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1.
Biomed Pharmacother ; 173: 116345, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38442670

RESUMO

Antagonists of the A2B adenosine receptor have recently emerged as targeted anticancer agents and immune checkpoint inhibitors within the realm of cancer immunotherapy. This study presents a comprehensive evaluation of novel Biginelli-assembled pyrimidine chemotypes, including mono-, bi-, and tricyclic derivatives, as A2BAR antagonists. We conducted a comprehensive examination of the adenosinergic profile (both binding and functional) of a large compound library consisting of 168 compounds. This approach unveiled original lead compounds and enabled the identification of novel structure-activity relationship (SAR) trends, which were supported by extensive computational studies, including quantum mechanical calculations and free energy perturbation (FEP) analysis. In total, 25 molecules showed attractive affinity (Ki < 100 nM) and outstanding selectivity for A2BAR. From these, five molecules corresponding to the new benzothiazole scaffold were below the Ki < 10 nM threshold, in addition to a novel dual A2A/A2B antagonist. The most potent compounds, and the dual antagonist, showed enantiospecific recognition in the A2BAR. Two A2BAR selective antagonists and the dual A2AAR/A2BAR antagonist reported in this study were assessed for their impact on colorectal cancer cell lines. The results revealed a significant and dose-dependent reduction in cell proliferation. Notably, the A2BAR antagonists exhibited remarkable specificity, as they did not impede the proliferation of non-tumoral cell lines. These findings support the efficacy and potential that A2BAR antagonists as valuable candidates for cancer therapy, but also that they can effectively complement strategies involving A2AAR antagonism in the context of immune checkpoint inhibition.


Assuntos
Antineoplásicos , Neoplasias Colorretais , Humanos , Antagonistas de Receptores Purinérgicos P1 , Receptor A2B de Adenosina/metabolismo , Antagonistas do Receptor A2 de Adenosina/farmacologia , Relação Estrutura-Atividade , Antineoplásicos/farmacologia , Neoplasias Colorretais/tratamento farmacológico
2.
Methods Mol Biol ; 2764: 179-203, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38393596

RESUMO

Cells within a tumor interact by generating, transmitting, and sensing mechanical forces. Among all the cells of the tumor microenvironment, cancer-associated fibroblasts (CAFs) are a paradigmatic example of mechanical communication. In different steps of tumor progression, CAFs pull and push on cancer cells, regulating cancer cell migration, invasion, compartmentalization, and signaling. There is thus an increasing need to experimentally address mechanical interactions within a tumor. A common technique to measure these interactions is laser ablation. Cutting a tissue region with a high-power laser triggers a sudden tissue displacement whose direction and magnitude reveal the local mechanical stresses. In this chapter, we provide a detailed protocol to perform laser ablations in vitro and ex vivo. First, we describe how to prepare cocultures of primary CAFs and cancer cells and tumor explants. Then, we explain how to perform laser ablations in these two systems and how to analyze the induced tissue displacements using particle image velocimetry (PIV). Overall, we provide a workflow to perform, analyze, and interpret laser ablations to explore tumor mechanical interactions.


Assuntos
Fibroblastos Associados a Câncer , Terapia a Laser , Neoplasias , Humanos , Fibroblastos Associados a Câncer/patologia , Fibroblastos/patologia , Neoplasias/patologia , Técnicas de Cocultura , Microambiente Tumoral , Linhagem Celular Tumoral , Movimento Celular
3.
Nat Commun ; 14(1): 6966, 2023 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-37907483

RESUMO

During tumor progression, cancer-associated fibroblasts (CAFs) accumulate in tumors and produce an excessive extracellular matrix (ECM), forming a capsule that enwraps cancer cells. This capsule acts as a barrier that restricts tumor growth leading to the buildup of intratumoral pressure. Combining genetic and physical manipulations in vivo with microfabrication and force measurements in vitro, we found that the CAFs capsule is not a passive barrier but instead actively compresses cancer cells using actomyosin contractility. Abrogation of CAFs contractility in vivo leads to the dissipation of compressive forces and impairment of capsule formation. By mapping CAF force patterns in 3D, we show that compression is a CAF-intrinsic property independent of cancer cell growth. Supracellular coordination of CAFs is achieved through fibronectin cables that serve as scaffolds allowing force transmission. Cancer cells mechanosense CAF compression, resulting in an altered localization of the transcriptional regulator YAP and a decrease in proliferation. Our study unveils that the contractile capsule actively compresses cancer cells, modulates their mechanical signaling, and reorganizes tumor morphology.


Assuntos
Fibroblastos Associados a Câncer , Neoplasias , Fibroblastos Associados a Câncer/patologia , Mecanotransdução Celular , Linhagem Celular Tumoral , Fibroblastos/patologia , Microambiente Tumoral , Neoplasias/patologia
4.
Int J Mol Sci ; 24(17)2023 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-37686015

RESUMO

Gynaecological serous carcinomas (GSCs) constitute a distinctive entity among female tumours characterised by a very poor prognosis. In addition to late-stage diagnosis and a high rate of recurrent disease associated with massive peritoneal carcinomatosis, the systematic acquisition of resistance to first-line chemotherapy based on platinum determines the unfavourable outcome of GSC patients. To explore the molecular mechanisms associated with platinum resistance, we generated patient-derived organoids (PDOs) from liquid biopsies of GSC patients. PDOs are emerging as a relevant preclinical model system to assist in clinical decision making, mainly from tumoural tissue and particularly for personalised therapeutic options. To approach platinum resistance in a GSC context, proficient PDOs were generated from the ascitic fluid of ovarian, primary peritoneal and uterine serous carcinoma patients in platinum-sensitive and platinum-resistant clinical settings from the uterine aspirate of a uterine serous carcinoma patient, and we also induced platinum resistance in vitro in a representative platinum-sensitive PDO. Histological and immunofluorescent characterisation of these ascites-derived organoids showed resemblance to the corresponding original tumours, and assessment of platinum sensitivity in these preclinical models replicated the clinical setting of the corresponding GSC patients. Differential gene expression profiling of a panel of 770 genes representing major canonical cancer pathways, comparing platinum-sensitive and platinum-resistant PDOs, revealed cellular response to DNA damage stimulus as the principal biological process associated with the acquisition of resistance to the first-line therapy for GSC. Additionally, candidate genes involved in regulation of cell adhesion, cell cycles, and transcription emerged from this proof-of-concept study. In conclusion, we describe the generation of PDOs from liquid biopsies in the context of gynaecological serous carcinomas to explore the molecular determinants of platinum resistance.


Assuntos
Ascite , Cistadenocarcinoma Seroso , Humanos , Feminino , Organoides , Peritônio , Líquido Ascítico , Cistadenocarcinoma Seroso/tratamento farmacológico , Cistadenocarcinoma Seroso/genética
5.
Sci Adv ; 9(33): eade2120, 2023 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-37585527

RESUMO

Fibroblasts play a fundamental role in tumor development. Among other functions, they regulate cancer cells' migration through rearranging the extracellular matrix, secreting soluble factors, and establishing direct physical contacts with cancer cells. Here, we report that migrating fibroblasts deposit on the substrate a network of tubular structures that serves as a guidance cue for cancer cell migration. Such membranous tubular network, hereafter called tracks, is stably anchored to the substrate in a ß5-integrin-dependent manner. We found that cancer cells specifically adhere to tracks by using clathrin-coated structures that pinch and engulf tracks. Tracks thus represent a spatial memory of fibroblast migration paths that is read and erased by cancer cells directionally migrating along them. We propose that fibroblast tracks represent a topography-based intercellular communication system capable of steering cancer cell migration.


Assuntos
Sinais (Psicologia) , Neoplasias , Humanos , Movimento Celular/fisiologia , Fibroblastos/fisiologia , Matriz Extracelular
6.
EMBO Mol Med ; 14(9): e15670, 2022 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-36069081

RESUMO

Centrosome amplification, the presence of more than two centrosomes in a cell is a common feature of most human cancer cell lines. However, little is known about centrosome numbers in human cancers and whether amplification or other numerical aberrations are frequently present. To address this question, we have analyzed a large cohort of primary human epithelial ovarian cancers (EOCs) from 100 patients. We found that rigorous quantitation of centrosome number in tumor samples was extremely challenging due to tumor heterogeneity and extensive tissue disorganization. Interestingly, even if centrosome clusters could be identified, the incidence of centrosome amplification was not comparable to what has been described in cultured cancer cells. Surprisingly, centrosome loss events where a few or many nuclei were not associated with centrosomes were clearly noticed and overall more frequent than centrosome amplification. Our findings highlight the difficulty of characterizing centrosome numbers in human tumors, while revealing a novel paradigm of centrosome number defects in EOCs.


Assuntos
Centrossomo , Neoplasias Ovarianas , Carcinoma Epitelial do Ovário/metabolismo , Carcinoma Epitelial do Ovário/patologia , Linhagem Celular , Centrossomo/metabolismo , Centrossomo/patologia , Feminino , Humanos , Neoplasias Ovarianas/patologia
7.
Biomed Pharmacother ; 153: 113408, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36076535

RESUMO

G-protein coupled receptors (GPCRs) have been largely targeted in a wide range of diseases, but few therapies have been directed against GPCRs in the field of cancer, partly because of the lack of effective target identification strategies. Here, using colorectal cancer (CRC) as a model, we explored the gene expression of a panel of GPCRs in tumor and stromal cells, identifying specific gene sets defining each cellular compartment. We selected the adenosine receptor 2B (A2BAR), specifically expressed in cancer cell lines compared with stromal cells, to explore the use of fluorescent ligands that can be used for target visualization. Fluorescent probes allowed semi-quantitative receptor mapping in living cells and validated the specific expression of A2BAR in CRC cell lines. As well, fluorescent ligands were effective at monitoring real-time A2BAR receptor labeling using live-imaging modalities, and displayed high efficiency when used to label complex 3D cellular systems such as tumor spheroids. Finally, we validated A2BAR as a potential pharmacological tool in CRC, using selective antagonists, finding a reduction in tumor cell proliferation. This proof-of-concept study suggests the use of fluorescent ligands for GPCR characterization through imaging, and as possible new tools used for target validation in drug screening methodologies.


Assuntos
Neoplasias Colorretais , Receptores Acoplados a Proteínas G , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Corantes Fluorescentes , Humanos , Ligantes , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais
8.
Cell ; 183(2): 411-428.e16, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32970988

RESUMO

The colon is primarily responsible for absorbing fluids. It contains a large number of microorganisms including fungi, which are enriched in its distal segment. The colonic mucosa must therefore tightly regulate fluid influx to control absorption of fungal metabolites, which can be toxic to epithelial cells and lead to barrier dysfunction. How this is achieved remains unknown. Here, we describe a mechanism by which the innate immune system allows rapid quality check of absorbed fluids to avoid intoxication of colonocytes. This mechanism relies on a population of distal colon macrophages that are equipped with "balloon-like" protrusions (BLPs) inserted in the epithelium, which sample absorbed fluids. In the absence of macrophages or BLPs, epithelial cells keep absorbing fluids containing fungal products, leading to their death and subsequent loss of epithelial barrier integrity. These results reveal an unexpected and essential role of macrophages in the maintenance of colon-microbiota interactions in homeostasis. VIDEO ABSTRACT.


Assuntos
Microbioma Gastrointestinal/fisiologia , Mucosa Intestinal/metabolismo , Macrófagos/metabolismo , Animais , Colo/metabolismo , Células Epiteliais/metabolismo , Epitélio , Feminino , Homeostase , Imunidade Inata/imunologia , Mucosa Intestinal/microbiologia , Macrófagos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microbiota , Transdução de Sinais
9.
Nat Commun ; 11(1): 404, 2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-31964880

RESUMO

Although fibroblast heterogeneity is recognized in primary tumors, both its characterization in and its impact on metastases remain unknown. Here, combining flow cytometry, immunohistochemistry and RNA-sequencing on breast cancer samples, we identify four Cancer-Associated Fibroblast (CAF) subpopulations in metastatic lymph nodes (LN). Two myofibroblastic subsets, CAF-S1 and CAF-S4, accumulate in LN and correlate with cancer cell invasion. By developing functional assays on primary cultures, we demonstrate that these subsets promote metastasis through distinct functions. While CAF-S1 stimulate cancer cell migration and initiate an epithelial-to-mesenchymal transition through CXCL12 and TGFß pathways, highly contractile CAF-S4 induce cancer cell invasion in 3-dimensions via NOTCH signaling. Patients with high levels of CAFs, particularly CAF-S4, in LN at diagnosis are prone to develop late distant metastases. Our findings suggest that CAF subset accumulation in LN is a prognostic marker, suggesting that CAF subsets could be examined in axillary LN at diagnosis.


Assuntos
Neoplasias da Mama/patologia , Fibroblastos Associados a Câncer/metabolismo , Metástase Linfática/patologia , Miofibroblastos/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Axila , Neoplasias da Mama/mortalidade , Neoplasias da Mama/terapia , Fibroblastos Associados a Câncer/patologia , Proliferação de Células , Separação Celular , Quimiocina CXCL12/metabolismo , Transição Epitelial-Mesenquimal , Feminino , Citometria de Fluxo , Seguimentos , Humanos , Estimativa de Kaplan-Meier , Linfonodos/citologia , Linfonodos/patologia , Pessoa de Meia-Idade , Miofibroblastos/patologia , Invasividade Neoplásica/patologia , Cultura Primária de Células , Prognóstico , Intervalo Livre de Progressão , Receptores Notch/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Células Tumorais Cultivadas , Microambiente Tumoral
10.
Cancers (Basel) ; 11(10)2019 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-31635093

RESUMO

Epigenetic modifications of glyco-genes have been documented in different types of cancer and are tightly linked to proliferation, invasiveness, metastasis, and drug resistance. This study aims to investigate the diagnostic, prognostic, and therapy-response predictive value of the glyco-gene B4GALT1 in colorectal cancer (CRC) patients. A Kaplan-Meier analysis was conducted in 1418 CRC patients (GEO and TCGA datasets) to assess the prognostic and therapy-response predictive values of the aberrant expression and methylation status of B4GALT1. Quantitative methylation-specific PCR (QMSP) and droplet digital quantitative methylation-specific PCR (dd-QMSP) were respectively used to detect hypermethylated B4GALT1 in metastasis and plasma in four cohorts of metastatic CRC cases (mCRC). Both the downregulated expression and promoter hypermethylation of B4GALT1 have a negative prognostic impact on CRC. Interestingly a low expression level of B4GALT1 was significantly associated with poor cetuximab response (progression-free survival (PFS) p = 0.01) particularly in wild-type (WT)-KRAS patients (p = 0.03). B4GALT1 promoter was aberrantly methylated in liver and lung metastases. The detection of hypermethylated B4GALT1 in plasma of mCRC patients showed a highly discriminative receiver operating characteristic (ROC) curve profile (area under curve (AUC) value 0.750; 95% CI: 0.592-0.908, p = 0.008), clearly distinguishing mCRC patients from healthy controls. Based on an optimal cut-off value defined by the ROC analysis, B4GALT1 yield a 100% specificity and a 50% sensitivity. These data support the potential value of B4GALT1 as an additional novel biomarker for the prediction of cetuximab response, and as a specific and sensitive diagnostic circulating biomarker that can be detected in CRC.

11.
Sci Rep ; 9(1): 12124, 2019 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-31431650

RESUMO

Previously, Aznar et al., showed that Daple/CCDC88C enables Wnt receptors to transactivate trimeric G-proteins during non-canonical Wnt signaling via a novel G-protein binding and activating (GBA) motif. By doing so, Daple serves two opposing roles; earlier during oncogenesis it suppresses neoplastic transformation and tumor growth, but later it triggers epithelial-to-mesenchymal-transition (EMT). We have identified and characterized two isoforms of the human Daple gene. While both isoforms cooperatively suppress tumor growth via their GBA motif, only the full-length transcript triggers EMT and invasion. Both isoforms are suppressed during colon cancer progression, and their reduced expression carries additive prognostic significance. These findings provide insights into the opposing roles of Daple during cancer progression and define the G-protein regulatory GBA motif as one of the minimal modules essential for Daple's role as a tumor suppressor.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas dos Microfilamentos/metabolismo , Neoplasias/metabolismo , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Células COS , Proliferação de Células/fisiologia , Chlorocebus aethiops , Estudos de Coortes , Colo/metabolismo , Genes Supressores de Tumor , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Proteínas dos Microfilamentos/genética , Células NIH 3T3 , Neoplasias/genética , Ligação Proteica , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo
12.
J Cell Sci ; 132(6)2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30765467

RESUMO

In the early stages of metastasis, cancer cells exit the primary tumor and enter the vasculature. Although most studies have focused on the tumor invasive front, cancer cells from the tumor core can also potentially metastasize. To address cell motility in the tumor core, we imaged tumor explants from spontaneously forming tumors in mice in real time using long-term two-photon microscopy. Cancer cells in the tumor core are remarkably dynamic and exhibit correlated migration patterns, giving rise to local 'currents' and large-scale tissue dynamics. Although cells exhibit stop-and-start migration with intermittent pauses, pausing does not appear to be required during division. Use of pharmacological inhibitors indicates that migration patterns in tumors are actively driven by the actin cytoskeleton. Under these conditions, we also observed a relationship between migration speed and correlation length, suggesting that cells in tumors are near a jamming transition. Our study provides new insight into the dynamics of cancer cells in the tumor core, opening new avenues of research in understanding the migratory properties of cancer cells and later metastasis.This article has an associated First Person interview with the first author of the paper.


Assuntos
Citoesqueleto de Actina/patologia , Movimento Celular , Células Neoplásicas Circulantes/patologia , Animais , Carcinogênese/induzido quimicamente , Camundongos , Microscopia de Fluorescência por Excitação Multifotônica , Neoplasias Experimentais , Cultura Primária de Células , Tamoxifeno/farmacologia
13.
Int J Cancer ; 145(7): 1852-1859, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30714617

RESUMO

Anal squamous cell carcinoma (ASCC) is a rare tumour, but its incidence is increasing. Standard chemoradiotherapy fails to cure 20% of patients and no targeted therapy is currently approved for recurrent ASCC. The PI3K/Akt/mTOR pathway is frequently altered in this poorly characterised carcinoma. IGF2 was identified here as a key factor in ASCC oncogenesis, as IGF2 was shown to play a crucial role in the PI3K pathway with frequent (~60%) and mutually exclusive genomic alterations in IGF2, IGF1R, PTEN and PIK3CA genes. We also demonstrated that IGF2 expression is mainly due to cancer-associated fibroblasts and that IGF2 secreted by cancer-associated fibroblasts from ASCC samples promotes proliferation of a human ASCC cell line via IGF2 paracrine signalling. Altogether, these results highlight the key role of the IGF2/PI3K axis, and the major role of cancer-associated fibroblasts in tumour growth via IGF2 secretion, suggesting a major role of IGF2/IGF1R inhibitors in ASCC therapies.


Assuntos
Neoplasias do Ânus/metabolismo , Fibroblastos Associados a Câncer/metabolismo , Carcinoma de Células Escamosas/metabolismo , Fator de Crescimento Insulin-Like II/genética , Fator de Crescimento Insulin-Like II/metabolismo , Animais , Neoplasias do Ânus/genética , Carcinoma de Células Escamosas/genética , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Mutação , Transplante de Neoplasias , Comunicação Parácrina , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais
14.
Curr Opin Cell Biol ; 56: 71-79, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30308331

RESUMO

The most abundant cell type in the tumor microenvironment are cancer-associated fibroblasts (CAFs). CAFs play an important role in tumor growth and progression. Besides direct communication with cancer cells via secreted molecules or cell-cell adhesions, CAFs also indirectly affect cancer cell behavior by remodeling the extracellular matrix (ECM). Here, we summarize recent findings on the distinct mechanisms that CAFs use to modify ECM, specifically, their proteolytic versus force-dependent activity. We then review the consequences of CAF force transmission on the physico-chemical properties of the matrix, focusing on the deposition of new matrix components, and the alteration of the organization and stiffness of the ECM. CAFs promote tumor invasion by creating the roads cancer cells use to escape the tumor mass. However, there is also evidence that CAFs can prevent invasion, possibly by forming a physical barrier around the tumor edge. We discuss the controversial role of CAFs in tumor progression.


Assuntos
Fibroblastos Associados a Câncer/patologia , Microambiente Tumoral , Animais , Fibroblastos Associados a Câncer/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Humanos , Neoplasias/patologia , Transdução de Sinais
15.
Methods Mol Biol ; 1749: 163-173, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29525997

RESUMO

Cell migration is a process that ensures correct cell localization and function in development and homeostasis. In disease such as cancer, cells acquire an upregulated migratory capacity that leads to their dissemination throughout the body. Live imaging of cell migration allows for better understanding of cell behaviors in development, adult tissue homeostasis and disease. We have optimized live imaging procedures to track cell migration in adult murine tissue explants derived from: (1) healthy gut; (2) primary intestinal carcinoma; and (3) the liver, a common metastatic site. To track epithelial cell migration in the gut, we generated an inducible fluorescent reporter mouse, enabling us to visualize and track individual cells in unperturbed gut epithelium. To image intratumoral cancer cells, we use a spontaneous intestinal cancer model based on the activation of Notch1 and deletion of p53 in the mouse intestinal epithelium, which gives rise to aggressive carcinoma. Interaction of cancer cells with a metastatic niche, the mouse liver, is addressed using a liver colonization model. In summary, we describe a method for long-term 3D imaging of tissue explants by two-photon excitation microscopy. Explant culturing and imaging can help understand dynamic behavior of cells in homeostasis and disease, and would be applicable to various tissues.


Assuntos
Movimento Celular/fisiologia , Imagem Óptica/métodos , Técnicas de Cultura de Órgãos/métodos , Animais , Células Cultivadas , Intestinos/citologia , Fígado/citologia , Neoplasias Hepáticas/patologia , Camundongos
16.
Int J Mol Sci ; 18(6)2017 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-28608814

RESUMO

Colorectal cancer (CRC) is one of the major causes of cancer-related deaths. Early detection of tumor relapse is crucial for determining the most appropriate therapeutic management. In clinical practice, computed tomography (CT) is routinely used, but small tumor changes are difficult to visualize, and reliable blood-based prognostic and monitoring biomarkers are urgently needed. The aim of this study was to prospectively validate a gene expression panel (composed of GAPDH, VIL1, CLU, TIMP1, TLN1, LOXL3 and ZEB2) for detecting circulating tumor cells (CTCs) as prognostic and predictive tool in blood samples from 94 metastatic CRC (mCRC) patients. Patients with higher gene panel expression before treatment had a reduced progression-free survival (PFS) and overall-survival (OS) rates compared with patients with low expression (p = 0.003 and p ≤ 0.001, respectively). Patients with increased expression of CTCs markers during treatment presented PFS and OS times of 8.95 and 11.74 months, respectively, compared with 14.41 and 24.7 for patients presenting decreased expression (PFS; p = 0.020; OS; p ≤ 0.001). Patients classified as non-responders by CTCs with treatment, but classified as responders by CT scan, showed significantly shorter survival times (PFS: 8.53 vs. 11.70; OS: 10.37 vs. 24.13; months). In conclusion, our CTCs detection panel demonstrated efficacy for early treatment response assessment in mCRC patients, and with increased reliability compared to CT scan.


Assuntos
Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , Regulação Neoplásica da Expressão Gênica , Células Neoplásicas Circulantes/patologia , Biomarcadores Tumorais/genética , Neoplasias Colorretais/patologia , Neoplasias Colorretais/terapia , Intervalo Livre de Doença , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Metástase Neoplásica/diagnóstico , Metástase Neoplásica/genética , Metástase Neoplásica/patologia , Metástase Neoplásica/terapia , Células Neoplásicas Circulantes/metabolismo , Prognóstico , Estudos Prospectivos , Resultado do Tratamento
17.
Cancer Res ; 77(13): 3431-3441, 2017 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-28536280

RESUMO

The interaction between circulating tumor cells (CTC) and endothelial cells during extravasation is a critical process during metastatic colonization, but its mechanisms remain poorly characterized. Here we report that the luminal side of liver blood vessels contains fibronectin deposits that are enriched in mice bearing primary tumors and are also present in vessels from human livers affected with metastases. Cancer cells attached to endothelial fibronectin deposits via talin1, a major component of focal adhesions. Talin1 depletion impaired cancer cell adhesion to the endothelium and transendothelial migration, resulting in reduced liver metastasis formation in vivo Talin1 expression levels in patient CTC's correlated with prognosis and therapy response. Together, our findings uncover a new mechanism for liver metastasis formation involving an active contribution of hepatic vascular fibronectin and talin1 in cancer cells. Cancer Res; 77(13); 3431-41. ©2017 AACR.


Assuntos
Fibronectinas/metabolismo , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/patologia , Células Neoplásicas Circulantes/patologia , Animais , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos Nus , Metástase Neoplásica , Migração Transendotelial e Transepitelial
18.
Sci Rep ; 6: 37820, 2016 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-27901069

RESUMO

Knowledge on the molecular mechanisms underlying metastasis colonization in Non-Small Cell Lung Cancer (NSCLC) remains incomplete. A complete overview integrating driver mutations, primary tumour heterogeneity and overt metastasis lacks the dynamic contribution of disseminating metastatic cells due to the inaccessibility to the molecular profiling of Circulating Tumour Cells (CTCs). By combining immunoisolation and whole genome amplification, we performed a global gene expression analysis of EpCAM positive CTCs from advanced NSCLC patients. We identified an EpCAM+ CTC-specific expression profile in NSCLC patients mostly associated with cellular movement, cell adhesion and cell-to-cell signalling mediated by PI3K/AKT, ERK1/2 and NF-kB pathways. NOTCH1 emerged as a driver connecting active signalling pathways, with a reduced number of related candidate genes (NOTCH1, PTP4A3, LGALS3 and ITGB3) being further validated by RT-qPCR on an independent cohort of NSCLC patients. In addition, these markers demonstrated high prognostic value for Progression-Free Survival (PFS). In conclusion, molecular characterization of EpCAM+ CTCs from advanced NSCLC patients provided with highly specific biomarkers with potential applicability as a "liquid biopsy" for monitoring of NSCLC patients and confirmed NOTCH1 as a potential therapeutic target to block lung cancer dissemination.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Células Neoplásicas Circulantes/metabolismo , Receptor Notch1/metabolismo , Células A549 , Idoso , Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Intervalo Livre de Doença , Molécula de Adesão da Célula Epitelial/metabolismo , Feminino , Humanos , Neoplasias Pulmonares/patologia , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Proteínas de Neoplasias/metabolismo , Células Neoplásicas Circulantes/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Prognóstico , Transdução de Sinais/fisiologia
19.
Sci Rep ; 6: 22112, 2016 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-26916336

RESUMO

The consequence of a loss of balance between G-protein activation and deactivation in cancers has been interrogated by studying infrequently occurring mutants of trimeric G-protein α-subunits and GPCRs. Prior studies on members of a newly identified family of non-receptor guanine nucleotide exchange factors (GEFs), GIV/Girdin, Daple, NUCB1 and NUCB2 have revealed that GPCR-independent hyperactivation of trimeric G proteins can fuel metastatic progression in a variety of cancers. Here we report that elevated expression of each GEF in circulating tumor cells (CTCs) isolated from the peripheral circulation of patients with metastatic colorectal cancer is associated with a shorter progression-free survival (PFS). The GEFs were stronger prognostic markers than two other markers of cancer progression, S100A4 and MACC1, and clustering of all GEFs together improved the prognostic accuracy of the individual family members; PFS was significantly lower in the high-GEFs versus the low-GEFs groups [H.R = 5, 20 (95% CI; 2,15-12,57)]. Because nucleotide exchange is the rate-limiting step in cyclical activation of G-proteins, the poor prognosis conferred by these GEFs in CTCs implies that hyperactivation of G-protein signaling by these GEFs is an important event during metastatic progression, and may be more frequently encountered than mutations in G-proteins and/or GPCRs.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/patologia , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células Neoplásicas Circulantes/patologia , Biomarcadores Tumorais/genética , Proteínas de Ligação ao Cálcio/metabolismo , Neoplasias Colorretais/mortalidade , Proteínas de Ligação a DNA/metabolismo , Intervalo Livre de Doença , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Metástase Neoplásica/genética , Metástase Neoplásica/patologia , Proteínas do Tecido Nervoso/metabolismo , Nucleobindinas , Estrutura Terciária de Proteína , Proteína A4 de Ligação a Cálcio da Família S100/genética , Proteína A4 de Ligação a Cálcio da Família S100/metabolismo , Transativadores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Transporte Vesicular/metabolismo
20.
Elife ; 4: e07091, 2015 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-26126266

RESUMO

Wnt signaling is essential for tissue homeostasis and its dysregulation causes cancer. Wnt ligands trigger signaling by activating Frizzled receptors (FZDRs), which belong to the G-protein coupled receptor superfamily. However, the mechanisms of G protein activation in Wnt signaling remain controversial. In this study, we demonstrate that FZDRs activate G proteins and trigger non-canonical Wnt signaling via the Dishevelled-binding protein, Daple. Daple contains a Gα-binding and activating (GBA) motif, which activates Gαi proteins and an adjacent domain that directly binds FZDRs, thereby linking Wnt stimulation to G protein activation. This triggers non-canonical Wnt responses, that is, suppresses the ß-catenin/TCF/LEF pathway and tumorigenesis, but enhances PI3K-Akt and Rac1 signals and tumor cell invasiveness. In colorectal cancers, Daple is suppressed during adenoma-to-carcinoma transformation and expressed later in metastasized tumor cells. Thus, Daple activates Gαi and enhances non-canonical Wnt signaling by FZDRs, and its dysregulation can impact both tumor initiation and progression to metastasis.


Assuntos
Receptores Frizzled/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas dos Microfilamentos/metabolismo , Via de Sinalização Wnt , Humanos
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