RESUMO
Intestinal tuft cells are critical for anti-helminth parasite immunity because they produce IL-25, which triggers IL-13 secretion by activated group 2 innate lymphoid cells (ILC2s) to expand both goblet and tuft cells. We show that epithelial Elp3, a tRNA-modifying enzyme, promotes tuft cell differentiation and is consequently critical for IL-25 production, ILC2 activation, goblet cell expansion and control of Nippostrongylus brasiliensis helminth infection in mice. Elp3 is essential for the generation of intestinal immature tuft cells and for the IL-13-dependent induction of glycolytic enzymes such as Hexokinase 1 and Aldolase A. Importantly, loss of epithelial Elp3 in the intestine blocks the codon-dependent translation of the Gator1 subunit Nprl2, an mTORC1 inhibitor, which consequently enhances mTORC1 activation and stabilizes Atf4 in progenitor cells. Likewise, Atf4 overexpression in mouse intestinal epithelium blocks tuft cell differentiation in response to intestinal helminth infection. Collectively, our data define Atf4 as a negative regulator of tuft cells and provide insights into promotion of intestinal type 2 immune response to parasites through tRNA modifications.
RESUMO
Dysregulation of messenger RNA (mRNA) translation, including preferential translation of mRNA with complex 5' untranslated regions such as the MYC oncogene, is recognized as an important mechanism in cancer. Here, we show that both human and murine chronic lymphocytic leukemia (CLL) cells display a high translation rate, which is inhibited by the synthetic flavagline FL3, a prohibitin (PHB)-binding drug. A multiomics analysis performed in samples from patients with CLL and cell lines treated with FL3 revealed the decreased translation of the MYC oncogene and of proteins involved in cell cycle and metabolism. Furthermore, inhibiting translation induced a proliferation arrest and a rewiring of MYC-driven metabolism. Interestingly, contrary to other models, the RAS-RAF-(PHBs)-MAPK pathway is neither impaired by FL3 nor implicated in translation regulation in CLL cells. Here, we rather show that PHBs are directly associated with the eukaryotic initiation factor (eIF)4F translation complex and are targeted by FL3. Knockdown of PHBs resembled FL3 treatment. Importantly, inhibition of translation controlled CLL development in vivo, either alone or combined with immunotherapy. Finally, high expression of translation initiation-related genes and PHBs genes correlated with poor survival and unfavorable clinical parameters in patients with CLL. Overall, we demonstrated that translation inhibition is a valuable strategy to control CLL development by blocking the translation of several oncogenic pathways including MYC. We also unraveled a new and direct role of PHBs in translation initiation, thus creating new therapeutic opportunities for patients with CLL.
Assuntos
Leucemia Linfocítica Crônica de Células B , Humanos , Camundongos , Animais , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Fator de Iniciação 4F em Eucariotos/genética , Proibitinas , Genes myc , RNA Mensageiro/genéticaRESUMO
Resistances to immunotherapies remains a major hurdle towards a cure for melanoma in numerous patients. An increase in the mesenchymal phenotype and a loss of differentiation have been clearly associated with resistance to targeted therapies. Similar phenotypes have been more recently also linked to resistance to immune checkpoint therapies. We demonstrated here that the loss of MIcrophthalmia associated Transcription Factor (MITF), a pivotal player in melanocyte differentiation, favors the escape of melanoma cells from the immune system. We identified Integrin beta-like protein 1 (ITGBL1), a secreted protein, upregulated in anti-PD1 resistant patients and in MITFlow melanoma cells, as the key immunomodulator. ITGBL1 inhibited immune cell cytotoxicity against melanoma cells by inhibiting NK cells cytotoxicity and counteracting beneficial effects of anti-PD1 treatment, both in vitro and in vivo. Mechanistically, MITF inhibited RUNX2, an activator of ITGBL1 transcription. Interestingly, VitaminD3, an inhibitor of RUNX2, improved melanoma cells to death by immune cells. In conclusion, our data suggest that inhibition of ITGBL1 might improve melanoma response to immunotherapies.
Assuntos
Carcinogênese/patologia , Citotoxicidade Imunológica , Fatores Imunológicos/metabolismo , Integrina beta1/metabolismo , Células Matadoras Naturais/imunologia , Melanoma/imunologia , Animais , Linhagem Celular Tumoral , Proliferação de Células , Melanoma/patologia , Camundongos Endogâmicos C57BL , Fator de Transcrição Associado à Microftalmia/metabolismoRESUMO
Transfer RNA dynamics contribute to cancer development through regulation of codon-specific messenger RNA translation. Specific aminoacyl-tRNA synthetases can either promote or suppress tumourigenesis. Here we show that valine aminoacyl-tRNA synthetase (VARS) is a key player in the codon-biased translation reprogramming induced by resistance to targeted (MAPK) therapy in melanoma. The proteome rewiring in patient-derived MAPK therapy-resistant melanoma is biased towards the usage of valine and coincides with the upregulation of valine cognate tRNAs and of VARS expression and activity. Strikingly, VARS knockdown re-sensitizes MAPK-therapy-resistant patient-derived melanoma in vitro and in vivo. Mechanistically, VARS regulates the messenger RNA translation of valine-enriched transcripts, among which hydroxyacyl-CoA dehydrogenase mRNA encodes for a key enzyme in fatty acid oxidation. Resistant melanoma cultures rely on fatty acid oxidation and hydroxyacyl-CoA dehydrogenase for their survival upon MAPK treatment. Together, our data demonstrate that VARS may represent an attractive therapeutic target for the treatment of therapy-resistant melanoma.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Melanoma , Animais , Humanos , Camundongos , Aminoacil-tRNA Sintetases/metabolismo , Aminoacil-tRNA Sintetases/genética , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Melanoma/genética , Melanoma/patologia , Melanoma/enzimologia , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Biossíntese de Proteínas , Inibidores de Proteínas Quinases/farmacologia , Valina/metabolismo , Valina/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The ubiquitination system plays a critical role in regulation of large array of biological processes and its alteration has been involved in the pathogenesis of cancers, among them cutaneous melanoma, which is responsible for the most deaths from skin cancers. Over the last decades, targeted therapies and immunotherapies became the standard therapeutic strategies for advanced melanomas. However, despite these breakthroughs, the prognosis of metastatic melanoma patients remains unoptimistic, mainly due to intrinsic or acquired resistances. Many avenues of research have been investigated to find new therapeutic targets for improving patient outcomes. Because of the pleiotropic functions of ubiquitination, and because each step of ubiquitination is amenable to pharmacological targeting, much attention has been paid to the role of this process in melanoma development and resistance to therapies. In this review, we summarize the latest data on ubiquitination and discuss the possible impacts on melanoma treatments.
RESUMO
Ubiquitination by serving as a major degradation signal of proteins, but also by controlling protein functioning and localization, plays critical roles in most key cellular processes. Here, we show that MITF, the master transcription factor in melanocytes, controls ubiquitination in melanoma cells. We identified FBXO32, a component of the SCF E3 ligase complex as a new MITF target gene. FBXO32 favors melanoma cell migration, proliferation, and tumor development in vivo. Transcriptomic analysis shows that FBXO32 knockdown induces a global change in melanoma gene expression profile. These include the inhibition of CDK6 in agreement with an inhibition of cell proliferation and invasion upon FBXO32 silencing. Furthermore, proteomic analysis identifies SMARC4, a component of the chromatin remodeling complexes BAF/PBAF, as a FBXO32 partner. FBXO32 and SMARCA4 co-localize at loci regulated by FBXO32, such as CDK6 suggesting that FBXO32 controls transcription through the regulation of chromatin remodeling complex activity. FBXO32 and SMARCA4 are the components of a molecular cascade, linking MITF to epigenetics, in melanoma cells.
Assuntos
Reprogramação Celular/genética , Epigênese Genética/genética , Melanoma/genética , Proteínas Musculares/metabolismo , Proteômica/métodos , Proteínas Ligases SKP Culina F-Box/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Melanoma/patologia , Camundongos , Camundongos Nus , Transfecção , Ubiquitinação , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Regulation of mRNA translation elongation impacts nascent protein synthesis and integrity and plays a critical role in disease establishment. Here, we investigate features linking regulation of codon-dependent translation elongation to protein expression and homeostasis. Using knockdown models of enzymes that catalyze the mcm5s2 wobble uridine tRNA modification (U34-enzymes), we show that gene codon content is necessary but not sufficient to predict protein fate. While translation defects upon perturbation of U34-enzymes are strictly dependent on codon content, the consequences on protein output are determined by other features. Specific hydrophilic motifs cause protein aggregation and degradation upon codon-dependent translation elongation defects. Accordingly, the combination of codon content and the presence of hydrophilic motifs define the proteome whose maintenance relies on U34-tRNA modification. Together, these results uncover the mechanism linking wobble tRNA modification to mRNA translation and aggregation to maintain proteome homeostasis.
Assuntos
Aminoácidos/química , Complexos Multienzimáticos/metabolismo , Elongação Traducional da Cadeia Peptídica , Processamento Pós-Transcricional do RNA , RNA de Transferência/metabolismo , Aminoácidos/genética , Aminoácidos/metabolismo , Linhagem Celular Tumoral , Uso do Códon , Técnicas de Silenciamento de Genes , Humanos , Interações Hidrofóbicas e Hidrofílicas , Complexos Multienzimáticos/genética , Agregados Proteicos/genética , Proteólise , Proteômica , RNA Mensageiro/metabolismo , RNA de Transferência/genética , Uridina/metabolismoRESUMO
The hematopoietic system is highly sensitive to perturbations in the translational machinery, of which an emerging level of regulation lies in the epitranscriptomic modification of transfer RNAs (tRNAs). Here, we interrogate the role of tRNA anticodon modifications in hematopoiesis by using mouse models of conditional inactivation of Elp3, the catalytic subunit of Elongator that modifies wobble uridine in specific tRNAs. Loss of Elp3 causes bone marrow failure by inducing death in committing progenitors and compromises the grafting activity of hematopoietic stem cells. Mechanistically, Elp3 deficiency activates a p53-dependent checkpoint in what resembles a misguided amino acid deprivation response that is accompanied by Atf4 overactivation and increased protein synthesis. While deletion of p53 rescues hematopoiesis, loss of Elp3 prompts the development of p53-mutated leukemia/lymphoma, and inactivation of p53 and Elongator cooperatively promotes tumorigenesis. Specific tRNA-modifying enzymes thus condition differentiation and antitumor fate decisions in hematopoietic stem cells and progenitors.
Assuntos
Hematopoese , Histona Acetiltransferases/metabolismo , RNA de Transferência/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Fator 4 Ativador da Transcrição/metabolismo , Aminoácidos/deficiência , Animais , Linhagem Celular , Sobrevivência Celular , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/ultraestrutura , Camundongos Endogâmicos C57BL , Biossíntese de Proteínas , Estresse Fisiológico , Resposta a Proteínas não Dobradas , Regulação para CimaRESUMO
HACE1 is an E3 ubiquitin ligase described as a tumour suppressor because HACE1-knockout mice develop multi-organ, late-onset cancers and because HACE1 expression is lost in several neoplasms, such as Wilms' tumours and colorectal cancer. However, a search of public databases indicated that HACE1 expression is maintained in melanomas. We demonstrated that HACE1 promoted melanoma cell migration and adhesion in vitro and was required for mouse lung colonisation by melanoma cells in vivo. Transcriptomic analysis of HACE1-depleted melanoma cells revealed an inhibition of ITGAV and ITGB1 as well changes in other genes involved in cell migration. We revealed that HACE1 promoted the K27 ubiquitination of fibronectin and regulated its secretion. Secreted fibronectin regulated ITGAV and ITGB1 expression, as well as melanoma cell adhesion and migration. Our findings disclose a novel molecular cascade involved in the regulation of fibronectin secretion, integrin expression and melanoma cell adhesion. By controlling this cascade, HACE1 displays pro-tumoural properties and is an important regulator of melanoma cell invasive properties.
Assuntos
Ubiquitina-Proteína Ligases/metabolismo , Animais , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Fibronectinas/metabolismo , Humanos , Integrinas/genética , Integrinas/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Camundongos Nus , Interferência de RNA , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/uso terapêutico , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Melanoma is one of the most lethal cancers when it reaches a metastatic stage. Despite advancements in targeted therapies (BRAF inhibitors) or immunotherapies (anti-CTLA-4 or anti-PD1), most patients with melanoma will need additional treatment. Thus, there is an urgent need to develop new therapeutical approaches to bypass resistance and achieve more prolonged responses. In this context, we were interested in E2F1, a transcription factor that plays a major role in the control of cell cycle under physiological and pathological conditions. Here we confirmed that E2F1 is highly expressed in melanoma cells. Inhibition of E2F1 activity further increased melanoma cell death and senescence, both in vitro and in vivo. Moreover, blocking E2F1 also induced death of melanoma cells resistant to BRAF inhibitors. In conclusion, our studies suggest that targeting the E2F1 signaling pathway may be therapeutically relevant for melanoma.