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1.
Am Heart J ; 253: 20-29, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-35728658

RESUMO

Presently, acute pharmacological termination of paroxysmal supraventricular tachycardia (PSVT) unresponsive to patient-initiated vagal maneuvers requires in-hospital intervention. Etripamil, a fast-acting, nondihydropyridine, L-type calcium channel blocker, is formulated as an intranasal spray to rapidly terminate atrioventricular (AV) nodal-dependent PSVT in a medically unsupervised setting. The NODE-301 study did not meet its prespecified primary end point of PSVT conversion over 5 hours following a single dose of etripamil 70 mg. However, analysis at earlier time points demonstrated etripamil treatment effect during the first 30 minutes, consistent with its expected rapid onset and short duration of action. This led to the design of the RAPID study, which includes a new dosing regimen (up to 2 etripamil 70 mg doses separated by 10 minutes) to increase the exposure and pharmacodynamic effect of etripamil. The primary objective of RAPID (NCT03464019) is to determine if etripamil self-administered by patients is superior to placebo in terminating PSVT in an at-home setting. The secondary objective is to evaluate the safety of etripamil when self-administered by patients without medical supervision. Additional efficacy end points include the proportion of patients requiring additional medical intervention in an emergency department to terminate PSVT and patient-reported outcomes. After successfully completing a test dose to assess the safety of 2 70 mg doses of etripamil during sinus rhythm, approximately 500 patients will be randomized 1:1 to etripamil or placebo to accrue 180 positively adjudicated AV nodal-dependent PSVT events for treatment with the study drug. Etripamil may offer a new alternative to the current in-hospital treatment modality, providing for safe and effective at-home termination of PSVT.


Assuntos
Taquicardia Paroxística , Taquicardia Supraventricular , Taquicardia Ventricular , Benzoatos/uso terapêutico , Humanos , Taquicardia Paroxística/tratamento farmacológico
2.
J Biol Chem ; 292(17): 7208-7222, 2017 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-28275053

RESUMO

The leucine-rich G protein-coupled receptor-5 (LGR5) is expressed in adult tissue stem cells of many epithelia, and its overexpression is negatively correlated with cancer prognosis. LGR5 potentiates WNT/ß-catenin signaling through its unique constitutive internalization property that clears negative regulators of the WNT-receptor complex from the membrane. However, both the mechanism and physiological relevance of LGR5 internalization are unclear. Therefore, a natural product library was screened to discover LGR5 internalization inhibitors and gain mechanistic insight into LGR5 internalization. The plant lignan justicidin B blocked the constitutive internalization of LGR5. Justicidin B is structurally similar to more potent vacuolar-type H+-ATPase inhibitors, which all inhibited LGR5 internalization by blocking clathrin-mediated endocytosis. We then tested the physiological relevance of LGR5 internalization blockade in vivo A LGR5-rainbow (LBOW) mouse line was engineered to express three different LGR5 isoforms along with unique fluorescent protein lineage reporters in the same mouse. In this manner, the effects of each isoform on cell fate can be simultaneously assessed through simple fluorescent imaging for each lineage reporter. LBOW mice express three different forms of LGR5, a wild-type form that constitutively internalizes and two mutant forms whose internalization properties have been compromised by genetic perturbations within the carboxyl-terminal tail. LBOW was activated in the intestinal epithelium, and a year-long lineage-tracing course revealed that genetic blockade of LGR5 internalization diminished cell fitness. Together these data provide proof-of-concept genetic evidence that blocking the clathrin-mediated endocytosis of LGR5 could be used to pharmacologically control cell behavior.


Assuntos
Clatrina/química , Endocitose , Leucina/química , Receptores Acoplados a Proteínas G/química , Adenosina Trifosfatases/química , Animais , Linhagem Celular Tumoral , Linhagem da Célula , Proliferação de Células , Dioxolanos/química , Epitélio/metabolismo , Feminino , Homeostase , Humanos , Lignanas/química , Camundongos , Camundongos Endogâmicos C57BL , Isoformas de Proteínas , Ratos , Células-Tronco/citologia , Processos Estocásticos , Via de Sinalização Wnt
3.
J Biol Chem ; 286(7): 5434-45, 2011 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-21127052

RESUMO

Cdc14 phosphatase regulates multiple events during anaphase and is essential for mitotic exit in budding yeast. Cdc14 is regulated in both a spatial and temporal manner. It is sequestered in the nucleolus for most of the cell cycle by the nucleolar protein Net1 and is released into the nucleus and cytoplasm during anaphase. To identify novel binding partners of Cdc14, we used affinity purification of Cdc14 and mass spectrometric analysis of interacting proteins from strains in which Cdc14 localization or catalytic activity was altered. To alter Cdc14 localization, we used a strain deleted for NET1, which causes full release of Cdc14 from the nucleolus. To alter Cdc14 activity, we generated mutations in the active site of Cdc14 (C283S or D253A), which allow binding of substrates, but not dephosphorylation, by Cdc14. Using this strategy, we identified new interactors of Cdc14, including multiple proteins involved in mitotic events. A subset of these proteins displayed increased affinity for catalytically inactive mutants of Cdc14 compared with the wild-type version, suggesting they are likely substrates of Cdc14. We have also shown that several of the novel Cdc14-interacting proteins, including Kar9 (a protein that orients the mitotic spindle) and Bni1 and Bnr1 (formins that nucleate actin cables and may be important for actomyosin ring contraction) are specifically dephosphorylated by Cdc14 in vitro and in vivo. Our findings suggest the dephosphorylation of the formins may be important for their observed localization change during exit from mitosis and indicate that Cdc14 targets proteins involved in wide-ranging mitotic events.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Mitose/fisiologia , Proteínas Tirosina Fosfatases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Substituição de Aminoácidos , Proteínas de Ciclo Celular/genética , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Mutação de Sentido Incorreto , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação/fisiologia , Transporte Proteico/fisiologia , Proteínas Tirosina Fosfatases/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
4.
Mol Cancer Res ; 19(10): 1699-1711, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34131071

RESUMO

HER2-positive breast cancers are among the most heterogeneous breast cancer subtypes. The early amplification of HER2 and its known oncogenic isoforms provide a plausible mechanism in which distinct programs of tumor heterogeneity could be traced to the initial oncogenic event. Here a Cancer rainbow mouse simultaneously expressing fluorescently barcoded wildtype (WTHER2), exon-16 null (d16HER2), and N-terminally truncated (p95HER2) HER2 isoforms is used to trace tumorigenesis from initiation to invasion. Tumorigenesis was visualized using whole-gland fluorescent lineage tracing and single-cell molecular pathology. We demonstrate that within weeks of expression, morphologic aberrations were already present and unique to each HER2 isoform. Although WTHER2 cells were abundant throughout the mammary ducts, detectable lesions were exceptionally rare. In contrast, d16HER2 and p95HER2 induced rapid tumor development. d16HER2 incited homogenous and proliferative luminal-like lesions which infrequently progressed to invasive phenotypes whereas p95HER2 lesions were heterogenous and invasive at the smallest detectable stage. Distinct cancer trajectories were observed for d16HER2 and p95HER2 tumors as evidenced by oncogene-dependent changes in epithelial specification and the tumor microenvironment. These data provide direct experimental evidence that intratumor heterogeneity programs begin very early and well in advance of screen or clinically detectable breast cancer. IMPLICATIONS: Although all HER2 breast cancers are treated equally, we show a mechanism by which clinically undetected HER2 isoforms program heterogenous cancer phenotypes through biased epithelial specification and adaptations within the tumor microenvironment.


Assuntos
Neoplasias da Mama/genética , Carcinogênese/genética , Isoformas de Proteínas/genética , Receptor ErbB-2/genética , Animais , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Camundongos , Camundongos Knockout , Microambiente Tumoral/genética
5.
Clin Cancer Res ; 25(9): 2725-2736, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30635338

RESUMO

PURPOSE: Immune-based therapy for metastatic breast cancer has had limited success, particularly in molecular subtypes with low somatic mutations rates. Strategies to augment T-cell infiltration of tumors include vaccines targeting established oncogenic drivers such as the genomic amplification of HER2. We constructed a vaccine based on a novel alphaviral vector encoding a portion of HER2 (VRP-HER2). PATIENTS AND METHODS: In preclinical studies, mice were immunized with VRP-HER2 before or after implantation of hHER2+ tumor cells and HER2-specific immune responses and antitumor function were evaluated. We tested VRP-HER2 in a phase I clinical trial where subjects with advanced HER2-overexpressing malignancies in cohort 1 received VRP-HER2 every 2 weeks for a total of 3 doses. In cohort 2, subjects received the same schedule concurrently with a HER2-targeted therapy. RESULTS: Vaccination in preclinical models with VRP-HER2 induced HER2-specific T cells and antibodies while inhibiting tumor growth. VRP-HER2 was well tolerated in patients and vaccination induced HER2-specific T cells and antibodies. Although a phase I study, there was 1 partial response and 2 patients with continued stable disease. Median OS was 50.2 months in cohort 1 (n = 4) and 32.7 months in cohort 2 (n = 18). Perforin expression by memory CD8 T cells post-vaccination significantly correlated with improved PFS. CONCLUSIONS: VRP-HER2 increased HER2-specific memory CD8 T cells and had antitumor effects in preclinical and clinical studies. The expansion of HER2-specific memory CD8 T cells in vaccinated patients was significantly correlated with increased PFS. Subsequent studies will seek to enhance T-cell activity by combining with anti-PD-1.


Assuntos
Neoplasias da Mama/terapia , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/administração & dosagem , Imunidade Humoral/imunologia , Memória Imunológica/imunologia , Receptor ErbB-2/imunologia , Vacinas de Subunidades Antigênicas/administração & dosagem , Adulto , Idoso , Alphavirus/genética , Animais , Apoptose , Neoplasias da Mama/imunologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Vacinas Anticâncer/imunologia , Proliferação de Células , Células Dendríticas/imunologia , Feminino , Seguimentos , Vetores Genéticos/administração & dosagem , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Prognóstico , Receptor ErbB-2/metabolismo , Taxa de Sobrevida , Células Tumorais Cultivadas , Vacinas de Subunidades Antigênicas/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Nat Commun ; 10(1): 5490, 2019 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-31792216

RESUMO

Field cancerization is a premalignant process marked by clones of oncogenic mutations spreading through the epithelium. The timescales of intestinal field cancerization can be variable and the mechanisms driving the rapid spread of oncogenic clones are unknown. Here we use a Cancer rainbow (Crainbow) modelling system for fluorescently barcoding somatic mutations and directly visualizing the clonal expansion and spread of oncogenes. Crainbow shows that mutations of ß-catenin (Ctnnb1) within the intestinal stem cell results in widespread expansion of oncogenes during perinatal development but not in adults. In contrast, mutations that extrinsically disrupt the stem cell microenvironment can spread in adult intestine without delay. We observe the rapid spread of premalignant clones in Crainbow mice expressing oncogenic Rspondin-3 (RSPO3), which occurs by increasing crypt fission and inhibiting crypt fixation. Crainbow modelling provides insight into how somatic mutations rapidly spread and a plausible mechanism for predetermining the intratumor heterogeneity found in colon cancers.


Assuntos
Neoplasias do Colo/genética , Modelos Animais de Doenças , Células-Tronco Neoplásicas/citologia , Animais , Carcinogênese , Proliferação de Células , Neoplasias do Colo/metabolismo , Neoplasias do Colo/fisiopatologia , Humanos , Camundongos , Mutação , Células-Tronco Neoplásicas/metabolismo , Oncogenes , Trombospondinas/genética , Trombospondinas/metabolismo
7.
Curr Biol ; 24(8): 868-74, 2014 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-24704076

RESUMO

Apoptotic extrusion is a multicellular process utilized by live cells to remove neighboring apoptotic cells. In epithelial tissues, this process has been shown to be critical for the preservation of tissue integrity and barrier function. Here we demonstrate that extrusion is driven by the retraction of the apoptotic cell, which, in turn, triggers a transient and coordinated elongation of the neighboring cells. The coordination of cell elongation requires E-cadherin-mediated cell-cell adhesion. Accordingly, cells that express low levels of E-cadherin are compromised in elongation and apoptotic extrusion, and furthermore, display loss of barrier function in response to apoptotic stimuli. These findings indicate that the maintenance of adhesive forces during apoptotic cell turnover might play an essential role in controlling tissue homeostasis.


Assuntos
Apoptose/fisiologia , Caderinas/fisiologia , Adesão Celular/fisiologia , Crescimento Celular , Epitélio/fisiologia , Animais , Caderinas/metabolismo , Cães , Células Madin Darby de Rim Canino , Microscopia Eletrônica de Varredura , Imagem com Lapso de Tempo
8.
Cancer Cell ; 25(2): 243-56, 2014 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-24525237

RESUMO

Mutations in KRAS are prevalent in human cancers and universally predictive of resistance to anticancer therapeutics. Although it is widely accepted that acquisition of an activating mutation endows RAS genes with functional autonomy, recent studies suggest that the wild-type forms of Ras may contribute to mutant Ras-driven tumorigenesis. Here, we show that downregulation of wild-type H-Ras or N-Ras in mutant K-Ras cancer cells leads to hyperactivation of the Erk/p90RSK and PI3K/Akt pathways and, consequently, the phosphorylation of Chk1 at an inhibitory site, Ser 280. The resulting inhibition of ATR/Chk1 signaling abrogates the activation of the G2 DNA damage checkpoint and confers specific sensitization of mutant K-Ras cancer cells to DNA damage chemotherapeutic agents in vitro and in vivo.


Assuntos
Transformação Celular Neoplásica/patologia , Dano ao DNA/genética , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Membrana/metabolismo , Neoplasias/patologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas ras/genética , Animais , Antineoplásicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Quinase 1 do Ponto de Checagem , Dano ao DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Feminino , Citometria de Fluxo , GTP Fosfo-Hidrolases/antagonistas & inibidores , GTP Fosfo-Hidrolases/genética , Humanos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Camundongos , Camundongos Nus , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Mutação/genética , Neoplasias/genética , Neoplasias/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Proteínas ras/metabolismo
9.
Curr Biol ; 20(15): 1372-7, 2010 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-20655225

RESUMO

Ras proteins play a central role in transducing signals that control cell proliferation, differentiation, motility, and survival. The location-specific signaling activity of Ras has been previously shown to be regulated by ubiquitination [1]. However, the molecular machinery that controls Ras ubiquitination has not been defined. Here we demonstrate through biochemical and functional analyses that Rabex-5 (also known as RabGEF1) [2, 3] functions as an E3 ligase for Ras. Rabex-5-mediated Ras ubiquitination promotes Ras endosomal localization and leads to the suppression of ERK activation. Moreover, the Ras effector RIN1 [4, 5] is required for Rabex-5-dependent Ras ubiquitination, suggesting a feedback mechanism by which Ras activation can be coupled to ubiquitination. These findings define new elements in the regulatory circuitry that link Ras compartmentalization to signaling output.


Assuntos
Endossomos/metabolismo , Retroalimentação Fisiológica , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas ras/metabolismo , Animais , Células COS , Chlorocebus aethiops , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Transdução de Sinais , Ubiquitinação
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