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1.
J Biol Chem ; 288(48): 34658-70, 2013 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-24114839

RESUMO

Advances in phosphoproteomics have made it possible to monitor changes in protein phosphorylation that occur at different steps in signal transduction and have aided the identification of new pathway components. In the present study, we applied this technology to advance our understanding of the responses of melanoma cells to signaling initiated by the secreted ligand WNT3A. We started by comparing the phosphopeptide patterns of cells treated with WNT3A for different periods of time. Next, we integrated these data sets with the results from a siRNA screen that targeted protein kinases. This integration of siRNA screening and proteomics enabled us to identify four kinases that exhibit altered phosphorylation in response to WNT3A and that regulate a luciferase reporter of ß-catenin-responsive transcription (ß-catenin-activated reporter). We focused on one of these kinases, an atypical PKC kinase, protein kinase N1 (PKN1). Reducing the levels of PKN1 with siRNAs significantly enhances activation of ß-catenin-activated reporter and increases apoptosis in melanoma cell lines. Using affinity purification followed by mass spectrometry, we then found that PKN1 is present in a protein complex with a WNT3A receptor, Frizzled 7, as well as with proteins that co-purify with Frizzled 7. These data establish that the protein kinase PKN1 inhibits Wnt/ß-catenin signaling and sensitizes melanoma cells to cell death stimulated by WNT3A.


Assuntos
Melanoma/metabolismo , Proteína Quinase C/genética , Via de Sinalização Wnt/genética , Proteína Wnt3A/metabolismo , Apoptose , Linhagem Celular Tumoral , Receptores Frizzled/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Melanoma/genética , Melanoma/patologia , Fosforilação , Proteína Quinase C/metabolismo , RNA Interferente Pequeno , Transdução de Sinais , Proteína Wnt3A/antagonistas & inibidores , Proteína Wnt3A/genética , beta Catenina/metabolismo
2.
Sci Signal ; 5(206): ra3, 2012 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-22234612

RESUMO

Because the Wnt/ß-catenin signaling pathway is linked to melanoma pathogenesis and to patient survival, we conducted a kinome small interfering RNA (siRNA) screen in melanoma cells to expand our understanding of the kinases that regulate this pathway. We found that BRAF signaling, which is constitutively activated in many melanomas by the BRAF(V600E) mutation, inhibits Wnt/ß-catenin signaling in human melanoma cells. Because inhibitors of BRAF(V600E) show promise in ongoing clinical trials, we investigated whether altering Wnt/ß-catenin signaling might enhance the efficacy of the BRAF(V600E) inhibitor PLX4720. We found that endogenous ß-catenin was required for PLX4720-induced apoptosis of melanoma cells and that activation of Wnt/ß-catenin signaling synergized with PLX4720 to decrease tumor growth in vivo and to increase apoptosis in vitro. This synergistic enhancement of apoptosis correlated with reduced abundance of an endogenous negative regulator of ß-catenin, AXIN1. In support of the hypothesis that AXIN1 is a mediator rather than a marker of apoptosis, siRNA directed against AXIN1 rendered resistant melanoma cell lines susceptible to apoptosis in response to treatment with a BRAF(V600E) inhibitor. Thus, Wnt/ß-catenin signaling and AXIN1 may regulate the efficacy of inhibitors of BRAF(V600E), suggesting that manipulation of the Wnt/ß-catenin pathway could be combined with BRAF inhibitors to treat melanoma.


Assuntos
Apoptose/fisiologia , Proteína Axina/fisiologia , Melanoma/metabolismo , Proteínas Proto-Oncogênicas B-raf/fisiologia , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Humanos , Melanoma/enzimologia , Melanoma/genética , Melanoma/patologia , Mutação
3.
Chem Biol ; 17(11): 1177-82, 2010 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-21095567

RESUMO

To identify new protein and pharmacological regulators of Wnt/ß-catenin signaling, we used a cell-based reporter assay to screen a collection of 1857 human-experienced compounds for their ability to enhance activation of the ß-catenin reporter by a low concentration of WNT3A. This identified 44 unique compounds, including the FDA-approved drug riluzole, which is presently in clinical trials for treating melanoma. We found that treating melanoma cells with riluzole in vitro enhances the ability of WNT3A to regulate gene expression, to promote pigmentation, and to decrease cell proliferation. Furthermore riluzole, like WNT3A, decreases metastases in a mouse melanoma model. Interestingly, siRNAs targeting the metabotropic glutamate receptor, GRM1, a reported indirect target of riluzole, enhance ß-catenin signaling. The unexpected regulation of ß-catenin signaling by both riluzole and GRM1 has implications for the future uses of this drug.


Assuntos
Antineoplásicos/uso terapêutico , Melanoma Experimental/metabolismo , Riluzol/uso terapêutico , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animais , Proliferação de Células , Regulação da Expressão Gênica , Genes Reporter , Melanoma Experimental/tratamento farmacológico , Camundongos , Interferência de RNA , RNA Interferente Pequeno , Receptores de Glutamato Metabotrópico/genética , Receptores de Glutamato Metabotrópico/metabolismo , Transdução de Sinais , Pigmentação da Pele , Proteína Wnt3 , Proteína Wnt3A
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