RESUMO
Phenotypic and metabolic heterogeneity within tumors is a major barrier to effective cancer therapy. How metabolism is implicated in specific phenotypes and whether lineage-restricted mechanisms control key metabolic vulnerabilities remain poorly understood. In melanoma, downregulation of the lineage addiction oncogene microphthalmia-associated transcription factor (MITF) is a hallmark of the proliferative-to-invasive phenotype switch, although how MITF promotes proliferation and suppresses invasion is poorly defined. Here, we show that MITF is a lineage-restricted activator of the key lipogenic enzyme stearoyl-CoA desaturase (SCD) and that SCD is required for MITFHigh melanoma cell proliferation. By contrast MITFLow cells are insensitive to SCD inhibition. Significantly, the MITF-SCD axis suppresses metastasis, inflammatory signaling, and an ATF4-mediated feedback loop that maintains de-differentiation. Our results reveal that MITF is a lineage-specific regulator of metabolic reprogramming, whereby fatty acid composition is a driver of melanoma phenotype switching, and highlight that cell phenotype dictates the response to drugs targeting lipid metabolism.
Assuntos
Adaptação Fisiológica/fisiologia , Ácidos Graxos/metabolismo , Melanoma/metabolismo , Fator de Transcrição Associado à Microftalmia/metabolismo , Estearoil-CoA Dessaturase/metabolismo , Animais , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Regulação para Baixo/fisiologia , Humanos , Camundongos , Invasividade Neoplásica/patologia , Fenótipo , Transdução de Sinais/fisiologiaRESUMO
P-TEFb, composed of CycT1 and CDK9, regulates the elongation of transcription by RNA polymerase II. In proliferating cells, it is regulated by 7SK snRNA in the 7SK snRNP complex. In resting cells, P-TEFb is absent, because CycT1 is dephosphorylated, released from CDK9 and rapidly degraded. In this study, we identified the mechanism of this degradation. We mapped the ubiquitination and degradation of free CycT1 to its N-terminal region from positions 1 to 280. This region is ubiquitinated at six lysines, where E3 ligases Siah1 and Siah2 bind and degrade these sequences. Importantly, the inhibition of Siah1/2 rescued the expression of free CycT1 in proliferating as well as resting primary cells. We conclude that Siah1/2 are the E3 ligases that bind and degrade the dissociated CycT1 in resting, terminally differentiated, anergic and/or exhausted cells.
Assuntos
Fator B de Elongação Transcricional Positiva , Fatores de Transcrição , Ubiquitina-Proteína Ligases/metabolismo , Fenômenos Fisiológicos Celulares , Ciclina T/genética , Fator B de Elongação Transcricional Positiva/metabolismo , RNA Polimerase II/metabolismo , RNA Nuclear Pequeno , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
A transplanted stem cell's engagement with a pathologic niche is the first step in its restoring homeostasis to that site. Inflammatory chemokines are constitutively produced in such a niche; their binding to receptors on the stem cell helps direct that cell's "pathotropism." Neural stem cells (NSCs), which express CXCR4, migrate to sites of CNS injury or degeneration in part because astrocytes and vasculature produce the inflammatory chemokine CXCL12. Binding of CXCL12 to CXCR4 (a G protein-coupled receptor, GPCR) triggers repair processes within the NSC. Although a tool directing NSCs to where needed has been long-sought, one would not inject this chemokine in vivo because undesirable inflammation also follows CXCL12-CXCR4 coupling. Alternatively, we chemically "mutated" CXCL12, creating a CXCR4 agonist that contained a strong pure binding motif linked to a signaling motif devoid of sequences responsible for synthetic functions. This synthetic dual-moity CXCR4 agonist not only elicited more extensive and persistent human NSC migration and distribution than did native CXCL 12, but induced no host inflammation (or other adverse effects); rather, there was predominantly reparative gene expression. When co-administered with transplanted human induced pluripotent stem cell-derived hNSCs in a mouse model of a prototypical neurodegenerative disease, the agonist enhanced migration, dissemination, and integration of donor-derived cells into the diseased cerebral cortex (including as electrophysiologically-active cortical neurons) where their secreted cross-corrective enzyme mediated a therapeutic impact unachieved by cells alone. Such a "designer" cytokine receptor-agonist peptide illustrates that treatments can be controlled and optimized by exploiting fundamental stem cell properties (e.g., "inflammo-attraction").
Assuntos
Quimiocina CXCL12/genética , Neurônios/metabolismo , Ligação Proteica/genética , Receptores CXCR4/genética , Astrócitos/metabolismo , Astrócitos/patologia , Movimento Celular/genética , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/patologia , Humanos , Células-Tronco Pluripotentes Induzidas , Inflamação/genética , Ligantes , Mutagênese/genética , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/transplante , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/terapia , Neurônios/patologiaRESUMO
Nutrient restriction reprograms cellular signaling and metabolic network to shape cancer phenotype. Lactate dehydrogenase A (LDHA) has a key role in aerobic glycolysis (the Warburg effect) through regeneration of the electron acceptor NAD+ and is widely regarded as a desirable target for cancer therapeutics. However, the mechanisms of cellular response and adaptation to LDHA inhibition remain largely unknown. Here, we show that LDHA activity supports serine and aspartate biosynthesis. Surprisingly, however, LDHA inhibition fails to impact human melanoma cell proliferation, survival, or tumor growth. Reduced intracellular serine and aspartate following LDHA inhibition engage GCN2-ATF4 signaling to initiate an expansive pro-survival response. This includes the upregulation of glutamine transporter SLC1A5 and glutamine uptake, with concomitant build-up of essential amino acids, and mTORC1 activation, to ameliorate the effects of LDHA inhibition. Tumors with low LDHA expression and melanoma patients acquiring resistance to MAPK signaling inhibitors, which target the Warburg effect, exhibit altered metabolic gene expression reminiscent of the ATF4-mediated survival signaling. ATF4-controlled survival mechanisms conferring synthetic vulnerability to the approaches targeting the Warburg effect offer efficacious therapeutic strategies.
Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Proliferação de Células , Glicólise , L-Lactato Desidrogenase/metabolismo , Melanoma/metabolismo , Proteínas de Neoplasias/metabolismo , Transdução de Sinais , Fator 4 Ativador da Transcrição/genética , Sistema ASC de Transporte de Aminoácidos/genética , Sistema ASC de Transporte de Aminoácidos/metabolismo , Ácido Aspártico/biossíntese , Ácido Aspártico/genética , Linhagem Celular Tumoral , Sobrevivência Celular , Humanos , L-Lactato Desidrogenase/antagonistas & inibidores , L-Lactato Desidrogenase/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Melanoma/genética , Melanoma/patologia , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismo , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Serina/biossíntese , Serina/genéticaRESUMO
Nuclear receptors (NRs) are implicated in the regulation of tumors and immune cells. We identify a tumor-intrinsic function of the orphan NR, NR2F6, regulating antitumor immunity. NR2F6 was selected from 48 candidate NRs based on an expression pattern in melanoma patient specimens (i.e., IFN-γ signature) associated with positive responses to immunotherapy and favorable patient outcomes. Correspondingly, genetic ablation of NR2F6 in a mouse melanoma model conferred a more effective response to PD-1 therapy. NR2F6 loss in B16F10 and YUMM1.7 melanoma cells attenuated tumor development in immune-competent but not -incompetent mice via the increased abundance of effector and progenitor-exhausted CD8+ T cells. Inhibition of NACC1 and FKBP10, identified as NR2F6 effectors, phenocopied NR2F6 loss. Inoculation of NR2F6 KO mice with NR2F6 KD melanoma cells further decreased tumor growth compared with NR2F6 WT mice. Tumor-intrinsic NR2F6 function complements its tumor-extrinsic role and justifies the development of effective anticancer therapies.
Assuntos
Linfócitos T CD8-Positivos , Melanoma , Animais , Camundongos , Imunoterapia , Melanoma/genética , Proteínas Repressoras/metabolismoRESUMO
The translation initiation complex 4F (eIF4F) is a rate-limiting factor in protein synthesis. Alterations in eIF4F activity are linked to several diseases, including cancer and infectious diseases. To this end, coronaviruses require eIF4F complex activity to produce proteins essential for their life cycle. Efforts to target coronaviruses by abrogating translation have been largely limited to repurposing existing eIF4F complex inhibitors. Here, we report the results of a high throughput screen to identify small molecules that disrupt eIF4F complex formation and inhibit coronavirus RNA and protein levels. Of 338,000 small molecules screened for inhibition of the eIF4F-driven, CAP-dependent translation, we identified SBI-1232 and two structurally related analogs, SBI-5844 and SBI-0498, that inhibit human coronavirus OC43 (HCoV-OC43; OC43) with minimal cell toxicity. Notably, gene expression changes after OC43 infection of Vero E6 or A549 cells were effectively reverted upon treatment with SBI-5844 or SBI-0498. Moreover, SBI-5844 or SBI-0498 treatment effectively impeded the eIF4F complex assembly, with concomitant inhibition of newly synthesized OC43 nucleocapsid protein and OC43 RNA and protein levels. Overall, we identify SBI-5844 and SBI-0498 as small molecules targeting the eIF4F complex that may limit coronavirus transcripts and proteins, thereby representing a basis for developing novel therapeutic modalities against coronaviruses.
RESUMO
Arginyl-tRNA-protein transferase 1 (ATE1) catalyses N-terminal protein arginylation, a post-translational modification implicated in cell migration, invasion and the cellular stress response. Herein, we report that ATE1 is overexpressed in NRAS-mutant melanomas, while it is downregulated in BRAF-mutant melanomas. ATE1 expression was higher in metastatic tumours, compared with primary tumours. Consistent with these findings, ATE1 depletion reduced melanoma cell viability, migration and colony formation. Reduced ATE1 expression also affected cell responses to mTOR and MEK inhibitors and to serum deprivation. Among putative ATE1 substrates is the tumour suppressor AXIN1, pointing to the possibility that ATE1 may fine-tune AXIN1 function in melanoma. Our findings highlight an unexpected role for ATE1 in melanoma cell aggressiveness and suggest that ATE1 constitutes a potential new therapeutic target.
Assuntos
Aminoaciltransferases , Melanoma , Aminoaciltransferases/genética , Aminoaciltransferases/metabolismo , Movimento Celular , Proliferação de Células , Humanos , Melanoma/genética , Processamento de Proteína Pós-Traducional , RNA de Transferência/metabolismoRESUMO
Tumour dependency on specific metabolic signals has been demonstrated and often guided numerous therapeutic approaches. We identify melanoma addiction to the mitochondrial protein glutaryl-CoA dehydrogenase (GCDH), which functions in lysine metabolism and controls protein glutarylation. GCDH knockdown induced cell death programmes in melanoma cells, an activity blocked by inhibition of the upstream lysine catabolism enzyme DHTKD1. The transcription factor NRF2 mediates GCDH-dependent melanoma cell death programmes. Mechanistically, GCDH knockdown induces NRF2 glutarylation, increasing its stability and DNA binding activity, with a concomitant transcriptional upregulation of ATF4, ATF3, DDIT3 and CHAC1, resulting in cell death. In vivo, inducible inactivation of GCDH effectively inhibited melanoma tumour growth. Correspondingly, reduced GCDH expression correlated with improved survival of patients with melanoma. These findings identify melanoma cell addiction to GCDH, limiting apoptotic signalling by controlling NRF2 glutarylation. Inhibiting the GCDH pathway could thus represent a therapeutic approach to treat melanoma.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos , Encefalopatias Metabólicas , Melanoma , Fator 2 Relacionado a NF-E2/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/genética , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Encefalopatias Metabólicas/genética , Encefalopatias Metabólicas/metabolismo , Encefalopatias Metabólicas/patologia , DNA , Glutaril-CoA Desidrogenase/genética , Glutaril-CoA Desidrogenase/metabolismo , Humanos , Complexo Cetoglutarato Desidrogenase , Lisina , Melanoma/genética , Proteínas Mitocondriais , Fator 2 Relacionado a NF-E2/genéticaRESUMO
A key hallmark of cancer, altered metabolism, is central to cancer pathogenesis and therapy resistance. Robust glutamine metabolism is among cellular processes regulating tumor progression and responsiveness to therapy in a number of cancers, including melanoma and breast cancer. Among mechanisms underlying the increase in glutamine metabolism in tumors is enhanced glutamine uptake mediated by the glutamine transporters, with SLC1A5 (also known as ASCT2) shown to play a predominant role. Correspondingly, increased SLC1A5 expression coincides with poorer survival in patients with breast cancer and melanoma. Therefore, we performed an image-based screen to identify small molecules that are able to prevent the localization of SLC1A5 to the plasma membrane without impacting cell shape. From 7,000 small molecules, nine were selected as hits, of which one (IMD-0354) qualified for further detailed functional assessment. IMD-0354 was confirmed as a potent inhibitor of glutamine uptake that attained sustained low intracellular glutamine levels. Concomitant with its inhibition of glutamine uptake, IMD-0354 attenuated mTOR signaling, suppressed two- and three-dimensional growth of melanoma cells, and induced cell-cycle arrest, autophagy, and apoptosis. Pronounced effect of IMD-0354 was observed in different tumor-derived cell lines, compared with nontransformed cells. RNA-sequencing analysis identified the unfolded protein response, cell cycle, and response (DNA damage response pathways) to be affected by IMD-0354. Combination of IMD-0354 with GLS1 or LDHA inhibitors enhanced melanoma cell death. In vivo, IMD-0354 suppressed melanoma growth in a xenograft model. As a modulator of glutamine metabolism, IMD-0354 may serve as an important therapeutic and experimental tool that deserves further examination.
Assuntos
Benzamidas/uso terapêutico , Proteínas de Transporte/antagonistas & inibidores , Glutamina/metabolismo , Melanoma/tratamento farmacológico , Animais , Benzamidas/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Masculino , Melanoma/patologia , Camundongos , TransfecçãoRESUMO
Acute myeloid leukemia (AML) remains incurable, largely due to its resistance to conventional treatments. Here, we find that increased abundance of the ubiquitin ligase RNF5 contributes to AML development and survival. High RNF5 expression in AML patient specimens correlates with poor prognosis. RNF5 inhibition decreases AML cell growth in culture, in patient-derived xenograft (PDX) samples and in vivo, and delays development of MLL-AF9-driven leukemogenesis in mice, prolonging their survival. RNF5 inhibition causes transcriptional changes that overlap with those seen upon histone deacetylase (HDAC)1 inhibition. RNF5 induces the formation of K29 ubiquitin chains on the histone-binding protein RBBP4, promoting its recruitment to and subsequent epigenetic regulation of genes involved in AML maintenance. Correspondingly, RNF5 or RBBP4 knockdown enhances AML cell sensitivity to HDAC inhibitors. Notably, low expression of both RNF5 and HDAC coincides with a favorable prognosis. Our studies identify an ERAD-independent role for RNF5, demonstrating that its control of RBBP4 constitutes an epigenetic pathway that drives AML, and highlight RNF5/RBBP4 as markers useful to stratify patients for treatment with HDAC inhibitors.
Assuntos
Predisposição Genética para Doença/genética , Inibidores de Histona Desacetilases/farmacologia , Leucemia Mieloide/genética , Ubiquitina-Proteína Ligases/genética , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Doença Aguda , Animais , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Células HEK293 , Células HL-60 , Humanos , Células K562 , Leucemia Mieloide/tratamento farmacológico , Leucemia Mieloide/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Células U937 , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Beta-galactosidase of Aspergillus sp. AF crude homogenate was immobilized in Ca-alginate gel beads and used for the production of galactooligosaccharides (GOS) from lactose. Optimum pH and temperature, thermal and storage stability of the enzyme activity were investigated and compared with those of the free enzyme. The study on the improvement of mechanical strength of the alginate beads was carried out through various methods, which demonstrates that the hardening process, where the alginate beads were treated with 0.225 M CaCl(2) solution after three batches to compensate the lost of calcium in the beads, provided a high mechanical stability for repeated use in large-scale production. The experiment results show that GOS yield increased with the increase of lactose concentration, and also increased with excessive addition of lactose (exceeding its solubility) at the beginning of the reaction. The immobilization of beta-galactosidase of Aspergillus sp. AF crude homogenate is cheap in processing cost and easy to carry out, and the immobilized enzyme possesses high performance for industrial application.
Assuntos
Aspergillus/enzimologia , Enzimas Imobilizadas/metabolismo , Proteínas Fúngicas/metabolismo , beta-Galactosidase/metabolismo , Alginatos , Cloreto de Cálcio/química , Cloreto de Cálcio/metabolismo , Misturas Complexas , Estabilidade Enzimática , Ácido Glucurônico , Ácidos Hexurônicos , Concentração de Íons de Hidrogênio , Microbiologia Industrial/métodos , Lactose/química , Lactose/metabolismo , Microesferas , Oligossacarídeos de Cadeias Ramificadas/química , Oligossacarídeos de Cadeias Ramificadas/metabolismo , TemperaturaRESUMO
Protein arginine methyltransferase 5 (PRMT5) controls diverse cellular processes and is implicated in cancer development and progression. Here, we report an inverse correlation between PRMT5 function and antitumor immunity. PRMT5 expression was associated with an antitumor immune gene signature in human melanoma tissue. Reducing PRMT5 activity antagonized melanoma growth in immunocompetent but not immunocompromised mice. PRMT5 methylation of IFI16 [interferon-γ (IFN-γ)-inducible protein 16] or its murine homolog IFI204, which are components of the cGAS/STING (stimulator of IFN genes) pathway, attenuated cytosolic DNA-induced IFN and chemokine expression in melanoma cells. PRMT5 also inhibited transcription of the gene encoding NLRC5 (nucleotide-binding oligomerization domain-like receptor family caspase recruitment domain containing 5), a protein that promotes the expression of genes implicated in major histocompatibility complex class I (MHCI) antigen presentation. PRMT5 knockdown augmented IFN and chemokine production and increased MHCI abundance in melanoma. Increased expression of IFI204 and NLRC5 was associated with decreased melanoma growth in murine models, and increased expression of IFI16 and NLRC5 correlated with prolonged survival of patients with melanoma. Combination of pharmacological (GSK3326595) or genetic (shRNA) inhibition of PRMT5 with immune checkpoint therapy limited growth of murine melanoma tumors (B16F10 and YUMM1.7) and enhanced therapeutic efficacy, compared with the effect of either treatment alone. Overall, our findings provide a rationale to test PRMT5 inhibitors in immunotherapy-based clinical trials as a means to enhance an antitumor immune response.
Assuntos
Melanoma , Nucleotidiltransferases , Animais , Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Melanoma/tratamento farmacológico , Melanoma/genética , Metilação , Camundongos , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , RNA Interferente PequenoRESUMO
Disrupted antiviral immune responses are associated with severe COVID-19, the disease caused by SAR-CoV-2. Here, we show that the 73-amino-acid protein encoded by ORF9c of the viral genome contains a putative transmembrane domain, interacts with membrane proteins in multiple cellular compartments, and impairs antiviral processes in a lung epithelial cell line. Proteomic, interactome, and transcriptomic analyses, combined with bioinformatic analysis, revealed that expression of only this highly unstable small viral protein impaired interferon signaling, antigen presentation, and complement signaling, while inducing IL-6 signaling. Furthermore, we showed that interfering with ORF9c degradation by either proteasome inhibition or inhibition of the ATPase VCP blunted the effects of ORF9c. Our study indicated that ORF9c enables immune evasion and coordinates cellular changes essential for the SARS-CoV-2 life cycle. ONE-SENTENCE SUMMARY: SARS-CoV-2 ORF9c is the first human coronavirus protein localized to membrane, suppressing antiviral response, resembling full viral infection.
RESUMO
Understanding the mechanisms underlying anti-tumor immunity is pivotal for improving immune-based cancer therapies. Here, we report that growth of BRAF-mutant melanoma cells is inhibited, up to complete rejection, in Siah2-/- mice. Growth-inhibited tumors exhibit increased numbers of intra-tumoral activated T cells and decreased expression of Ccl17, Ccl22, and Foxp3. Marked reduction in Treg proliferation and tumor infiltration coincide with G1 arrest in tumor infiltrated Siah2-/- Tregs in vivo or following T cell stimulation in culture, attributed to elevated expression of the cyclin-dependent kinase inhibitor p27, a Siah2 substrate. Growth of anti-PD-1 therapy resistant melanoma is effectively inhibited in Siah2-/- mice subjected to PD-1 blockade, indicating synergy between PD-1 blockade and Siah2 loss. Low SIAH2 and FOXP3 expression is identified in immune responsive human melanoma tumors. Overall, Siah2 regulation of Treg recruitment and cell cycle progression effectively controls melanoma development and Siah2 loss in the host sensitizes melanoma to anti-PD-1 therapy.
Assuntos
Melanoma/imunologia , Proteínas Nucleares/imunologia , Linfócitos T Reguladores/imunologia , Ubiquitina-Proteína Ligases/imunologia , Animais , Quimiocina CCL17/genética , Quimiocina CCL17/imunologia , Quimiocina CCL22/genética , Quimiocina CCL22/imunologia , Proteína Forkhead Box O3/genética , Proteína Forkhead Box O3/imunologia , Humanos , Melanoma/genética , Camundongos , Camundongos Knockout , Proteínas Nucleares/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
Growing evidence supports the importance of gut microbiota in the control of tumor growth and response to therapy. Here, we select prebiotics that can enrich bacterial taxa that promote anti-tumor immunity. Addition of the prebiotics inulin or mucin to the diet of C57BL/6 mice induces anti-tumor immune responses and inhibition of BRAF mutant melanoma growth in a subcutaneously implanted syngeneic mouse model. Mucin fails to inhibit tumor growth in germ-free mice, indicating that the gut microbiota is required for the activation of the anti-tumor immune response. Inulin and mucin drive distinct changes in the microbiota, as inulin, but not mucin, limits tumor growth in syngeneic mouse models of colon cancer and NRAS mutant melanoma and enhances the efficacy of a MEK inhibitor against melanoma while delaying the emergence of drug resistance. We highlight the importance of gut microbiota in anti-tumor immunity and the potential therapeutic role for prebiotics in this process.
Assuntos
Microbioma Gastrointestinal/efeitos dos fármacos , Inulina/uso terapêutico , Melanoma/tratamento farmacológico , Mucinas/uso terapêutico , Prebióticos/análise , Animais , Inulina/farmacologia , Melanoma/patologia , Camundongos , Mucinas/farmacologiaRESUMO
Among the hallmarks of melanoma are impaired proteostasis and rapid development of resistance to targeted therapy that represent a major clinical challenge. However, the molecular machinery that links these processes is unknown. Here we describe that by stabilizing key melanoma oncoproteins, the ubiquitin ligase RNF4 promotes tumorigenesis and confers resistance to targeted therapy in melanoma cells, xenograft mouse models, and patient samples. In patients, RNF4 protein and mRNA levels correlate with poor prognosis and with resistance to MAPK inhibitors. Remarkably, RNF4 tumorigenic properties, including therapy resistance, require the translation initiation factor initiation elongation factor alpha (eIF2α). RNF4 binds, ubiquitinates, and stabilizes the phosphorylated eIF2α (p-eIF2α) but not activating transcription factor 4 or C/EBP homologous protein that mediates the eIF2α-dependent integrated stress response. In accordance, p-eIF2α levels were significantly elevated in high-RNF4 patient-derived melanomas. Thus, RNF4 and p-eIF2α establish a positive feed-forward loop connecting oncogenic translation and ubiquitin-dependent protein stabilization in melanoma.
Assuntos
Fator de Iniciação 2 em Eucariotos/metabolismo , Melanoma/genética , Proteínas Nucleares/metabolismo , Neoplasias Cutâneas/genética , Fatores de Transcrição/metabolismo , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Melanoma/tratamento farmacológico , Melanoma/mortalidade , Melanoma/patologia , Camundongos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Oncogenes/genética , Prognóstico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Estabilidade Proteica , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Pele/patologia , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/mortalidade , Neoplasias Cutâneas/patologia , Ubiquitinação/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Mechanisms regulating nuclear organization control fundamental cellular processes, including the cell and chromatin organization. Their disorganization, including aberrant nuclear architecture, has been often implicated in cellular transformation. Here, we identify Lamin A, among proteins essential for nuclear architecture, as SPANX (sperm protein associated with the nucleus on the X chromosome), a cancer testis antigen previously linked to invasive tumor phenotypes, interacting protein in melanoma. SPANX interaction with Lamin A was mapped to the immunoglobulin fold-like domain, a region critical for Lamin A function, which is often mutated in laminopathies. SPANX downregulation in melanoma cell lines perturbed nuclear organization, decreased cell viability, and promoted senescence-associated phenotypes. Moreover, SPANX knockdown (KD) in melanoma cells promoted proliferation arrest, a phenotype mediated in part by IRF3/IL1A signaling. SPANX KD in melanoma cells also prompted the secretion of IL1A, which attenuated the proliferation of naïve melanoma cells. Identification of SPANX as a nuclear architecture complex component provides an unexpected insight into the regulation of Lamin A and its importance in melanoma. IMPLICATIONS: SPANX, a testis protein, interacts with LMNA and controls nuclear architecture and melanoma growth.
Assuntos
Lamina Tipo A/metabolismo , Laminas/metabolismo , Melanoma/genética , Proteínas Nucleares/genética , Humanos , Melanoma/patologia , TransfecçãoRESUMO
Based on tetrapeptide AVPI, we were able to design and synthesize a new simplified scaffold to inhibit the BIR3 domain of the XIAP protein at low micromolar range. The uncomplicated synthesis and the binding activity of the molecule disclosed here represent an attractive alternative to develop new compounds targeting the protein-protein interaction of XIAP/caspase9.
Assuntos
Proteínas Inibidoras de Apoptose , Oligopeptídeos/química , Relação Estrutura-Atividade , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/administração & dosagem , Apoptose , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Proteínas Mitocondriais , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidoresRESUMO
Bacterial-derived CRISPR-Cas9 nucleases have become a common tool in genome engineering. However, the editing efficiency by even the best-crafted Cas9 nucleases varies considerably with different genomic sites, and efforts to explore the vast natural Cas9 diversity have often met with mixed or little success. Here, we show that modification of the widely used Streptococcus pyogenes Cas9 by fusion with chromatin-modulating peptides (CMPs), derived from high mobility group proteins HMGN1 and HMGB1, histone H1, and chromatin remodeling complexes, improves its activity by up to several fold, particularly on refractory target sites. We further show that this CMP fusion strategy (termed CRISPR-chrom) is also effective in improving the activities of smaller Cas9 nucleases from Streptococcus pasteurianus and Campylobacter jejuni, as well as four newly characterized Cas9 orthologs from Bacillus smithii, Lactobacillus rhamnosus, Mycoplasma canis, and Parasutterella excrementihominis. Our findings suggest that this CRISPR-chrom strategy can be used to improve established Cas9 nucleases and facilitate exploration of novel Cas9 orthologs for genome modification.
Assuntos
Proteína 9 Associada à CRISPR/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Edição de Genes/métodos , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Cromatina/genética , Endonucleases/genética , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Proteína HMGN1/genética , Proteína HMGN1/metabolismo , Humanos , Células K562 , Peptídeos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Streptococcus pyogenes/genéticaRESUMO
While amino acid restriction remains an attractive strategy for cancer therapy, metabolic adaptations limit its effectiveness. Here we demonstrate a role of translational reprogramming in the survival of asparagine-restricted cancer cells. Asparagine limitation in melanoma and pancreatic cancer cells activates receptor tyrosine kinase-MAPK signalling as part of a feedforward mechanism involving mammalian target of rapamycin complex 1 (mTORC1)-dependent increase in MAPK-interacting kinase 1 (MNK1) and eukaryotic translation initiation factor 4E (eIF4E), resulting in enhanced translation of activating transcription factor 4 (ATF4) mRNA. MAPK inhibition attenuates translational induction of ATF4 and the expression of its target asparagine synthetase (ASNS), sensitizing melanoma and pancreatic tumours to asparagine restriction, reflected in inhibition of their growth. Correspondingly, low ASNS expression is among the top predictors of response to inhibitors of MAPK signalling in patients with melanoma and is associated with favourable prognosis when combined with low MAPK signalling activity. These studies reveal an axis of adaptation to asparagine deprivation and present a rationale for clinical evaluation of MAPK inhibitors in combination with asparagine restriction approaches.