RESUMO
The molecular connections between homeostatic systems that maintain both genome integrity and proteostasis are poorly understood. Here we identify the selective activation of the unfolded protein response transducer IRE1α under genotoxic stress to modulate repair programs and sustain cell survival. DNA damage engages IRE1α signaling in the absence of an endoplasmic reticulum (ER) stress signature, leading to the exclusive activation of regulated IRE1α-dependent decay (RIDD) without activating its canonical output mediated by the transcription factor XBP1. IRE1α endoribonuclease activity controls the stability of mRNAs involved in the DNA damage response, impacting DNA repair, cell cycle arrest and apoptosis. The activation of the c-Abl kinase by DNA damage triggers the oligomerization of IRE1α to catalyze RIDD. The protective role of IRE1α under genotoxic stress is conserved in fly and mouse. Altogether, our results uncover an important intersection between the molecular pathways that sustain genome stability and proteostasis.
Assuntos
Sobrevivência Celular/genética , Reparo do DNA , Proteínas de Drosophila/metabolismo , Endorribonucleases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Estabilidade de RNA/genética , Animais , Dano ao DNA , Proteínas de Drosophila/genética , Drosophila melanogaster , Endorribonucleases/genética , Feminino , Fibroblastos , Instabilidade Genômica , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Multimerização Proteica , Proteínas Serina-Treonina Quinases/genética , Proteostase/genética , Proteínas Proto-Oncogênicas c-abl/metabolismo , RNA Mensageiro/metabolismoRESUMO
RNA editing has emerged as a novel mechanism in cancer progression. The double stranded RNA-specific adenosine deaminase (ADAR) modifies the expression of an important proportion of genes involved in cell cycle control, DNA damage response (DDR) and transcriptional processing, suggesting an important role of ADAR in transcriptome regulation. Despite the phenotypic implications of ADAR deregulation in several cancer models, the role of ADAR on DDR and proliferation in breast cancer has not been fully addressed. Here, we show that ADAR expression correlates significantly with clinical outcomes and DDR, cell cycle and proliferation mRNAs of previously reported edited transcripts in breast cancer patients. ADAR's knock-down in a breast cancer cell line produces stability changes of mRNAs involved in DDR and DNA replication. Breast cancer cells with reduced levels of ADAR show a decreased viability and an increase in apoptosis, displaying a significant decrease of their DDR activation, compared to control cells. These results suggest that ADAR plays an important role in breast cancer progression through the regulation of mRNA stability and expression of those genes involved in proliferation and DDR impacting the viability of breast cancer cells.
Assuntos
Adenosina Desaminase/metabolismo , Neoplasias da Mama/metabolismo , Ciclo Celular/fisiologia , Dano ao DNA/fisiologia , Edição de RNA , Proteínas de Ligação a RNA/metabolismo , Transcriptoma , Adenosina Desaminase/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Progressão da Doença , Feminino , Humanos , Células MCF-7 , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genéticaRESUMO
Transient Receptor Potential Melastatin 4 (TRPM4) is a Ca2+ -activated and voltage-dependent monovalent cation channel, which depolarizes the plasma cell membrane, thereby modulating Ca2+ influx across Ca2+ -permeable pathways. TRPM4 is involved in different physiological processes such as T cell activation and the migration of endothelial and certain immune cells. Overexpression of this channel has been reported in various types of tumors including prostate cancer. In this study, a significant overexpression of TRPM4 was found only in samples from cancer with a Gleason score higher than 7, which are more likely to spread. To evaluate whether TRPM4 overexpression was related to the spreading capability of tumors, TRPM4 was knockdown by using shRNAs in PC3 prostate cancer cells and the effect on cellular migration and invasion was analyzed. PC3 cells with reduced levels of TRPM4 (shTRPM4) display a decrease of the migration/invasion capability. A reduction in the expression of Snail1, a canonical epithelial to mesenchymal transition (EMT) transcription factor, was also observed. Consistently, these cells showed a significant change in the expression of key EMT markers such as MMP9, E-cadherin/N-cadherin, and vimentin, indicating a partial reversion of the EMT process. Whereas, the overexpression of TRPM4 in LnCaP cells resulted in increased levels of Snail1, reduction in the expression of E-cadherin and increase in their migration potential. This study suggests a new and indirect mechanism of regulation of migration/invasion process by TRPM4 in prostate cancer cells, by inducing the expression of Snail1 gene and consequently, increasing the EMT.
Assuntos
Transição Epitelial-Mesenquimal/fisiologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Canais de Cátion TRPM/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Masculino , Modelos Biológicos , Gradação de Tumores , Invasividade Neoplásica , Células PC-3 , Neoplasias da Próstata/genética , Fatores de Transcrição da Família Snail/genética , Fatores de Transcrição da Família Snail/metabolismo , Canais de Cátion TRPM/antagonistas & inibidores , Canais de Cátion TRPM/genética , Regulação para CimaRESUMO
BACKGROUND: Whole transcriptome RNA variant analyses have shown that adenosine deaminases acting on RNA (ADAR) enzymes modify a large proportion of cellular RNAs, contributing to transcriptome diversity and cancer evolution. Despite the advances in the understanding of ADAR function in breast cancer, ADAR RNA editing functional consequences are not fully addressed. RESULTS: We characterized A to G(I) mRNA editing in 81 breast cell lines, showing increased editing at 3'UTR and exonic regions in breast cancer cells compared to immortalized non-malignant cell lines. In addition, tumors from the BRCA TCGA cohort show a 24% increase in editing over normal breast samples when looking at 571 well-characterized UTRs targeted by ADAR1. Basal-like subtype breast cancer patients with high level of ADAR1 mRNA expression shows a worse clinical outcome and increased editing in their 3'UTRs. Interestingly, editing was particularly increased in the 3'UTRs of ATM, GINS4 and POLH transcripts in tumors, which correlated with their mRNA expression. We confirmed the role of ADAR1 in this regulation using a shRNA in a breast cancer cell line (ZR-75-1). CONCLUSIONS: Altogether, these results revealed a significant association between the mRNA editing in genes related to cancer-relevant pathways and clinical outcomes, suggesting an important role of ADAR1 expression and function in breast cancer.
Assuntos
Regiões 3' não Traduzidas/genética , Adenosina Desaminase/genética , Neoplasias da Mama/genética , Edição de RNA/genética , Estabilidade de RNA/genética , Proteínas de Ligação a RNA/genética , Adenosina Desaminase/metabolismo , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Estabilidade de RNA/fisiologia , Proteínas de Ligação a RNA/metabolismoRESUMO
Increased expression of the TRPM4 channel has been reported to be associated with the progression of prostate cancer. However, the molecular mechanism underlying its effect remains unknown. This work found that decreasing TRPM4 levels leads to the reduced proliferation of PC3 cells. This effect was associated with a decrease in total ß-catenin protein levels and its nuclear localization, and a significant reduction in Tcf/Lef transcriptional activity. Moreover, TRPM4 silencing increases the Ser33/Ser37/Thr41 ß-catenin phosphorylated population and reduces the phosphorylation of GSK-3ß at Ser9, suggesting an increase in ß-catenin degradation as the underlying mechanism. Conversely, TRPM4 overexpression in LNCaP cells increases the Ser9 inhibitory phosphorylation of GSK-3ß and the total levels of ß-catenin and its nonphosphorylated form. Finally, PC3 cells with reduced levels of TRPM4 showed a decrease in basal and stimulated phosphoactivation of Akt1, which is likely responsible for the decrease in GSK-3ß activity in these cells. Our results also suggest that the effect of TRPM4 on Akt1 is probably mediated by an alteration in the calcium/calmodulin-EGFR axis, linking TRPM4 activity with the observed effects in ß-catenin-related signaling pathways. These results suggest a role for TRPM4 channels in ß-catenin oncogene signaling and underlying mechanisms, highlighting this ion channel as a new potential target for future therapies in prostate cancer.
Assuntos
Proliferação de Células/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Neoplasias da Próstata/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Canais de Cátion TRPM/metabolismo , beta Catenina/metabolismo , Cálcio/metabolismo , Calmodulina/metabolismo , Linhagem Celular Tumoral , Progressão da Doença , Glicogênio Sintase Quinase 3 beta/genética , Células HEK293 , Humanos , Masculino , Células PC-3 , Fosforilação/genética , Neoplasias da Próstata/genética , Proteínas Proto-Oncogênicas c-akt/genética , Canais de Cátion TRPM/genética , beta Catenina/genéticaRESUMO
BACKGROUND: Whole transcriptome RNA variant analyses have shown that adenosine deaminases acting on RNA ( ADAR ) enzymes modify a large proportion of cellular RNAs, contributing to transcriptome diversity and cancer evolution. Despite the advances in the understanding of ADAR function in breast cancer, ADAR RNA editing functional consequences are not fully addressed. RESULTS: We characterized A to G(I) mRNA editing in 81 breast cell lines, showing increased editing at 3'UTR and exonic regions in breast cancer cells compared to immortalized non-malignant cell lines. In addition, tumors from the BRCA TCGA cohort show a 24% increase in editing over normal breast samples when looking at 571 well-characterized UTRs targeted by ADAR1. Basal-like subtype breast cancer patients with high level of ADAR1 mRNA expression shows a worse clinical outcome and increased editing in their 3'UTRs. Interestingly, editing was particularly increased in the 3'UTRs of ATM, GINS4 and POLH transcripts in tumors, which correlated with their mRNA expression. We confirmed the role of ADAR1 in this regulation using a shRNA in a breast cancer cell line (ZR-75-1). CONCLUSIONS: Altogether, these results revealed a significant association between the mRNA editing in genes related to cancer-relevant pathways and clinical outcomes, suggesting an important role of ADAR1 expression and function in breast cancer.
