RESUMO
Signal transducer and activator of transcription 3 (STAT3) is an important transcription factor involved in many physiological functions including embryonic development and immune responses and is often activated under pathological conditions such as cancer. Strategies to inactivate STAT3 are being pursued as potential anticancer therapies and have led to the identification of Stattic (6-nitrobenzo[b]thiophene-1,1-dioxide) as a "specific" STAT3 inhibitor that is often used to interrogate STAT3-mediated gene expression in vitro and in vivo. Here, we show that Stattic exerts many STAT3-independent effects on cancer cells, calling for reassessment of results previously ascribed to STAT3 functions. Studies of the STAT3-deficient prostate cancer cell line PC-3 (PC3) along with STAT3-proficient breast cancer cell lines (MDA-MB-231, SUM149) revealed that Stattic attenuated histone acetylation and neutralized effects of the histone deacetylase (HDAC) inhibitor romidepsin. In PC3 cells, Stattic alone inhibited gene expression of CCL20 and CCL2, but activated expression of TNFA, CEBPD, SOX2, and MYC. In addition, we found that Stattic promoted autophagy and caused cell death. These data point to profound epigenetic effects of Stattic that are independent of its function as a STAT3 inhibitor. Our results demonstrate that Stattic directly or indirectly reduces histone acetylation and suggest reevaluation of Stattic and related compounds as polypharmacological agents through multipronged cytotoxic effects on cancer cells.
Assuntos
Antineoplásicos/farmacologia , Óxidos S-Cíclicos/farmacologia , Regulação Neoplásica da Expressão Gênica , Histonas/genética , Processamento de Proteína Pós-Traducional , Fator de Transcrição STAT3/genética , Acetilação/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Autofagia/genética , Proteína delta de Ligação ao Facilitador CCAAT/agonistas , Proteína delta de Ligação ao Facilitador CCAAT/genética , Proteína delta de Ligação ao Facilitador CCAAT/metabolismo , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Linhagem Celular Tumoral , Quimiocina CCL2/antagonistas & inibidores , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Quimiocina CCL20/antagonistas & inibidores , Quimiocina CCL20/genética , Quimiocina CCL20/metabolismo , Feminino , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Histonas/antagonistas & inibidores , Histonas/metabolismo , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Células PC-3 , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-myc/agonistas , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição SOXB1/agonistas , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/agonistas , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Proteína Vermelha FluorescenteRESUMO
Uncontrolled proliferation and defective apoptosis are two major factors responsible for maintaining the malignant properties of melanoma cells. Our previous study demonstrated that induced expression of four reprogramming factors remodeled the phenotype of B16F10 mouse melanoma cells into melanoma stem cells. The present study was conducted to investigate the effect of the four Yamanaka reprogramming factors, namely Oct4, Sox2, Klf4 and cMyc (OSKM), on the proliferation and apoptosis of melanoma cells, and to identify the responsible molecular signals. The results identified that expression of the four reprogramming factors was highly induced by doxycycline treatment in the stable melanoma cell clone that was transfected with a plasmid expressing these factors, driven by the TetOn element. It was further confirmed that induced expression of these factors enhanced the proliferation and suppressed the apoptosis of the melanoma cells. In addition, induced OSKM expression increased cell proliferation, accelerated the progression of the cell cycle, and upregulated the mRNA expression levels of Janus kinase 2 (JAK2) and CyclinB1. Induced expression of these factors also decreased the apoptosis, as well as upregulated Bcell lymphoma 2 (BCL2) and downregulated BCL2associated X (BAX) mRNA expression levels. Taken together, the results suggested that upregulated JAK2 and CyclinB1 may be responsible for the enhanced proliferation of melanoma cells, and that BCL2 upregulation and BAX downregulation may account for the suppressed apoptosis of these cells.
Assuntos
Reprogramação Celular , Doxiciclina/farmacologia , Regulação Neoplásica da Expressão Gênica , Fatores de Transcrição Kruppel-Like/genética , Fator 3 de Transcrição de Octâmero/genética , Proteínas Proto-Oncogênicas c-myc/genética , Fatores de Transcrição SOXB1/genética , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclina B1/genética , Ciclina B1/metabolismo , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/agonistas , Fatores de Transcrição Kruppel-Like/metabolismo , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fator 3 de Transcrição de Octâmero/agonistas , Fator 3 de Transcrição de Octâmero/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-myc/agonistas , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição SOXB1/agonistas , Fatores de Transcrição SOXB1/metabolismo , Transfecção , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
Prenatal folic acid (FA) supplementation prevents neural tube defects. Folate receptor alpha (FRα) is critical for embryonic development, including neural crest (NC) development. Previously we showed that FRα translocates to the nucleus in response to FA, where it acts as a transcription factor. In this study, we examined if FA through interaction with FRα regulates stem cell characteristics of cranial neural crest cells (CNCCs)-critical for normal development. We hypothesized that FRα upregulates coding genes and simultaneously downregulates non-coding miRNA which targets coding genes in CNCCs. Quantitative RT-PCR and chromatin immunoprecipitation showed that FRα upregulates Oct4, Sox2, and Klf4 by binding to their cis-regulator elements-5' enhancer/promoters defined by H3K27Ac and p300 occupancy. FA via FRα downregulates miRNAs, miR-138 and miR-let-7, which target Oct4 and Trim71 (an Oct4 downstream effector), respectively. Co-immunoprecipitation data suggests that FRα interacts with the Drosha-DGCR8 complex to affect pre-miRNA processing. Transfecting anti-miR-138 or anti-miR-let-7 into non-proliferating neural crest cells (NCCs) derived from Splotch (Sp-/- ), restored their proliferation potential. In summary, these results suggest a novel pleiotropic role of FRα: (a) direct activation of Oct4, Sox2, and Klf4 genes; and (b) repression of biogenesis of miRNAs that target these genes or their effector molecules. Stem Cells 2016;34:2721-2732.
