease. Here, we investigated the MYCN-driven transcriptome of 20 primary neuroblastomas with and without MYCN amplification using next-generation RNA sequencing and compared the results to those from an in vitro cell model for inducible MYCN (SH-EP MYCN-ER). Transcriptome sequencing produced 30-90 million mappable reads for each dataset. The most abundant RNA species was mRNA, but snoRNAs, pseudogenes and processed transcripts were also recovered. A total of 223 genes were significantly differentially expressed between MYCN-amplified and single-copy tumors. Of those genes associated with MYCN both in vitro and in vivo, 32% of MYCN upregulated and 37% of MYCN downregulated genes were verified either as previously identified MYCN targets or as having MYCN-binding motifs. Pathway analyses suggested transcriptomal upregulation of mTOR-related genes by MYCN. MYCN-driven neuroblastomas in mice displayed activation of the mTOR pathway on the protein level and activation of MYCN in SH-EP MYCN-ER cells resulted in high sensitivity toward mTOR inhibition in vitro. We conclude that next-generation RNA sequencing allows for the identification of MYCN regulated transcripts in neuroblastoma. As our results suggest MYCN involvement in mTOR pathway activation on the transcriptional level, mTOR inhibitors should be further evaluated for the treatment of MYCN-amplified neuroblastoma.
Assuntos
Regulação Neoplásica da Expressão Gênica , Neuroblastoma/genética , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Transcriptoma , Animais , Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Redes Reguladoras de Genes , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , Proteína Proto-Oncogênica N-Myc , Neuroblastoma/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Mensageiro/genética , RNA não Traduzido , Análise de Sequência de RNA , Células Tumorais CultivadasRESUMO
OBJECTIVES: The chemokine receptor antagonists maraviroc and vicriviroc and the integrase inhibitors elvitegravir and raltegravir are novel antiretroviral agents for the treatment of HIV-1 infections. ATP-binding cassette (ABC) transporters as modulators of the effectiveness and safety of therapy can mediate viral resistance and drug-drug interactions. To expand knowledge on drug-drug interactions of these antiretrovirals we investigated whether these compounds are substrates, inhibitors or inducers of important ABC transporters. METHODS: We evaluated P-glycoprotein (P-gp/ABCB1) inhibition by the calcein assay in P388/dx and L-MDR1 cells, breast cancer resistance protein (BCRP/ABCG2) inhibition in MDCKII-BCRP cells by pheophorbide A efflux, and inhibition of the multidrug resistance-associated protein 2 (MRP2/ABCC2) by using the MRP2 PREDIVEZ™ Vesicular Transport Kit. Substrate characteristics were evaluated by growth inhibition assays in MDCKII cells overexpressing particular ABC transporters. Induction of transporters was quantified by real-time RT-PCR in LS180 cells and for ABCB1 also at the functional level. RESULTS: Elvitegravir and vicriviroc inhibited ABCB1 in P388/dx and L-MDR1 cells (f2 values 1.9±0.2 µmol/L and 8.5±3.6 µmol/L, respectively). The IC50 for ABCG2 inhibition was 15.7±5.7 µmol/L for elvitegravir and 236.7±93.3 µmol/L for vicriviroc. Raltegravir and maraviroc showed no evidence of ABCB1 or ABCG2 inhibition. Maraviroc and vicriviroc stimulated ABCC2 transport function. Growth inhibition assays suggest that elvitegravir, raltegravir and vicriviroc are substrates of ABCB1. Induction assays demonstrate that mRNA expression of several ABC transporters is induced by these antiretrovirals in LS180 cells. CONCLUSIONS: The new antiretrovirals bear the potential to modulate expression and function of several ABC transporters, with elvitegravir revealing the highest interaction potential.