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1.
J Pathol ; 248(4): 501-513, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30957234

RESUMO

Glioblastoma (GBM) is a highly invasive brain neoplasia with an elevated recurrence rate after surgical resection. The cyclin D1 (Ccnd1)/Cdk4-retinoblastoma 1 (RB1) axis is frequently altered in GBM, leading to overproliferation by RB1 deletion or by Ccnd1-Cdk4 overactivation. High levels of Ccnd1-Cdk4 also promote GBM cell invasion by mechanisms that are not so well understood. The purpose of this work is to elucidate the in vivo role of cytoplasmic Ccnd1-Cdk4 activity in the dissemination of GBM. We show that Ccnd1 activates the invasion of primary human GBM cells through cytoplasmic RB1-independent mechanisms. By using GBM mouse models, we observed that evaded GBM cells showed cytoplasmic Ccnd1 colocalizing with regulators of cell invasion such as RalA and paxillin. Our genetic data strongly suggest that, in GBM cells, the Ccnd1-Cdk4 complex is acting upstream of those regulators. Accordingly, expression of Ccnd1 induces focal adhesion kinase, RalA and Rac1 activities. Finally, in vivo experiments demonstrated increased GBM dissemination after expression of membrane-targeted Ccnd1. We conclude that Ccnd1-Cdk4 activity promotes GBM dissemination through cytoplasmic and RB1-independent mechanisms. Therefore, inhibition of Ccnd1-Cdk4 activity may be useful to hinder the dissemination of recurrent GBM. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

2.
Gynecol Oncol ; 153(2): 425-435, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30853360

RESUMO

OBJECTIVES: The PI3K/AKT/mTOR pathway is frequently overactivated in endometrial cancer (EC). We assessed the efficacy of ABTL0812, a novel first-in-class molecule presenting a unique mechanism of action inhibiting this pathway. METHODS: We investigated the effects of ABTL0812 on proliferation, cell death and modulation of intracellular signaling pathways in a wide panel of endometrioid and non-endometrioid cell lines, an inducible PTEN knock-out murine model, and two patient-derived xenograft murine models of EC. Then, TRIB3 expression was evaluated as potential ABTL0812 pharmacodynamic biomarker in a Phase 1b/2a clinical trial. RESULTS: ABTL0812 induced an upregulation of TRIB3 expression, resulting in the PI3K/AKT/mTOR axis inhibition and autophagy cell death induction on EC cells but not in healthy endometrial cells. ABTL0812 treatment also impaired PTEN knock-out cells to progress from hyperplasia to cancer. The therapeutic effects of ABTL0812 were demonstrated in vivo. ABTL0812 increased TRIB3 mRNA levels in whole blood samples of eight EC patients, demonstrating that TRIB3 mRNA could be used as a pharmacodynamic biomarker to monitor the ABTL0812 treatment. CONCLUSIONS: ABTL0812 may represent a novel and highly effective therapeutic agent by inducing TRIB3 expression and autophagy in EC patients, including those with poorer prognosis.


Assuntos
Antineoplásicos/farmacologia , Proteínas de Ciclo Celular/metabolismo , Neoplasias do Endométrio/tratamento farmacológico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Repressoras/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Idoso , Animais , Autofagia/efeitos dos fármacos , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios Clínicos Fase I como Assunto , Ensaios Clínicos Fase II como Assunto , Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/patologia , Feminino , Humanos , Camundongos , Pessoa de Meia-Idade , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/biossíntese , Proteínas Repressoras/genética , Regulação para Cima/efeitos dos fármacos
3.
J Pathol ; 2018 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-30206933

RESUMO

Many human cancers present Phosphatase and tensin homolog (PTEN) deficiency and between 20 and 30% of colorectal tumors show PTEN loss. The transcription factor, E2 promoter binding factor 1 (E2F-1), exhibits tumor promoter or suppressive functions depending on cellular type and tissue context, but its role in the progression and development of colorectal carcinogenesis was largely unknown. Here, using a tamoxifen-inducible PTEN knockout mouse model, we have demonstrated that loss of PTEN leads to the development of colorectal tumorigenesis through the serrated pathway. Next, we studied PTEN loss-driven colorectal lesions in the context of E2F-1 deficiency in vivo. Our results revealed that monoallelic and biallelic absence of E2F-1 led to an increased incidence and progression of serrated tumorigenesis induced by PTEN loss. Finally, we investigated the mechanisms by which double PTEN/E2F-1 deficiency leads to enhanced tumorigenesis. We found that colorectal tumors from PTEN/E2F-1 double knockout mice and the human colorectal carcinoma cell line HT29 with shRNA-mediated downregulation of PTEN and E2F-1 exhibit hyperactivation of the RAS-MAPK pathway, accumulation of DNA damage and resistance to apoptosis. To date, this is the first preclinical study evaluating the effect of genetic deletion of E2F-1 in colorectal malignancies driven by PTEN deficiency. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

4.
J Pathol ; 242(2): 152-164, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28349562

RESUMO

PTEN is one of the most frequently mutated genes in human cancers. The frequency of PTEN alterations is particularly high in endometrial carcinomas. Loss of PTEN leads to dysregulation of cell division, and promotes the accumulation of cell cycle complexes such as cyclin D1-CDK4/6, which is an important feature of the tumour phenotype. Cell cycle proteins have been presented as key targets in the treatment of the pathogenesis of cancer, and several CDK inhibitors have been developed as a strategy to generate new anticancer drugs. Palbociclib (PD-332991) specifically inhibits CDK4/6, and it has been approved for use in metastatic breast cancer in combination with letrazole. Here, we used a tamoxifen-inducible Pten knockout mouse model to assess the antitumour effects of cyclin D1 knockout and CDK4/6 inhibition by palbociclib on endometrial tumours. Interestingly, both cyclin D1 deficiency and palbociclib treatment triggered shrinkage of endometrial neoplasias. In addition, palbociclib treatment significantly increased the survival of Pten-deficient mice, and, as expected, had a general effect in reducing tumour cell proliferation. To further analyse the effects of palbociclib on endometrial carcinoma, we established subcutaneous tumours with human endometrial cancer cell lines and primary endometrial cancer xenografts, which allowed us to provide more translational and predictive data. To date, this is the first preclinical study evaluating the response to CDK4/6 inhibition in endometrial malignancies driven by PTEN deficiency, and it reveals an important role of cyclin D-CDK4/6 activity in their development. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Assuntos
Antineoplásicos/farmacologia , Ciclina D1/genética , Neoplasias do Endométrio/tratamento farmacológico , PTEN Fosfo-Hidrolase/genética , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Animais , Carcinogênese , Ciclina D1/antagonistas & inibidores , Ciclina D1/metabolismo , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/genética , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/genética , Modelos Animais de Doenças , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/patologia , Feminino , Humanos , Camundongos , Camundongos Knockout , Tamoxifeno/efeitos adversos , Transplante Heterólogo
5.
Autophagy ; 13(3): 608-624, 2017 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-28055301

RESUMO

Targeted therapies in endometrial cancer (EC) using kinase inhibitors rarely result in complete tumor remission and are frequently challenged by the appearance of refractory cell clones, eventually resulting in disease relapse. Dissecting adaptive mechanisms is of vital importance to circumvent clinical drug resistance and improve the efficacy of targeted agents in EC. Sorafenib is an FDA-approved multitarget tyrosine and serine/threonine kinase inhibitor currently used to treat hepatocellular carcinoma, advanced renal carcinoma and radioactive iodine-resistant thyroid carcinoma. Unfortunately, sorafenib showed very modest effects in a multi-institutional phase II trial in advanced uterine carcinoma patients. Here, by leveraging RNA-sequencing data from the Cancer Cell Line Encyclopedia and cell survival studies from compound-based high-throughput screenings we have identified the lysosomal pathway as a potential compartment involved in the resistance to sorafenib. By performing additional functional biology studies we have demonstrated that this resistance could be related to macroautophagy/autophagy. Specifically, our results indicate that sorafenib triggers a mechanistic MAPK/JNK-dependent early protective autophagic response in EC cells, providing an adaptive response to therapeutic stress. By generating in vivo subcutaneous EC cell line tumors, lung metastatic assays and primary EC orthoxenografts experiments, we demonstrate that targeting autophagy enhances sorafenib cytotoxicity and suppresses tumor growth and pulmonary metastasis progression. In conclusion, sorafenib induces the activation of a protective autophagic response in EC cells. These results provide insights into the unopposed resistance of advanced EC to sorafenib and highlight a new strategy for therapeutic intervention in recurrent EC.


Assuntos
Autofagia , Neoplasias do Endométrio/tratamento farmacológico , Neoplasias do Endométrio/patologia , Terapia de Alvo Molecular , Animais , Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Progressão da Doença , Neoplasias do Endométrio/enzimologia , Neoplasias do Endométrio/ultraestrutura , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Feminino , Humanos , Camundongos Nus , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Niacinamida/análogos & derivados , Niacinamida/farmacologia , Niacinamida/uso terapêutico , Compostos de Fenilureia/farmacologia , Compostos de Fenilureia/uso terapêutico , Sorafenibe , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Adv Exp Med Biol ; 943: 149-207, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27910068

RESUMO

Endometrial cancer (EC) is the most common gynecologic malignancy in the western world with more than 280,000 cases per year worldwide. Prognosis for EC at early stages, when primary surgical resection is the most common initial treatment, is excellent. Five-year survival rate is around 70 %.Several molecular alterations have been described in the different types of EC. They occur in genes involved in important signaling pathways. In this chapter, we will review the most relevant altered pathways in EC, including PI3K/AKT/mTOR, RAS-RAF-MEK-ERK, Tyrosine kinase, WNT/ß-Catenin, cell cycle, and TGF-ß signaling pathways. At the end of the chapter, the most significant clinical trials will be briefly discussed.This information is important to identify specific targets for therapy.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias do Endométrio/tratamento farmacológico , Terapia de Alvo Molecular/métodos , Transdução de Sinais/efeitos dos fármacos , Neoplasias do Endométrio/metabolismo , Feminino , Humanos , Terapia de Alvo Molecular/tendências , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Fator de Crescimento Transformador beta/metabolismo , beta Catenina/metabolismo
7.
Int J Gynecol Cancer ; 2016 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-27258723

RESUMO

OBJECTIVE: The PI3K/AKT/mTOR pathway is frequently aberrantly activated in endometrial carcinoma (EC). Temsirolimus is an mTOR inhibitor that has shown clinical activity in EC. We aimed to characterize the biological effects on mTOR pathway of temsirolimus in treatment-naive patients with primary EC, and to identify potential biomarkers associated with a short-term exposure to temsirolimus. MATERIALS AND METHODS: Patients with EC were treated with 4 doses of temsirolimus previous to surgery. The primary objective was the analysis of paired endometrial aspirates and posttreatment (hysterectomy specimens) tumor tissue samples for mTOR downstream effectors p-S6K1 and p-4BEP1 levels by immunohistochemistry. Secondary objectives included analysis of expression of other mTOR-related biomarkers by immunohistochemistry, as well as analysis of the predictive value of mutations in mTOR-related genes. Toxicity was also assessed. RESULTS: Eleven patients were included in the study. p-S6K1 expression was reduced after treatment with temsirolimus in all patients. Variations of the expression of other mTOR-related proteins including p-4BEP1, PTEN, p-AKT, p53, p27, BAD, Bcl-2, Ki67, and cyclin D1 were also observed. Interestingly, the biological effects of the drug were more evident 1 week after the last dose of temsirolimus. Effects were less evident on tumors harboring mutations in NRAS. Toxicity was acceptable, being mucositis the most frequent adverse event. CONCLUSIONS: Short temsirolimus exposure effectively inhibits mTOR pathway in patients with endometrial cancer. p-S6K1 expression is a promising biomarker of sensitivity. The preoperative window opportunity in EC is a realistic scenario for biological knowledge and target development.

8.
Eur J Cancer ; 63: 74-87, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27288872

RESUMO

The phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) axis is frequently dysregulated in cancer due to mutations in different nodes of the pathway or constitutive activation of receptor tyrosine kinases. Multikinase inhibitors as sorafenib and regorafenib represent a therapeutic approach for the treatment of these types of tumours. In the present study, we have evaluated the anti-tumoural effects of Sorafenib and Regorafenib on endometrial, prostate and thyroid neoplasias. Both inhibitors reduced cell viability in vitro and lead to a disruption of the PI3K/AKT/mTOR pathway. In vivo, we have demonstrated that Sorafenib and Regorafenib reduce thyroid hyperplasias induced by the loss of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), although none of the treatments eliminated the disease. Altogether, we present the first study that correlates the response to multikinase inhibitors with a specific mutation. Moreover, this is the first report characterising the response to Regorafenib in thyroid, prostate and endometrial neoplasias.


Assuntos
Antineoplásicos/uso terapêutico , Carcinoma/tratamento farmacológico , Neoplasias do Endométrio/tratamento farmacológico , Niacinamida/análogos & derivados , Compostos de Fenilureia/uso terapêutico , Neoplasias da Próstata/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Piridinas/uso terapêutico , Neoplasias da Glândula Tireoide/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Niacinamida/farmacologia , Niacinamida/uso terapêutico , PTEN Fosfo-Hidrolase/deficiência , Compostos de Fenilureia/farmacologia , Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Sorafenibe
9.
Nat Commun ; 7: 11581, 2016 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-27181366

RESUMO

Cyclin D1 (Ccnd1) together with its binding partner Cdk4 act as a transcriptional regulator to control cell proliferation and migration, and abnormal Ccnd1·Cdk4 expression promotes tumour growth and metastasis. While different nuclear Ccnd1·Cdk4 targets participating in cell proliferation and tissue development have been identified, little is known about how Ccnd1·Cdk4 controls cell adherence and invasion. Here, we show that the focal adhesion component paxillin is a cytoplasmic substrate of Ccnd1·Cdk4. This complex phosphorylates a fraction of paxillin specifically associated to the cell membrane, and promotes Rac1 activation, thereby triggering membrane ruffling and cell invasion in both normal fibroblasts and tumour cells. Our results demonstrate that localization of Ccnd1·Cdk4 to the cytoplasm does not simply act to restrain cell proliferation, but constitutes a functionally relevant mechanism operating under normal and pathological conditions to control cell adhesion, migration and metastasis through activation of a Ccnd1·Cdk4-paxillin-Rac1 axis.


Assuntos
Ciclina D1/metabolismo , Citoplasma/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Paxilina/metabolismo , Animais , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Ciclina D1/deficiência , Quinase 4 Dependente de Ciclina/metabolismo , Regulação para Baixo/genética , Fibroblastos/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Camundongos , Invasividade Neoplásica , Metástase Neoplásica , Fosforilação , Fosfosserina/metabolismo , Ligação Proteica , Ratos , Especificidade por Substrato , Proteínas rac1 de Ligação ao GTP/metabolismo
10.
Oncotarget ; 7(19): 26979-91, 2016 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-27105504

RESUMO

Cyclin D1 (Ccnd1) is a proto-oncogen amplified in many different cancers and nuclear accumulation of Ccnd1 is a characteristic of tumor cells. Ccnd1 activates the transcription of a large set of genes involved in cell cycle progress and proliferation. However, Ccnd1 also targets cytoplasmic proteins involved in the regulation of cell migration and invasion. In this work, we have analyzed by immunohistochemistry the localization of Ccnd1 in endometrial, breast, prostate and colon carcinomas with different types of invasion. The number of cells displaying membranous or cytoplasmic Ccnd1 was significantly higher in peripheral cells than in inner cells in both collective and pushing invasion patterns of endometrial carcinoma, and in collective invasion pattern of colon carcinoma. Also, the cytoplasmic localization of Ccnd1 was higher when tumors infiltrated as single cells, budding or small clusters of cells. To evaluate cytoplasmic function of cyclin D1, we have built a variant (Ccnd1-CAAX) that remains attached to the cell membrane therefore sequestering this cyclin in the cytoplasm. Tumor cells harboring Ccnd1-CAAX showed high levels of invasiveness and metastatic potential compared to those containing the wild type allele of Ccnd1. However, Ccnd1-CAAX expression did not alter proliferative rates of tumor cells. We hypothesize that the role of Ccnd1 in the cytoplasm is mainly associated with the invasive capability of tumor cells. Moreover, we propose that subcellular localization of Ccnd1 is an interesting guideline to measure cancer outcome.


Assuntos
Biomarcadores Tumorais/metabolismo , Ciclina D1/metabolismo , Citoplasma/metabolismo , Neoplasias/metabolismo , Motivos de Aminoácidos/genética , Animais , Biomarcadores Tumorais/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Células Cultivadas , Neoplasias do Colo/metabolismo , Ciclina D1/genética , Neoplasias do Endométrio/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Masculino , Camundongos Nus , Camundongos SCID , Microscopia Confocal , Invasividade Neoplásica , Neoplasias da Próstata/metabolismo
11.
Pigment Cell Melanoma Res ; 29(3): 352-71, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26988132

RESUMO

Heat shock proteins (HSPs), are molecular chaperones that assist the proper folding of nascent proteins. This study aims to evaluate the antitumour effects of the hsp90 inhibitor NVP-AUY922 in melanoma, both in vitro and in vivo. Our results show that NVP-AUY922 inhibits melanoma cell growth in vitro, with down regulation of multiple signalling pathways involved in melanoma progression such as NF-ĸB and MAPK/ERK. However, NVP-AUY922 was unable to limit tumour growth in vivo. Cotreatment of A375M xenografts with NVP-AUY922 and PFT-µ, a dual inhibitor of both hsp70 and autophagy, induced a synergistic increase of cell death in vitro, and delayed tumour formation in A375M xenografts. PFT-µ depleted cells from the reduced form of glutathione (GSH) and increased oxidative stress. The oxidative stress induced by PFT-µ further enhanced NVP-AUY922-induced cytotoxic effects. These data suggest a potential therapeutic role for NVP-AUY922 used in combination with PFT-µ, in melanoma.


Assuntos
Antineoplásicos/uso terapêutico , Glutationa/metabolismo , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Isoxazóis/uso terapêutico , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Resorcinóis/uso terapêutico , Sulfonamidas/uso terapêutico , Animais , Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Isoxazóis/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Melanoma/genética , Camundongos SCID , NF-kappa B/metabolismo , Metástase Neoplásica , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Resorcinóis/farmacologia , Pele/efeitos dos fármacos , Pele/metabolismo , Pele/patologia , Sulfonamidas/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Blood ; 127(15): 1907-11, 2016 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-26773036

RESUMO

Since its discovery in the late 1990s, Pten has turned out to be one of the most important tumor suppressor genes. Pten loss results in increased activation of the phosphatidylinositol 3-kinase/Akt signaling pathway, which is associated with increased proliferation, survival, and neoplastic growth. Here, we have addressed the effects of conditional deletion of Pten in hematopoietic cells by crossing Pten conditional knockout mice with a knock-in mouse expressing the Cre recombinase in the CD45 locus. CD45 is also known as leukocyte common antigen, and it is expressed in virtually all white cells and in hematopoietic stem cells. Using a reporter mouse, we demonstrate that CD45:Cre mouse displays recombinase activity in both myeloid and lymphoid cells. However, deletion of Pten in CD45-expressing cells induces development of T-cell acute lymphoblastic leukemia and lymphoma, but not other hematologic malignancies.


Assuntos
Antígenos Comuns de Leucócito/metabolismo , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Animais , Células da Medula Óssea/metabolismo , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Deleção de Genes , Células-Tronco Hematopoéticas/citologia , Integrases/metabolismo , Estimativa de Kaplan-Meier , Masculino , Camundongos , Camundongos Knockout
13.
Mol Imaging Biol ; 18(4): 545-56, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-26604096

RESUMO

PURPOSE: In this study, we first aimed to evaluate the effects in vitro and in vivo, of the Hsp90 inhibitor NVP-AUY922, in endometrial cancer (EC). We also aimed to track nuclear factor kappa B (NF-κB) signalling, a key pathway involved in endometrial carcinogenesis and to check whether NVP-AUY922 treatment modulates it both in vitro and in vivo. PROCEDURES: I n vitro effects of NVP-AUY922 on EC cell growth and the signalling pathways were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), clonogenic assays, Western Blot and luciferase assay. NVP-AUY922 effect on Ishikawa (IK) xenograft growth was evaluated in vivo, and NF-κB activity was monitored using bioluminescence imaging. RESULTS: NVP-AUY922 inhibited the growth of three endometrial cell lines tested in vitro. In vivo, NVP-AUY922 reduced tumour growth of 47 % (p = 0.042) compared to control condition. Moreover, the bioluminescence signal of the tumours harbouring IK NF-κB-LUC cells was significantly reduced in NVP-AUY922-treated animals compared to untreated ones. CONCLUSIONS: NVP-AUY922 reduced EC tumour growth and NF-κB signalling both in vitro and in vivo. As therapeutic resistance of EC remains a challenge for oncologists nowadays, we think that NVP-AUY922 represents a valid alternative to conventional chemotherapy, and we believe that this approach for assessing and tracking the activation of NF-κB pathway may be of therapeutic benefit.


Assuntos
Diagnóstico por Imagem/métodos , Neoplasias do Endométrio/diagnóstico , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Isoxazóis/farmacologia , Medições Luminescentes/métodos , NF-kappa B/metabolismo , Resorcinóis/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Clonais , Neoplasias do Endométrio/patologia , Feminino , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Luciferases/metabolismo , Camundongos , Ensaios Antitumorais Modelo de Xenoenxerto
14.
PLoS One ; 10(8): e0136863, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26322890

RESUMO

Endothelial cell activation leading to leukocyte recruitment and adhesion plays an essential role in the initiation and progression of atherosclerosis. Vitamin D has cardioprotective actions, while its deficiency is a risk factor for the progression of cardiovascular damage. Our aim was to assess the role of basal levels of vitamin D receptor (VDR) on the early leukocyte recruitment and related endothelial cell-adhesion-molecule expression, as essential prerequisites for the onset of atherosclerosis. Knockdown of VDR in endothelial cells (shVDR) led to endothelial cell activation, characterized by upregulation of VCAM-1, ICAM-1 and IL-6, decreased peripheral blood mononuclear cell (PBMC) rolling velocity and increased PBMC rolling flux and adhesion to the endothelium. shVDR cells showed decreased IκBα levels and accumulation of p65 in the nucleus compared to shRNA controls. Inhibition of NF-κB activation with super-repressor IκBα blunted all signs of endothelial cell activation caused by downregulation of VDR in endothelial cells. In vivo, deletion of VDR led to significantly larger aortic arch and aortic root lesions in apoE-/- mice, with higher macrophage content. apoE-/-VDR-/-mice showed higher aortic expression of VCAM-1, ICAM-1 and IL-6 when compared to apoE-/-VDR+/+ mice. Our data demonstrate that lack of VDR signaling in endothelial cells leads to a state of endothelial activation with increased leukocyte-endothelial cell interactions that may contribute to the more severe plaque accumulation observed in apoE-/-VDR-/- mice. The results reveal an important role for basal levels of endothelial VDR in limiting endothelial cell inflammation and atherosclerosis.


Assuntos
Aterosclerose/metabolismo , Aterosclerose/patologia , Comunicação Celular/fisiologia , Células Endoteliais/metabolismo , Endotélio/metabolismo , Leucócitos Mononucleares/metabolismo , Vitamina D/metabolismo , Animais , Adesão Celular/fisiologia , Linhagem Celular , Regulação para Baixo/fisiologia , Feminino , Humanos , Proteínas I-kappa B/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-6/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Inibidor de NF-kappaB alfa , NF-kappa B/metabolismo , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patologia , Receptores de Calcitriol/metabolismo , Transdução de Sinais/fisiologia , Regulação para Cima/fisiologia , Molécula 1 de Adesão de Célula Vascular/metabolismo
15.
Methods ; 77-78: 31-40, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25461816

RESUMO

PTEN is an important tumor suppressor gene. Interpreting PTEN deficiency in the appropriate microscopic context of cancer may be important to understand its role in tumor development and progression. This may be particularly relevant in heterogeneous tumors. Here, we discuss the usefulness of 3D cultures in understanding the consequences of PTEN inactivation in tissue architecture. Afterwards, we discuss the role of immunohistochemistry and fluorescent in situ hybridization in assessing PTEN loss in tumors. In this review, endometrial carcinoma is used as a model.


Assuntos
Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/patologia , PTEN Fosfo-Hidrolase/deficiência , Técnicas de Cultura de Tecidos/métodos , Proteínas Supressoras de Tumor/deficiência , Animais , Neoplasias do Endométrio/genética , Feminino , Humanos , Mutação/genética , PTEN Fosfo-Hidrolase/genética , Proteínas Supressoras de Tumor/genética
16.
Int J Cancer ; 136(8): 1863-73, 2015 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-25219463

RESUMO

Endometrial carcinomas, the most common malignant tumour of the female genital tract, are usually diagnosed at an early stage with uterine-confined disease and an overall favourable prognosis. However, up to 20% of endometrial carcinomas will end up in recurrent disease, associated with a drop in survival and representing the major clinical challenge. Management of this group of risk patients relies on robust biomarkers that may predict which endometrial carcinomas will relapse. For this, we performed a proteomic analysis comparing primary lesions with recurrences and identified ANXA2 as a potential biomarker associated with recurrent disease that we further validated in an independent series of samples by immunohistochemistry. We demonstrated in vitro a role for ANXA2 in the promotion of metastasis rather than interfering with sensitivity to radio/chemotherapy. In addition, ANXA2 silencing resulted in a reduced metastatic pattern in a mice model of endometrial cancer dissemination, with a limited presence of circulating tumor cells. Finally, a retrospective study in a cohort of 93 patients showed that ANXA2 effectively predicted those endometrioid endometrial carcinomas that finally recurred. Importantly, ANXA2 demonstrated a predictive value also among low risk Stage I endometrioid endometrial carcinomas, highlighting the clinical utility of ANXA2 biomarker as predictor of recurrent disease in endometrial cancer. Retrospective and prospective studies are ongoing to validate ANXA2 as a potential tool for optimal stratification of patients susceptible to receive radical surgery and radio/chemotherapy.


Assuntos
Anexina A2/sangue , Biomarcadores Tumorais/sangue , Neoplasias do Endométrio/sangue , Neoplasias do Endométrio/diagnóstico , Recidiva Local de Neoplasia/sangue , Recidiva Local de Neoplasia/diagnóstico , Idoso , Animais , Feminino , Humanos , Camundongos , Camundongos Nus , Metástase Neoplásica/diagnóstico , Células Neoplásicas Circulantes/metabolismo , Proteômica/métodos , Estudos Retrospectivos
17.
Hum Pathol ; 45(12): 2394-403, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25282036

RESUMO

Histologic typing may be difficult in a subset of endometrial carcinoma (EC) cases. In these cases, interobserver agreement improves when immunohistochemistry (IHC) is used. A series of endometrioid type (EEC) grades 1, 2, and 3 and serous type (SC) were immunostained for p53, p16, estrogen receptor, PTEN, IMP2, IMP3, HER2, cyclin B2 and E1, HMGA2, FolR1, MSLN, Claudins 3 and 4, and NRF2. Nine biomarkers showed significant differences with thresholds in IHC value scale between both types (p53 ≥ 20, IMP2 ≥ 115, IMP3 ≥ 2, cyclin E1 ≥ 220, HMGA2 ≥ 30, FolR1 ≥ 50, p16 ≥ 170, nuclear PTEN ≥ 2 and estrogen receptor ≤ 50; P < .005). This combination led to increased discrimination when considering cases satisfying 0 to 5 conditions predicted as EEC and those satisfying 6 to 9 conditions predicted as SC. This signature correctly predicted all 48 EEC grade 1-2 cases and 18 SC cases, but 3 SC cases were wrongly predicted as EEC. Sensitivity was 86% (95% confidence interval [CI], 64%-97%), and specificity was 100% (95% CI, 89%-100%). The classifier correctly predicted all 28 EEC grade 3 cases but only identified the EEC and SC components in 4 of 9 mixed EEC-SC. An independent validation series (29 EEC grades 1-2, 28 EEC grade 3, and 31 SC) showed 100% sensitivity (95% CI, 84%-100%) and 83% specificity (95% CI, 64%-94%). We propose an internally and externally validated 9-protein biomarker signature to predict the histologic type of EC (EEC or SC) by IHC. The results also suggest that mixed EEC-SC is molecularly ambiguous.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma Endometrioide/metabolismo , Carcinoma/metabolismo , Neoplasias do Endométrio/metabolismo , Carcinoma/patologia , Carcinoma Endometrioide/patologia , Neoplasias do Endométrio/patologia , Feminino , Humanos , Imuno-Histoquímica , Sensibilidade e Especificidade
18.
Histopathology ; 65(3): 371-88, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25353038

RESUMO

AIMS: To check the usefulness of a standardized protocol of PTEN FISH in 31 endometrial carcinomas (ECs) in comparison with SNP array (SNPA), multiplex ligation-dependent probe amplification (MLPA), and immunohistochemistry. METHODS AND RESULTS: Fluorescence in-situ hybridization analysis showed two PTEN copies in 17 cases, three copies in nine cases, hemizygous deletion in two cases, and diverse cell populations with different PTEN copy number in three cases. A good correlation was seen between FISH and SNPA, particularly in cases with three copies. FISH identified two cases with entire deletion of chromosome 10, but did not identify a focal deletion of PTEN. Five cases with PTEN deletion and duplication of the second allele by SNPA were interpreted as normal by FISH. Concordance between FISH and MLPA was seen in 15 cases with two copies, and in two cases with PTEN deletion. Six cases were interpreted as amplified by MLPA, but showed polyploidy by FISH. FISH was superior to SNPA and MLPA in assessing the tumours with diverse cell populations with different PTEN copies. CONCLUSIONS: The results show good concordance between FISH, SNPA and MLPA. SNPA was superior in tumours with deletion of one copy and duplication of the second allele. FISH was superior in assessing tumour heterogeneity.


Assuntos
Neoplasias do Endométrio/genética , Hibridização in Situ Fluorescente , PTEN Fosfo-Hidrolase/genética , Carcinoma Endometrioide/genética , Carcinoma Endometrioide/metabolismo , Carcinoma Endometrioide/patologia , Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/patologia , Feminino , Dosagem de Genes , Genes Supressores de Tumor , Humanos , Imuno-Histoquímica , Reação em Cadeia da Polimerase Multiplex , PTEN Fosfo-Hidrolase/metabolismo , Polimorfismo de Nucleotídeo Único , Reprodutibilidade dos Testes
19.
Carcinogenesis ; 35(12): 2679-86, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25233929

RESUMO

Myometrial infiltration represents a main clinical determinant of endometrial carcinomas (EC) presenting as aggressive high-grade deeply invasive neoplasms, substantially associated with risk of recurrence and death. The up-regulation of ETV5 transcription factor linked to the promotion of epithelial to mesenchymal transition is considered as a basic mechanism underlying the initial steps of EC invasion. In this work, we aimed to investigate the transcription program of tumor invasion regulated by ETV5. We performed a comparative Chip-on-chip analysis at invasive front and superficial area of human EC. ETV5 specific binding to promoter regions of genes related to cellular migration, adhesion and invasion at deep invasion tumor areas highlighted the relevance of neural networks associated with cellular plasticity. Interestingly, brain-derived neurotrophic factor (BDNF) demonstrated a principal role orchestrating ETV5-mediated epithelial-to-mesenchymal transition in endometrial cancer. Impairment of the BDNF/tropomyosin-related kinase B (TrkB)/extracellular signal-regulated kinase axis in endometrial cancer cell lines reversed the aggressive and invasive phenotype promoted by the up-regulation of ETV5 at the invasive front of EC. Likewise, BDNF directly impacted on the efficiency of ETV5 promoted metastasis in a mice model of endometrial distant dissemination. These results translate the recognized role of BDNF/TrkB on neural plasticity into a relevant cancer metastasis event; suggest common mechanisms shared by neural development and tumor invasion; and offer new therapeutic opportunities specifically directed against disseminated disease in endometrial cancer.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Movimento Celular , Proteínas de Ligação a DNA/genética , Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/patologia , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Fatores de Transcrição/genética , Animais , Western Blotting , Fator Neurotrófico Derivado do Encéfalo/genética , Proliferação de Células , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/metabolismo , Neoplasias do Endométrio/genética , Feminino , Imunofluorescência , Humanos , Camundongos , Invasividade Neoplásica , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/metabolismo , Células Tumorais Cultivadas
20.
Mol Cancer ; 13: 223, 2014 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-25261936

RESUMO

BACKGROUND: About 20% of patients diagnosed with endometrial cancer (EC) are considered high-risk with unfavorable prognosis. In the framework of the European Network for Individualized Treatment in EC (ENITEC), we investigated the presence and phenotypic features of Circulating Tumor Cells (CTC) in high-risk EC patients. METHODS: CTC isolation was carried out in peripheral blood samples from 34 patients, ranging from Grade 3 Stage IB to Stage IV carcinomas and recurrences, and 27 healthy controls using two methodologies. Samples were subjected to EpCAM-based immunoisolation using the CELLection™ Epithelial Enrich kit (Invitrogen, Dynal) followed by RTqPCR analysis. The phenotypic determinants of endometrial CTC in terms of pathogenesis, hormone receptor pathways, stem cell markers and epithelial to mesenchymal transition (EMT) drivers were asked. Kruskal-Wallis analysis followed by Dunn's post-test was used for comparisons between groups. Statistical significance was set at p < 0.05. RESULTS: EpCAM-based immunoisolation positively detected CTC in high-risk endometrial cancer patients. CTC characterization indicated a remarkable plasticity phenotype defined by the expression of the EMT markers ETV5, NOTCH1, SNAI1, TGFB1, ZEB1 and ZEB2. In addition, the expression of ALDH and CD44 pointed to an association with stemness, while the expression of CTNNB1, STS, GDF15, RELA, RUNX1, BRAF and PIK3CA suggested potential therapeutic targets. We further recapitulated the EMT phenotype found in endometrial CTC through the up-regulation of ETV5 in an EC cell line, and validated in an animal model of systemic dissemination the propensity of these CTC in the accomplishment of metastasis. CONCLUSIONS: Our results associate the presence of CTC with high-risk EC. Gene-expression profiling characterized a CTC-plasticity phenotype with stemness and EMT features. We finally recapitulated this CTC-phenotype by over-expressing ETV5 in the EC cell line Hec1A and demonstrated an advantage in the promotion of metastasis in an in vivo mouse model of CTC dissemination and homing.


Assuntos
Neoplasias do Endométrio/genética , Neoplasias do Endométrio/patologia , Perfilação da Expressão Gênica , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patologia , Idoso , Animais , Separação Celular , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos Nus , Metástase Neoplásica , Fenótipo , Fatores de Risco , Fatores de Transcrição/metabolismo
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