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1.
Oncogene ; 39(15): 3056-3074, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32066881

RESUMO

The Bcl-xL apoptosis inhibitor plays a major role in vertebrate development. In addition to its effect on apoptosis, Bcl-xL is also involved in cell migration and mitochondrial metabolism. These effects may favour the onset and dissemination of metastasis. However, the underlying molecular mechanisms remain to be fully understood. Here we focus on the control of cell migration by Bcl-xL in the context of breast cancer cells. We show that Bcl-xL silencing led to migration defects in Hs578T and MDA-MB231 cells. These defects were rescued by re-expressing mitochondria-addressed, but not endoplasmic reticulum-addressed, Bcl-xL. The use of BH3 mimetics, such as ABT-737 and WEHI-539 confirmed that the effect of Bcl-xL on migration did not depend on interactions with BH3-containing death accelerators such as Bax or BH3-only proteins. In contrast, the use of a BH4 peptide that disrupts the Bcl-xL/VDAC1 complex supports that Bcl-xL by acting on VDAC1 permeability contributes to cell migration through the promotion of reactive oxygen species production by the electron transport chain. Collectively our data highlight the key role of Bcl-xL at the interface between cell metabolism, cell death, and cell migration, thus exposing the VDAC1/Bcl-xL interaction as a promising target for anti-tumour therapy in the context of metastatic breast cancer.

2.
Eur Urol Oncol ; 2019 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-31676281

RESUMO

BACKGROUND: In metastatic castration-resistant prostate cancer (mCRPC), androgen receptor splice variant 7 (AR-V7) expression is associated with a low response to androgen receptor signaling (ARS) inhibitors such as abiraterone or enzalutamide. OBJECTIVE: To perform a highly sensitive assay for detecting AR-V7 (hsAR-V7) in circulating tumor cells (CTCs) and evaluate its ability to predict response to ARS inhibitors. DESIGN, SETTING, AND PARTICIPANTS: From 41 mCRPC patients, CTCs were prospectively enriched using AdnaTest platform and analyzed for AR-V7 with and without the highly sensitive assay. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The first objective of the study was to compare AR-V7 detection rates with and without the highly sensitive assay. Next, we investigated how AR-V7 (detected without the highly sensitive assay) and hsAR-V7 status influenced prostate-specific antigen (PSA) response and long-term clinical outcomes (PSA progression-free survival [PFS] and radiological PFS) after ARS-inhibitor treatment. Finally, discriminatory abilities of the assays were assessed by C-index to compare their impact on long-term clinical outcomes. RESULTS AND LIMITATIONS: AR-V7 detection rates increased from 22% to 56% when the highly sensitive assay was used. The discriminatory abilities of hsAR-V7 for PSA PFS (C-index, 0.74; 95% confidence interval [CI], 0.60-0.88) and radiological PFS (0.70; 95% CI, 0.55-0.85) were higher than those of AR-V7 detected without the highly sensitive assay (0.60, 0.51-0.72, and 0.56, 0.44-0.67, respectively). After ARS-inhibitor treatment, PSA response was lower in hsAR-V7+ (53%) than in hsAR-V7- (93%) patients (p = 0.016). AR-V7+ patients had shorter median PSA PFS (3.0 vs 10.6 mo, p = 0.032) and nonsignificantly shorter median radiological PFS (6.0 vs 14.8 mo, p = 0.24) compared with AR-V7- patients. The hsAR-V7+ status was associated with shorter median PSA PFS (3.0 mo vs not reached, p = 0.0001) and radiological PFS (median, 6.0 mo vs not reached, p = 0.0026). CONCLUSIONS: The hsAR-V7 assay achieved the highest AR-V7 detection rates among those reported in mCRPC. Discriminatory abilities for long-term clinical outcomes were better with hsAR-V7 assay. PATIENT SUMMARY: We prospectively analyzed circulating tumor cells from men with metastatic castration-resistant prostate cancer for androgen receptor splice variant 7 (AR-V7) status using a highly sensitive assay. It yielded higher AR-V7 detection rates and predicted resistance to androgen receptor signaling inhibitors with better discriminatory abilities for long-term clinical outcomes.

3.
Nat Commun ; 9(1): 4545, 2018 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-30382089

RESUMO

Aerotaxis or chemotaxis to oxygen was described in bacteria 130 years ago. In eukaryotes, the main adaptation to hypoxia currently described relies on HIF transcription factors. To investigate whether aerotaxis is conserved in higher eukaryotes, an approach based on the self-generation of hypoxia after cell confinement was developed. We show that epithelial cells from various tissues migrate with an extreme directionality towards oxygen to escape hypoxia, independently of the HIF pathway. We provide evidence that, concomitant to the oxygen gradient, a gradient of reactive oxygen species (ROS) develops under confinement and that antioxidants dampen aerotaxis. Finally, we establish that in mammary cells, EGF receptor, the activity of which is potentiated by ROS and inhibited by hypoxia, represents the molecular target that guides hypoxic cells to oxygen. Our results reveals that aerotaxis is a property of higher eukaryotic cells and proceeds from the conversion of oxygen into ROS.


Assuntos
Movimento Celular , Receptores ErbB/metabolismo , Glândulas Mamárias Humanas/citologia , Oxigênio/farmacologia , Hipóxia Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Modelos Biológicos , Oxirredução , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos
5.
Assay Drug Dev Technol ; 16(6): 350-360, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30088945

RESUMO

In response to a variety of insults the unfolded protein response (UPR) is a major cell program quickly engaged to promote either cell survival or if stress levels cannot be relieved, apoptosis. UPR relies on three major pathways, named from the endoplasmic reticulum (ER) resident proteins IRE1α, PERK, and ATF6 that mediate response. Current tools to measure the activation of these ER stress response pathways in mammalian cells are cumbersome and not compatible with high-throughput imaging. In this study, we present IRE1α and PERK sensors with improved sensitivity, based on the canonical events of xbp1 splicing and ATF4 translation at ORF3. These sensors can be integrated into host cell genomes through lentiviral transduction, opening the way for use in a wide array of immortalized or primary mammalian cells. We demonstrate that high-throughput single-cell analysis offers unprecedented kinetic details compared with endpoint measurement of IRE1α and PERK activity. Finally, we point out the limitations of dye-based nuclear segmentation for live cell imaging applications, as we show that these dyes induce UPR and can strongly affect both the kinetic and dynamic responses of IRE1α and PERK pathways.


Assuntos
Corantes/química , Endorribonucleases/análise , Imagem Óptica , Proteínas Serina-Treonina Quinases/análise , eIF-2 Quinase/análise , Células Cultivadas , Estresse do Retículo Endoplasmático , Endorribonucleases/metabolismo , Células HCT116 , Células HEK293 , Células HeLa , Ensaios de Triagem em Larga Escala , Humanos , Proteínas Serina-Treonina Quinases/metabolismo , Análise de Célula Única , eIF-2 Quinase/metabolismo
6.
Cancer Res ; 78(6): 1404-1417, 2018 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-29330143

RESUMO

Drug resistance and metastatic relapse remain a top challenge in breast cancer treatment. In this study, we present preclinical evidence for a strategy to eradicate advanced breast cancers by targeting the BCL-2 homolog Nrh/BCL2L10, which we discovered to be overexpressed in >45% of a large cohort of breast invasive carcinomas. Nrh expression in these tumors correlated with reduced metastasis-free survival, and we determined it to be an independent marker of poor prognosis. Nrh protein localized to the endoplasmic reticulum. Mechanistic investigations showed that Nrh made BH4 domain-dependent interactions with the ligand-binding domain of the inositol-1,4,5-triphosphate receptor (IP3R), a type 1/3 Ca2+ channel, allowing Nrh to negatively regulate ER-Ca2+ release and to mediate antiapoptosis. Notably, disrupting Nrh/IP3R complexes by BH4 mimetic peptides was sufficient to inhibit the growth of breast cancer cells in vitro and in vivo Taken together, our results highlighted Nrh as a novel prognostic marker and a candidate therapeutic target for late stage breast cancers that may be addicted to Nrh.Significance: These findings offer a comprehensive molecular model for the activity of Nrh/BCL2L10, a little studied antiapoptotic molecule, prognostic marker, and candidate drug target in breast cancer. Cancer Res; 78(6); 1404-17. ©2018 AACR.


Assuntos
Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Retículo Endoplasmático/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Apoptose/fisiologia , Sítios de Ligação , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Cálcio/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Retículo Endoplasmático/efeitos dos fármacos , Feminino , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Camundongos SCID , Terapia de Alvo Molecular/métodos , Fragmentos de Peptídeos/metabolismo , Peptídeos/farmacologia , Prognóstico , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Sci Rep ; 6: 36570, 2016 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-27827394

RESUMO

Intracellular Ca2+ signaling regulates cell migration by acting on cytoskeleton architecture, cell directionality and focal adhesions dynamics. In migrating cells, cytosolic Ca2+ pool and Ca2+ pulses are described as key components of these effects. Whereas the role of the mitochondrial calcium homeostasis and the Mitochondria Cacium Uniporter (MCU) in cell migration were recently highlighted in vivo using the zebrafish model, their implication in actin cystokeleton dynamics and cell migration in mammals is not totally characterized. Here, we show that mcu silencing in two human cell lines compromises their migration capacities. This phenotype is characterized by actin cytoskeleton stiffness, a cell polarization loss and an impairment of the focal adhesion proteins dynamics. At the molecular level, these effects appear to be mediated by the reduction of the ER and cytosolic Ca2+ pools, which leads to a decrease in Rho-GTPases, RhoA and Rac1, and Ca2+-dependent Calpain activites, but seem to be independent of intracellular ATP levels. Together, this study highlights the fundamental and evolutionary conserved role of the mitochondrial Ca2+ homeostasis in cytoskeleton dynamics and cell migration.


Assuntos
Citoesqueleto de Actina/metabolismo , Cálcio/metabolismo , Movimento Celular , Mitocôndrias/metabolismo , Animais , Polaridade Celular , Regulação para Baixo , Adesões Focais , Modelos Animais , Peixe-Zebra
8.
Oncotarget ; 7(28): 44023-44038, 2016 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-27281619

RESUMO

At the time of diagnosis, 60% of patients with head and neck squamous cell carcinoma (HNSCC) present tumors in an advanced stage (III-IV) of disease and 80% will relapse within the first two years post-treatment, due to their frequent radio(chemo)resistance. To identify new molecular targets and companion biomarkers, we have investigated the miRNome of 75 stage III-IV oropharynx tumors without relapse (R) or with loco-regional relapse (non-responder, NR) within two years post-treatment. Interestingly, miR-422a was significantly downregulated in NR tumors, in agreement with the increase in cell proliferation and adhesion induced by miR-422a inhibition in vitro. Furthermore, we identified CD73/NT5E oncogene as target of miR-422a. Indeed, modulation of the endogenous level of miR-422a inversely influences the expression and the enzymatic activity of CD73. Moreover, knocking down CD73 mimics the effects of miR-422a upregulation. Importantly, in tumors, miR-422a and CD73 expression levels are inversely correlated, and both are predictive of relapse free survival - especially considering loco(regional) recurrence - in vitro two independent cohorts of advanced oropharynx or HNSCC (N=255) tumors. In all, we reported, for the first time, that MiR-422a and its target CD73 are involved in early loco(regional) recurrence of HNSCC tumors and are new targets for personalized medicine.


Assuntos
5'-Nucleotidase/genética , Carcinoma de Células Escamosas/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/genética , MicroRNAs/genética , 5'-Nucleotidase/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Adesão Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/genética , Feminino , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia , Interferência de RNA
9.
Histopathology ; 68(2): 279-85, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26033501

RESUMO

AIMS: FOXL2 mutation has been consistently identified in adult granulosa cell tumours (A-GCTs). DICER1 mutations have been described predominantly in Sertoli-Leydig cell tumours (SLCTs). The prognostic implication of these mutations remains uncertain, as moderately sized studies have yielded variable outcomes. Our aim was to determine the implications of DICER1 and FOXL2 mutations in 156 ovarian sex cord-stromal tumours (SCSTs). METHODS AND RESULTS: FOXL2 mutations were found in 94% of pathologically confirmed A-GCTs (95/101), in one of eight juvenile granulosa cell tumours (J-GCTs), and in two of 19 SLCTs. DICER1 mutations in the RNase IIIb domain were found in six of 19 SLCTs, two of eight J-GCTs, and one of 12 undifferentiated SCSTs (Und-SCSTs). Comparison of DICER1-mutated SLCTs with DICER1-non-mutated SLCTs showed that patient age at diagnosis was lower and oestrogen receptor expression was more frequent in DICER1-mutated tumours. With a median follow-up of 22 months, two of five DICER1-mutated SLCTs relapsed, in contrast to none of eight DICER1-non-mutated tumours. CONCLUSIONS: Our results suggest that, in contrast to FOXL2 mutations in A-GCT, DICER1 mutations in SLCT might be more useful for prognosis than for diagnosis. However, study of a larger cohort of patients is necessary to establish this. Identification of genetic alterations in SCST offers promising therapeutic options.


Assuntos
RNA Helicases DEAD-box/genética , Proteína Forkhead Box L2/genética , Tumor de Células da Granulosa/genética , Neoplasias Ovarianas/genética , Ribonuclease III/genética , Tumor de Células de Sertoli-Leydig/genética , Tumores do Estroma Gonadal e dos Cordões Sexuais/genética , Adolescente , Adulto , Idoso , Feminino , Tumor de Células da Granulosa/diagnóstico , Tumor de Células da Granulosa/patologia , Humanos , Pessoa de Meia-Idade , Mutação , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/patologia , Inclusão em Parafina , Prognóstico , Tumor de Células de Sertoli-Leydig/patologia , Tumores do Estroma Gonadal e dos Cordões Sexuais/diagnóstico , Tumores do Estroma Gonadal e dos Cordões Sexuais/patologia , Adulto Jovem
10.
PLoS One ; 10(9): e0139179, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26407179

RESUMO

The increased expression of the Disintegrin and Metalloprotease ADAM12 has been associated with human cancers, however its role remain unclear. We have previously reported that ADAM12 expression is induced by the transforming growth factor, TGF-ß and promotes TGF-ß-dependent signaling through interaction with the type II receptor of TGF-ß. Here we explore the implication of ADAM12 in TGF-ß-mediated epithelial to mesenchymal transition (EMT), a key process in cancer progression. We show that ADAM12 expression is correlated with EMT markers in human breast cancer cell lines and biopsies. Using a non-malignant breast epithelial cell line (MCF10A), we demonstrate that TGF-ß-induced EMT increases expression of the membrane-anchored ADAM12L long form. Importantly, ADAM12L overexpression in MCF10A is sufficient to induce loss of cell-cell contact, reorganization of actin cytoskeleton, up-regulation of EMT markers and chemoresistance. These effects are independent of the proteolytic activity but require the cytoplasmic tail and are specific of ADAM12L since overexpression of ADAM12S failed to induce similar changes. We further demonstrate that ADAM12L-dependent EMT is associated with increased phosphorylation of Smad3, Akt and ERK proteins. Conversely, inhibition of TGF-ß receptors or ERK activities reverses ADAM12L-induced mesenchymal phenotype. Together our data demonstrate that ADAM12L is associated with EMT and contributes to TGF-ß-dependent EMT by favoring both Smad-dependent and Smad-independent pathways.


Assuntos
Proteínas ADAM/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Proteína ADAM12 , Adulto , Idoso , Idoso de 80 Anos ou mais , Biocatálise/efeitos dos fármacos , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Citoplasma/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Mesoderma/metabolismo , Pessoa de Meia-Idade , Fenótipo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética
11.
BMC Cancer ; 15: 453, 2015 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-26040677

RESUMO

BACKGROUND: The Transforming growth factor ß (TGFß) signaling has a paradoxical role in cancer development and outcome. Besides, the prognostic significance of the TGFß1, SMAD4 in breast cancer patients is an area of many contradictions. The transcriptional intermediary factor 1γ (TIF1γ) is thought to interact with the TGFß/SMAD signaling through different mechanisms. Our study aims to define the prognostic significance of TGFß1, SMAD4 and TIF1γ expression in breast cancer patients and to detect possible interactions among those markers that might affect the outcome. METHODS: Immunohistochemistry was performed on tissue microarray (TMA) blocks prepared from samples of 248 operable breast cancer patients who presented at Centre Léon Bérard (CLB) between 1998 and 2001. The intensity and the percentage of stained tumor cells were integrated into a single score (0-6) and a cutoff was defined for high or low expression for each marker. Correlation was done between TGFß1, SMAD4 and TIF1γ expression with the clinico-pathologic parameters using Pearson's chi-square test. Kaplan-Meier method was used to estimate distant metastasis free survival (DMFS), disease free survival (DFS) and overall survival (OS) and the difference between the groups was evaluated with log-rank test. RESULTS: 223 cases were assessable for TIF1γ, 204 for TGFß1 and 173 for SMAD4. Median age at diagnosis was 55.8 years (range: 27 to 89 years). Tumors were larger than 20 mm in 49.2% and 45.2% had axillary lymph node (LN) metastasis (N1a to N3). 19.4% of the patients had SBR grade I tumors, 46.8% grade II tumors and 33.9% grade III tumors. ER was positive in 85.4%, PR in 75.5% and Her2-neu was over-expressed in 10% of the cases. Nuclear TIF1γ, cytoplasmic TGFß1, nuclear and cytoplasmic SMAD4 stainings were high in 35.9%, 30.4%, 27.7% and 52.6% respectively. TIF1γ expression was associated with younger age (p=0.006), higher SBR grade (p<0.001), more ER negativity (p=0.035), and tumors larger than 2 cm (p=0.081), while TGFß1 was not associated with any of the traditional prognostic factors. TGFß1 expression in tumor cells was a marker of poor prognosis regarding DMFS (HR=2.28; 95% CI: 1.4 to 3.8; p=0.002), DFS (HR=2.00; 95% CI: 1.25 to 3.5; p=0.005) and OS (HR=1.89; 95 % CI: 1.04 to 3.43; p=0.037). TIF1γ expression carried a tendency towards poorer DMFS (p=0.091), DFS (p=0.143) and OS (p=0.091). In the multivariate analysis TGFß1 remained an independent predictor of shorter DMFS, DFS and OS. Moreover, the prognostic significance of TGFß1 was more obvious in the TIF1γ high patient subgroup than in the patients with TIF1γ low expression. The subgroup expressing both markers had the worst DMFS (HR=3.2; 95% CI: 1.7 to 5.9; p<0.0001), DFS (HR=3.02; 95 % CI: 1.6 to 5.6; p<0.0001) and OS (HR=2.7; 95 % CI: 1.4 to 5.4; p=0.005). CONCLUSION: There is a crosstalk between the TIF1γ and the TGFß1/SMAD4 signaling that deteriorates the outcome of operable breast cancer patients and when combined together they can serve as an effective prognostic tool for those patients.


Assuntos
Neoplasias da Mama/química , Neoplasias da Mama/patologia , Carcinoma/química , Proteína Smad4/análise , Fatores de Transcrição/análise , Fator de Crescimento Transformador beta1/análise , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/cirurgia , Carcinoma/secundário , Carcinoma/cirurgia , Núcleo Celular/química , Citoplasma/química , Intervalo Livre de Doença , Feminino , Humanos , Metástase Linfática , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Receptor ErbB-2/análise , Receptores Estrogênicos/análise , Receptores de Progesterona/análise , Transdução de Sinais , Taxa de Sobrevida , Carga Tumoral
12.
Carcinogenesis ; 36(5): 564-73, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25823895

RESUMO

Granulosa cell tumor (GCT) is a rare and severe form of sex-cord stromal ovarian tumor that is characterized by its long natural history and tendency to recur years after surgical ablation. Because there is no efficient curative treatment beyond surgery, ~20% of patients die of the consequences of their tumor. However, very little is known of the molecular etiology of this pathology. About 70% of GCT patients present with elevated circulating estradiol (E2). Because this hormone is known to increase tumor growth and progression in a number of cancers, we investigated the possible role of E2 in GCTs. Cell-based studies with human GCT metastases and primary tumor-derived cells, ie KGN and COV434 cells, respectively, aimed at evaluating E2 effect on cell growth, migration and invasion. Importantly, we found that E2 did not affect GCT cell growth, but that it significantly decreased the migration and matrix invasion of metastatic GCT cells. Noteworthy, our molecular studies revealed that this effect was accompanied by the inhibition through non-genomic mechanisms of extracellular signal-regulated kinase 1/2 (ERK1/2), which is constitutively activated in GCTs. By using pharmacological and RNA silencing approaches, we found that E2 action was mediated by G protein-coupled estrogen receptor 1 (GPER1) signaling pathway. Analyses of GPER1 expression on tissue microarrays from human GCTs confirmed its expression in ~90% of GCTs. Overall, our study reveals that E2 would act via non-classical pathways to prevent metastasis spreading in GCTs and also reveals GPER1 as a possible target in this disease.


Assuntos
Movimento Celular/efeitos dos fármacos , Estradiol/farmacologia , Estrogênios/farmacologia , Tumor de Células da Granulosa/tratamento farmacológico , Recidiva Local de Neoplasia/tratamento farmacológico , Receptores Estrogênicos/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Adulto , Idoso , Apoptose/efeitos dos fármacos , Western Blotting , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Imunofluorescência , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Tumor de Células da Granulosa/metabolismo , Tumor de Células da Granulosa/patologia , Humanos , Técnicas Imunoenzimáticas , Pessoa de Meia-Idade , Metástase Neoplásica , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/patologia , Prognóstico , RNA Interferente Pequeno/genética , Receptores Estrogênicos/antagonistas & inibidores , Receptores Estrogênicos/genética , Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Receptores Acoplados a Proteínas-G/genética , Análise Serial de Tecidos , Células Tumorais Cultivadas , Adulto Jovem
13.
J Proteomics ; 110: 183-94, 2014 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-25242195

RESUMO

UNLABELLED: Disease phenotype reorganizations are the consequences of signaling pathway perturbations and protein abundance modulations. Characterizing the protein signature of a biological event allows the identification of new candidate biomarkers, new targets for treatments and selective patient therapy. The combination of discovery LC-MS/MS analyses and targeted mass spectrometry using selected reaction monitoring (SRM) mode has emerged as a powerful technology for biomarker identification and quantification owing to faster development time and multiplexing capability. The epithelial-mesenchymal transition (EMT) is a process that controls local invasion and metastasis generation by stimulating changes in adhesion and migration of cells but also in metabolic pathways. In this study, the non-transformed human breast epithelial cell line MCF10A, treated by TGFß or overexpressing mutant K-Ras(v12), two EMT inducers frequently involved in cancer progression, was used to characterize protein abundance changes during an EMT event. The LC-MS/MS analysis and label-free quantification revealed that TGFß and K-Ras(v12) induce a similar pattern of protein regulation and that besides the expected cytoskeletal changes, a strong increase in the anabolism and energy production machinery was observed. BIOLOGICAL SIGNIFICANCE: To our knowledge, this is the first proteomic analysis combining a label-free quantification with an SRM validation of proteins regulated by TGFß and K-Rasv12. This study reveals new insights in the characterization of the changes occurring during an epithelial-mesenchymal transition (EMT) event. Notably, a strong increase in the anabolism and energy production machinery was observed upon both EMT inducers.


Assuntos
Mama/metabolismo , Cromatografia Líquida/métodos , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Espectrometria de Massas/métodos , Fator de Crescimento Transformador beta/metabolismo , Proteínas ras/metabolismo , Linhagem Celular , Feminino , Humanos , Mapeamento de Peptídeos/métodos , Coloração e Rotulagem , Fator de Crescimento Transformador beta/química , Proteínas ras/química
14.
Front Oncol ; 4: 222, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25184116

RESUMO

A key question for urologic practitioners is whether an apparently organ-confined prostate cancer (PCa) is actually aggressive or not. The dilemma is to specifically identify among all prostate tumors the very aggressive high-grade cancers that will become life-threatening by developing extra-prostatic invasion and metastatic potential and the indolent cancers that will never modify a patient's life expectancy. A choice must be made between several therapeutic options to achieve the optimal personalized management of the disease that causes as little harm as possible to patients. Reliable clinical, biological, or pathological markers that would enable distinctions to be made between aggressive and indolent PCas in routine practice at the time of initial diagnosis are still lacking. The molecular mechanisms that explain why a PCa is aggressive or not are also poorly understood. Among the potential markers and/or actors in PCa aggressiveness, Src and other members of the Src kinase family, are valuable candidates. Activation of Src-dependent intracellular pathways is frequently observed in PCa. Indeed, Src is at the cross-roads of several pathways [including androgen receptor (AR), TGFbeta, Bcl-2, Akt/PTEN or MAPK, and ERK …], and is now known to influence some of the cellular and tissular events that accompany tumor progression: cell proliferation, cell motility, invasion, epithelial-to-mesenchymal transition, resistance to apoptosis, angiogenesis, neuroendocrine differentiation, and metastatic spread. Recent work even suggests that Src could also play a part in PCa initiation in coordination with the AR. The aim of this review is to gather data that explore the links between the Src kinase family and PCa progression and aggressiveness.

15.
Cell Rep ; 7(6): 1900-13, 2014 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-24910439

RESUMO

The RNA helicases DDX5 and DDX17 are members of a large family of highly conserved proteins that are involved in gene-expression regulation; however, their in vivo targets and activities in biological processes such as cell differentiation, which requires reprogramming of gene-expression programs at multiple levels, are not well characterized. Here, we uncovered a mechanism by which DDX5 and DDX17 cooperate with heterogeneous nuclear ribonucleoprotein (hnRNP) H/F splicing factors to define epithelial- and myoblast-specific splicing subprograms. We then observed that downregulation of DDX5 and DDX17 protein expression during myogenesis and epithelial-to-mesenchymal transdifferentiation contributes to the switching of splicing programs during these processes. Remarkably, this downregulation is mediated by the production of miRNAs induced upon differentiation in a DDX5/DDX17-dependent manner. Since DDX5 and DDX17 also function as coregulators of master transcriptional regulators of differentiation, we propose to name these proteins "master orchestrators" of differentiation that dynamically orchestrate several layers of gene expression.


Assuntos
RNA Helicases DEAD-box/genética , MicroRNAs/genética , Processamento Alternativo , Animais , Diferenciação Celular/genética , RNA Helicases DEAD-box/metabolismo , Regulação para Baixo , Células Epiteliais/enzimologia , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Transição Epitelial-Mesenquimal/genética , Éxons , Regulação da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Células MCF-7 , Camundongos , MicroRNAs/biossíntese , MicroRNAs/metabolismo , Mioblastos/enzimologia , Mioblastos/metabolismo , Mioblastos/fisiologia , Transcrição Genética
16.
Sci Signal ; 7(312): ra14, 2014 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-24518293

RESUMO

Members of the Bcl-2 protein family regulate mitochondrial membrane permeability and also localize to the endoplasmic reticulum where they control Ca(2+) homeostasis by interacting with inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs). In zebrafish, Bcl-2-like 10 (Nrz) is required for Ca(2+) signaling during epiboly and gastrulation. We characterized the mechanism by which Nrz controls IP3-mediated Ca(2+) release during this process. We showed that Nrz was phosphorylated during early epiboly, and that in embryos in which Nrz was knocked down, reconstitution with Nrz bearing mutations designed to prevent its phosphorylation disrupted cyclic Ca(2+) transients and the assembly of the actin-myosin ring and led to epiboly arrest. In cultured cells, wild-type Nrz, but not Nrz with phosphomimetic mutations, interacted with the IP3 binding domain of IP3R1, inhibited binding of IP3 to IP3R1, and prevented histamine-induced increases in cytosolic Ca(2+). Collectively, these data suggest that Nrz phosphorylation is necessary for the generation of IP3-mediated Ca(2+) transients and the formation of circumferential actin-myosin cables required for epiboly. Thus, in addition to their role in apoptosis, by tightly regulating Ca(2+) signaling, Bcl-2 family members participate in the cellular events associated with early vertebrate development, including cytoskeletal dynamics and cell movement.


Assuntos
Sinalização do Cálcio/fisiologia , Movimento Celular/fisiologia , Embrião não Mamífero/fisiologia , Inositol 1,4,5-Trifosfato/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Sequência de Bases , Western Blotting , Biologia Computacional , Embrião não Mamífero/citologia , Transferência Ressonante de Energia de Fluorescência , Técnicas de Silenciamento de Genes , Vetores Genéticos/genética , Células HeLa , Humanos , Imunoprecipitação , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Simulação de Dinâmica Molecular , Morfolinos/genética , Fosforilação , Proteínas Proto-Oncogênicas/genética , Alinhamento de Sequência , Estatísticas não Paramétricas , Proteínas de Peixe-Zebra/genética
17.
Gynecol Oncol ; 132(1): 181-7, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24157616

RESUMO

OBJECTIVE: Ovarian sex cord-stromal tumors (SCSTs) are rare and their diagnosis is often difficult to establish. Recently, immunostaining and molecular analysis for Forkhead box L2 (FOXL2) have been developed in this pathology. This study aims to assess the benefit of an algorithm incorporating these new tools for a better diagnosis and classification of SCSTs METHODS: Seventy-two tumors with a potential diagnosis of SCSTs were addressed by 37 different pathologists to one French rare ovarian tumor expert center, member of the Rare Malignant Ovarian Tumor network (TMRO). Then a "second opinion" (SO) through an algorithm incorporating immunostaining (IHC) and molecular analysis of FOXL2 was performed for all these cases. This algorithm was then validated by all pathologists of the TMRO network. RESULTS: After a second opinion including molecular analysis and immunostaining for FOXL2 the initial diagnosis was changed in 15 of 72 samples (21%). FOXL2 mutation was present in 44 out of 47 adult granulosa cell tumors (94%), in 3 out of 8 Thecomas (37%), in 1 out of 10 Sertoli-Leydig cell tumors (SLSTs) (10%) and in 3 out of 5 undifferentiated-SCSTs (Und-SCSTs) (60%). Immunoexpression of FOXL2 was available in 45 cases of SCSTs: FOXL2 was expressed in 44 of them (98%). CONCLUSIONS: A second opinion in an expert center for all cases of SCSTs is fundamental to get an optimal classification of these rare tumors. This second opinion could be performed with an algorithm which integrates FOXL2 mutation and expression status of FOXL2 in order to standardize the practice.


Assuntos
Fatores de Transcrição Forkhead/análise , Fatores de Transcrição Forkhead/genética , Mutação , Neoplasias Ovarianas/diagnóstico , Tumores do Estroma Gonadal e dos Cordões Sexuais/diagnóstico , Algoritmos , Feminino , Proteína Forkhead Box L2 , Humanos , Imuno-Histoquímica , Neoplasias Ovarianas/genética , Encaminhamento e Consulta , Tumores do Estroma Gonadal e dos Cordões Sexuais/genética , Análise Serial de Tecidos
18.
Cancer Res ; 73(22): 6621-31, 2013 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-24078802

RESUMO

Epithelial-to-mesenchymal transition (EMT) is a transdifferentiation process that converts epithelial cells into highly motile mesenchymal cells. This physiologic process occurs largely during embryonic development but is aberrantly reactivated in different pathologic situations, including fibrosis and cancer. We conducted a siRNA screening targeted to the human kinome with the aim of discovering new EMT effectors. With this approach, we have identified mTOR complex 1 (mTORC1), a nutrient sensor that controls protein and lipid synthesis, as a key regulator of epithelial integrity. Using a combination of RNAi and pharmacologic approaches, we report here that inhibition of either mTOR or RPTOR triggers EMT in mammary epithelial cells. This EMT was characterized by the induction of the mesenchymal markers such as fibronectin, vimentin, and PAI-1, together with the repression of epithelial markers such as E-cadherin and ZO-3. In addition, mTORC1 blockade enhanced in vivo migratory properties of mammary cells and induced EMT independent of the TGF-ß pathway. Finally, among the transcription factors known to activate EMT, both ZEB1 and ZEB2 were upregulated following mTOR repression. Their increased expression correlated with a marked reduction in miR-200b and miR-200c mRNA levels, two microRNAs known to downregulate ZEB1 and ZEB2 expression. Taken together, our findings unravel a novel function for mTORC1 in maintaining the epithelial phenotype and further indicate that this effect is mediated through the opposite regulation of ZEB1/ZEB2 and miR-200b and miR-200c. Furthermore, these results suggest a plausible etiologic explanation for the progressive pulmonary fibrosis, a frequent adverse condition associated with the therapeutic use of mTOR inhibitors.


Assuntos
Transição Epitelial-Mesenquimal/fisiologia , Complexos Multiproteicos/fisiologia , Serina-Treonina Quinases TOR/fisiologia , Animais , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Células Cultivadas , Embrião de Galinha , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Humanos , Células MCF-7 , Alvo Mecanístico do Complexo 1 de Rapamicina , MicroRNAs/metabolismo , Complexos Multiproteicos/antagonistas & inibidores , Complexos Multiproteicos/genética , Invasividade Neoplásica , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , Proteínas Repressoras/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genética , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/fisiologia , Homeobox 2 de Ligação a E-box com Dedos de Zinco , Homeobox 1 de Ligação a E-box em Dedo de Zinco
19.
Nat Commun ; 4: 2330, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23942336

RESUMO

Bcl-2 proteins are acknowledged as key regulators of programmed cell death. However, increasing data suggest additional roles, including regulation of the cell cycle, metabolism and cytoskeletal dynamics. Here we report the discovery and characterization of a new Bcl-2-related multidomain apoptosis accelerator, Bcl-wav, found in fish and frogs. Genetic and molecular studies in zebrafish indicate that Bcl-wav and the recently identified mitochondrial calcium uniporter (MCU) contribute to the formation of the notochord axis by controlling blastomere convergence and extension movements during gastrulation. Furthermore, we found that Bcl-wav controls intracellular Ca(2+) trafficking by acting on the mitochondrial voltage-dependent anion channel, and possibly on MCU, with direct consequences on actin microfilament dynamics and blastomere migration guidance. Thus, from an evolutionary point of view, the original function of Bcl-2 proteins might have been to contribute in controlling the global positioning system of blastomeres during gastrulation, a critical step in metazoan development.


Assuntos
Canais de Cálcio/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Peixe-Zebra/embriologia , Actinas/genética , Sequência de Aminoácidos , Animais , Apoptose/genética , Transporte Biológico/genética , Blastômeros/metabolismo , Cálcio/metabolismo , Canais de Cálcio/genética , Movimento Celular/genética , Células Cultivadas , Gástrula/embriologia , Gastrulação/genética , Técnicas de Silenciamento de Genes , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Camundongos , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Morfogênese , Morfolinos/genética , Notocorda/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Canal de Ânion 1 Dependente de Voltagem/genética
20.
J Cell Sci ; 126(Pt 16): 3713-23, 2013 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-23788427

RESUMO

TIF1γ, a new regulator of TGFß signaling, inhibits the Smad4-mediated TGFß response by interaction with Smad2/3 or ubiquitylation of Smad4. We have shown that TIF1γ participates in TGFß signaling as a negative regulator of Smad4 during the TGFß-induced epithelial-to-mesenchymal transition (EMT) in mammary epithelial cells, and during terminal differentiation of mammary alveolar epithelial cells and lactation. We demonstrate here that TIF1γ is sumoylated and interacts with Ubc9, the only known SUMO-conjugating enzyme. Four functional sumoylation sites lie within the middle domain of TIF1γ, the Smad interaction domain. We show that a sumoylation-defective TIF1γ mutant significantly reduces TIF1γ inhibition of Smad complexes and that of the Smad-mediated TGFß transcriptional response. Moreover, chromatin immunoprecipitation experiments indicate that TIF1γ sumoylation is required to limit Smad4 binding on the PAI-1 TGFß target gene promoter. Ectopic expression of TIF1γ in mammary epithelial cells inhibits TGFß-induced EMT, an effect relieved by expression of non-sumoylated TIF1γ. Taken together, our results identify a new TGFß regulatory layer, whereby sumoylation strengthens the TIF1γ repressive action on canonical TGFß signaling.


Assuntos
Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Sequência de Aminoácidos , Animais , Diferenciação Celular/fisiologia , Humanos , Dados de Sequência Molecular , Inibidor 1 de Ativador de Plasminogênio/genética , Regiões Promotoras Genéticas , Transdução de Sinais , Proteínas Smad/genética , Proteínas Smad/metabolismo , Sumoilação , Transfecção
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