RESUMO
The MYCN oncoprotein drives the development of numerous neuroendocrine and pediatric tumors. Here we show that MYCN interacts with the nuclear RNA exosome, a 3'-5' exoribonuclease complex, and recruits the exosome to its target genes. In the absence of the exosome, MYCN-directed elongation by RNA polymerase II (RNAPII) is slow and non-productive on a large group of cell-cycle-regulated genes. During the S phase of MYCN-driven tumor cells, the exosome is required to prevent the accumulation of stalled replication forks and of double-strand breaks close to the transcription start sites. Upon depletion of the exosome, activation of ATM causes recruitment of BRCA1, which stabilizes nuclear mRNA decapping complexes, leading to MYCN-dependent transcription termination. Disruption of mRNA decapping in turn activates ATR, indicating transcription-replication conflicts. We propose that exosome recruitment by MYCN maintains productive transcription elongation during S phase and prevents transcription-replication conflicts to maintain the rapid proliferation of neuroendocrine tumor cells.
Assuntos
Núcleo Celular/enzimologia , Proliferação de Células , Replicação do DNA , Exossomos/enzimologia , Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/enzimologia , RNA Polimerase II/metabolismo , Transcrição Gênica , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/genética , Quebras de DNA de Cadeia Dupla , Exorribonucleases/genética , Exorribonucleases/metabolismo , Exossomos/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Masculino , Camundongos , Proteína Proto-Oncogênica N-Myc/genética , Células NIH 3T3 , Neuroblastoma/genética , Neuroblastoma/patologia , Regiões Promotoras Genéticas , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , RNA Polimerase II/genética , Terminação da Transcrição GenéticaRESUMO
Oncoproteins of the MYC family drive the development of numerous human tumours1. In unperturbed cells, MYC proteins bind to nearly all active promoters and control transcription by RNA polymerase II2,3. MYC proteins can also coordinate transcription with DNA replication4,5 and promote the repair of transcription-associated DNA damage6, but how they exert these mechanistically diverse functions is unknown. Here we show that MYC dissociates from many of its binding sites in active promoters and forms multimeric, often sphere-like structures in response to perturbation of transcription elongation, mRNA splicing or inhibition of the proteasome. Multimerization is accompanied by a global change in the MYC interactome towards proteins involved in transcription termination and RNA processing. MYC multimers accumulate on chromatin immediately adjacent to stalled replication forks and surround FANCD2, ATR and BRCA1 proteins, which are located at stalled forks7,8. MYC multimerization is triggered in a HUWE16 and ubiquitylation-dependent manner. At active promoters, MYC multimers block antisense transcription and stabilize FANCD2 association with chromatin. This limits DNA double strand break formation during S-phase, suggesting that the multimerization of MYC enables tumour cells to proliferate under stressful conditions.
Assuntos
RNA Polimerases Dirigidas por DNA , Humanos , Cromatina/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Regiões Promotoras Genéticas/genética , RNA Polimerase II/metabolismo , Transcrição Gênica , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Quebras de DNA de Cadeia Dupla , Fase S , Sítios de Ligação , RNA Mensageiro/biossínteseRESUMO
Deregulated expression of MYC induces a dependence on the NUAK1 kinase, but the molecular mechanisms underlying this dependence have not been fully clarified. Here, we show that NUAK1 is a predominantly nuclear protein that associates with a network of nuclear protein phosphatase 1 (PP1) interactors and that PNUTS, a nuclear regulatory subunit of PP1, is phosphorylated by NUAK1. Both NUAK1 and PNUTS associate with the splicing machinery. Inhibition of NUAK1 abolishes chromatin association of PNUTS, reduces spliceosome activity, and suppresses nascent RNA synthesis. Activation of MYC does not bypass the requirement for NUAK1 for spliceosome activity but significantly attenuates transcription inhibition. Consequently, NUAK1 inhibition in MYC-transformed cells induces global accumulation of RNAPII both at the pause site and at the first exon-intron boundary but does not increase mRNA synthesis. We suggest that NUAK1 inhibition in the presence of deregulated MYC traps non-productive RNAPII because of the absence of correctly assembled spliceosomes.
Assuntos
Núcleo Celular/metabolismo , Cromatina/metabolismo , Proteínas Quinases/metabolismo , Proteína Fosfatase 1/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Repressoras/metabolismo , Spliceossomos/metabolismo , Transcrição Gênica , Animais , Núcleo Celular/genética , Cromatina/genética , Regulação da Expressão Gênica , Células HeLa , Humanos , Camundongos , Células NIH 3T3 , Fosforilação , Proteínas Quinases/genética , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Splicing de RNA , Proteínas Repressoras/genética , Spliceossomos/genéticaRESUMO
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
In pancreatic ductal adenocarcinoma (PDAC), endogenous MYC is required for S-phase progression and escape from immune surveillance. Here we show that MYC in PDAC cells is needed for the recruitment of the PAF1c transcription elongation complex to RNA polymerase and that depletion of CTR9, a PAF1c subunit, enables long-term survival of PDAC-bearing mice. PAF1c is largely dispensable for normal proliferation and regulation of MYC target genes. Instead, PAF1c limits DNA damage associated with S-phase progression by being essential for the expression of long genes involved in replication and DNA repair. Surprisingly, the survival benefit conferred by CTR9 depletion is not due to DNA damage, but to T-cell activation and restoration of immune surveillance. This is because CTR9 depletion releases RNA polymerase and elongation factors from the body of long genes and promotes the transcription of short genes, including MHC class I genes. The data argue that functionally distinct gene sets compete for elongation factors and directly link MYC-driven S-phase progression to tumor immune evasion.
Assuntos
Fenômenos Bioquímicos , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Proteínas Proto-Oncogênicas c-myc , Animais , Camundongos , Carcinoma Ductal Pancreático/patologia , Proliferação de Células , RNA Polimerases Dirigidas por DNA/metabolismo , Evasão da Resposta Imune , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas c-myc/metabolismoRESUMO
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
Oncogenic transformation of lung epithelial cells is a multistep process, frequently starting with the inactivation of tumour suppressors and subsequent development of activating mutations in proto-oncogenes, such as members of the PI3K or MAPK families. Cells undergoing transformation have to adjust to changes, including altered metabolic requirements. This is achieved, in part, by modulating the protein abundance of transcription factors. Here, we report that the ubiquitin carboxyl-terminal hydrolase 28 (USP28) enables oncogenic reprogramming by regulating the protein abundance of proto-oncogenes such as c-JUN, c-MYC, NOTCH and ∆NP63 at early stages of malignant transformation. USP28 levels are increased in cancer compared with in normal cells due to a feed-forward loop, driven by increased amounts of oncogenic transcription factors such as c-MYC and c-JUN. Irrespective of oncogenic driver, interference with USP28 abundance or activity suppresses growth and survival of transformed lung cells. Furthermore, inhibition of USP28 via a small-molecule inhibitor resets the proteome of transformed cells towards a 'premalignant' state, and its inhibition synergizes with clinically established compounds used to target EGFRL858R -, BRAFV600E - or PI3KH1047R -driven tumour cells. Targeting USP28 protein abundance at an early stage via inhibition of its activity is therefore a feasible strategy for the treatment of early-stage lung tumours, and the observed synergism with current standard-of-care inhibitors holds the potential for improved targeting of established tumours.
Assuntos
Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas B-raf , Carcinogênese/genética , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Receptores ErbB/genética , Humanos , Proteínas Proto-Oncogênicas B-raf/metabolismo , Fatores de Transcrição , Ubiquitina Tiolesterase/genéticaRESUMO
Squamous cell carcinomas (SCC) frequently have an exceptionally high mutational burden. As consequence, they rapidly develop resistance to platinum-based chemotherapy and overall survival is limited. Novel therapeutic strategies are therefore urgently required. SCC express ∆Np63, which regulates the Fanconi Anemia (FA) DNA-damage response in cancer cells, thereby contributing to chemotherapy-resistance. Here we report that the deubiquitylase USP28 is recruited to sites of DNA damage in cisplatin-treated cells. ATR phosphorylates USP28 and increases its enzymatic activity. This phosphorylation event is required to positively regulate the DNA damage repair in SCC by stabilizing ∆Np63. Knock-down or inhibition of USP28 by a specific inhibitor weakens the ability of SCC to cope with DNA damage during platin-based chemotherapy. Hence, our study presents a novel mechanism by which ∆Np63 expressing SCC can be targeted to overcome chemotherapy resistance. Limited treatment options and low response rates to chemotherapy are particularly common in patients with squamous cancer. The SCC specific transcription factor ∆Np63 enhances the expression of Fanconi Anemia genes, thereby contributing to recombinational DNA repair and Cisplatin resistance. Targeting the USP28-∆Np63 axis in SCC tones down this DNA damage response pathways, thereby sensitizing SCC cells to cisplatin treatment.
Assuntos
Carcinoma de Células Escamosas , Anemia de Fanconi , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Cisplatino/farmacologia , Anemia de Fanconi/tratamento farmacológico , Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Humanos , Fatores de Transcrição/metabolismo , Ubiquitina Tiolesterase/metabolismoRESUMO
BACKGROUND: Despite advances in treatment of patients with non-small cell lung cancer, carriers of certain genetic alterations are prone to failure. One such factor frequently mutated, is the tumor suppressor PTEN. These tumors are supposed to be more resistant to radiation, chemo- and immunotherapy. RESULTS: We demonstrate that loss of PTEN led to altered expression of transcriptional programs which directly regulate therapy resistance, resulting in establishment of radiation resistance. While PTEN-deficient tumor cells were not dependent on DNA-PK for IR resistance nor activated ATR during IR, they showed a significant dependence for the DNA damage kinase ATM. Pharmacologic inhibition of ATM, via KU-60019 and AZD1390 at non-toxic doses, restored and even synergized with IR in PTEN-deficient human and murine NSCLC cells as well in a multicellular organotypic ex vivo tumor model. CONCLUSION: PTEN tumors are addicted to ATM to detect and repair radiation induced DNA damage. This creates an exploitable bottleneck. At least in cellulo and ex vivo we show that low concentration of ATM inhibitor is able to synergise with IR to treat PTEN-deficient tumors in genetically well-defined IR resistant lung cancer models.
RESUMO
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
The transcription factor ∆Np63 is a master regulator of epithelial cell identity and essential for the survival of squamous cell carcinoma (SCC) of lung, head and neck, oesophagus, cervix and skin. Here, we report that the deubiquitylase USP28 stabilizes ∆Np63 and maintains elevated ∆NP63 levels in SCC by counteracting its proteasome-mediated degradation. Impaired USP28 activity, either genetically or pharmacologically, abrogates the transcriptional identity and suppresses growth and survival of human SCC cells. CRISPR/Cas9-engineered in vivo mouse models establish that endogenous USP28 is strictly required for both induction and maintenance of lung SCC. Our data strongly suggest that targeting ∆Np63 abundance via inhibition of USP28 is a promising strategy for the treatment of SCC tumours.
Assuntos
Carcinoma de Células Escamosas , Transativadores/metabolismo , Ubiquitina Tiolesterase/metabolismo , Animais , Carcinoma de Células Escamosas/metabolismo , Células Epiteliais , Humanos , Camundongos , Estabilidade Proteica , Fatores de Transcrição , Proteínas Supressoras de TumorRESUMO
Tumours depend on altered rates of protein synthesis for growth and survival, which suggests that mechanisms controlling mRNA translation may be exploitable for therapy. Here, we show that loss of APC, which occurs almost universally in colorectal tumours, strongly enhances the dependence on the translation initiation factor eIF2B5. Depletion of eIF2B5 induces an integrated stress response and enhances translation of MYC via an internal ribosomal entry site. This perturbs cellular amino acid and nucleotide pools, strains energy resources and causes MYC-dependent apoptosis. eIF2B5 limits MYC expression and prevents apoptosis in APC-deficient murine and patient-derived organoids and in APC-deficient murine intestinal epithelia in vivo. Conversely, the high MYC levels present in APC-deficient cells induce phosphorylation of eIF2α via the kinases GCN2 and PKR. Pharmacological inhibition of GCN2 phenocopies eIF2B5 depletion and has therapeutic efficacy in tumour organoids, which demonstrates that a negative MYC-eIF2α feedback loop constitutes a targetable vulnerability of colorectal tumours.
Assuntos
Neoplasias Colorretais/genética , Fator de Iniciação 2 em Eucariotos/genética , Regulação Neoplásica da Expressão Gênica , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteína da Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Apoptose/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Colo/metabolismo , Colo/patologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Fator de Iniciação 2B em Eucariotos/antagonistas & inibidores , Fator de Iniciação 2B em Eucariotos/genética , Fator de Iniciação 2B em Eucariotos/metabolismo , Retroalimentação Fisiológica , Feminino , Células HCT116 , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Biossíntese de Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transdução de Sinais , Análise de Sobrevida , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Fbw7, the substrate recognition subunit of SCF(Fbw7) ubiquitin ligase, mediates the turnover of multiple proto-oncoproteins and promotes its own degradation. Fbw7-dependent substrate ubiquitination is antagonized by the Usp28 deubiquitinase. Here, we show that Usp28 preferentially antagonizes autocatalytic ubiquitination and stabilizes Fbw7, resulting in dose-dependent effects in Usp28 knockout mice. Monoallelic deletion of Usp28 maintains stable Fbw7 but drives Fbw7 substrate degradation. In contrast, complete knockout triggers Fbw7 degradation and leads to the accumulation of Fbw7 substrates in several tissues and embryonic fibroblasts. On the other hand, overexpression of Usp28 stabilizes both Fbw7 and its substrates. Consequently, both complete loss and ectopic expression of Usp28 promote Ras-driven oncogenic transformation. We propose that dual regulation of Fbw7 activity by Usp28 is a safeguard mechanism for maintaining physiological levels of proto-oncogenic Fbw7 substrates, which is equivalently disrupted by loss or overexpression of Usp28.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Transformação Celular Neoplásica/metabolismo , Proteínas F-Box/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Alelos , Animais , Biocatálise , Proliferação de Células , Transformação Celular Neoplásica/patologia , Proteína 7 com Repetições F-Box-WD , Deleção de Genes , Células HeLa , Humanos , Camundongos Knockout , Modelos Biológicos , Especificidade de Órgãos , Estabilidade Proteica , Proteólise , Especificidade por Substrato , Transcrição GênicaRESUMO
Colorectal cancer is the third most common cancer worldwide. Although the transcription factor c-MYC is misregulated in the majority of colorectal tumors, it is difficult to target directly. The deubiquitinase USP28 stabilizes oncogenic factors, including c-MYC; however, the contribution of USP28 in tumorigenesis, particularly in the intestine, is unknown. Here, using murine genetic models, we determined that USP28 antagonizes the ubiquitin-dependent degradation of c-MYC, a known USP28 substrate, as well as 2 additional oncogenic factors, c-JUN and NOTCH1, in the intestine. Mice lacking Usp28 had no apparent adverse phenotypes, but exhibited reduced intestinal proliferation and impaired differentiation of secretory lineage cells. In a murine model of colorectal cancer, Usp28 deletion resulted in fewer intestinal tumors, and importantly, in established tumors, Usp28 deletion reduced tumor size and dramatically increased lifespan. Moreover, we identified Usp28 as a c-MYC target gene highly expressed in murine and human intestinal cancers, which indicates that USP28 and c-MYC form a positive feedback loop that maintains high c-MYC protein levels in tumors. Usp28 deficiency promoted tumor cell differentiation accompanied by decreased proliferation, which suggests that USP28 acts similarly in intestinal homeostasis and colorectal cancer models. Hence, inhibition of the enzymatic activity of USP28 may be a potential target for cancer therapy.