RESUMO
BACKGROUND: Patients with ulcerative colitis (UC) are at risk for colorectal neoplasia. Challenges associated with surveillance colonoscopy with random biopsies for detection of dysplasia/cancer are well-documented. This study extended our findings in UC-associated colorectal cancer to include low-grade dysplasia (LGD) patients, testing whether our biomarker panel detects any UC-associated neoplasm. METHODS: DNA from the LGD area and the corresponding nonadjacent, non-dysplastic section from 171 UC-LGD patients was extracted. TaqMan SNP Genotyping Assays for TNF-α, IL-1ß, and IL23R were used to evaluate polymorphisms for each gene. Bisulfite-treated DNA was used for methylation testing of RUNX3, COX2, and MINT1. LGD data were combined with UC-cancer patient data for statistical testing. Logistic regression analyses determined associations between genetic/epigenetic/clinical variables and UC-associated neoplasia. Receiver operating characteristic analyses were performed to determine the final synchronous neoplasm detection panel. RESULTS: Comparison of nonadjacent, non-dysplastic DNA from UC-neoplasm patients versus UC-controls indicated that TNF-α, IL-1ß, and methylation of RUNX3, MINT1, and COX2 were significantly different (P < 0.0001). In multivariable analysis, all remained significant with an area under the curve of 0.85, exceeding the clinical variable panel area under the curve. Combining clinical and experimental variables yielded a neoplasm biomarker panel with an area under the curve of 0.95 (sensitivity and specificity of 82% and 91%, respectively). Analysis of DNA from LGD with known progression compared with LGD without progression indicated a significant difference in RUNX3 methylation. CONCLUSIONS: A combined clinical, genetic, and epigenetic model for detecting synchronous neoplasm by testing of non-neoplastic colonic tissue had favorable operating characteristics and could complement current patient care.
Assuntos
Colite Ulcerativa/patologia , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , DNA/análise , Mucosa Intestinal/patologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Área Sob a Curva , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Biópsia , Estudos de Casos e Controles , Colite Ulcerativa/complicações , Colonoscopia , Neoplasias Colorretais/patologia , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Ciclo-Oxigenase 2/genética , Metilação de DNA , Feminino , Genótipo , Humanos , Interleucina-1beta/genética , Mucosa Intestinal/química , Masculino , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/genética , Polimorfismo de Nucleotídeo Único , Curva ROC , Receptores de Interleucina/genética , Fator de Necrose Tumoral alfa/genéticaRESUMO
BACKGROUND: Aberrant methylation of the EYA4 gene (mEYA4) highly discriminates ulcerative colitis (UC) cases with colorectal neoplasia from UC controls in both tissue and stool. It is not known if mEYA4 is also present in nonadjacent non-neoplastic mucosa (NNM) of UC patients with colorectal neoplasia. METHODS: Formalin-fixed tissues from 25 UC cases with colorectal cancer (CRC) and 25 UC controls with neither CRC nor dysplasia were matched on gender, age, disease duration, disease extent, and coexistence of primary sclerosing cholangitis. DNA was extracted from sections of CRC and NNM from cases and UC control mucosae. Bisulfite-treated DNA was amplified using real-time methylation-specific PCR. The Wilcoxon rank-sum test assessed differences in mEYA4 levels from CRC, NNM, and control samples. Logistic regression was used to estimate sensitivity and specificity. RESULTS: Sufficient DNA was available for 20 cases and 20 controls. The median mEYA4 level (with interquartile range) was 2 (0-5.7) in control mucosae but 93 (38.5-306) in CRC (P < 0.0001) and 27.4 (3-140) in NNM (P = 0.0009). At 95% specificity, mEYA4 was present in 80% of CRC and 50% of NNM tissue samples. The odds ratio of mEYA4 in NNM as an indicator of synchronous CRC was 19 (95% confidence interval, 2-170). CONCLUSIONS: The authors demonstrate significantly higher mEYA4 levels in NNM and synchronous CRC from UC cases than in colorectal mucosae of UC controls without neoplasia. The finding of this CRC-associated field change has important implications to the use of mEYA4 as a potential UC surveillance marker in tissue or stool.
Assuntos
Biomarcadores Tumorais/genética , Colite Ulcerativa/genética , Colo/patologia , Neoplasias Colorretais/etiologia , Metilação de DNA , Regiões Promotoras Genéticas/genética , Transativadores/genética , Estudos de Casos e Controles , Colite Ulcerativa/complicações , Colite Ulcerativa/patologia , Colo/metabolismo , Neoplasias Colorretais/patologia , DNA/genética , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Lesões Pré-Cancerosas/etiologia , Lesões Pré-Cancerosas/patologia , Prognóstico , Curva ROCRESUMO
UNLABELLED: Heparan sulfate proteoglycans (HSPGs) act as coreceptors or storage sites for growth factors and cytokines such as fibroblast growth factor and Wnts. Glypican 3 (GPC3) is the most highly expressed HSPG in hepatocellular carcinoma (HCC). Sulfatase 2 (SULF2), an enzyme with 6-O-desulfatase activity on HSPGs, is up-regulated in 60% of primary HCCs and is associated with a worse prognosis. We have previously shown that the oncogenic effect of SULF2 in HCC may be mediated in part through up-regulation of GPC3. Here we demonstrate that GPC3 stimulates the Wnt/ß-catenin pathway and mediates the oncogenic function of SULF2 in HCC. Wnt signaling in vitro and in vivo was assessed in SULF2-negative Hep3B HCC cells transfected with SULF2 and in SULF2-expressing Huh7 cells transfected with short hairpin RNA targeting SULF2. The interaction between GPC3, SULF2, and Wnt3a was assessed by coimmunoprecipitation and flow cytometry. ß-catenin-dependent transcriptional activity was assessed with the TOPFLASH (T cell factor reporter plasmid) luciferase assay. In HCC cells, SULF2 increased cell surface GPC3 and Wnt3a expression, stabilized ß-catenin, and activated T cell factor transcription factor activity and expression of the Wnt/ß-catenin target gene cyclin D1. Opposite effects were observed in SULF2-knockdown models. In vivo, nude mouse xenografts established from SULF2-transfected Hep3B cells showed enhanced GPC3, Wnt3a, and ß-catenin levels. CONCLUSION: Together, these findings identify a novel mechanism mediating the oncogenic function of SULF2 in HCC that includes GPC3-mediated activation of Wnt signaling via the Wnt3a/glycogen synthase kinase 3 beta axis.
Assuntos
Carcinoma Hepatocelular/enzimologia , Neoplasias Hepáticas/enzimologia , Sulfotransferases/sangue , Animais , Carcinoma Hepatocelular/sangue , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Caspase 3/metabolismo , Linhagem Celular Tumoral , Ativação Enzimática , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica , Genes Reporter , Glipicanas/sangue , Glipicanas/genética , Humanos , Antígeno Ki-67/genética , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Luciferases/genética , Camundongos , Camundongos Nus , Plasmídeos/genética , Sulfatases , Transfecção , Proteínas Wnt/genética , Proteína Wnt3 , Proteína Wnt3ARESUMO
Inflammatory bowel disease is characterized by chronic inflammation which predisposes to colorectal cancer. The mechanisms by which inflammation promotes tumorigenesis are not fully known. We aimed to investigate the links between colonic inflammation and tumorigenesis via epigenetic gene silencing. Colon cancer specimens were assessed for the expression of DNA methyltransferase-1 (DNMT-1) using immunohistochemistry. Colorectal carcinoma cell lines were assessed for DNMT1 expression, methylcytosine content, promoter methylation, gene expression, and tumorigenesis in response to interleukin (IL)-6. DNMT1 was expressed at higher levels in both the peritumoral stroma and tumor in inflammatory bowel disease-associated cancers compared with sporadic colon cancers. IL-6 treatment of colon cancer cells resulted in an increase in DNMT1 expression, independent of de novo gene expression. IL-6 increased the methylation of promoter regions of genes associated with tumor suppression, adhesion, and apoptosis resistance. Expression of a subset of these genes was downregulated by IL-6, an effect that was prevented by preincubation with 5-azadeoxycytidine, a DNMT1 inhibitor. Anchorage-independent growth and migration of colon cancer cells was also increased by IL-6 in a 5-azadeoxycytidine-sensitive manner. Our results indicate that DNMT-mediated gene silencing may play a role in inflammation-associated colon tumorigenesis.
Assuntos
Carcinoma/genética , Colite/genética , Neoplasias do Colo/genética , DNA (Citosina-5-)-Metiltransferases/genética , Inativação Gênica/fisiologia , Interleucina-6/metabolismo , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Testes de Carcinogenicidade , Carcinoma/enzimologia , Carcinoma/imunologia , Linhagem Celular Tumoral , Movimento Celular/genética , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/imunologia , Transformação Celular Neoplásica/metabolismo , Colite/enzimologia , Colite/imunologia , Neoplasias do Colo/enzimologia , Neoplasias do Colo/imunologia , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/genética , Decitabina , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Inibidores Enzimáticos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Genes Supressores de Tumor/efeitos dos fármacos , Genes Supressores de Tumor/fisiologia , Humanos , Imuno-Histoquímica , Mediadores da Inflamação/metabolismo , Mediadores da Inflamação/farmacologia , Interleucina-6/farmacologia , Metástase Neoplásica/genética , Regiões Promotoras Genéticas/genética , Regulação para Cima/fisiologiaRESUMO
OBJECTIVES: Patients with ulcerative colitis (UC) are at increased risk for developing colorectal cancer (CRC). Surveillance in this at-risk population remains challenging. We assessed the methylation status of genes in the non-neoplastic mucosa of UC-CRC patients and controls to identify potential biomarkers of CRC. METHODS: We evaluated the methylation status of 10 genes (p16, p14, runt-related transcript factor-3 (RUNX3), cyclooxygenase-2 (COX-2), E-cadherin, methylated-in-tumor-1 (MINT1), MINT31, HPP1, estrogen receptor, SLC5A8) in UC-CRC tumors and non-neoplastic sections from both UC-CRC cases and UC controls (n=114 for each) using methylation-specific PCR. RESULTS: Amplification was successful for 96 UC controls, 83 tumors, and 66 non-adjacent, non-neoplastic samples. The prevalence of methylation was significantly greater in UC-CRC tumors for p16, RUNX3, MINT1, MINT31, and HPP1. Methylation of COX-2 and E-cadherin was greater in UC controls than in tumors. Univariate testing of these genes using non-adjacent, non-neoplastic sections from UC-CRC cases indicated that associations between p16, RUNX3, MINT1, MINT31, E-cadherin, and COX-2 and UC-CRC remained significant. In multivariable analysis of the six genes, only RUNX3, MINT1, and COX-2 remained significantly associated with the UC-CRC cases (odds ratio=12.6, 9.0, and 0.2, respectively). The results remained unaffected by the presence of PSC or severity of inflammation. Logistic regression modeling with the three genes showed interactions that increased the odds ratio for each gene. CONCLUSIONS: RUNX3, MINT1, and COX-2 are potential biomarkers for detecting the presence of CRC in patients with UC. These genes should be evaluated as biomarkers for colorectal dysplasia.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Colite Ulcerativa/genética , Neoplasias Colorretais/genética , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Ciclo-Oxigenase 2/genética , Proteínas do Tecido Nervoso/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Estudos de Casos e Controles , Distribuição de Qui-Quadrado , Colite Ulcerativa/complicações , Colite Ulcerativa/patologia , Neoplasias Colorretais/complicações , Neoplasias Colorretais/patologia , Progressão da Doença , Feminino , Predisposição Genética para Doença , Humanos , Mucosa Intestinal/patologia , Modelos Logísticos , Masculino , Metilação , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/patologia , Fatores de RiscoRESUMO
UNLABELLED: It has been shown that the heparin-degrading endosulfatase, sulfatase 1 (SULF1), functions as a liver tumor suppressor, but the role of the related sulfatase, sulfatase 2 (SULF2), in liver carcinogenesis remains to be elucidated. We investigated the effect of SULF2 on liver tumorigenesis. Expression of SULF2 was increased in 79 (57%) of 139 hepatocellular carcinomas (HCCs) and 8 (73%) of 11 HCC cell lines. Forced expression of SULF2 increased HCC cell growth and migration, whereas knockdown of SULF2 using short hairpin RNA targeting SULF2 abrogated HCC cell proliferation and migration in vitro. Because SULF1 and SULF2 desulfate heparan sulfate proteoglycans (HSPGs) and the HSPG glypican 3 (GPC3) is up-regulated in HCC, we investigated the effects of SULF2 on GPC3 expression and the association of SULF2 with GPC3. SULF2-mediated cell growth was associated with increased binding of fibroblast growth factor 2 (FGF2), phosphorylation of extracellular signal-regulated kinase and AKT, and expression of GPC3. Knockdown of GPC3 attenuated FGF2 binding in SULF2-expressing HCC cells. The effects of SULF2 on up-regulation of GPC3 and tumor growth were confirmed in nude mouse xenografts. Moreover, HCC patients with increased SULF2 expression in resected HCC tissues had a worse prognosis and a higher rate of recurrence after surgery. CONCLUSION: In contrast to the tumor suppressor effect of SULF1, SULF2 has an oncogenic effect in HCC mediated in part through up-regulation of FGF signaling and GPC3 expression.
Assuntos
Carcinoma Hepatocelular/enzimologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Glipicanas/metabolismo , Neoplasias Hepáticas/enzimologia , Sulfotransferases/metabolismo , Animais , Carcinoma Hepatocelular/diagnóstico , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células , Humanos , Neoplasias Hepáticas/diagnóstico , Camundongos , Camundongos Nus , Prognóstico , Transdução de Sinais/fisiologia , Sulfatases , Regulação para CimaRESUMO
OBJECTIVES: Ulcerative colitis (UC) is characterized by chronic recurrent mucosal inflammation. Genetic studies in UC have indicated linkage to chromosome 6 in the region of the tumor necrosis factor-alpha (TNF-alpha) gene, a potent proinflammatory cytokine. TNF-alpha production is influenced by multiple factors including the type of immune cell and its level of activation. However, several single nucleotide polymorphisms (SNP) in the promoter region of TNF-alpha have been correlated with either increased or decreased production, indicating that regulation of TNF-alpha is in part genetic. Because UC patients are at increased risk for developing colorectal cancer (CRC), we investigated if there was an association between SNPs in the promoter of the TNF-alpha gene and UC-CRC. METHODS: DNA was extracted from formalin-fixed, paraffin-embedded tissue from 114 UC-CRC cases and 114 UC-no CRC controls. Controls were frequency matched on duration and extent of colitis, age, ethnicity, and gender. All 228 tissue samples were analyzed for five TNF-alpha promoter polymorphisms (-238[G-->A], -308[G-->A], -857[C-->T], -863[C-->A], and -1031[T-->C]) using PCR and sequencing. RESULTS: The -308(G-->A) SNP was significantly associated with UC-CRC cases at both the allele and genotype level (P < 0.0001). No other SNPs were significantly associated with UC-CRC. CONCLUSION: We report a novel finding of a strong association between the -308(G-->A) SNP and UC-CRC. Complete elucidation of the mechanism of UC-CRC carcinogenesis will require investigation of other genes involved in modulating inflammation, but our results suggest that some UC patients may have additional genetic predispositions toward developing UC-CRC.
Assuntos
Colite Ulcerativa/complicações , Colite Ulcerativa/genética , Neoplasias Colorretais/complicações , Polimorfismo Genético , Fator de Necrose Tumoral alfa/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Colon cancers with microsatellite instability (MSI) demonstrate a host immune response characterized by tumor infiltrating lymphocytes (TILs) that may exert effects upon tumor cell apoptosis and cell proliferation. Accordingly, we compared rates of apoptosis and cell proliferation in colon cancers with defective DNA mismatch repair and their association with phenotypic features and clinical outcome. Primary Astler-Coller stage B2 and C colon carcinomas (n = 329) were analyzed for MSI and for hMLH1 and hMSH2 protein expression. Apoptosis (TUNEL assay) and p53 expression were also analyzed by immunohistochemistry, and TILs were quantified by morphology. DNA ploidy and proliferation (PI: S phase + G(2)M) were evaluated using flow cytometry. MSI-H (n = 58) colon cancers showed increased TILs that were significantly associated with increased apoptosis, higher apoptosis to proliferation (AI/PI) ratios, reduced proliferative indices (PI) and diploid DNA content. Increased TILs (p = 0.036) and reduced PI (p = 0.042), but not AI or AI/PI, were associated with improved disease-free survival. Tumors with MSI-H (p = 0.032) or loss of hMLH1 or hMSH2 proteins (p = 0.040), or diploidy (p = 0.0015), had better adjusted overall survival rates. Interestingly, similar rates of cell turnover and overlapping survival rates were found in diploid MSS/MSI-L tumors and in MSI-H cases. In conclusion, higher apoptosis/proliferation ratios and reduced cell proliferation are phenotypic features of MSI-H tumors that are associated with increased TILs, indicating an activated immune response that may contribute to their favorable survival rates.
Assuntos
Apoptose/genética , Carcinoma/patologia , Proliferação de Células , Neoplasias do Colo/patologia , Linfócitos do Interstício Tumoral/imunologia , Instabilidade de Microssatélites , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma/genética , Carcinoma/imunologia , Neoplasias do Colo/genética , Neoplasias do Colo/imunologia , Reparo de Erro de Pareamento de DNA , Feminino , Humanos , Masculino , Repetições de Microssatélites , Pessoa de Meia-Idade , Ploidias , Proteína Supressora de Tumor p53/metabolismoRESUMO
BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is the third most common cause of cancer death worldwide. Improved treatments for advanced HCC are urgently needed. The recently identified human sulfatase 1 enzyme (SULF1) desulfates cell surface heparan sulfate glycosaminoglycans and down-regulates cell growth signaling in HCC cells in vitro. While investigating the epigenetic regulation of SULF1, we discovered that histone H4 acetylation is up-regulated by SULF1 in HCC cells. Histone deacetylase (HDAC) inhibitors reprogram cellular gene expression through the acetylation of nucleosomal histones and promote cell growth arrest and apoptosis. Hence, they are a promising modality for cancer treatment. METHODS: To explore the interaction between SULF1 expression and HDAC inhibitor action, we examined the effects of SULF1 expression on HCC cells and xenografts treated with HDAC inhibitors. RESULTS: (1) Forced expression of SULF1 significantly delayed the growth of Huh7 and Hep3B xenografts in nude mice in vivo. (2) SULF1 increased histone H4 acetylation by modulation of cellular HDAC and histone acetyltransferase activities. (3) SULF1 enhanced the induction of apoptosis by the HDAC inhibitors apicidin and scriptaid. (4) SULF1 enhanced the inhibition of tumor growth, migration, and angiogenesis by HDAC inhibitors. We also demonstrate that knockdown of SULF1 with shRNA constructs up-regulates phosphorylation of AKT and Erk and attenuates apicidin-induced apoptosis. The interaction between SULF1 and apicidin was confirmed in vivo in Huh7 and Hep3B xenografts. CONCLUSIONS: These results show that SULF1 promotes histone H4 acetylation, potentiates the effects of HDAC inhibitors, and inhibits HCC tumorigenesis.