COX-2 (PTGS2) gene methylation in epithelial, subepithelial lymphocyte and stromal tissue compartments in a spectrum of esophageal squamous neoplasia

Background: Previous studies have shown important effects of stromal elements in carcinogenesis. To explore the tumor–stromal relationship in esophageal neoplasia, we examined methylation of COX-2 (PTGS2), a gene etiologically associated with the development of gastrointestinal cancers, in adjacent foci of epithelium, subepithelial lymphocytes and non-lymphocytic stromal cells found in sections of normal squamous epithelium, squamous dysplasia and invasive esophageal squamous cell carcinoma. Methods: Adjacent foci of epithelium, subepithelial lymphocytic aggregates and non-lymphocytic stromal tissues were laser microdissected from six fully embedded, ethanol fixed, esophagectomy samples from Shanxi, China, a high-risk region for esophageal cancer. Promoter CpG site-specific hypermethylation status of COX-2 was determined using real-time methylation-specific PCR (qMS-PCR) based on Taqman Chemistry. The methylation status of a subset of samples was confirmed by pyrosequencing. Results: Forty-nine microdissected foci were analyzed. COX-2 gene methylation was significantly more common in subepithelial lymphocytes (12/16 (75% of all foci)) than in epithelial foci (3/16 (19%)) or foci of non-lymphocytic stromal tissues (3/17 (18%)) (Fisher's exact p = 0.05). Two of three epithelial samples and all three stromal samples that showed COX-2 methylation were adjacent to foci of methylated subepithelial lymphocytes. Pyrosequencing confirmed the methylation status in a subset of samples. Conclusions: In these esopohageal cancer patients, COX-2 gene methylation was more common in subepithelial lymphocytes than in adjacent epithelial or stromal cells in both grades of dysplasia and in foci of invasive cancer. These findings raise the possibility that methylation of subepithelial lymphocytes may be important for tumorigenesis. Future studies of gene methylation should consider separate evaluation of epithelial and non-epithelial cell populations.     

ERα and ERK1/2 MAP kinase expression in microdissected stromal and epithelial endometrial cells

Our previous published data detected higher expression of total and active mitogen activated protein kinase (MAPK) in the epithelial vs. stromal cells of the endometrium. In the present work we compared the expression of ERK1/2 MAPK and estrogen receptor α (ERα) in epithelial versus stromal cells in benign human endometrial tissues. Laser capture microdissection was used to separate glandular epithelium and stromal cells from six frozen, proliferative phase endometrial specimens.Total and phosphorylated levels for ERK1/2 and ERα were measured by quantitation of signals from Western blots using specific antibodies against the active and total forms of ERK1/2 and against ERα. When the level of the proteins was quantitated and normalized to β actin from microdissected stroma and epithelium, no significant difference was detected in the levels of these proteins between the two tissue compartments. There was a trend toward higher expression in the stroma vs. epithelium, respectively (active ERK1/2 0.45 ± 0.17 vs. 0.2 ± 0.65; total ERK1/2 0.54 ± 0.35 vs. 0.28 ± 0.23; ERα 0.82 ± 0.28 vs. 0.54 ± 0.18; n = 6). These data demonstrate that there are comparable levels of ERα (P = 0.41), total ERK1/2 (P = 0.18) and active ERK1/2 (P = 0.13) in the stroma and epithelium of proliferative phase endometrium with a trend toward higher expression of these proteins in the stromal compartment.     

p16, MGMT, RARbeta2, CLDN3, CRBP and MT1G gene methylation in esophageal squamous cell carcinoma and its precursor lesions

Esophageal squamous cell carcinoma (ESCC) is a common cancer with a very poor prognosis. New methods are needed to screen high-risk populations and identify curable tumors and precursor lesions early. Molecular markers may be useful in such screening efforts. This study was designed to determine the prevalence of p16, MGMT, RARbeta2, CLDN3, CRBP and MT1G gene methylation in patients with ESCC to evaluate the variation of gene methylation across a spectrum of preneoplastic lesions, and assess the feasibility of using gene methylation in a primary screening test utilizing frozen esophageal cells collected by balloon cytology samplers. Samples were obtained from high-risk subjects from north central China. These samples included 11 foci of histologically normal mucosa, 8 foci of low-grade squamous dysplasia, 7 foci of high-grade squamous dysplasia, and 13 foci of ESCC from 6 fully embedded resection specimens; endoscopic biopsies from 6 individuals with no histological evidence of disease; and frozen esophageal balloon samples from 12 asymptomatic subjects. Promoter CpG site-specific hypermethylation status was determined for each gene using real-time methylation-specific PCR (qMS-PCR) based on Taqman chemistry. Of the 6 ESCC patients, 5 showed methylation of at least one gene. For most genes, methylation occurred with increasing frequency during neoplastic progression, with the largest increase found between low- and high-grade dysplasia. There was considerable variation in methylation patterns among different foci of the same histological grade, even within individual patients, but 16/20 (80%) of high-grade dysplastic and cancer foci had >or= 2 methylated genes, while 17/19 (89%) of normal and low-grade dysplastic foci had <2 methylated genes. These genes were rarely methylated in histologically normal mucosa from patients with or without ESCC. Gene methylation was common and easily detectable in the frozen esophageal cells collected by balloon cytology samplers. Our data suggest that methylation of p16, MGMT, RARbeta2, CLDN3, CRBP, and MT1G is common in the esophageal mucosa of patients with ESCC in this high-risk population, and tends to increase in prevalence in foci with increasing histological severity of disease. Methylation data from panels of genes may be able to identify patients with high-grade lesions. Balloon cytology may be able to screen the length of the esophagus effectively for a subset of cells with abnormal methylation, and may be useful in a primary screening test for ESCC and its precursor lesions.     

Promoter methylation regulates Helicobacter pylori-stimulated cyclooxygenase-2 expression in gastric epithelial cells

Cyclooxygenase (COX)-2, the inducible form of the rate-limiting enzyme for prostaglandin synthesis, is up-regulated in gastrointestinal cancers and is a key mediator of epithelial cell growth. Helicobacter pylori is causally linked to gastric cancer. In H. pylori gastritis, COX-2 expression localizes to the subepithelial region, with variable levels in the epithelium. In contrast, in gastric cancer, COX-2 strongly predominates in the epithelium, suggesting that the transition to consistent epithelial COX-2 overexpression may be a critical molecular event in gastric carcinogenesis. Because aberrant promoter methylation inhibits expression of a variety of genes in gastrointestinal cancers, we sought to determine whether methylation of the COX-2 promoter could regulate the response to H. pylori in gastric epithelial cells. We assessed COX-2 expression and promoter methylation status in six gastric epithelial cell lines. In all four of the cell lines that exhibited basal expression of COX-2 and a significant increase in expression in response to H. pylori, the COX-2 promoter was unmethylated, whereas in the two cell lines that did not express COX-2, the COX-2 promoter was methylated. Treatment of COX-2-methylated cells with the demethylating agent 5-azacytidine had a modest effect on COX-2 expression, but when 5-azacytidine-treated cells were subsequently stimulated with H. pylori, there was a significant, 5-10-fold enhancement of both COX-2 mRNA and protein expression and release of the COX-2 product, prostaglandin E2. In contrast, in COX-2-expressing cell lines that were unmethylated at the COX-2 promoter, 5-azacytidine had no effect on H. pylori-stimulated COX-2 expression. These findings suggest that loss of COX-2 methylation may facilitate COX-2 expression and promote gastric carcinogenesis associated with H. pylori infection.     

Frequent gene dosage alterations in stromal cells of epithelial ovarian carcinomas

Stromal cells are an active and integral part of epithelial neoplasms. We have previously observed allelic imbalance on chromosome 3p21 in both stromal and epithelial cells of ovarian tumors. This study was designed to explore gene dosage alterations throughout human chromosomes from stromal and epithelial cells of epithelial ovarian carcinomas. Thirteen stromal and 24 epithelial samples, microdissected from epithelial ovarian carcinomas, were analyzed using multiplex ligation-dependent probe amplification technique. Analysis covered 110 cancer related genes. Frequent genetic alterations were detected both in the stroma and epithelium of ovarian carcinomas. The mean number of altered genes per tumor was 10.8 in stroma and 23.6 in epithelium. In the stroma, the mean number of gains was 6.6 and of losses 4.2 and in the epithelium 13.7 and 9.9. The high number of changes associated with advanced tumor stage (p = 0.035) and death due to ovarian cancer (p = 0.032). The most frequent alteration was the deletion of the deleted in colorectal carcinoma (DCC) on chromosome 18q21.3 in 62% of samples. Loss of DCC was related to endometrioid subtype (p = 0.033). Large chromosomal aberrations were detected on the basis of alterations in adjacent genes. Most importantly, 38 genes showed similar genetic alterations (gain-gain or loss-loss) in stromal and epithelial compartments of 11 tumor pairs. Thus, frequent genetic alterations in stromal cells of epithelial ovarian carcinomas resembled those of malignant epithelial cells and may indicate a common precursor cell type. Epithelial-mesenchymal transition may generate transformed cancer cells and modify the tumor microenvironment with distinct properties.     

Significance of COX-2 expression in human esophageal squamous cell carcinoma

Cyclooxygenase-2 (COX-2) is well established to play an important role in the tumorigenesis of a variety of human cancers; however, the function of COX-2 in the development of esophageal squamous cell carcinoma (ESCC) remains less clear. Here, we determined, first, the pattern of COX-2 expression in normal esophageal mucosa, dysplasia, carcinoma in situ (CIS) and invasive SCC. Immunohistochemical analysis showed that, while COX-2 was weakly expressed, if at all, in normal squamous epithelium, strong COX-2 expression was detected as early as the stage of dysplasia and frequently in 20 of 26 (77%) CIS and 86 of 111 (77%) invasive SCC. Upregulation of COX-2 in ESCC was found to be significantly associated with tumor progression (R = 0.493, P < 0.01). Further, treatment of human ESCC cell lines (KYSE450 and KYSE510) with NS-398, a COX-2 specific chemical inhibitor, suppressed the production of prostaglandin E2 (PGE2) and induced cell growth inhibition, cell cycle arrest at the G1-S checkpoint, and the expression of cyclin-dependent kinase inhibitors p21waf1/cip1 and p27kip1. Finally, knockdown expression of COX-2 in KYSE450 cells by a specific COX-2 siRNA dramatically inhibited PGE2 production, cell growth and, more importantly, colony formation and tumorigenesis in nude mice. Together, this study suggested that COX-2 may be involved in an early stage of squamous cell carcinogenesis of the esophagus and has a non-redundant role in the regulation of cellular proliferation and tumorigenesis of esophageal epithelial cells.     

COX-2在Barrett食管与食管腺癌发病中的意义

目的：研究环氧化酶（COX-2）在Barrett食管（BE）及食管腺癌（EA）中的表达，探讨BE及EA的发病机制。方法：采用免疫组化染色及real—timePCR检测正常食管上皮、BE粘膜及EA标本中COX-2的蛋白及mRNA表达情况。结果：COX-2蛋白在正常鳞状上皮中几乎不表达，而在BE中呈轻中度阳性表达，在EA中呈弥漫强阳性表达，三者之间差异显著（P〈0．01），且呈线性趋势（r=0．810，P〈0．01）。COX-2mRNA在BE及EA中表达均增强，其表达率分别为正常鳞状上皮的1．3728倍（P〈0．01）和1．8312倍（P〈0．01）。结论：COX-2在BE及EA中表达升高，提示其表达的上调可能在BE及EA的发生发展中起重要作用。     

CNTN-1基因在食管鳞癌中的表达

目的研究CNTN-1基因在人食管鳞癌中的mRNA表达及其与肿瘤发生的关系。方法采用SYBR Green实时定量PCR技术和免疫组织化学技术检测20例正常食管上皮组织和60例食管鳞癌及癌旁组织中的CNTN-1基因mRNA水平。结果与正常食管上皮组织和癌旁组织相比,CNTN-1基因在食管鳞癌组织中mRNA水平较正常食管上皮组织显著上调6.7倍,较癌旁组织上调2.3倍。免疫组织化学的结果也表明CNTN-1基因在食管鳞癌组织中的mRNA表达要高于正常食管上皮组织和癌旁组织。结论CNTN-1基因在人食管鳞癌中较正常食管上皮组织和癌旁组织mRNA表达显著上调。CNTN-1基因的表达与食管鳞癌的发生可能有一定关系。     

食管鳞状细胞癌CD40COX-2 表达及其与血管生成关系的研究*

目的:探讨CD40和COX-2 在食管鳞状细胞癌组织中的表达情况、临床病理意义及其与肿瘤血管生成的关系。方法:采用免疫组织化学方法研究79例食管癌和28例正常食管黏膜上皮组织中CD40和COX-2 的表达情况,根据CD34表达计算食管癌组织平均微血管密度（MVD）;分析CD40、COX-2 的表达与食管癌发生部位、肿瘤大小、分化程度、MVD及淋巴结转移的关系。同时采用免疫细胞化学染色及Western Blot方法检测食管癌Eca109 细胞和原代培养的正常食管上皮细胞CD40和COX-2 在蛋白水平上表达的差异。结果:免疫组化结果表明,食管癌组织中CD40和COX-2 的阳性表达率均明显高于正常食管鳞状上皮（54.43% vs 10.71% ,69.62% vs 17.86% ,P 均<0.05）。CD40在食管癌中的表达与淋巴结转移有关,伴有淋巴结转移者CD40阳性表达率明显高于无淋巴结转移者（70.37% vs 46.15% ,P<0.05）,但CD40表达与其他临床病理特征无明显相关。COX-2 在食管癌中的阳性表达与肿瘤大小、部位、分化程度、有无淋巴结转移等均无明显相关（P>0.05）。 CD40与COX-2 在食管癌组织中表达明显相关（P<0.05）,相关系数（Φ）为0.446。食管癌组织中MVD明显高于正常食管组织（25.02± 5.52vs 12.09± 4.55,P<0.05）。 CD40和COX-2 阳性表达组食管癌组织MVD明显高于阴性表达组（26.37± 6.02vs 22.58± 5.25,P<0.05）。 体外研究结果表明,食管癌Eca109 细胞CD40和COX-2 表达均高于正常食管上皮细胞（P<0.05）。 结论:CD40表达与食管鳞状细胞癌的发生、发展密切相关,CD40可能通过影响COX-2 的表达促进食管癌组织微血管生成。      

Ⅱ型环氧合酶和CDK4在食管鳞癌中的表达及意义

目的 探讨Ⅱ型环氧合酶(COX-2)和CDK4在食管鳞癌中的表达与食管鳞癌的发生、浸润及转移的关系.方法 采用免疫组化S-P法检测49例食管鳞癌及癌旁黏膜组织中的表达.结果 COX-2在癌旁正常上皮、单纯增生上皮、轻度不典型增生上皮、重度不典型增生上皮、原位癌和浸润癌组织中的阳性表达率分别为0%(0/25)、7.1%(2/28)、33.3%(7/21)、50.0%(5/10)、69.2%(9/13)和73.5%(36/49).正常上皮组织与单纯增生上皮比较差异没有统计学意义(P＞0.05),但与轻度不典型增生上皮、重度不典型增生上皮、原位癌和浸润癌组织比较差异有统计学意义(P＜0.01).其在食管鳞癌中的阳性率与癌组织分级有关(P＜0.05).CDK4蛋白在癌旁正常上皮、单纯增生上皮、轻度不典型增生上皮、重度不典型增生上皮、原位癌和浸润癌组织中的阳性表达率分别为8%(2/25)、21.4%(6/28)、42.9%(9/21)、70%(7/10)、61.5%(8/13)和91.8%(45/49).正常上皮组织与单纯增生上皮比较差异没有统计学意义(P＞0.05),但与轻度不典型增生上皮、重度不典型增生上皮、原位癌和浸润癌组织比较差异有统计学意义(P＜0.01).CDK4阳性率与鳞癌分级无关(P＞0.05).COX-2、CDK4蛋白表达与食管鳞癌的浸润深度和淋巴结转移无关(P＞0.05).食管鳞癌组织中COX-2、CDK4蛋白阳性率有正相关性(P＜0.01).结论 COX-2、CDK4蛋白表达与食管鳞癌的发生有关.     

新疆哈萨克族和汉族食管癌患者金属硫蛋白-3 CpG岛甲基化的研究

目的： 检测分析和比较新疆哈萨克族和汉族食管癌及癌旁组织中金属硫蛋白-3 (metallothionein-3,MT-3)基因启动区CpG岛的甲基化情况,为寻找新疆哈萨克族食管癌诊断治疗的分子标记物提供线索。方法：采用甲基化特异性聚合酶链反应 (methylation specific polymerase chain reaction,MSP)的方法,检测33例哈萨克族和30例汉族食管癌患者食管癌组织和癌旁组织中MT-3 基因启动子区CpG岛的甲基化发生状况。 结果：33例哈萨克族食管癌组织和癌旁组织标本中MT-3基因启动子区甲基化发生率分别为66.7% (22/33)和27.3% (9/33),差异有统计学意义 (χ2=10.280,P=0.001);30例汉族食管癌组织和癌旁组织标本中MT-3基因启动子区甲基化发生率分别为56.7% (17/30)和26.7% (8/30),差异亦有统计学意义 (χ2=5.554,P=0.018)。哈、汉民族之间癌组织和癌旁组织MT-3基因启动子区甲基化率的差异均无统计学意义 (χ2=0.666,P=0. 414;χ2=0.003,P=0.957)。 结论：MT-3基因启动子区CpG岛的甲基化可能与食管癌的发生与发展有关,但不是哈族食管癌患者的特异性致病因素。     

食管鳞癌组织中TSLC1基因启动子甲基化状态检测的临床意义

目的:探讨肺癌抑癌基因1（TSLC1）在食管鳞癌组织中基因启动子甲基化状态及其与临床病理参数的关系。方法:应用甲基化特异性PCR检测TSLC1基因启动子在98例食管鳞癌组织及相应癌旁组织中的甲基化状态并结合临床病理资料进行分析。结果:在食管鳞癌组织中TSLC1基因甲基化阳性率（55.1%,54/98）显著高于癌旁组织（4.1%,4/98）（P<0.01）。TSLC1甲基化与食管鳞癌临床分期和浸润深度显著相关（P=0.035和0.001）。结论:TSLC1基因甲基化可能是参与食管鳞癌发展的重要分子事件。     

Loss of annexin 1 correlates with early onset of tumorigenesis in esophageal and prostate carcinoma

Annexin I protein expression was evaluated in patient-matched longitudinal study sets of laser capture microdissected normal, premalignant, and invasive epithelium from human esophageal squamous cell cancer and prostatic adenocarcinoma. In 25 esophageal cases (20 by Western blot and 5 by immunohistochemistry) and 17 prostate cases (3 by Western blot and 14 by immunohistochemistry), both tumor types showed either complete loss or a dramatic reduction in the level of annexin I protein expression compared with patient-matched normal epithelium (P < or = 0.05). Moreover, by using Western blot analysis of laser capture microdissected, patient-matched longitudinal study sets of both tumor types, the loss of protein expression occurred in premalignant lesions. Concordance of this result with immunohistochemical analysis suggests that annexin I may be an essential component for maintenance of the normal epithelial phenotype. Additional studies investigating the mechanism(s) and functional consequences of annexin I protein loss in tumor cells are warranted.     

FHIT基因甲基化检测对肺癌早期诊断的临床意义

目的探讨人原发性肺癌中FHIT基因启动子区的甲基化状态及其临床意义。方法采用甲基化特异性聚合酶链技术（MSP）,对59例人原发性肺癌组织（肺癌组）、32例肺正常组织（正常组）、15例癌旁组织（癌旁组）及11例支气管鳞状化生组织进行FHIT基因5’-CpG岛甲基化状况检测,甲基化阳性标本采用基因克隆测序技术进行序列测定。结果肺癌组、癌旁组和正常组的FHIT基因甲基化率分别为37．3％（22／59）、20．0％（3／15）、0（0／32）,三组相比差异有统计学意义（P〈0．01）;支气管鳞状化生组织的甲基化阳性率为18．1％（2／11）。FHIT基因甲基化率肺癌吸烟者与非吸烟者差异无统计学意义（P〉0．05）。FHIT甲基化扩增产物测序结果显示,所有CpG岛中的胞嘧啶均保持不变,而CpG岛二核苷酸以外的胞嘧啶经过PCR扩增后,则被胸腺嘧啶取代。结论FHIT基因5’-CpG岛甲基化参与了肺癌的演变,可能在肺癌发生的早期阶段,采用MsP方法进行FHIT甲基化检测,可能对肺癌的早期诊断具有一定的临床意义。     

Insulin-like growth factor 2 messenger RNA binding protein 3 (IGF2BP3) is a marker of unfavourable prognosis in colorectal cancer

BackgroundEvidence suggests that insulin-like growth factor 2 messenger RNA binding protein 3 (IGF2BP3, also known as IMP3) represents a promising cancer biomarker. However, the clinical, pathological, molecular and prognostic features of IGF2BP3-positive colorectal cancers remain uncertain.Materials and methodsWe evaluated IGF2BP3 expression by immunohistochemistry in 671 rectal and colon cancer cases that form part of a molecular pathological epidemiology database. Cox proportional hazards regression models were used to compute mortality hazard ratio (HR), adjusting for clinical, pathological and molecular features, including microsatellite instability, the CpG island methylator phenotype, LINE-1 methylation and KRAS, BRAF and PIK3CA mutations.ResultsAmong 671 colorectal cancers, 234 (35%) tumours were positive for IGF2BP3. In contrast, normal colorectal epithelium was negative for IGF2BP3 in all 403 specimens of normal mucosa adjacent to carcinoma. IGF2BP3 positivity was associated with poor differentiation (p = 0.0003), stage III-IV disease (p = 0.0081), BRAF mutation (p = 0.031), and LINE-1 hypomethylation (p = 0.020). IGF2BP3 positivity was significantly associated with shorter colorectal cancer-specific [log-rank p < 0.0001; multivariate HR, 1.37; 95% confidence interval (CI), 1.02–1.84] and overall survival (log-rank p = 0.0004; multivariate HR, 1.32; 95% CI, 1.05–1.66).ConclusionsIGF2BP3 expression in colorectal cancer is associated with adverse clinical outcome. Our findings support a role for IGF2BP3 as a diagnostic and/or prognostic biomarker in colorectal cancer.     

Subepithelial myofibroblasts express cyclooxygenase-2 in colorectal tubular adenomas.

PURPOSE: Recent data support the hypothesis that the inducible isoform of cyclooxygenase (COX-2) plays a role in the early stages of colonic carcinogenesis and that nonsteroidal anti-inflammatory drugs (NSAIDs) retard the development of colon cancer by modulating COX-2. However, the cell types responsible for producing COX-2 in colorectal adenomas remain a subject of controversy. EXPERIMENTAL DESIGN: COX-2 expression in normal colonic mucosa (n = 50), hyperplastic polyps (n = 43), sporadic adenomas (n = 67), and invasive colonic adenocarcinoma (n = 39) was studied in formalin-fixed and paraffin-embedded tissue sections from endoscopy biopsy and colonic resection specimens. Immunohistochemistry (avidin-biotin complex technique with double immunolabeling) was used to identify the phenotypes of COX-2-producing cells. RESULTS: In colorectal adenomas, increased expression of COX-2 was detected and localized to alpha smooth muscle actin ( proportional, variant SMA)-positive subepithelial stromal cells (myofibroblasts) in the periluminal region of the lamina propria in 63 (94%) of 67 cases. In contrast, in normal colonic mucosa and in hyperplastic polyps with intact epithelium, COX-2 expression was found only in macrophages and endothelial cells. In areas in which the surface epithelium was ulcerated in normal mucosa as well as hyperplastic or neoplastic polyps, COX-2 expression was increased in granulation tissue (and present in macrophages, endothelium, and myofibroblasts). In invasive carcinoma, COX-2 expression in myofibroblasts was limited to the adenomatous portion of the tumor and was detected in 62% of cases (n = 39). In addition, focal expression of COX-2 by malignant epithelial cells was observed in 23% of invasive adenocarcinoma. CONCLUSIONS: These results show that increased COX-2 expression in sporadic adenoma of the colon is common and is localized specifically to subepithelial intestinal myofibroblasts. These findings further support the hypothesis that myofibroblasts are important target cells for NSAID-mediated chemoprevention of colorectal cancer.