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1.
Nat Commun ; 15(1): 4096, 2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38750019

RESUMO

The presence of heterogeneity in responses to oncolytic virotherapy poses a barrier to clinical effectiveness, as resistance to this treatment can occur through the inhibition of viral spread within the tumor, potentially leading to treatment failures. Here we show that 4-octyl itaconate (4-OI), a chemical derivative of the Krebs cycle-derived metabolite itaconate, enhances oncolytic virotherapy with VSVΔ51 in various models including human and murine resistant cancer cell lines, three-dimensional (3D) patient-derived colon tumoroids and organotypic brain tumor slices. Furthermore, 4-OI in combination with VSVΔ51 improves therapeutic outcomes in a resistant murine colon tumor model. Mechanistically, we find that 4-OI suppresses antiviral immunity in cancer cells through the modification of cysteine residues in MAVS and IKKß independently of the NRF2/KEAP1 axis. We propose that the combination of a metabolite-derived drug with an oncolytic virus agent can greatly improve anticancer therapeutic outcomes by direct interference with the type I IFN and NF-κB-mediated antiviral responses.


Assuntos
Terapia Viral Oncolítica , Vírus Oncolíticos , Succinatos , Animais , Humanos , Terapia Viral Oncolítica/métodos , Succinatos/farmacologia , Camundongos , Linhagem Celular Tumoral , Interferon Tipo I/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Neoplasias do Colo/terapia , Neoplasias do Colo/imunologia , Neoplasias do Colo/tratamento farmacológico , Antivirais/farmacologia , NF-kappa B/metabolismo , Quinase I-kappa B/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Inflamação/tratamento farmacológico , Feminino , Vírus da Estomatite Vesicular Indiana/fisiologia , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
2.
Free Radic Biol Med ; 208: 478-493, 2023 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-37714439

RESUMO

Transcription factor nuclear factor erythroid 2 p45-related factor 2 (Nrf2) is the principal determinant of the cellular redox homeostasis, contributing to mitochondrial function, integrity and bioenergetics. The main negative regulator of Nrf2 is Kelch-like ECH associated protein 1 (Keap1), a substrate adaptor for Cul3/Rbx1 ubiquitin ligase, which continuously targets Nrf2 for ubiquitination and proteasomal degradation. Loss-of-function mutations in Keap1 occur frequently in lung cancer, leading to constitutive Nrf2 activation. We used the human lung cancer cell line A549 and its CRISPR/Cas9-generated homozygous Nrf2-knockout (Nrf2-KO) counterpart to assess the role of Nrf2 on mitochondrial health. To confirm that the observed effects of Nrf2 deficiency are not due to clonal selection or long-term adaptation to the absence of Nrf2, we also depleted Nrf2 by siRNA (siNFE2L2), thus creating populations of Nrf2-knockdown (Nrf2-KD) A549 cells. Nrf2 deficiency decreased mitochondrial respiration, but increased the mitochondrial membrane potential, mass, DNA content, and the number of mitolysosomes. The proportion of ATG7 and ATG3 within their respective LC3B conjugates was increased in Nrf2-deficient cells with mutant Keap1, whereas the formation of new autophagosomes was not affected. Thus, in lung cancer cells with loss-of-function Keap1, Nrf2 facilitates mitolysosome degradation thereby ensuring timely clearance of damaged mitochondria.


Assuntos
Neoplasias Pulmonares , Fator 2 Relacionado a NF-E2 , Humanos , Proteínas Culina/genética , Proteínas Culina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Neoplasias Pulmonares/genética , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Mitocôndrias/metabolismo , Células A549
3.
Biosci Rep ; 43(1)2023 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-36622366

RESUMO

Preserving proteostasis is a major survival mechanism for cancer. Dual specificity tyrosine phosphorylation-regulated kinase 2 (DYRK2) is a key oncogenic kinase that directly activates the transcription factor heat-shock factor 1 (HSF1) and the 26S proteasome. Targeting DYRK2 has proven to be a tractable strategy to target cancers sensitive to proteotoxic stress; however, the development of HSF1 inhibitors remains in its infancy. Importantly, multiple other kinases have been shown to redundantly activate HSF1 that promoted ideas to directly target HSF1. The eventual development of direct HSF1 inhibitor KRIBB11 suggests that the transcription factor is indeed a druggable target. The current study establishes that concurrent targeting of HSF1 and DYRK2 can indeed impede cancer by inducing apoptosis faster than individual targetting. Furthermore, targeting the DYRK2-HSF1 axis induces death in proteasome inhibitor-resistant cells and reduces triple-negative breast cancer (TNBC) burden in ectopic and orthotopic xenograft models. Together the data indicate that cotargeting of kinase DYRK2 and its substrate HSF1 could prove to be a beneficial strategy in perturbing neoplastic malignancies.


Assuntos
Neoplasias , Humanos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fosforilação , Regulação da Expressão Gênica , Inibidores de Proteassoma/farmacologia
4.
Free Radic Biol Med ; 191: 203-211, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36084789

RESUMO

The transcription factor BACH1 regulates the expression of a variety of genes including genes involved in oxidative stress responses, inflammation, cell motility, cancer cell invasion and cancer metabolism. Based on this, BACH1 has become a promising therapeutic target in cancer (as anti-metastatic target) and also in chronic conditions linked to oxidative stress and inflammation, where BACH1 inhibitors share a therapeutic space with activators of transcription factor NRF2. However, while there is a growing number of NRF2 activators, there are only a few described BACH1 inhibitors/degraders. The synthetic acetylenic tricyclic bis(cyanoenone),(±)-(4bS,8aR,10aS)-10a-ethynyl-4b,8,8-trimethyl-3,7-dioxo-3.4b,7,8,8a,9,10, 10a-octahydrophenanthrene-2,6-dicarbonitrile, TBE31 is a potent activator of NRF2 without any BACH1 activity. Herein we found that biotinylation of TBE31 greatly reduces its potency as NRF2 activator (50-75-fold less active) while acquiring a novel activity as a BACH1 degrader (100-200-fold more active). We demonstrate that TBE56, the biotinylated TBE31, interacts and promotes the degradation of BACH1 via a mechanism involving the E3 ligase FBXO22. TBE56 is a potent and sustained BACH1 degrader (50-fold more potent than hemin) and accordingly a powerful HMOX1 inducer. TBE56 degrades BACH1 in lung and breast cancer cells, impairing breast cancer cell migration and invasion in a BACH1-dependent manner, while TBE31 has no significant effect. Altogether, our study identifies that the biotinylation of TBE31 provides novel activities with potential therapeutic value, providing a rationale for further characterisation of this and related compounds.


Assuntos
Neoplasias da Mama , Proteínas F-Box , Acetileno , Alcinos , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Biotinilação , Proteínas F-Box/metabolismo , Feminino , Hemina , Humanos , Inflamação , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
5.
Redox Biol ; 51: 102291, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35313207

RESUMO

The transcription factor BACH1 is a potential therapeutic target for a variety of chronic conditions linked to oxidative stress and inflammation, as well as cancer metastasis. However, only a few BACH1 degraders/inhibitors have been described. BACH1 is a transcriptional repressor of heme oxygenase 1 (HMOX1), which is positively regulated by transcription factor NRF2 and is highly inducible by derivatives of the synthetic oleanane triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO). Most of the therapeutic activities of these compounds are due to their anti-inflammatory and antioxidant properties, which are widely attributed to their ability to activate NRF2. However, with such a broad range of action, these compounds have other molecular targets that have not been fully identified and could also be of importance for their therapeutic profile. Herein we identified BACH1 as a target of two CDDO-derivatives (CDDO-Me and CDDO-TFEA), but not of CDDO. While both CDDO and CDDO-derivatives activate NRF2 similarly, only CDDO-Me and CDDO-TFEA inhibit BACH1, which explains the much higher potency of these CDDO-derivatives as HMOX1 inducers compared with unmodified CDDO. Notably, we demonstrate that CDDO-Me and CDDO-TFEA inhibit BACH1 via a novel mechanism that reduces BACH1 nuclear levels while accumulating its cytoplasmic form. In an in vitro model, both CDDO-derivatives impaired lung cancer cell invasion in a BACH1-dependent and NRF2-independent manner, while CDDO was inactive. Altogether, our study identifies CDDO-Me and CDDO-TFEA as dual KEAP1/BACH1 inhibitors, providing a rationale for further therapeutic uses of these drugs.


Assuntos
Ácido Oleanólico , Triterpenos , Proteína 1 Associada a ECH Semelhante a Kelch , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Ácido Oleanólico/análogos & derivados , Ácido Oleanólico/farmacologia , Estresse Oxidativo , Triterpenos/farmacologia
6.
Antioxidants (Basel) ; 11(2)2022 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-35204145

RESUMO

The evolutionary conserved non-heme Fe-containing protein pirin has been implicated as an important factor in cell proliferation, migration, invasion, and tumour progression of melanoma, breast, lung, cervical, prostate, and oral cancers. Here we found that pirin is overexpressed in human colorectal cancer in comparison with matched normal tissue. The overexpression of pirin correlates with activation of transcription factor nuclear factor erythroid 2 p45-related factor 2 (Nrf2) and increased expression of the classical Nrf2 target NAD(P)H:quinone oxidoreductase 1 (NQO1), but interestingly and unexpectedly, not with expression of the aldo-keto reductase (AKR) family members AKR1B10 and AKR1C1, which are considered to be the most overexpressed genes in response to Nrf2 activation in humans. Using pharmacologic and genetic approaches to either downregulate or upregulate Nrf2, we show that pirin is regulated by Nrf2 in human and mouse cells and in the mouse colon in vivo. The small molecule pirin inhibitor TPhA decreased the viability of human colorectal cancer (DLD1) cells, but this decrease was independent of the levels of pirin. Our study demonstrates the Nrf2-dependent regulation of pirin and encourages the pursuit for specific pirin inhibitors.

7.
Cell Death Differ ; 29(1): 105-117, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34363019

RESUMO

The cell division cycle 25A (CDC25A) phosphatase is a key regulator of cell cycle progression that acts on the phosphorylation status of Cyclin-Cyclin-dependent kinase complexes, with an emergent role in the DNA damage response and cell survival control. The regulation of CDC25A activity and its protein level is essential to control the cell cycle and maintain genomic integrity. Here we describe a novel ubiquitin/proteasome-mediated pathway negatively regulating CDC25A stability, dependent on its phosphorylation by the serine/threonine kinase DYRK2. DYRK2 phosphorylates CDC25A on at least 7 residues, resulting in its degradation independent of the known CDC25A E3 ubiquitin ligases. CDC25A in turn is able to control the phosphorylation of DYRK2 at several residues outside from its activation loop, thus affecting DYRK2 localization and activity. An inverse correlation between DYRK2 and CDC25A protein amounts was observed during cell cycle progression and in response to DNA damage, with CDC25A accumulation responding to the manipulation of DYRK2 levels or activity in either physiological scenario. Functional data show that the pro-survival activity of CDC25A and the pro-apoptotic activity of DYRK2 could be partly explained by the mutual regulation between both proteins. Moreover, DYRK2 modulation of CDC25A expression and/or activity contributes to the DYRK2 role in cell cycle regulation. Altogether, we provide evidence suggesting that DYRK2 and CDC25A mutually control their activity and stability by a feedback regulatory loop, with a relevant effect on the genotoxic stress pathway, apoptosis, and cell cycle regulation.


Assuntos
Proteínas Serina-Treonina Quinases , Fosfatases cdc25 , Ciclo Celular , Dano ao DNA , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Fosfatases cdc25/genética , Fosfatases cdc25/metabolismo
8.
Cancers (Basel) ; 13(14)2021 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-34298830

RESUMO

The ubiquitin E3 ligase TNF Receptor Associated Factor 6 (TRAF6) participates in a large number of different biological processes including innate immunity, differentiation and cell survival, raising the need to specify and shape the signaling output. Here, we identify a lipopolysaccharide (LPS)-dependent increase in TRAF6 association with the kinase IKKε (inhibitor of NF-κB kinase subunit ε) and IKKε-mediated TRAF6 phosphorylation at five residues. The reconstitution of TRAF6-deficient cells, with TRAF6 mutants representing phosphorylation-defective or phospho-mimetic TRAF6 variants, showed that the phospho-mimetic TRAF6 variant was largely protected from basal ubiquitin/proteasome-mediated degradation, and also from autophagy-mediated decay in autolysosomes induced by metabolic perturbation. In addition, phosphorylation of TRAF6 and its E3 ligase function differentially shape basal and LPS-triggered signaling networks, as revealed by phosphoproteome analysis. Changes in LPS-triggered phosphorylation networks of cells that had experienced autophagy are partially dependent on TRAF6 and its phosphorylation status, suggesting an involvement of this E3 ligase in the interplay between metabolic and inflammatory circuits.

9.
J Biol Chem ; 296: 100233, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33376136

RESUMO

Over the last decade, the CMGC kinase DYRK2 has been reported as a tumor suppressor across various cancers triggering major antitumor and proapoptotic signals in breast, colon, liver, ovary, brain, and lung cancers, with lower DYRK2 expression correlated with poorer prognosis in patients. Contrary to this, various medicinal chemistry studies reported robust antiproliferative properties of DYRK2 inhibitors, whereas unbiased 'omics' and genome-wide association study-based studies identified DYRK2 as a highly overexpressed kinase in various patient tumor samples. A major paradigm shift occurred in the last 4 years when DYRK2 was found to regulate proteostasis in cancer via a two-pronged mechanism. DYRK2 phosphorylated and activated the 26S proteasome to enhance degradation of misfolded/tumor-suppressor proteins while also promoting the nuclear stability and transcriptional activity of its substrate, heat-shock factor 1 triggering protein folding. Together, DYRK2 regulates proteostasis and promotes protumorigenic survival for specific cancers. Indeed, potent and selective small-molecule inhibitors of DYRK2 exhibit in vitro and in vivo anti-tumor activity in triple-negative breast cancer and myeloma models. However, with conflicting and contradictory reports across different cancers, the overarching role of DYRK2 remains enigmatic. Specific cancer (sub)types coupled to spatiotemporal interactions with substrates could decide the procancer or anticancer role of DYRK2. The current review aims to provide a balanced and critical appreciation of the literature to date, highlighting top substrates such as p53, c-Myc, c-Jun, heat-shock factor 1, proteasome, or NOTCH1, to discuss DYRK2 inhibitors available to the scientific community and to shed light on this duality of protumorigenic and antitumorigenic roles of DYRK2.


Assuntos
Neoplasias/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Fatores de Transcrição de Choque Térmico/metabolismo , Humanos , Neoplasias/patologia , Fosforilação , Prognóstico , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Tirosina Quinases/fisiologia , Proteostase/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Quinases Dyrk
10.
Phytomedicine ; 86: 153062, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31409554

RESUMO

BACKGROUND: The isothiocyanate sulforaphane (SFN) has multiple protein targets in mammalian cells, affecting processes of fundamental importance for the maintenance of cellular homeostasis, among which are those regulated by the stress response transcription factor nuclear factor erythroid 2 p45-related factor 2 (NRF2) and the serine/threonine protein kinase mechanistic target of rapamycin (mTOR). Whereas the way by which SFN activates NRF2 is well established, the molecular mechanism(s) of how SFN inhibits mTOR is not understood. HYPOTHESIS/PURPOSE: The aim of this study was to investigate the mechanism(s) by which SFN inhibits mTOR STUDY DESIGN AND METHODS: We used the human osteosarcoma cell line U2OS and its CRISPR/Cas9-generated NRF2-knockout counterpart to test the requirement for NRF2 and the involvement of mTOR regulators in the SFN-mediated inhibition of mTOR. RESULTS: SFN inhibits mTOR in a concentration- and time-dependent manner, and this inhibition occurs in the presence or in the absence of NRF2. The phosphatidylinositol 3-kinase (PI3K)-AKT/protein kinase B (PKB) is a positive regulator of mTOR, and treatment with SFN caused an increase in the phosphorylation of AKT at T308 and S473, two phosphorylation sites associated with AKT activation. Interestingly however, the levels of pS552 ß-catenin, an AKT phosphorylation site, were decreased, suggesting that the catalytic activity of AKT was inhibited. In addition, SFN inhibited the activity of the cytoplasmic histone deacetylase 6 (HDAC6), the inhibition of which has been reported to promote the acetylation and decreases the kinase activity of AKT. CONCLUSION: SFN inhibits HDAC6 and decreases the catalytic activity of AKT, and this partially explains the mechanism by which SFN inhibits mTOR.


Assuntos
Isotiocianatos/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , Sulfóxidos/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo
11.
Cell Death Differ ; 28(5): 1563-1578, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33268814

RESUMO

To survive proteotoxic stress, cancer cells activate the proteotoxic-stress response pathway, which is controlled by the transcription factor heat shock factor 1 (HSF1). This pathway supports cancer initiation, cancer progression and chemoresistance and thus is an attractive therapeutic target. As developing inhibitors against transcriptional regulators, such as HSF1 is challenging, the identification and targeting of upstream regulators of HSF1 present a tractable alternative strategy. Here we demonstrate that in triple-negative breast cancer (TNBC) cells, the dual specificity tyrosine-regulated kinase 2 (DYRK2) phosphorylates HSF1, promoting its nuclear stability and transcriptional activity. DYRK2 depletion reduces HSF1 activity and sensitises TNBC cells to proteotoxic stress. Importantly, in tumours from TNBC patients, DYRK2 levels positively correlate with active HSF1 and associates with poor prognosis, suggesting that DYRK2 could be promoting TNBC. These findings identify DYRK2 as a key modulator of the HSF1 transcriptional programme and a potential therapeutic target.


Assuntos
Fatores de Transcrição de Choque Térmico/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Humanos , Prognóstico , Fatores de Transcrição/metabolismo , Transfecção , Quinases Dyrk
12.
iScience ; 23(10): 101638, 2020 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-33103077

RESUMO

Transcription factor nuclear factor erythroid 2 p45-related factor 2 (Nrf2) and its main negative regulator, Kelch-like ECH-associated protein 1 (Keap1), are at the interface between redox and intermediary metabolism, allowing adaptation and survival under conditions of oxidative, inflammatory, and metabolic stress. Nrf2 is the principal determinant of redox homeostasis, and contributes to mitochondrial function and integrity and cellular bioenergetics. Using proteomics and lipidomics, we show that genetic downregulation of Keap1 in mice, and the consequent Nrf2 activation to pharmacologically relevant levels, leads to upregulation of carboxylesterase 1 (Ces1) and acyl-CoA oxidase 2 (Acox2), decreases triglyceride levels, and alters the lipidome. This is accompanied by downregulation of hepatic ATP-citrate lyase (Acly) and decreased levels of acetyl-CoA, a trigger for autophagy. These findings suggest that downregulation of Keap1 confers features of a fasted metabolic state, which is an important consideration in the drug development of Keap1-targeting pharmacologic Nrf2 activators.

13.
Redox Biol ; 37: 101689, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32863231

RESUMO

Oxidative stress and inflammation in the brain are two key hallmarks of neurodegenerative diseases (NDs) such as Alzheimer's, Parkinson's, Huntington's and multiple sclerosis. The axis NRF2-BACH1 has anti-inflammatory and anti-oxidant properties that could be exploited pharmacologically to obtain neuroprotective effects. Activation of NRF2 or inhibition of BACH1 are, individually, promising therapeutic approaches for NDs. Compounds with dual activity as NRF2 activators and BACH1 inhibitors, could therefore potentially provide a more robust antioxidant and anti-inflammatory effects, with an overall better neuroprotective outcome. The phytocannabinoid cannabidiol (CBD) inhibits BACH1 but lacks significant NRF2 activating properties. Based on this scaffold, we have developed a novel CBD derivative that is highly effective at both inhibiting BACH1 and activating NRF2. This new CBD derivative provides neuroprotection in cell models of relevance to Huntington's disease, setting the basis for further developments in vivo.


Assuntos
Fator 2 Relacionado a NF-E2 , Doenças Neurodegenerativas , Estresse Oxidativo , Animais , Antioxidantes/farmacologia , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Camundongos , Fator 2 Relacionado a NF-E2/metabolismo
14.
Biomolecules ; 10(10)2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32992842

RESUMO

Aberrant hyperactivation of nuclear factor erythroid 2 (NF-E2) p45-related factor 2 (NRF2) is a common event in many tumour types and associates with resistance to therapy and poor patient prognosis; however, its relevance in colorectal tumours is not well-established. Measuring the expression of surrogate genes for NRF2 activity in silico, in combination with validation in patients' samples, we show that the NRF2 pathway is upregulated in colorectal tumours and that high levels of nuclear NRF2 correlate with a poor patient prognosis. These results highlight the need to overcome the protection provided by NRF2 and present an opportunity to selectively kill cancer cells with hyperactive NRF2. Exploiting the CRISPR/Cas9 technology, we generated colorectal cancer cell lines with hyperactive NRF2 and used them to perform a drug screen. We identified AT9283, an Aurora kinase inhibitor, for its selectivity towards killing cancer cells with hyperactive NRF2 as a consequence to either genetic or pharmacological activation. Our results show that hyperactivation of NRF2 in colorectal cancer cells might present a vulnerability that could potentially be therapeutically exploited by using the Aurora kinase inhibitor AT9283.


Assuntos
Benzimidazóis/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Fator 2 Relacionado a NF-E2/genética , Inibidores de Proteínas Quinases/farmacologia , Ureia/análogos & derivados , Benzimidazóis/efeitos adversos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Avaliação Pré-Clínica de Medicamentos , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Inibidores de Proteínas Quinases/efeitos adversos , Transdução de Sinais/efeitos dos fármacos , Ureia/efeitos adversos , Ureia/farmacologia
15.
Redox Biol ; 28: 101321, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31518892

RESUMO

Cannabidiol (CBD) is a major non-psychotropic phytocannabinoid that attracted a great attention for its therapeutic potential against different pathologies including skin diseases. However, although the efficacy in preclinical models and the clinical benefits of CBD in humans have been extensively demonstrated, the molecular mechanism(s) and targets responsible for these effects are as yet unknown. Herein we characterized at the molecular level the effects of CBD on primary human keratinocytes using a combination of RNA sequencing (RNA-Seq) and sequential window acquisition of all theoretical mass spectrometry (SWATH-MS). Functional analysis revealed that CBD regulated pathways involved in keratinocyte differentiation, skin development and epidermal cell differentiation among other processes. In addition, CBD induced the expression of several NRF2 target genes, with heme oxygenase 1 (HMOX1) being the gene and the protein most upregulated by CBD. CRISPR/Cas9-mediated genome editing, RNA interference and biochemical studies demonstrated that the induction of HMOX1 mediated by CBD, involved nuclear export and proteasomal degradation of the transcriptional repressor BACH1. Notably, we showed that the effect of BACH1 on HMOX1 expression in keratinocytes is independent of NRF2. In vivo studies showed that topical CBD increased the levels of HMOX1 and of the proliferation and wound-repair associated keratins 16 and 17 in the skin of mice. Altogether, our study identifies BACH1 as a molecular target for CBD in keratinocytes and sets the basis for the use of topical CBD for the treatment of different skin diseases including atopic dermatitis and keratin disorders.


Assuntos
Antioxidantes/farmacologia , Fatores de Transcrição de Zíper de Leucina Básica/genética , Canabidiol/farmacologia , Heme Oxigenase-1/genética , Queratinócitos/citologia , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Heme Oxigenase-1/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Espectrometria de Massas , Proteólise , Análise de Sequência de RNA , Transdução de Sinais/efeitos dos fármacos
16.
Cell Mol Life Sci ; 77(13): 2621-2639, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31605148

RESUMO

NOTCH proteins constitute a receptor family with a widely conserved role in cell cycle, growing and development regulation. NOTCH1, the best characterised member of this family, regulates the expression of key genes in cell growth and angiogenesis, playing an essential role in cancer development. These observations provide a relevant rationale to propose the inhibition of the intracellular domain of NOTCH1 (Notch1-IC) as a strategy for treating various types of cancer. Notch1-IC stability is mainly controlled by post-translational modifications. FBXW7 ubiquitin E3 ligase-mediated degradation is considered one of the most relevant, being the previous phosphorylation at Thr-2512 residue required. In the present study, we describe for the first time a new regulation mechanism of the NOTCH1 signalling pathway mediated by DYRK2. We demonstrate that DYRK2 phosphorylates Notch1-IC in response to chemotherapeutic agents and facilitates its proteasomal degradation by FBXW7 ubiquitin ligase through a Thr-2512 phosphorylation-dependent mechanism. We show that DYRK2 regulation by chemotherapeutic agents has a relevant effect on the viability, motility and invasion capacity of cancer cells expressing NOTCH1. In summary, we reveal a novel mechanism of regulation for NOTCH1 which might help us to better understand its role in cancer biology.


Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptor Notch1/metabolismo , Linhagem Celular , Dano ao DNA , Humanos , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Domínios Proteicos , Receptor Notch1/química , TYK2 Quinase , Quinases Dyrk
17.
Proc Natl Acad Sci U S A ; 116(49): 24881-24891, 2019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31754034

RESUMO

Dependence on the 26S proteasome is an Achilles' heel for triple-negative breast cancer (TNBC) and multiple myeloma (MM). The therapeutic proteasome inhibitor, bortezomib, successfully targets MM but often leads to drug-resistant disease relapse and fails in breast cancer. Here we show that a 26S proteasome-regulating kinase, DYRK2, is a therapeutic target for both MM and TNBC. Genome editing or small-molecule mediated inhibition of DYRK2 significantly reduces 26S proteasome activity, bypasses bortezomib resistance, and dramatically delays in vivo tumor growth in MM and TNBC thereby promoting survival. We further characterized the ability of LDN192960, a potent and selective DYRK2-inhibitor, to alleviate tumor burden in vivo. The drug docks into the active site of DYRK2 and partially inhibits all 3 core peptidase activities of the proteasome. Our results suggest that targeting 26S proteasome regulators will pave the way for therapeutic strategies in MM and TNBC.


Assuntos
Bortezomib/farmacologia , Processos Neoplásicos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , TYK2 Quinase/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , Animais , Linhagem Celular Tumoral , Feminino , Edição de Genes , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mieloma Múltiplo , Fosforilação , Complexo de Endopeptidases do Proteassoma/genética , Inibidores de Proteassoma/farmacologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Neoplasias de Mama Triplo Negativas/patologia , Quinases Dyrk
18.
Chem Res Toxicol ; 32(12): 2538-2551, 2019 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-31746589

RESUMO

3-Nitrobenzanthrone (3-NBA) is a suspected human carcinogen present in diesel exhaust. It requires metabolic activation via nitroreduction in order to form DNA adducts and promote mutagenesis. We have determined that human aldo-keto reductases (AKR1C1-1C3) and NAD(P)H:quinone oxidoreductase 1 (NQO1) contribute equally to the nitroreduction of 3-NBA in lung epithelial cell lines and collectively represent 50% of the nitroreductase activity. The genes encoding these enzymes are induced by the transcription factor NF-E2 p45-related factor 2 (NRF2), which raises the possibility that NRF2 activation exacerbates 3-NBA toxification. Since A549 cells possess constitutively active NRF2, we examined the effect of heterozygous (NRF2-Het) and homozygous NRF2 knockout (NRF2-KO) by CRISPR-Cas9 gene editing on the activation of 3-NBA. To evaluate whether NRF2-mediated gene induction increases 3-NBA activation, we examined the effects of NRF2 activators in immortalized human bronchial epithelial cells (HBEC3-KT). Changes in AKR1C1-1C3 and NQO1 expression by NRF2 knockout or use of NRF2 activators were confirmed by qPCR, immunoblots, and enzyme activity assays. We observed decreases in 3-NBA activation in the A549 NRF2 KO cell lines (53% reduction in A549 NRF2-Het cells and 82% reduction in A549 NRF2-KO cells) and 40-60% increases in 3-NBA bioactivation due to NRF2 activators in HBEC3-KT cells. Together, our data suggest that activation of the transcription factor NRF2 exacerbates carcinogen metabolism following exposure to diesel exhaust which may lead to an increase in 3-NBA-derived DNA adducts.


Assuntos
Poluentes Atmosféricos/toxicidade , Benzo(a)Antracenos/toxicidade , Regulação da Expressão Gênica/fisiologia , Mutagênicos/toxicidade , Fator 2 Relacionado a NF-E2/metabolismo , 20-Hidroxiesteroide Desidrogenases/genética , Células A549 , Ativação Metabólica , Poluentes Atmosféricos/metabolismo , Membro C3 da Família 1 de alfa-Ceto Redutase/genética , Benzo(a)Antracenos/metabolismo , Brônquios/citologia , Células Epiteliais/efeitos dos fármacos , Técnicas de Inativação de Genes , Humanos , Hidroxiesteroide Desidrogenases/genética , Imidazóis/farmacologia , Isotiocianatos/farmacologia , Mutagênicos/metabolismo , NAD(P)H Desidrogenase (Quinona)/genética , Fator 2 Relacionado a NF-E2/agonistas , Fator 2 Relacionado a NF-E2/genética , Ácido Oleanólico/análogos & derivados , Ácido Oleanólico/farmacologia , Estresse Oxidativo/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sulfóxidos
19.
Sci Rep ; 7(1): 11023, 2017 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-28887499

RESUMO

Autophagy plays a critical role in the maintenance of cellular homeostasis by degrading proteins, lipids and organelles. Autophagy is activated in response to stress, but its regulation in the context of other stress response pathways, such as those mediated by heat shock factor 1 (HSF1) and nuclear factor-erythroid 2 p45-related factor 2 (NRF2), is not well understood. We found that the Michael acceptor bis(2-hydoxybenzylidene)acetone (HBB2), a dual activator of NRF2 and HSF1, protects against the development of UV irradiation-mediated cutaneous squamous cell carcinoma in mice. We further show that HBB2 is an inducer of autophagy. In cells, HBB2 increases the levels of the autophagy-cargo protein p62/sequestosome 1, and the lipidated form of microtubule-associated protein light chain 3 isoform B. Activation of autophagy by HBB2 is impaired in NRF2-deficient cells, which have reduced autophagic flux and low basal and induced levels of p62. Conversely, HSF1-deficient cells have increased autophagic flux under both basal as well as HBB2-induced conditions, accompanied by increased p62 levels. Our findings suggest that NRF2 and HSF1 have opposing roles during autophagy, and illustrate the existence of tight mechanistic links between the cellular stress responses.


Assuntos
Autofagia , Regulação da Expressão Gênica , Fatores de Transcrição de Choque Térmico/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Animais , Linhagem Celular , Humanos , Camundongos , Neoplasias Experimentais/patologia , Osteoblastos/fisiologia , Neoplasias Cutâneas/patologia
20.
Mol Cell Biol ; 36(18): 2403-17, 2016 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-27354066

RESUMO

Heat shock factor 1 (HSF1) monitors the structural integrity of the proteome. Phosphorylation at S326 is a hallmark for HSF1 activation, but the identity of the kinase(s) phosphorylating this site has remained elusive. We show here that the dietary agent phenethyl isothiocyanate (PEITC) inhibits heat shock protein 90 (Hsp90), the main negative regulator of HSF1; activates p38 mitogen-activated protein kinase (MAPK); and increases S326 phosphorylation, trimerization, and nuclear translocation of HSF1, and the transcription of a luciferase reporter, as well as the endogenous prototypic HSF1 target Hsp70. In vitro, all members of the p38 MAPK family rapidly and stoichiometrically catalyze the S326 phosphorylation. The use of stable knockdown cell lines and inhibitors indicated that among the p38 MAPKs, p38γ is the principal isoform responsible for the phosphorylation of HSF1 at S326 in cells. A protease-mass spectrometry approach confirmed S326 phosphorylation and unexpectedly revealed that p38 MAPK also catalyzes the phosphorylation of HSF1 at S303/307, previously known repressive posttranslational modifications. Thus, we have identified p38 MAPKs as highly efficient catalysts for the phosphorylation of HSF1. Furthermore, our findings suggest that the magnitude and persistence of activation of p38 MAPK are important determinants of the extent and duration of the heat shock response.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Choque Térmico HSP70/metabolismo , Serina/metabolismo , Fatores de Transcrição/genética , Transcrição Gênica , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Linhagem Celular , Proteínas de Ligação a DNA/química , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Células HeLa , Fatores de Transcrição de Choque Térmico , Humanos , Isotiocianatos/farmacologia , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Fosforilação , Multimerização Proteica , Transporte Proteico , Fatores de Transcrição/química
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