RESUMO
Oral squamous cell carcinoma (oral SCC) is an aggressive disease and despite intensive treatments, 5-year survival rates for patients have remained low in the last 20 years. Enhancer of zeste homolog 2 (EZH2), part of polycomb repressive complex 2 (PRC2), is highly expressed in human oral SCC samples and cell lines and has been associated with greater epithelia-to-mesenchymal transition (EMT), invasion and metastasis. Here, we developed a tamoxifen-regulated, transgenic mouse line (KcEZH2) in which EZH2 is selectively knocked out (KO) in some tongue epithelial basal stem cells (SCs) in adult mice. EZH2 KO SCs do not show the H3K27me3 mark, as assessed by double-label immunofluorescence. We used this mouse line to assess EZH2 actions during oral tumorigenesis with our immunocompetent 4-nitroquinoline 1-oxide model of oral SCC. We report that higher percentages of mice with invasive SCCs and high-grade neoplastic lesions are observed in mice containing EZH2 KO SCs (KcEZH2-2TΔ and KcEZH2-5TΔ mice). Moreover, EZH2 expression does not correlate with the expression of markers of invasive SCCs. Finally, EZH2 KO cells that are E-cadherin+ are present at invasion fronts infiltrating underlying muscle tissue. Our findings indicate that the knockout of EZH2 in basal SCs of tongue epithelia results in more aggressive carcinomas, and this should be considered when targeting EZH2 as a therapeutic strategy.
Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste/deficiência , Mucosa Bucal/metabolismo , Mucosa Bucal/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Animais , Biomarcadores Tumorais , Linhagem Celular Tumoral , Metilação de DNA , Modelos Animais de Doenças , Suscetibilidade a Doenças , Epigênese Genética/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Predisposição Genética para Doença , Histonas/metabolismo , Humanos , Receptores de Hialuronatos/metabolismo , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Invasividade Neoplásica , Fosforilação , Fator de Transcrição STAT3/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Tamoxifeno/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We previously showed that mRNA 3' end cleavage reaction in cell extracts is strongly but transiently inhibited under DNA-damaging conditions. The cleavage stimulation factor-50 (CstF-50) has a role in this response, providing a link between transcription-coupled RNA processing and DNA repair. In this study, we show that CstF-50 interacts with nuclear poly(A)-specific ribonuclease (PARN) using in vitro and in extracts of UV-exposed cells. The CstF-50/PARN complex formation has a role in the inhibition of 3' cleavage and activation of deadenylation upon DNA damage. Extending these results, we found that the tumour suppressor BARD1, which is involved in the UV-induced inhibition of 3' cleavage, strongly activates deadenylation by PARN in the presence of CstF-50, and that CstF-50/BARD1 can revert the cap-binding protein-80 (CBP80)-mediated inhibition of PARN activity. We also provide evidence that PARN along with the CstF/BARD1 complex participates in the regulation of endogenous transcripts under DNA-damaging conditions. We speculate that the interplay between polyadenylation, deadenylation and tumour-suppressor factors might prevent the expression of prematurely terminated messengers, contributing to control of gene expression under different cellular conditions.
Assuntos
Núcleo Celular/metabolismo , Dano ao DNA , Poliadenilação , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Reparo do DNA , Exorribonucleases/metabolismo , Regulação da Expressão Gênica , Glutationa Transferase/metabolismo , Células HeLa , Humanos , Modelos Biológicos , RNA/metabolismo , RNA Mensageiro/metabolismo , Proteínas Recombinantes/química , Raios UltravioletaRESUMO
The tongue epithelium is maintained by a proliferative basal layer. This layer contains long-lived stem cells (SCs), which produce progeny cells that move up to the surface as they differentiate. B-lymphoma Mo-MLV insertion region 1 (BMI1), a protein in mammalian Polycomb Repressive Complex 1 (PRC1) and a biomarker of oral squamous cell carcinoma, is expressed in almost all basal epithelial SCs of the tongue, and single, Bmi1-labelled SCs give rise to cells in all epithelial layers. We previously developed a transgenic mouse model (KrTB) containing a doxycycline- (dox) controlled, Tet-responsive element system to selectively overexpress Bmi1 in the tongue basal epithelial SCs. Here, we used this model to assess BMI1 actions in tongue epithelia. Genome-wide transcriptomics revealed increased levels of transcripts involved in the cellular response to hypoxia in Bmi1-overexpressing (KrTB+DOX) oral epithelia even though these mice were not subjected to hypoxia conditions. Ectopic Bmi1 expression in tongue epithelia increased the levels of hypoxia inducible factor-1 alpha (HIF1α) and HIF1α targets linked to metabolic reprogramming during hypoxia. We used chromatin immunoprecipitation (ChIP) to demonstrate that Bmi1 associates with the promoters of HIF1A and HIF1A-activator RELA (p65) in tongue epithelia. We also detected increased SC proliferation and oxidative stress in Bmi1-overexpressing tongue epithelia. Finally, using a human oral keratinocyte line (OKF6-TERT1R), we showed that ectopic BMI1 overexpression decreases the oxygen consumption rate while increasing the extracellular acidification rate, indicative of elevated glycolysis. Thus, our data demonstrate that high BMI1 expression drives hypoxic signaling, including metabolic reprogramming, in normal oral cavity epithelia.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia , Camundongos Transgênicos , Complexo Repressor Polycomb 1 , Transdução de Sinais , Complexo Repressor Polycomb 1/metabolismo , Complexo Repressor Polycomb 1/genética , Animais , Camundongos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Humanos , Língua/metabolismo , Língua/patologia , Mucosa Bucal/metabolismo , Mucosa Bucal/patologia , Hipóxia Celular , Epitélio/metabolismo , Boca/metabolismo , Boca/patologia , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Neoplasias Bucais/genética , Proteínas Proto-OncogênicasRESUMO
While nuclear tau plays a role in DNA damage response (DDR) and chromosome relaxation, the mechanisms behind these functions are not fully understood. Here, we show that tau forms complex(es) with factors involved in nuclear mRNA processing such as tumor suppressor p53 and poly(A)-specific ribonuclease (PARN) deadenylase. Tau induces PARN activity in different cellular models during DDR, and this activation is further increased by p53 and inhibited by tau phosphorylation at residues implicated in neurological disorders. Tau's binding factor Pin1, a mitotic regulator overexpressed in cancer and depleted in Alzheimer's disease (AD), also plays a role in the activation of nuclear deadenylation. Tau, Pin1 and PARN target the expression of mRNAs deregulated in AD and/or cancer. Our findings identify novel biological roles of tau and toxic effects of hyperphosphorylated-tau. We propose a model in which factors involved in cancer and AD regulate gene expression by interactions with the mRNA processing machinery, affecting the transcriptome and suggesting insights into alternative mechanisms for the initiation and/or developments of these diseases.
RESUMO
The cellular response to DNA damage is an intricate mechanism that involves the interplay among several pathways. In this study, we provide evidence of the roles of the polyadenylation factor cleavage stimulation factor 50 (CstF-50) and the ubiquitin (Ub) escort factor p97 as cofactors of BRCA1/BARD1 E3 Ub ligase, facilitating chromatin remodeling during the DNA damage response (DDR). CstF-50 and p97 formed complexes with BRCA1/BARD1, Ub, and some BRCA1/BARD1 substrates, such as RNA polymerase (RNAP) II and histones. Furthermore, CstF-50 and p97 had an additive effect on the activation of the ubiquitination of these BRCA1/BARD1 substrates during DDR. Importantly, as a result of these functional interactions, BRCA1/BARD1/CstF-50/p97 had a specific effect on the chromatin structure of genes that were differentially expressed. This study provides new insights into the roles of RNA processing, BRCA1/BARD1, the Ub pathway, and chromatin structure during DDR.
Assuntos
Adenosina Trifosfatases/genética , Proteína BRCA1/genética , Montagem e Desmontagem da Cromatina , Fator Estimulador de Clivagem/genética , Dano ao DNA , Reparo do DNA , Proteínas Nucleares/genética , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genética , Adenosina Trifosfatases/metabolismo , Proteína BRCA1/metabolismo , Fator Estimulador de Clivagem/metabolismo , Proteínas de Ligação a DNA/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Proteínas Nucleares/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Fatores de Poliadenilação e Clivagem de mRNA/metabolismoRESUMO
The DNA damage response involves coordinated control of gene expression and DNA repair. Using deep sequencing, we found widespread changes of alternative cleavage and polyadenylation site usage on ultraviolet-treatment in mammalian cells. Alternative cleavage and polyadenylation regulation in the 3' untranslated region is substantial, leading to both shortening and lengthening of 3' untranslated regions of genes. Interestingly, a strong activation of intronic alternative cleavage and polyadenylation sites is detected, resulting in widespread expression of truncated transcripts. Intronic alternative cleavage and polyadenylation events are biased to the 5' end of genes and affect gene groups with important functions in DNA damage response and cancer. Moreover, intronic alternative cleavage and polyadenylation site activation during DNA damage response correlates with a decrease in U1 snRNA levels, and is reversible by U1 snRNA overexpression. Importantly, U1 snRNA overexpression mitigates ultraviolet-induced apoptosis. Together, these data reveal a significant gene regulatory scheme in DNA damage response where U1 snRNA impacts gene expression via the U1-alternative cleavage and polyadenylation axis.