RESUMO
Pediatric diffuse midline gliomas are devastating diseases. Among them, diffuse midline gliomas H3K27M-mutant are associated with worse prognosis. However, recent studies have highlighted significant differences in clinical behavior and biological alterations within this specific subgroup. In this context, simple markers are needed to refine the prognosis of diffuse midline gliomas H3K27M-mutant and guide the clinical management of patients. The aims of this study were (i) to describe the molecular, immunohistochemical and, especially, chromosomal features of a cohort of diffuse midline gliomas and (ii) to focus on H3K27M-mutant tumors to identify new prognostic markers. Patients were retrospectively selected from 2001 to 2017. Tumor samples were analyzed by immunohistochemistry (including H3K27me3, EGFR, c-MET and p53), next-generation sequencing and comparative genomic hybridization array. Forty-nine patients were included in the study. The median age at diagnosis was 9 years, and the median overall survival (OS) was 9.4 months. H3F3A or HIST1H3B mutations were identified in 80% of the samples. Within the H3K27M-mutant tumors, PDGFRA amplification, loss of 17p and a complex chromosomal profile were significantly associated with worse survival. Three prognostic markers were identified in diffuse midline gliomas H3K27M-mutant: PDGFRA amplification, loss of 17p and a complex chromosomal profile. These markers are easy to detect in daily practice and should be considered to refine the prognosis of this entity.
Assuntos
Glioma/genética , Glioma/patologia , Adolescente , Neoplasias Encefálicas/patologia , Criança , Pré-Escolar , Estudos de Coortes , Hibridização Genômica Comparativa/métodos , Feminino , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Histonas/genética , Humanos , Imuno-Histoquímica/métodos , Lactente , Masculino , Mutação , Prognóstico , Estudos Retrospectivos , Adulto JovemRESUMO
Pancreatic cancer cells express high levels of MUC1, MUC4 and MUC16 mRNAs that encode membrane-bound mucins. These mRNAs share unusual features such as a long half-life. However, it remains unknown how mucin mRNA stability is regulated. Galectin-3 (Gal-3) is an endogenous lectin playing important biological functions in epithelial cells. Gal-3 is encoded by LGALS3 which is up-regulated in pancreatic cancer. Despite the absence of a RNA-recognition motif, Gal-3 interacts indirectly with pre-mRNAs in the nucleus and promotes constitutive splicing. However a broader role of Gal-3 in mRNA fate is unexplored. We report herein that Gal-3 increases MUC4 mRNA stability through an intermediate, hnRNP-L which binds to a conserved CA repeat element in the 3'UTR in a Gal-3 dependent manner and also controls Muc4 mRNA levels in epithelial tissues of Gal3-/- mice. Gal-3 interacts with hnRNP-L in the cytoplasm, especially during cell mitosis, but only partly associates with protein markers of P-Bodies or Stress Granules. By RNA-IP plus RNA-seq analysis and imaging, we demonstrate that Gal-3 binds to mature spliced MUC4 mRNA in the perinuclear region, probably in hnRNP-L-containing RNA granules. Our findings highlight a new role for Gal-3 as a non-classic RNA-binding protein that regulates MUC4 mRNA post-transcriptionally.
Assuntos
Citoplasma/metabolismo , Células Epiteliais/metabolismo , Galectina 3/metabolismo , Mucina-4/genética , Neoplasias Pancreáticas/patologia , RNA Mensageiro/metabolismo , Animais , Proteínas Sanguíneas , Citoplasma/química , Células Epiteliais/química , Galectinas , Regulação da Expressão Gênica , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estabilidade de RNARESUMO
Colorectal cancers (CRC) with microsatellite instability (MSI) display unique clinicopathologic features including a mucinous pattern with frequent expression of the secreted mucins MUC2 and MUC5AC. The mechanisms responsible for this altered pattern of expression remain largely unknown. We quantified DNA methylation of mucin genes (MUC2, MUC5AC, MUC4) in colonic cancers and examined the association with clinicopathological characteristics and molecular (MSI, KRAS, BRAF, and TP53 mutations) features. A control cohort was used for validation. We detected frequent hypomethylation of MUC2 and MUC5AC in CRC. MUC2 and MUC5AC hypomethylation was associated with MUC2 and MUC5AC protein expression (p = 0.004 and p < 0.001, respectively), poor differentiation (p = 0.001 and p = 0.007, respectively) and MSI status (p < 0.01 and p < 0.001, respectively). Interestingly, MUC5AC hypomethylation was specific to MSI cancers. Moreover, it was significantly associated with BRAF mutation and CpG island methylator phenotype (p < 0.001 and p < 0.001, respectively). All these results were confirmed in the control cohort. In the multivariate analysis, MUC5AC hypomethylation was a highly predictive biomarker for MSI cancers. MUC5AC demethylation appears to be a hallmark of MSI in CRC. Determination of MUC5AC methylation status may be useful for understanding and predicting the natural history of CRC.
Assuntos
Neoplasias Colorretais/genética , Metilação de DNA , Instabilidade de Microssatélites , Mucina-5AC/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Ilhas de CpG/genética , Decitabina , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HCT116 , Células HT29 , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Mucina-5AC/metabolismo , Mutação , Prognóstico , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras) , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Supressora de Tumor p53/genética , Proteínas ras/genéticaRESUMO
PURPOSE: Metastasis and drug resistance are the major limitations in the survival and management of patients with cancer. This study aimed to identify the mechanisms underlying HT29 colon cancer cell chemoresistance acquired after sequential exposure to 5-fluorouracil (5FU), a classical anticancer drug for treatment of epithelial solid tumors. We examined its clinical relevance in a cohort of patients with colon cancer with liver metastases after 5FU-based neoadjuvant chemotherapy and surgery. RESULTS: We show that a clonal 5F31 cell population, resistant to 1 µmol/L 5FU, express a typical cancer stem cell-like phenotype and enter into a reversible quiescent G0 state upon reexposure to higher 5FU concentrations. These quiescent cells overexpressed the tyrosine kinase c-Yes that became activated and membrane-associated upon 5FU exposure. This enhanced signaling pathway induced the dissociation of the Yes/YAP (Yes-associated protein) molecular complex and depleted nuclear YAP levels. Consistently, YES1 silencing decreased nuclear YAP accumulation and induced cellular quiescence in 5F31 cells cultured in 5FU-free medium. Importantly, YES1 and YAP transcript levels were higher in liver metastases of patients with colon cancer after 5FU-based neoadjuvant chemotherapy. Moreover, the YES1 and YAP transcript levels positively correlated with colon cancer relapse and shorter patient survival (P < 0.05 and P < 0.025, respectively). CONCLUSIONS: We identified c-Yes and YAP as potential molecular targets to eradicate quiescent cancer cells and dormant micrometastases during 5FU chemotherapy and resistance and as predictive survival markers for colon cancer.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Neoplasias do Colo/metabolismo , Fluoruracila/farmacologia , Neoplasias Hepáticas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-yes/metabolismo , Fatores de Transcrição/metabolismo , Antimetabólitos Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/metabolismo , Pontos de Checagem do Ciclo Celular , Proteínas de Ciclo Celular , Núcleo Celular/metabolismo , Proliferação de Células , Quinase do Ponto de Checagem 2/metabolismo , Quimioterapia Adjuvante , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/mortalidade , Neoplasias do Colo/patologia , Intervalo Livre de Doença , Resistencia a Medicamentos Antineoplásicos , Fluoruracila/uso terapêutico , Expressão Gênica , Células HT29 , Humanos , Estimativa de Kaplan-Meier , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/secundário , Terapia Neoadjuvante , Micrometástase de Neoplasia/prevenção & controle , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Proteínas Nucleares/genética , Modelos de Riscos Proporcionais , Transporte Proteico , Proteínas Proto-Oncogênicas c-yes/genética , Fatores de Transcrição/genéticaRESUMO
Metastasis and drug resistance are major problems in cancer chemotherapy. The purpose of this work was to analyze the molecular mechanisms underlying the invasive potential of drug-resistant colon carcinoma cells. Cellular models included the parental HT-29 cell line and its drug-resistant derivatives selected after chronic treatment with either 5-fluorouracil, methotrexate, doxorubicin, or oxaliplatin. Drug-resistant invasive cells were compared with noninvasive cells using cDNA microarray, quantitative reverse transcription-PCR, flow cytometry, immunoblots, and ELISA. Functional and cellular signaling analyses were undertaken using pharmacologic inhibitors, function-blocking antibodies, and silencing by retrovirus-mediated RNA interference. 5-Fluorouracil- and methotrexate-resistant HT-29 cells expressing an invasive phenotype in collagen type I and a metastatic behavior in immunodeficient mice exhibited high expression of the chemokine receptor CXCR4. Macrophage migration-inhibitory factor (MIF) was identified as the critical autocrine CXCR4 ligand promoting invasion in drug-resistant colon carcinoma HT-29 cells. Silencing of CXCR4 and impairing the MIF-CXCR4 signaling pathways by ISO-1, pAb FL-115, AMD-3100, monoclonal antibody 12G5, and BIM-46187 abolished this aggressive phenotype. Induction of CXCR4 was associated with the upregulation of two genes encoding transcription factors previously shown to control CXCR4 expression (HIF-2alpha and ASCL2) and maintenance of intestinal stem cells (ASCL2). Enhanced CXCR4 expression was detected in liver metastases resected from patients with colon cancer treated by the standard FOLFOX regimen. Combination therapies targeting the CXCR4-MIF axis could potentially counteract the emergence of the invasive metastatic behavior in clonal derivatives of drug-resistant colon cancer cells.
Assuntos
Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Oxirredutases Intramoleculares/metabolismo , Fatores Inibidores da Migração de Macrófagos/metabolismo , Receptores CXCR4/biossíntese , Animais , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Fluoruracila/farmacologia , Perfilação da Expressão Gênica , Inativação Gênica , Células HT29 , Humanos , Metotrexato/farmacologia , Camundongos , Invasividade Neoplásica , Metástase Neoplásica , Compostos Organoplatínicos/farmacologia , Oxaliplatina , Fenótipo , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Transdução de Sinais , Regulação para CimaRESUMO
We have previously reported that galectin-4, a tandem repeat-type galectin, regulates the raft-dependent delivery of glycoproteins to the apical brush border membrane of enterocyte-like HT-29 cells. N-Acetyllactos-amine-containing glycans, known as galectin ligands, were found enriched in detergent-resistant membranes. Here, we analyzed the potential contribution of N- and/or O-glycans in this mechanism. Structural studies were carried out on the brush border membrane-enriched fraction using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and nano-ESI-QTOF-MS/MS. The pattern of N-glycans was very heterogeneous, with the presence of high mannose- and hybrid-type glycans as well as a multitude of complex-type glycans. In contrast, the pattern of O-glycans was very simple with the presence of two major core type 1 O-glycans, sialylated and bisialylated T-antigen structures [Neu5Acalpha2-3Galbeta1-3GalNAc-ol and Neu5Acalpha2- 3Galbeta1-3(Neu5Acalpha2-6)GalNAc-ol]. Thus, N-glycans rather than O-glycans contain the N-acetyllactosamine recognition signals for the lipid raft-based galectin-4-dependent apical delivery. In the presence of 1-deoxymannojirimycin, a drug which inhibits the generation of hybrid-type or complex type N-glycans, the extensively O-glycosylated mucin-like MUC1 glycoprotein was not delivered to the apical brush border but accumulated inside the cells. Altogether, our data demonstrate the crucial role of complex N-glycans in the galectin-4-dependent delivery of glycoproteins to the apical brush border membrane of enterocytic HT-29 cells.
Assuntos
Enterócitos/citologia , Glicoproteínas de Membrana/metabolismo , Microvilosidades/metabolismo , Polissacarídeos/metabolismo , Sequência de Carboidratos , Epitopos/metabolismo , Glicosídeo Hidrolases/metabolismo , Glicosilação , Células HT29 , Humanos , Ácido Fluorídrico/metabolismo , Espectrometria de Massas , Glicoproteínas de Membrana/química , Microscopia Confocal , Dados de Sequência Molecular , Mucina-1/metabolismo , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Polissacarídeos/química , Transporte ProteicoRESUMO
We have previously reported that silencing of galectin-4 expression in polarized HT-29 cells perturbed apical biosynthetic trafficking and resulted in a phenotype similar to the inhibitor of glycosylation, 1-benzyl-2-acetamido-2-deoxy-beta-d-galactopyranoside (GalNAcalpha-O-bn). We now present evidence of a lipid raft-based galectin-4-dependent mechanism of apical delivery of glycoproteins in these cells. First, galectin-4 recruits the apical glycoproteins in detergent-resistant membranes (DRMs) because these glycoproteins were depleted in DRMs isolated from galectin-4-knockdown (KD) HT-29 5M12 cells. DRM-associated glycoproteins were identified as ligands for galectin-4. Structural analysis showed that DRMs were markedly enriched in a series of complex N-glycans in comparison to detergent-soluble membranes. Second, in galectin-4-KD cells, the apical glycoproteins still exit the Golgi but accumulated inside the cells, showing that their recruitment within lipid rafts and their apical trafficking required the delivery of galectin-4 at a post-Golgi level. This lectin that is synthesized on free cytoplasmic ribosomes is externalized from HT-29 cells mostly in the apical medium and follows an apical endocytic-recycling pathway that is required for the apical biosynthetic pathway. Together, our data show that the pattern of N-glycosylation of glycoproteins serves as a recognition signal for endocytosed galectin-4, which drives the raft-dependent apical pathway of glycoproteins in enterocyte-like HT-29 cells.