RESUMO
Laser capture microdissection (LCM) allows microscopic procurement of specific cell types from tissue sections. Here, we present an optimized workflow for coupling LCM to LCâ¿¿MS/MS including: sectioning of tissue, a standard LCM workflow, protein digestion and advanced LCâ¿¿MS/MS. Soluble proteins extracted from benign epithelial cells, their associated stroma, tumor epithelial cells and their associated stromal cells from a single patient tissue sample were digested and profiled using advanced LCâ¿¿MS/MS. The correlation between technical replicates was R2 = 0.99 with a mean % CV of 9.55% ± 8.73. The correlation between sample replicates was R2 = 0.97 with a mean % CV of 13.83% ± 10.17. This represents a robust, systematic approach for profiling of the tumor microenvironment using LCM coupled to label-free LCâ¿¿MS/MS.
RESUMO
Diet is hypothesized to be a critical environmentally related risk factor for prostate cancer (PCa) development, and specific diets and dietary components can also affect PCa progression; however, the mechanisms underlying these associations remain elusive. As for a maturing organism, PCa's epigenome is plastic and evolves from the pre-neoplastic to the metastatic stage. In particular, epigenetic remodeling relies on substrates or cofactors obtained from the diet. Here we review the evidence that bridges dietary modulation to alterations in the prostate epigenome. We propose that such diet-related effects offer a mechanistic link between the impact of different diets and the course of PCa development and progression.
Assuntos
Epigênese Genética , Código das Histonas/genética , Neoplasias da Próstata/dietoterapia , Neoplasias da Próstata/genética , Acetilação , Metilação de DNA/genética , Genoma Humano , Humanos , Masculino , Neoplasias da Próstata/patologia , Fatores de RiscoRESUMO
BACKGROUND: Key challenges of biopsy-based determination of prostate cancer aggressiveness include tumour heterogeneity, biopsy-sampling error, and variations in biopsy interpretation. The resulting uncertainty in risk assessment leads to significant overtreatment, with associated costs and morbidity. We developed a performance-based strategy to identify protein biomarkers predictive of prostate cancer aggressiveness and lethality regardless of biopsy-sampling variation. METHODS: Prostatectomy samples from a large patient cohort with long follow-up were blindly assessed by expert pathologists who identified the tissue regions with the highest and lowest Gleason grade from each patient. To simulate biopsy-sampling error, a core from a high- and a low-Gleason area from each patient sample was used to generate a 'high' and a 'low' tumour microarray, respectively. RESULTS: Using a quantitative proteomics approach, we identified from 160 candidates 12 biomarkers that predicted prostate cancer aggressiveness (surgical Gleason and TNM stage) and lethal outcome robustly in both high- and low-Gleason areas. Conversely, a previously reported lethal outcome-predictive marker signature for prostatectomy tissue was unable to perform under circumstances of maximal sampling error. CONCLUSIONS: Our results have important implications for cancer biomarker discovery in general and development of a sampling error-resistant clinical biopsy test for prediction of prostate cancer aggressiveness.
Assuntos
Biomarcadores Tumorais/análise , Próstata/patologia , Neoplasias da Próstata/mortalidade , Neoplasias da Próstata/patologia , Actinina/análise , Idoso , Alquil e Aril Transferases/análise , Área Sob a Curva , Biópsia por Agulha Fina , Proteínas Culina/análise , Proteínas de Ligação a DNA/análise , Seguimentos , Proteínas de Choque Térmico HSP70/análise , Humanos , Processamento de Imagem Assistida por Computador , Masculino , Proteínas de Membrana/análise , Pessoa de Meia-Idade , Proteínas Mitocondriais/análise , Gradação de Tumores , Estadiamento de Neoplasias , Fosforilação , Próstata/química , Neoplasias da Próstata/química , Proteômica , Proteína FUS de Ligação a RNA , Curva ROC , Proteína S6 Ribossômica/análise , Proteína S6 Ribossômica/metabolismo , Viés de Seleção , Proteína Smad2/análise , Proteína Smad4/análise , Análise Serial de Tecidos , Canal de Ânion 1 Dependente de Voltagem/análise , Proteína 1 de Ligação a Y-Box/análiseRESUMO
Cancer cells are characterized by altered ubiquitination of many proteins. The ubiquitin-specific protease 2a (USP2a) is a deubiquitinating enzyme overexpressed in prostate adenocarcinomas, where it exhibits oncogenic behavior in a variety of ways including targeting c-Myc via the miR-34b/c cluster. Here we demonstrate that USP2a induces drug resistance in both immortalized and transformed prostate cells. Specifically, it confers resistance to typically pro-oxidant agents, such as cisplatin (CDDP) and doxorubicin (Doxo), and to taxanes. USP2a overexpression protects from drug-induced oxidative stress by reducing reactive oxygen species (ROS) production and stabilizing the mitochondrial membrane potential (ΔΨ), thus impairing downstream p38 activation and triggering of apoptosis. The molecular mediator of the USP2a protective function is the glutathione (GSH). Through miR-34b/c-driven c-Myc regulation, USP2a increases intracellular GSH content, thus interfering with the oxidative cascade triggered by chemotherapeutic agents. In light of these findings, targeting Myc and/or miR-34b/c might revert chemo-resistance.
Assuntos
Antineoplásicos/farmacologia , Endopeptidases/metabolismo , Antioxidantes/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Sequestradores de Radicais Livres/metabolismo , Glutationa/metabolismo , Humanos , Masculino , MicroRNAs/metabolismo , Modelos Biológicos , Oxidantes/toxicidade , Oxirredução/efeitos dos fármacos , Próstata/efeitos dos fármacos , Próstata/metabolismo , Próstata/patologia , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ubiquitina TiolesteraseRESUMO
Ubiquitination of epidermal growth factor receptor (EGFR) is required for downregulation of the receptor by endocytosis. Impairment of this pathway results in constitutively active EGFR, which is associated with carcinogenesis, particularly in lung cancer. We previously demonstrated that the deubiquitinating enzyme ubiquitin-specific protease 2a (USP2a) has oncogenic properties. Here, we show a new role for USP2a as a regulator of EGFR endocytosis. USP2a localizes to early endosomes and associates with EGFR, stabilizing the receptor, which retains active downstream signaling. HeLa cells transiently expressing catalytically active, but not mutant (MUT), USP2a show increased plasma membrane-localized EGFR, as well as decreased internalized and ubiquitinated EGFR. Conversely, USP2a silencing reverses this phenotype. Importantly, USP2a prevents the degradation of MUT in addition to wild-type EGFR. Finally, we observed that USP2a and EGFR proteins are coordinately overexpressed in non-small cell lung cancers. Taken together, our data indicate that USP2a antagonizes EGFR endocytosis and thus amplifies signaling activity from the receptor. Our findings suggest that regulation of deubiquitination could be exploited therapeutically in cancers overexpressing EGFR.
Assuntos
Endocitose/fisiologia , Endopeptidases/fisiologia , Receptores ErbB/metabolismo , Proteólise , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Regulação para Baixo/genética , Endocitose/genética , Endopeptidases/genética , Endopeptidases/metabolismo , Receptores ErbB/genética , Regulação Neoplásica da Expressão Gênica , Células HeLa , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Estabilidade Proteica , Ubiquitina Tiolesterase , Proteases Específicas de Ubiquitina , Ubiquitinação/genéticaRESUMO
BACKGROUND: AMP-activated protein kinase (AMPK, PRKA) has central roles in cellular metabolic sensing and energy balance homeostasis, and interacts with various pathways (e.g., TP53 (p53), FASN, MTOR and MAPK3/1 (ERK)). AMP-activated protein kinase activation is cytotoxic to cancer cells, supporting AMPK as a tumour suppressor and a potential therapeutic target. However, no study has examined its prognostic role in colorectal cancers. METHODS: Among 718 colon and rectal cancers, phosphorylated AMPK (p-AMPK) and p-MAPK3/1 expression was detected in 409 and 202 tumours, respectively, by immunohistochemistry. Cox proportional hazards model was used to compute mortality hazard ratio (HR), adjusting for clinical and tumoral features, including microsatellite instability, CpG island methylator phenotype, LINE-1 methylation, and KRAS, BRAF and PIK3CA mutations. RESULTS: Phosphorylated AMPK expression was not associated with survival among all patients. Notably, prognostic effect of p-AMPK significantly differed by p-MAPK3/1 status (P(interaction)=0.0017). Phosphorylated AMPK expression was associated with superior colorectal cancer-specific survival (adjusted HR 0.42; 95% confidence interval (CI), 0.24-0.74) among p-MAPK3/1-positive cases, but not among p-MAPK3/1-negative cases (adjusted HR 1.22; 95% CI: 0.85-1.75). CONCLUSION: Phosphorylated AMPK expression in colorectal cancer is associated with superior prognosis among p-MAPK3/1-positive cases, but not among p-MAPK3/1-negative cases, suggesting a possible interaction between the AMPK and MAPK pathways influencing tumour behaviour.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Neoplasias Colorretais/epidemiologia , Neoplasias Colorretais/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Idoso , Biomarcadores Tumorais/metabolismo , Classe I de Fosfatidilinositol 3-Quinases , Neoplasias Colorretais/mortalidade , Metilação de DNA , Feminino , Humanos , Masculino , Instabilidade de Microssatélites , Pessoa de Meia-Idade , Mutação , Proteínas do Tecido Nervoso/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Prognóstico , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
BACKGROUND: Circulating tumour cells (CTCs) offer a non-invasive approach to obtain and characterise metastatic tumour cells, but their usefulness has been limited by low CTC yields from conventional isolation methods. METHODS: To improve CTC yields and facilitate their molecular characterisation we compared the Food and Drug Administration-approved CellSearch Epithelial Kit (CEK) to a simplified CTC capture method, CellSearch Profile Kit (CPK), on paired blood samples from patients with metastatic breast (n=75) and lung (n=71) cancer. Molecular markers including Human Epidermal growth factor Receptor 2 (HER2) were evaluated on CTCs by fluorescence in situ hybridisation (FISH) and compared to patients' primary and metastatic cancer. RESULTS: The median cell count from patients with breast cancer using the CPK was 117 vs 4 for CEK (P<0.0001). Lung cancer samples were similar; CPK: 145 cells vs CEK:4 cells (P<0.0001). Recovered CTCs were relatively pure (60-70%) and were evaluable by FISH and immunofluorescence. A total of 10 of 30 (33%) breast cancer patients with HER2-negative primary and metastatic tissue had HER2-amplified CTCs. CONCLUSION: The CPK method provides a high yield of relatively pure CTCs, facilitating their molecular characterisation. Circulating tumour cells obtained using CPK technology demonstrate that significant discordance exists between HER2 amplification of a patient's CTCs and that of the primary and metastatic tumour.
Assuntos
Neoplasias da Mama/genética , Genes erbB-2/genética , Separação Imunomagnética/métodos , Células Neoplásicas Circulantes , Neoplasias da Mama/sangue , Feminino , Imunofluorescência , Amplificação de Genes , Humanos , Hibridização in Situ FluorescenteRESUMO
Micro-RNAs are a group of small noncoding RNAs approximately 22 nucleotides in length. Recent work has shown differential expression of mature micro-RNAs in human cancers. We characterized the alteration in expression of miR-29b in ovarian serous carcinoma. miR-29b expression was analyzed using quantitative stem-loop reverse transcriptase polymerase chain reaction on a set of 50 formalin-fixed, paraffin-embedded ovarian serous carcinoma samples. Protein expression of p53, estrogen receptor, progesterone receptor, human epidermal growth factor receptor 2, Ki-67, and insulinlike growth factor 1 was quantified in the corresponding tissue microarray. The expression profile of miR-29b was correlated with clinicopathological and patient survival data. We provide definitive evidence that miR-29b is down-regulated in a significant proportion of ovarian serous carcinomas and is associated with specific clinicopathological features, most notably high miR-29b expression being associated with reduced disease-free survival.
Assuntos
Cistadenocarcinoma Seroso/genética , MicroRNAs/biossíntese , Neoplasias Ovarianas/genética , Cistadenocarcinoma Seroso/metabolismo , Cistadenocarcinoma Seroso/patologia , Intervalo Livre de Doença , Feminino , Humanos , Imuno-Histoquímica , MicroRNAs/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BRAF((V600E)) mutation is the most frequent genetic alteration in papillary thyroid carcinomas (PTCs) that are 80-90% of all thyroid cancers. We evaluated the relationship between BRAF((V600E)) and tumor, host, and environmental factors in PTCs from all geographical areas of Sicily. By PCR, BRAF((V600E)) was investigated in a series of 323 PTCs diagnosed in 2002-2005. The correlation between clinicopathological tumor, host, and environmental characteristics and the presence of BRAF((V600E)) were evaluated by both univariate and multivariate analyses. BRAF((V600E)) was found in 38.6% PTCs, with a 52% frequency in the classical PTCs and 26.4% in the tall cell variant. Univariate analysis indicated that BRAF((V600E)) was associated with greater tumor size (P=0.0048), extra-thyroid invasion (P<0.0001), and cervical lymph nodal metastases (P=0.0001). Multivariate logistic regression analysis confirmed that BRAF((V600E)) was an independent predictor of extra-thyroid invasion (P=0.0001) and cervical lymph nodal metastasis (P=0.0005). The association between BRAF((V600E)) and extra-thyroid invasion was also found in micro-PTCs (P=0.006). In 60 classical PTCs, BRAF((V600E)) was positively correlated with matrix metalloproteinase-9 expression (P=0.0047), suggesting a possible mechanism for BRAF((V600E)) effect on PTC invasiveness. No association was found between BRAF((V600E)) and patient age, gender, or iodine intake. In contrast, a strong association was found with residency in Eastern Sicily (P<0.0001 compared with Western Sicily). These results indicate that BRAF((V600E)) mutation is a marker of aggressive disease in both micro- and macro-PTCs. Moreover, for the first time, a possible link between BRAF((V600E)) mutation and environmental carcinogens is suggested.
Assuntos
Carcinoma Papilar/genética , Mutação/genética , Proteínas Proto-Oncogênicas B-raf/genética , Neoplasias da Glândula Tireoide/genética , Primers do DNA , Progressão da Doença , Feminino , Seguimentos , Geografia , Humanos , Técnicas Imunoenzimáticas , Lasers , Metástase Linfática , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Microdissecção , Pessoa de Meia-Idade , Invasividade Neoplásica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sicília/epidemiologiaRESUMO
p21 (CDKN1A/CIP1/WAF1), one of the cyclin-dependent kinase inhibitors, plays a key role in regulating the cell cycle and is transcriptionally regulated by p53. Down-regulation of p21 is caused by TP53 mutations in colorectal cancer. CpG island methylator phenotype (CIMP) appears to be a distinct subtype of colorectal cancer with concordant methylation of multiple gene promoters and is associated with a high degree of microsatellite instability (MSI-H) and BRAF mutations. However, no study to date has evaluated the relationship between p21 expression and CIMP in colorectal cancer. The purpose of this study was to examine the inter-relationships between p21, p53, CIMP, MSI and KRAS/BRAF status in colorectal cancer. We utilized 737 relatively unbiased samples of colorectal cancers from two large prospective cohort studies. Using quantitative real-time PCR (MethyLight), we measured DNA methylation in five CIMP-specific gene promoters [CACNA1G, CDKN2A (p16/INK4A), CRABP1, MLH1 and NEUROG1]. CIMP-high (>or=4/5 methylated promoters) was diagnosed in 118 (16%) of the 737 tumours. We also assessed expression of p21 and p53 by immunohistochemistry. Among the 737 tumours, 371 (50%) showed p21 loss. Both p21 loss and p53 positivity were inversely associated with CIMP-high, MSI-H and BRAF mutations. The associations of p21 with these molecular features were still present after tumours were stratified by p53 status. In contrast, the associations of p53 positivity with the molecular features were no longer present after tumours were stratified by p21 status. When CIMP-high and non-CIMP-high tumours were stratified by MSI or KRAS/BRAF status, CIMP-high and MSI-H (but not BRAF mutations) were still inversely associated with p21 loss. In conclusion, down-regulation of p21 is inversely correlated with CIMP-high and MSI-H in colorectal cancer, independent of TP53 and BRAF status.
Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Ilhas de CpG/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Instabilidade de Microssatélites , Metilação de DNA , Bases de Dados como Assunto , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Mutação , Reação em Cadeia da Polimerase/métodos , Estudos Prospectivos , Análise Serial de Proteínas/métodos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras) , Proteína Supressora de Tumor p53/metabolismo , Proteínas ras/genéticaRESUMO
Fatty acid synthase (FASN), a key enzyme for de novo lipogenesis, is overexpressed in many malignant tumours and is associated with aggressive biological behaviour. FASN expression and its possible relationship with more aggressive behaviour in gastrointestinal stromal tumours (GISTs) have not been addressed to date. Here, FASN expression was assessed by immunohistochemistry in 60 primary GISTs (28 low/intermediate risk and 32 high risk) and seven metastatic GISTs. Sixteen smooth muscle gastrointestinal tumours were used as controls. FASN was overexpressed in 36 of 60 GISTs (60%): in 12 of 28 (42%) low/intermediate-risk GISTs and in 24 of 32 (75%) high-risk GISTs (p<0.05). Two primary and seven metastatic GISTs and five GIST cell lines (GIST882, GIST430, GIST522, GIST62, and GIST48), analysed by western blot, showed variable FASN expression. Most metastatic samples expressed high levels of FASN protein. Additionally, seven of 60 GISTs showed a proliferation rate higher than 10% by Ki67 and all of them expressed FASN (p<0.04). Finally, proliferation and apoptosis were investigated after FASN silencing in GIST882 cells, which displayed the highest FASN expression. siRNA-mediated FASN knock-down inhibited expression of the proliferation marker cyclin A, whereas no changes in p27 and cleaved PARP expression were seen. It is concluded that FASN is preferentially overexpressed in high-risk and metastatic GISTs, and that its overexpression likely contributes to cell proliferation.
Assuntos
Biomarcadores Tumorais/metabolismo , Ácido Graxo Sintases/metabolismo , Tumores do Estroma Gastrointestinal/enzimologia , Biomarcadores Tumorais/genética , Western Blotting/métodos , Proliferação de Células , Ácido Graxo Sintases/genética , Tumores do Estroma Gastrointestinal/patologia , Tumores do Estroma Gastrointestinal/secundário , Inativação Gênica , Humanos , RNA Neoplásico/genética , RNA Interferente Pequeno/genética , Células Tumorais CultivadasRESUMO
BACKGROUND: The concept of CpG island methylator phenotype (CIMP) is not universally accepted. Even if specific clinicopathological features have been associated with CIMP, investigators often failed to demonstrate a bimodal distribution of the number of methylated markers, which would suggest CIMP as a distinct subtype of colorectal cancer. Previous studies primarily used methylation specific polymerase chain reaction which might detect biologically insignificant low levels of methylation. AIM: To demonstrate a distinct genetic profile of CIMP colorectal cancer using quantitative DNA methylation analysis that can distinguish high from low levels of DNA methylation. MATERIALS AND METHODS: We developed quantitative real time polymerase chain reaction (MethyLight) assays and measured DNA methylation (percentage of methylated reference) of five carefully selected loci (promoters of CACNA1G, CDKN2A (p16), CRABP1, MLH1, and NEUROG1) in 460 colorectal cancers from large prospective cohorts. RESULTS: There was a clear bimodal distribution of 80 microsatellite instability-high (MSI-H) tumours according to the number of methylated promoters, with no tumours showing 3/5 methylated loci. Thus we defined CIMP as having >or=4/5 methylated loci, and 17% (78) of the 460 tumours were classified as CIMP. CIMP was significantly associated with female sex, MSI, BRAF mutations, and wild-type KRAS. Both CIMP MSI-H tumours and CIMP microsatellite stable (MSS) tumours showed much higher frequencies of BRAF mutations (63% and 54%) than non-CIMP counterparts (non-CIMP MSI-H (0%, p<10(-5)) and non-CIMP MSS tumours (6.6%, p<10(-4)), respectively). CONCLUSION: CIMP is best characterised by quantitative DNA methylation analysis. CIMP is a distinct epigenotype of colorectal cancer and may be less frequent than previously reported.
Assuntos
Neoplasias Colorretais/genética , Ilhas de CpG , Metilação de DNA , Marcadores Genéticos , Predisposição Genética para Doença , Inquéritos Epidemiológicos , Humanos , Repetições de Microssatélites , Estudos Prospectivos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras) , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas rasRESUMO
The rising prevalence of Barrett's esophagus and Barrett's associated adenocarcinoma in the Western world has stimulated increasing interest in this disease. This has resulted in a plethora of articles concerning its molecular biology, but the tumor suppressor gene, p27, has received little attention. In this article, we review the literature concerning the role of p27 in Barrett's esophagus and its malignant transformation, and we evaluate its possible role as an important clinical biomarker, as well as potential chemopreventive clinical agents aimed at substituting its antitumoral activity.
Assuntos
Adenocarcinoma/genética , Esôfago de Barrett/genética , Proteínas de Ciclo Celular/genética , Neoplasias Esofágicas/genética , Genes Supressores de Tumor/fisiologia , Proteínas Supressoras de Tumor/genética , Adenocarcinoma/patologia , Adenocarcinoma/terapia , Animais , Esôfago de Barrett/patologia , Esôfago de Barrett/terapia , Biomarcadores Tumorais , Proteínas de Ciclo Celular/fisiologia , Transformação Celular Neoplásica/genética , Inibidor de Quinase Dependente de Ciclina p27 , Ciclo-Oxigenase 2 , Modelos Animais de Doenças , Neoplasias Esofágicas/patologia , Neoplasias Esofágicas/terapia , Esôfago/enzimologia , Esôfago/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas de Membrana , Camundongos , Mucosa/patologia , Mutação , Prostaglandina-Endoperóxido Sintases/metabolismo , Ratos , Proteínas Supressoras de Tumor/fisiologiaRESUMO
Lung metastases from colorectal carcinomas (CRC) can be resected with improved survival. The distinction between primary lung adenocarcinomas and metastases from CRC may sometimes be difficult, especially on cytologic specimens or small bronchoscopic biopsies. Immunohistochemistry may be of help in this setting: available markers include TTF-1 and SP-A, which are markers of lung origin, whereas there are no good markers of intestinal origin, besides cytokeratin 7 and 20 coexpression pattern, which is not very specific. The nuclear CDX-2 transcription factor, which is the product of a homeobox gene necessary for intestinal organogenesis, is expressed in normal colonic epithelia and most colorectal adenocarcinomas, and could potentially be of diagnostic usefulness. Our aim was to investigate CDX-2 immunohistochemical expression using a new monoclonal antibody and to verify if CDX-2 can be a reliable marker to identify the colorectal origin of lung metastases. CDX-2 expression was evaluated in formalin-fixed, paraffin-embedded samples of normal adult human tissues (50 samples) and in 299 surgically resected carcinomas of different origins, including 125 non-lung adenocarcinomas, 117 primary lung tumors, 5 mesotheliomas, and 52 adenocarcinomas metastatic to the lung. CDX-2 was also evaluated on a series of 20 bioptic and 10 cytologic specimens (5 cases of colorectal metastases to the lung, 5 cases of metastases from other organs, and 10 primary lung adenocarcinomas). In normal tissues CDX-2 immunoreactivity was observed only in ileal and colorectal epithelia. CDX-2 was expressed in almost all primary and metastatic CRC (88 of 90) and was never observed in primary lung tumors. CDX-2 was also expressed in a limited group of adenocarcinomas of other sites (gastric, biliopancreatic, and mucinous ovarian adenocarcinomas). CDX-2 could be easily detected in all bioptic and cytologic samples of CRC metastases. CDX-2 is a reliable, specific, and sensitive immunohistochemical marker of normal and neoplastic intestinal epithelium. CDX-2 can be easily applied to routine histologic and cytologic material and is therefore a useful marker in the differential diagnosis of primary versus metastatic adenocarcinomas in the lung, and among metastases from an unknown primary, supports intestinal origin.
Assuntos
Adenocarcinoma/secundário , Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/patologia , Genes Homeobox , Proteínas de Homeodomínio/metabolismo , Neoplasias Pulmonares/secundário , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adulto , Biomarcadores Tumorais/genética , Fator de Transcrição CDX2 , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Feminino , Expressão Gênica , Proteínas de Homeodomínio/genética , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , TransativadoresRESUMO
The optimal treatment of patients with cancer depends on establishing accurate diagnoses by using a complex combination of clinical and histopathological data. In some instances, this task is difficult or impossible because of atypical clinical presentation or histopathology. To determine whether the diagnosis of multiple common adult malignancies could be achieved purely by molecular classification, we subjected 218 tumor samples, spanning 14 common tumor types, and 90 normal tissue samples to oligonucleotide microarray gene expression analysis. The expression levels of 16,063 genes and expressed sequence tags were used to evaluate the accuracy of a multiclass classifier based on a support vector machine algorithm. Overall classification accuracy was 78%, far exceeding the accuracy of random classification (9%). Poorly differentiated cancers resulted in low-confidence predictions and could not be accurately classified according to their tissue of origin, indicating that they are molecularly distinct entities with dramatically different gene expression patterns compared with their well differentiated counterparts. Taken together, these results demonstrate the feasibility of accurate, multiclass molecular cancer classification and suggest a strategy for future clinical implementation of molecular cancer diagnostics.
Assuntos
Perfilação da Expressão Gênica , Neoplasias/classificação , Neoplasias/diagnóstico , Biomarcadores Tumorais , Análise por Conglomerados , Humanos , Família Multigênica , Neoplasias/genéticaRESUMO
Most large bowel cancers are moderately to well-differentiated adenocarcinomas comprised chiefly or entirely of glands lined by tall columnar cells. We have identified a subset of poorly differentiated colon carcinomas with a distinctive histopathological appearance that we term large cell minimally differentiated carcinomas (LCMDCs). These tumors likely include a group of poorly differentiated carcinomas previously described by others as medullary adenocarcinomas. To better understand the pathogenesis of these uncommon neoplasms, we compared molecular features of 15 LCMDCs to those present in 25 differentiated adenocarcinomas (DACs) of the colon. Tumors were examined for alterations commonly seen in typical colorectal carcinomas, including increased p53 and beta-catenin immunoreactivity, K-ras gene mutations, microsatellite instability, and loss of heterozygosity of markers on chromosomes 5q, 17p, and 18q. In addition, tumors were evaluated by immunohistochemistry for CDX2, a homeobox protein whose expression in normal adult tissues is restricted to intestinal and colonic epithelium. Markedly reduced or absent CDX2 expression was noted in 13 of 15 (87%) LCMDCs, whereas only 1 of the 25 (4%) DACs showed reduced CDX2 expression (P < 0.001). Nine of 15 (60%) LCMDCs had the high-frequency microsatellite instability phenotype, but only 2 of 25 (8%) DACs had the high-frequency microsatellite instability phenotype (P = 0.002). Our findings provide support for the hypothesis that the molecular pathogenesis of LCMDCs is distinct from that of most DACs. CDX2 alterations and DNA mismatch repair defects have particularly prominent roles in the development of LCMDCs.
Assuntos
Carcinoma de Células Grandes/patologia , Neoplasias do Colo/patologia , Proteínas de Ligação a DNA , Proteínas de Homeodomínio/biossíntese , Repetições de Microssatélites/genética , Transativadores , Proteínas Adaptadoras de Transdução de Sinal , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Fator de Transcrição CDX2 , Carcinoma de Células Grandes/genética , Carcinoma de Células Grandes/metabolismo , Proteínas de Transporte , Cromossomos Humanos Par 17/genética , Cromossomos Humanos Par 18/genética , Cromossomos Humanos Par 5/genética , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Proteínas do Citoesqueleto/análise , Feminino , Genes ras/genética , Humanos , Imuno-Histoquímica , Perda de Heterozigosidade , Masculino , Pessoa de Meia-Idade , Proteína 1 Homóloga a MutL , Proteína 2 Homóloga a MutS , Mutação , Proteínas de Neoplasias/análise , Proteínas Nucleares , Proteínas Proto-Oncogênicas/análise , Proteína Supressora de Tumor p53/análise , beta CateninaRESUMO
We have generated a molecular taxonomy of lung carcinoma, the leading cause of cancer death in the United States and worldwide. Using oligonucleotide microarrays, we analyzed mRNA expression levels corresponding to 12,600 transcript sequences in 186 lung tumor samples, including 139 adenocarcinomas resected from the lung. Hierarchical and probabilistic clustering of expression data defined distinct subclasses of lung adenocarcinoma. Among these were tumors with high relative expression of neuroendocrine genes and of type II pneumocyte genes, respectively. Retrospective analysis revealed a less favorable outcome for the adenocarcinomas with neuroendocrine gene expression. The diagnostic potential of expression profiling is emphasized by its ability to discriminate primary lung adenocarcinomas from metastases of extra-pulmonary origin. These results suggest that integration of expression profile data with clinical parameters could aid in diagnosis of lung cancer patients.
Assuntos
Adenocarcinoma/classificação , Expressão Gênica , Neoplasias Pulmonares/classificação , RNA Mensageiro , RNA Neoplásico , Adenocarcinoma/genética , Adenocarcinoma/patologia , Carcinoma de Células Pequenas/classificação , Carcinoma de Células Pequenas/genética , Carcinoma de Células Escamosas/classificação , Carcinoma de Células Escamosas/genética , Progressão da Doença , Perfilação da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Metástase Neoplásica , RNA Mensageiro/análise , RNA Neoplásico/análise , Estudos Retrospectivos , Fumar/efeitos adversos , Taxa de Sobrevida , Fatores de TempoRESUMO
OBJECTIVE: In a previous study, we showed that experimentally induced gastroduodenal-esophageal reflux in mice treated with a carcinogen can result in Barrett esophagus and Barrett-associated adenocarcinoma. Since we have shown that most Barrett-associated adenocarcinomas in human beings have lost the tumor suppressor gene p27, we sought to determine whether cancer would be more likely to develop in p27 knockout mice than in p27 heterozygous or p27 wild type mice. METHODS: Three groups of mice were treated by esophagojejunostomy resulting in gastroduodenal-esophageal reflux and by a carcinogen (N -methyl-N -benzylnitrosamine): group I (50 wild type), group II (45 p27 heterozygous), and group III (50 p27 knockout). The mice were killed 18 to 20 weeks after operation and studied macroscopically and histopathologically. RESULTS: Barrett esophagus developed in 7 (14%) mice in group I, 4 (8.9%) mice in group II, and 13 (26%) mice in group III. Cancers developed in 30 (60%) mice in group I, 31 (68%) mice in group II, and 43 (86%) mice in group III. Ten percent of the cancers in group I were adenocarcinomas, as were 16.1% in group II, and 23.3% in group III. The difference between rates of Barrett esophagus in groups I and II compared with group III was statistically significant (P =.035), as was true of the cancer rates (P =.006). The percentage of cancers that were adenocarcinomas was highest in group III, but not significantly different from groups I and II. CONCLUSIONS: This experimental mouse model of Barrett esophagus and Barrett- associated adenocarcinoma is similar to what occurs in human beings and may be useful in developing methods to inhibit malignant transformation of Barrett esophagus.
Assuntos
Proteínas de Ciclo Celular/genética , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Esôfago/patologia , Genes Supressores de Tumor , Proteínas Supressoras de Tumor/genética , Animais , Inibidor de Quinase Dependente de Ciclina p27 , Camundongos , Camundongos Knockout , Mucosa/patologiaRESUMO
The ubiquitin pathway is involved in the proteolytic turnover of many short-lived cellular regulatory proteins. Since selective degradation of substrates of this system requires the covalent attachment of a polyubiquitin chain to the substrates, degradation could be counteracted by de-ubiquitinating enzymes (or isopeptidases) which selectively remove the polyubiquitin chain. Unp is a human isopeptidase with still poorly understood biological functions. Here, we show that cellular Unp specifically interacts with the retinoblastoma gene product (pRb).
Assuntos
Proteínas Oncogênicas/metabolismo , Proteína do Retinoblastoma/metabolismo , Motivos de Aminoácidos , Anticorpos/imunologia , Ciclo Celular , Linhagem Celular , Humanos , Células Jurkat , Proteínas Oncogênicas/química , Proteínas Oncogênicas/imunologia , Células Tumorais Cultivadas , Ubiquitina Tiolesterase , Proteases Específicas de Ubiquitina , Ubiquitinas/metabolismoRESUMO
We used our palindromic polymerase chain reaction (PCR)-driven cDNA differential display technique to identify and isolate a gene, designated periostin, from cancer tissues and found it to be overexpressed in several human tumors. We attempted to determine the influence of periostin expression on clinical outcome in patients with non-small cell lung cancer (NSCLC) by reverse transcriptase (RT)-PCR analysis. Periostin gene was highly expressed at the tumor periphery of lung cancer tissue but not within the tumor by in situ RNA hybridization, suggesting that expression of periostin may be involved in the process of tumor invasion. Periostin transcripts were detected in 50 (49.0%) of the tumor samples, although some paired normal lung samples showed weak expression. There was no relationship between periostin gene expression and gender, N- or T-status. The NSCLC patients with periostin expression had significantly poorer survival than the patients without periostin expression (P = 0.0338).