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1.
Semin Cancer Biol ; 60: 1-13, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31362074

RESUMO

Metastasis still poses the highest challenge for personalized therapy in cancer, partly due to a still incomplete understanding of its molecular evolution. We recently presented the most comprehensive whole-genome study of colorectal metastasis vs. matched primary tumors and suggested novel components of disease progression and metastasis evolution, some of them potentially relevant for targeted therapy. In this review, we try to put these findings into perspective with latest discoveries of colleagues and recent literature, and propose a systematic international team effort to collectively define the "metastasome", a term we introduce to summarize all genomic, epigenomic, transcriptomic, further -omic, molecular and functional characteristics rendering metastases different from primary tumors. Based on recent discoveries, we propose a revised metastasis model for colorectal cancer which is based on a common ancestor clone, early dissemination but flexible early or late stage clonal separation paralleling stromal interactions. Furthermore, we discuss hypotheses on site-specific metastasis, colorectal cancer progression, metastasis-targeted diagnosis and therapy, and metastasis prevention based on latest metastasome data.


Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Suscetibilidade a Doenças , Genômica , Metástase Neoplásica/genética , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Evolução Clonal/genética , Neoplasias Colorretais/mortalidade , Progressão da Doença , Genômica/métodos , Humanos , Células-Tronco Neoplásicas , Prognóstico
2.
Adv Exp Med Biol ; 1277: 1-31, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33119862

RESUMO

The tumor microenvironment (TME) is decisive for the eradication or survival of any tumor mass. Moreover, it plays a pivotal role for metastasis and for providing the metastatic niche. The TME offers special physiological conditions and is composed of, for example, surrounding blood vessels, the extracellular matrix (ECM), diverse signaling molecules, exosomes and several cell types including, but not being limited to, infiltrated immune cells, cancer-associated endothelial cells (CAEs), and cancer-associated fibroblasts (CAFs). These cells can additionally and significantly contribute to tumor and metastasis progression, especially also by acting via their own deregulated micro (mi) RNA expression or activity. Thus, miRNAs are essential players in the crosstalk between cancer cells and the TME. MiRNAs are small non-coding (nc) RNAs that typically inhibit translation and stability of messenger (m) RNAs, thus being able to regulate several cell functions including proliferation, migration, differentiation, survival, invasion, and several steps of the metastatic cascade. The dynamic interplay between miRNAs in different cell types or organelles such as exosomes, ECM macromolecules, and the TME plays critical roles in many aspects of cancer development. This chapter aims to give an overview on the multiple contributions of miRNAs as players within the TME, to summarize the role of miRNAs in the crosstalk between different cell populations found within the TME, and to illustrate how they act on tumorigenesis and the behavior of cells in the TME context. Lastly, the potential clinical utility of miRNAs for cancer therapy is discussed.


Assuntos
MicroRNAs , Neoplasias/genética , Microambiente Tumoral , Fibroblastos Associados a Câncer , Carcinogênese , Células Endoteliais , Humanos , MicroRNAs/genética
3.
Hepatology ; 68(5): 1817-1832, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-29790588

RESUMO

The identification of viability-associated long noncoding RNAs (lncRNAs) might be a promising rationale for new therapeutic approaches in liver cancer. Here, we applied an RNA interference screening approach in hepatocellular carcinoma (HCC) cell lines to find viability-associated lncRNAs. Among the multiple identified lncRNAs with a significant impact on HCC cell viability, we selected cancer susceptibility 9 (CASC9) due to the strength of its phenotype, expression, and up-regulation in HCC versus normal liver. CASC9 regulated viability across multiple HCC cell lines as shown by clustered regularly interspaced short palindromic repeats interference and single small interfering RNA (siRNA)-mediated and siRNA pool-mediated depletion of CASC9. Further, CASC9 depletion caused an increase in apoptosis and a decrease of proliferation. We identified the RNA binding protein heterogeneous nuclear ribonucleoprotein L (HNRNPL) as a CASC9 interacting protein by RNA affinity purification and validated it by native RNA immunoprecipitation. Knockdown of HNRNPL mimicked the loss-of-viability phenotype observed upon CASC9 depletion. Analysis of the proteome (stable isotope labeling with amino acids in cell culture) of CASC9-depleted and HNRNPL-depleted cells revealed a set of coregulated genes which implied a role of the CASC9:HNRNPL complex in AKT signaling and DNA damage sensing. CASC9 expression levels were elevated in patient-derived tumor samples compared to normal control tissue and had a significant association with overall survival of HCC patients. In a xenograft chicken chorioallantoic membrane model, we measured decreased tumor size after knockdown of CASC9. Conclusion: Taken together, we provide a comprehensive list of viability-associated lncRNAs in HCC; we identified the CASC9:HNRNPL complex as a clinically relevant viability-associated lncRNA/protein complex which affects AKT signaling and DNA damage sensing in HCC.


Assuntos
Carcinoma Hepatocelular/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo L/metabolismo , Neoplasias Hepáticas/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Longo não Codificante/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Galinhas , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/metabolismo , RNA Interferente Pequeno , Transdução de Sinais
4.
Int J Cancer ; 141(9): 1879-1890, 2017 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-28656629

RESUMO

The tumor suppressor P53 is a critical regulator of normal cellular homeostasis whose function is either distorted or lost in several cancer types including colorectal cancer (CRC). A small group of microRNAs have come to be recognized as essential mediators of P53 function. In a genome-wide systematic approach, we explored miRNAs that are substantially altered by P53 loss and found miR-30e to be the most significantly deregulated miRNA in P53-knockout human CRC cells. We identified miR-30e-5p to be a novel direct transcriptional target of P53 with gain and loss of function experiments revealing miR-30e-5p to be a significant regulator of tumor cell migration, invasion and in vivo metastasis mediated in part by integrins alpha-6 and beta-1 as novel targets. MiR-30e-5p also significantly reduced tumor cell proliferation by causing G1/S cell cycle arrest, which was achieved by inducing P21 and P27 expression. Finally, we found miR-30e-5p to be lost in resected CRC tumors as compared to normal colon tissues. Taken together, miR-30e-5p is a novel effector of P53-induced suppression of migration, invasion and metastasis.


Assuntos
Neoplasias Colorretais/genética , Integrina alfa6/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , MicroRNAs/genética , Proteína Supressora de Tumor p53/genética , Proteínas Adaptadoras de Transdução de Sinal , Animais , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Neoplasias Colorretais/patologia , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p27/genética , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Camundongos , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Metástase Neoplásica , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Semin Cancer Biol ; 35: 200-11, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26192966

RESUMO

MicroRNAs now not only represent a significant mechanism for post-transcriptional gene regulation, but have come to be appreciated as molecules with far reaching tentacles affecting diverse processes and pathologies by modulating amongst others, cellular gene expression, epigentic mechanisms, complex signaling cascades, cell-cell communication, the immune system and microenvironmental interactions between several cell types, tissues and organ systems. In this review, we systematically reflect on the impact of miRNAs on all types of benign and malignant human stem cells, looking at the roles they play in maintaining or changing the stem cell state, and review how aberrations of their expression and function within diverse types of stem cells orchestrate carcinogenesis and metastasis. As a conclusion, we consider it striking to see how similar some miR-driven mechanisms are between different types of stem cells and cancer cells, and how this might support hypotheses of miR-driven embryologic pathway reactivation in metastasis or propose putative functions of miRs in important novel cross-topic fields such as obesity and cancer.


Assuntos
Regulação da Expressão Gênica , MicroRNAs/genética , Células-Tronco Neoplásicas/metabolismo , Células-Tronco/metabolismo , Animais , Diferenciação Celular/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Metástase Neoplásica , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Células-Tronco Neoplásicas/patologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Células-Tronco/citologia , Microambiente Tumoral/genética
6.
Org Biomol Chem ; 14(22): 5001-6, 2016 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-27114268

RESUMO

Two molecular beacons were designed as complementary fluorescent imaging probes for miRNA-21 and miRNA-31. Both beacons were prepared by a combination of solid-phase protocol and Cu(i)-catalyzed cycloaddition chemistry. The four photostable and bright fluorophores were attached to 2'-positions in the stem part of the two beacons. One beacon was labeled by a green-to-red emitting and the other by a blue-to-yellow emitting energy transfer pair. This two by two combination yields the four color emission readout. In vitro experiments demonstrate rapid and highly selective opening of both molecular beacons upon addition of the complementary target RNA and excellent green : red and blue : yellow emission color contrasts. Confocal microscopy of selected cancer cell lines provides evidence that a four color imaging of versicular miRNA-21 and miRNA-31 can be achieved both selectively and simultaneously upon transfection by the beacons, and that the fluorescent readouts track well with miRNA levels determined by PCR.


Assuntos
MicroRNAs/metabolismo , Sondas de Oligonucleotídeos/metabolismo , Imagem Óptica/métodos , Linhagem Celular Tumoral , Sobrevivência Celular , Humanos , Espaço Intracelular/metabolismo , Sondas de Oligonucleotídeos/química , Fatores de Tempo
7.
Mol Cancer ; 14: 34, 2015 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-25743773

RESUMO

Cancer is an exceedingly complex disease that is orchestrated and driven by a combination of multiple aberrantly regulated processes. The nature and depth of involvement of individual events vary between cancer types, and in lung cancer, the deregulation of the epigenetic machinery, the tumor microenvironment and the immune system appear to be especially relevant. The contribution of microRNAs to carcinogenesis and cancer progression is well established with many reports and investigations describing the involvement of microRNAs in lung cancer, however most of these studies have concentrated on single microRNA-target relations and have not adequately addressed the complexity of their interactions. In this review, we focus, in part, on the role of microRNAs in the epigenetic regulation of lung cancer where they act as active molecules modulating enzymes that take part in methylation-mediated silencing and chromatin remodeling. Additionally, we highlight their contribution in controlling and modulating the tumor microenvironment and finally, we describe their role in the critical alteration of essential molecules that influence the immune system in lung cancer development and progression.


Assuntos
Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , MicroRNAs/genética , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia , Animais , Humanos , Imunomodulação , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia
8.
Int J Cancer ; 136(9): 2228-40, 2015 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-25303768

RESUMO

Antibody therapy of solid cancers is well established, but suffers from unsatisfactory tumor penetration of large immunoglobulins or from low serum retention of antibody fragments. Oncolytic viruses are in advanced clinical development showing excellent safety, but suboptimal potency due to limited virus spread within tumors. Here, by developing an immunoRNase-encoding oncolytic adenovirus, we combine viral oncolysis with intratumoral genetic delivery of a small antibody-fusion protein for targeted bystander killing of tumor cells (viro-antibody therapy). Specifically, we explore genetic delivery of a small immunoRNase consisting of an EGFR-binding scFv antibody fragment fused to the RNase Onconase (ONC(EGFR)) that induces tumor cell death by RNA degradation after cellular internalization. Onconase is a frog RNase that combines lack of immunogenicity and excellent safety in patients with high tumor killing potency due to its resistance to the human cytosolic RNase inhibitor. We show that ONC(EGFR) expression by oncolytic adenoviruses is feasible with an optimized, replication-dependent gene expression strategy. Virus-encoded ONC(EGFR) induces potent and EGFR-dependent bystander killing of tumor cells. Importantly, the ONC(EGFR)-encoding oncolytic adenovirus showed dramatically increased cytotoxicity specifically to EGFR-positive tumor cells in vitro and significantly enhanced therapeutic activity in a mouse xenograft tumor model. The latter demonstrates that ONC(EGFR) is expressed at levels sufficient to trigger tumor cell killing in vivo. The established ONC(EGFR)-encoding oncolytic adenovirus represents a novel agent for treatment of EGFR-positive tumors. This viro-antibody therapy platform can be further developed for targeted/personalized cancer therapy by exploiting antibody diversity to target further established or emerging tumor markers or combinations thereof.


Assuntos
Adenoviridae/genética , Antineoplásicos/administração & dosagem , Antineoplásicos/metabolismo , Vetores Genéticos/genética , Vírus Oncolíticos/genética , Ribonucleases/administração & dosagem , Ribonucleases/metabolismo , Animais , Anticorpos Antivirais , Linhagem Celular Tumoral , Receptores ErbB/metabolismo , Feminino , Terapia Genética/métodos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Terapia Viral Oncolítica/métodos , RNA/metabolismo , Replicação Viral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
9.
RNA ; 19(12): 1711-25, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24129493

RESUMO

MicroRNAs (miRNAs) have been widely studied in order to elucidate their biological functions. MicroRNA microarrays or miRNA overexpression libraries generated by synthesis and cloning of individual miRNAs have been used to study their different roles. In this work, we have developed a novel methodology to express mature miRNAs and other small RNAs from a double convergent RNA polymerase III promoter. We show that the generated miRNAs function similarly to those processed from primary transcripts or pri-miRNAs. This system allowed us to produce a lentiviral library expressing the whole population of small RNAs present in a metastatic cell line. A functional screening using this library led to the identification of hsa-miR-30b and hsa-miR-30c as negative regulators of cell death induced by loss of attachment (anoikis). Importantly, we demonstrated that the acquisition of anoikis resistance via these miRNAs is achieved through down-regulation of caspase 3 expression. Moreover, overexpression of these miRNAs resulted in a decrease of other types of caspase 3-dependent cell death and enhanced the survival of MCF10A acinar cells in morphogenesis assays, suggesting a putative role as oncomirs. In summary, this novel methodology provides a powerful and effective way for identifying novel small RNAs involved in a particular biological process.


Assuntos
Anoikis/genética , Caspase 3/genética , MicroRNAs/genética , Regiões 3' não Traduzidas , Sequência de Bases , Sítios de Ligação , Caspase 3/metabolismo , Técnicas de Cultura de Células , Forma Celular , Repressão Enzimática , Feminino , Expressão Gênica , Biblioteca Gênica , Células HCT116 , Células HEK293 , Humanos , Glândulas Mamárias Humanas/citologia , MicroRNAs/metabolismo , Morfogênese , Interferência de RNA
10.
Clin Sci (Lond) ; 128(11): 761-74, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25602745

RESUMO

Transforming growth factor ß (TGF-ß) is cytostatic towards damage-induced compensatory hepatocyte proliferation. This function is frequently lost during hepatocarcinogenesis, thereby switching the TGF-ß role from tumour suppressor to tumour promoter. In the present study, we investigate Smad7 overexpression as a pathophysiological mechanism for cytostatic TGF-ß inhibition in liver damage and hepatocellular carcinoma (HCC). Transgenic hepatocyte-specific Smad7 overexpression in damaged liver of fumarylacetoacetate hydrolase (FAH)-deficient mice increased compensatory proliferation of hepatocytes. Similarly, modulation of Smad7 expression changed the sensitivity of Huh7, FLC-4, HLE and HLF HCC cell lines for cytostatic TGF-ß effects. In our cohort of 140 HCC patients, Smad7 transcripts were elevated in 41.4% of HCC samples as compared with adjacent tissue, with significant positive correlation to tumour size, whereas low Smad7 expression levels were significantly associated with worse clinical outcome. Univariate and multivariate analyses indicate Smad7 levels as an independent predictor for overall (P<0.001) and disease-free survival (P=0.0123). Delineating a mechanism for Smad7 transcriptional regulation in HCC, we identified cold-shock Y-box protein-1 (YB-1), a multifunctional transcription factor. YB-1 RNAi reduced TGF-ß-induced and endogenous Smad7 expression in Huh7 and FLC-4 cells respectively. YB-1 and Smad7 mRNA expression levels correlated positively (P<0.0001). Furthermore, nuclear co-localization of Smad7 and YB-1 proteins was present in cancer cells of those patients. In summary, the present study provides a YB-1/Smad7-mediated mechanism that interferes with anti-proliferative/tumour-suppressive TGF-ß actions in a subgroup of HCC cells that may facilitate aspects of tumour progression.


Assuntos
Carcinoma Hepatocelular/genética , Proliferação de Células , Hepatócitos/metabolismo , Hepatopatias/genética , Neoplasias Hepáticas/genética , Proteína Smad7/genética , Animais , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Células Cultivadas , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Hepatopatias/metabolismo , Hepatopatias/patologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Camundongos da Linhagem 129 , Camundongos Knockout , Camundongos Transgênicos , Microscopia Confocal , Pessoa de Meia-Idade , Análise Multivariada , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Smad7/metabolismo , Análise de Sobrevida , Fator de Crescimento Transformador beta/farmacologia , Proteína 1 de Ligação a Y-Box/genética , Proteína 1 de Ligação a Y-Box/metabolismo
11.
Int J Cancer ; 134(12): 2808-19, 2014 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-24285420

RESUMO

The FUS-CHOP fusion protein has been found to be instrumental for specific oncogenic processes in liposarcoma, but its ability to induce metastasis and the underlying mechanisms by which this can be achieved remain unknown. To dissect its functional role in this context, we stably overexpressed this protein in SW872 liposarcoma and HT1080 fibrosarcoma cell lines, and were able to demonstrate that forced expression of FUS-CHOP significantly increases migration and invasion, as well as enhances lung and liver metastasis in the in vivo chicken chorioallantoic membrane (CAM) model, that is proliferation independent. Additionally, FUS-CHOP enhances the expression of matrix-metalloproteinases -2 and -9, and transactivates their promoters in vitro. Mutational analysis showed that C/EBP-ß- (-769/-755), NF-κB (-525/-516) and CREB/AP-1 (-218/-207) sites were important for MMP-2 and NF-κB (-604/-598), AP-1 (-539/-532) and AP-1 (-81/-72) for MMP-9 transactivation. Moreover, a direct in vivo interaction of FUS-CHOP was observed in case of the MMP-2 promoter within region (-769/-207). siRNA data revealed that MMP-2 expression is essential in the FUS-CHOP induced metastatic phenotype. MMP-2-mRNA and protein expression correlated significantly with FUS-CHOP positivity in 46 resected patient liposarcoma tissues. We have for the first time provided substantial evidence for the FUS-CHOP oncoprotein as an inducer of metastasis that is due to the transcriptional induction of specific tumor-associated proteases. Insights gained from this study not only support a deeper understanding of the mechanistic properties of FUS-CHOP, but also open up new avenues for targeted therapy.


Assuntos
Lipossarcoma/patologia , Metaloproteinase 2 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/biossíntese , Proteínas de Fusão Oncogênica/metabolismo , Proteína FUS de Ligação a RNA/metabolismo , Fator de Transcrição CHOP/metabolismo , Células 3T3 , Idoso , Animais , Proteína beta Intensificadora de Ligação a CCAAT/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Galinhas , Membrana Corioalantoide/citologia , Membrana Corioalantoide/patologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Células HEK293 , Humanos , Lipossarcoma/genética , Lipossarcoma/metabolismo , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/genética , Camundongos , Pessoa de Meia-Idade , NF-kappa B/genética , Invasividade Neoplásica , Metástase Neoplásica , Proteínas de Fusão Oncogênica/biossíntese , Proteínas de Fusão Oncogênica/genética , Regiões Promotoras Genéticas/genética , Interferência de RNA , RNA Interferente Pequeno , Proteína FUS de Ligação a RNA/biossíntese , Proteína FUS de Ligação a RNA/genética , Fator de Transcrição AP-1/genética , Fator de Transcrição CHOP/biossíntese , Fator de Transcrição CHOP/genética
12.
J Surg Oncol ; 109(6): 597-605, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24391050

RESUMO

BACKGROUND: Increased activity of Src has been found in several human cancers, and often is associated with poor clinical outcome. The present study aimed to determine whether Src activity is increased in gastrointestinal stromal tumors (GIST), and whether it correlates with established tumor or patient characteristics and prognosis. METHODS: Tumor/normal tissues of 29 patients were analyzed for Src activity/protein with kinase assays, and for VEGF/VEGFR with immunohistochemical staining. RESULTS: Src activity was higher in tumor than in normal tissues (P = 0.093). However, when imatinib responders were excluded from the analyses, it was significantly higher in the tumor tissue (P = 0.017). Additionally, it was higher in primary compared to recurrent tumors or metastasis (P = 0.04). Univariate survival analysis showed a longer overall survival for patients with high Src activity (P = 0.038). In multivariate analysis, the response to imatinib treatment was the only survival-influencing factor (P = 0.072). CONCLUSIONS: Src activity is increased in GIST. In contrast to most other tumor entities, it does not correlate with poor clinical outcome, but decreases during the progression from primary tumor to recurrence and metastasis, especially under therapy with imatinib. Additionally, our results show that higher Src activity is associated with longer overall survival.


Assuntos
Neoplasias Gastrointestinais/metabolismo , Tumores do Estroma Gastrointestinal/metabolismo , Quinases da Família src/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/uso terapêutico , Benzamidas/uso terapêutico , Éxons , Neoplasias Gastrointestinais/tratamento farmacológico , Neoplasias Gastrointestinais/mortalidade , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Tumores do Estroma Gastrointestinal/mortalidade , Humanos , Mesilato de Imatinib , Imuno-Histoquímica , Pessoa de Meia-Idade , Análise Multivariada , Mutação , Metástase Neoplásica , Recidiva Local de Neoplasia/metabolismo , Piperazinas/uso terapêutico , Prognóstico , Proteínas Proto-Oncogênicas c-kit/genética , Pirimidinas/uso terapêutico , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
13.
Cell Mol Life Sci ; 69(3): 435-48, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21710320

RESUMO

Expression of the glycosylphosphatidylinositol-anchored membrane protein CD24 correlates with a poor prognosis for many human cancers, and in experimental tumors can promote metastasis. However, the mechanism by which CD24 contributes to tumor progression remains unclear. Here we report that in MTLy breast cancer cells CD24 interacts with and augments the kinase activity of c-src, a protein strongly implicated in promoting invasion and metastasis. This occurs within and is dependent upon intact lipid rafts. CD24-augmented c-src kinase activity increased formation of focal adhesion complexes, accelerated phosphorylation of FAK and paxillin and consequently enhanced integrin-mediated adhesion. Loss and gain of function approaches showed that c-src activity is necessary and sufficient to mediate the effects of CD24 on integrin-dependent adhesion and cell spreading, as well as on invasion. Together these results indicate that c-src is a CD24-activated mediator that promotes integrin-mediated adhesion and invasion, and suggest a mechanism by which CD24 might contribute to tumor progression through stimulating the activity of c-src or another member of the Src family.


Assuntos
Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Antígeno CD24/metabolismo , Integrinas/metabolismo , Microdomínios da Membrana/enzimologia , Microdomínios da Membrana/metabolismo , Proteínas Tirosina Quinases/metabolismo , Antibacterianos/farmacologia , Neoplasias da Mama/metabolismo , Proteína Tirosina Quinase CSK , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Doxiciclina/farmacologia , Feminino , Fibronectinas/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Humanos , Paxilina/metabolismo , Fosforilação , Ligação Proteica , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirimidinas/farmacologia , Quinases da Família src
14.
Cancers (Basel) ; 15(23)2023 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-38067284

RESUMO

Olaparib suppresses DNA damage repair by inhibiting the poly ADP ribose polymerase (PARP), especially in cancers with BRCA1/2 mutations or the BRCA-ness phenotype. However, the first trials showed that some patients with defective DNA damage repair are still resistant to olaparib. The recovery of the wildtype BRCA is a prominent mechanism of PARP inhibitor (PARPi) resistance in BRCA-deficient tumors, but additional molecular features of olaparib resistance remain poorly understood. The objective of our study was to find molecular parameters that contribute to olaparib response or resistance in CRC. We report that histone acetyltransferase KAT2B decreases BRCA2 expression by reducing the acetylation of the 27th amino acid in histone H3 (H3K27) at the promoter of the BRCA2 gene in colorectal cancer (CRC). This increases the sensitivity of CRC cells toward olaparib treatment. The H3K27ac binding domain of BRCA2 may be required for its transcription. Low endogenous KAT2B expression, which we identify in a subset of cultured BRCA2-expressing CRC cells, leads to an accumulation of γH2AX (more DNA damage), resulting in low PARPi resistance in BRCA-expressing cells. Our results reveal KAT2B and histone acetylation as regulators of BRCA2 expression and PARPi responses in BRCA2-expressing CRC cells, providing further insights into molecular prerequisites for targeting BRCA-functional tumors.

15.
Cancers (Basel) ; 16(1)2023 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-38201452

RESUMO

MiR-494-5p expression has been suggested to be associated with colorectal cancer (CRC) and its metastases in our previous studies. However, functional investigations on the molecule-mediating actions of this miR in CRC are lacking. In silico analysis in the present study revealed a putative binding sequence within the 3'UTR of JAK1. Overexpression of miR-494-5p in cultured CRC significantly reduced the luciferase activity of a reporter plasmid containing the wild-type JAK1-3'UTR, which was abolished by seed sequence mutation. Furthermore, the overexpression of miR-494-5p in CRC cell lines led to a significant reduction in JAK1 expression, proliferation, in vitro migration, and invasion. These effects were abolished by co-transfection with a specific double-stranded RNA that inhibits endogenous miR-494-5p. Moreover, IL-4-induced migration, invasion, and phosphorylation of JAK1, STAT6, and AKT proteins were reduced after an overexpression of this miR, suggesting that this miR affects one of the most essential pathways in CRC. A Kaplan-Meier plotter analysis revealed that patients with high JAK1 expression show reduced survival. Together, these data suggest that miR-494-5p physically inhibits the expression of JAK1 at the translational level as well as in migration and invasion, supporting the hypothesis of miR-494-5p as an early tumor suppressor and inhibitor of early steps of metastasis in CRC.

16.
Cancers (Basel) ; 16(1)2023 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-38201598

RESUMO

Understanding molecular networks of CRLM is an ongoing area of research. In this study, paired CRC tissue and adjacent noncancerous tissue from 15 non-metastatic CRC patients and paired CRC tissue and matched liver metastatic tissues from 15 CRLM patients along with their adjacent noncancerous tissues were evaluated. We assessed Rap1 pathway-related genes including NRAS, FGF-1, NGF, and KDR expression by qRT-PCR and their protein status by Western blot. In CRLM patients, NRAS, FGF1, and KDR mRNA and protein were expressed at higher levels in metastatic than in CRC primary tumor and adjacent noncancerous tissue (p < 0.05). In non-metastatic patients, NRAS, FGF1, KDR, and NGF gene expression did not differ between CRC primary tumor-and adjacent noncancerous tissue (p > 0.05). ROC curve analysis showed a reasonable diagnostic accuracy of NRAS, FGF1, KDR, and FGF for the discrimination of metastatic patients from non- metastatic ones on analysis of their primary tumors. The data suggest that further functional studies on Rap1-related genes' role in CRLM are needed. In conclusion, the present data broaden our knowledge about specific molecular characteristics of CRLM. An increased understanding of the molecular features of metastasis has the potential to create more successful treatment, or prevention, of metastasis, especially in multimodal primary tumor treatment.

17.
J Biol Chem ; 286(6): 4150-64, 2011 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-21118818

RESUMO

MicroRNAs (miRNAs) are an emerging class of non-coding endogenous RNAs involved in multiple cellular processes, including cell differentiation. Treatment with retinoic acid (RA) results in neural differentiation of neuroblastoma cells. We wanted to elucidate whether miRNAs contribute to the gene expression changes induced by RA in neuroblastoma cells and whether miRNA regulation is involved in the transduction of the RA signal. We show here that RA treatment of SH-SY5Y neuroblastoma cells results in profound changes in the expression pattern of miRNAs. Up to 42 different miRNA species significantly changed their expression (26 up-regulated and 16 down-regulated). Among them, the closely related miR-10a and -10b showed the most prominent expression changes. Induction of miR-10a and -10b by RA also could be detected in LA-N-1 neuroblastoma cells. Loss of function experiments demonstrated that miR-10a and -10b are essential mediators of RA-induced neuroblastoma differentiation and of the associated changes in migration, invasion, and in vivo metastasis. In addition, we found that the SR-family splicing factor SFRS1 (SF2/ASF) is a target for miR-10a -and -10b in HeLa and SH-SY5Y neuroblastoma cells. We show here that changes in miR-10a and -10b expression levels may regulate SFRS1-dependent alternative splicing and translational functions. Taken together, our results give support to the idea that miRNA regulation plays a key role in RA-induced neuroblastoma cell differentiation. The discovery of SFRS1 as direct target of miR-10a and -10b supports the emerging functional interaction between two post-transcriptional mechanisms, microRNAs and splicing, in the neuronal differentiation context.


Assuntos
Antineoplásicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Glicoproteínas de Membrana/metabolismo , Neuroblastoma/metabolismo , Proteínas Nucleares/metabolismo , Receptores Imunológicos/metabolismo , Tretinoína/farmacologia , Processamento Alternativo/efeitos dos fármacos , Processamento Alternativo/genética , Animais , Diferenciação Celular/genética , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Embrião de Galinha , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Células HeLa , Humanos , Glicoproteínas de Membrana/genética , Invasividade Neoplásica , Metástase Neoplásica , Neuroblastoma/genética , Neuroblastoma/patologia , Proteínas Nucleares/genética , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/genética , Proteínas de Ligação a RNA , Receptores Imunológicos/genética , Fatores de Processamento de Serina-Arginina
18.
Mol Cancer ; 11: 38, 2012 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-22682314

RESUMO

BACKGROUND: Tri- and tetra-nucleotide repeats in mammalian genomes can induce formation of alternative non-B DNA structures such as triplexes and guanine (G)-quadruplexes. These structures can induce mutagenesis, chromosomal translocations and genomic instability. We wanted to determine if proteins that bind triplex DNA structures are quantitatively or qualitatively different between colorectal tumor and adjacent normal tissue and if this binding activity correlates with patient clinical characteristics. METHODS: Extracts from 63 human colorectal tumor and adjacent normal tissues were examined by gel shifts (EMSA) for triplex DNA-binding proteins, which were correlated with clinicopathological tumor characteristics using the Mann-Whitney U, Spearman's rho, Kaplan-Meier and Mantel-Cox log-rank tests. Biotinylated triplex DNA and streptavidin agarose affinity binding were used to purify triplex-binding proteins in RKO cells. Western blotting and reverse-phase protein array were used to measure protein expression in tissue extracts. RESULTS: Increased triplex DNA-binding activity in tumor extracts correlated significantly with lymphatic disease, metastasis, and reduced overall survival. We identified three multifunctional splicing factors with biotinylated triplex DNA affinity: U2AF65 in cytoplasmic extracts, and PSF and p54nrb in nuclear extracts. Super-shift EMSA with anti-U2AF65 antibodies produced a shifted band of the major EMSA H3 complex, identifying U2AF65 as the protein present in the major EMSA band. U2AF65 expression correlated significantly with EMSA H3 values in all extracts and was higher in extracts from Stage III/IV vs. Stage I/II colon tumors (p=0.024). EMSA H3 values and U2AF65 expression also correlated significantly with GSK3 beta, beta-catenin, and NF- B p65 expression, whereas p54nrb and PSF expression correlated with c-Myc, cyclin D1, and CDK4. EMSA values and expression of all three splicing factors correlated with ErbB1, mTOR, PTEN, and Stat5. Western blots confirmed that full-length and truncated beta-catenin expression correlated with U2AF65 expression in tumor extracts. CONCLUSIONS: Increased triplex DNA-binding activity in vitro correlates with lymph node disease, metastasis, and reduced overall survival in colorectal cancer, and increased U2AF65 expression is associated with total and truncated beta-catenin expression in high-stage colorectal tumors.


Assuntos
Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/mortalidade , Proteínas de Ligação a DNA/metabolismo , Proteômica , Neoplasias Colorretais/patologia , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Linfonodos/patologia , Masculino , Metástase Neoplásica , Estadiamento de Neoplasias , Proteínas Associadas à Matriz Nuclear/genética , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fatores de Transcrição de Octâmero/genética , Fatores de Transcrição de Octâmero/metabolismo , Fator de Processamento Associado a PTB , Ligação Proteica , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , RecQ Helicases/genética , RecQ Helicases/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Fator de Processamento U2AF , Helicase da Síndrome de Werner , beta Catenina/genética , beta Catenina/metabolismo
19.
Int J Cancer ; 130(9): 2044-53, 2012 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-21633953

RESUMO

MicroRNAs (miRNAs) are small non-coding RNAs which regulate gene expression by base-pairing to the 3'-UTR of the target mRNA. Recently, miRNAs have been shown to regulate cancer metastasis, however, central molecular mechanisms of this ability still need to be investigated. Epithelial to mesenchymal transition (EMT), which is characterized especially by repression of E-cadherin expression and increased cell motility, is an essential component of cancer metastasis and progression. In the present study, we found that Snai1, a known transcriptional repressor of E-cadherin and modulator of EMT, is post-transcriptionally targeted by miRNA-30a in non-small cell lung cancer (NSCLC). Consistent with this, microRNA-30a expression was found inversely proportional to the invasive potential of various NSCLC cell lines, correlating positively with E-cadherin (epithelial marker) and negatively with N-cadherin (mesenchymal marker) expression. Forced re-introduction of miR-30a significantly altered cell morphology, in vitro invasion and migration of invasive cell lines, this being paralleled by a downregulation of Snai1 and upregulation of E-cadherin expression. Using a chicken embryonic metastasis assay, we found that miR-30a suppresses in vivo distant metastasis to the lungs and liver. Finally, we screened the expression of miR-30a in 64 consecutively resected NSCLC patients and found that, in 81% of the patients, expression of miR-30a was downregulated significantly (p < 0.0001) in tumors compared to corresponding normal tissues. These results suggest that miR-30a targets Snai1, inhibits invasion and metastasis, and is downregulated in NSCLC.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Transição Epitelial-Mesenquimal/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , MicroRNAs/genética , MicroRNAs/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Caderinas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Movimento Celular , Embrião de Galinha , Regulação Neoplásica da Expressão Gênica , Vetores Genéticos , Humanos , Neoplasias Pulmonares/genética , Invasividade Neoplásica , Metástase Neoplásica , Fatores de Transcrição da Família Snail , Transfecção
20.
Cancers (Basel) ; 14(4)2022 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-35205718

RESUMO

Colorectal cancer (CRC) represents one of the most frequent human cancer entities and is still amongst the "top killers" in human cancer, although fundamental progress has been made in recent years in CRC prevention, early diagnosis, basic and translational research, and (targeted) therapy [...].

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