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1.
Mol Cancer ; 18(1): 170, 2019 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-31771591

RESUMO

BACKGROUND: The gene encoding the E3 ubiquitin ligase substrate-binding adaptor SPOP is frequently mutated in primary prostate cancer, but how SPOP mutations contribute to prostate cancer pathogenesis remains poorly understood. Stress granules (SG) assembly is an evolutionarily conserved strategy for survival of cells under stress, and often upregulated in human cancers. We investigated the role of SPOP mutations in aberrant activation of the SG in prostate cancer and explored the relevanve of the mechanism in therapy resistance. METHODS: We identified SG nucleating protein Caprin1 as a SPOP interactor by using the yeast two hybrid methods. A series of functional analyses in cell lines, patient samples, and xenograft models were performed to investigate the biological significance and clinical relevance of SPOP regulation of SG signaling in prostate cancer. RESULTS: The cytoplasmic form of wild-type (WT) SPOP recognizes and triggers ubiquitin-dependent degradation of Caprin1. Caprin1 abundance is elevated in SPOP-mutant expressing prostate cancer cell lines and patient specimens. SPOP WT suppresses SG assembly, while the prostate cancer-associated mutants enhance SG assembly in a Caprin1-dependent manner. Knockout of SPOP or expression of prostate cancer-associated SPOP mutants conferred resistance to death caused by SG inducers (e.g. docetaxel, sodium arsenite and H2O2) in prostate cancer cells. CONCLUSIONS: SG assembly is aberrantly elevated in SPOP-mutated prostate cancer. SPOP mutations cause resistance to cellular stress induced by chemtherapeutic drug such as docetaxel in prostate cancer.

2.
Leukemia ; 2019 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-31776466

RESUMO

Recurrent oncogenic mutations of MyD88 have been identified in a variety of lymphoid malignancies. Gain-of-function mutations of MyD88 constitutively activate downstream NF-κB signaling pathways, resulting in increased cellular proliferation and survival. However, whether MyD88 activity can be aberrantly regulated in MyD88-wild-type lymphoid malignancies remains poorly understood. SPOP is an adaptor protein of CUL3-based E3 ubiquitin ligase complex and frequently mutated genes in prostate and endometrial cancers. In this study, we reveal that SPOP binds to and induces the nondegradative ubiquitination of MyD88 by recognizing an atypical SPOP-binding motif in MyD88. This modification blocks Myddosome assembly and downstream NF-κB activation. SPOP is mutated in a subset of lymphoid malignancies, including diffuse large B-cell lymphoma (DLBCL). Lymphoid malignancies-associated SPOP mutants exhibited impaired binding to MyD88 and suppression of NF-κB activation. The DLBCL-associated, SPOP-binding defective mutants of MyD88 escaped from SPOP-mediated ubiquitination, and their effect on NF-κB activation is stronger than that of wild-type MyD88. Moreover, SPOP suppresses DLBCL cell growth in vitro and tumor xenograft in vivo by inhibiting the MyD88/NF-κB signaling. Therefore, SPOP acts as a tumor suppressor in DLBCL. Mutations in the SPOP-MyD88 binding interface may disrupt the SPOP-MyD88 regulatory axis and promote aberrant MyD88/NF-κB activation and cell growth in DLCBL.

3.
Cell Commun Signal ; 17(1): 4, 2019 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-30642362

RESUMO

BACKGROUND: As the first member of the metastasis-associated protein (MTA) family, MTA1 and another MTA family member, MTA2, have both been reported to promote breast cancer progression and metastasis. However, the difference and relationship between MTA1 and MTA2 have not been fully elucidated. METHODS: Transwell assays were used to assess the roles of MTA1 and MTA2 in the metastasis of ZR-75-30 luminal B breast cancer cells in vitro. Immunoblotting and qRT-PCR were used to evaluate the effect of MTA1 overexpression on MTA2. Proteases that cleave MTA2 were predicted using an online web server. The role of neutrophil elastase (NE) in MTA1 overexpression-induced MTA2 downregulation was confirmed by specific inhibitor treatment, knockdown, overexpression and immunocytochemistry, and NE cleavage sites in MTA2 were confirmed by MTA2 truncation and mutation. The effect of MTA1 overexpression on the intrinsic inhibitor of NE, elafin, was detected by qRT-PCR, immunoblotting and treatment with inhibitors. RESULTS: MTA1 overexpression inhibited, while MTA2 promoted the metastasis of ZR-75-30 cells in vitro. MTA1 overexpression downregulated MTA2 expression at the protein level rather than the mRNA level. NE was predicted to cleave MTA2 and was responsible for MTA1 overexpression-induced MTA2 degradation. NE was found to cleave MTA2 in the C-terminus at the 486, 497, 542, 583 and 621 sites. MTA1 overexpression activated NE by downregulating elafin in a histone deacetylase- and DNA methyltransferase-dependent manner. CONCLUSIONS: MTA1 and MTA2 play opposing roles in the metastasis of ZR-75-30 luminal B breast cancer cells in vitro. MTA1 downregulates MTA2 at the protein level by epigenetically repressing the expression of elafin and releasing the inhibition of neutrophil elastase, which cleaves MTA2 in the C-terminus at multiple specific sites.


Assuntos
Histona Desacetilases/metabolismo , Proteólise , Proteínas Repressoras/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Regulação para Baixo/genética , Elafina/farmacologia , Histona Desacetilases/química , Humanos , Elastase de Leucócito/antagonistas & inibidores , Elastase de Leucócito/metabolismo , Modelos Biológicos , Metástase Neoplásica , Proteínas Repressoras/química
4.
Cell Prolif ; 52(2): e12553, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30485581

RESUMO

OBJECTIVES: We aimed to elucidate the role and molecular mechanisms of FOXM1 in regulating metastasis in oesophageal squamous cell carcinoma (ESCC) as well as its clinical implications. MATERIALS AND METHODS: The expression levels of four isoforms of FOXM1 were analysed by real-time PCR. Next, genetically modification using overexpression and RNAi systems and transwell were employed to examine FOXM1c function in invasion and migration. Dual luciferase and ChIP assays were performed to decipher the underlying mechanism for transcriptional regulation. The expression levels of FOXM1 and IRF1 were determined by immunohistochemistry staining in ESCC specimens. RESULTS: The FOXM1c was predominantly overexpressed in ESCC cell lines compared to the other FOXM1 isoforms. Ectopic expression of FOXM1c promoted invasion and migration of ESCC cells lines, whereas downregulation of FOXM1c inhibited these processes. Moreover, FOXM1c expression was positively correlated with IRF1 expression in ESCC cell lines and tumour specimens. IRF1 is, at least in part, responsible for FOXM1c-mediated invasion and migration. Mechanistically, we identified IRF1 as a transcriptional target of FOXM1c and found a FOXM1c-binding site in the IRF1 promoter region. Furthermore, high expression levels of both FOXM1c and IRF1 were positively associated with low survival rate and predicted a poor prognosis of oesophageal cancer patients. CONCLUSION: FOXM1c promotes the metastasis by transcriptionally targeting IRF1 and may serve as a potential prognostic predictor for oesophageal cancer.


Assuntos
Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas do Esôfago/genética , Proteína Forkhead Box M1/genética , Regulação Neoplásica da Expressão Gênica , Fator Regulador 1 de Interferon/genética , Invasividade Neoplásica/genética , Linhagem Celular Tumoral , Movimento Celular , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/patologia , Humanos , Invasividade Neoplásica/patologia
5.
Cell Death Dis ; 9(9): 887, 2018 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-30166523

RESUMO

Radiation therapy is an important treatment modality for esophageal cancer. However, acquisition of radioresistance ultimately results in esophageal cancer relapse. CD59, a membrane-bound complement regulatory protein, can transduce signals via a Src kinase in the lipid raft, thus playing a complement-independent role. However, the effect of CD59 on the esophageal cancer response to ionizing radiation remains unclear. In this study, we found that the expression level of CD59 was positively correlated with the radioresistance of esophageal cancer cell lines and clinical specimens. High CD59 expression indicated poor overall survival (OS) and disease-free survival (DFS) in esophageal squamous cell carcinoma (ESCC) patients who received radiotherapy. Genetic alteration of CD59 expression modulated the radiosensitivity of esophageal cancer cells to ionizing radiation. CD59 deficiency exacerbated DNA damage, hindered cell proliferation, and induced G2/M cell cycle arrest and cellular senescence, leading to an impaired DNA damage repair ability. In addition, CD59 deficiency almost completely reduced the phosphorylation of Src at Y416 despite ionizing radiation. A Src inhibitor saracatinib sensitized esophageal cancer cells to irradiation. Therefore, CD59 may be a potential biomarker for predicting the radioresistance of ESCC to radiotherapy.


Assuntos
Antígenos CD59/genética , Neoplasias Esofágicas/radioterapia , Carcinoma de Células Escamosas do Esôfago/radioterapia , Tolerância a Radiação/genética , Animais , Benzodioxóis/farmacologia , Biomarcadores Tumorais/genética , Antígenos CD59/antagonistas & inibidores , Linhagem Celular Tumoral , Proliferação de Células/genética , Senescência Celular/genética , Dano ao DNA/genética , Reparo do DNA/genética , Intervalo Livre de Doença , Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas do Esôfago/genética , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Quinazolinas/farmacologia , Transplante Heterólogo
6.
J Exp Clin Cancer Res ; 37(1): 145, 2018 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-29996942

RESUMO

BACKGROUND: Next-generation sequencing of the exome and genome of prostate cancers has identified numerous genetic alterations. SPOP (Speckle-type POZ Protein) is one of the most frequently mutated genes in primary prostate cancer, suggesting that SPOP may be a potential driver of prostate cancer. The aim of this work was to investigate how SPOP mutations contribute to prostate cancer development and progression. METHODS: To identify molecular mediators of the tumor suppressive function of SPOP, we performed a yeast two-hybrid screen in a HeLa cDNA library using the full-length SPOP as bait. Immunoprecipitation and Western Blotting were used to analyze the interaction between SPOP and ATF2. Cell migration and invasion were determined by Transwell assays. Immunohistochemistry were used to analyze protein levels in patients' tumor samples. RESULTS: Here we identified ATF2 as a bona fide substrate of the SPOP-CUL3-RBX1 E3 ubiquitin ligase complex. SPOP recognizes multiple Ser/Thr (S/T)-rich degrons in ATF2 and triggers ATF2 degradation via the ubiquitin-proteasome pathway. Strikingly, prostate cancer-associated mutants of SPOP are defective in promoting ATF2 degradation in prostate cancer cells and contribute to facilitating prostate cancer cell proliferation, migration and invasion. CONCLUSION: SPOP promotes ATF2 ubiquitination and degradation, and ATF2 is an important mediator of SPOP inactivation-induced cell proliferation, migration and invasion.


Assuntos
Fator 2 Ativador da Transcrição/genética , Proteínas Nucleares/genética , Neoplasias da Próstata/genética , Proteínas Repressoras/genética , Fator 2 Ativador da Transcrição/metabolismo , Progressão da Doença , Humanos , Masculino , Proteínas Nucleares/metabolismo , Neoplasias da Próstata/patologia , Proteínas Repressoras/metabolismo , Transfecção , Ubiquitinação
7.
Cell Death Dis ; 9(5): 528, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29743530

RESUMO

Cytokinesis is the last step of cell division and is concluded by the abscission of the intercellular bridge that connects two daughter cells. The tight regulation of cytokinesis completion is essential because cytokinesis failure is associated with various human diseases. Here, we report that iASPP, a member of the apoptosis-stimulating proteins of p53 (ASPP) family, is required for proper cell division. iASPP depletion results in abnormal midbody structure and failed cytokinesis. We used protein affinity purification methods to identify the functional partners of iASPP. We found that iASPP associates with centrosomal protein of 55 kDa (CEP55), an important cytokinetic abscission regulator. Mechanically, iASPP acts as a PP1-targeting subunit to facilitate the interaction between PP1 and CEP55 and to remove PLK1-mediated Ser436 phosphorylation in CEP55 during late mitosis. The latter step is critical for the timely recruitment of CEP55 to the midbody. The present observations revealed a previously unrecognized function of iASPP in cytokinesis. This function, in turn, likely contributes to the roles of iASPP in tumor development and genetic diseases.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Citocinese , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Proteína Fosfatase 1/metabolismo , Proteínas Repressoras/metabolismo , Células A549 , Proteínas de Ciclo Celular/genética , Células HCT116 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Mitose , Complexos Multiproteicos/genética , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patologia , Proteínas Nucleares/genética , Fosforilação/genética , Proteína Fosfatase 1/genética , Proteínas Repressoras/genética
8.
Nat Med ; 23(9): 1055-1062, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28805822

RESUMO

Bromodomain and extraterminal domain (BET) protein inhibitors are emerging as promising anticancer therapies. The gene encoding the E3 ubiquitin ligase substrate-binding adaptor speckle-type POZ protein (SPOP) is the most frequently mutated in primary prostate cancer. Here we demonstrate that wild-type SPOP binds to and induces ubiquitination and proteasomal degradation of BET proteins (BRD2, BRD3 and BRD4) by recognizing a degron motif common among them. In contrast, prostate cancer-associated SPOP mutants show impaired binding to BET proteins, resulting in decreased proteasomal degradation and accumulation of these proteins in prostate cancer cell lines and patient specimens and causing resistance to BET inhibitors. Transcriptome and BRD4 cistrome analyses reveal enhanced expression of the GTPase RAC1 and cholesterol-biosynthesis-associated genes together with activation of AKT-mTORC1 signaling as a consequence of BRD4 stabilization. Our data show that resistance to BET inhibitors in SPOP-mutant prostate cancer can be overcome by combination with AKT inhibitors and further support the evaluation of SPOP mutations as biomarkers to guide BET-inhibitor-oriented therapy in patients with prostate cancer.


Assuntos
Azepinas/farmacologia , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Proteínas Nucleares/genética , Neoplasias da Próstata/genética , Proteínas Repressoras/genética , Triazóis/farmacologia , Proteínas rac1 de Ligação ao GTP/genética , Azepinas/uso terapêutico , Western Blotting , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Perfilação da Expressão Gênica , Humanos , Imunoprecipitação , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Terapia de Alvo Molecular , Complexos Multiproteicos/efeitos dos fármacos , Complexos Multiproteicos/metabolismo , Mutação , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/efeitos dos fármacos , Proteínas Nucleares/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/efeitos dos fármacos , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serina-Treonina Quinases TOR/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Triazóis/uso terapêutico , Proteínas rac1 de Ligação ao GTP/metabolismo
9.
PLoS Genet ; 13(4): e1006748, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28448495

RESUMO

Next-generation sequencing of the exome and genome of prostate cancers has identified numerous genetic alternations. SPOP (Speckle-type POZ Protein) was one of the most frequently mutated genes in primary prostate cancer, suggesting SPOP is a potential driver of prostate cancer development and progression. However, how SPOP mutations contribute to prostate cancer pathogenesis remains poorly understood. SPOP acts as an adaptor protein of the CUL3-RBX1 E3 ubiquitin ligase complex that generally recruits substrates for ubiquitination and subsequent degradation. ER-localized isoform of the formin protein inverted formin 2 (INF2) mediates actin polymerization at ER-mitochondria intersections and facilitates DRP1 recruitment to mitochondria, which is a critical step in mitochondrial fission. Here, we revealed that SPOP recognizes a Ser/Thr (S/T)-rich motif in the C-terminal region of INF2 and triggers atypical polyubiquitination of INF2. These ubiquitination modifications do not lead to INF2 instability, but rather reduces INF2 localization in ER and mitochondrially associated DRP1 puncta formation, therefore abrogates its ability to facilitate mitochondrial fission. INF2 mutant escaping from SPOP-mediated ubiquitination is more potent in prompting mitochondrial fission. Moreover, prostate cancer-associated SPOP mutants increase INF2 localization in ER and promote mitochondrial fission, probably through a dominant-negative effect to inhibit endogenous SPOP. Moreover, INF2 is important for SPOP inactivation-induced prostate cancer cell migration and invasion. These findings reveal novel molecular events underlying the regulation of INF2 function and localization, and provided insights in understanding the relationship between SPOP mutations and dysregulation of mitochondrial dynamics in prostate cancer.


Assuntos
Movimento Celular/genética , Proteínas dos Microfilamentos/genética , Proteínas Nucleares/genética , Neoplasias da Próstata/genética , Proteínas Repressoras/genética , Linhagem Celular Tumoral , Exoma , GTP Fosfo-Hidrolases/genética , Regulação Neoplásica da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Proteínas dos Microfilamentos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Mitocôndrias/genética , Mitocôndrias/patologia , Dinâmica Mitocondrial/genética , Proteínas Mitocondriais/genética , Mutação , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Proteínas Nucleares/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas Repressoras/metabolismo
10.
Cancer Lett ; 375(1): 133-141, 2016 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-26926424

RESUMO

The oncoprotein MDM2 is an E3 ubiquitin ligase that targets tumor suppressor p53 for ubiquitination and proteasomal degradation, restraining the potent activity of p53 and enabling cell survival and proliferation. Dysregulation of MDM2-p53 axis was frequently observed in human cancers. Originally, it is proposed that MDM2 degradation was mainly achieved by destructive self-ubiquitination. However, recent study suggests that MDM2 may be targeted for degradation by an external E3 ubiquitin ligase(s) under physiological levels. Here, we identified E3 ubiquitin ligase RNF12 as an MDM2-interacting protein through yeast two hybrid methods. We demonstrated that RNF12 targets MDM2 for ubiquitination and proteasomal-dependent degradation, which is independent of MDM2's self-ubiquitination activity. Accordingly, RNF12 elevates p53 protein level by abrogating MDM2-mediated p53 degradation and ubiquitination. Finally, we showed that RNF12 regulates cell growth suppression and DNA damage-induced apoptosis in a p53-dependent manner. Taken together, we establish RNF12 as a novel positive regulator of p53 pathway and an external E3 ubiquitin ligase for MDM2 destruction. These data shed light on the potential roles of RNF12 in MDM2-p53 axis and tumor suppression.


Assuntos
Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Antineoplásicos Fitogênicos/farmacologia , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Etoposídeo/farmacologia , Humanos , Proteólise , Ubiquitinação
11.
Cancer Lett ; 372(1): 101-9, 2016 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-26739059

RESUMO

CCT3 was one of the subunits of molecular chaperone CCT/TRiC complex, which plays a central role in maintaining cellular proteostasis. We demonstrated that expressions of CCT3 mRNA and protein are highly up-regulated in hepatocellular carcinoma (HCC) tissues, and high level of CCT3 is correlated with poor survival in cancer patients. In HCC cell lines, CCT3 depletion suppresses cell proliferation by inducing mitotic arrest at prometaphase and apoptosis eventually. We also identified CCT3 as a novel regulator of spindle integrity and as a requirement for proper kinetochore-microtubule attachment during mitosis. Moreover, we found that CCT3 depletion sensitizes HCC cells to microtubule destabilizing drug Vincristine. Collectively, our study suggests that CCT3 is indispensible for HCC cell proliferation, and provides a potential drug target for treatment of HCC.


Assuntos
Carcinoma Hepatocelular/metabolismo , Proliferação de Células , Chaperonina com TCP-1/metabolismo , Neoplasias Hepáticas/metabolismo , Mitose , Antineoplásicos Fitogênicos/farmacologia , Apoptose , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/patologia , Pontos de Checagem do Ciclo Celular , Proliferação de Células/efeitos dos fármacos , Chaperonina com TCP-1/genética , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Humanos , Estimativa de Kaplan-Meier , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , Mitose/efeitos dos fármacos , Interferência de RNA , RNA Mensageiro/metabolismo , Transdução de Sinais , Fatores de Tempo , Transfecção , Moduladores de Tubulina/farmacologia , Vincristina/farmacologia
12.
Am J Cancer Res ; 6(12): 2901-2909, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-28042509

RESUMO

VGLL4 is a transcriptional repressor that interacts with transcription factors TEADs and inhibits YAP-induced overgrowth and tumorigenesis. VGLL4 protein was dramatically reduced in various types of human cancers. But how VGLL4 protein is post-transcriptional regulated is poorly understood. In this study, we identify deubiquitinating enzyme USP11 as a novel VGLL4 interactor. We reveal that the USP domain of USP11 and the N-terminal region of VGLL4 are required for mutual binding. USP11 controls VGLL4 protein stability by promoting its deubiquitination. Furthermore, our results show that knockdown of USP11 promotes cell growth, migration, and invasion in a YAP-dependent manner. Together, our results suggest that USP11 may exert its tumor suppressor role by modulating VGLL4/YAP-TEADs regulatory loop.

13.
Am J Cancer Res ; 5(10): 3210-20, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26693071

RESUMO

Progesterone induces proliferation of breast cancer cells and contributes to the development of breast cancer. The effects of progesterone are mediated by progesterone receptors (PRs). However, it is still not fully understood how the proliferative effects of PR is regulated in vivo. Increasing amount of evidence strongly suggests that dysregulation of ubiquitin-proteasome system is closely associated with cancer pathogenesis. Speckle-type POZ protein (SPOP) is an adaptor protein of the CUL3-based E3 ubiquitin ligase complexes. SPOP represents one of the highest loci for loss of heterozygosity (LOH) in breast cancer. SPOP downregulation contributes to breast cancer cell growth and invasion. In this study, we revealed PR as a bona fide substrate for SPOP. SPOP interacts with PR in vivo and targets PR for ubiquitin-dependent proteasomal degradation. Moreover, SPOP suppresses progesteroneinduced PR transactivation, S phase entry, and Erk1/2 activation. Our study revealed novel molecular mechanisms underlying the regulation of PR protein homeostasis in breast cancer cells, and provided insights in understanding the relationship between SPOP inactivation and the development of breast cancer.

14.
Oncotarget ; 6(39): 41550-65, 2015 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-26595804

RESUMO

Regulated interactions between kinetochores and spindle microtubules are critical for maintaining genomic stability during chromosome segregation. Defects in chromosome segregation are widespread phenomenon in human cancers that are thought to serve as the fuel for tumorigenic progression. Tumor suppressor proteins ASPP1 and ASPP2, two members of the apoptosis stimulating proteins of p53 (ASPP) family, are frequently down-regulated in human cancers. Here we report that ASPP1/2 are required for proper mitotic progression. In ASPP1/2 co-depleted cells, the persistence of unaligned chromosomes and the reduction of tension across sister kinetochores on aligned chromosomes resulted in persistent spindle assembly checkpoint (SAC) activation. Using protein affinity purification methods, we searched for functional partners of ASPP1/2, and found that ASPP1/2 were associated with a subset of kinetochore proteins (Hec1, KNL-1, and CENP-F). It was found that ASPP1/2 act as PP1-targeting subunits to facilitate the interaction between PP1 and Hec1, and catalyze Hec1 (Ser165) dephosphorylation during late mitosis. These observations revealed a previously unrecognized function of ASPP1/2 in chromosome segregation and kinetochore-microtubule attachments that likely contributes to their roles in chromosome stability and tumor suppression.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Segregação de Cromossomos , Cinetocoros/enzimologia , Microtúbulos/enzimologia , Mitose , Proteína Fosfatase 1/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Reguladoras de Apoptose/genética , Ciclo Celular , Proteínas Cromossômicas não Histona/metabolismo , Proteínas do Citoesqueleto , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Proteínas dos Microfilamentos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Complexos Multiproteicos , Proteínas Nucleares/metabolismo , Fosforilação , Ligação Proteica , Proteína Fosfatase 1/genética , Interferência de RNA , Transdução de Sinais , Fatores de Tempo , Transfecção , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
15.
Cancer Lett ; 369(1): 167-74, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26300492

RESUMO

Characterization of the exome and genome of carcinoma (ccRCC) by next-generation sequencing identified numerous genetic alternations. BRCA1-associated protein-1 (BAP1) was identified as one of the most frequently mutated genes in ccRCC, suggesting that BAP1 is a potential key driver for ccRCC cancer initiation and progression. However, how BAP1 mutations contribute to ccRCC remains to be elucidated. BAP1 is a nuclear de-ubiquitinating enzyme and cleaves the ubiquitin chain from the substrates. Here, we identified MCRS1 as a bona fide substrate for BAP1. MCRS1 is a component of the centrosome proteins, and plays an essential role in spindle assembly. BAP1 binds to MCRS1 and stabilizes MCRS1 by de-ubiquitination. BAP1 contributes to chromosome stability partially via MCRS1. A positive correlation was identified between BAP1 and MCRS1 expression in ccRCC tissues. Both BAP1 loss and MCRS1 down-regulation in ccRCC were associated with adverse clinicopathological features. This study revealed a novel mechanism for BAP1 involved in MCRS1 stability regulation, and provided insight in understanding the relationship between BAP1 mutations and chromosome instability in ccRCC.


Assuntos
Carcinoma de Células Renais/metabolismo , Instabilidade Cromossômica , Neoplasias Renais/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Supressoras de Tumor/fisiologia , Ubiquitina Tiolesterase/fisiologia , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Regulação para Baixo , Feminino , Células HEK293 , Humanos , Neoplasias Renais/genética , Neoplasias Renais/patologia , Masculino , Pessoa de Meia-Idade , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Ubiquitinação
16.
Biochem Biophys Res Commun ; 458(3): 494-500, 2015 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-25660448

RESUMO

Centrosome linker tethers interphase centrosomes together allowing them to function as a single microtubule organization center. The centrosome linker is disrupted at the onset of mitosis to ensure timely centrosome disjunction and bipolar spindle formation and is reassembled at the end of mitosis. While the mechanism controlling centrosome linker disassembly at early mitosis has been well explored, little is known about how the linker is subsequently reassembled before mitotic exit. Here we report that ASPP1 and ASPP2, two members of the apoptosis stimulating proteins of p53 (ASPP) family, are involved in centrosome linker reassembly. We showed that ASPP1/2 interacted with centrosome linker protein C-Nap1. Co-depletion of ASPP1 and ASPP2 inhibited re-association of C-Nap1 with centrosome at the end of mitosis. Moreover, ASPP1/2 facilitated the interaction between C-Nap1 and PP1α, and this interaction was significantly reduced by co-depletion of ASPP1/2. ASPP1/2 antagonized the NEK2A-mediated C-Nap1 Ser2417/2421 phosphorylation in a PP1-dependent manner. Co-depletion of ASPP1 and ASPP2 inhibited dephosphorylation of C-Nap1 (Ser2417/2421) at the end of mitosis. Based on these findings, we propose that ASPP1/2 act as PP1-targeting subunits to facilitate C-Nap1 dephosphorylation and centrosome linker reassembly at the end of mitosis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Autoantígenos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centrossomo/metabolismo , Células HeLa , Humanos , Mitose , Mapas de Interação de Proteínas
17.
Biochem Biophys Res Commun ; 458(4): 849-55, 2015 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-25689719

RESUMO

HDGFRP2 (HRP-2) belongs to the Hepatoma-derived growth factor (HDGF)-related proteins (HRPs) family, which are characterized by a conserved HATH/PWWP domain at a well-conserved region of the N-terminus. However, the cellular function of HRP-2 remains unknown. In this study, we showed for the first time that HRP-2 is frequently overexpressed in human HCC tissues at mRNA and protein levels. We further showed that HRP-2 can promote HCC cells growth in vitro and xenograft tumors in vivo. Using protein affinity purification methods, we searched for functional partners of HRP-2, and found that HRP-2 interacts with various proteins known to be involved in transcription elongation and processing. Furthermore, we demonstrate HRP-2 interacts and co-localizes with RNA processing regulator IWS1, and positively regulated the mRNA level of Cyclin D1. Together, our study suggests HRP-2 may act as an mRNA processing co-factor to promote cells growth by regulating the mRNA of key oncogenes, which can be explored further for cancer treatment.


Assuntos
Carcinoma Hepatocelular/genética , Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular/genética , Neoplasias Hepáticas/genética , Fígado/patologia , Animais , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células , Ciclina D/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Fígado/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Nus , Oncogenes , Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regulação para Cima
18.
J Cell Sci ; 128(5): 992-1000, 2015 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-25588844

RESUMO

A hallmark of neutrophil polarization is the back localization of active RHOA and phosphorylated myosin light chain (pMLC, also known as MYL2). However, the mechanism for the polarization is not entirely clear. Here, we show that FAM65B, a newly identified RHOA inhibitor, is important for the polarization. When FAM65B is phosphorylated, it binds to 14-3-3 family proteins and becomes more stable. In neutrophils, chemoattractants stimulate FAM65B phosphorylation largely depending on the signals from the front of the cells that include those mediated by phospholipase Cß (PLCß) and phosphoinositide 3-kinase γ (PI3Kγ), leading to FAM65B accumulation at the leading edge. Concordantly, FAM65B deficiency in neutrophils resulted in an increase in RHOA activity and localization of pMLC to the front of cells, as well as defects in chemotaxis directionality and adhesion to endothelial cells under flow. These data together elucidate a mechanism for RHOA and pMLC polarization in stimulated neutrophils through direct inhibition of RHOA by FAM65B at the leading edge.


Assuntos
Neutrófilos/metabolismo , Proteínas/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Adesão Celular/fisiologia , Quimiotaxia/fisiologia , Classe Ib de Fosfatidilinositol 3-Quinase , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Cadeias Leves de Miosina/genética , Cadeias Leves de Miosina/metabolismo , Neutrófilos/citologia , Fosfolipase C beta/genética , Fosfolipase C beta/metabolismo , Fosforilação/fisiologia , Proteínas/genética , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Proteínas rho de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/antagonistas & inibidores , Proteína rhoA de Ligação ao GTP/genética
19.
FEBS Lett ; 589(1): 94-101, 2015 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-25436413

RESUMO

ASPP2 is an important tumor suppressor protein promoting p53-dependent and-independent apoptosis. However, it has been unclear how ASPP2 protein is regulated. Here, we identified Itch as the E3 ubiquitin ligase for ASPP2. Itch interacts with ASPP2 and mediates its degradation and ubiquitination in vivo. The PPXY motif of ASPP2 interacts with the WW domains of Itch. Yap1 competes with Itch for binding to ASPP2, and prevents Itch-mediated degradation and ubiquitination of ASPP2. Together, these observations reveal that Itch and Yap1 have antagonistic roles in the regulation of ASPP2 protein stability through competing post-translational regulatory mechanism of ASPP2.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Fosfoproteínas/metabolismo , Proteólise , Proteínas Repressoras/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Reguladoras de Apoptose/genética , Linhagem Celular , Humanos , Fosfoproteínas/genética , Estabilidade Proteica , Proteínas Repressoras/genética , Fatores de Transcrição , Ubiquitina-Proteína Ligases/genética
20.
PLoS One ; 9(10): e110296, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25329897

RESUMO

Rap1GAP is a GTPase-activating protein (GAP) that specifically stimulates the GTP hydrolysis of Rap1 GTPase. Although Rap1GAP is recognized as a tumor suppressor gene and downregulated in various cancers, little is known regarding the regulation of Rap1GAP ubiquitination and degradation under physiological conditions. Here, we demonstrated that Rap1GAP is ubiquitinated and degraded through proteasome pathway in mitosis. Proteolysis of Rap1GAP requires the PLK1 kinase and ß-TrCP ubiquitin ligase complex. We revealed that PLK1 interacts with Rap1GAP in vivo through recognition of an SSP motif within Rap1GAP. PLK1 phosphorylates Ser525 in conserved 524DSGHVS529 degron of Rap1GAP and promotes its interaction with ß-TrCP. We also showed that Rap1GAP was a cell cycle regulator and that tight regulation of the Rap1GAP degradation in mitosis is required for cell proliferation.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proliferação de Células , Proteínas Ativadoras de GTPase/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteólise , Proteínas Proto-Oncogênicas/metabolismo , Ubiquitinação , Proteínas Contendo Repetições de beta-Transducina/metabolismo , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/genética , Células HEK293 , Células HeLa , Humanos , Mitose , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Contendo Repetições de beta-Transducina/genética
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