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1.
Int J Mol Sci ; 21(7)2020 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-32244500

RESUMO

MicroRNAs are tiny but powerful regulators of gene expression at the post-transcriptional level. Aberrant expression of oncogenic and tumor-suppressor microRNAs has been recognized as a common feature of human cancers. Colorectal cancer represents a major clinical challenge in the developed world and the design of innovative therapeutic approaches relies on the identification of novel biological targets. Here, we perform a functional screening in colorectal cancer cells using a library of locked nucleic acid (LNA)-modified anti-miRs in order to unveil putative oncogenic microRNAs whose inhibition yields a cytotoxic effect. We identify miR-1285-3p and further explore the effect of its targeting in both commercial cell lines and primary colorectal cancer stem cells, finding induction of cell cycle arrest and apoptosis. We show that DAPK2, a known tumor-suppressor, is a novel miR-1285 target and mediates both the anti-proliferative and the pro-apoptotic effects of miR-1285 depletion. Altogether, our findings uncover a novel oncogenic microRNA in colorectal cancer and lay the foundation for further studies aiming at the development of possible therapeutic strategies based on miR-1285 targeting.


Assuntos
Apoptose/fisiologia , Proliferação de Células/fisiologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Proteínas Quinases Associadas com Morte Celular/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Proteínas Quinases Associadas com Morte Celular/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células-Tronco Neoplásicas , Oligonucleotídeos
2.
Mol Cancer ; 18(1): 70, 2019 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-30927908

RESUMO

In the last decades, the role of the microenvironment in tumor progression and therapeutic outcome has gained increasing attention. Cancer-associated fibroblasts (CAFs) have emerged as key players among stromal cells, owing to their abundance in most solid tumors and their diverse tumor-restraining/promoting roles. The interplay between tumor cells and neighboring CAFs takes place by both paracrine signals (cytokines, exosomes and metabolites) or by the multifaceted functions of the surrounding extracellular matrix. Here, we dissect the most recent identified mechanisms underlying CAF-mediated control of tumor progression and therapy resistance, which include induction of the epithelial-to-mesenchymal transition (EMT), activation of survival pathways or stemness-related programs and metabolic reprogramming in tumor cells. Importantly, the recently unveiled heterogeneity in CAFs claims tailored therapeutic efforts aimed at eradicating the specific subset facilitating tumor progression, therapy resistance and relapse. However, despite the large amount of pre-clinical data, much effort is still needed to translate CAF-directed anti-cancer strategies from the bench to the clinic.


Assuntos
Fibroblastos Associados a Câncer/metabolismo , Resistencia a Medicamentos Antineoplásicos , Neoplasias/metabolismo , Antineoplásicos/farmacologia , Fibroblastos Associados a Câncer/efeitos dos fármacos , Progressão da Doença , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias/tratamento farmacológico , Comunicação Parácrina , Transdução de Sinais , Microambiente Tumoral
3.
Int J Mol Sci ; 19(4)2018 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-29570632

RESUMO

In the last few years, non-coding RNAs (ncRNAs) have been a hot topic in cancer research. Many ncRNAs were found to regulate the apoptotic process and to play a role in tumor cell resistance to treatment. The apoptotic program is on the frontline as self-defense from cancer onset, and evasion of apoptosis has been classified as one of the hallmarks of cancer responsible for therapy failure. The B-cell lymphoma 2 (BCL-2) family members are key players in the regulation of apoptosis and mediate the activation of the mitochondrial death machinery in response to radiation, chemotherapeutic agents and many targeted therapeutics. The balance between the pro-survival and the pro-apoptotic BCL-2 proteins is strictly controlled by ncRNAs. Here, we highlight the most common mechanisms exerted by microRNAs, long non-coding RNAs and circular RNAs on the main mediators of the intrinsic apoptotic cascade with particular focus on their significance in cancer biology.


Assuntos
Apoptose/fisiologia , Neoplasias/genética , RNA não Traduzido/genética , Animais , Apoptose/genética , Humanos , MicroRNAs/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia
4.
Cell Death Dis ; 9(2): 49, 2018 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-29352138

RESUMO

Treatment of lung cancer is an unmet need as it accounts for the majority of cancer deaths worldwide. The development of new therapies urges the identification of potential targets. MicroRNAs' expression is often deregulated in cancer and their modulation has been proposed as a successful strategy to interfere with tumor cell growth and spread. We recently reported on an unbiased high-content approach to identify miRNAs regulating cell proliferation and tumorigenesis in non-small cell lung cancer (NSCLC). Here we studied the oncogenic role of miR-663 in NSCLC biology and analyzed the therapeutic potential of miR-663 targeting. We found that miR-663 regulates apoptosis by controlling mitochondrial outer membrane permeabilization (MOMP) through the expression of two novel direct targets PUMA/BBC3 and BTG2. Specifically, upon miR-663 knockdown the BH3-only protein PUMA/BBC3 directly activates mitochondrial depolarization and cell death, while BTG2 accumulation further enhances this effect by triggering p53 mitochondrial localization. Moreover, we show that miR-663 depletion is sufficient to elicit cell death in NSCLC cells and to impair tumor growth in vivo.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Proteínas Imediatamente Precoces/metabolismo , Neoplasias Pulmonares/genética , MicroRNAs/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Células A549 , Animais , Proteínas Reguladoras de Apoptose/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/terapia , Linhagem Celular Tumoral , Feminino , Células HeLa , Xenoenxertos , Humanos , Proteínas Imediatamente Precoces/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/terapia , Camundongos , Camundongos Nus , MicroRNAs/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Permeabilidade , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais , Transfecção , Proteínas Supressoras de Tumor/genética
5.
Curr Opin Pharmacol ; 35: 1-11, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28527911

RESUMO

Cancer stem cells (CSCs) represent the main target of the current efforts to eradicate cancer, because of their ability to promote metastatic dissemination and survive cytotoxic therapies. Here, we highlight the potential of patient-derived CSCs as an in vitro and in vivo pre-clinical model and of liquid biopsy as a diagnostic, prognostic and predictive tool. We discuss recently developed therapeutic strategies aiming at specifically targeting the cancer stem cell population, particularly focusing on the latest advances in cancer immunotherapy. In fact, it is now widely accepted that the microenvironment plays an active role in supporting tumor progression. Hence, the crosstalk between CSCs and the host immune system is nowadays object of intensive study, with the aim to develop effective therapeutic strategies targeting the ability of CSCs to escape immune-surveillance through immunoediting.


Assuntos
Células-Tronco Neoplásicas , Animais , Humanos , Imunoterapia , Neoplasias/terapia , Medicina de Precisão
6.
Hum Pathol ; 46(11): 1760-8, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26362204

RESUMO

Cardiac dysfunction of Fabry disease (FD) has been associated with myofilament damage and cell death as result of α-galactosidase A deficiency and globotriaosylceramide accumulation. We sought to evaluate the role of oxidative stress in FD cardiomyocyte dysfunction. Myocardial tissue from 18 patients with FD was investigated for the expression of inducible nitric oxide synthase (iNOS) and nitrotyrosine by immunohistochemistry. Western blot analysis for nitrotyrosine was also performed. Oxidative damage to DNA was investigated by immunostaining for 8-hydroxydeoxyguanosine (8-OHdG), whereas apoptosis was evaluated by in situ ligation with hairpin probes. iNOS and nitrotyrosine expression was increased in FD hearts compared with hypertrophic cardiomyopathy and normal controls. Remarkably, immunostaining was homogeneously expressed in FD male cardiomyocytes, whereas it was only detected in the affected cardiomyocytes of FD females. Western blot analysis confirmed an increase in FD cardiomyocyte protein nitration compared with controls. 8-OHdG was expressed in 25% of cardiomyocyte nuclei from FD patients, whereas it was absent in controls. The intensity of immunostaining for iNOS/nitrotyrosine correlated with 8-OHdG expression in cardiomyocyte nuclei. Apoptosis of FD cardiomyocytes was 187-fold higher than in controls, and apoptotic nuclei were positive for 8-OHdG. Cardiac dysfunction of FD reflects increased myocardial nitric oxide production with oxidative damage of cardiomyocyte myofilaments and DNA, causing cell dysfunction and death.


Assuntos
Doença de Fabry/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Estresse Oxidativo/fisiologia , Adulto , Idoso , Apoptose/fisiologia , Doença de Fabry/mortalidade , Doença de Fabry/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Miocárdio/patologia , Miócitos Cardíacos/patologia , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismo
7.
Sci Rep ; 5: 9841, 2015 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-25923013

RESUMO

Metastasis is responsible for over 90% of cancer-associated mortality. In epithelial carcinomas, a key process in metastatic progression is the epigenetic reprogramming of an epithelial-to-mesenchymal transition-like (EMT) change towards invasive cellular phenotypes. In non-epithelial cancers, different mechanisms must underlie metastatic change, but relatively little is known about the factors involved. Here, we identify the chromatin regulatory Sirtuin factor SIRT7 as a key regulator of metastatic phenotypes in both epithelial and mesenchymal cancer cells. In epithelial prostate carcinomas, high SIRT7 levels are associated with aggressive cancer phenotypes, metastatic disease, and poor patient prognosis, and depletion of SIRT7 can reprogram these cells to a less aggressive phenotype. Interestingly, SIRT7 is also important for maintaining the invasiveness and metastatic potential of non-epithelial sarcoma cells. Moreover, SIRT7 inactivation dramatically suppresses cancer cell metastasis in vivo, independent of changes in primary tumor growth. Mechanistically, we also uncover a novel link between SIRT7 and its family member SIRT1, providing the first demonstration of direct interaction and functional interplay between two mammalian sirtuins. Together with previous work, our findings highlight the broad role of SIRT7 in maintaining the metastatic cellular phenotype in diverse cancers.


Assuntos
Células Epiteliais/patologia , Transição Epitelial-Mesenquimal/genética , Metástase Neoplásica/genética , Metástase Neoplásica/patologia , Sarcoma/genética , Sirtuínas/genética , Linhagem Celular Tumoral , Cromatina/genética , Progressão da Doença , Epigênese Genética/genética , Humanos , Fenótipo , Prognóstico , Sarcoma/patologia
8.
Autophagy ; 11(2): 253-70, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25700560

RESUMO

In liver the mitochondrial sirtuin, SIRT5, controls ammonia detoxification by regulating CPS1, the first enzyme of the urea cycle. However, while SIRT5 is ubiquitously expressed, urea cycle and CPS1 are only present in the liver and, to a minor extent, in the kidney. To address the possibility that SIRT5 is involved in ammonia production also in nonliver cells, clones of human breast cancer cell lines MDA-MB-231 and mouse myoblast C2C12, overexpressing or silenced for SIRT5 were produced. Our results show that ammonia production increased in SIRT5-silenced and decreased in SIRT5-overexpressing cells. We also obtained the same ammonia increase when using a new specific inhibitor of SIRT5 called MC3482. SIRT5 regulates ammonia production by controlling glutamine metabolism. In fact, in the mitochondria, glutamine is transformed in glutamate by the enzyme glutaminase, a reaction producing ammonia. We found that SIRT5 and glutaminase coimmunoprecipitated and that SIRT5 inhibition resulted in an increased succinylation of glutaminase. We next determined that autophagy and mitophagy were increased by ammonia by measuring autophagic proteolysis of long-lived proteins, increase of autophagy markers MAP1LC3B, GABARAP, and GABARAPL2, mitophagy markers BNIP3 and the PINK1-PARK2 system as well as mitochondrial morphology and dynamics. We observed that autophagy and mitophagy increased in SIRT5-silenced cells and in WT cells treated with MC3482 and decreased in SIRT5-overexpressing cells. Moreover, glutaminase inhibition or glutamine withdrawal completely prevented autophagy. In conclusion we propose that the role of SIRT5 in nonliver cells is to regulate ammonia production and ammonia-induced autophagy by regulating glutamine metabolism.


Assuntos
Amônia/farmacologia , Autofagia/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitofagia/efeitos dos fármacos , Sirtuínas/metabolismo , Autofagia/fisiologia , Glutaminase/metabolismo , Humanos , Mitocôndrias/metabolismo , Mitofagia/fisiologia , Proteólise/efeitos dos fármacos , Ubiquitina-Proteína Ligases/metabolismo
9.
Clin Cancer Res ; 20(7): 1741-6, 2014 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-24536059

RESUMO

SIRT7 belongs to the Sirtuin family of NAD-dependent enzymes, the members of which play diverse roles in aging, metabolism, and disease biology. Increased SIRT7 expression is observed in human cancers and growing evidence suggests important SIRT7 functions in fundamental cellular programs with an impact on oncogenic transformation and tumor biology. SIRT7 associates with chromatin, where it catalyzes selective deacetylation of lysine 18 on histone H3 (H3K18), an emerging epigenetic biomarker of aggressive tumors and poor clinical outcome in patients with cancer. Through H3K18 deacetylation at specific promoters, SIRT7 controls a tumor-suppressive gene expression program that stabilizes the transformed state of cancer cells. SIRT7 also orchestrates several molecular processes, including rRNA and tRNA synthesis, which ultimately promote the increased ribosome biogenesis necessary for tumor cell growth and proliferation. Remarkably, inactivation of SIRT7 can reverse the transformed phenotype of cancer cells and reduce their tumorigenicity in vivo. These findings place SIRT7 at the crossroads of chromatin signaling, metabolic, and tumor-regulatory pathways. Thus, SIRT7 is a promising pharmacologic target for epigenetic cancer therapy. The development of SIRT7 modulators may allow new therapeutic strategies that control tumor progression by reprogramming the chromatin landscape and biosynthetic machinery of cancer cells.


Assuntos
Carcinogênese/genética , Redes e Vias Metabólicas/genética , Neoplasias/genética , Sirtuínas/genética , Acetilação , Proliferação de Células , Cromatina/genética , Histonas/metabolismo , Humanos , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Regiões Promotoras Genéticas , Transdução de Sinais/genética
10.
Cell Rep ; 5(3): 654-665, 2013 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-24210820

RESUMO

Nonalcoholic fatty liver disease is the most common chronic liver disorder in developed countries. Its pathogenesis is poorly understood, and therapeutic options are limited. Here, we show that SIRT7, an NAD(+)-dependent H3K18Ac deacetylase, functions at chromatin to suppress ER stress and prevent the development of fatty liver disease. SIRT7 is induced upon ER stress and is stabilized at the promoters of ribosomal proteins through its interaction with the transcription factor Myc to silence gene expression and to relieve ER stress. SIRT7-deficient mice develop chronic hepatosteatosis resembling human fatty liver disease. Myc inactivation or pharmacological suppression of ER stress alleviates fatty liver caused by SIRT7 deficiency. Importantly, SIRT7 suppresses ER stress and reverts the fatty liver disease in diet-induced obese mice. Our study identifies SIRT7 as a cofactor of Myc for transcriptional repression and delineates a druggable regulatory branch of the ER stress response that prevents and reverts fatty liver disease.


Assuntos
Estresse do Retículo Endoplasmático/fisiologia , Fígado Gorduroso/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Sirtuínas/metabolismo , Animais , Feminino , Genes myc , Células HEK293 , Células Hep G2 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/genética , Sirtuínas/deficiência , Sirtuínas/genética , Transfecção
11.
Sci Rep ; 3: 3085, 2013 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-24169447

RESUMO

The chromatin regulatory factor SIRT6 plays pivotal roles in metabolism, tumor suppression, and aging biology. Despite the fundamental roles of SIRT6 in physiology and disease, only a handful of molecular and functional interactions of SIRT6 have been reported. Here, we characterize the SIRT6 interactome and identify 80+ novel SIRT6-interacting proteins. The discovery of these SIRT6-associations considerably expands knowledge of the SIRT6 interaction network, and suggests previously unknown functional interactions of SIRT6 in fundamental cellular processes. These include chromatin remodeling, mitotic chromosome segregation, protein homeostasis, and transcriptional elongation. Extended analysis of the SIRT6 interaction with G3BP1, a master stress response factor, uncovers an unexpected role and mechanism of SIRT6 in regulating stress granule assembly and cellular stress resistance.


Assuntos
Proteínas de Transporte/metabolismo , Mapas de Interação de Proteínas , Transdução de Sinais/genética , Sirtuínas/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , DNA Helicases , Células HEK293 , Células HeLa , Humanos , Camundongos , Proteínas de Ligação a Poli-ADP-Ribose , Ligação Proteica , Proteômica , RNA Helicases , Interferência de RNA , Proteínas com Motivo de Reconhecimento de RNA , RNA Interferente Pequeno , Sirtuínas/genética , Ubiquitina Tiolesterase/genética
12.
Intern Emerg Med ; 8 Suppl 1: S23-5, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23462891

RESUMO

The research of the last decade highlighted the existence of a family of genes activated by cellular stresses that allow the cells to reactivate defense and repair activities regardless of age. The prolonged activation of these genes enhances the organism health and lifespan. Members of this gene family are called sirtuins (SIRT). The founding member of the SIRT protein family, Sir2 is a limiting component of yeast longevity. Many members of this family have been also identified as key longevity regulators in species ranging from yeast to fly. On the other hand, the role of SIRTs in the regulation of mammalian ageing has been questioned. While SIRTs' effects on lifespan are still a matter of scientific debate, the beneficial effects of SIRTs in terms of physical health and quality of aging are widely accepted. Increasing evidence suggests a pivotal role for SIRTs in mediating the adaptive response to physical exercise. The following review summarizes the knowledge so far acquired on sirtuins' role in mediating beneficial effects of physical exercise. In particular, the first paragraph gives an overture on mammalian sirtuins defining their localization, function when possible, and substrates. In the second paragraph, we discuss recent data regarding alteration of sirtuins expression and activity after physical exercise collected by our laboratory and others'.


Assuntos
Exercício Físico/fisiologia , Sirtuínas/fisiologia , Adaptação Fisiológica , Envelhecimento/fisiologia , Animais , Humanos
13.
Front Pharmacol ; 4: 13, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23408731

RESUMO

HIF1α and NFkB are two transcription factors very frequently activated in tumors and involved in tumor growth, progression, and resistance to chemotherapy. In fact, HIF1α and NFkB together regulate transcription of over a thousand genes that, in turn, control vital cellular processes such as adaptation to the hypoxia, metabolic reprograming, inflammatory reparative response, extracellular matrix digestion, migration and invasion, adhesion, etc. Because of this wide involvement they could control in an integrated manner the origin of the malignant phenotype. Interestingly, hypoxia and inflammation have been sequentially bridged in tumors by the discovery that alarmin receptors genes such as RAGE, P2X7, and some TLRs, are activated by HIF1α; and that, in turn, alarmin receptors strongly activate NFkB and proinflammatory gene expression, evidencing all the hallmarks of the malignant phenotype. Recently, a large number of drugs have been identified that inhibit one or both transcription factors with promising results in terms of controlling tumor progression. In addition, many of these molecules are natural compounds or off-label drugs already used to cure other pathologies. Some of them are undergoing clinical trials and soon they will be used alone or in combination with standard anti-tumoral agents to achieve a better treatment of tumors with reduction of metastasis formation and, more importantly, with a net increase in survival. This review highlights the central role of HIF1α activated in hypoxic regions of the tumor, of NFkB activation and proinflammatory gene expression in transformed cells to understand their progression toward malignancy. Different molecules and strategies to inhibit these transcription factors will be reviewed. Finally, the central role of a new class of deacetylases called Sirtuins in regulating HIF1α and NFkB activity will be outlined.

14.
J Cell Physiol ; 228(8): 1754-61, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23359486

RESUMO

The following study demonstrated that, in in vitro differentiated neurons, SIRT1 silencing induced an increase of IGF-1 protein expression and secretion and of IGF-1R protein levels which, in turn, prolonged neuronal cell survival in presence of an apoptotic insult. On the contrary, SIRT1 overexpression increased cell death. In particular, IGF-1 and IGF-1R expression levels were negatively regulated by SIRT1. In SIRT1 silenced cells, the increase in IGF-1 and IGF-1R expression was associated to an increase in AKT and ERK1/2 phosphorylation. Moreover, neuronal differentiation was reduced in SIRT1 overexpressing cells and increased in SIRT1 silenced cells. We conclude that SIRT1 silenced neurons appear more committed to differentiation and more resistant to cell death through the activation of IGF-1 survival pathway.


Assuntos
Fator de Crescimento Insulin-Like I/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Transdução de Sinais , Sirtuína 1/antagonistas & inibidores , Sirtuína 1/genética , Animais , Morte Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Sobrevivência Celular , Regulação para Baixo/genética , Camundongos , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores , RNA Interferente Pequeno/genética , Ratos , Receptor IGF Tipo 1/antagonistas & inibidores , Receptor IGF Tipo 1/metabolismo , Transdução de Sinais/genética , Regulação para Cima/genética
15.
Carcinogenesis ; 32(8): 1167-75, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21642357

RESUMO

The role of hypoxia in regulating tumor progression is still controversial. Here, we demonstrate that, similarly to what previously observed by us in human prostate and breast tumor samples, hypoxia increases expression of the receptor for advanced glycation end products (RAGE) and the purinergic receptor P2X7 (P2X7R). The role of hypoxia was shown by the fact that hypoxia-inducible factor (HIF)-1α silencing downregulated RAGE and P2X7R protein levels as well as nuclear factor-kappaB (NF-κB) expression. In contrast, NF-κB silencing reduced P2X7R expression without affecting RAGE protein levels or nuclear accumulation of HIF-1α. Treatment of hypoxic tumor cells with HMGB1 and BzATP ligands, respectively, of RAGE and P2X7R, activated a signaling pathway that, through Akt and Erk phosphorylation, determines nuclear accumulation of NF-κB and increases cell invasion. Inhibition of Akt by SH5 and Erk by INH1 prevented both nuclear translocation of NF-κB and cell invasion. Moreover, silencing RAGE and P2X7R abolished nuclear accumulation of NF-κB as well as cell invasion without affecting HIF-1α stabilization. Once in the nucleus, NF-κB would contribute to cell survival and invasion under hypoxia, by maintaining RAGE and P2X7R expression levels and matrix metalloproteinases 2 and 9 synthesis. These results show that, hypoxia can upregulate expression levels of membrane receptors that, by binding extracellular molecules eventually released by necrotic cells, contribute to the increased invasiveness of transformed tumor cells. Moreover, these observations strengthen our working hypothesis that upregulation of damage-associated molecular patterns receptors by HIF-1α represents the crucial event bridging hypoxia and inflammation in obtaining the malignant phenotype.


Assuntos
Neoplasias da Mama/patologia , Movimento Celular , Núcleo Celular/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Imunológicos/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Quinase I-kappa B/antagonistas & inibidores , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Immunoblotting , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , NF-kappa B/genética , Fosforilação , Transporte Proteico , RNA Interferente Pequeno/genética , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/genética , Receptores Purinérgicos P2X7/química , Receptores Purinérgicos P2X7/genética , Transdução de Sinais
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