RESUMO
Lung cancer is still the leading cause of cancer-related death worldwide. Interest is growing towards early detection and advances in liquid biopsy to isolate circulating tumor cells (CTCs). This pilot study aimed to detect epithelial CTCs in the peripheral blood of early-stage non-small cell lung cancer (NSCLC) patients. We used Smart BioSurface® (SBS) slide, a nanoparticle-coated slide able to immobilize viable nucleated cellular fraction without pre-selection and preserve cell integrity. Forty patients undergoing lung resection for NSCLC were included; they were divided into two groups according to CTC value, with a cut-off of three CTCs/mL. All patients were positive for CTCs. The mean CTC value was 4.7(± 5.8 S.D.) per ml/blood. In one patient, next generation sequencing (NGS) analysis of CTCs revealed v-raf murine sarcoma viral oncogene homolog B(BRAF) V600E mutation, which has also been identified in tissue biopsy. CTCs count affected neither overall survival (OS, p = 0.74) nor progression-free survival (p = 0.829). Multivariable analysis confirmed age (p = 0.020) and pNodal-stage (p = 0.028) as negative predictors of OS. Preliminary results of this pilot study suggest the capability of this method in detecting CTCs in all early-stage NSCLC patients. NGS on single cell, identified as CTC by immunofluorescence staining, is a powerful tool for investigating the molecular landscape of cancer, with the aim of personalized therapies.
RESUMO
Drug development efforts that focused on single targets failed to provide effective treatment for Alzheimer's disease (AD). Therefore, we designed cholinesterase inhibition (ChEI)-based multi-target-directed ligands (MTDLs) to simultaneously target AD-related receptors. We built a library of 70 compounds, sequentially screened for ChEI, and determined σ1R, σ2R, NMDAR-GluN2B binding affinities, and P2X7R antagonistic activities. Nine fulfilled in silico drug-likeness criteria and did not display toxicity in three cell lines. Seven displayed cytoprotective activity in two stress-induced cellular models. Compared to donepezil, six showed equal/better synaptic protection in a zebrafish model of acute amyloidosis-induced synaptic degeneration. Two P2X7R antagonists alleviated the activation state of microglia in vivo. Permeability studies were performed, and four did not inhibit CYP450 3A4, 2D6, and 2C9. Therefore, four ChEI-based lead MTDLs are promising drug candidates for synaptic integrity protection and could serve as disease-modifying AD treatment. Our study also proposes zebrafish as a useful preclinical tool for drug discovery and development.
Assuntos
Doença de Alzheimer , Acetilcolinesterase/metabolismo , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Animais , Inibidores da Colinesterase/química , Inibidores da Colinesterase/farmacologia , Inibidores da Colinesterase/uso terapêutico , Colinesterases , Donepezila/uso terapêutico , Chumbo/uso terapêutico , Ligantes , Peixe-Zebra/metabolismoRESUMO
The chromatin reader protein Spindlin1 plays an important role in epigenetic regulation, through which it has been linked to several types of malignant tumors. In the current work, we report on the development of novel analogs of the previously published lead inhibitor A366. In an effort to improve the activity and explore the structure-activity relationship (SAR), a series of 21 derivatives was synthesized, tested in vitro, and investigated by means of molecular modeling tools. Docking studies and molecular dynamics (MD) simulations were performed to analyze and rationalize the structural differences responsible for the Spindlin1 activity. The analysis of MD simulations shed light on the important interactions. Our study highlighted the main structural features that are required for Spindlin1 inhibitory activity, which include a positively charged pyrrolidine moiety embedded into the aromatic cage connected via a propyloxy linker to the 2-aminoindole core. Of the latter, the amidine group anchor the compounds into the pocket through salt bridge interactions with Asp184. Different protocols were tested to identify a fast in silico method that could help to discriminate between active and inactive compounds within the A366 series. Rescoring the docking poses with MM-GBSA calculations was successful in this regard. Because A366 is known to be a G9a inhibitor, the most active developed Spindlin1 inhibitors were also tested over G9a and GLP to verify the selectivity profile of the A366 analogs. This resulted in the discovery of diverse selective compounds, among which 1s and 1t showed Spindlin1 activity in the nanomolar range and selectivity over G9a and GLP. Finally, future design hypotheses were suggested based on our findings.
Assuntos
Fenômenos Biofísicos , Proteínas de Ciclo Celular/química , Epigênese Genética , Proteínas Associadas aos Microtúbulos/química , Fosfoproteínas/química , Conformação Proteica , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/ultraestrutura , Entropia , Humanos , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/ultraestrutura , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/genética , Fosfoproteínas/ultraestrutura , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
Metastasis is facilitated by the formation of a "premetastatic niche," which is fostered by primary tumor-derived factors. Colorectal cancer (CRC) metastasizes mainly to the liver. We show that the premetastatic niche in the liver is induced by bacteria dissemination from primary CRC. We report that tumor-resident bacteria Escherichia coli disrupt the gut vascular barrier (GVB), an anatomical structure controlling bacterial dissemination along the gut-liver axis, depending on the virulence regulator VirF. Upon GVB impairment, bacteria disseminate to the liver, boost the formation of a premetastatic niche, and favor the recruitment of metastatic cells. In training and validation cohorts of CRC patients, we find that the increased levels of PV-1, a marker of impaired GVB, is associated with liver bacteria dissemination and metachronous distant metastases. Thus, PV-1 is a prognostic marker for CRC distant recurrence and vascular impairment, leading to liver metastases.
Assuntos
Neoplasias Colorretais/irrigação sanguínea , Neoplasias Colorretais/patologia , Neoplasias Hepáticas/patologia , Metástase Neoplásica/patologia , Recidiva Local de Neoplasia/patologia , Bactérias/isolamento & purificação , Neoplasias do Colo/irrigação sanguínea , Neoplasias do Colo/patologia , Humanos , Fígado/patologia , Neoplasias Hepáticas/secundárioRESUMO
The biological players involved in angiogenesis are only partially defined. Here, we report that endothelial cells (ECs) express a novel isoform of the cell-surface adhesion molecule L1CAM, termed L1-ΔTM. The splicing factor NOVA2, which binds directly to L1CAM pre-mRNA, is necessary and sufficient for the skipping of L1CAM transmembrane domain in ECs, leading to the release of soluble L1-ΔTM. The latter exerts high angiogenic function through both autocrine and paracrine activities. Mechanistically, L1-ΔTM-induced angiogenesis requires fibroblast growth factor receptor-1 signaling, implying a crosstalk between the two molecules. NOVA2 and L1-ΔTM are overexpressed in the vasculature of ovarian cancer, where L1-ΔTM levels correlate with tumor vascularization, supporting the involvement of NOVA2-mediated L1-ΔTM production in tumor angiogenesis. Finally, high NOVA2 expression is associated with poor outcome in ovarian cancer patients. Our results point to L1-ΔTM as a novel, EC-derived angiogenic factor which may represent a target for innovative antiangiogenic therapies.
Assuntos
Processamento Alternativo , Proteínas Angiogênicas/metabolismo , Células Endoteliais/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Células Cultivadas , Humanos , Antígeno Neuro-Oncológico VentralRESUMO
How cells move chemotactically remains a major unmet challenge in cell biology. Emerging evidence indicates that for interpreting noisy, shallow gradients of soluble cues a system must behave as an excitable process. Here, through an RNAi-based, high-content screening approach, we identify RAB35 as necessary for the formation of growth factors (GFs)-induced waves of circular dorsal ruffles (CDRs), apically restricted actin-rich migratory protrusions. RAB35 is sufficient to induce recurrent and polarized CDRs that travel as propagating waves, thus behaving as an excitable system that can be biased to control cell steering. Consistently, RAB35 is essential for promoting directed chemotactic migration and chemoinvasion of various cells in response to gradients of motogenic GFs. Molecularly, RAB35 does so by directly regulating the activity of p85/PI3K polarity axis. We propose that RAB35 is a molecular determinant for the control of an excitable, oscillatory system that acts as a steering wheel for GF-mediated chemotaxis and chemoinvasion.
Assuntos
Quimiotaxia/genética , Classe Ia de Fosfatidilinositol 3-Quinase/genética , Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Proteínas rab de Ligação ao GTP/genética , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Animais , Linhagem Celular Tumoral , Quimiotaxia/efeitos dos fármacos , Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/ultraestrutura , Fibroblastos/efeitos dos fármacos , Fibroblastos/ultraestrutura , Expressão Gênica , Células HeLa , Humanos , Camundongos , Imagem Molecular , Fator de Crescimento Derivado de Plaquetas/farmacologia , Cultura Primária de Células , Transdução de Sinais , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
USP6NL, also named RN-tre, is a GTPase-activating protein involved in control of endocytosis and signal transduction. Here we report that USP6NL is overexpressed in breast cancer, mainly of the basal-like/integrative cluster 10 subtype. Increased USP6NL levels were accompanied by gene amplification and were associated with worse prognosis in the METABRIC dataset, retaining prognostic value in multivariable analysis. High levels of USP6NL in breast cancer cells delayed endocytosis and degradation of the EGFR, causing chronic AKT (protein kinase B) activation. In turn, AKT stabilized the glucose transporter GLUT1 at the plasma membrane, increasing aerobic glycolysis. In agreement, elevated USP6NL sensitized breast cancer cells to glucose deprivation, indicating that their glycolytic capacity relies on this protein. Depletion of USP6NL accelerated EGFR/AKT downregulation and GLUT1 degradation, impairing cell proliferation exclusively in breast cancer cells that harbored increased levels of USP6NL. Overall, these findings argue that USP6NL overexpression generates a metabolic rewiring that is essential to foster the glycolytic demand of breast cancer cells and promote their proliferation.Significance: USP6NL overexpression leads to glycolysis addiction of breast cancer cells and presents a point of metabolic vulnerability for therapeutic targeting in a subset of aggressive basal-like breast tumors.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/13/3432/F1.large.jpg Cancer Res; 78(13); 3432-44. ©2018 AACR.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias da Mama/patologia , Proteínas Ativadoras de GTPase/metabolismo , Transportador de Glucose Tipo 1/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Linhagem Celular Tumoral , Proliferação de Células , Conjuntos de Dados como Assunto , Receptores ErbB/metabolismo , Feminino , Proteínas Ativadoras de GTPase/genética , Amplificação de Genes , Glicólise , Humanos , Fosforilação , Prognóstico , Estabilidade Proteica , Proteólise , Análise de SobrevidaRESUMO
Neprilysin is also known as skin fibroblast-derived elastase, and its up-regulation during aging is associated with impairments of the elastic fiber network, loss of skin elasticity and wrinkle formation. However, information on its elastase activity is still limited. The aim of this study was to investigate the degradation of fibrillar skin elastin by neprilysin and the influence of the donor's age on the degradation process using mass spectrometry and bioinformatics approaches. The results showed that cleavage by neprilysin is dependent on previous damage of elastin. While neprilysin does not cleave young and intact skin elastin well, it degrades elastin fibers from older donors, which may further promote aging processes. With regards to the cleavage behavior of neprilysin, a strong preference for Gly at P1 was found, while Gly, Ala and Val were well accepted at P1' upon cleavage of tropoelastin and skin elastin. The results of the study indicate that the progressive release of bioactive elastin peptides by neprilysin upon skin aging may enhance local tissue damage and accelerate extracellular matrix aging processes.
Assuntos
Elastina/metabolismo , Neprilisina/metabolismo , Proteólise , Pele/metabolismo , Idoso , Sequência de Aminoácidos , Criança , Elastina/química , Humanos , Masculino , Simulação de Acoplamento Molecular , Conformação ProteicaRESUMO
The methyllysine reader protein Spindlin1 has been implicated in the tumorigenesis of several types of cancer and may be an attractive novel therapeutic target. Small-molecule inhibitors of Spindlin1 should be valuable as chemical probes as well as potential new therapeutics. We applied an iterative virtual screening campaign, encompassing structure- and ligand-based approaches, to identify potential Spindlin1 inhibitors from databases of commercially available compounds. Our inâ silico studies coupled with inâ vitro testing were successful in identifying novel Spindlin1 inhibitors. Several 4-aminoquinazoline and quinazolinethione derivatives were among the active hit compounds, which indicated that these scaffolds represent promising lead structures for the development of Spindlin1 inhibitors. Subsequent lead optimization studies were hence carried out, and numerous derivatives of both lead scaffolds were synthesized. This resulted in the discovery of novel inhibitors of Spindlin1 and helped explore the structure-activity relationships of these inhibitor series.
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Fosfoproteínas/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
CCDC6 gene product is a pro-apoptotic protein substrate of ATM, whose loss or inactivation enhances tumour progression. In primary tumours, the impaired function of CCDC6 protein has been ascribed to CCDC6 rearrangements and to somatic mutations in several neoplasia. Recently, low levels of CCDC6 protein, in NSCLC, have been correlated with tumor prognosis. However, the mechanisms responsible for the variable levels of CCDC6 in primary tumors have not been described yet.We show that CCDC6 turnover is regulated in a cell cycle dependent manner. CCDC6 undergoes a cyclic variation in the phosphorylated status and in protein levels that peak at G2 and decrease in mitosis. The reduced stability of CCDC6 in the M phase is dependent on mitotic kinases and on degron motifs that are present in CCDC6 and direct the recruitment of CCDC6 to the FBXW7 E3 Ubl. The de-ubiquitinase enzyme USP7 appears responsible of the fine tuning of the CCDC6 stability, affecting cells behaviour and drug response.Thus, we propose that the amount of CCDC6 protein in primary tumors, as reported in lung, may depend on the impairment of the CCDC6 turnover due to altered protein-protein interaction and post-translational modifications and may be critical in optimizing personalized therapy.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Resistencia a Medicamentos Antineoplásicos/fisiologia , Proteínas F-Box/metabolismo , Neoplasias Pulmonares/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Western Blotting , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Proteína 7 com Repetições F-Box-WD , Feminino , Imunofluorescência , Técnicas de Inativação de Genes , Humanos , Masculino , Pessoa de Meia-Idade , RNA Interferente Pequeno , Reação em Cadeia da Polimerase em Tempo Real , Análise Serial de Tecidos , Transfecção , Peptidase 7 Específica de UbiquitinaRESUMO
Non-small cell lung cancer (NSCLC) is the main cause of cancer-related death worldwide and new therapeutic strategies are urgently needed. In this study, we have characterized a panel of NSC lung cancer cell lines for the expression of coiled-coil-domain containing 6 (CCDC6), a tumor suppressor gene involved in apoptosis and DNA damage response. We show that low CCDC6 protein levels are associated with a weak response to DNA damage and a low number of Rad51 positive foci. Moreover, CCDC6 deficient lung cancer cells show defects in DNA repair via homologous recombination. In accordance with its role in the DNA damage response, CCDC6 attenuation confers resistance to cisplatinum, the current treatment of choice for NSCLC, but sensitizes the cells to olaparib, a small molecule inhibitor of the repair enzymes PARP1/2. Remarkably, the combination of the two drugs is more effective than each agent individually, as demonstrated by a combination index <1. Finally, CCDC6 is expressed at low levels in about 30% of the NSCL tumors we analyzed by TMA immunostaining. The weak CCDC6 protein staining is significatively correlated with the presence of lymph node metastasis (p ≤ 0.02) and negatively correlated to the disease free survival (p ≤ 0.01) and the overall survival (p ≤ 0.05). Collectively, the data indicate that CCDC6 levels provide valuable insight for OS. CCDC6 could represent a predictive biomarker of resistance to conventional single mode therapy and yield insight on tumor sensitivity to PARP inhibitors in NSCLC.
Assuntos
Antineoplásicos/farmacologia , Proteínas do Citoesqueleto/deficiência , Neoplasias Pulmonares/tratamento farmacológico , Idoso , Idoso de 80 Anos ou mais , Apoptose/efeitos dos fármacos , Apoptose/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Cisplatino/farmacologia , Proteínas do Citoesqueleto/genética , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Intervalo Livre de Doença , Feminino , Humanos , Neoplasias Pulmonares/genética , Metástase Linfática/genética , Masculino , Pessoa de Meia-Idade , Ftalazinas , Piperazinas , Rad51 Recombinase/genéticaRESUMO
The role of endocytic proteins and the molecular mechanisms underlying epithelial cell cohesion and tumor dissemination are not well understood. Here, we report that the endocytic F-BAR-containing CDC42-interacting protein 4 (CIP4) is required for ERBB2- and TGF-ß1-induced cell scattering, breast cancer (BC) cell motility and invasion into 3D matrices, and conversion from ductal breast carcinoma in situ to invasive carcinoma in mouse xenograft models. CIP4 promotes the formation of an E-cadherin-CIP4-SRC complex that controls SRC activation, E-cadherin endocytosis, and localized phosphorylation of the myosin light chain kinase, thereby impinging on the actomyosin contractility required to generate tangential forces to break cell-cell junctions. CIP4 is upregulated in ERBB2-positive human BC, correlates with increased distant metastasis, and is an independent predictor of poor disease outcome in subsets of BC patients. Thus, it critically controls cell-cell cohesion and is required for the acquisition of an invasive phenotype in breast tumors.
Assuntos
Células Epiteliais/citologia , Regulação Neoplásica da Expressão Gênica , Proteínas Associadas aos Microtúbulos/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Actomiosina/metabolismo , Animais , Caderinas/metabolismo , Carcinoma Ductal de Mama/patologia , Carcinoma Intraductal não Infiltrante/patologia , Linhagem Celular Tumoral , Endocitose , Feminino , Humanos , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Antígenos de Histocompatibilidade Menor , Transplante de Neoplasias , Receptor ErbB-2/metabolismo , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Maspin, a member of the serpin family of protease inhibitors, is involved in key processes of cancer progression. Its biological activity seems to be cancer and compartment specific, with the protein acting either as a suppressor or as a tumor promoter in different cancer types. Characterization of maspin expression and its sub-cellular localization in melanoma is missing, hence, we aim to investigate its possible association with melanoma prognostic factors and disease progression. Nuclear and cytoplasmic maspin expression were evaluated on 60 nevi, 152 primary lesions, and 106 melanoma metastases using tissue microarrays and immunohistochemistry. The association between maspin immunoreactivity and patient's clinic-pathological features was evaluated. Multivariate logistic models and survival analyses were performed for maspin expression in primary melanomas. Nuclear maspin was detected in 8% nevi, 49% primary melanomas, and 28% metastases, whereas cytoplasmic maspin in 12% nevi, 18% primary lesions, and 9% metastases. In univariate analysis, nuclear maspin expression in primary melanomas was significantly associated with melanoma prognostic factors (nodular histotype, tumor thickness, mitotic rate, and ulceration) and disease stage, whereas cytoplasmic maspin was observed at higher frequency in thin superficial spreading melanomas, without mitosis. In multivariate analysis, nuclear maspin remained significantly associated with risk of developing a tumor prone to disease progression and, accordingly, with significantly shorter disease-free and overall survival. In this study, maspin was expressed at highest frequency in primary lesions and when expressed in the nuclei, was significantly associated with poor prognostic markers, melanoma recurrence, and worse survival. The present study suggests a tumor-suppressive effect of cytoplasmic maspin and a tumor-promoting effect of nuclear maspin, which open the discussion on its potential use in cancer therapy.
Assuntos
Biomarcadores Tumorais/análise , Melanoma/patologia , Serpinas/biossíntese , Neoplasias Cutâneas/patologia , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Progressão da Doença , Humanos , Estimativa de Kaplan-Meier , Melanoma/metabolismo , Melanoma/mortalidade , Metástase Neoplásica/patologia , Modelos de Riscos Proporcionais , Serpinas/análise , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/mortalidade , Análise Serial de TecidosRESUMO
BACKGROUND: DNA damage response has been clearly described as an anti-cancer barrier in early human tumorigenesis. Moreover, interestingly, testicular germ cell tumors (TGCTs) have been reported to lack the DNA Damage Response (DDR) pathway activation. CCDC6 is a pro-apoptotic phosphoprotein substrate of the kinase ataxia telangectasia mutated (ATM) able to sustain DNA damage checkpoint in response to genotoxic stress and is commonly rearranged in malignancies upon fusion with different partners. In our study we sought to determine whether CCDC6 could have a role in the patho-genesis of testicular germ cell tumors. METHODS: To achieve this aim, analysis for CCDC6 expression has been evaluated on serial sections of the mouse testis by immunohistochemistry and on separate populations of murine testicular cells by western blot. Next, the resistance to DNA damage-induced apoptosis and the production of reactive oxygen species has been investigated in GC1 cells, derived from immortalized type B murine germ cells, following CCDC6 silencing. Finally, the CCDC6 expression in normal human testicular cells, in Intratubular Germ Cell Neoplasia Unclassified (IGCNU), in a large series of male germ cell tumours and in the unique human seminoma TCam2 cell line has been evaluated by immunohistochemistry and by Western Blot analyses. RESULTS: The analysis of the CCDC6 expression revealed its presence in Sertoli cells and in spermatogonial cells. CCDC6 loss was the most consistent feature among the primary tumours and TCam2 cells. Interestingly, following treatment with low doses of H2O2, the silencing of CCDC6 in GC1 cells caused a decrease in the oxidized form of cytochrome c and low detection of Bad, PARP-1 and Caspase 3 proteins. Moreover, in the silenced cells, upon oxidative damage, the cell viability was protected, the γH2AX activation was impaired and the Reactive Oxygen Species (ROS) release was decreased. CONCLUSIONS: Therefore, our results suggest that the loss of CCDC6 could aid the spermatogonial cells to be part of a pro-survival pathway that helps to evade the toxic effects of endogenous oxidants and contributes to testicular neoplastic growth.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Neoplasias Embrionárias de Células Germinativas/metabolismo , Neoplasias Embrionárias de Células Germinativas/patologia , Neoplasias Testiculares/metabolismo , Neoplasias Testiculares/patologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Citocromos c/metabolismo , Proteínas do Citoesqueleto/genética , Expressão Gênica , Inativação Gênica , Humanos , Masculino , Camundongos , Neoplasias Embrionárias de Células Germinativas/genética , Peróxidos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Seminoma/genética , Seminoma/metabolismo , Seminoma/patologia , Neoplasias Testiculares/genética , Testículo/metabolismo , Testículo/patologiaRESUMO
CCDC6 was originally identified in chimeric genes as caused by chromosomal translocation involving the RET protooncogene in some thyroid tumors. Recognised as a 65 kDa pro-apoptotic phosphoprotein, CCDC6 has been enrolled as an ATM substrate that contribute to protect genome integrity by modulating PP4c activity in response to genotoxic stress. Recently, CCDC6 has been identified as a repressor of CREB1-dependent transcription. Sumoylation has emerged as an important mechanism in transcriptional control. Here, we report the identification and characterization of three sites of sumoylation in CCDC6 (K74, K266 and K424) which are highly conserved in vertebrates. We demonstrate that the post-translational modifications by SUMO2 constrain most of the CCDC6 protein in the cytosol and affect its functional interaction with CREB1 with a decrease of CCDC6 repressive function on CREB1 transcriptional activity. Indeed, the impairment of functional outcome of sumoylated CCDC6 is obtained knocking down all three the sumoylation sites. Interestingly, in thyroid cells the SUMO2-mediated CCDC6 post-translational modifications are induced by Forskolin, a cAMP analog. Signal transduction via the cAMP pathway is known to be ubiquitous and represents a major line of communication between many organisms and their environment. We believe that CCDC6 could be an important player in the dynamics of cAMP signaling by fine regulating CREB1 transcriptional activity in normal and transformed thyroid cells.
Assuntos
Carcinoma/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteínas do Citoesqueleto/química , Proteínas Proto-Oncogênicas c-ret/genética , Sumoilação , Neoplasias da Glândula Tireoide/genética , Animais , Carcinoma Papilar , Linhagem Celular Tumoral , Proliferação de Células , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas do Citoesqueleto/análise , Proteínas do Citoesqueleto/metabolismo , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Lisina/química , Lisina/metabolismo , Transporte Proteico , Proteínas Proto-Oncogênicas c-ret/metabolismo , Ratos , Câncer Papilífero da Tireoide , Transcrição GênicaRESUMO
AIMS: The natural history of oral squamous cell carcinomas (OSCCs) is variable and difficult to predict. This study aimed to assess the value of the expression of poly(ADP-ribose) polymerase 1 (PARP-1), chromatin assembly factor-1 (CAF-1)/p60 and the stem cell markers CD133, CD166, CD44, CD44v6 and nestin as markers of outcome and progression-free survival in OSCC patients. METHODS: Clinical data were collected from 66 patients (41 male and 25 female, aged 29-92 years) who underwent surgery for OSCC of the tongue, floor, lips, and palate. During follow-up (range: 12-131 months), 14 patients experienced relapse/metastasis and/or death. The study was performed by immunohistochemistry on paraffin-embedded tumour tissues, western blot analysis of tumour protein lysates and human cell lines, and RNA silencing assays. In addition, the human papillomavirus (HPV) status of primary tumours was evaluated by immunohistochemistry and viral subtyping. Univariate and multivariate analyses were performed to determine the correlation between these parameters and the clinical and pathological variables of the study population. RESULTS AND CONCLUSIONS: We found that a PARP-1(high) /CAF-1 p60(high) /nestin(high) phenotype characterized the OSCCs with the worst prognosis (all HPV-negative). This may be of benefit in clinical management, since radio-enhancing anti-PARP-1 and/or anti-CAF-1/p60 agents may allow radioresistance to be bypassed in the nestin-overexpressing, metastasizing OSCC cells.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Fator 1 de Modelagem da Cromatina/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Neoplasias Bucais/metabolismo , Metástase Neoplásica/diagnóstico , Proteínas do Tecido Nervoso/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Regulação para Cima , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/mortalidade , Progressão da Doença , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/diagnóstico , Neoplasias Bucais/mortalidade , Nestina , Valor Preditivo dos Testes , Prognóstico , Taxa de Sobrevida , Fatores de TranscriçãoRESUMO
CCDC6 was originally identified in chimeric genes caused by chromosomal translocation involving the RET proto-oncogene in some thryoid tumors mostly upon ionizing radiation exposure. Recognised as a pro-apoptotic phosphoprotein that negatively regulates CREB1-dependent transcription, CCDC6 is an ATM substrate that is responsive to genotoxic stress. Here we report that following genotoxic stress, loss or inactivation of CCDC6 in cancers that carry the CCDC6 fusion, accelerates the dephosphorylation of pH2AX S139, resulting in defective G2 arrest and premature mitotic entry. Moreover, we show that CCDC6 depleted cells appear to repair DNA damaged in a shorter time compared to controls, based on reporter assays in cells. High-troughput proteomic screening predicted the interaction between the CCDC6 gene product and the catalytic subunit of Serin-Threonin Protein Phosphatase 4 (PP4c) recently identified as the evolutionarily conserved pH2AX S139 phosphatase that is activated upon DNA Damage. We describe the interaction between CCDC6 and PP4c and we report the modulation of PP4c enzymatic activity in CCDC6 depleted cells. We discuss the functional significance of CCDC6-PP4c interactions and hypothesize that CCDC6 may act in the DNA Damage Response by negatively modulating PP4c activity. Overall, our data suggest that in primary tumours the loss of CCDC6 function could influence genome stability and thereby contribute to carcinogenesis.
Assuntos
Proteínas do Citoesqueleto/genética , Dano ao DNA , Histonas/metabolismo , Mitose , Neoplasias/genética , Linhagem Celular , Proteínas do Citoesqueleto/metabolismo , Fase G2 , Inativação Gênica , Células HeLa , Humanos , Neoplasias/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Proto-Oncogene MasRESUMO
Cellular senescence has been widely recognized as a tumor suppressing mechanism that acts as a barrier to cancer development after oncogenic stimuli. A prominent in vivo model of the senescence barrier is represented by nevi, which are composed of melanocytes that, after an initial phase of proliferation induced by activated oncogenes (most commonly BRAF), are blocked in a state of cellular senescence. Transformation to melanoma occurs when genes involved in controlling senescence are mutated or silenced and cells reacquire the capacity to proliferate. Pirin (PIR) is a highly conserved nuclear protein that likely functions as a transcriptional regulator whose expression levels are altered in different types of tumors. We analyzed the expression pattern of PIR in adult human tissues and found that it is expressed in melanocytes and has a complex pattern of regulation in nevi and melanoma: it is rarely detected in mature nevi, but is expressed at high levels in a subset of melanomas. Loss of function and overexpression experiments in normal and transformed melanocytic cells revealed that PIR is involved in the negative control of cellular senescence and that its expression is necessary to overcome the senescence barrier. Our results suggest that PIR may have a relevant role in melanoma progression.
Assuntos
Proteínas de Transporte/metabolismo , Transformação Celular Neoplásica/metabolismo , Senescência Celular/fisiologia , Melanócitos/metabolismo , Proteínas Nucleares/metabolismo , Adulto , Western Blotting , Dioxigenases , Humanos , Imuno-Histoquímica , Melanoma/metabolismo , Melanoma/patologia , Pessoa de Meia-Idade , Nevo Pigmentado/metabolismo , Nevo Pigmentado/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Análise Serial de TecidosRESUMO
BACKGROUND: Deubiquitinating enzymes (DUBs) are proteases that process ubiquitin (Ub) or ubiquitin-like gene products, remodel polyubiquitin(-like) chains on target proteins, and counteract protein ubiquitination exerted by E3 ubiquitin-ligases. A wealth of studies has established the relevance of DUBs to the control of physiological processes whose subversion is known to cause cellular transformation, including cell cycle progression, DNA repair, endocytosis and signal transduction. Altered expression of DUBs might, therefore, subvert both the proteolytic and signaling functions of the Ub system. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we report the first comprehensive screening of DUB dysregulation in human cancers by in situ hybridization on tissue microarrays (ISH-TMA). ISH-TMA has proven to be a reliable methodology to conduct this kind of study, particularly because it allows the precise identification of the cellular origin of the signals. Thus, signals associated with the tumor component can be distinguished from those associated with the tumor microenvironment. Specimens derived from various normal and malignant tumor tissues were analyzed, and the "normal" samples were derived, whenever possible, from the same patients from whom tumors were obtained. Of the â¼90 DUBs encoded by the human genome, 33 were found to be expressed in at least one of the analyzed tissues, of which 22 were altered in cancers. Selected DUBs were subjected to further validation, by analyzing their expression in large cohorts of tumor samples. This analysis unveiled significant correlations between DUB expression and relevant clinical and pathological parameters, which were in some cases indicative of aggressive disease. CONCLUSIONS/SIGNIFICANCE: The results presented here demonstrate that DUB dysregulation is a frequent event in cancer, and have implications for therapeutic approaches based on DUB inhibition.