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1.
Oncogene ; 40(45): 6321-6328, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34625709

RESUMO

Induced waves of calcium fluxes initiate multiple signalling pathways that play an important role in the differentiation and maturation of B-cells. Finely tuned transient Ca+2 fluxes from the endoplasmic reticulum in response to B-cell receptor (BCR) or chemokine receptor activation are followed by more sustained calcium influxes from the extracellular environment and contribute to the mechanisms responsible for the proliferation of B-cells, their migration within lymphoid organs and their differentiation. Dysregulation of these well-balanced mechanisms in B-cell lymphomas results in uncontrolled cell proliferation and resistance to apoptosis. Consequently, several cytotoxic drugs (and anti-proliferative compounds) used in standard chemotherapy regimens for the treatment of people with lymphoma target calcium-dependent pathways. Furthermore, ~10% of lymphoma associated mutations are found in genes with functions in calcium-dependent signalling, including those affecting B-cell receptor signalling pathways. In this review, we provide an overview of the Ca2+-dependent signalling network and outline the contribution of its key components to B cell lymphomagenesis. We also consider how the oncogenic Epstein-Barr virus, which is causally linked to the pathogenesis of a number of B-cell lymphomas, can modify Ca2+-dependent signalling.

2.
Proc Natl Acad Sci U S A ; 118(39)2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34521767

RESUMO

Early stages of colorectal cancer (CRC) development are characterized by a complex rewiring of transcriptional networks resulting in changes in the expression of multiple genes. Here, we demonstrate that the deletion of a poorly studied tetraspanin protein Tspan6 in Apcmin/+ mice, a well-established model for premalignant CRC, resulted in increased incidence of adenoma formation and tumor size. We demonstrate that the effect of Tspan6 deletion results in the activation of EGF-dependent signaling pathways through increased production of the transmembrane form of TGF-α (tmTGF-α) associated with extracellular vesicles. This pathway is modulated by an adaptor protein syntenin-1, which physically links Tspan6 and tmTGF-α. In support of this, the expression of Tspan6 is frequently decreased or lost in CRC, and this correlates with poor survival. Furthermore, the analysis of samples from the epidermal growth factor receptor (EGFR)-targeting clinical trial (COIN trial) has shown that the expression of Tspan6 in CRC correlated with better patient responses to EGFR-targeted therapy involving Cetuximab. Importantly, Tspan6-positive patients with tumors in the proximal colon (right-sided) and those with KRAS mutations had a better response to Cetuximab than the patients that expressed low Tspan6 levels. These results identify Tspan6 as a regulator of CRC development and a potential predictive marker for EGFR-targeted therapies in CRC beyond RAS pathway mutations.


Assuntos
Biomarcadores Tumorais/metabolismo , Cetuximab/farmacologia , Neoplasias Colorretais/patologia , Regulação Neoplásica da Expressão Gênica , Tetraspaninas/metabolismo , Tetraspaninas/fisiologia , Animais , Antineoplásicos Imunológicos/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Prognóstico , Taxa de Sobrevida , Tetraspaninas/genética , Células Tumorais Cultivadas
3.
Int J Mol Sci ; 22(16)2021 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-34445169

RESUMO

Tetraspanins are a family of transmembrane proteins that form a network of protein-protein interactions within the plasma membrane. Within this network, tetraspanin are thought to control the lateral segregation of their partners at the plasma membrane through mechanisms involving specific lipids. Here, we used a single molecule tracking approach to study the membrane behavior of tetraspanins in mammary epithelial cells and demonstrate that despite a common overall behavior, each tetraspanin (CD9, CD81 and CD82) has a specific signature in terms of dynamics. Furthermore, we demonstrated that tetraspanin dynamics on the cell surface are dependent on gangliosides. More specifically, we found that CD82 expression increases the dynamics of CD81 and alters its localization at the plasma membrane, this has no effect on the behavior of CD9. Our results provide new information on the ability of CD82 and gangliosides to differentially modulate the dynamics and organization of tetraspanins at the plasma membrane and highlight that its lipid and protein composition is involved in the dynamical architecture of the tetraspanin web. We predict that CD82 may act as a regulator of the lateral segregation of specific tetraspanins at the plasma membrane while gangliosides could play a crucial role in establishing tetraspanin-enriched areas.


Assuntos
Membrana Celular/metabolismo , Células Epiteliais/metabolismo , Gangliosídeos/metabolismo , Proteína Kangai-1/metabolismo , Tetraspanina 28/metabolismo , Membrana Celular/química , Células Cultivadas , Células Epiteliais/química , Células Epiteliais/citologia , Gangliosídeos/análise , Humanos , Proteína Kangai-1/análise , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismo , Tetraspanina 28/análise
4.
Cells ; 10(6)2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34207462

RESUMO

The plasma membrane is a key actor of cell migration. For instance, its tension controls persistent cell migration and cell surface caveolae integrity. Then, caveolae constituents such as caveolin-1 can initiate a mechanotransduction loop that involves actin- and focal adhesion-dependent control of the mechanosensor YAP to finely tune cell migration. Tetraspanin CD82 (also named KAI-1) is an integral membrane protein and a metastasis suppressor. Its expression is lost in many cancers including breast cancer. It is a strong inhibitor of cell migration by a little-known mechanism. We demonstrated here that CD82 controls persistent 2D migration of EGF-induced single cells, stress fibers and focal adhesion sizes and dynamics. Mechanistically, we found that CD82 regulates membrane tension, cell surface caveolae abundance and YAP nuclear translocation in a caveolin-1-dependent manner. Altogether, our data show that CD82 controls 2D cell migration using membrane-driven mechanics involving caveolin and the YAP pathway.


Assuntos
Membrana Celular/metabolismo , Movimento Celular/fisiologia , Proteína Kangai-1/metabolismo , Metástase Neoplásica/patologia , Neoplasias/metabolismo , Fibras de Estresse/metabolismo , Tetraspaninas/metabolismo , Caveolina 1/metabolismo , Adesão Celular/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Mecanotransdução Celular/fisiologia , Proteínas de Membrana/metabolismo , Neoplasias/patologia , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismo
5.
Biomol NMR Assign ; 14(2): 221-225, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32535836

RESUMO

The CaMK subfamily of Ser/Thr kinases are regulated by calmodulin interactions with their C-terminal regions. They are exemplified by Ca2+/calmodulin dependent protein kinase 1δ which is known as CaMK1D, CaMKIδ or CKLiK. CaMK1D mediates intracellular signalling downstream of Ca2+ influx and thereby exhibits amplifications of Ca2+signals and polymorphisms that have been implicated in breast cancer and diabetes. Here we report the backbone 1H, 13C, 15N assignments of the 38 kDa human CaMK1D protein in its free state, including both the canonical bi-lobed kinase fold as well as the autoinhibitory and calmodulin binding domains.


Assuntos
Biocatálise , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/química , Ressonância Magnética Nuclear Biomolecular , Sequência de Aminoácidos , Humanos , Domínios Proteicos , Estrutura Secundária de Proteína
6.
J Med Chem ; 63(13): 6784-6801, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32433887

RESUMO

Polymorphisms in the region of the calmodulin-dependent kinase isoform D (CaMK1D) gene are associated with increased incidence of diabetes, with the most common polymorphism resulting in increased recognition by transcription factors and increased protein expression. While reducing CaMK1D expression has a potentially beneficial effect on glucose processing in human hepatocytes, there are no known selective inhibitors of CaMK1 kinases that can be used to validate or translate these findings. Here we describe the development of a series of potent, selective, and drug-like CaMK1 inhibitors that are able to provide significant free target cover in mouse models and are therefore useful as in vivo tool compounds. Our results show that a lead compound from this series improves insulin sensitivity and glucose control in the diet-induced obesity mouse model after both acute and chronic administration, providing the first in vivo validation of CaMK1D as a target for diabetes therapeutics.


Assuntos
Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Dieta/efeitos adversos , Descoberta de Drogas , Resistência à Insulina , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Animais , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/química , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Obesidade/induzido quimicamente , Conformação Proteica , Inibidores de Proteínas Quinases/uso terapêutico
7.
J Pathol ; 251(1): 63-73, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32129471

RESUMO

The immune microenvironment in inflammatory breast cancer (IBC) is poorly characterised, and molecular and cellular pathways that control accumulation of various immune cells in IBC tissues remain largely unknown. Here, we discovered a novel pathway linking the expression of the tetraspanin protein CD151 in tumour cells with increased accumulation of macrophages in cancerous tissues. It is notable that elevated expression of CD151 and a higher number of tumour-infiltrating macrophages correlated with better patient responses to chemotherapy. Accordingly, CD151-expressing IBC xenografts were characterised by the increased infiltration of macrophages. In vitro migration experiments demonstrated that CD151 stimulates the chemoattractive potential of IBC cells for monocytes via mechanisms involving midkine (a heparin-binding growth factor), integrin α6ß1, and production of extracellular vesicles (EVs). Profiling of chemokines secreted by IBC cells demonstrated that CD151 increases production of midkine. Purified midkine specifically stimulated migration of monocytes, but not other immune cells. Further experiments demonstrated that the chemoattractive potential of IBC-derived EVs is blocked by anti-midkine antibodies. These results demonstrate for the first time that changes in the expression of a tetraspanin protein by tumour cells can affect the formation of the immune microenvironment by modulating recruitment of effector cells to cancerous tissues. Therefore, a CD151-midkine pathway can be considered as a novel target for controlled changes of the immune landscape in IBC. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Assuntos
Neoplasias Inflamatórias Mamárias/patologia , Macrófagos/patologia , Tetraspanina 24/metabolismo , Microambiente Tumoral/fisiologia , Linhagem Celular Tumoral , Quimiocinas/metabolismo , Humanos , Neoplasias Inflamatórias Mamárias/metabolismo , Macrófagos/metabolismo , Midkina/metabolismo , Tetraspanina 24/imunologia
8.
Pathobiology ; 87(2): 61-74, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31715606

RESUMO

The prognostic value of the immune cell infiltrate in the breast carcinoma microenvironment is still uncertain. We reviewed published articles analysing the infiltration of inflammatorycells in the microenvironment of breast carcinoma. Data revealed the importance of infiltration of these immune cells in the prognosis of breast carcinoma, particularly the triple-negative and HER2-positive phenotypes. Tumour-infiltrating lymphocytes and their subtypes play a fundamental role in predicting the pathological complete response (pCR) to neoadjuvant chemotherapy. More research aiming to dissect a complex network of communication between cancer cells and other cellular components of the tumour microenvironment is necessary to develop more effective therapeutic approaches.


Assuntos
Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Linfócitos do Interstício Tumoral/imunologia , Terapia Neoadjuvante , Microambiente Tumoral/imunologia , Biomarcadores Tumorais , Neoplasias da Mama/terapia , Feminino , Humanos , Linfócitos do Interstício Tumoral/classificação , Prognóstico , Transdução de Sinais/imunologia , Neoplasias de Mama Triplo Negativas/imunologia
9.
J Cell Sci ; 131(21)2018 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-30257985

RESUMO

Expression of the tetraspanin CD151 is frequently upregulated in epithelial malignancies and correlates with poor prognosis. Here, we report that CD151 is involved in regulation of the expression of fibroblast growth factor receptor 2 (FGFR2). Depletion of CD151 in breast cancer cells resulted in an increased level of FGFR2. Accordingly, an inverse correlation between CD151 and FGFR2 was observed in breast cancer tissues. CD151-dependent regulation of the FGFR2 expression relies on post-transcriptional mechanisms involving HuR (also known as ELAVL1), a multifunctional RNA-binding protein, and the assembly of processing bodies (P-bodies). Depletion of CD151 correlated with inhibition of PKC, a well-established downstream target of CD151. Accordingly, the levels of dialcylglycerol species were decreased in CD151-negative cells, and inhibition of PKC resulted in the increased expression of FGFR2. Whereas expression of FGFR2 itself did not correlate with any of the clinicopathological data, we found that FGFR2-/CD151+ patients were more likely to have developed lymph node metastasis. Conversely, FGFR2-/CD151- patients demonstrated better overall survival. These results illustrate functional interdependency between CD151 complexes and FGFR2, and suggest a previously unsuspected role of CD151 in breast tumorigenesis.


Assuntos
Neoplasias da Mama/metabolismo , Proteína Quinase C/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/biossíntese , Tetraspanina 24/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carcinogênese , Linhagem Celular Tumoral , Feminino , Humanos , Células MCF-7 , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Transdução de Sinais , Tetraspanina 24/biossíntese , Tetraspanina 24/genética , Transcrição Genética
10.
Am J Physiol Gastrointest Liver Physiol ; 313(2): G138-G149, 2017 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-28473332

RESUMO

CD151, a member of the tetraspanin family of receptors, is a lateral organizer and modulator of activity of several families of transmembrane proteins. It has been implicated in the development and progression of several cancers, but its role in chronic inflammatory disease is less well understood. Here we show that CD151 is upregulated by distinct microenvironmental signals in a range of chronic inflammatory liver diseases and in primary liver cancer, in which it supports lymphocyte recruitment. CD151 was highly expressed in endothelial cells of the hepatic sinusoids and neovessels developing in fibrotic septa and tumor margins. Primary cultures of human hepatic sinusoidal endothelial cells (HSECs) expressed CD151 at the cell membrane and in intracellular vesicles. CD151 was upregulated by VEGF and HepG2 conditioned media but not by proinflammatory cytokines. Confocal microscopy confirmed that CD151 colocalized with the endothelial adhesion molecule/immunoglobulin superfamily member, VCAM-1. Functional flow-based adhesion assays with primary human lymphocytes and HSECs demonstrated a 40% reduction of lymphocyte adhesion with CD151 blockade. Inhibition of lymphocyte adhesion was similar between VCAM-1 blockade and a combination of CD151/VCAM-1 blockade, suggesting a collaborative role between the two receptors. These studies demonstrate that CD151 is upregulated within the liver during chronic inflammation, where it supports lymphocyte recruitment via liver endothelium. We propose that CD151 regulates the activity of VCAM-1 during lymphocyte recruitment to the human liver and could be a novel anti-inflammatory target in chronic liver disease and hepatocellular cancer prevention.NEW & NOTEWORTHY Chronic hepatitis is characterized by lymphocyte accumulation in liver tissue, which drives fibrosis and carcinogenesis. Here, we demonstrate for the first time that the tetraspanin CD151 supports lymphocyte adhesion to liver endothelium. We show that CD151 is upregulated in chronic liver disease and hepatocellular carcinoma (HCC) and is regulated on endothelium by tissue remodeling and procarcinogenic factors. These regulatory and functional studies identify CD151 as a potential therapeutic target to treat liver fibrosis and HCC.


Assuntos
Carcinoma Hepatocelular/metabolismo , Adesão Celular/fisiologia , Doença Hepática Terminal/metabolismo , Neoplasias Hepáticas/metabolismo , Fígado/metabolismo , Linfócitos/metabolismo , Tetraspanina 24/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo , Carcinoma Hepatocelular/patologia , Doença Hepática Terminal/patologia , Humanos , Fígado/efeitos dos fármacos , Fígado/patologia , Neoplasias Hepáticas/patologia , Linfócitos/efeitos dos fármacos , Linfócitos/patologia , Regulação para Cima/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/farmacologia
11.
Mol Neurodegener ; 12(1): 25, 2017 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-28279219

RESUMO

BACKGROUND: The mechanisms behind Aß-peptide accumulation in non-familial Alzheimer's disease (AD) remain elusive. Proteins of the tetraspanin family modulate Aß production by interacting to γ-secretase. METHODS: We searched for tetraspanins with altered expression in AD brains. The function of the selected tetraspanin was studied in vitro and the physiological relevance of our findings was confirmed in vivo. RESULTS: Tetraspanin-6 (TSPAN6) is increased in AD brains and overexpression in cells exerts paradoxical effects on Amyloid Precursor Protein (APP) metabolism, increasing APP-C-terminal fragments (APP-CTF) and Aß levels at the same time. TSPAN6 affects autophagosome-lysosomal fusion slowing down the degradation of APP-CTF. TSPAN6 recruits also the cytosolic, exosome-forming adaptor syntenin which increases secretion of exosomes that contain APP-CTF. CONCLUSIONS: TSPAN6 is a key player in the bifurcation between lysosomal-dependent degradation and exosome mediated secretion of APP-CTF. This corroborates the central role of the autophagosomal/lysosomal pathway in APP metabolism and shows that TSPAN6 is a crucial player in APP-CTF turnover.


Assuntos
Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Tetraspaninas/metabolismo , Animais , Western Blotting , Exossomos/metabolismo , Exossomos/ultraestrutura , Humanos , Imageamento Tridimensional , Imuno-Histoquímica , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Neurônios/metabolismo , Reação em Cadeia da Polimerase em Tempo Real
12.
Oncotarget ; 8(8): 13277-13292, 2017 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-28129652

RESUMO

The transmembrane 4 L six family proteins TM4SF1, TM4SF4, and TM4SF5 share 40-50% overall sequence identity, but their C-terminus identity is limited. It may be likely that the C-termini of the members are important and unique for own regulatory functions. We thus examined how the TM4SF5 C-terminus affected cellular functions differentially from other family members. Using colon cancer cells expressing wildtype (WT), C-terminus-deleted, or chimeric mutants, diverse cellular functions were explored in 2-dimensional (2D) and 3-dimensional (3D) condition. The C-termini of the proteins were relatively comparable with respect to 2D cell proliferation, although each C-terminal-deletion mutant exhibited increased proliferation relative to the WT. Using chimeric constructs, we found that the TM4SF5 C-terminus was critical for regulating the diverse metastatic functions of TM4SF5, and could positively replace the C-termini of other family members. Replacement of the TM4SF1 or TM4SF4 C-terminus with that of TM4SF5 increased spheroids growth, transwell migration, and invasive dissemination from spheroids in 3D collagen gels. TM4SF5-mediated effects required its extracellular loop 2 linked to the C-terminus via the transmembrane domain 4, with causing c-Src activation. Altogether, the C-terminus of TM4SF5 appears to mediate pro-migratory roles, depending on a structural relay from the second extracellular loop to the C-terminus.


Assuntos
Antígenos de Superfície/genética , Proliferação de Células/genética , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Mutação , Proteínas de Neoplasias/genética , Animais , Antígenos de Superfície/metabolismo , Western Blotting , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Movimento Celular/genética , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Células HCT116 , Células HT29 , Humanos , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Proteínas de Neoplasias/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esferoides Celulares/metabolismo , Transplante Heterólogo
13.
J Cancer ; 8(17): 3607-3614, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29667990

RESUMO

In 2006, a remarkable collaboration between University of Texas MD Anderson Cancer Center clinicians and Texas and New Mexico State legislators led to the formation of a dedicated IBC Research Program and Clinic at MD Anderson. This initiative provided funding and infrastructure to foster coordination of an IBC World Consortium of national and international experts, and launch the first ever IBC international conference in 2008, which brought together experts from around the world to facilitate collaborations and accelerate progress. Indeed great progress has been made since then. National and international experts in IBC convened at the 10th Anniversary Conference of the MD Anderson IBC Clinic and Research Program and presented the most extensive sequencing analysis to date comparing IBC to non-IBC, gene- and protein-based immunoprofiling of IBC versus non-IBC patients, and converging lines of evidence on the specific role of the microenvironment in IBC. Novel models, unique metabolic mechanisms, and prominent survival pathways have been identified and were presented. Multiple clinical trials based on the work of the last decade are in progress or in development. The important challenges ahead were discussed. This progress and a coordinated summary of these works are presented herein.

14.
Platelets ; 28(7): 629-642, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28032533

RESUMO

The tetraspanins are a superfamily of four-transmembrane proteins, which regulate the trafficking, lateral diffusion and clustering of the transmembrane proteins with which they interact. We have previously shown that tetraspanin Tspan9 is expressed on platelets. Here we have characterised gene-trap mice lacking Tspan9. The mice were viable with normal platelet numbers and size. Tspan9-deficient platelets were specifically defective in aggregation and secretion induced by the platelet collagen receptor GPVI, despite normal surface GPVI expression levels. A GPVI activation defect was suggested by partially impaired GPVI-induced protein tyrosine phosphorylation. In mechanistic experiments, Tspan9 and GPVI co-immunoprecipitated and co-localised, but super-resolution imaging revealed no defects in collagen-induced GPVI clustering on Tspan9-deficient platelets. However, single particle tracking using total internal reflection fluorescence microscopy showed that GPVI lateral diffusion was reduced by approximately 50% in the absence of Tspan9. Therefore, Tspan9 plays a fine-tuning role in platelet activation by regulating GPVI membrane dynamics.


Assuntos
Plaquetas/metabolismo , Ativação Plaquetária/efeitos dos fármacos , Glicoproteínas da Membrana de Plaquetas/genética , Tetraspaninas/genética , Difosfato de Adenosina/farmacologia , Animais , Ácido Araquidônico/farmacologia , Plaquetas/patologia , Proteínas de Transporte/farmacologia , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peptídeos/farmacologia , Fosforilação , Agregação Plaquetária/efeitos dos fármacos , Glicoproteínas da Membrana de Plaquetas/metabolismo , Cultura Primária de Células , Ligação Proteica , Transporte Proteico , Transdução de Sinais , Tetraspaninas/química , Tetraspaninas/deficiência
15.
Sci Rep ; 6: 32337, 2016 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-27578500

RESUMO

Human papillomaviruses enter host cells via a clathrin-independent endocytic pathway involving tetraspanin proteins. However, post-endocytic trafficking required for virus capsid disassembly remains unclear. Here we demonstrate that the early trafficking pathway of internalised HPV particles involves tetraspanin CD63, syntenin-1 and ESCRT-associated adaptor protein ALIX. Following internalisation, viral particles are found in CD63-positive endosomes recruiting syntenin-1, a CD63-interacting adaptor protein. Electron microscopy and immunofluorescence experiments indicate that the CD63-syntenin-1 complex controls delivery of internalised viral particles to multivesicular endosomes. Accordingly, infectivity of high-risk HPV types 16, 18 and 31 as well as disassembly and post-uncoating processing of viral particles was markedly suppressed in CD63 or syntenin-1 depleted cells. Our analyses also present the syntenin-1 interacting protein ALIX as critical for HPV infection and CD63-syntenin-1-ALIX complex formation as a prerequisite for intracellular transport enabling viral capsid disassembly. Thus, our results identify the CD63-syntenin-1-ALIX complex as a key regulatory component in post-endocytic HPV trafficking.


Assuntos
Proteínas de Ligação ao Cálcio/genética , Proteínas de Ciclo Celular/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Infecções por Papillomavirus/genética , Sinteninas/genética , Tetraspanina 30/genética , Neoplasias do Colo do Útero/genética , Proteínas de Ligação ao Cálcio/química , Carcinogênese/genética , Proteínas de Ciclo Celular/química , Endocitose/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Feminino , Células HeLa , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/patogenicidade , Papillomavirus Humano 18/genética , Papillomavirus Humano 18/patogenicidade , Papillomavirus Humano 31/genética , Papillomavirus Humano 31/patogenicidade , Humanos , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Infecções por Papillomavirus/patologia , Infecções por Papillomavirus/virologia , Ligação Proteica , Transporte Proteico/genética , Tetraspanina 30/química , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/virologia
17.
Biochem J ; 473(12): 1703-18, 2016 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-27048593

RESUMO

Septins are a family of cytoskeletal GTP-binding proteins that assemble into membrane-associated hetero-oligomers and organize scaffolds for recruitment of cytosolic proteins or stabilization of membrane proteins. Septins have been implicated in a diverse range of cancers, including gastric cancer, but the underlying mechanisms remain unclear. The hypothesis tested here is that septins contribute to cancer by stabilizing the receptor tyrosine kinase ErbB2, an important target for cancer treatment. Septins and ErbB2 were highly over-expressed in gastric cancer cells. Immunoprecipitation followed by MS analysis identified ErbB2 as a septin-interacting protein. Knockdown of septin-2 or cell exposure to forchlorfenuron (FCF), a well-established inhibitor of septin oligomerization, decreased surface and total levels of ErbB2. These treatments had no effect on epidermal growth factor receptor (EGFR), emphasizing the specificity and functionality of the septin-ErbB2 interaction. The level of ubiquitylated ErbB2 at the plasma membrane was elevated in cells treated with FCF, which was accompanied by a decrease in co-localization of ErbB2 with septins at the membrane. Cathepsin B inhibitor, but not bafilomycin or lactacystin, prevented FCF-induced decrease in total ErbB2 by increasing accumulation of ubiquitylated ErbB2 in lysosomes. Therefore, septins protect ErbB2 from ubiquitylation, endocytosis and lysosomal degradation. The FCF-induced degradation pathway is distinct from and additive with the degradation induced by inhibiting ErbB2 chaperone Hsp90. These results identify septins as novel regulators of ErbB2 expression that contribute to the remarkable stabilization of the receptor at the plasma membrane of cancer cells and may provide a basis for the development of new ErbB2-targeting anti-cancer therapies.


Assuntos
Receptor ErbB-2/metabolismo , Septinas/metabolismo , Neoplasias Gástricas/metabolismo , Western Blotting , Linhagem Celular Tumoral , Cromatografia Líquida , Citoesqueleto/metabolismo , Humanos , Imunoprecipitação , Compostos de Fenilureia/farmacologia , Ligação Proteica/efeitos dos fármacos , Piridinas/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/fisiologia , Receptor ErbB-2/genética , Septinas/antagonistas & inibidores , Septinas/genética , Transdução de Sinais/fisiologia , Espectrometria de Massas em Tandem , Ubiquitinação/efeitos dos fármacos
18.
Br J Cancer ; 113(9): 1350-7, 2015 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-26418423

RESUMO

BACKGROUND: The proposed involvement of CD151 in breast cancer (BCa) progression is based on findings from studies in invasive ductal carcinoma (IDC). The IDC and invasive lobular carcinoma (ILC) represent distinct disease entities. Here we evaluated clinical significance of CD151 alone and in association with integrin α3ß1 in patients with ILC in context of the data of our recent IDC study. METHODS: Expression of CD151 and/or integrin α3ß1 was evaluated in ILC samples (N=117) using immunohistochemistry. The findings were analysed in relation to our results from an IDC cohort (N=182) demonstrating a prognostic value of an expression of CD151/integrin α3ß1 complex in patients with HER2-negative tumours. RESULTS: Unlike in the IDCs, neither CD151 nor CD151/α3ß1 complex showed any correlation with any of the ILC characteristics. Lack of both CD151 and α3ß1 was significantly correlated with poor survival (P=0.034) in lymph node-negative ILC N(-) cases. The CD151(-)/α3ß1(-) patients had 3.12-fold higher risk of death from BCa in comparison with the rest of the ILC N(-) patients. CONCLUSIONS: Biological role of CD151/α3ß1 varies between ILC and IDC. Assessment of CD151/α3ß1 might help to identify ILC N(-) patients with increased risk of distant metastases.


Assuntos
Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Carcinoma Lobular/metabolismo , Integrina alfa3beta1/metabolismo , Linfonodos/patologia , Tetraspanina 24/metabolismo , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/patologia , Carcinoma Lobular/patologia , Feminino , Humanos , Imuno-Histoquímica/métodos , Pessoa de Meia-Idade , Prognóstico , Receptor ErbB-2/metabolismo
19.
Oncotarget ; 6(15): 13731-41, 2015 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-25915532

RESUMO

Multicellular 3-dimensional (3D) in vitro models of normal human breast tissue to study cancer initiation are required. We present a model incorporating three of the major functional cell types of breast, detail the phenotype and document our breast cancer initiation studies. Myoepithelial cells and fibroblasts were isolated and immortalised from breast reduction mammoplasty samples. Tri-cultures containing non-tumorigenic luminal epithelial cells HB2, or HB2 overexpressing different HER proteins, together with myoepithelial cells and fibroblasts were established in collagen I. Phenotype was assessed morphologically and immunohistochemically and compared to normal breast tissue. When all three cell types were present, polarised epithelial structures with lumens and basement membrane production were observed, akin to normal human breast tissue. Overexpression of HER2 or HER2/3 caused a significant increase in size, while HER2 overexpression resulted in development of a DCIS-like phenotype. In summary, we have developed a 3D tri-cellular model of normal human breast, amenable to comparative analysis after genetic manipulation and with potential to dissect the mechanisms behind the early stages of breast cancer initiation.


Assuntos
Neoplasias da Mama/patologia , Mama/citologia , Técnicas de Cultura de Células/métodos , Mama/enzimologia , Neoplasias da Mama/enzimologia , Feminino , Humanos , Imageamento Tridimensional/métodos , Imuno-Histoquímica , Radioimunodetecção/métodos , Receptor ErbB-2/biossíntese , Receptor ErbB-3/biossíntese
20.
Viruses ; 6(2): 893-908, 2014 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-24553111

RESUMO

Human papillomaviruses (HPV) are non-enveloped DNA tumor viruses that infect skin and mucosa. The most oncogenic subtype, HPV16, causes various types of cancer, including cervical, anal, and head and neck cancers. During the multistep process of infection, numerous host proteins are required for the delivery of virus genetic information into the nucleus of target cells. Over the last two decades, many host-cell proteins such as heparan sulfate proteoglycans, integrins, growth factor receptors, actin and the tetraspanin CD151 have been described to be involved in the process of infectious entry of HPV16. Tetraspanins have the ability to organize membrane microdomains and to directly influence the function of associated molecules, including binding of receptors to their ligands, receptor oligomerization and signal transduction. Here, we summarize the current knowledge on CD151, and CD151-associated partners during HPV infection and discuss the underlying mechanisms.


Assuntos
Interações Hospedeiro-Patógeno , Papillomaviridae/fisiologia , Receptores Virais/metabolismo , Tetraspanina 24/metabolismo , Internalização do Vírus , Humanos , Modelos Biológicos
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