RESUMO
Rhabdomyosarcoma (RMS) is the most common type of pediatric soft tissue sarcoma. It is classified into two main subtypes: embryonal (eRMS) and alveolar (aRMS). MYC family proteins are frequently highly expressed in RMS tumors, with the highest levels correlated with poor prognosis. A pharmacological approach to inhibit MYC in cancer cells is represented by Bromodomain and Extra-Terminal motif (BET) protein inhibitors. In this paper, we evaluated the effects of BET inhibitor (+)-JQ1 (JQ1) on the viability of aRMS and eRMS cells. Interestingly, we found that the drug sensitivity of RMS cell lines to JQ1 was directly proportional to the expression of MYC. JQ1 induces G1 arrest in cells with the highest steady-state levels of MYC, whereas apoptosis is associated with MYC downregulation. These findings suggest BET inhibition as an effective strategy for the treatment of RMS alone or in combination with other drugs.
Assuntos
Azepinas , Rabdomiossarcoma , Apoptose , Azepinas/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Criança , Humanos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Rabdomiossarcoma/tratamento farmacológico , Fatores de Transcrição/metabolismo , Triazóis/farmacologiaRESUMO
In this study, the immunomodulatory effects of a sequential micro-immunotherapy medicine, referred as MIM-seq, were appraised in human primary M1 and M2 macrophages, in which the secretion of pro-inflammatory cytokines, such as interleukin (IL)-1ß, IL-6, IL-12, IL-23, and tumor necrosis factor (TNF)-alpha, was inhibited. In addition, the potential anti-proliferative effects of MIM-seq on tumor cells was assessed in three models of colorectal cancer (CRC): an in vitro two-dimensions (2D) model of HCT-116 cells, an in vitro tri-dimensional (3D) model of spheroids, and an in vivo model of subcutaneous xenografted mice. In these models, MIM-seq displayed anti-proliferative effects when compared with the vehicle. In vivo, the tumor growth was slightly reduced in MIM-seq-treated animals. Moreover, MIM-seq could slightly reduce the growth of our spheroid models, especially under serum-deprivation. When MIM-seq was combined with two well-known anti-cancerogenic agents, either resveratrol or etoposide, MIM-seq could even further reduce the spheroid's volume, pointing up the need to further assess whether MIM-seq could be beneficial for CRC patients as an adjuvant therapy. Altogether, these data suggest that MIM-seq could have anti-tumor properties against CRC and an immunomodulatory effect towards the mediators of inflammation, whose systemic dysregulation is considered to be a poor prognosis for patients.
Assuntos
Carcinoma , Neoplasias do Colo , Animais , Neoplasias do Colo/tratamento farmacológico , Modelos Animais de Doenças , Xenoenxertos , Humanos , Fatores Imunológicos/farmacologia , Imunoterapia , Macrófagos , Camundongos , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Burkitt lymphoma is a very aggressive B-cell non-Hodgkin lymphoma. Although remarkable progress has been made in the therapeutic scenario for patients with Burkitt lymphoma, search and development of new effective anticancer agents to improve patient outcome and minimize toxicity has become an urgent issue. In this study, the antitumoral activity of Inula viscosa, a traditional herb obtained from plants collected on the Asinara Island, Italy, was evaluated in order to explore potential antineoplastic effects of its metabolites on Burkitt lymphoma. Raji human cell line was treated with increasing Inula viscosa extract concentration for cytotoxicity screening and subsequent establishment of cell cycle arrest and apoptosis. Moreover, gene expression profiles were performed to identify molecular mechanisms involved in the anticancer activities of this medical plant. The Inula viscosa extract exhibited powerful antiproliferative and cytotoxic activities on Raji cell line, showing a dose- and time-dependent decrease in cell viability, obtained by cell cycle arrest in the G2/M phase and an increase in cell apoptosis. The treatment with Inula viscosa caused downregulation of genes involved in cell cycle and proliferation (c-MYC, CCND1) and inhibition of cell apoptosis (BCL2, BCL2L1, BCL11A). The Inula viscosa extract causes strong anticancer effects on Burkitt lymphoma cell line. The molecular mechanisms underlying such antineoplastic activity are based on targeting and downregulation of genes involved in cell cycle and apoptosis. Our data suggest that Inula viscosa natural metabolites should be further exploited as potential antineoplastic agents against Burkitt lymphoma.
Assuntos
Linfoma de Burkitt/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Inula/química , Proteínas de Neoplasias/genética , Apoptose/efeitos dos fármacos , Linfoma de Burkitt/genética , Linfoma de Burkitt/patologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Extratos Vegetais/química , Extratos Vegetais/farmacologiaRESUMO
Small cell lung cancer (SCLC) is an aggressive type of lung cancer with high mortality that is caused by frequent relapses and acquired resistance. Despite that several target-based approaches with potential therapeutic impact on SCLC have been identified, numerous targeted drugs have not been successful in providing improvements in cancer patients when used as single agents. A combination of targeted therapies could be a strategy to induce maximum lethal effects on cancer cells. As a starting point in the development of new drug combination strategies for the treatment of SCLC, we performed a mid-throughput screening assay by treating a panel of SCLC cell lines with BETi or AKi in combination with PARPi or EZH2i. We observed drug synergy between I-BET762 and Talazoparib, BETi and PARPi, respectively, in SCLC cells. Combinatorial efficacy was observed in MYCs-amplified and MYCs-wt SCLC cells over SCLC cells with impaired MYC signaling pathway or non-tumor cells. We indicate that drug synergy between I-BET762 and Talazoparib is associated with the attenuation HR-DSBR process and the downregulation of various players of DNA damage response by BET inhibition, such as CHEK2, PTEN, NBN, and FANCC. Our results provide a rationale for the development of new combinatorial strategies for the treatment of SCLC.
Assuntos
Antineoplásicos/farmacologia , Benzodiazepinas/farmacologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Ftalazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Linhagem Celular Tumoral , Células Cultivadas , Dano ao DNA/efeitos dos fármacos , Sinergismo Farmacológico , HumanosRESUMO
Iodine deficiency remains a major public health concern in many countries, including some European regions. This study aimed at understanding the effect of a supplement of marine alga Ascophyllum nodosum as a iodine fortifier in the cow diet, on the compositional and microbiological quality of milk. The results obtained in this work indicated that the dietary inclusion of A. nodosum exerted significant effects on cow milk composition. In particular, it increased iodine content and reduced the quantity of free amino acids without modifying the free fatty acid content. From a microbiological point of view, statistically significant differences were found in presumptive mesophilic lactobacilli, mesophilic lactococci, and Pseudomonas spp. counts. Based on a culture-independent method, milk obtained after dietary inclusion of A. nodosum harbored the highest number of Firmicutes (e.g., Lactococcus lactis) and the lowest number of Proteobacteria (e.g., Pseudomonas). In addition to changes in bacterial population, diet supplementation with A. nodosum changed the catabolic profiles of the milk community, according to Biolog Ecoplate (Biolog Inc., Hayward, CA) results. The results of this study suggest that the dietary inclusion of the marine alga A. nodosum led to an improvement of the iodine content in milk, and to a modification of its microbiota with a positive effect on milk hygiene and transformation.
Assuntos
Ascophyllum , Dieta/veterinária , Microbiota , Leite/química , Leite/microbiologia , Aminoácidos/análise , Ração Animal/análise , Animais , Bovinos , Contagem de Colônia Microbiana , DNA Bacteriano/genética , Ácidos Graxos não Esterificados/análise , Feminino , Contaminação de Alimentos , Microbiologia de Alimentos , Qualidade dos Alimentos , Iodo/administração & dosagem , Lactação , Lactobacillus/isolamento & purificação , Lactococcus/isolamento & purificação , Proteobactérias/isolamento & purificação , Pseudomonas/isolamento & purificação , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
Multi-drug resistance (MDR) is a serious challenge in contemporary clinical practice and is mostly responsible for the failure of cancer medication therapies. Several experimental evidence links MDR to the overexpression of the drug efflux transporter P-gp, therefore, the discovery of novel P-glycoprotein inhibitors is required to treat or prevent MDR and to improve the absorption of chemotherapy drugs via the gastrointestinal system. In this work, we explored a series of novel pyridoquinoxaline-based derivatives designed from parental compounds, previously proved active in enhancing anticancer drugs in MDR nasopharyngeal carcinoma (KB). Among them, derivative 10d showed the most potent and selective inhibition of fluorescent dye efflux, if compared to reference compounds (MK-571, Novobiocin, Verapamil), and the highest MDR reversal activity when co-administered with the chemotherapeutic agents Vincristine and Etoposide, at non-cytotoxic concentrations. Molecular modelling predicted the two compound 10d binding mode in a ratio of 2:1 with the target protein. No cytotoxicity was observed in healthy microglia cells and off-target investigations showed the absence of CaV1.2 channel blockade. In summary, our findings indicated that 10d could potentially be a novel therapeutic coadjutant by inhibiting P-gp transport function in vitro, thereby reversing cancer multidrug resistance.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Antineoplásicos , Descoberta de Drogas , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Quinoxalinas , Humanos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Quinoxalinas/farmacologia , Quinoxalinas/química , Quinoxalinas/síntese química , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Relação Estrutura-Atividade , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Estrutura Molecular , Ensaios de Seleção de Medicamentos Antitumorais , Relação Dose-Resposta a Droga , Proliferação de Células/efeitos dos fármacos , Modelos MolecularesRESUMO
RB family members are negative regulators of the cell cycle, involved in numerous biological processes such as cellular senescence, development and differentiation. Disruption of RB family pathways are linked to loss of cell cycle control, cellular immortalization and cancer. RB family, and in particular the most studied member RB/p105, has been considered a tumor suppressor gene by more than three decades, and numerous efforts have been done to understand his molecular activity. However, the epigenetic mechanisms behind Rb-mediated tumor suppression have been uncovered only in recent years. In this review, the role of RB family members in cancer epigenetics will be discussed. We start with an introduction to epigenomes, chromatin modifications and cancer epigenetics. In order to provide a clear picture of the involvement of RB family in the epigenetic field, we describe the RB family role in the epigenetic landscape dynamics based on the heterochromatin variety involved, facultative or constitutive. We want to stress that, despite dissimilar modulations, RB family is involved in both mammalian varieties of heterochromatin establishment and maintenance and that disruption of RB family pathways drives to alterations of both heterochromatin structures, thus to the global epigenetic landscape.
Assuntos
Epigênese Genética , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Cromatina/genética , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina/genética , Montagem e Desmontagem da Cromatina/fisiologia , Metilação de DNA/genética , Metilação de DNA/fisiologia , Feminino , Histonas/metabolismo , Humanos , Camundongos , Neoplasias/genética , Neoplasias/metabolismo , Processamento de Proteína Pós-Traducional/genética , Processamento de Proteína Pós-Traducional/fisiologiaRESUMO
CTCF is an evolutionary conserved and ubiquitously expressed protein that binds thousands of sites in the human genome. Ectopic expression of CTCF in various normal and tumoral human cell lines inhibits cell division and clonogenicity, with the consequence to consider CTCF a potential tumor-suppressor factor. In this review article, we focused on the molecular mechanisms engaged by CTCF to modulate the expression of several key-regulators of differentiation, cellular senescence, cell cycle control and progression, whose expression is frequently altered in tumors. Moreover, we discussed common features of CTCF at each tumor-related DNA-binding sequence, such as protein-partners, post-translational modifications, and distinctive epigenetic marks establishment. The investigation of the molecular mechanisms engaged by CTCF to modulate tumor-related genes emphasizes the cell-type dependency of its tumor suppressor role. Indeed, the ability of CTCF to bind their promoters strictly depends by cell-type features as DNA methylation, BORIS-binding and post-translational modifications as PARYlation.
Assuntos
Regulação da Expressão Gênica/fisiologia , Proteínas Repressoras/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Fator de Ligação a CCCTC , DNA , Humanos , Ligação Proteica , Proteínas Repressoras/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
(1) Tomentosin is the most representative sesquiterpene lactone extracted by I. viscosa. Recently, it has gained particular attention in therapeutic oncologic fields due to its anti-tumor properties. (2) In this study, the potential anticancer features of tomentosin were evaluated on human Burkitt's lymphoma (BL) cell line, treated with increasing tomentosin concentration for cytotoxicity screening. (3) Our data showed that both cell cycle arrest and cell apoptosis induction are responsible of the antiproliferative effects of tomentosin and may end in the inhibition of BL cell viability. Moreover, a microarray gene expression profile was performed to assess differentially expressed genes contributing to tomentosin activity. Seventy-five genes deregulated by tomentosin have been identified. Downregulated genes are enriched in immune-system pathways, and PI3K/AKT and JAK/STAT pathways which favor proliferation and growth processes. Importantly, different deregulated genes identified in tomentosin-treated BL cells are prevalent in molecular pathways known to lead to cellular death, specifically by apoptosis. Tomentosin-treatment in BL cells induces the downregulation of antiapoptotic genes such as BCL2A1 and CDKN1A and upregulation of the proapoptotic PMAIP1 gene. (4) Overall, our results suggest that tomentosin could be taken into consideration as a potential natural product with limited toxicity and relevant anti-tumoral activity in the therapeutic options available to BL patients.
RESUMO
Multiple myeloma (MM) is an aggressive B cell malignancy. Substantial progress has been made in the therapeutic context for patients with MM, however it still represents an incurable disease due to drug resistance and recurrence. Development of more effective or synergistic therapeutic approaches undoubtedly represents an unmet clinical need. Tomentosin is a bioactive natural sesquiterpene lactone extracted by various plants with therapeutic properties, including antineoplastic effects. In the present study, the potential antitumor activity of tomentosin was evaluated on the human RPMI8226 cell line, treated with increasing tomentosin concentration for cytotoxicity screening. The data suggested that both cell cycle arrest and cell apoptosis could explain the antiproliferative effects of tomentosin and may result in the inhibition of RPMI8226 cell viability. To assess differentially expressed genes contributing to tomentosin activity and identify its mechanism of action, a microarray gene expression profile was performed, identifying 126 genes deregulated by tomentosin. To address the systems biology and identify how tomentosin deregulates gene expression in MM from a systems perspective, all deregulated genes were submitted to enrichment and molecular network analysis. The ProteinProtein Interaction (PPI) network analysis showed that tomentosin in human MM induced the downregulation of genes involved in several pathways known to lead immunesystem processes, such as cytokinecytokine receptor interaction, chemokine or NFκB signaling pathway, as well as genes involved in pathways playing a central role in cellular neoplastic processes, such as growth, proliferation, migration, invasion and apoptosis. Tomentosin also induced endoplasmic reticulum stress via upregulation of cyclic AMPdependent transcription factor ATF4 and DNA damageinducible transcript 3 protein genes, suggesting that in the presence of tomentosin the protective unfolded protein response signaling may induce cell apoptosis. The functional connections analysis executed using the Connectivity Map tool, suggested that the effects of tomentosin on RPMI8226 cells might be similar to those exerted by heat shock proteins inhibitors. Taken together, these data suggested that tomentosin may be a potential drug candidate for the treatment of MM.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Lactonas/farmacologia , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Sesquiterpenos/farmacologia , Apoptose/genética , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Mieloma Múltiplo/patologia , Mapas de Interação de Proteínas/efeitos dos fármacos , Mapas de Interação de Proteínas/genética , Sesquiterpenos/químicaRESUMO
Effectively treating KRAS-driven tumors remains an unsolved challenge. The inhibition of downstream signaling effectors is a way of overcoming the issue of direct targeting of mutant KRAS, which has shown limited efficacy so far. Bromodomain and Extra-Terminal (BET) protein inhibition has displayed anti-tumor activity in a wide range of cancers, including KRAS-driven malignancies. Here, we preclinically evaluate the effect of BET inhibition making use of a new BET inhibitor, BAY 1238097, against Pancreatic Ductal Adenocarcinoma (PDAC) and Non-Small Cell Lung Cancer (NSCLC) models harboring RAS mutations both in vivo and in vitro. Our results demonstrate that BET inhibition displays significant therapeutic impact in genetic mouse models of KRAS-driven PDAC and NSCLC, reducing both tumor area and tumor grade. The same approach also causes a significant reduction in cell number of a panel of RAS-mutated human cancer cell lines (8 PDAC and 6 NSCLC). In this context, we demonstrate that while BET inhibition by BAY 1238097 decreases MYC expression in some cell lines, at least in PDAC cells its anti-tumorigenic effect is independent of MYC regulation. Together, these studies reinforce the use of BET inhibition and prompt the optimization of more efficient and less toxic BET inhibitors for the treatment of KRAS-driven malignancies, which are in urgent therapeutic need.
RESUMO
Lentiviral vectors are powerful tools for gene expression studies. Here we report the construction of pTIJ, a vector for inducible gene expression. pTIJ was generated from pTRIPZ backbone, which is designed for the inducible expression of shRNA sequences, by the introducing of a multiple cloning site upstream of the Tet promoter and the removal of miR30 flanking sequences. To evaluate pTIJ as a tool for the inducible expression of genes of interest, we introduced MYC cDNA into pTIJ and infected two small cell lung cancer cell lines, H209 and H345. Induction of MYC expression by doxycycline was detectable in both cell lines by real-time PCR and western blot analysis. This study highlights the relevance of pTIJ vector to allow the inducible expression of any gene of interest. In our belief, pTIJ will be an extremely useful tool to simplify the generation of genetically engineered cell lines for the inducible expression of cDNA sequences in biological studies. Furthermore, we report the generation of a pTIJ-MYC vector for the inducible expression of the oncogene MYC.
Assuntos
Vetores Genéticos/genética , Lentivirus/genética , Western Blotting , Linhagem Celular Tumoral , DNA Complementar/genética , Humanos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
We aimed to elucidate the effect of JQ1, a BET inhibitor, on small cell lung cancers (SCLCs) with MYCL amplification and/or expression. Fourteen SCLC cell lines, including four with MYCL amplification, were examined for the effects of JQ1 on protein and gene expression by Western blot and mRNA microarray analyses. The sensitivity of SCLC cells to JQ1 was assessed by cell growth and apoptosis assays. MYCL was expressed in all the 14 cell lines, whereas MYC/MYCN expression was restricted mostly to cell lines with gene amplification. ASCL1, a transcription factor shown to play a role in SCLC, was also expressed in 11/14 cell lines. All SCLC cell lines were sensitive to JQ1 with GI50 values ≤1.23 µM, with six of them showing GI50 values <0.1 µM. Expression of MYCL as well as MYCN, ASCL1 and other driver oncogenes including CDK6 was reduced by JQ1 treatment, in particular in the cell lines with high expression of the respective genes; however, no association was observed between the sensitivity to JQ1 and the levels of MYCL, MYCN and ASCL1 expression. In contrast, levels of CDK6 expression and its reduction rates by JQ1 were associated with JQ1 sensitivity. Therefore, we concluded that CDK6 is a novel target of JQ1 and predictive marker for JQ1 sensitivity in SCLC cells.
Assuntos
Antineoplásicos/farmacologia , Azepinas/farmacologia , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogênicas c-myc/genética , Carcinoma de Pequenas Células do Pulmão/genética , Triazóis/farmacologia , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quinase 6 Dependente de Ciclina/genética , Quinase 6 Dependente de Ciclina/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Amplificação de Genes , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/patologia , TranscriptomaRESUMO
Small cell lung cancer (SCLC) is the most aggressive type of lung cancer with high mortality. One of the MYC family genes, MYC, MYCL or MYCN, is amplified in ~20% of the SCLCs; therefore, MYC proteins are potential therapeutic targets in SCLC patients. We investigated the therapeutic impact of Omomyc, a MYC dominant negative, in a panel of SCLC cell lines. Strikingly, Omomyc suppressed the growth of all tested cell lines by inducing cell cycle arrest and/or apoptosis. Induction of G1 arrest by Omomyc was found to be dependent on the activation of CDKN1A, in part, through the TP73 pathway. Our results strongly indicate that SCLC cells carrying amplification of MYC, MYCL or MYCN are addicted to MYC function, suggesting that MYC targeting would be an efficient therapeutic option for SCLC patients.
Assuntos
Neoplasias Pulmonares/genética , Neoplasias Pulmonares/terapia , Fragmentos de Peptídeos/biossíntese , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/biossíntese , Proteínas de Ligação a Retinoblastoma/genética , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Pequenas Células do Pulmão/terapia , Proteína Supressora de Tumor p53/genética , Ubiquitina-Proteína Ligases/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Pontos de Checagem do Ciclo Celular/genética , Morte Celular/genética , Processos de Crescimento Celular/genética , Amplificação de Genes , Inativação Gênica , Genes p53 , Terapia Genética/métodos , Células HEK293 , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Terapia de Alvo Molecular , Fragmentos de Peptídeos/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas de Ligação a Retinoblastoma/metabolismo , Carcinoma de Pequenas Células do Pulmão/metabolismo , Carcinoma de Pequenas Células do Pulmão/patologia , Transfecção , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Rhabdomyosarcoma (RMS) is a pediatric tumor that arises from muscle precursor cells. RMS cells express several markers of early myogenic differentiation, but they fail to complete both differentiation program and cell cycle arrest, resulting in uncontrolled proliferation and incomplete myogenesis. Previous studies showed that EZH2, which is involved in both differentiation and cancer progression, is overexpressed in RMS, but a functional binding between its expression and its functional role in tumor formation or progression has not yet been demonstrated. We hypothesized that EZH2 is a key regulator of muscular differentiation program in RMS cells. In this study, we demonstrated that EZH2 directly binds muscle specific genes in RD cells. Silencing of EZH2 promotes the recruitment of a multiprotein complex at muscle-specific promoters, their transcriptional activation and protein expression. Moreover, we demonstrated that EZH2 is directly involved in transcriptional repression of MyoD, the main factor promoting myogenesis. EZH2 ablation induces MyoD activation the recovery of its binding on muscle-specific genes.
Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias Musculares/genética , Proteína MyoD/genética , Mioblastos/metabolismo , Complexo Repressor Polycomb 2/antagonistas & inibidores , Complexo Repressor Polycomb 2/genética , Rabdomiossarcoma/genética , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Criança , Proteína Potenciadora do Homólogo 2 de Zeste , Técnicas de Silenciamento de Genes , Vetores Genéticos , Humanos , Lentivirus , Camundongos , Neoplasias Musculares/metabolismo , Neoplasias Musculares/patologia , Proteína MyoD/metabolismo , Mioblastos/patologia , Complexo Repressor Polycomb 2/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Rabdomiossarcoma/metabolismo , Rabdomiossarcoma/patologia , Ativação TranscricionalRESUMO
Although innumerable investigations regarding the biology of lung cancer have been carried out, many aspects thereof remain to be addressed, including the role played by the retinoblastoma-related protein Rb2/p130 during the evolution of this disease. Here we report novel findings on the mechanisms that control Rb2/p130 gene expression in lung fibroblasts and characterize the effects of Rb2/p130 deregulation on the proliferative features of lung cancer cells. We revealed for the first time that in lung fibroblasts the expression of Rb2/p130 gene is directly controlled by the chromatin insulator CCCTC-binding factor, CTCF, which by binding to the Rb2/p130 gene promoter induces, and/or maintains, a specific local chromatin organization that in turn governs the transcriptional activity of Rb2/p130 gene. However, in lung cancer cells the activity of CTCF in controlling Rb2/p130 gene expression is impaired by BORIS, a CTCF-paralogue, which by binding to the Rb2/p130 gene could trigger changes in the chromatin asset established by CTCF, thereby affecting CTCF regulatory activity on Rb2/p130 transcription. These studies not only provide essential basic insights into the molecular mechanisms that control Rb2/p130 gene expression in lung cancer, but also offer a potential paradigm for the actions of other activators and/or corepressors, such as CTCF and BORIS, that could be crucial in explaining how alterations in the mechanism regulating Rb2/p130 gene expression may accelerate the progression of lung tumors, or favor the onset of recurrence after cancer treatment.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Proteínas Repressoras/metabolismo , Proteína p130 Retinoblastoma-Like/genética , Transcrição Gênica , Sítios de Ligação , Fator de Ligação a CCCTC , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma de Células Pequenas/genética , Carcinoma de Células Pequenas/patologia , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Posicionamento Cromossômico/genética , Fibroblastos/metabolismo , Humanos , Neoplasias Pulmonares/patologia , Fases de Leitura Aberta/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteína p130 Retinoblastoma-Like/metabolismoRESUMO
A prospective study was performed to assess the relationship between human papillomavirus (HPV) infection in 199 infertile couples and outcome of assisted reproductive technologies (ARTs). A highly statistically significant correlation between pregnancy loss rate (proportion of pregnancies detected by ß-hCG that did not progress beyond 20 weeks) and positive HPV DNA testing in the male partner of infertile couples, compared with HPV negatives, was observed (66.7% vs. 15%).