Li-Dong Wang

Deciphering the Signature of Selective Constraints on Cancerous Mitochondrial Genome

In accordance with the hypothesis that cancer formation is a process of somatic evolution driven by natural selection, signature of positive selection has been detected on a number of cancer-related nuclear genes. It remains, however, controversial whether a similar selective pressure has also acted on mitochondrial DNA (mtDNA), a small molecule in mitochondrion that may play an important role in tumorigenesis by altering oxidative phosphorylation. To better understand the mutational pattern on cancerous mtDNA and decipher the genetic signature left by natural selection, a total of 186 entire mitochondrial genomes of cancerous and adjacent normal tissues from 93 esophageal cancer patients were obtained and extensively studied. Our results revealed that the observed mutational pattern on the cancerous mtDNAs might be best explained as relaxation of negative selection. Taking into account an additional 1,235 cancerous (nearly) complete mtDNA sequences retrieved from the literature, our results suggested that the relaxed selective pressure was the most likely explanation for the accumulation of mtDNA variation in different types of cancer. This notion is in good agreement with the observation that aerobic glycolysis, instead of mitochondrial respiration, plays the key role in generating energy in cancer cells. Furthermore, our study provided solid evidence demonstrating that problems in some of the published cancerous mtDNA data adequately explained the previously contradictory conclusions about the selective pressure on cancer mtDNA, thus serving as a paradigm emphasizing the importance of data quality in affecting our understanding on the role of mtDNA in tumorigenesis.

Autoantibody detection to tumor-associated antigens of P53, IMP1, P16, cyclin B1, P62, C-myc, Survivn, and Koc for the screening of high-risk subjects and early detection of esophageal squamous cell carcinoma

The aim of this study was to evaluate the diagnostic values by detecting sera autoantibodies to eight tumor-associated antigens (TAAs) of P53, IMP1, P16, cyclin B1, P62, C-myc, Survivn and Koc full-length recombinant proteins for the screening of high-risk subjects and early detection of esophageal squamous cell carcinoma (ESCC). Enzyme-linked immunosorbent assay was used to detect autoantibodies against the eight selected TAAs in 567 sera samples from four groups, including 200 individuals with normal esophageal epithelia (NOR), 214 patients with esophageal basal cell hyperplasia (BCH), 65 patients with esophageal dysplasia (DYS), and 88 patients with ESCC. In addition, the expression of the eight antigens in esophageal tissues was analyzed by immunohistochemistry. Statistically significant distribution differences were identified among the four groups for each of the individual autoantibodies to six TAAs (P53, IMP1, P16, cyclin B1, P62, and C-myc); the detection rates of antoantibodies were positively correlated with the progression of ESCC. When autoantibody assay successively accumulated to six TAAs (P53, IMP1, P16, cyclin B1, P62, and C-myc), a stepwise increased detection frequency of autoantibodies was found in the four sera groups (6% in NOR, 18% in BCH, 38% in DYS, and 64% in ESCC, respectively), the risks to BHC, DYS, and ESCC steadily increased about 3-, 9-, and 27-folds. The sensitivity and the specificity for autoantibodies against the six TAAs in diagnosing ESCC reached up to 64% and 94%, respectively. The area under the receiver operating characteristic curve for the six anti-TAA autoantibodies was 0.78 (95% confidence interval 0.74-0.83). No more increasing in sensitivity was found with the addition of new anti-TAA autoantibodies. A combination detection of autoantibodies to TAAs might distinguish ESCC patients from normal individuals and the patients with esophageal precancerous lesions.

ANO1 protein as a potential biomarker for esophageal cancer prognosis and precancerous lesion development prediction

Objectives Anoctamin 1 (ANO1) has been found to be overexpressed in esophageal squamous cell carcinoma (ESCC) in our previous study. Herein we showed the clinical relevance of ANO1 alterations with ESCC and esophageal precancerous lesion progression. Results ANO1 was detected in 38.1% (109/286) and 25.4% (77/303) of tumors in the two cohorts, but in none of morphologically normal operative margin tissues. ANO1 expression was significantly associated with a shorter overall survival (OS), especially in patients with moderately differentiated and stage IIA tumors. In 499 iodine-unstained biopsies from the endoscopic screening cohort in 2005-2007, all the 72 pathologically normal epithelial mucosa presented negative immunostaining, whereas ANO1 expression was observed in 3/11 tumors and 5/231 intraepithelial lesions. 7/8 ANO1-positive cases had developed unfavorable outcomes revealed by endoscopic follow-up in 2012. Analysis of another independent cohort of 148 intraepithelial lesions further confirmed the correlation between ANO1 expression and progression of precancerous lesions. 3/4 intraepithelial lesions with ANO1 expression had developed ESCC within 4-9 years after the initial endoscopic examination. Methods Immunohistochemistry (IHC) was performed to examine ANO1 expression in surgical ESCC specimens and two independent cohorts of esophageal biopsies from endoscopic screening in high-incidence area of ESCC in northern China. Association between ANO1 expression, clinico-pathologic parameters, and the impact on overall survival was analyzed. Conclusions Positive ANO1 is a promising biomarker to predict the unfavorable outcome for ESCC patients. More importantly, it can predict disease progression of precancerous lesions.

Detection of autoantibodies to a panel of tumor-associated antigens for the diagnosis values of gastric cardia adenocarcinoma.

To evaluate the diagnostic values of using autoantibodies in sera to a panel of eight tumor-associated antigens (TAAs) of P53, Koc, P62, C-myc, IMP1, Survivn, P16 and Cyclin B1 full-length recombinant proteins for early detection of patients with gastric cardia adenocarcinoma (GCA) and high-risk subjects screening. Enzyme-linked immunosorbent assay was used to detect autoantibodies against the eight selected TAAs in 383 sera samples from four groups, including 140 subjects with normal gastric cardia epithelia (NOR), 76 patients with chronic atrophic gastritis (CAG), 79 patients with gastric cardia dysplasia (DYS) and 88 patients with GCA. In addition, the expression of the eight antigens was analyzed in gastric cardia tissues by immunohistochemical method. The individual autoantibodies to six TAAs (P53, P62, IMP1, Survivn P16 and Cyclin B1) were significantly higher in sera from patients with GCA than that in normal subjects (P < 0.05). When autoantibody assay successively accumulated to seven TAAs (P53, Koc, P62, C-myc, IMP1, Survivn and P16), a stepwise increased detection frequency of autoantibodies was found in the four sera groups (13% in NOR, 39% in CAG, 46% in DYS, and 64% in GCA, respectively), the risks to CAG, DYS and GCA steadily increased about 4.4-, 5.7- and 12.0-fold. The sensitivity and the specificity for autoantibodies against the seven TAAs in diagnosing GCA reached up to 64% and 87%, respectively. The area under the receiver operating characteristic curve for the seven anti-TAA autoantibodies was 0.73 (95%CI: 0.68-0.78) No more increase in sensitivity was found with the addition of new anti-TAA autoantibodies. A combination detection of autoantibodies to TAAs might be helpful to distinguish GCA patients from normal subjects and the patients with gastric cardia precancerous lesions. In addition, further studies in patients with GCA and precancerous lesions using enlarged TAA panels might improve the sensitivity and specificity of cancer detection and high-risk subjects screening.

Correlation of telomere length shortening with TP53 somatic mutations, polymorphisms and allelic loss in breast tumors and esophageal cancer.

Genomic instability caused by telomere erosion is an important mechanism of tumorigenesis. p53 plays a key role in cellular senescence and/or apoptosis associated with telomere erosion which positions p53 as a guard against tumorigenesis. The present study was undertaken to investigate the potential interactions between p53 functional mutations, polymorphisms, allelic loss and telomere erosion in 126 breast tumor patients and 68 esophageal cancer patients. Telomere length (TL) was measured by real-time quantitative PCR. Somatic mutations, polymorphisms and allelic loss in the TP53 gene were detected by direct sequencing of both tumor and normal tissue samples. Our results showed that telomeres were significantly shorter in tumors with somatic p53 mutations compared with tumors with wild-type p53 in both breast tumors (P=0.007) and esophageal cancer (P=0.001). Telomeres of patients with minor genotype CC of rs12951053 and GG of rs1042522 were significantly shorter compared to patients with other genotypes of this single nucleotide polymorphism in esophageal cancer tissue. Furthermore, TP53 allelic loss was detected and significantly associated with somatic mutations in both types of tumor tissues. These findings suggest that somatic p53 mutations, rs12951053 genotype CC and rs1042522 genotype GG contribute to erosion of telomeres, and TP53 allelic loss may be one of the representations of chromosomal instability caused by telomere erosion combined with somatic p53 mutations. These results support that the TP53 gene has a strong interaction with TL erosion in tumorigenesis.

Variations in the MHC Region Confer Risk to Esophageal Squamous Cell Carcinoma on the Subjects from High-Incidence Area in Northern China

Background The human major histocompatibility complex (MHC) is the most important region in vertebrate genome, and is crucial in innate immunity. Recent studies have demonstrated the possible role of polymorphisms in the MHC region to high risk for esophageal squamous cell carcinoma (ESCC). Our previous genome-wide association study (GWAS) has indicated that the MHC region may confer important risk loci for ESCC, but without further fine mapping. The aim of this study is to further identify the risk loci in the MHC region for ESCC in Chinese population. Methods Conditional logistic regression analysis (CLRA) was performed on 24 single nucleotide polymorphisms (SNPs) within the MHC region, which were obtained from the genetically matched 937 cases and 692 controls of Chinese Han population. The identified promising SNPs were further correlated with clinical and clinicopathology characteristics. Immunohistochemistry was performed to explore the protein expression pattern of the related genes in ESCC and neighboring normal tissues. Results Of the 24 promising SNPs analyzed, we identified three independent SNPs in the MHC region associated with ESCC: rs35399661 (Pu200a=u200a6.07E-06, ORu200a=u200a1.71, 95%CIu200a=u200a1.36–2.17), rs3763338 (Pu200a=u200a1.62E-05, ORu200a=u200a0.63, 95%CIu200a=u200a0.50–0.78) and rs2844695 (Pu200a=u200a7.60E-05, ORu200a=u200a0.74, 95%CIu200a=u200a0.64–0.86). These three SNPs were located at the genes of HLA-DQA1, TRIM27, and DPCR1, respectively. Further analyses showed that rs2844695 was preferentially associated with younger ESCC cases (Pu200a=u200a0.009). The positive immunostaining rates both for HLA-DQA1 and TRIM27 were much higher in ESCC tissues than in neighboring normal tissues (69.4% vs. 26.8% for HLA-DQA1 and 77.6% vs. 47.8% for TRIM27, P<0.001). Furthermore, the overexpression of HLA-DQA1 is correlated significantly with age (Pu200a=u200a0.001) and family history (P<0.001). Conclusion This study for the first time provides evidence that multiple genetic factors within the MHC region confer risk to ESCC on the subjects from high-risk area in northern China.

MicroRNA-200c regulates cisplatin resistance by targeting ZEB2 in human gastric cancer cells

This study was specifically designed to confirm the hypothesis that microRNA-200c (miR-200c) affects the development of cisplatin (DDP) resistance in human gastric cancer cells by targeting zinc finger E-box binding homeoboxxa02 (ZEB2). A total of 50 gastric cancer tissues and their corresponding normal adjacent tissue samples were collected. Then, the expression levels of miR-200c and ZEB2 in both gastric cancer specimens and cells were detected using the quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) and immunohistochemical methods. A dual‑luciferase reporter gene assay was conducted to evaluate the effect of miR-200c on the 3-untranslated region (3UTR) luciferase activity of ZEB2. SGC7901/DDP cells were transfected with miR-200c mimics and ZEB2 siRNA, respectively. Subsequently, changes in cellular proliferation and apoptosis were detected through the methyl thiazolyl tetrazolium assay and flow cytometric analysis, respectively. We also carried out a western blot analysis assay in order to detect the expression of apoptosis-related genes and ZEB2. miR-200c was significantly downregulated and ZEB2 was significantly upregulated in both gastric cancer tissues and SGC7901/DDP cells when compared with those in normal tissues and SGC7901 cells (P<0.01). The dual luciferase reporter gene assay showed that miR-200c could specifically bind with the 3UTR of ZEB2 and significantly suppress the luciferase activity by 42% (P<0.01). Upregulation of miR-200c or downregulation of ZEB2 enhanced the sensitivity of SGC7901/DDP cells to DDP. miR‑200c was significantly downregulated in both gastric cancer tissues and cells, while the expression of ZEB2 exhibited the opposite trend. Our study further demonstrated that miR-200c could enhance the sensitivity of SGC7901/DDP cells to DDP through targeted regulation of ZEB2 expression in gastric cancer tissues.

SLC52A3 expression is activated by NF-κB p65/Rel-B and serves as a prognostic biomarker in esophageal cancer

The human riboflavin transporter-3 (encoded by SLC52A3) plays a prominent role in riboflavin absorption. Interestingly, abnormal expression patterns of SLC52A3 in multiple types of human cancers have been recently noted. However, the molecular mechanisms underlying its dysregulation remain unclear. In this study, we find that SLC52A3 has two transcript variants that differ in the transcriptional start site, and encode different proteins: SLC52A3a and SLC52A3b. Importantly, aberrant expressions of SLC52A3 are associated with stepwise development of esophageal squamous cell carcinoma (ESCC) as well as the survival rates of ESCC patients. Functionally, SLC52A3a, but not SLC52A3b, strongly promotes the proliferation and colony formation of ESCC cells. Furthermore, SLC52A3 5′-flanking regions contain NF-κB p65/Rel-B-binding sites, which are crucial for mediating SLC52A3 transcriptional activity in ESCC cells. Chromatin immunoprecipitation and electrophoretic mobility shift assay reveal that p65/Rel-B bind to 5′-flanking regions of SLC52A3. Accordingly, NF-κB signaling upregulates SLC52A3 transcription upon TNFα stimulation. Taken together, these results elucidate the mechanisms underlying SLC52A3 overexpression in ESCC. More importantly, our findings identify SLC52A3 as both a predictive and prognostic biomarker for this deadly cancer.

Characterization of serum estradiol level and tissue estrogen receptor immunostaining with clinical response and reproductive factor changes in Chinese female patients with esophageal squamous cell carcinoma

Pre-menopausal female patients have a prolonged survival than post-menopausal patients, indicating that estrogen and/or estrogen receptor (ER) may have some biological effects on prognosis. ER expression in cancer tissue has been reported to be a significant prognostic marker in multiple human cancers. However, the prognostic value of estrogen and/or ER on female patients with esophageal squamous cell carcinoma (ESCC) is rarely reported. The present study was undertaken to elucidate the associations of serum estradiol level, tissue estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ) expression with clinical response and reproductive factor changes in 387 female ESCC patients. Radioimmunoassay revealed that serum estradiol level was higher in pre-menopausal than those in peri-menopausal and post-menopausal patients. Furthermore, patients with higher serum estradiol level appeared to have a better survival. Immunostaining results suggested that ERα positive (+) expression was mainly located in cytoplasm of tumor cells with a positive rate of 69.9% and ERβ (+) was mainly located in nucleus of tumor cells with a positive rate of 64.9%. We did not find the relations of ER expression with tumor invasion (P>0.05), lymph node metastasis (P>0.05), TNM staging (P>0.05) and treatment method (P>0.05). Surprisingly, ERα (+) expression was higher in post-menopausal patients than those in pre-menopausal patients (P<0.05). Patients with number of pregnancy≥4 have a higher ERβ (+) expression than those patients with≤3 (P<0.05). Univariate and multivariate survival analysis showed that ERβ (+) expression in addition to ERα (-) expression are favorable prognostic markers in female ESCC patients (P<0.05). Further related study is needed to in-depth explore the potential mechanisms of ERα and ERβ in survival of female patients with ESCC.

1 058 例食管良性肿瘤临床病理特征

目的n 探讨食管良性肿瘤临床病理特征。n 方法1 058例食管良性肿瘤均来自1973年1月至2015年1月郑州大学第一附属医院河南省食管癌重点开放实验室50万例食管和贲门癌生物样本和临床信息数据库。采用SPSS 21.0软件进行统计学分析。n 结果本组50万例数据库中，临床病理信息完整的食管肿瘤患者共249 246例，食管良性肿瘤占0.42%（1058/249 246），其中男性544例，年龄（50±11）岁；女性514例，年龄（52±11）岁。在组织病理学诊断的10种良性肿瘤中食管平滑肌瘤最常见为84.50%（894/1 058），其次为乳头状瘤6.90%（73/1 058），腺瘤最少为0.38%（4/1 058）。平滑肌瘤、间质瘤、神经纤维瘤以男性为主；脂肪瘤、颗粒细胞瘤、神经鞘瘤和血管瘤以女性为主。此外，本组发现的5例错构瘤全部发生于女性。以发生率≥50%为易发标准，青年男性易发食管良性瘤依次为平滑肌瘤和间质瘤，而青年女性依次为颗粒细胞瘤和脂肪瘤；老年男性依次为乳头状瘤、间质瘤和平滑肌瘤；老年女性依次为神经鞘瘤、乳头状瘤、平滑肌瘤、间质瘤和错构瘤；此外，男性脂肪瘤、血管瘤、神经纤维瘤和腺瘤，女性神经纤维瘤均发生在老年；不同年龄良性肿瘤总体发生率（P=0.034）和平滑肌瘤发生率（P=0.004）差异均具有统计学意义。本组平滑肌瘤、乳头状瘤、间质瘤和神经鞘瘤好发部位以中段为主，脂肪瘤以下段为主。本组良性肿瘤治疗以单纯手术为主为57.54%（492/855），其次为内镜下切除38.01%（325/855）和其他为4.45%（38/855）。n 结论食管良性肿瘤发生率低，组织类型较多，且不同性别和年龄患者易发类型明显不同，肿瘤组织类型不同，好发部位不同，手术及内镜治疗为主要治疗方式。