RESUMO
The most prevalent genetic cause of both amyotrophic lateral sclerosis and frontotemporal dementia is a (GGGGCC)n nucleotide repeat expansion (NRE) occurring in the first intron of the C9orf72 gene (C9). Brain glucose hypometabolism is consistently observed in C9-NRE carriers, even at pre-symptomatic stages, but its role in disease pathogenesis is unknown. Here, we show alterations in glucose metabolic pathways and ATP levels in the brains of asymptomatic C9-BAC mice. We find that, through activation of the GCN2 kinase, glucose hypometabolism drives the production of dipeptide repeat proteins (DPRs), impairs the survival of C9 patient-derived neurons, and triggers motor dysfunction in C9-BAC mice. We also show that one of the arginine-rich DPRs (PR) could directly contribute to glucose metabolism and metabolic stress by inhibiting glucose uptake in neurons. Our findings provide a potential mechanistic link between energy imbalances and C9-ALS/FTD pathogenesis and suggest a feedforward loop model with potential opportunities for therapeutic intervention.
Assuntos
Esclerose Lateral Amiotrófica , Proteína C9orf72 , Demência Frontotemporal , Glucose , Fenótipo , Proteína ran de Ligação ao GTP , Animais , Camundongos , Trifosfato de Adenosina/metabolismo , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Modelos Animais de Doenças , Expansão das Repetições de DNA/genética , Demência Frontotemporal/genética , Demência Frontotemporal/metabolismo , Demência Frontotemporal/patologia , Glucose/metabolismo , Camundongos Transgênicos , Neurônios/metabolismo , Biossíntese de Proteínas , Proteína ran de Ligação ao GTP/metabolismoRESUMO
Circular RNAs (circRNAs), which are increasingly being implicated in a variety of functions in normal and cancerous cells1-5, are formed by back-splicing of precursor mRNAs in the nucleus6-10. circRNAs are predominantly localized in the cytoplasm, indicating that they must be exported from the nucleus. Here we identify a pathway that is specific for the nuclear export of circular RNA. This pathway requires Ran-GTP, exportin-2 and IGF2BP1. Enhancing the nuclear Ran-GTP gradient by depletion or chemical inhibition of the major protein exporter CRM1 selectively increases the nuclear export of circRNAs, while reducing the nuclear Ran-GTP gradient selectively blocks circRNA export. Depletion or knockout of exportin-2 specifically inhibits nuclear export of circRNA. Analysis of nuclear circRNA-binding proteins reveals that interaction between IGF2BP1 and circRNA is enhanced by Ran-GTP. The formation of circRNA export complexes in the nucleus is promoted by Ran-GTP through its interactions with exportin-2, circRNA and IGF2BP1. Our findings demonstrate that adaptors such as IGF2BP1 that bind directly to circular RNAs recruit Ran-GTP and exportin-2 to export circRNAs in a mechanism that is analogous to protein export, rather than mRNA export.
Assuntos
Transporte Ativo do Núcleo Celular , Núcleo Celular , Transporte de RNA , RNA Circular , Transporte Ativo do Núcleo Celular/fisiologia , Núcleo Celular/metabolismo , Guanosina Trifosfato/metabolismo , Carioferinas/antagonistas & inibidores , Carioferinas/deficiência , Carioferinas/genética , Carioferinas/metabolismo , Proteínas Nucleares/metabolismo , Proteína ran de Ligação ao GTP/metabolismo , RNA Circular/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteína Exportina 1/metabolismo , Transporte ProteicoRESUMO
Exportin receptors are concentrated in the nucleus to transport essential cargoes out of it. A mislocalization of exportins to the cytoplasm is linked to disease. Hence, it is important to understand how their containment within the nucleus is regulated. Here, we have studied the nuclear efflux of exportin2 (cellular apoptosis susceptibility protein or CAS) that delivers karyopherinα (Kapα or importinα), the cargo adaptor for karyopherinß1 (Kapß1 or importinß1), to the cytoplasm in a Ran guanosine triphosphate (RanGTP)-mediated manner. We show that the N-terminus of CAS attenuates the interaction of RanGTPase activating protein 1 (RanGAP1) with RanGTP to slow GTP hydrolysis, which suppresses CAS nuclear exit at nuclear pore complexes (NPCs). Strikingly, a single phosphomimetic mutation (T18D) at the CAS N-terminus is sufficient to abolish its nuclear retention and coincides with metastatic cellular behavior. Furthermore, downregulating Kapß1 disrupts CAS nuclear retention, which highlights the balance between their respective functions that is essential for maintaining the Kapα transport cycle. Therefore, NPCs play a functional role in selectively partitioning exportins in the cell nucleus.
Assuntos
Núcleo Celular , Proteína de Suscetibilidade a Apoptose Celular , Carioferinas , Proteína ran de Ligação ao GTP , Transporte Ativo do Núcleo Celular/fisiologia , Transporte Biológico , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Carioferinas/metabolismo , Poro Nuclear/metabolismo , Proteína ran de Ligação ao GTP/metabolismo , Humanos , Proteína de Suscetibilidade a Apoptose Celular/genética , Proteína de Suscetibilidade a Apoptose Celular/metabolismoRESUMO
Nuclear protein trafficking requires the soluble transport factor RanBP1. The subcellular distribution of RanBP1 is dynamic, as the protein shuttles between the nucleus and cytoplasm. To date, the signaling pathways regulating RanBP1 subcellular localization are poorly understood. During interphase, RanBP1 resides mostly in the cytoplasm. We show here that oxidative stress concentrates RanBP1 in the nucleus, and our study defines the underlying mechanisms. Specifically, RanBP1's cysteine residues are not essential for its oxidant-induced relocation. Furthermore, our pharmacological approaches uncover that signaling mediated by epidermal growth factor receptor (EGFR) and protein kinase A (PKA) control RanBP1 localization during stress. In particular, pharmacological inhibitors of EGFR or PKA diminish the oxidant-dependent relocation of RanBP1. Mutant analysis identified serine 60 and tyrosine 103 as regulators of RanBP1 nuclear accumulation during oxidant exposure. Taken together, our results define RanBP1 as a target of oxidative stress and a downstream effector of EGFR and PKA signaling routes. This positions RanBP1 at the intersection of important cellular signaling circuits.
Assuntos
Núcleo Celular , Proteína ran de Ligação ao GTP , Núcleo Celular/metabolismo , Transporte Ativo do Núcleo Celular , Proteína ran de Ligação ao GTP/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Estresse Oxidativo , Receptores ErbB/metabolismo , Oxidantes/metabolismoAssuntos
Fosfatidilinositol 3-Quinases , Transdução de Sinais , Fosforilação , Transporte Ativo do Núcleo Celular , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Proteína ran de Ligação ao GTP/genética , Proteína ran de Ligação ao GTP/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
RAS-related nuclear protein(RAN) is a nuclear shuttle and normally regulates events in the cell cycle. When overexpressed in cultured cells, it causes increases in cell migration/invasion in vitro and its overexpression is associated with early breast cancer patient deaths in vivo. However, the underlying mechanism is unknown. The effect of RAN overexpression on potential targets MMP2, ATF3, CXCR3 was investigated by Real-Time PCR/Western blots in the triple receptor negative breast cancer(TRNBC) cell line MDA-MB231 and consequent biological effects were measured by cell adhesion, cell migration and cell invasion assays. Results showed that knockdown of RAN lead to a reduction of MMP2 and its potential regulators ATF3 and CXCR3. Moreover, knockdown of ATF3 or CXCR3 downregulated MMP2 without affecting RAN, indicating that RAN regulates MMP2 through ATF3 and CXCR3. Knockdown of RAN and MMP2 reduced cell adhesion, cell migration and cell growth in agar, whilst overexpression of MMP2 reversed the knockdown of RAN. Furthermore, immunohistochemical staining for RAN and MMP2 are positively associated with each other in the same tumour and separately with patient survival times in breast cancer specimens, suggesting that a high level of RAN may be a pre-requisite for MMP2 overexpression and metastasis. Moreover, positive immunohistochemical staining for both RAN and MMP-2 reduces further patient survival times over that for either protein separately. Our results suggest that MMP2 expression can stratify progression of breast cancers with a high and low incidence of RAN, both RAN and MMP2 in combination can be used for a more accurate patient prognosis. SIMPLE SUMMARY: Ran is an important regulator of normal cell growth and behaviour. We have established in cell line models of breast cancer (BC) a molecular pathway between RAN and its protein-degrading effector MMP-2 and properties related to metastasis in culture. Using immunohistochemistry (IHC) staining of primary BCs, we have shown that RAN and MMP-2 are on their own significantly associated with patient demise from metastatic BC. Moreover, when staining for MMP-2 is added to that for RAN in the primary tumours, there is a significant decrease in patient survival time over that for either protein alone. Thus a combination of staining for RAN and MMP2 is an excellent marker for poor prognosis in breast cancer.
Assuntos
Neoplasias da Mama , Metaloproteinase 2 da Matriz , Neoplasias de Mama Triplo Negativas , Proteína ran de Ligação ao GTP , Feminino , Humanos , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular , Regulação Neoplásica da Expressão Gênica , Guanosina Trifosfato , Metaloproteinase 2 da Matriz/metabolismo , Invasividade Neoplásica , Proteína ran de Ligação ao GTP/genética , Proteína ran de Ligação ao GTP/metabolismo , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
BACKGROUND: More than twenty years after its discovery, the role of the importin beta superfamily member Ran GTP-binding protein (RanBP) 17 is still ill defined. Previously, we observed notable RanBP17 RNA expression levels in head and neck squamous cell carcinoma (HNSCC) cell lines with disruptive TP53 mutations. METHODS: We deployed HNSCC cell lines as well as cell lines from other tumor entities such as HCT116, MDA-MB-231 and H460, which were derived from colon, breast and lung cancers respectively. RNAi was used to evaluate the effect of RanBP17 on cell proliferation. FACS analysis was used for cell sorting according to their respective cell cycle phase and for BrdU assays. Immunocytochemistry was deployed for colocalization studies of RanBP17 with Nucleolin and SC35 (nuclear speckles) domains. TCGA analysis was performed for prognostic assessment and correlation analysis of RanBP17 in HNSCC patients. RESULTS: RNAi knockdown of RanBP17, significantly reduced cell proliferation in HNSCC cell lines. This effect was also seen in the HNSCC unrelated cell lines HCT116 and MDA-MB-231. Similarly, inhibiting cell proliferation with cisplatin reduced RanBP17 in keratinocytes but lead to induction in tumor cell lines. A similar observation was made in tumor cell lines after treatment with the EGFR kinase inhibitor AG1478. In addition to previous reports, showing colocalization of RanBP17 with SC35 domains, we observed colocalization of RanBP17 to nuclear bodies that are distinct from nucleoli and SC35 domains. Interestingly, for HPV positive but not HPV negative HNSCC, TCGA data base analysis revealed a strong positive correlation of RanBP17 RNA with patient survival and CDKN2A. CONCLUSIONS: Our data point to a role of RanBP17 in proliferation of HNSCC and other epithelial cells. Furthermore, RanBP17 could potentially serve as a novel prognostic marker for HNSCC patients. However, we noted a major discrepancy between RanBP17 RNA and protein expression levels with the used antibodies. These observations could be explained by the presence of additional RanBP17 splice isoforms and more so of non-coding circular RanBP17 RNA species. These aspects need to be addressed in more detail by future studies.
Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias de Cabeça e Pescoço/genética , Humanos , Inibidores de Proteínas Quinases/farmacologia , RNA , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , beta Carioferinas/genética , Proteína ran de Ligação ao GTP/genética , Proteína ran de Ligação ao GTP/metabolismo , Proteína ran de Ligação ao GTP/farmacologiaRESUMO
RSL1D1 (ribosomal L1 domain containing 1), a member of the universal ribosomal protein uL1 family, was suggested to be a new candidate target for colorectal cancer (CRC). However, the role of RSL1D1 in cancer, including CRC, remains largely elusive. Here, we demonstrated that RSL1D1 expression was significantly elevated in tumors from CRC patients and that high expression of RSL1D1 was correlated with poorer survival of CRC patients. Functionally, RSL1D1 promoted the proliferation, invasion, and metastasis of CRC cells by suppressing autophagy. Interestingly, RSL1D1 interacted with RAN and inhibited its deacetylation by competitively binding with Sirt7. By affecting the acetylation of RAN, RSL1D1 inhibited the accumulation of nuclear STAT3 and the STAT3-regulated autophagic program. Taken together, our study uncovered the key role of the RSL1D1/RAN/STAT3 regulatory axis in autophagy and tumor progression in CRC, providing a new candidate target for CRC treatment.
Assuntos
Autofagia , Neoplasias Colorretais/patologia , Proteínas da Gravidez/metabolismo , Proteínas Ribossômicas/metabolismo , Proteína ran de Ligação ao GTP/metabolismo , Acetilação , Animais , Linhagem Celular Tumoral , Movimento Celular , Núcleo Celular/metabolismo , Proliferação de Células , Neoplasias Colorretais/metabolismo , Humanos , Camundongos , Metástase Neoplásica , Proteínas da Gravidez/genética , Prognóstico , Ligação Proteica , Proteínas Ribossômicas/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Sirtuínas/metabolismoRESUMO
AIMS: Ran GTPase is involved in nucleocytoplasmic shuttling of proteins and is overexpressed in several cancers. The expression of Ran in malignant melanoma (MM) and its functional activity have not been described and were investigated in this study. METHODS: The prognostic value of Ran expression was tested in a series of 185 primary cutaneous MM cases using immunohistochemistry. The functional activity of Ran was investigated in the two melanoma cell lines. Ran expression was knocked down using two siRNAs and the effect on the expression of the c-Met oncogene, a potential downstream target of Ran, was tested. Functional effects of Ran knockdown on cell motility and cell proliferation were also assessed. RESULTS: Positive Ran expression was seen in 12.4% of MM and was associated with advanced clinical stage and greater Breslow thickness. Positive expression was an independent marker of shorter overall survival (p=0.023). Knockdown of Ran results in decreased expression of c-Met and the downstream c-met signalling targets ERK1/2. There was a significant reduction in cell migration (p<0.001) and cell invasion (p<0.001). c-Met knockdown decreased the expression of Ran through MAPK and PI3K-AKT in A375 cell line, inhibited the cell viability and migration of both A375 and G361 melanoma cell lines while invasion was enhanced. CONCLUSION: Ran is a poor prognostic marker in cutaneous MM. It upregulates expression of the oncogene c-Met and, possibly through this, it promotes cell motility which may in turn promote metastasis.
Assuntos
Melanoma/diagnóstico , Proteínas Proto-Oncogênicas c-met/metabolismo , Transdução de Sinais , Neoplasias Cutâneas/diagnóstico , Proteína ran de Ligação ao GTP/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Melanoma/patologia , Invasividade Neoplásica , Prognóstico , Proteínas Proto-Oncogênicas c-met/genética , Neoplasias Cutâneas/patologia , Proteína ran de Ligação ao GTP/genética , Melanoma Maligno CutâneoRESUMO
As oncogenes and tumor suppressor genes, long non-coding RNAs (lncRNAs) regulate the biological behavior of gastric cancer (GC) cells such as proliferation, invasion, and metastasis through various signal pathways. At present, although numerous lncRNAs that significantly influence the development and progression of GC have been identified, a considerable number of them have not been found and studied yet. In this study, we identified a new lncRNA derived from pseudogenes WFDC21P, which have not been reported in any previous GC study. LncRNA WFDC21P was significantly upregulated in GC cells and tissues, and clinically associated with the pathological stages of advanced GC. WFDC21P promoted proliferation and metastasis of GC cells both in vitro and in vivo. LncRNA WFDC21P was directly bound to GTPase Ran and it promoted the activity of the Akt/GSK3ß/ß-catenin pathway. Forkhead Box P3 (FOXP3), as a transcription factor of WFDC21P, was directly bound to the promoter region and it positively regulated the transcription of WFDC21P. This finding may provide a novel biomarker and therapeutic target for GC.
Assuntos
Progressão da Doença , Oncogenes , Pseudogenes , RNA Longo não Codificante/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Animais , Sequência de Bases , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Feminino , Fatores de Transcrição Forkhead/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Invasividade Neoplásica , Metástase Neoplásica , Ligação Proteica , RNA Longo não Codificante/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Frações Subcelulares/metabolismo , Regulação para Cima/genética , Proteína ran de Ligação ao GTP/metabolismoRESUMO
Acute myeloid leukemia (AML) harboring inv(16)(p13q22) expresses high levels of miR-126. Here we show that the CBFB-MYH11 (CM) fusion gene upregulates miR-126 expression through aberrant miR-126 transcription and perturbed miR-126 biogenesis via the HDAC8/RAN-XPO5-RCC1 axis. Aberrant miR-126 upregulation promotes survival of leukemia-initiating progenitors and is critical for initiating and maintaining CM-driven AML. We show that miR-126 enhances MYC activity through the SPRED1/PLK2-ERK-MYC axis. Notably, genetic deletion of miR-126 significantly reduces AML rate and extends survival in CM knock-in mice. Therapeutic depletion of miR-126 with an anti-miR-126 (miRisten) inhibits AML cell survival, reduces leukemia burden and leukemia stem cell (LSC) activity in inv(16) AML murine and xenograft models. The combination of miRisten with chemotherapy further enhances the anti-leukemia and anti-LSC activity. Overall, this study provides molecular insights for the mechanism and impact of miR-126 dysregulation in leukemogenesis and highlights the potential of miR-126 depletion as a therapeutic approach for inv(16) AML.
Assuntos
Antineoplásicos/uso terapêutico , Cromossomos Humanos Par 16/genética , Leucemia Mieloide Aguda/tratamento farmacológico , MicroRNAs/antagonistas & inibidores , Células-Tronco Neoplásicas/efeitos dos fármacos , Animais , Antineoplásicos/farmacologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ciclo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Inversão Cromossômica/genética , Família de Proteínas EGF/genética , Fator de Transcrição GATA2/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Histona Desacetilases/metabolismo , Humanos , Carioferinas/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Terapia de Alvo Molecular , Células Progenitoras Mieloides/efeitos dos fármacos , Células Progenitoras Mieloides/metabolismo , Células Progenitoras Mieloides/patologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Proteínas Nucleares/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Repressoras/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína ran de Ligação ao GTP/metabolismoRESUMO
Oral squamous cell carcinoma (OSCC) is one of the most common malignancies in the world, with a high mortality rate. RAN is a member of the Ras GTPase family and is overexpressed in a range of cancers, however, the relationship between RAN and OSCC is rarely reported. In this study, we found that RAN is overexpressed in OSCC tissues. RAN inhibition retarded OSCC cell proliferation and led to apoptosis and cell cycle arrest. Knockdown of RAN inhibited tumor growth in vivo. Strikingly, we found that RAN and oncogene Y-box binding protein-1 (YBX1) are positively associated with the immune infiltrates of CD4+ Th2 cells in multiple types of cancer, and can promote IL-4 expression. IL-4 treatment can partially rescue RAN knockdown-induced cell apoptosis in OSCC cells. Moreover, overexpression of RAN could rescue cell growth inhibition caused by knockdown of YBX1. Furthermore, patients with low expression of both RAN and YBX1 had better overall survival than others. Collectively, these findings indicate that RAN is a target of YBX1. RAN and YBX1 are required for cell proliferation and IL-4 expression. RAN and YBX1 are co-expressed and can serve as potential co-biomarkers for poor prognosis in OSCC.
Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/patologia , Regulação Neoplásica da Expressão Gênica , Interleucina-4/metabolismo , Neoplasias Bucais/patologia , Proteína 1 de Ligação a Y-Box/metabolismo , Proteína ran de Ligação ao GTP/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Ciclo Celular , Movimento Celular , Proliferação de Células , Feminino , Humanos , Interleucina-4/genética , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Neoplasias Bucais/metabolismo , Prognóstico , Estudos Retrospectivos , Taxa de Sobrevida , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína 1 de Ligação a Y-Box/genética , Proteína ran de Ligação ao GTP/genéticaRESUMO
CRM1 is a nuclear export receptor that has been intensively targeted over the last decade for the development of antitumor and antiviral drugs. Structural analysis of several inhibitor compounds bound to CRM1 revealed that their mechanism of action relies on the covalent modification of a critical cysteine residue (Cys528 in the human receptor) located in the nuclear export signal-binding cleft. This study presents the crystal structure of human CRM1, covalently modified by 2-mercaptoethanol on Cys528, in complex with RanGTP at 2.58â Å resolution. The results demonstrate that buffer components can interfere with the characterization of cysteine-dependent inhibitor compounds.
Assuntos
Cisteína/química , Carioferinas/química , Carioferinas/metabolismo , Mercaptoetanol/química , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteína ran de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Humanos , Modelos Moleculares , Sinais de Exportação Nuclear , Proteína ran de Ligação ao GTP/química , Proteína Exportina 1RESUMO
Senescence is a key barrier to neoplastic transformation. To identify senescence regulators relevant to cancer, we screened a genome-wide shRNA library. Here, we describe exportin 7 (XPO7) as a novel regulator of senescence and validate its function in telomere-induced, replicative, and oncogene-induced senescence (OIS). XPO7 is a bidirectional transporter that regulates the nuclear-cytoplasmic shuttling of a broad range of substrates. Depletion of XPO7 results in reduced levels of TCF3 and an impaired induction of the cyclin-dependent kinase inhibitor p21CIP1 during OIS. Deletion of XPO7 correlates with poorer overall survival in several cancer types. Moreover, depletion of XPO7 alleviated OIS and increased tumor formation in a mouse model of liver cancer. Our results suggest that XPO7 is a novel tumor suppressor that regulates p21CIP1 expression to control senescence and tumorigenesis.
Assuntos
Senescência Celular/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Carioferinas/genética , Carioferinas/metabolismo , Proteína ran de Ligação ao GTP/genética , Proteína ran de Ligação ao GTP/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Neoplasias/fisiopatologia , Proteína 2 de Ligação a Repetições Teloméricas/genéticaRESUMO
PURPOSE: Radiation therapy is a viable treatment option for patients with unresectable hepatocellular carcinoma (HCC). However, radiation resistance and adverse effects are issues that needs to be addressed. Herein, for the first time, we investigated the ability of collectrin (CLTRN) to enhance radiosensitivity in patients with HCC. METHODS AND MATERIALS: Transcriptome sequencing technology (RNA-seq technology) was used to analyze the transcription-level changes in the genes in HepG2 cells before and after x-ray irradiation. Combining the results with the HCC tissue RNA-seq data, we determined the ultimate target gene through bioinformatics analysis and cellular verification. A series of cellular and molecular biology techniques were applied in vitro and in vivo to confirm whether CLTRN can enhance radiosensitivity in HCC cells. Subsequently, the downstream action mechanism, the upstream transcription factor, and the interaction proteins of CLTRN were determined. RESULTS: First, we confirmed that CLTRN is the target gene for radiation therapy and verified the association between CLTRN and radiosensitivity. In vivo and in vitro experiments were performed. Investigation of the gene regulatory mechanism revealed that the genes analyzed at the transcriptome level after CLTRN overexpression were mostly enriched in the glutathione metabolic pathway. As glutathione metabolism forms a vital link in ferroptosis, we surmised that CLTRN is associated with ferroptosis. This was confirmed through detection of cellular iron, determination of reactive oxygen species levels, use of transmission electron microscopy, and monitoring of ferroptosis-related protein indicators. Lastly, we investigated whether nuclear respiratory factor 1 is the upstream transcription factor of CLTRN and whether dihydrolipoamide dehydrogenase and members of the RAS oncogene family are its interacting proteins. CONCLUSIONS: CLTRN is a vital regulator of radiation sensitivity and could serve as a novel therapeutic target or prognostic marker in HCC treatment.
Assuntos
Carcinoma Hepatocelular/patologia , Ferroptose , Neoplasias Hepáticas/patologia , Glicoproteínas de Membrana/metabolismo , Fator 1 Nuclear Respiratório/metabolismo , Tolerância a Radiação , Proteína ran de Ligação ao GTP/metabolismo , Células Hep G2 , HumanosRESUMO
Artesunate (ART) is a clinically approved antimalarial drug and was revealed as a candidate of colorectal cancer chemopreventive agents in our drug screening system. Here, we aimed to understand the suppressive effects of ART on intestinal tumorigenesis. In vitro, ART reduced T-cell factor/lymphoid enhancer factor (TCF/LEF) promoter transcriptional activity. In vivo, ART inhibited intestinal polyp development. We found that ART reduces TCF1/TCF7 nuclear translocation by binding the Ras-related nuclear protein (RAN), suggesting that ART inhibits TCF/LEF transcriptional factor nuclear translocation by binding to RAN, thereby inhibiting Wnt signaling. Our results provide a novel mechanism through which artesunate inhibits intestinal tumorigenesis.
Assuntos
Polipose Adenomatosa do Colo/prevenção & controle , Artesunato/farmacologia , Carcinogênese/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/patologia , Proteína da Polipose Adenomatosa do Colo/genética , Animais , Artesunato/uso terapêutico , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Técnicas de Silenciamento de Genes , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , Masculino , Camundongos , Camundongos Transgênicos , Mutação , Regiões Promotoras Genéticas , Fator 1 de Transcrição de Linfócitos T/genética , Fator 1 de Transcrição de Linfócitos T/metabolismo , Ativação Transcricional/efeitos dos fármacos , Via de Sinalização Wnt/genética , Proteína ran de Ligação ao GTP/antagonistas & inibidores , Proteína ran de Ligação ao GTP/genética , Proteína ran de Ligação ao GTP/metabolismoRESUMO
Spindle assembly is spatially regulated by a chromosome-derived Ran- GTP gradient. Previous work proposed that Ran-GTP activates spindle assembly factors (SAFs) around chromosomes by dissociating inhibitory importins from SAFs. However, it is unclear whether the Ran-GTP gradient equivalently activates SAFs that localize at distinct spindle regions. In addition, Ran's dual functions in interphase nucleocytoplasmic transport and mitotic spindle assembly have made it difficult to assess its mitotic roles in somatic cells. Here, using auxin-inducible degron technology in human cells, we developed acute mitotic depletion assays to dissect Ran's mitotic roles systematically and separately from its interphase function. In contrast to the prevailing model, we found that the Ran pathway is not essential for spindle assembly activities that occur at sites spatially separated from chromosomes, including activating NuMA for spindle-pole focusing or for targeting TPX2. On the other hand, Ran-GTP is required to localize HURP and HSET specifically at chromosome-proximal regions to set proper spindle length during prometaphase. We demonstrated that Ran-GTP and importin-ß coordinately promote HURP's dynamic microtubule binding-dissociation cycle, which maintains HURP near chromosomes during metaphase. Together, we propose that the Ran pathway acts on spindle assembly independently of its interphase functions in mitotic human cells but does not equivalently regulate all Ran-regulated SAFs. Ran-dependent spindle assembly is likely coupled with additional parallel pathways that activate SAFs distantly located from the chromosomes.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Mitose , Proteínas de Neoplasias/metabolismo , Polos do Fuso/metabolismo , Proteína ran de Ligação ao GTP/metabolismo , Proteínas de Ciclo Celular/genética , Cromossomos , Técnicas de Introdução de Genes , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Guanosina Trifosfato/metabolismo , Células HCT116 , Células HEK293 , Humanos , Microscopia Intravital , Cinesinas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismoRESUMO
Nucleocytoplasmic transport (NCT) defects have been implicated in neurodegenerative diseases such as C9ORF72-associated amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/FTD). Here, we identify a neuroprotective pathway of like-Sm protein 12 (LSM12) and exchange protein directly activated by cyclic AMP 1 (EPAC1) that sustains the nucleocytoplasmic RAN gradient and thereby suppresses NCT dysfunction by the C9ORF72-derived poly(glycine-arginine) protein. LSM12 depletion in human neuroblastoma cells aggravated poly(GR)-induced impairment of NCT and nuclear integrity while promoting the nuclear accumulation of poly(GR) granules. In fact, LSM12 posttranscriptionally up-regulated EPAC1 expression, whereas EPAC1 overexpression rescued the RAN gradient and NCT defects in LSM12-deleted cells. C9-ALS patient-derived neurons differentiated from induced pluripotent stem cells (C9-ALS iPSNs) displayed low expression of LSM12 and EPAC1. Lentiviral overexpression of LSM12 or EPAC1 indeed restored the RAN gradient, mitigated the pathogenic mislocalization of TDP-43, and suppressed caspase-3 activation for apoptosis in C9-ALS iPSNs. EPAC1 depletion biochemically dissociated RAN-importin ß1 from the cytoplasmic nuclear pore complex, thereby dissipating the nucleocytoplasmic RAN gradient essential for NCT. These findings define the LSM12-EPAC1 pathway as an important suppressor of the NCT-related pathologies in C9-ALS/FTD.
Assuntos
Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Proteína ran de Ligação ao GTP/metabolismo , Transporte Ativo do Núcleo Celular , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Núcleo Celular/metabolismo , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/genética , AMP Cíclico/metabolismo , Citoplasma/metabolismo , Demência Frontotemporal/genética , Demência Frontotemporal/metabolismo , Demência Frontotemporal/patologia , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Poro Nuclear/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genéticaRESUMO
The glycoprotein M of herpes simplex virus 1 (HSV-1) is dynamically relocated from nuclear membranes to the trans-Golgi network (TGN) during infection, but molecular partners that promote this relocalization are unknown. Furthermore, while the presence of the virus is essential for this phenomenon, it is not clear if this is facilitated by viral or host proteins. Past attempts to characterize glycoprotein M (gM) interacting partners identified the viral protein gN by coimmunoprecipitation and the host protein E-Syt1 through a proteomics approach. Interestingly, both proteins modulate the activity of gM on the viral fusion machinery. However, neither protein is targeted to the nuclear membrane and consequently unlikely explains the dynamic regulation of gM nuclear localization. We thus reasoned that gM may transiently interact with other molecules. To resolve this issue, we opted for a proximity-dependent biotin identification (BioID) proteomics approach by tagging gM with a BirA* biotinylation enzyme and purifying BirA substrates on a streptavidin column followed by mass spectrometry analysis. The data identified gM and 170 other proteins that specifically and reproducibly were labeled by tagged gM at 4 or 12 h postinfection. Surprisingly, 35% of these cellular proteins are implicated in protein transport. Upon testing select candidate proteins, we discovered that XPO6, an exportin, is required for gM to be released from the nucleus toward the TGN. This is the first indication of a host or viral protein that modulates the presence of HSV-1 gM on nuclear membranes.IMPORTANCE The mechanisms that enable integral proteins to be targeted to the inner nuclear membrane are poorly understood. Herpes simplex virus 1 (HSV-1) glycoprotein M (gM) is an interesting candidate, as it is dynamically relocalized from nuclear envelopes to the trans-Golgi network (TGN) in a virus- and time-dependent fashion. However, it was, until now, unclear how gM was directed to the nucleus or evaded that compartment later on. Through a proteomic study relying on a proximity-ligation assay, we identified several novel gM interacting partners, many of which are involved in vesicular transport. Analysis of select proteins revealed that XPO6 is required for gM to leave the nuclear membranes late in the infection. This was unexpected, as XPO6 is an exportin specifically associated with actin/profilin nuclear export. This raises some very interesting questions about the interaction of HSV-1 with the exportin machinery and the cargo specificity of XPO6.
Assuntos
Herpesvirus Humano 1/metabolismo , Carioferinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Membrana Nuclear/metabolismo , Proteínas do Envelope Viral/metabolismo , Proteínas Virais/metabolismo , Proteína ran de Ligação ao GTP/metabolismo , Rede trans-Golgi/metabolismo , Biotinilação , Carbono-Nitrogênio Ligases/química , Carbono-Nitrogênio Ligases/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Herpesvirus Humano 1/genética , Interações Hospedeiro-Patógeno/genética , Humanos , Carioferinas/genética , Glicoproteínas de Membrana/genética , Membrana Nuclear/virologia , Ligação Proteica , Transporte Proteico , Proteômica/métodos , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Coloração e Rotulagem/métodos , Estreptavidina/química , Sinaptotagminas/genética , Sinaptotagminas/metabolismo , Proteínas do Envelope Viral/genética , Proteínas Virais/genética , Proteína ran de Ligação ao GTP/genética , Rede trans-Golgi/virologiaRESUMO
The Ran GTPase plays critical roles in multiple cellular processes including interphase nucleocytoplasmic transport and mitotic spindle assembly. During mitosis in mammalian cells, GTP-bound Ran (Ran-GTP) is concentrated near mitotic chromatin while GDP-bound Ran (Ran-GDP) is more abundant distal to chromosomes. This pattern spatially controls spindle formation because Ran-GTP locally releases spindle assembly factors (SAFs), such as Hepatoma Up-Regulated Protein (HURP), from inhibitory interactions near chromosomes. Regulator of Chromatin Condensation 1 (RCC1) is Ran's chromatin-bound exchange factor, and RanBP1 is a conserved Ran-GTP-binding protein that has been implicated as a mitotic regulator of RCC1 in embryonic systems. Here, we show that RanBP1 controls mitotic RCC1 dynamics in human somatic tissue culture cells. In addition, we observed the re-localization of HURP in metaphase cells after RanBP1 degradation, consistent with the idea that altered RCC1 dynamics functionally modulate SAF activities. Together, our findings reveal an important mitotic role for RanBP1 in human somatic cells, controlling the spatial distribution and magnitude of mitotic Ran-GTP production and thereby ensuring the accurate execution of Ran-dependent mitotic events. ABBREVIATIONS: AID: Auxin-induced degron; FLIP: Fluorescence loss in photobleaching; FRAP: Fluorescence recovery after photobleaching; GDP: guanosine diphosphate; GTP: guanosine triphosphate; HURP: Hepatoma Up-Regulated Protein; NE: nuclear envelope; NEBD: Nuclear Envelope Breakdown; RanBP1: Ran-binding protein 1; RanGAP1: Ran GTPase-Activating Protein 1; RCC1: Regulator of Chromatin Condensation 1; RRR complex: RCC1/Ran/RanBP1 heterotrimeric complex; SAF: Spindle Assembly Factor; TIR1: Transport Inhibitor Response 1 protein; XEE: Xenopus egg extract.