RESUMO
Human doublecortin (DCX) mutations are associated with severe brain malformations leading to aberrant neuron positioning (heterotopia), intellectual disability and epilepsy. The Dcx protein plays a key role in neuronal migration, and hippocampal pyramidal neurons in Dcx knockout (KO) mice are disorganized. The single CA3 pyramidal cell layer observed in wild type (WT) is present as two abnormal layers in the KO, and CA3 KO pyramidal neurons are more excitable than WT. Dcx KO mice also exhibit spontaneous epileptic activity originating in the hippocampus. It is unknown, however, how hyperexcitability arises and why two CA3 layers are observed.Transcriptome analyses were performed to search for perturbed postnatal gene expression, comparing Dcx KO CA3 pyramidal cell layers with WT. Gene expression changes common to both KO layers indicated mitochondria and Golgi apparatus anomalies, as well as increased cell stress. Intriguingly, gene expression analyses also suggested that the KO layers differ significantly from each other, particularly in terms of maturity. Layer-specific molecular markers and BrdU birthdating to mark the final positions of neurons born at distinct timepoints revealed inverted layering of the CA3 region in Dcx KO animals. Notably, many early-born 'outer boundary' neurons are located in an inner position in the Dcx KO CA3, superficial to other pyramidal neurons. This abnormal positioning likely affects cell morphology and connectivity, influencing network function. Dissecting this Dcx KO phenotype sheds light on coordinated developmental mechanisms of neuronal subpopulations, as well as gene expression patterns contributing to a bi-layered malformation associated with epilepsy.
Assuntos
Hipocampo/metabolismo , Hipocampo/patologia , Proteínas Associadas aos Microtúbulos/fisiologia , Neurônios/metabolismo , Neurônios/patologia , Neuropeptídeos/fisiologia , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/ultraestrutura , Região CA1 Hipocampal/metabolismo , Região CA1 Hipocampal/patologia , Região CA1 Hipocampal/ultraestrutura , Região CA3 Hipocampal/metabolismo , Região CA3 Hipocampal/patologia , Região CA3 Hipocampal/ultraestrutura , Proteínas do Domínio Duplacortina , Proteína Duplacortina , Feminino , Hipocampo/ultraestrutura , Processamento de Imagem Assistida por Computador , Microdissecção e Captura a Laser , Masculino , Camundongos , Camundongos Knockout , Microscopia Confocal , Neurônios/ultraestruturaRESUMO
BACKGROUND: Ampullary adenocarcinoma (AA) originates from either intestinal (INT) or pancreaticobiliary (PB) epithelium. Different prognostic factors of recurrence have been identified in previous studies. METHODS: In 91 AA patients of the AGEO retrospective multicentre cohort, we evaluated the centrally reviewed morphological classification, panel markers of Ang et al. including CK7, CK20, MUC1, MUC2 and CDX2, the 50-gene panel mutational analysis, and the clinicopathological AGEO prognostic score. RESULTS: Forty-three (47%) of the 91 tumours were Ang-INT, 29 (32%) were Ang-PB, 18 (20%) were ambiguous (Ang-AMB) and one could not be classified. Among these 90 tumours, 68.7% of INT tumours were Ang-INT and 78.2% of PB tumours were Ang-PB. MUC5AC expression was detected in 32.5% of the 86 evaluable cases. Among 71 tumours, KRAS, TP53, APC and PIK3CA were the most frequently mutated genes. The KRAS mutation was significantly more frequent in the PB subtype. In multivariate analysis, only AGEO prognostic score and tumour subtype were associated with relapse-free survival. Only AGEO prognostic score was associated with overall survival. CONCLUSIONS: Mutational analysis and MUC5AC expression provide no additional value in the prognostic evaluation of AA patients. Ang et al. classification and the AGEO prognostic score were confirmed as a strong prognosticator for disease recurrence.
Assuntos
Adenocarcinoma/genética , Ampola Hepatopancreática , Neoplasias do Ducto Colédoco/genética , Neoplasias Duodenais/genética , Adenocarcinoma/classificação , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Proteína da Polipose Adenomatosa do Colo/genética , Fator de Transcrição CDX2/metabolismo , Classe I de Fosfatidilinositol 3-Quinases/genética , Neoplasias do Ducto Colédoco/classificação , Neoplasias do Ducto Colédoco/metabolismo , Neoplasias do Ducto Colédoco/patologia , Neoplasias Duodenais/classificação , Neoplasias Duodenais/metabolismo , Neoplasias Duodenais/patologia , Feminino , Humanos , Imuno-Histoquímica , Queratina-20/metabolismo , Queratina-7/metabolismo , Masculino , Pessoa de Meia-Idade , Mucina-5AC/metabolismo , Mucina-1/metabolismo , Mucina-2/metabolismo , Prognóstico , Proteínas Proto-Oncogênicas p21(ras)/genética , Estudos Retrospectivos , Proteína Supressora de Tumor p53/genéticaRESUMO
Microsatellite instability (MSI) caused by mismatch repair deficiency (dMMR) is detected in a small proportion of pancreatic ductal adenocarcinomas (PDACs). dMMR and MSI have been associated with responses of metastatic tumors, including PDACs, to immune checkpoint inhibitor therapy. We performed immunohistochemical analyses of a 445 PDAC specimens, collected from consecutive patients at multiple centers, to identify those with dMMR, based on loss of mismatch repair proteins MLH1, MSH2, MSH6, and/or PMS2. We detected dMMR in 1.6% of tumor samples; we found dMMR in a larger proportion of intraductal papillary mucinous neoplasms-related tumors (4/58, 6.9%) than non- intraductal papillary mucinous neoplasms PDAC (5/385, 1.3%) (P = .02). PDACs with dMMR contained potentially immunogenic mutations because of MSI in coding repeat sequences. PDACs with dMMR or MSI had a higher density of CD8+ T cells at the invasive front than PDACs without dMMR or MSI (P = .08; Fisher exact test). A higher proportion of PDACs with dMMR or MSI expressed the CD274 molecule (PD-L1, 8/9) than PDACs without dMMR or MSI (4/10) (P = .05). Times of disease-free survival and overall survival did not differ significantly between patients with PDACs with dMMR or MSI vs without dMMR or MSI. Studies are needed to determine whether these features of PDACs with dMMR or MSI might serve as prognostic factors.
Assuntos
Carcinoma Ductal Pancreático/genética , Instabilidade de Microssatélites , Neoplasias Císticas, Mucinosas e Serosas/genética , Neoplasias Pancreáticas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/análise , Linfócitos T CD8-Positivos/imunologia , Carcinoma Ductal Pancreático/química , Carcinoma Ductal Pancreático/imunologia , Carcinoma Ductal Pancreático/patologia , Proteínas de Ligação a DNA/análise , Intervalo Livre de Doença , Feminino , Predisposição Genética para Doença , Humanos , Linfócitos do Interstício Tumoral/imunologia , Masculino , Pessoa de Meia-Idade , Endonuclease PMS2 de Reparo de Erro de Pareamento/análise , Proteína 1 Homóloga a MutL/análise , Proteína 2 Homóloga a MutS/análise , Neoplasias Císticas, Mucinosas e Serosas/química , Neoplasias Císticas, Mucinosas e Serosas/imunologia , Neoplasias Císticas, Mucinosas e Serosas/patologia , Neoplasias Pancreáticas/química , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/patologia , Fenótipo , Fatores de TempoRESUMO
AIMS: To investigate the status of somatostatin receptors (SSTRs) in resected hepatocellular carcinoma (HCC). METHODS AND RESULTS: Transcript and protein levels of SSTR2, SSTR3 and SSTR5 were investigated, with real-time polymerase chain reaction (PCR) and manual and automated immunohistochemistry (IHC), in 53 resected HCCs and paired non-tumour tissues. SSTR1, SSTR4, SSTR5TMD4 and SSTR5TMD5 were analysed with real-time PCR. SSTR3 and SSTR5 transcripts were expressed in ~25% of HCCs, but not in adjacent non-tumour tissues. SSTR1 and SSTR2 transcripts were overexpressed in 42% and 32% of HCCs, respectively. SSTR4, SSTR5TMD4 and SSTR5TMD5 were not detected. Membrane staining for SSTR2 was detected in 38% of HCCs, whereas SSTR5 protein was detectable in only 11% of HCCs. SSTR3 protein was detected in the majority of HCCs and adjacent non-tumour liver tissues, but membrane staining was <20% of that in HCCs. The results obtained with the two IHC methods were highly correlated (P < 0.0001). Statistical analyses also showed a positive correlation between SSTR2 membrane staining and cytokeratin 19 expression (P = 0.04), serum α-fetoprotein level (P = 0.002), and poor differentiation (P = 0.05). CONCLUSIONS: Membrane SSTR2 is detected reliably in HCCs by IHC, and is a potential therapeutic target, as it is coexpressed with markers of poor prognosis.
Assuntos
Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Receptores de Somatostatina/biossíntese , Biomarcadores Tumorais/análise , Carcinoma Hepatocelular/mortalidade , Feminino , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/mortalidade , Masculino , Pessoa de Meia-Idade , Prognóstico , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Somatostatina/análiseRESUMO
Microsatellite instability (MSI) due to mismatch repair (MMR) deficiency is reported in 5-10% of colorectal cancers (CRCs) complicating inflammatory bowel diseases (IBD). The molecular mechanisms underlying MMR deficiency may be different in IBD CRCs, and in sporadic and hereditary MSI tumors. Here, we hypothesize that overexpression of miR-155 and miR-21, two inflammation-related microRNAs that target core MMR proteins, may constitute a pre-neoplastic event for the development of MSI IBD CRCs. We studied miR-155 and miR-21 expression using real-time quantitative PCR in MSI (n = 10) and microsatellite stable (n = 10) IBD CRCs, and in MSI (n = 32) and microsatellite stable (n = 30) non-IBD CRCs. We also screened colonic samples from IBD patients without cancer (n = 18) and used healthy colonic mucosa as controls (n = 20). MiR-155 and miR-21 appeared significantly overexpressed not only in the colonic mucosa of IBD subjects without CRC but also in neoplastic tissues of IBD patients compared with non-IBD controls (P < 0.001). Importantly, in patients with IBD CRCs, miR-155 and miR-21 overexpression extended to the distant non-neoplastic mucosa (P < 0.001). Ratios of expressions in tumors versus matched distant mucosa revealed a nearly significant association between miR-155 overexpression and MSI in IBDs (P = 0.057). These results show a strong deregulation of both MMR-targeting microRNAs in IBD subjects with or without cancer. MiR-155 overexpression being particularly associated to MSI IBD CRCs and extending to distant non-neoplastic mucosa, strongly suggests that a pre-neoplastic miR-155 field defect may promote MSI-driven transformation of the colonic mucosa. The detection and monitoring of miR-155 field defect may, therefore, have implications for the prevention and treatment of MSI IBD CRCs.
Assuntos
Neoplasias Colorretais/genética , Reparo de Erro de Pareamento de DNA , Doenças Inflamatórias Intestinais/genética , MicroRNAs/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Mucosa Intestinal/citologia , Masculino , MicroRNAs/biossíntese , Instabilidade de Microssatélites , Pessoa de Meia-Idade , Adulto JovemRESUMO
Some LMNA mutations responsible for insulin-resistant lipodystrophic syndromes are associated with peripheral subcutaneous lipoatrophy and faciocervical fat accumulation. Their pathophysiologic characteristics are unknown. We compared histologic, immunohistologic, ultrastructural, and protein expression features of enlarged cervical subcutaneous adipose tissue (scAT) obtained during plastic surgery from four patients with LMNA p.R482W, p.R439C, or p.H506D mutations versus cervical fat from eight control subjects, buffalo humps from five patients with HIV infection treated or not with protease inhibitors, and dorsocervical lipomas from two patients with mitochondrial DNA mutations. LMNA-mutated cervical scAT and HIV-related buffalo humps were dystrophic, with an increased percentage of small adipocytes, increased fibrosis without inflammatory features, and decreased number of blood vessels, as compared with control samples. Samples from patients with LMNA mutations or protease inhibitor-based therapy demonstrated accumulation of prelamin A, altered expression of adipogenic proteins and brown fat-like features, with an increased number of mitochondria and overexpression of uncoupling protein 1 (UCP1). These features were absent in samples from control subjects and from patients with HIV not treated with protease inhibitors. Mitochondrial DNA-mutated cervical lipomas demonstrated inflammatory fibrosis with distinct mitochondrial abnormalities but neither UCP1 expression nor prelamin A accumulation. In conclusion, Enlarged cervical scAT from patients with lipodystrophy demonstrated small adipocytes, fibrosis, and decreased vessel numbers. However, only cervical fat from patients with LMNA mutations or who had received protease inhibitor therapy accumulated prelamin A and exhibited similar remodeling toward a brown-like phenotype with UCP1 overexpression and mitochondrial alterations.
Assuntos
Tecido Adiposo Marrom/patologia , Lamina Tipo A/genética , Lipodistrofia/genética , Mutação/genética , Adipócitos/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Fibrose/patologia , Humanos , Lipodistrofia/patologia , Masculino , Pessoa de Meia-Idade , PescoçoRESUMO
BACKGROUND: Antiprogestins are of growing interest for the development of new treatments in the gynecological field. Ulipristal acetate (UPA) is a progesterone receptor (PR) modulator considered for long-term administration in contraception and is currently being registered for the treatment of uterine fibroids. In light of the influences of hormonal dysfunction in breast pathologies, the secondary consequences of chronic UPA therapy need to be established. The aim of this study was to determine UPA actions mediated by PR and glucocorticoid receptor (GR) in normal and transformed breast. METHODS: UPA, progesterone (P) and dexamethasone (DEX) effects were observed on PR and GR responsive genes and on proliferation and apoptosis of normal human breast epithelial (HBE) and breast cancer cells. Human normal breast tissue samples were xenografted in athymic mice and treated with estradiol (E2), or E2 + P, or E2 + P + UPA. RESULTS: Analysis of PR and GR reporter gene transactivation and their respective endogenous target genes indicated that UPA exerted anti-progestational and anti-glucocorticoid activity in both types of cells with a more pronounced effect in cancer cells. When combined with P or DEX, UPA limits the proliferation of HBE cells but increases growth in breast cancer cell lines. UPA administration had no impact on the mitotic index on xenografted human breast tissue exposed to gonadal hormones at similar concentrations to those present in normal women. CONCLUSIONS: Although further clinical trials are required to confirm that the results from our experimental models can be extrapolated to women treated with UPA, they suggest that such treatment would not be deleterious to normal breast tissue at least for a cycle (28 days) of continuous administration.
Assuntos
Mama/efeitos dos fármacos , Mama/patologia , Anticoncepcionais/farmacologia , Norpregnadienos/farmacologia , Adolescente , Adulto , Animais , Proteínas Reguladoras de Apoptose/biossíntese , Mama/metabolismo , Ciclina A/biossíntese , Dexametasona/farmacologia , Células Epiteliais/efeitos dos fármacos , Estradiol/metabolismo , Ácido Graxo Sintase Tipo I/biossíntese , Feminino , Genes Reporter , Humanos , Antígeno Ki-67/biossíntese , Leiomioma/metabolismo , Glândulas Mamárias Humanas/metabolismo , Glândulas Mamárias Humanas/patologia , Proteínas de Membrana/biossíntese , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Progesterona/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Receptores de Glucocorticoides/metabolismo , Ativação Transcricional , Transplante HeterólogoAssuntos
Azatioprina/efeitos adversos , Neoplasias Colorretais/induzido quimicamente , Neoplasias Colorretais/genética , Instabilidade de Microssatélites , Adulto , Idoso , Idoso de 80 Anos ou mais , Azatioprina/uso terapêutico , Feminino , Humanos , Doenças Inflamatórias Intestinais/complicações , Doenças Inflamatórias Intestinais/tratamento farmacológico , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND AND AIMS: O(6)-Methylguanine-DNA methyltransferase (MGMT) removes methyl adducts from O(6)-guanine. Known as methylation tolerance, selection for mismatch repair (MMR)-deficient cells that are unable to initiate lethal processing of O(6)-methylguanine-induced mismatches in DNA is observed in vitro as a consequence of MGMT deficiency. It was therefore hypothesised that an MGMT field defect may constitute a preneoplastic event for the development of MMR-deficient tumours displaying microsatellite instability (MSI). METHODS: MGMT expression was investigated by immunohistochemistry and the methylation status of the gene promoter by PCR in neoplastic, adjacent and distant mucosal tissues of patients with MSI or non-MSI (MSS) colorectal cancer (CRC). The cancers were familial (42 MSI, 13 MSS) or sporadic (40 MSI, 49 MSS) in origin, or arose in the context of inflammatory bowel disease (IBD; 13 MSI, 36 MSS). Colonic mucosa from patients with diverticulitis (n=20) or IBD (n=39 in 27 patients) without cancer served as controls. RESULTS: Loss of MGMT expression was more frequent in MSI than MSS CRC (p=0.047). In comparison with MSS tumours, MSI CRC occurred more frequently adjacent to patches of mucosa that lacked MGMT expression (p=0.002). Overall, loss of MGMT expression was associated with MGMT gene promoter methylation (p=0.03). CONCLUSION: MGMT field defects are more frequently associated with MSI than MSS CRC. These findings indicate that methylation tolerance may be a crucial initiating step prior to MMR deficiency in the development of MSI CRC in familial, sporadic and IBD settings.
Assuntos
Neoplasias Colorretais/genética , Metilação de DNA , Reparo de Erro de Pareamento de DNA , DNA de Neoplasias/genética , O(6)-Metilguanina-DNA Metiltransferase/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/patologia , Feminino , Humanos , Mucosa Intestinal/enzimologia , Masculino , Instabilidade de Microssatélites , Pessoa de Meia-Idade , Proteína 1 Homóloga a MutL , Mutação , Estadiamento de Neoplasias , Proteínas Nucleares/genética , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Lesões Pré-Cancerosas/genética , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras) , Adulto Jovem , Proteínas ras/genéticaRESUMO
Exogenous overexpression of the metastasis suppressor gene Nm23-H1 reduces the metastatic potential of multiple types of cancer cells and suppresses in vitro tumor cell motility and invasion. Mutational analysis of Nm23-H1 revealed that substitution mutants P96S and S120G did not inhibit motility and invasion. To elucidate the molecular mechanism of Nm23-H1 motility suppression, expression microarray analysis of an MDA-MB-435 cancer cell line overexpressing wild-type Nm23-H1 was done and cross-compared with expression profiles from lines expressing the P96S and S120G mutants. Nine genes, MET, PTN, SMO, FZD1, L1CAM, MMP2, NETO2, CTGF, and EDG2, were down-regulated by wild-type but not by mutant Nm23-H1 expression. Reduced expression of these genes coincident with elevated Nm23-H1 expression was observed in human breast tumor cohorts, a panel of breast carcinoma cell lines, and hepatocellular carcinomas from control versus Nm23-M1 knockout mice. The functional significance of the down-regulated genes was assessed by transfection and in vitro motility assays. Only EDG2 overexpression significantly restored motility to Nm23-H1-suppressed cancer cells, enhancing motility by 60-fold in these cells. In addition, silencing EDG2 expression with small interfering RNA reduced the motile phenotype of metastatic breast cancer cells. These data suggest that Nm23-H1 suppresses metastasis, at least in part, through down-regulation of EDG2 expression.
Assuntos
Neoplasias da Mama/patologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Núcleosídeo-Difosfato Quinase/fisiologia , Receptores de Ácidos Lisofosfatídicos/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Movimento Celular , Estudos de Coortes , Colágeno/metabolismo , Regulação para Baixo , Combinação de Medicamentos , Perfilação da Expressão Gênica , Inativação Gênica , Humanos , Immunoblotting , Técnicas Imunoenzimáticas , Laminina/metabolismo , Camundongos , Camundongos Knockout , Nucleosídeo NM23 Difosfato Quinases , Análise de Sequência com Séries de Oligonucleotídeos , Proteoglicanas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Ácidos Lisofosfatídicos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
The aim of this study was to evaluate two RGD radiotracers radiolabelled with fluorine-18 or gallium-68, in detecting angiogenesis in grafted human tumours and monitoring their treatment with the anti-angiogenic agent bevacizumab. Sixteen mice bearing an U87MG tumour in one flank and a contralateral A549 tumour were treated with intravenous injections of bevacizumab twice a week for 3 weeks. PET images with 18F-RGD-K5 and 68Ga-RGD were acquired before treatment (baseline), after three bevacizumab injections (t1) and after seven bevacizumab injections (t2). In A549 tumours, the treatment stopped the tumour growth, with a tumour volume measured by calliper remaining between 0.28 and 0.40 cm3. The decrease in tumour uptake of both RGD tracers was non-significant. Therefore it was not possible to predict this efficacy on tumour growth based on RGD PET results, whereas ex vivo measurements showed a significantly lower tumour uptake of both tracers in mice sacrificed at t2 vs. at baseline. In U87MG tumours, the uptake measured on PET decreased during treatment, reflecting the partial therapeutic effect observed on tumour volume, consisting in a decrease in the slope of tumour growth. Using 18F-RGD-K5, this decrease in tumour SUVmax became significant at t1, whereas it was also observed with the 68Ga-RGD tracer, but only at t2. 18F-RGD-K5 appeared more efficient than 68Ga-RGD in the visualisation and follow-up of U87MG tumours. The comparison of those results with those of immunohistochemistry at baseline and at t2 favoured the hypothesis that tumour RGD uptake reflects other cancer properties than just its angiogenic capacity.
RESUMO
Background & Aims: Recent studies have shown that cancers arise as a result of the positive selection of driver somatic events in tumor DNA, with negative selection playing only a minor role, if any. However, these investigations were concerned with alterations at nonrepetitive sequences and did not take into account mutations in repetitive sequences that have very high pathophysiological relevance in the tumors showing microsatellite instability (MSI) resulting from mismatch repair deficiency investigated in the present study. Methods: We performed whole-exome sequencing of 47 MSI colorectal cancers (CRCs) and confirmed results in an independent cohort of 53 MSI CRCs. We used a probabilistic model of mutational events within microsatellites, while adapting pre-existing models to analyze nonrepetitive DNA sequences. Negatively selected coding alterations in MSI CRCs were investigated for their functional and clinical impact in CRC cell lines and in a third cohort of 164 MSI CRC patients. Results: Both positive and negative selection of somatic mutations in DNA repeats was observed, leading us to identify the expected true driver genes associated with the MSI-driven tumorigenic process. Several coding negatively selected MSI-related mutational events (n = 5) were shown to have deleterious effects on tumor cells. In the tumors in which deleterious MSI mutations were observed despite the negative selection, they were associated with worse survival in MSI CRC patients (hazard ratio, 3; 95% CI, 1.1-7.9; P = .03), suggesting their anticancer impact should be offset by other as yet unknown oncogenic processes that contribute to a poor prognosis. Conclusions: The present results identify the positive and negative driver somatic mutations acting in MSI-driven tumorigenesis, suggesting that genomic instability in MSI CRC plays a dual role in achieving tumor cell transformation. Exome sequencing data have been deposited in the European genome-phenome archive (accession: EGAS00001002477).
Assuntos
Carcinogênese/genética , Neoplasias Colorretais/genética , Instabilidade de Microssatélites , Mutação/genética , Sequências Repetitivas de Ácido Nucleico/genética , Animais , Sequência de Bases , Linhagem Celular Tumoral , Estudos de Coortes , Feminino , Xenoenxertos , Humanos , Masculino , Camundongos , Camundongos Nus , Modelos Estatísticos , Sequenciamento do ExomaRESUMO
Mismatch-repair (MMR)-deficient cells show increased in vitro tolerance to thiopurines because they escape apoptosis resulting from MMR-dependent signaling of drug-induced DNA damage. Prolonged treatment with immunosuppressants including azathioprine (Aza), a thiopurine prodrug, has been suggested as a risk factor for the development of late onset leukemias/lymphomas displaying a microsatellite instability (MSI) phenotype, the hallmark of a defective MMR system. We performed a dose effect study in mice to investigate the development of MSI lymphomas associated with long term Aza treatment. Over two years, Aza was administered to mice that were wild type, null or heterozygous for the MMR gene Msh2. Ciclosporin A, an immunosuppressant with an MMR-independent signaling, was also administered to Msh2(wt) mice as controls. Survival, lymphoma incidence and MSI tumor phenotype were investigated. Msh2(+/-) mice were found more tolerant than Msh2(wt) mice to the cytotoxicity of Aza. In Msh2(+/-) mice, Aza induced a high incidence of MSI lymphomas in a dose-dependent manner. In Msh2(wt) mice, a substantial lifespan was only observed at the lowest Aza dose. It was associated with the development of lymphomas, one of which displayed the MSI phenotype, unlike the CsA-induced lymphomas. Our findings define Aza as a risk factor for an MSI-driven lymphomagenesis process.
Assuntos
Azatioprina/toxicidade , Linfoma/genética , Instabilidade de Microssatélites , Proteína 2 Homóloga a MutS/genética , Adulto , Idoso , Animais , Reparo de Erro de Pareamento de DNA/genética , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Humanos , Imuno-Histoquímica , Imunossupressores/toxicidade , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/metabolismo , Estimativa de Kaplan-Meier , Linfoma/induzido quimicamente , Linfoma/metabolismo , Masculino , Camundongos Knockout , Pessoa de Meia-Idade , Proteína 2 Homóloga a MutS/metabolismo , Fenótipo , Medição de Risco/métodos , Fatores de Risco , Fatores de Tempo , Adulto JovemRESUMO
BACKGROUND: Crohn's disease (CD) is associated with an increased risk of small bowel adenocarcinoma (SBA). However, there are no guidelines for the screening and early diagnosis of SBA. Colorectal cancer associated with chronic colitis arises from dysplasia. High-risk patients benefit from surveillance colonoscopies aimed to detect dysplasia. The dysplasia-carcinoma sequence remains poorly documented in CD-associated SBA. Moreover, molecular data about SBA complicating CD and associated dysplasia are very limited. We therefore assessed dysplasia and several key molecular markers of carcinogenesis in SBA and dysplasia developed in patients with CD. METHODS: Forty-five SBA complicating CD and 4 specimens with dysplasia without SBA were screened. In SBA, we looked for dysplasia and determined their pathological characteristics (type, grade, distribution). We also stained for mismatch repair proteins (MLH1, MSH2, MSH6, PMS2), p53, ß-catenin, and p16 and looked for KRAS, BRAF and PIK3CA mutations. RESULTS: All neoplastic lesions, except 1 lesion, were found in inflamed mucosal areas. Dysplasia was found in 20 of 41 patients with SBA (49%). Dysplasia was flat or raised, low grade or high grade, and adjacent or distant to concomitant SBA. Molecular markers of SBA carcinogenesis complicating CD were similar to those observed in chronic colitis-related colorectal cancer (KRAS, BRAF, p53, MSI), although differences were observed for ß-catenin and p16. No PIK3CA mutations were observed. CONCLUSIONS: These results suggest that there is an inflammation-dysplasia-adenocarcinoma sequence in at least half of CD-related SBA, similar to what is observed in chronic colitis-related colorectal cancer and may have implications for the prevention and treatment of this cancer.
Assuntos
Adenocarcinoma/etiologia , Neoplasias Colorretais/etiologia , Doença de Crohn/complicações , Displasia Fibromuscular/etiologia , Inflamação/etiologia , Intestino Delgado/patologia , Adenocarcinoma/diagnóstico , Adenocarcinoma/mortalidade , Adulto , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/mortalidade , Feminino , Displasia Fibromuscular/diagnóstico , Displasia Fibromuscular/mortalidade , Seguimentos , Humanos , Inflamação/diagnóstico , Inflamação/mortalidade , Masculino , Pessoa de Meia-Idade , Prognóstico , Fatores de Risco , Taxa de Sobrevida , Adulto JovemRESUMO
Evidence suggests that signalling through lipopolysaccharide (LPS) has a significant role in the development of gastrointestinal malignancies. We previously demonstrated the critical role of myeloid differentiation (MD)-2, the essential co-receptor of LPS, for induction of cyclooxygenase (Cox)-2 in intestinal epithelial cells. Cyclooxigenase-2 was suggested to play a key role in colorectal cancer through the effects of prostaglandin (PG) E(2) generated. We, therefore, addressed the role of MD-2 in several parameters related to malignancy, namely cell proliferation and migration, using colon cancer cells (HT-29). We found that overexpression of MD-2 confers a significantly greater proliferation and migration capacity to these cells. MD-2-dependent proliferation and migration appeared independent of Cox-2 activity but was reduced by endothelial growth factor receptor (EGFR) neutralizing antibodies as well as by pharmacological inhibition of EGFR tyrosine phosphorylation. We propose that MD-2 overexpression contributes to tumour aggressiveness via a Cox-2-independent excessive EGFR signalling. Moreover, MD-2 expression levels were higher in tissue from patients with colorectal cancer as compared with paired control colorectal mucosa. Our data attest to a role of MD-2 activity in colon cancer epithelial cell proliferation and migration, which may be important in the general correlation between innate immune response, chronic inflammation, and cancer.
Assuntos
Carcinoma/metabolismo , Neoplasias do Colo/metabolismo , Ciclo-Oxigenase 2/metabolismo , Antígeno 96 de Linfócito/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Adulto , Anticorpos Bloqueadores/farmacologia , Carcinoma/imunologia , Carcinoma/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/imunologia , Neoplasias do Colo/patologia , Ciclo-Oxigenase 2/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imunidade Inata , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/metabolismo , Antígeno 96 de Linfócito/genética , Antígeno 96 de Linfócito/imunologia , Masculino , Pessoa de Meia-Idade , Receptores de Fatores de Crescimento do Endotélio Vascular/imunologia , Transgenes/genéticaRESUMO
CCN5 is a member of the CCN (connective tissue growth factor/cysteine-rich 61/nephroblastoma overexpressed) family and was identified as an estrogen-inducible gene in estrogen receptor-positive cell lines. However, the role of CCN5 in breast carcinogenesis remains unclear. We report here that the CCN5 protein is localized mostly in the cytoplasm and in part in the nucleus of human tumor breast tissue. Using a heterologous transcription assay, we demonstrate that CCN5 can act as a transcriptional repressor presumably through association with histone deacetylase 1 (HDAC1). Microarray gene expression analysis showed that CCN5 represses expression of genes associated with epithelial-mesenchymal transition (EMT) as well as expression of key components of the transforming growth factor ß (TGF-ß) signaling pathway, prominent among them TGF-ßRII receptor. We show that CCN5 is recruited to the TGF-ßRII promoter, thereby providing a mechanism by which CCN5 restricts transcription of the TGF-ßRII gene. Consistent with this finding, CCN5, we found, functions to suppress TGF-ß-induced transcriptional responses and invasion that is concomitant with EMT. Thus, our data uncovered CCN5 as a novel transcriptional repressor that plays an important role in regulating tumor progression functioning, at least in part, by inhibiting the expression of genes involved in the TGF-ß signaling cascade that is known to promote EMT.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Transcrição Gênica , Fator de Crescimento Transformador beta/metabolismo , Proteínas de Sinalização Intercelular CCN , Caderinas/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo/genética , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Histona Desacetilases/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/química , Invasividade Neoplásica , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Estrutura Terciária de Proteína , Transporte Proteico , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/genética , Proteínas Repressoras/química , Frações Subcelulares/metabolismo , Fatores de Transcrição/química , Fator de Crescimento Transformador beta/genéticaRESUMO
PURPOSE: Epidermal growth factor receptor (EGFR) and VEGF(R) signaling show extensive cross-talk, providing a rationale for joint targeting of the two pathways. However, combinations of monoclonal antibodies (mAb) targeting EGFR and VEGF showed disappointing activity in patients with colorectal cancer (CRC). We speculated that inhibition of surface receptors and ligands might only partly prevent oncogenic signaling whereas small-molecule tyrosine kinase inhibitors (TKI) would also influence intracellular signaling. EXPERIMENTAL DESIGN: Mice with CRC xenografts were treated with two TKIs, vargatef and afatinib, or with two mAbs, bevacizumab and cetuximab, and their influence on tumor growth, viability, in vivo DNA synthesis, and the presence of phosphorylated EGFR and VEGFR was determined. The activity of the TKIs was further characterized in CRC cells with different KRAS status. RESULTS: Vargatef and afatinib together showed strong tumor growth inhibition toward HT-29 xenografts compared with either drug alone, which was associated with a 5-fold increase in apoptotic tumor cell death. In comparison, bevacizumab and cetuximab together were exclusively cytostatic with no more activity than either drug alone. Exposure to the two TKIs was accompanied by a marked decrease of tumor-associated intracellular phospho-VEGFR1 and phospho-EGFR, whereas similar exposure to the two mAbs had no detectable effect. A synergistic activity of vargatef plus afatinib was observed in all eight CRC cell lines examined, independent of KRAS status. CONCLUSIONS: Our results indicate that attenuation of intracellular EGFR and/or VEGF signaling is required for cytotoxic activity. These findings provide a rationale for trials of the TKIs, even in patients with mutant KRAS.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Afatinib , Animais , Anticorpos Monoclonais Humanizados/uso terapêutico , Bevacizumab , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cetuximab , Neoplasias Colorretais/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Receptores ErbB/antagonistas & inibidores , Feminino , Células HT29 , Humanos , Indóis/farmacologia , Camundongos , Camundongos Nus , Proteínas Tirosina Quinases/antagonistas & inibidores , Quinazolinas/farmacologia , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: The enthesis, which attaches the tendon to the bone, naturally disappears with aging, thus limiting joint mobility. Surgery is frequently needed but the clinical outcome is often poor due to the decreased natural healing capacity of the elderly. This study explored the benefits of a treatment based on injecting chondrocyte and mesenchymal stem cells (MSC) in a new rat model of degenerative enthesis repair. METHODOLOGY: The Achilles' tendon was cut and the enthesis destroyed. The damage was repaired by classical surgery without cell injection (group G1, n = 52) and with chondrocyte (group G2, n = 51) or MSC injection (group G3, n = 39). The healing rate was determined macroscopically 15, 30 and 45 days later. The production and organization of a new enthesis was assessed by histological scoring of collagen II immunostaining, glycoaminoglycan production and the presence of columnar chondrocytes. The biomechanical load required to rupture the bone-tendon junction was determined. PRINCIPAL FINDINGS: The spontaneous healing rate in the G1 control group was 40%, close to those observed in humans. Cell injection significantly improved healing (69%, p = 0.0028 for G2 and p = 0.006 for G3) and the load-to-failure after 45 days (p<0.05) over controls. A new enthesis was clearly produced in cell-injected G2 and G3 rats, but not in the controls. Only the MSC-injected G3 rats had an organized enthesis with columnar chondrocytes as in a native enthesis 45 days after surgery. CONCLUSIONS: Cell therapy is an efficient procedure for reconstructing degenerative entheses. MSC treatment produced better organ regeneration than chondrocyte treatment. The morphological and biomechanical properties were similar to those of a native enthesis.
Assuntos
Osso e Ossos/fisiologia , Transplante de Células-Tronco Mesenquimais/métodos , Regeneração , Tendões/fisiologia , Animais , Osso e Ossos/citologia , Bovinos , Condrócitos/transplante , Humanos , Injeções , Masculino , Ratos , Ratos Wistar , Reprodutibilidade dos Testes , Tendões/citologia , Fatores de TempoRESUMO
BACKGROUND: The thiopurine prodrug azathioprine is used extensively in cancer therapy. Exposure to this drug results in the selection of DNA mismatch repair-deficient cell clones in vitro. It has also been suggested that thiopurine drugs might constitute a risk factor for the emergence of human neoplasms displaying microsatellite instability (MSI) because of deficient DNA mismatch repair. METHODS: Azathioprine was administered via drinking water (6-20 mg/kg body weight per day) to mice that were null (Msh2â»(/)â»; n = 27), heterozygous (Msh2(+/)â»; n = 22), or wild type (Msh2(WT); n = 18) for the DNA mismatch repair gene Msh2. Control mice (45 Msh2â»(/)â», 38 Msh2(+/)â», and 12 Msh2(WT)) received drinking water lacking azathioprine. The effect of azathioprine on tumorigenesis and survival of the mice was evaluated by Kaplan-Meier curves using log-rank and Gehan-Breslow-Wilcoxon tests. Mouse tumor samples were characterized by histology and immunophenotyping, and their MSI status was determined by polymerase chain reaction analysis of three noncoding microsatellite markers and by immunohistochemistry. Msh2 status of tumor samples was assessed by loss of heterozygosity analyses and sequencing after reverse transcription-polymerase chain reaction of the entire Msh2 coding sequence. All statistical tests were two-sided. RESULTS: Most untreated Msh2(WT) and Msh2(+/)â» mice remained asymptomatic and alive at 250 days of age, whereas azathioprine-treated Msh2(WT) and Msh2(+/)â» mice developed lymphomas and died prematurely (median survival of 71 and 165 days of age, respectively). Azathioprine-treated Msh2(+/)â» mice developed diffuse lymphomas lacking Msh2 expression and displaying MSI due to somatic inactivation of the functional Msh2 allele by loss of heterozygosity or mutation. By contrast, azathioprine-treated Msh2(WT) mice displayed no obvious tumor phenotype, but histological examination showed microscopic splenic foci of neoplastic lymphoid cells that retained Msh2 expression and did not display MSI. Both untreated and azathioprine-treated Msh2â»(/)â» mice had a reduced lifespan compared with untreated Msh2(WT) mice (median survival of 127 and 107 days of age, respectively) and developed lymphomas with MSI. CONCLUSION: Azathioprine-induced carcinogenesis in mice depends on the number of functional copies of the Msh2 gene.
Assuntos
Antimetabólitos Antineoplásicos/toxicidade , Azatioprina/toxicidade , Carcinógenos/toxicidade , Reparo de Erro de Pareamento de DNA/genética , Imunossupressores/toxicidade , Linfoma/induzido quimicamente , Linfoma/genética , Proteína 2 Homóloga a MutS/genética , Administração Oral , Animais , DNA de Neoplasias/genética , Modelos Animais de Doenças , Genótipo , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Perda de Heterozigosidade , Linfoma/patologia , Camundongos , Instabilidade de Microssatélites/efeitos dos fármacos , Reação em Cadeia da Polimerase , Projetos de PesquisaRESUMO
Liver fibrosis is produced by myofibroblasts of different origins. In culture models, rat myofibroblasts derived from hepatic stellate cells (HSCs) and from periductal portal mesenchymal cells, show distinct proliferative and immunophenotypic evolutive profiles, in particular regarding desmin microfilament (overexpressed vs shut-down, respectively). Here, we examined the contributions of both cell types, in two rat models of cholestatic injury, arterial liver ischemia and bile duct ligation (BDL). Serum and (immuno)histochemical hepatic analyses were performed at different time points (2 days, 1, 2 and 6 weeks) after injury induction. Cholestatic liver injury, as attested by serum biochemical tests, was moderate/resolutive in ischemia vs severe and sustained in BDL. Spatio-temporal and morphometric analyses of cytokeratin-19 and Sirius red stainings showed that in both models, fibrosis accumulated around reactive bile ductules, with a significant correlation between the progression rates of fibrosis and of the ductular reaction (both higher in BDL). After 6 weeks, fibrosis was stabilized and did not exceed F2 (METAVIR) in arterial ischemia, whereas micronodular cirrhosis (F4) was established in BDL. Immuno-analyses of alpha-smooth muscle actin and desmin expression profiles showed that intralobular HSCs underwent early phenotypic changes marked by desmin overexpression in both models and that the accumulation of fibrosis coincided with that of alpha-SMA-labeled myofibroblasts around portal/septal ductular structures. With the exception of desmin-positive myofibroblasts located at the portal/septal-lobular interface at early stages, and of myofibroblastic HSCs detected together with fine lobular septa in BDL cirrhotic liver, the vast majority of myofibroblasts were desmin-negative. These findings suggest that both in resolutive and sustained cholestatic injury, fibrosis is produced by myofibroblasts that derive predominantly from portal/periportal mesenchymal cells. While HSCs massively undergo phenotypic changes marked by desmin overexpression, a minority fully converts into matrix-producing myofibroblasts, at sites, which however may be important in the healing process that circumscribes wounded hepatocytes.