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1.
J Biol Chem ; 299(4): 103023, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36805338

RESUMO

Raf kinase inhibitor protein (RKIP) is a multifunctional modulator of intracellular signal transduction. Although most of its functions have been considered cytosolic, we show here that the localization of RKIP is primarily nuclear in both growing and quiescent Madin-Darby canine kidney epithelial cells and in Cal-51 and BT-20 human breast cancer cells. We have identified a putative bipartite nuclear localization signal (NLS) in RKIP that maps to the surface of the protein surrounding a known regulatory region. Like classical NLS sequences, the putative NLS of RKIP is rich in arginine and lysine residues. Deletion of and point mutations in the putative NLS lead to decreased nuclear localization. Point mutation of all the basic residues in the putative NLS of RKIP particularly strongly reduces nuclear localization. We found consistent results in reexpression experiments with wildtype or mutant RKIP in RKIP-silenced cells. A fusion construct of the putative NLS of RKIP alone to a heterologous reporter protein leads to nuclear localization of the fusion protein, demonstrating that this sequence alone is sufficient for import into the nucleus. We found that RKIP interacts with the nuclear transport factor importin α in BT-20 and MDA-MB-231 human breast cancer cells, suggesting importin-mediated active nuclear translocation. Evaluating the biological function of nuclear localization of RKIP, we found that the presence of the putative NLS is important for the role of RKIP in mitotic checkpoint regulation in MCF-7 human breast cancer cells. Taken together, these findings suggest that a bipartite NLS in RKIP interacts with importin α for active transport of RKIP into the nucleus and that this process may be involved in the regulation of mitotic progression.


Assuntos
Sinais de Localização Nuclear , Proteína de Ligação a Fosfatidiletanolamina , alfa Carioferinas , Animais , Cães , Humanos , Transporte Ativo do Núcleo Celular , alfa Carioferinas/genética , alfa Carioferinas/metabolismo , Núcleo Celular/metabolismo , Sinais de Localização Nuclear/genética , Sinais de Localização Nuclear/metabolismo , Proteína de Ligação a Fosfatidiletanolamina/genética , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Células Madin Darby de Rim Canino
2.
Nature ; 559(7715): 637-641, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-30022161

RESUMO

Diabetes is a complex metabolic syndrome that is characterized by prolonged high blood glucose levels and frequently associated with life-threatening complications1,2. Epidemiological studies have suggested that diabetes is also linked to an increased risk of cancer3-5. High glucose levels may be a prevailing factor that contributes to the link between diabetes and cancer, but little is known about the molecular basis of this link and how the high glucose state may drive genetic and/or epigenetic alterations that result in a cancer phenotype. Here we show that hyperglycaemic conditions have an adverse effect on the DNA 5-hydroxymethylome. We identify the tumour suppressor TET2 as a substrate of the AMP-activated kinase (AMPK), which phosphorylates TET2 at serine 99, thereby stabilizing the tumour suppressor. Increased glucose levels impede AMPK-mediated phosphorylation at serine 99, which results in the destabilization of TET2 followed by dysregulation of both 5-hydroxymethylcytosine (5hmC) and the tumour suppressive function of TET2 in vitro and in vivo. Treatment with the anti-diabetic drug metformin protects AMPK-mediated phosphorylation of serine 99, thereby increasing TET2 stability and 5hmC levels. These findings define a novel 'phospho-switch' that regulates TET2 stability and a regulatory pathway that links glucose and AMPK to TET2 and 5hmC, which connects diabetes to cancer. Our data also unravel an epigenetic pathway by which metformin mediates tumour suppression. Thus, this study presents a new model for how a pernicious environment can directly reprogram the epigenome towards an oncogenic state, offering a potential strategy for cancer prevention and treatment.


Assuntos
Adenilato Quinase/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Diabetes Mellitus/metabolismo , Glucose/metabolismo , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/metabolismo , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Animais , DNA/química , DNA/metabolismo , Metilação de DNA , Diabetes Mellitus/genética , Dioxigenases , Estabilidade Enzimática , Epigênese Genética , Hemoglobinas Glicadas/análise , Humanos , Hiperglicemia/metabolismo , Metformina/farmacologia , Metformina/uso terapêutico , Camundongos , Camundongos Nus , Neoplasias/tratamento farmacológico , Neoplasias/genética , Fosforilação , Fosfosserina/metabolismo , Especificidade por Substrato , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Biochem Biophys Res Commun ; 589: 240-246, 2022 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-34929447

RESUMO

Estrogen signaling plays important roles in diverse physiological and pathophysiological processes. However, the relationship between estrogen signaling and epigenetic regulation is not fully understood. Here, we explored the effect of estrogen signaling on the expression of Ten-Eleven Translocation (TET) family genes and DNA hydroxylmethylation in estrogen receptor alpha positive (ERα+) breast cancer cells. By analyzing the RNA-seq data, we identified TET2 as an estradiol (E2)-responsive gene in ERα+ MCF7 cells. RT-qPCR and Western blot analyses confirmed that both the mRNA and protein levels of TET2 gene were upregulated in MCF7 cells by E2 treatment. ChIP-seq and qPCR analyses showed that the enrichment of ERα and H3K27ac on the upstream regulatory regions of TET2 gene was increased in MCF7 cells upon E2 treatment. Moreover, E2 treatment also led to a significant increase in the global 5-hydroxymethylcytosine (5hmC) level, while knockout of TET2 abolished such E2-induced 5hmC increase. Conversely, treatment with ICI 182780, a potent and selective estrogen receptor degrader (SERD), inhibited TET2 gene expression and down-regulated the 5hmC level in MCF7 cells. Taken together, our study identified an ERα/TET2/5hmC epigenetic pathway, which may participate in the estrogen-associated physiological and pathophysiological processes.


Assuntos
5-Metilcitosina/metabolismo , Neoplasias da Mama/genética , Proteínas de Ligação a DNA/genética , Dioxigenases/genética , Estrogênios/metabolismo , Regulação Neoplásica da Expressão Gênica , Transdução de Sinais , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Dioxigenases/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Elementos Facilitadores Genéticos/genética , Estradiol/farmacologia , Receptor alfa de Estrogênio/metabolismo , Feminino , Fulvestranto/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Oxirredução , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos
4.
Rev Esp Enferm Dig ; 114(1): 59-61, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34470460

RESUMO

Acute cholangitis is a life-threatening infection, and several features are associated with a worse prognosis if prompt treatment is not started. Eighty-six subjects with acute cholangitis were included in a retrospective analysis. Patients had a median age of 53 years (range: 34.5 to 70 years) and 55 (64.0 %) were female. Of the entire cohort, 16 patients had a history of diabetes mellitus. Regarding acute cholangitis severity, 17 (19.8 %) cases were classified as severe, 41 (47.7 %) as moderate, and 28 (32.6 %) as mild.


Assuntos
Infecções Bacterianas , Colangite , Diabetes Mellitus Tipo 2 , Infecções Intra-Abdominais , Doença Aguda , Adulto , Idoso , Bactérias , Infecções Bacterianas/complicações , Colangite/etiologia , Diabetes Mellitus Tipo 2/complicações , Feminino , Humanos , Infecções Intra-Abdominais/complicações , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Fatores de Risco , Índice de Gravidade de Doença
5.
Blood ; 129(16): 2233-2245, 2017 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-28096095

RESUMO

Dysregulated oncogenic serine/threonine kinases play a pathological role in diverse forms of malignancies, including multiple myeloma (MM), and thus represent potential therapeutic targets. Here, we evaluated the biological and functional role of p21-activated kinase 4 (PAK4) and its potential as a new target in MM for clinical applications. PAK4 promoted MM cell growth and survival via activation of MM survival signaling pathways, including the MEK-extracellular signal-regulated kinase pathway. Furthermore, treatment with orally bioavailable PAK4 allosteric modulator (KPT-9274) significantly impacted MM cell growth and survival in a large panel of MM cell lines and primary MM cells alone and in the presence of bone marrow microenvironment. Intriguingly, we have identified FGFR3 as a novel binding partner of PAK4 and observed significant activity of KPT-9274 against t(4;14)-positive MM cells. This set of data supports PAK4 as an oncogene in myeloma and provide the rationale for the clinical evaluation of PAK4 modulator in myeloma.


Assuntos
Regulação Neoplásica da Expressão Gênica , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Inibidores de Proteínas Quinases/farmacologia , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Quinases Ativadas por p21/genética , Regulação Alostérica , Animais , Apoptose/efeitos dos fármacos , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/enzimologia , Células da Medula Óssea/patologia , Caspases/genética , Caspases/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cromossomos Humanos Par 14 , Cromossomos Humanos Par 4 , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/enzimologia , Leucócitos Mononucleares/patologia , Camundongos , Camundongos Nus , Terapia de Alvo Molecular , Mieloma Múltiplo/enzimologia , Mieloma Múltiplo/patologia , Cultura Primária de Células , Ligação Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Translocação Genética , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases Ativadas por p21/antagonistas & inibidores , Quinases Ativadas por p21/metabolismo
6.
Am J Hematol ; 91(9): 923-30, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27312795

RESUMO

Primary mediastinal B-cell lymphoma (PMBL) is an entity of B-cell lymphoma distinct from the other molecular subtypes of diffuse large B-cell lymphoma (DLBCL). We investigated the prevalence, specificity, and clinical relevance of mutations of XPO1, which encodes a member of the karyopherin-ß nuclear transporters, in a large cohort of PMBL. PMBL cases defined histologically or by gene expression profiling (GEP) were sequenced and the XPO1 mutational status was correlated to genetic and clinical characteristics. The XPO1 mutational status was also assessed in DLBCL, Hodgkin lymphoma (HL) and mediastinal gray-zone lymphoma (MGZL).The biological impact of the mutation on Selective Inhibitor of Nuclear Export (SINE) compounds (KPT-185/330) sensitivity was investigated in vitro. XPO1 mutations were present in 28/117 (24%) PMBL cases and in 5/19 (26%) HL cases but absent/rare in MGZL (0/20) or DLBCL (3/197). A higher prevalence (50%) of the recurrent codon 571 variant (p.E571K) was observed in GEP-defined PMBL and was associated with shorter PFS. Age, International Prognostic Index and bulky mass were similar in XPO1 mutant and wild-type cases. KPT-185 induced a dose-dependent decrease in cell proliferation and increased cell-death in PMBL cell lines harboring wild type or XPO1 E571K mutant alleles. Experiments in transfected U2OS cells further confirmed that the XPO1 E571K mutation does not have a drastic impact on KPT-330 binding. To conclude the XPO1 E571K mutation represents a genetic hallmark of the PMBL subtype and serves as a new relevant PMBL biomarker. SINE compounds appear active for both mutated and wild-type protein. Am. J. Hematol. 91:923-930, 2016. © 2016 Wiley Periodicals, Inc.


Assuntos
Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Carioferinas/genética , Linfoma de Células B/genética , Mutação , Receptores Citoplasmáticos e Nucleares/genética , Acrilatos/farmacologia , Adolescente , Adulto , Idoso , Biomarcadores , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Doença de Hodgkin/genética , Humanos , Hidrazinas/farmacologia , Carioferinas/antagonistas & inibidores , Carioferinas/fisiologia , Linfoma de Células B/mortalidade , Linfoma de Células B/patologia , Masculino , Neoplasias do Mediastino/genética , Neoplasias do Mediastino/mortalidade , Pessoa de Meia-Idade , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/fisiologia , Análise de Sequência de DNA , Triazóis/farmacologia , Adulto Jovem , Proteína Exportina 1
7.
Epigenetics ; 17(10): 1180-1194, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-34689714

RESUMO

Aberrant DNA methylation is an epigenetic hallmark of malignant tumours. The DNA methylation level is regulated by not only DNA methyltransferases (DNMTs) but also Ten-Eleven Translocation (TET) family proteins. However, the exact role of TET genes in breast cancer remains controversial. Here, we uncover that the ERα-positive breast cancer patients with high TET2 mRNA expression had better overall survival rates. Consistently, knockout of TET2 promotes the tumorigenesis of ERα-positive MCF7 breast cancer cells. Mechanistically, TET2 loss leads to aberrant DNA methylation (gain of 5mC) at a large proportion of enhancers, accompanied by significant reduction in H3K4me1 and H3K27ac enrichment. By analysing the epigenetically reprogrammed enhancers, we identify oestrogen responsive element (ERE) as one of the enriched motifs of transcriptional factors. Importantly, TET2 loss impairs 17beta-oestradiol (E2)-induced transcription of the epigenetically reprogrammed EREs-associated genes through attenuating the binding of ERα. Taken together, these findings shed light on our understanding of the epigenetic mechanisms underlying the enhancer reprogramming during breast cancer pathogenesis.


Assuntos
Neoplasias da Mama , Dioxigenases , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , DNA/metabolismo , Metilação de DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dioxigenases/metabolismo , Elementos Facilitadores Genéticos , Epigênese Genética , Estradiol , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Estrogênios , Feminino , Humanos , Metiltransferases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro/metabolismo
8.
Arthritis Rheumatol ; 74(8): 1363-1375, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35333447

RESUMO

OBJECTIVE: To investigate the hypothesis that selective inhibitors of nuclear export (SINE compounds), recently approved for treatment of refractory plasma cell (PC) malignancy, may have potential in the treatment of lupus. METHODS: Female NZB/NZW mice were treated with the SINE compound KPT-350 or vehicle control. Tissue specimens were harvested and analyzed by flow cytometry, using standard markers. Nephritis was monitored by determining the proteinuria score and by histologic analysis of kidney specimens. Serum anti-double-stranded DNA (anti-dsDNA) levels were measured by enzyme-linked immunosorbent assay, and total numbers of IgG-secreting and dsDNA-specific antibody-secreting cells were assessed by enzyme-linked immunospot assay. RESULTS: KPT-350 abrogated murine lupus nephritis at both early and late stages of the disease and rapidly impaired generation of autoreactive PCs in germinal centers (GCs). SINE compounds inhibited the production of NF-κB-driven homeostatic chemokines by stromal cells, altering splenic B and T cell strategic positioning and significantly reducing follicular helper T cell, GC B cell, and autoreactive PC counts. KPT-350 also decreased levels of cytokines and chemokines involved in PC survival and recruitment in the kidney of lupus-prone mice. Exportin 1, the target of SINE compounds, was detected in GCs of human tonsils, splenic B cells of lupus patients, and multiple B cell subsets in the kidneys of patients with lupus nephritis. CONCLUSION: Collectively, our results provide support for the therapeutic potential of SINE compounds, via their targeting of several molecular and cellular pathways critical in lupus pathogenesis, including autoantibody production by plasma cells.


Assuntos
Lúpus Eritematoso Sistêmico , Nefrite Lúpica , Transporte Ativo do Núcleo Celular , Animais , Autoanticorpos , Modelos Animais de Doenças , ELISPOT , Feminino , Humanos , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Camundongos , Camundongos Endogâmicos NZB , Plasmócitos
9.
Chemistry ; 17(2): 649-54, 2011 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-21207585

RESUMO

A library of 91 heterocyclic compounds composed of 16 distinct scaffolds has been synthesized through a sequence of phosphine-catalyzed ring-forming reactions, Tebbe reactions, Diels-Alder reactions, and, in some cases, hydrolysis. This effort in diversity-oriented synthesis produced a collection of compounds that exhibited high levels of structural variation both in terms of stereochemistry and the range of scaffolds represented. A simple but powerful sequence of reactions thus led to a high-diversity library of relatively modest size with which to explore biologically relevant regions of chemical space. From this library, several molecules were identified that inhibit the migration and invasion of breast cancer cells and may serve as leads for the development of antimetastatic agents.


Assuntos
Antineoplásicos/síntese química , Compostos Heterocíclicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Catálise , Técnicas de Química Combinatória , Ciclização , Feminino , Compostos Heterocíclicos/química , Compostos Heterocíclicos/farmacologia , Humanos , Estrutura Molecular , Fosfinas/química
10.
Cells ; 9(10)2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-33023194

RESUMO

Triple-negative breast cancer (TNBC) is an aggressive disease with poor prognosis and limited therapeutic options. Recent advances in the immunotherapy field have enabled the development of new treatment strategies, among which the use of bispecific antibodies (BsAbs), able to redirect T cells against tumors, has shown promising results. In particular, a BsAb that uses TNF-related apoptosis-inducing ligand receptor 2 (TRAIL-R2) as a target was constructed and demonstrated good results in redirecting CD3+ T cells to kill TRAIL-R2-expressing TNBC cells. In the present study, we investigated whether treatment with selinexor, a selective inhibitor of nuclear export (SINE) targeting exportin-1/chromosome maintenance protein 1 (XPO1/CRM1), could potentiate the antitumor activity of this BsAb. In combination experiments, we found that selinexor-exposed TNBC cells exhibited greater growth inhibition when treated with the TRAIL-R2xCD3 BsAb than that expected by simple additivity. Similarly, the apoptosis rate in selinexor/TRAIL-R2xCD3 BsAb-treated TNBC cells was significantly higher than that observed after exposure to either single agent. Together, our results suggest that the combination of selinexor and TRAIL-R2xCD3 BsAb can be a viable anticancer strategy and indicate this treatment as a promising therapeutic option for TNBC patients.


Assuntos
Anticorpos Biespecíficos/fisiologia , Hidrazinas/uso terapêutico , Triazóis/uso terapêutico , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Linhagem Celular Tumoral , Feminino , Humanos , Hidrazinas/farmacologia , Triazóis/farmacologia
11.
Blood Adv ; 4(3): 586-598, 2020 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-32045477

RESUMO

The selective inhibitor of nuclear export (SINE) compounds selinexor (KPT-330) and eltanexor (KPT-8602) are from a novel class of small molecules that target exportin-1 (XPO1 [CRM1]), an essential nucleo-cytoplasmic transport protein responsible for the nuclear export of major tumor suppressor proteins and growth regulators such as p53, p21, and p27. XPO1 also affects the translation of messenger RNAs for critical oncogenes, including MYC, BCL2, MCL1, and BCL6, by blocking the export of the translation initiation factor eIF4E. Early trials with venetoclax (ABT-199), a potent, selective inhibitor of BCL2, have revealed responses across a variety of hematologic malignancies. However, many tumors are not responsive to venetoclax. We used models of acute myeloid leukemia (AML) and diffuse large B-cell lymphoma (DLBCL) to determine in vitro and in vivo responses to treatment with venetoclax and SINE compounds combined. Cotreatment with venetoclax and SINE compounds demonstrated loss of viability in multiple cell lines. Further in vitro analyses showed that this enhanced cell death was the result of an increase in apoptosis that led to a loss of clonogenicity in methylcellulose assays, coinciding with activation of p53 and loss of MCL1. Treatment with SINE compounds and venetoclax combined led to a reduction in tumor growth in both AML and DLBCL xenografts. Immunohistochemical analysis of tissue sections revealed that the reduction in tumor cells was partly the result of an induction of apoptosis. The enhanced effects of this combination were validated in primary AML and DLBCL patient cells. Our studies reveal synergy with SINE compounds and venetoclax in aggressive hematologic malignancies and provide a rationale for pursuing this approach in a clinical trial.


Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes , Neoplasias Hematológicas , Transporte Ativo do Núcleo Celular , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular Tumoral , Neoplasias Hematológicas/tratamento farmacológico , Humanos , Sulfonamidas
12.
Sci Adv ; 5(8): eaaw2880, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31489368

RESUMO

Alzheimer's disease (AD), a progressive neurodegenerative disorder, is the most common untreatable form of dementia. Identifying molecular biomarkers that allow early detection remains a key challenge in the diagnosis, treatment, and prognostic evaluation of the disease. Here, we report a novel experimental and analytical model characterizing epigenetic alterations during AD onset and progression. We generated the first integrated base-resolution genome-wide maps of the distribution of 5-methyl-cytosine (5mC), 5-hydroxymethyl-cytosine (5hmC), and 5-formyl/carboxy-cytosine (5fC/caC) in normal and AD neurons. We identified 27 AD region-specific and 39 CpG site-specific epigenetic signatures that were independently validated across our familial and sporadic AD models, and in an independent clinical cohort. Thus, our work establishes a new model and strategy to study the epigenetic alterations underlying AD onset and progression and provides a set of highly reliable AD-specific epigenetic signatures that may have early diagnostic and prognostic implications.


Assuntos
Doença de Alzheimer/genética , Metilação de DNA/genética , DNA/genética , Epigênese Genética/genética , 5-Metilcitosina/metabolismo , Idoso , Doença de Alzheimer/metabolismo , Biomarcadores/metabolismo , Citosina/metabolismo , Progressão da Doença , Epigenômica/métodos , Feminino , Humanos , Masculino , Neurônios/metabolismo
14.
Oncotarget ; 9(56): 30773-30786, 2018 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-30112106

RESUMO

INTRODUCTION: The goal of this study was to examine the effects of selinexor, an inhibitor of exportin-1 mediated nuclear export, on DNA damage repair and to evaluate the cytotoxic effects of selinexor in combination with DNA damaging agents (DDAs) in cancer cells. RESULTS: Selinexor reduced the expression of DNA damage repair (DDR) proteins. This did not induce significant DNA damage in tested cell lines. Inhibition of DDR protein expression resulted in enhanced cancer cell death when cells were pretreated with DDAs. In contrast, enhanced cell death was not detected in cells that were pretreated with selinexor then with DDAs. In vivo, single-agent selinexor, docetaxel, or cisplatin treatment resulted in 66.7%, 51.5%, and 26.6% tumor growth inhibition (TGI), respectively, in an MDA-MB-231 xenograft model. Consequently, combination treatment with docetaxel or cisplatin followed by selinexor in vivo resulted in 93.9% and 103.4% TGI, respectively. Immunohistochemical staining and immunoblot analysis of tumor sections confirmed reduced expression of DDR proteins. CONCLUSION: Selinexor treatment inhibited DDR mechanisms in cancer cell lines and therefore potentiated DNA damage-based therapy. The sequential combination of DDAs followed by selinexor increased cancer cell death. This combination is superior to each individual therapy and has a mechanistic rationale as a novel anticancer strategy. METHODS: Cancer cells treated with selinexor ± DDAs were analyzed using reverse phase protein arrays, immunoblots, quantitative PCR and immunofluorescence. Mice bearing MDA-MB-231 tumors were treated with subtherapeutic doses of selinexor, cisplatin, docetaxel and selinexor in combination with either cisplatin or docetaxel. Tumor growth was evaluated for 25 days.

15.
Biochem Pharmacol ; 147: 93-103, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29155058

RESUMO

The XPO1/CRM1 inhibitor selinexor (KPT-330), is currently being evaluated in multiple clinical trials as an anticancer agent. XPO1 participates in the nuclear export of FoxO-1, which we previously found to be decreased in platinum-resistant ovarian carcinoma. The aim of this study was to determine whether enriching FoxO-1 nuclear localization using selinexor would increase ovarian cancer cell sensitivity to cisplatin. Selinexor, as a single agent, displayed a striking antiproliferative effect in different ovarian carcinoma cell lines. A schedule-dependent synergistic effect of selinexor in combination with cisplatin was found in cisplatin-sensitive IGROV-1, the combination efficacy being more evident in sensitive than in the resistant cells. In IGROV-1 cells, the combination was more effective when selinexor followed cisplatin exposure. A modulation of proteins involved in apoptosis (p53, Bax) and in cell cycle progression (p21WAF1) was found by Western blotting. Selinexor-treated cells exhibited enriched FoxO-1 nuclear staining. Knock-down experiments with RNA interference indicated that FOXO1-silenced cells displayed a reduced sensitivity to selinexor. FOXO1 silencing also tended to reduce the efficacy of the drug combination at selected cisplatin concentrations. Selinexor significantly inhibited tumor growth, induced FoxO-1 nuclear localization and improved the efficacy of cisplatin in IGROV-1 xenografts. Taken together, our results support FoxO-1 as one of the key factors promoting sensitivity towards selinexor and the synergistic interaction between cisplatin and selinexor in ovarian carcinoma cells with selected molecular backgrounds, highlighting the need for treatment regimens tailored to the molecular tumor features.


Assuntos
Cisplatino/administração & dosagem , Proteína Forkhead Box O1/metabolismo , Hidrazinas/administração & dosagem , Carioferinas/antagonistas & inibidores , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Triazóis/administração & dosagem , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Apoptose , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Proteína Forkhead Box O1/genética , Humanos , Camundongos , Camundongos Nus , Neoplasias Ovarianas/genética , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Proteína Exportina 1
16.
Oncotarget ; 9(39): 25529-25544, 2018 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-29876006

RESUMO

Multiple myeloma (MM) is a plasma cell neoplasm that results in over 11,000 deaths in the United States annually. The backbone therapy for the treatment of MM patients almost always includes combinations with corticosteroids such as dexamethasone (DEX). We found that DEX in combination with selinexor, an inhibitor of exportin-1 (XPO1) activity, synergistically inhibits the mTOR pathway and subsequently promotes cell death in MM cells. Specifically, we show that selinexor induces the expression of the glucocorticoid receptor (GR) and when combined with dexamethasone increases GR transcriptional activity. Moreover, we found that key downstream targets of the mTOR pathway are deregulated by the combination and identified a mechanism in which GR enhances the expression of REDD1 in GR positive cells while suppressing mTOR activity and cell viability. While the single agent activity of selinexor in MM cells appears to be GR-independent, synergy with DEX depends on GR expression. These data suggest that patients with tumor cells that are GR positive will benefit substantially from the combination. The current findings are consistent with the beneficial therapeutic outcome in patients with MM when treated with the combination of selinexor and DEX. In addition, they provide a rationale for testing GR and REDD1 as predictive and prognostic markers of response, respectively, for patients treated with this beneficial combination.

17.
Oncotarget ; 9(82): 35327-35342, 2018 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-30450161

RESUMO

Emerging studies have shown that the expression of AR splice variants (ARv) lacking ligand-binding domain is associated with castrate-resistant prostate cancer (CRPC) and higher risk of tumor metastasis and recurrence. Nuclear export protein XPO1 regulates the nuclear localization of many proteins including tumor suppressor proteins. Increased XPO1 in prostate cancer is associated with a high Gleason score and bone metastasis. In this study, we found that high expression of AR splice variant 7 (AR-v7) was correlated with increased XPO1 expression. Silencing of XPO1 by RNAi or treatment with Selective Inhibitor of Nuclear Export (SINE) compounds selinexor and eltanexor (KPT-8602) down-regulated the expression of AR, AR-v7 and ARv567es at mRNA and protein levels. XPO1 silencing also inhibited the expression of AR and ARv regulators including FOXA1, Src, Vav3, MED1 and Sam68, leading to the suppression of ARv and AR target genes, UBE2C and PSA. By targeting XPO1/ARv signaling, SINE suppressed prostate cancer (PCa) growth in vitro and in vivo and potentiated the anti-cancer activity of anti-AR agents, enzalutamide and abiraterone. Therefore, XPO1 inhibition could be a novel promising agent used in combination with conventional chemotherapeutics and AR-targeted therapy for the better treatment of PCa, especially CRPC.

18.
Sci Transl Med ; 10(447)2018 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-29950445

RESUMO

Patient mortality rates have remained stubbornly high (40%) for the past 35 years in head and neck squamous cell carcinoma (HNSCC) due to inherent or acquired drug resistance. Thus, a critical issue in advanced SCC is to identify and target the mechanisms that contribute to therapy resistance. We report that the transcriptional inhibitor, E2F7, is mislocalized to the cytoplasm in >80% of human HNSCCs, whereas the transcriptional activator, E2F1, retains localization to the nucleus in SCC. This results in an imbalance in the control of E2F-dependent targets such as SPHK1, which is derepressed and drives resistance to anthracyclines in HNSCC. Specifically, we show that (i) E2F7 is subject to exportin 1 (XPO1)-dependent nuclear export, (ii) E2F7 is selectively mislocalized in most of SCC and multiple other tumor types, (iii) mislocalization of E2F7 in HNSCC causes derepression of Sphk1 and drives anthracycline resistance, and (iv) anthracycline resistance can be reversed with a clinically available inhibitor of XPO1, selinexor, in xenotransplant models of HNSCC. Thus, we have identified a strategy to repurpose anthracyclines for use in SCC. More generally, we provide a strategy to restore the balance of E2F1 (activator) and E2F7 (inhibitor) activity in cancer.


Assuntos
Antraciclinas/farmacologia , Núcleo Celular/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Fator de Transcrição E2F7/metabolismo , Carioferinas/antagonistas & inibidores , Terapia de Alvo Molecular , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Doxorrubicina/farmacologia , Fator de Transcrição E2F1/metabolismo , Humanos , Carioferinas/metabolismo , Camundongos Endogâmicos NOD , Camundongos SCID , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteína Exportina 1
19.
J Clin Invest ; 128(10): 4260-4279, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30015632

RESUMO

Nucleophosmin (NPM1) is among the most frequently mutated genes in acute myeloid leukemia (AML). It is not known, however, how the resulting oncoprotein mutant NPM1 is leukemogenic. To reveal the cellular machinery in which NPM1 participates in myeloid cells, we analyzed the endogenous NPM1 protein interactome by mass spectrometry and discovered abundant amounts of the master transcription factor driver of monocyte lineage differentiation PU.1 (also known as SPI1). Mutant NPM1, which aberrantly accumulates in cytoplasm, dislocated PU.1 into cytoplasm with it. CEBPA and RUNX1, the master transcription factors that collaborate with PU.1 to activate granulomonocytic lineage fates, remained nuclear; but without PU.1, their coregulator interactions were toggled from coactivators to corepressors, repressing instead of activating more than 500 granulocyte and monocyte terminal differentiation genes. An inhibitor of nuclear export, selinexor, by locking mutant NPM1/PU.1 in the nucleus, activated terminal monocytic fates. Direct depletion of the corepressor DNA methyltransferase 1 (DNMT1) from the CEBPA/RUNX1 protein interactome using the clinical drug decitabine activated terminal granulocytic fates. Together, these noncytotoxic treatments extended survival by more than 160 days versus vehicle in a patient-derived xenotransplant model of NPM1/FLT3-mutated AML. In sum, mutant NPM1 represses monocyte and granulocyte terminal differentiation by disrupting PU.1/CEBPA/RUNX1 collaboration, a transforming action that can be reversed by pharmacodynamically directed dosing of clinical small molecules.


Assuntos
Granulócitos/metabolismo , Leucemia Mieloide Aguda/metabolismo , Monócitos/metabolismo , Mutação , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Granulócitos/patologia , Xenoenxertos , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Camundongos , Monócitos/patologia , Proteínas de Neoplasias/genética , Transplante de Neoplasias , Proteínas Nucleares/genética , Nucleofosmina , Células THP-1 , Fatores de Transcrição/genética
20.
Oncotarget ; 8(67): 111225-111245, 2017 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-29340049

RESUMO

BACKGROUND AND AIMS: Docetaxel (DTX) modestly increases patient survival of metastatic castration-resistant prostate cancer (mCRPC) due to insurgence of pharmacological resistance. Deregulation of Chromosome Region Maintenance (CRM-1)/ exportin-1 (XPO-1)-mediated nuclear export may play a crucial role in this phenomenon. MATERIAL AND METHODS: Here, we evaluated the effects of two Selective Inhibitor of Nuclear Export (SINE) compounds, selinexor (KPT-330) and KPT-251, in association with DTX by using 22rv1, PC3 and DU145 cell lines with their. DTX resistant derivatives. RESULTS AND CONCLUSIONS: We show that DTX resistance may involve overexpression of ß-III tubulin (TUBB3) and P-glycoprotein as well as increased cytoplasmic accumulation of Foxo3a. Increased levels of XPO-1 were also observed in DTX resistant cells suggesting that SINE compounds may modulate DTX effectiveness in sensitive cells as well as restore the sensitivity to DTX in resistant ones. Pretreatment with SINE compounds, indeed, sensitized to DTX through increased tumor shrinkage and apoptosis by preventing DTX-induced cell cycle arrest. Basally SINE compounds induce FOXO3a activation and nuclear accumulation increasing the expression of FOXO-responsive genes including p21, p27 and Bim causing cell cycle arrest. SINE compounds-catenin and survivin supporting apoptosis. ßdown-regulated Cyclin D1, c-myc, Nuclear sequestration of p-Foxo3a was able to reduce ABCB1 and TUBB3 H2AX levels, prolonged γ expression. Selinexor treatment increased DTX-mediated double strand breaks (DSB), and reduced the levels of DNA repairing proteins including DNA PKc and Topo2A. Our results provide supportive evidence for the therapeutic use of SINE compounds in combination with DTX suggesting their clinical use in mCRPC patients.

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