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1.
J Biol Chem ; 288(48): 34658-70, 2013 Nov 29.
Artículo en Inglés | MEDLINE | ID: mdl-24114839

RESUMEN

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.


Asunto(s)
Melanoma/metabolismo , Proteína Quinasa C/genética , Vía de Señalización Wnt/genética , Proteína Wnt3A/metabolismo , Apoptosis , Línea Celular Tumoral , Receptores Frizzled/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Melanoma/genética , Melanoma/patología , Fosforilación , Proteína Quinasa C/metabolismo , ARN Interferente Pequeño , Transducción de Señal , Proteína Wnt3A/antagonistas & inhibidores , Proteína Wnt3A/genética , beta Catenina/metabolismo
2.
Clin Cancer Res ; 30(6): 1189-1199, 2024 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-37851052

RESUMEN

PURPOSE: Merkel cell carcinoma (MCC) is a highly immunogenic skin cancer. Although essentially all MCCs are antigenic through viral antigens or high tumor mutation burden, MCC has a response rate of only approximately 50% to PD-(L)1 blockade suggesting barriers to T-cell responses. Prior studies of MCC immunobiology have focused on CD8 T-cell infiltration and their exhaustion status, while the role of innate immunity, particularly myeloid cells, in MCC remains underexplored. EXPERIMENTAL DESIGN: We utilized single-cell transcriptomics from 9 patients with MCC and multiplex IHC staining of 54 patients' preimmunotherapy tumors, to identify myeloid cells and evaluate association with immunotherapy response. RESULTS: Single-cell transcriptomics identified tumor-associated macrophages (TAM) as the dominant myeloid component within MCC tumors. These TAMs express an immunosuppressive gene signature characteristic of monocytic myeloid-derived suppressor cells and importantly express several targetable immune checkpoint molecules, including PD-L1 and LILRB receptors, that are not present on tumor cells. Analysis of 54 preimmunotherapy tumor samples showed that a subset of TAMs (CD163+, CD14+, S100A8+) selectively infiltrated tumors that had significant CD8 T cells. Indeed, higher TAM prevalence was associated with resistance to PD-1 blockade. While spatial interactions between TAMs and CD8 T cells were not associated with response, myeloid transcriptomic data showed evidence for cytokine signaling and expression of LILRB receptors, suggesting potential immunosuppressive mechanisms. CONCLUSIONS: This study further characterizes TAMs in MCC tumors and provides insights into their possible immunosuppressive mechanism. TAMs may reduce the likelihood of treatment response in MCC by counteracting the benefit of CD8 T-cell infiltration. See related commentary by Silk and Davar, p. 1076.


Asunto(s)
Carcinoma de Células de Merkel , Neoplasias Cutáneas , Humanos , Carcinoma de Células de Merkel/tratamiento farmacológico , Carcinoma de Células de Merkel/genética , Carcinoma de Células de Merkel/metabolismo , Receptor de Muerte Celular Programada 1 , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Linfocitos T CD8-positivos , Células Mieloides/metabolismo
3.
Proc Natl Acad Sci U S A ; 106(4): 1193-8, 2009 Jan 27.
Artículo en Inglés | MEDLINE | ID: mdl-19144919

RESUMEN

This study demonstrates that in malignant melanoma, elevated levels of nuclear beta-catenin in both primary tumors and metastases correlate with reduced expression of a marker of proliferation and with improved survival, in contrast to colorectal cancer. The reduction in proliferation observed in vivo is recapitulated in B16 murine melanoma cells and in human melanoma cell lines cultured in vitro with either WNT3A or small-molecule activators of beta-catenin signaling. Consistent with these results, B16 melanoma cells expressing WNT3A also exhibit decreased tumor size and decreased metastasis when implanted into mice. Genome-wide transcriptional profiling reveals that WNT3A up-regulates genes implicated in melanocyte differentiation, several of which are down-regulated with melanoma progression. These findings suggest that WNT3A can mediate transcriptional changes in melanoma cells in a manner reminiscent of the known role of Wnt/beta-catenin signaling in normal melanocyte development, thereby altering melanoma cell fate to one that may be less proliferative and potentially less aggressive. Our results may explain the observed loss of nuclear beta-catenin with melanoma progression in human tumors, which could reflect a dysregulation of cellular differentiation through a loss of homeostatic Wnt/beta-catenin signaling.


Asunto(s)
Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Transducción de Señal , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animales , Biomarcadores/metabolismo , Diferenciación Celular , Línea Celular Tumoral , Linaje de la Célula , Núcleo Celular/metabolismo , Proliferación Celular , Modelos Animales de Enfermedad , Humanos , Melanocitos/metabolismo , Melanocitos/patología , Ratones , Análisis de Supervivencia , Regulación hacia Arriba , Proteína Wnt3 , Proteína Wnt3A
4.
Cancer Immunol Res ; 8(5): 648-659, 2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-32179557

RESUMEN

Merkel cell carcinoma (MCC) is often caused by persistent expression of Merkel cell polyomavirus (MCPyV) T-antigen (T-Ag). These non-self proteins comprise about 400 amino acids (AA). Clinical responses to immune checkpoint inhibitors, seen in about half of patients, may relate to T-Ag-specific T cells. Strategies to increase CD8+ T-cell number, breadth, or function could augment checkpoint inhibition, but vaccines to augment immunity must avoid delivery of oncogenic T-antigen domains. We probed MCC tumor-infiltrating lymphocytes (TIL) with an artificial antigen-presenting cell (aAPC) system and confirmed T-Ag recognition with synthetic peptides, HLA-peptide tetramers, and dendritic cells (DC). TILs from 9 of 12 (75%) subjects contained CD8+ T cells recognizing 1-8 MCPyV epitopes per person. Analysis of 16 MCPyV CD8+ TIL epitopes and prior TIL data indicated that 97% of patients with MCPyV+ MCC had HLA alleles with the genetic potential that restrict CD8+ T-cell responses to MCPyV T-Ag. The LT AA 70-110 region was epitope rich, whereas the oncogenic domains of T-Ag were not commonly recognized. Specific recognition of T-Ag-expressing DCs was documented. Recovery of MCPyV oncoprotein-specific CD8+ TILs from most tumors indicated that antigen indifference was unlikely to be a major cause of checkpoint inhibition failure. The myriad of epitopes restricted by diverse HLA alleles indicates that vaccination can be a rational component of immunotherapy if tumor immune suppression can be overcome, and the oncogenic regions of T-Ag can be modified without impacting immunogenicity.


Asunto(s)
Antígenos Virales de Tumores/inmunología , Linfocitos T CD8-positivos/inmunología , Carcinoma de Células de Merkel/inmunología , Epítopos/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Poliomavirus de Células de Merkel/inmunología , Neoplasias Cutáneas/inmunología , Adulto , Anciano , Antígenos Virales de Tumores/metabolismo , Carcinogénesis/inmunología , Carcinoma de Células de Merkel/terapia , Femenino , Humanos , Inmunoterapia/métodos , Masculino , Persona de Mediana Edad , Neoplasias Cutáneas/terapia , Adulto Joven
5.
Curr Biol ; 16(7): 702-9, 2006 Apr 04.
Artículo en Inglés | MEDLINE | ID: mdl-16581517

RESUMEN

The precise coordination of signals that control proliferation is a key feature of growth regulation in developing tissues . While much has been learned about the basic components of signal transduction pathways, less is known about how receptor localization, compartmentalization, and trafficking affect signaling in developing tissues. Here we examine the mechanism by which the Drosophila Neurofibromatosis 2 (NF2) tumor suppressor ortholog Merlin (Mer) and the related tumor suppressor expanded (ex) regulate proliferation and differentiation in imaginal epithelia. Merlin and Expanded are members of the FERM (Four-point one, Ezrin, Radixin, Moesin) domain superfamily, which consists of membrane-associated cytoplasmic proteins that interact with transmembrane proteins and may function as adapters that link to protein complexes and/or the cytoskeleton . We demonstrate that Merlin and Expanded function to regulate the steady-state levels of signaling and adhesion receptors and that loss of these proteins can cause hyperactivation of associated signaling pathways. In addition, pulse-chase labeling of Notch in living tissues indicates that receptor levels are upregulated at the plasma membrane in Mer; ex double mutant cells due to a defect in receptor clearance from the cell surface. We propose that these proteins control proliferation by regulating the abundance, localization, and turnover of cell-surface receptors and that misregulation of these processes may be a key component of tumorigenesis.


Asunto(s)
Proteínas de Drosophila/fisiología , Drosophila/metabolismo , Endocitosis/fisiología , Proteínas de la Membrana/fisiología , Neurofibromina 2/fisiología , Receptores de Superficie Celular/metabolismo , Transducción de Señal , Animales , Membrana Celular/metabolismo , Proliferación Celular , Drosophila/anatomía & histología , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Glicoproteínas de Membrana , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mutación , Neurofibromina 2/genética , Complejo GPIb-IX de Glicoproteína Plaquetaria , Transporte de Proteínas/fisiología , Receptores de Superficie Celular/genética , Receptores Notch/metabolismo , Regulación hacia Arriba , Alas de Animales/anatomía & histología , Alas de Animales/metabolismo
6.
Cancer Immunol Res ; 7(10): 1727-1739, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31405946

RESUMEN

Although CD4+ T cells likely play key roles in antitumor immune responses, most immuno-oncology studies have been limited to CD8+ T-cell responses due to multiple technical barriers and a lack of shared antigens across patients. Merkel cell carcinoma (MCC) is an aggressive skin cancer caused by Merkel cell polyomavirus (MCPyV) oncoproteins in 80% of cases. Because MCPyV oncoproteins are shared across most patients with MCC, it is unusually feasible to identify, characterize, and potentially augment tumor-specific CD4+ T cells. Here, we report the identification of CD4+ T-cell responses against six MCPyV epitopes, one of which included a conserved, essential viral oncogenic domain that binds/disables the cellular retinoblastoma (Rb) tumor suppressor. We found that this epitope (WEDLT209-228) could be presented by three population-prevalent HLA class II alleles, making it a relevant target in 64% of virus-positive MCC patients. Cellular staining with a WEDLT209-228-HLA-DRB1*0401 tetramer indicated that specific CD4+ T cells were detectable in 78% (14 of 18) of evaluable MCC patients, were 250-fold enriched within MCC tumors relative to peripheral blood, and had diverse T-cell receptor sequences. We also identified a modification of this domain that still allowed recognition by these CD4+ T cells but disabled binding to the Rb tumor suppressor, a key step in the detoxification of a possible therapeutic vaccine. The use of these new tools for deeper study of MCPyV-specific CD4+ T cells may provide broader insight into cancer-specific CD4+ T-cell responses.


Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Carcinogénesis/inmunología , Carcinoma de Células de Merkel/inmunología , Epítopos/inmunología , Poliomavirus de Células de Merkel/inmunología , Neoplasias Cutáneas/inmunología , Carcinogénesis/metabolismo , Carcinogénesis/patología , Carcinoma de Células de Merkel/tratamiento farmacológico , Carcinoma de Células de Merkel/metabolismo , Carcinoma de Células de Merkel/patología , Línea Celular Tumoral , Voluntarios Sanos , Humanos , Oligopéptidos/inmunología , Proteína de Retinoblastoma/metabolismo , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología
7.
PLoS One ; 9(3): e90853, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24595234

RESUMEN

The proper control of tissue growth is essential during normal development and an important problem in human disease. Merlin, the product of the Neurofibromatosis 2 tumor suppressor gene, has been extensively studied to understand its functions in growth control. Here we describe experiments in which we used Drosophila as an in vivo system to test the functions of the normal human NF2 gene products and patient-derived mutant alleles. Although the predominant NF2 gene isoform, isoform 1, could functionally replace the Drosophila Merlin gene, a second isoform with a distinct C-terminal tail could not. Immunofluorescence studies show that the two isoforms have distinct subcellular localizations when expressed in the polarized imaginal epithelium, and function in genetic rescue assays correlates with apical localization of the NF2 protein. Interestingly, we found that a patient-derived missense allele, NF2L64P, appears to be temperature sensitive. These studies highlight the utility of Drosophila for in vivo functional analysis of highly conserved human disease genes.


Asunto(s)
Drosophila/genética , Genes de la Neurofibromatosis 2 , Neurofibromina 2/genética , Alelos , Animales , Humanos , Mutación , Neurofibromina 2/análisis , Isoformas de Proteínas/análisis , Isoformas de Proteínas/genética
8.
PLoS One ; 9(4): e94748, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24733413

RESUMEN

Unprecedented clinical responses have been reported in advanced stage metastatic melanoma patients treated with targeted inhibitors of constitutively activated mutant BRAF, which is present in approximately half of all melanomas. We and others have previously observed an association of elevated nuclear ß-catenin with improved survival in molecularly-unselected melanoma patients. This study sought to determine whether levels of Wnt/ß-catenin signaling in melanoma tumors prior to treatment might predict patient responses to BRAF inhibitors (BRAFi). We performed automated quantification of ß-catenin immunohistochemical expression in pretreatment BRAF-mutant tumors from 32 BRAFi-treated melanoma patients. Unexpectedly, patients with higher nuclear ß-catenin in their tumors did not exhibit the survival advantage previously observed in molecularly-unselected melanoma patients who did not receive BRAFi. In cultured melanoma cells treated with long-term BRAFi, activation of Wnt/ß-catenin signaling is markedly inhibited, coinciding with a loss of the enhancement of BRAFi-induced apoptosis by WNT3A observed in BRAFi-naïve cells. Together, these observations suggest that long-term treatment with BRAFi can impact the interaction between BRAF/MAPK and Wnt/ß-catenin signaling to affect patient outcomes. Studies with larger patient cohorts are required to determine whether nuclear ß-catenin expression correlates with clinical responses to BRAFi and to specific mechanisms of acquired resistance to BRAFi. Understanding these pathway interactions will be necessary to facilitate efforts to individualize therapies for melanoma patients.


Asunto(s)
Melanoma/tratamiento farmacológico , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Neoplasias Cutáneas/tratamiento farmacológico , Vía de Señalización Wnt , Proteína Wnt3A/metabolismo , beta Catenina/metabolismo , Adulto , Anciano , Línea Celular Tumoral , Quinasas MAP Reguladas por Señal Extracelular , Femenino , Regulación Enzimológica de la Expresión Génica , Humanos , Inmunohistoquímica , Masculino , Melanocitos/enzimología , Melanoma/mortalidad , Persona de Mediana Edad , Metástasis de la Neoplasia , Estudios Retrospectivos , Neoplasias Cutáneas/mortalidad , Tasa de Supervivencia , Resultado del Tratamiento , Adulto Joven
9.
J Clin Invest ; 124(7): 2877-90, 2014 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-24865425

RESUMEN

About half of all melanomas harbor a mutation that results in a constitutively active BRAF kinase mutant (BRAF(V600E/K)) that can be selectively inhibited by targeted BRAF inhibitors (BRAFis). While patients treated with BRAFis initially exhibit measurable clinical improvement, the majority of patients eventually develop drug resistance and relapse. Here, we observed marked elevation of WNT5A in a subset of tumors from patients exhibiting disease progression on BRAFi therapy. WNT5A transcript and protein were also elevated in BRAFi-resistant melanoma cell lines generated by long-term in vitro treatment with BRAFi. RNAi-mediated reduction of endogenous WNT5A in melanoma decreased cell growth, increased apoptosis in response to BRAFi challenge, and decreased the activity of prosurvival AKT signaling. Conversely, overexpression of WNT5A promoted melanoma growth, tumorigenesis, and activation of AKT signaling. Similarly to WNT5A knockdown, knockdown of the WNT receptors FZD7 and RYK inhibited growth, sensitized melanoma cells to BRAFi, and reduced AKT activation. Together, these findings suggest that chronic BRAF inhibition elevates WNT5A expression, which promotes AKT signaling through FZD7 and RYK, leading to increased growth and therapeutic resistance. Furthermore, increased WNT5A expression in BRAFi-resistant melanomas correlates with a specific transcriptional signature, which identifies potential therapeutic targets to reduce clinical BRAFi resistance.


Asunto(s)
Melanoma/tratamiento farmacológico , Melanoma/genética , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Resistencia a Antineoplásicos/genética , Receptores Frizzled/antagonistas & inhibidores , Receptores Frizzled/genética , Receptores Frizzled/metabolismo , Humanos , Indoles/farmacología , Melanoma/metabolismo , Mutación , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Interferencia de ARN , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal , Sulfonamidas/farmacología , Regulación hacia Arriba/efectos de los fármacos , Proteínas Wnt/antagonistas & inhibidores , Vía de Señalización Wnt/efectos de los fármacos , Proteína Wnt-5a , beta Catenina/metabolismo
10.
PLoS One ; 8(7): e69593, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23869245

RESUMEN

While the TRAIL pathway represents a promising therapeutic target in melanoma, resistance to TRAIL-mediated apoptosis remains a barrier to its successful adoption. Since the Wnt/ß-catenin pathway has been implicated in facilitating melanoma cell apoptosis, we investigated the effect of Wnt/ß-catenin signaling on regulating the responses of melanoma cells to TRAIL. Co-treatment of melanoma cell lines with WNT3A-conditioned media and recombinant TRAIL significantly enhanced apoptosis compared to treatment with TRAIL alone. This apoptosis correlates with increased abundance of the pro-apoptotic proteins BCL2L11 and BBC3, and with decreased abundance of the anti-apoptotic regulator Mcl1. We then confirmed the involvement of the Wnt/ß-catenin signaling pathway by demonstrating that siRNA-mediated knockdown of an intracellular ß-catenin antagonist, AXIN1, or treating cells with an inhibitor of GSK-3 also enhanced melanoma cell sensitivity to TRAIL. These studies describe a novel regulation of TRAIL sensitivity in melanoma by Wnt/ß-catenin signaling, and suggest that strategies to enhance Wnt/ß-catenin signaling in combination with TRAIL agonists warrant further investigation.


Asunto(s)
Apoptosis , Melanoma/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Vía de Señalización Wnt , Proteína Wnt3A/farmacología , beta Catenina/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína Axina/antagonistas & inhibidores , Proteína 11 Similar a Bcl2 , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Medios de Cultivo Condicionados , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Humanos , Melanoma/patología , Proteínas de la Membrana/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas/metabolismo , Interferencia de ARN , Ligando Inductor de Apoptosis Relacionado con TNF/genética , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo
11.
Cell Cycle ; 11(20): 3724-30, 2012 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-22895053

RESUMEN

The limitations of revolutionary new mutation-specific inhibitors of BRAF(V600E) include the universal recurrence seen in melanoma patients treated with this novel class of drugs. Recently, our lab showed that simultaneous activation of the Wnt/ß-catenin signaling pathway and targeted inhibition of BRAF(V600E) by PLX4720 synergistically induces apoptosis across a spectrum of BRAF(V600E) melanoma cell lines. As a follow-up to that study, treatment of BRAF-mutant and NRAS-mutant melanoma lines with WNT3A and the MEK inhibitor AZD6244 also induces apoptosis. The susceptibility of BRAF-mutant lines and NRAS-mutant lines to apoptosis correlates with negative regulation of Wnt/ß-catenin signaling by ERK/MAPK signaling and dynamic decreases in abundance of the downstream scaffolding protein, AXIN1. Apoptosis-resistant NRAS-mutant lines can sensitize to AZD6244 by pretreatment with AXIN1 siRNA, similar to what we previously reported in BRAF-mutant cell lines. Taken together, these findings indicate that NRAS-mutant melanoma share with BRAF-mutant melanoma the potential to regulate apoptosis upon MEK inhibition through WNT3A and dynamic regulation of cellular AXIN1. Understanding the cellular context that makes melanoma cells susceptible to this combination treatment will contribute to the study and development of novel therapeutic combinations that may lead to more durable responses.


Asunto(s)
Antineoplásicos/farmacología , Bencimidazoles/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Indoles/farmacología , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Sulfonamidas/farmacología , Proteína Wnt3A/farmacología , Apoptosis/efectos de los fármacos , Proteína Axina/antagonistas & inhibidores , Proteína Axina/genética , Proteína Axina/metabolismo , Línea Celular Tumoral , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/metabolismo , Melanoma/patología , Mutación , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , ARN Interferente Pequeño/genética , Transducción de Señal/efectos de los fármacos , beta Catenina/agonistas , beta Catenina/genética , beta Catenina/metabolismo
12.
Sci Signal ; 5(240): ra64, 2012 Sep 04.
Artículo en Inglés | MEDLINE | ID: mdl-22949735

RESUMEN

The FAM123 gene family comprises three members: FAM123A, the tumor suppressor WTX (also known as FAM123B), and FAM123C. WTX is required for normal development and causally contributes to human disease, in part through its regulation of ß-catenin-dependent WNT signaling. The roles of FAM123A and FAM123C in signaling, cell behavior, and human disease remain less understood. We defined and compared the protein-protein interaction networks for each member of the FAM123 family by affinity purification and mass spectrometry. Protein localization and functional studies suggest that the FAM123 family members have conserved and divergent cellular roles. In contrast to WTX and FAM123C, we found that microtubule-associated proteins were enriched in the FAM123A protein interaction network. FAM123A interacted with and tracked with the plus end of dynamic microtubules. Domain interaction experiments revealed a "SKIP" amino acid motif in FAM123A that mediated interaction with the microtubule tip tracking proteins end-binding protein 1 (EB1) and EB3--and therefore with microtubules. Cells depleted of FAM123A showed compartment-specific effects on microtubule dynamics, increased actomyosin contractility, larger focal adhesions, and decreased cell migration. These effects required binding of FAM123A to and inhibition of the guanine nucleotide exchange factor ARHGEF2, a microtubule-associated activator of RhoA. Together, these data suggest that the SKIP motif enables FAM123A, but not the other FAM123 family members, to bind to EB proteins, localize to microtubules, and coordinate microtubule dynamics and actomyosin contractility.


Asunto(s)
Actomiosina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Factores de Intercambio de Guanina Nucleótido/antagonistas & inhibidores , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Secuencias de Aminoácidos/genética , Cromatografía de Afinidad , Adhesiones Focales/metabolismo , Humanos , Espectrometría de Masas , Mapeo de Interacción de Proteínas , Factores de Intercambio de Guanina Nucleótido Rho
13.
Sci Signal ; 5(206): ra3, 2012 Jan 10.
Artículo en Inglés | MEDLINE | ID: mdl-22234612

RESUMEN

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.


Asunto(s)
Apoptosis/fisiología , Proteína Axina/fisiología , Melanoma/metabolismo , Proteínas Proto-Oncogénicas B-raf/fisiología , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Humanos , Melanoma/enzimología , Melanoma/genética , Melanoma/patología , Mutación
14.
Chem Biol ; 17(11): 1177-82, 2010 Nov 24.
Artículo en Inglés | MEDLINE | ID: mdl-21095567

RESUMEN

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.


Asunto(s)
Antineoplásicos/uso terapéutico , Melanoma Experimental/metabolismo , Riluzol/uso terapéutico , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animales , Proliferación Celular , Regulación de la Expresión Génica , Genes Reporteros , Melanoma Experimental/tratamiento farmacológico , Ratones , Interferencia de ARN , ARN Interferente Pequeño , Receptores de Glutamato Metabotrópico/genética , Receptores de Glutamato Metabotrópico/metabolismo , Transducción de Señal , Pigmentación de la Piel , Proteína Wnt3 , Proteína Wnt3A
15.
Nature ; 421(6918): 83-7, 2003 Jan 02.
Artículo en Inglés | MEDLINE | ID: mdl-12511959

RESUMEN

Two prominent characteristics of epithelial cells, apical-basal polarity and a highly ordered cytoskeleton, depend on the existence of precisely localized protein complexes associated with the apical plasma membrane, and on a separate machinery that regulates the spatial order of actin assembly. ERM (ezrin, radixin, moesin) proteins have been proposed to link transmembrane proteins to the actin cytoskeleton in the apical domain, suggesting a structural role in epithelial cells, and they have been implicated in signalling pathways. Here, we show that the sole Drosophila ERM protein Moesin functions to promote cortical actin assembly and apical-basal polarity. As a result, cells lacking Moesin lose epithelial characteristics and adopt invasive migratory behaviour. Our data demonstrate that Moesin facilitates epithelial morphology not by providing an essential structural function, but rather by antagonizing activity of the small GTPase Rho. Thus, Moesin functions in maintaining epithelial integrity by regulating cell-signalling events that affect actin organization and polarity. Furthermore, our results show that there is negative feedback between ERM activation and activity of the Rho pathway.


Asunto(s)
Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Proteínas de Microfilamentos/metabolismo , Transducción de Señal , Proteínas de Unión al GTP rho/metabolismo , Actinas/metabolismo , Animales , Animales Modificados Genéticamente , Línea Celular , Movimiento Celular , Polaridad Celular , Tamaño de la Célula , Proteínas del Citoesqueleto , Proteínas de Drosophila , Drosophila melanogaster/genética , Retroalimentación Fisiológica , Proteínas de Microfilamentos/genética , Datos de Secuencia Molecular , Mutación , Fenotipo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Porcinos , Proteínas de Unión al GTP rho/genética
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