RESUMO
XAF1 is a tumor suppressor gene with low or absent expression in cancer. Since transcriptional reactivation or ectopic-mediated expression of XAF1 inhibits tumor growth, it is of great interest to elucidate the molecular mechanisms leading to XAF1 silencing. YY1 is a transcription factor that acts as a repressor or an activator to modulate several cancer-associated cellular processes. Both YY1 and XAF1 have key roles in prostate cancer (PCa) progression and are associated with worse clinical outcomes. To assess whether YY1 regulates the transcriptional activation of the XAF1 gene, we performed gene-reporter assays coupled with site-directed mutagenesis, which showed that YY1 is able to mediate XAF1 silencing. Concordantly, ChIP-qPCR assays showed that YY1 interacts with the XAF1 promoter in PC3 cells that lacks XAF1 expression. This association was lost after exposure to epigenetic modulators that induce XAF1 expression. Further supporting the YY1's repressive role, we found transcriptional reactivation of the XAF1 gene by YY1 downregulation. As expected by previous reports showing that HDAC1 is needed for YY1-mediated repressive actions, we observed XAF1 re-expression after either inhibition or downregulation of the HDAC1 gene. Finally, expression data retrieved from the TCGA consortium showed that PCa samples presented lower XAF1 and higher HDAC expression levels than normal tissues. Thus, our results support a model in which YY1 is able to silence tumor suppressor genes such as XAF1 through HDAC1 in PCa.
Assuntos
Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Neoplasias/genética , Neoplasias da Próstata/genética , Fator de Transcrição YY1/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Reguladoras de Apoptose , Sítios de Ligação , Linhagem Celular Tumoral , Histona Desacetilase 1/genética , Histona Desacetilase 1/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Masculino , Proteínas de Neoplasias/biossíntese , Regiões Promotoras Genéticas , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/metabolismo , Proteínas Repressoras/metabolismo , Fator de Transcrição YY1/genéticaRESUMO
MicroRNAs (miRNAs) are known to mediate post-transcriptional gene silencing in the cytoplasm and recent evidence indicates that may also possess nuclear roles in regulating gene expression. A previous study showed that miR-138 is involved in the multidrug resistance of leukemia cells through down-regulation of the drug efflux pump P-glycoprotein (P-gp), the protein encoded by the human multidrug-resistant ABCB1/MDR1 gene. However, the transcriptional regulatory mechanisms responsible remain to be elucidated. To deepen the description of the mechanism of transcriptional gene silencing on the MDR1 promoter, we initially performed a bioinformatics search for potential miR-138 binding sites in the MDR1 gene promoter sequence. Interestingly, we did not find miR-138 binding sites in this region, suggesting an indirect regulation. From six representative transcriptional factors involved in MDR1 gene regulation, an in silico analysis revealed that NF-κB/p65 has a specific binding site for miR-138. The results of luciferase reporter assay, western blot and flow cytometry shown here suggest that miR-138 might modulate the human MDR1 expression by inhibiting NF-κB/p65 as an indirect mechanism of MDR1 regulation. Furthermore, employing the human macrophage-like cell line U937 we observed comparable results with NF-κB/p65 down-regulation and we also observed a significant reduction in the IL-6 and TNF-α mRNA, as well as in their secreted pro-inflammatory cytokines following miR-138 expression, suggesting that canonical NF-κB target genes might also be potential targets for miR-138 in leukemia cells.
Assuntos
Proteínas de Transporte/genética , MicroRNAs/genética , NF-kappa B/genética , Proteínas de Neoplasias/genética , Regiões Promotoras Genéticas/genética , Interferência de RNA/fisiologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Células Cultivadas , Simulação por Computador , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Modelos GenéticosRESUMO
BACKGROUND: Cancer stem cells (CSCs) are regulated by several signaling pathways that ultimately control their maintenance and expansion. NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) forms a protein complex that controls DNA transcription and, as such, plays an important role in proliferation, inflammation, angiogenesis, invasion and metastasis. The NF-κB signaling pathway, which has been found to be constitutively activated in CSCs from a variety of cancers, participates in the maintenance, expansion, proliferation and survival of CSCs. Targeted disruption of this pathway may profoundly impair the adverse phenotype of CSCs and may provide a therapeutic opportunity to remove the CSC fraction. In particular, it may be attractive to use specific NF-κB inhibitors in chronic therapeutic schemes to reduce disease progression. Exceptional low toxicity profiles of these inhibitors are a prerequisite for use in combined treatment regimens and to avoid resistance. CONCLUSION: Although still preliminary, recent evidence shows that such targeted strategies may be useful in adjuvant chemo-preventive settings.
