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1.
Genes Chromosomes Cancer ; 63(8): e23256, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-39193983

RESUMO

Papillary thyroid carcinoma (PTC), the most common malignancy of follicular cell derivation, is generally associated with good prognosis. Nevertheless, it is important to identify patients with aggressive PTCs and unfavorable outcome. Molecular markers such as BRAFV600E mutation and TERT promoter mutations have been proposed for risk stratification. While TERT promoter mutations have been frequently associated with aggressive PTCs, the association of BRAFV600E mutation with increased recurrence and mortality is less clear and has been controversially discussed. The aim of the present study was to analyze whether differentially expressed genes can predict BRAFV600E mutations as well as TERT promoter mutations in PTCs. RNA sequencing identified a large number of differentially expressed genes between BRAFV600E and BRAFwildtype PTCs. Of those, AHNAK2, DCSTAMP, and FN1 could be confirmed in a larger cohort (n = 91) to be significantly upregulated in BRAFV600E mutant PTCs using quantitative RT-PCR. Moreover, individual PTC expression values of DCSTAMP and FN1 were able to predict the BRAFV600E mutation status with high sensitivity and specificity. The expression of TERT was detected in all PTCs harboring TERT promoter mutations and in 19% of PTCs without TERT promoter mutations. Tumors with both TERT expression and TERT promoter mutations were particularly associated with aggressive clinicopathological features and a shorter recurrence-free survival. Altogether, it will be interesting to explore the biological function of AHNAK2, DCSTAMP, and FN1 in PTC in more detail. The analysis of their expression patterns could allow the characterization of PTC subtypes and thus enabling a more individualized surgical and medical treatment.


Assuntos
Mutação , Recidiva Local de Neoplasia , Telomerase , Câncer Papilífero da Tireoide , Neoplasias da Glândula Tireoide , Humanos , Telomerase/genética , Feminino , Masculino , Câncer Papilífero da Tireoide/genética , Câncer Papilífero da Tireoide/patologia , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Pessoa de Meia-Idade , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Adulto , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas de Membrana/genética , Idoso , Transcriptoma , Biomarcadores Tumorais/genética , Regulação Neoplásica da Expressão Gênica , Regiões Promotoras Genéticas , Proteínas do Citoesqueleto , Fibronectinas
2.
Cells ; 12(9)2023 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-37174657

RESUMO

Primary liver cancer is the third leading cause of cancer-related death worldwide. An increasing body of evidence suggests that the Hippo tumor suppressor pathway plays a critical role in restricting cell proliferation and determining cell fate during physiological and pathological processes in the liver. Merlin (Moesin-Ezrin-Radixin-like protein) encoded by the NF2 (neurofibromatosis type 2) gene is an upstream regulator of the Hippo signaling pathway. Targeting of Merlin to the plasma membrane seems to be crucial for its major tumor-suppressive functions; this is facilitated by interactions with membrane-associated proteins, including CD44 (cluster of differentiation 44). Mutations within the CD44-binding domain of Merlin have been reported in many human cancers. This study evaluated the relative contribution of CD44- and Merlin-dependent processes to the development and progression of liver tumors. To this end, mice with a liver-specific deletion of the Nf2 gene were crossed with Cd44-knockout mice and subjected to extensive histological, biochemical and molecular analyses. In addition, cells were isolated from mutant livers and analyzed by in vitro assays. Deletion of Nf2 in the liver led to substantial liver enlargement and generation of hepatocellular carcinomas (HCCs), intrahepatic cholangiocarcinomas (iCCAs), as well as mixed hepatocellular cholangiocarcinomas. Whilst deletion of Cd44 had no influence on liver size or primary liver tumor development, it significantly inhibited metastasis formation in Nf2-mutant mice. CD44 upregulates expression of integrin ß2 and promotes transendothelial migration of liver cancer cells, which may facilitate metastatic spreading. Overall, our results suggest that CD44 may be a promising target for intervening with metastatic spreading of liver cancer.


Assuntos
Neoplasias dos Ductos Biliares , Carcinoma Hepatocelular , Colangiocarcinoma , Receptores de Hialuronatos , Neoplasias Hepáticas , Neurofibromatose 2 , Animais , Humanos , Camundongos , Neoplasias dos Ductos Biliares/genética , Ductos Biliares Intra-Hepáticos , Carcinoma Hepatocelular/genética , Colangiocarcinoma/genética , Genes da Neurofibromatose 2 , Receptores de Hialuronatos/genética , Neoplasias Hepáticas/genética , Neurofibromatose 2/genética , Neurofibromina 2/genética , Neurofibromina 2/metabolismo
3.
Int J Mol Sci ; 23(15)2022 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-35955749

RESUMO

Osteosarcoma is the most common type of pediatric bone tumor. Despite great advances in chemotherapy during the past decades, the survival rates of osteosarcoma patients remain unsatisfactory. Drug resistance is one of the main reasons, leading to treatment failure and poor prognosis. Previous reports correlated expression of cluster of differentiation 44 (CD44) with drug resistance and poor survival of osteosarcoma patients, however the underlying mechanisms are poorly defined. Here, we investigated the role of CD44 in the regulation of drug chemoresistance, using osteosarcoma cells isolated from mice carrying a mutation of the tumor suppressor neurofibromatosis type 2 (Nf2) gene. CD44 expression was knocked-down in the cells using CRISPR/Cas9 approach. Subsequently, CD44 isoforms and mutants were re-introduced to investigate CD44-dependent processes. Sensitivity to doxorubicin was analyzed in the osteosarcoma cells with modified CD44 expression by immunoblot, colony formation- and WST-1 assay. To dissect the molecular alterations induced by deletion of Cd44, RNA sequencing was performed on Cd44-positive and Cd44-negative primary osteosarcoma tissues isolated from Nf2-mutant mice. Subsequently, expression of candidate genes was evaluated by quantitative reverse transcription PCR (qRT-PCR). Our results indicate that CD44 increases the resistance of osteosarcoma cells to doxorubicin by up-regulating the levels of multidrug resistance (MDR) 1 protein expression, and suggest the role of proteolytically released CD44 intracellular domain, and hyaluronan interactions in this process. Moreover, high throughput sequencing analysis identified differential regulation of several apoptosis-related genes in Cd44-positive and -negative primary osteosarcomas, including p53 apoptosis effector related to PMP-22 (Perp). Deletion of Cd44 in osteosarcoma cells led to doxorubicin-dependent p53 activation and a profound increase in Perp mRNA expression. Overall, our results suggest that CD44 might be an important regulator of drug resistance and suggest that targeting CD44 can sensitize osteosarcoma to standard chemotherapy.


Assuntos
Neoplasias Ósseas , Osteossarcoma , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Camundongos , Osteossarcoma/tratamento farmacológico , Osteossarcoma/genética , Osteossarcoma/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
4.
Sci Rep ; 11(1): 16745, 2021 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-34408162

RESUMO

The current standard for molecular profiling of colorectal cancer (CRC) is using resected or biopsied tissue specimens. However, they are limited regarding sampling frequency, representation of tumor heterogeneity, and sampling can expose patients to adverse side effects. The analysis of cell-free DNA (cfDNA) from blood plasma, which is part of a liquid biopsy, is minimally invasive and in principle enables detection of all tumor-specific mutations. Here, we analyzed cfDNA originating from nucleus and mitochondria and investigated their characteristics and mutation status in a cohort of 18 CRC patients and 10 healthy controls using targeted next-generation sequencing (NGS) and digital PCR. Longitudinal analyses of nuclear cfDNA level and size during chemotherapy revealed a decreasing cfDNA content and a shift from short to long fragments, indicating an appropriate therapy response, while shortened cfDNAs and increased cfDNA content corresponded with tumor recurrence. Comparative NGS analysis of nuclear tissue and plasma DNA demonstrated a good patient-level concordance and cfDNA revealed additional variants in three of the cases. Analysis of mitochondrial cfDNA surprisingly revealed a higher plasma copy number in healthy subjects than in CRC patients. These results highlight the potential clinical utility of liquid biopsies in routine diagnostics and surveillance of CRC patients as complementation to tissue biopsies or as an attractive alternative in cases where tissue biopsies are risky or the quantity/quality does not allow testing.


Assuntos
Neoplasias Colorretais/genética , DNA Mitocondrial/genética , DNA de Neoplasias/genética , Sequenciamento de Nucleotídeos em Larga Escala , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Colorretais/patologia , Feminino , Humanos , Biópsia Líquida , Masculino , Pessoa de Meia-Idade
5.
Int J Cancer ; 147(9): 2564-2577, 2020 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-32525563

RESUMO

Merlin is a versatile tumor suppressor protein encoded by the NF2 gene. Several lines of evidence suggest that Merlin exerts its tumor suppressor activity, at least in part, by forming an inhibitory complex with cluster of differentiation 44 (CD44). Consistently, numerous NF2 mutations in cancer patients are predicted to perturb the interaction of Merlin with CD44. We hypothesized that disruption of the Merlin-CD44 complex through loss of Merlin, unleashes putative tumor- or metastasis-promoting functions of CD44. To evaluate the relevance of the Merlin-CD44 interaction in vivo, we compared tumor growth and progression in Cd44-positive and Cd44-negative Nf2-mutant mice. Heterozygous Nf2-mutant mice were prone to developing highly metastatic osteosarcomas. Importantly, while the absence of the Cd44 gene had no effect on the frequency of primary osteosarcoma development, it strongly diminished osteosarcoma metastasis formation in the Nf2-mutant mice. In vitro assays identified transendothelial migration as the most prominent cellular phenotype dependent on CD44. Adhesion to endothelial cells was blocked by interfering with integrin α4ß1 (very late antigen-4, VLA-4) on osteosarcoma cells and CD44 upregulated levels of integrin VLA-4 ß1 subunit. Among other putative functions of CD44, which may contribute to the metastatic behavior, the passage through the endothelial cells also appears to be critical in vivo, as CD44 significantly promoted formation of lung metastasis upon intravenous injection of osteosarcoma cells into immunocompromised mice. Altogether, our results strongly suggest that CD44 plays a metastasis-promoting role in the absence of Merlin.


Assuntos
Neoplasias Ósseas/genética , Receptores de Hialuronatos/metabolismo , Neoplasias Pulmonares/genética , Neurofibromina 2/genética , Osteossarcoma/genética , Animais , Neoplasias Ósseas/patologia , Osso e Ossos/patologia , Adesão Celular/genética , Linhagem Celular Tumoral/transplante , Proliferação de Células/genética , Modelos Animais de Doenças , Progressão da Doença , Humanos , Receptores de Hialuronatos/genética , Pulmão/patologia , Neoplasias Pulmonares/secundário , Masculino , Camundongos , Camundongos Knockout , Osteossarcoma/secundário
6.
Int J Cancer ; 144(11): 2782-2794, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30485423

RESUMO

Primary liver cancer (PLC) ranks among the most lethal solid cancers worldwide due to lack of effective biomarkers for early detection and limited treatment options in advanced stages. Development of primary culture models that closely recapitulate phenotypic and molecular diversities of PLC is urgently needed to improve the patient outcome. Long-term cultures of 7 primary liver cancer cell lines of hepatocellular and cholangiocellular origin were established using defined culture conditions. Morphological and histological characteristics of obtained cell lines and xenograft tumors were analyzed and compared to original tumors. Time course analyses of transcriptomic and genomic changes were performed using next-generation sequencing (NGS). Key oncogenic alterations were identified by targeted NGS and cell lines carrying potentially actionable mutations were treated with corresponding specific inhibitors. PDCL fully resembled morphological features of the primary cancers in vitro and in vivo over extended period in culture. Genomic alterations as well as transcriptome profiles showed high similarity with primary tumors and remained stable during long-term culturing. Targeted-NGS confirmed that key oncogenic mutations such as TP53, KRAS, CTNNB1 as well as actionable mutations (e.g. MET, cKIT, KDR) were highly conserved in PDCL and amenable for individualized therapeutic approaches. Integrative genomic and transcriptomic approaches further demonstrated that PDCL more closely resemble molecular and prognostic features of PLC than established cell lines and are valuable tool for direct target evaluation. Our integrative analysis demonstrates that PDCL represents refined model for discovery of relevant molecular subgroups and exploration of precision medicine approaches for the treatment of this deadly disease.


Assuntos
Linhagem Celular Tumoral/patologia , Neoplasias Hepáticas/patologia , Medicina de Precisão/métodos , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinogênese/genética , Análise Mutacional de DNA , Perfilação da Expressão Gênica/métodos , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Camundongos , Mutação , Cultura Primária de Células/métodos , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
7.
J Biol Chem ; 290(28): 17041-54, 2015 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-25925953

RESUMO

Ectodomain shedding of transmembrane precursor proteins generates numerous life-essential molecules, such as epidermal growth factor receptor ligands. This cleavage not only releases the regulatory growth factor, but it is also the required first step for the subsequent processing by γ-secretase and the release of gene regulatory intracellular fragments. Signaling within the cell modifies the cytoplasmic tails of substrates, a step important in starting the specific and regulated cleavage of a large number of studied substrates. Ectodomain cleavage occurs, however, on the outside of the plasma membrane and is carried out by membrane-bound metalloproteases. How the intracellular domain modification communicates with the ectodomain of the substrate to allow for cleavage to occur is unknown. Here, we show that homodimerization of a cluster-of-differentiation-44 or of pro-neuregulin-1 monomers represents an essential pre-condition for their regulated ectodomain cleavage. Both substrates are associated with their respective metalloproteases under both basal or cleavage-stimulated conditions. These interactions only turn productive by specific intracellular signal-induced intracellular domain modifications of the substrates, which in turn regulate metalloprotease access to the substrates' ectodomain and cleavage. We propose that substrate intracellular domain modification induces a relative rotation or other positional change of the dimerization partners that allow metalloprotease cleavage in the extracellular space. Our findings fill an important gap in understanding substrate-specific inside-out signal transfer along cleaved transmembrane proteins and suggest that substrate dimerization (homo- or possibly heterodimerization) might represent a general principle in ectodomain shedding.


Assuntos
Receptores de Hialuronatos/metabolismo , Neuregulina-1/metabolismo , Proteínas ADAM/química , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAM10 , Proteína ADAM17 , Secretases da Proteína Precursora do Amiloide/química , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Bovinos , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Células Cultivadas , Células HEK293 , Humanos , Receptores de Hialuronatos/química , Receptores de Hialuronatos/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Modelos Biológicos , Mutagênese Sítio-Dirigida , Células NIH 3T3 , Neuregulina-1/química , Neuregulina-1/genética , Multimerização Proteica , Estrutura Terciária de Proteína , Proteólise , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato
8.
Obes Facts ; 8(1): 77-86, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25765165

RESUMO

OBJECTIVE: To assess factors determining the reaction of individuals to the threats of overweight and obesity and to examine the interdependencies between weight-reducing strategies. METHODS: Cross-country survey covering 19 countries and 13,155 interviews. Data were analysed using a bivariate probit model that allows simultaneously analysing two weight-reducing strategies. RESULTS: Results show that weight-reducing strategies chosen are not independent from each other. Findings also reveal that different strategies are chosen by different population segments. Women are more likely to change their dietary patterns and less likely to become physically active after surpassing a weight threshold. In addition, the probability of a dietary change in case of overweight differs considerably between countries. The study also reveals that attitudes are an important factor for the strategy choice. CONCLUSIONS: It is vital for public health policies to understand determinants of citizens' engagement in weight reduction strategies once a certain threshold is reached. Thus, results can support the design of public health campaigns and programmes that aim to change community or national health behaviour trends taking into account, e.g., national differences.


Assuntos
Comportamento de Escolha , Dieta , Exercício Físico , Comportamentos Relacionados com a Saúde , Motivação , Obesidade/prevenção & controle , Redução de Peso , Adulto , Atitude Frente a Saúde , Etnicidade , Feminino , Saúde Global , Humanos , Masculino , Pessoa de Meia-Idade , Sobrepeso , Fatores Sexuais , Aumento de Peso
9.
Mol Cancer Res ; 13(5): 879-90, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25652588

RESUMO

UNLABELLED: Ectodomain cleavage (shedding) of transmembrane proteins by metalloproteases (MMP) generates numerous essential signaling molecules, but its regulation is not totally understood. CD44, a cleaved transmembrane glycoprotein, exerts both antiproliferative or tumor-promoting functions, but whether proteolysis is required for this is not certain. CD44-mediated contact inhibition and cellular proliferation are regulated by counteracting CD44 C-terminal interacting proteins, the tumor suppressor protein merlin (NF2) and ERM proteins (ezrin, radixin, moesin). We show here that activation or overexpression of constitutively active merlin or downregulation of ERMs inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced [as well as serum, hepatocyte growth factor (HGF), or platelet-derived growth factor (PDGF)] CD44 cleavage by the metalloprotease ADAM10, whereas overexpressed ERM proteins promoted cleavage. Merlin- and ERM-modulated Ras or Rac activity was not required for this function. However, latrunculin (an actin-disrupting toxin) or an ezrin mutant which is unable to link CD44 to actin, inhibited CD44 cleavage, identifying a cytoskeletal C-terminal link as essential for induced CD44 cleavage. Cellular migration, an important tumor property, depended on CD44 and its cleavage and was inhibited by merlin. These data reveal a novel function of merlin and suggest that CD44 cleavage products play a tumor-promoting role. Neuregulin, an EGF ligand released by ADAM17 from its pro-form NRG1, is predominantly involved in regulating cellular differentiation. In contrast to CD44, release of neuregulin from its pro-form was not regulated by merlin or ERM proteins. Disruption of the actin cytoskeleton however, also inhibited NRG1 cleavage. This current study presents one of the first examples of substrate-selective cleavage regulation. IMPLICATIONS: Investigating transmembrane protein cleavage and their regulatory pathways have provided new molecular insight into their important role in cancer formation and possible treatment.


Assuntos
Movimento Celular/fisiologia , Receptores de Hialuronatos/metabolismo , Neurofibromina 2/metabolismo , Animais , Proliferação de Células/fisiologia , Células Cultivadas , Fibroblastos , Humanos , Camundongos , Camundongos Transgênicos , Células NIH 3T3 , Células Tumorais Cultivadas
10.
FEBS Lett ; 587(16): 2698-704, 2013 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-23856463

RESUMO

The cell surface glycoprotein CD44 enhances phorbol-12-myristate 13-acetate (TPA)-induced expression of p21WAF1 by stabilizing its mRNA and enhancing the protein's half-life in several cell lines. Only the plasma membrane-anchored cytoplasmic tail of CD44 and its interacting ezrin, radixin, moesin (ERM) proteins are required for this effect. A mitogen activated kinase (MEK) inhibitor abolishes the action of CD44 on p21. Down-regulation of p21 dramatically decreased anchorage-independence of a cancer cell line, whereas CD44 expression in this background could partially rescue the phenotype.


Assuntos
Inibidor de Quinase Dependente de Ciclina p21/química , Regulação Enzimológica da Expressão Gênica , Receptores de Hialuronatos/química , Ésteres de Forbol/química , Animais , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Células NIH 3T3 , Regiões Promotoras Genéticas , Ligação Proteica , RNA/química , Transdução de Sinais , Proteínas ras/metabolismo
11.
Proc Natl Acad Sci U S A ; 110(24): 9776-81, 2013 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-23720309

RESUMO

Ectodomain cleavage of cell-surface proteins by A disintegrin and metalloproteinases (ADAMs) is highly regulated, and its dysregulation has been linked to many diseases. ADAM10 and ADAM17 cleave most disease-relevant substrates. Broad-spectrum metalloprotease inhibitors have failed clinically, and targeting the cleavage of a specific substrate has remained impossible. It is therefore necessary to identify signaling intermediates that determine substrate specificity of cleavage. We show here that phorbol ester or angiotensin II-induced proteolytic release of EGF family members may not require a significant increase in ADAM17 protease activity. Rather, inducers activate a signaling pathway using PKC-α and the PKC-regulated protein phosphatase 1 inhibitor 14D that is required for ADAM17 cleavage of TGF-α, heparin-binding EGF, and amphiregulin. A second pathway involving PKC-δ is required for neuregulin (NRG) cleavage, and, indeed, PKC-δ phosphorylation of serine 286 in the NRG cytosolic domain is essential for induced NRG cleavage. Thus, signaling-mediated substrate selection is clearly distinct from regulation of enzyme activity, an important mechanism that offers itself for application in disease.


Assuntos
Proteínas ADAM/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador alfa/metabolismo , Proteínas ADAM/genética , Proteína ADAM17 , Anfirregulina , Angiotensina II/farmacologia , Western Blotting , Linhagem Celular Tumoral , Família de Proteínas EGF , Ativação Enzimática/efeitos dos fármacos , Citometria de Fluxo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Isoenzimas/metabolismo , Células Jurkat , Ligantes , Fosforilação , Proteína Quinase C/metabolismo , Proteínas/metabolismo , Proteólise/efeitos dos fármacos , Interferência de RNA , Serina/genética , Serina/metabolismo , Especificidade por Substrato , Acetato de Tetradecanoilforbol/farmacologia , Fator de Crescimento Transformador alfa/genética
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