Shutian Zhang

Integrated Analysis Identifies Molecular Signatures and Specific Prognostic Factors for Different Gastric Cancer Subtypes

BACKGROUND: Gastric cancer (GC) is the fifth leading cause of cancer-related deaths worldwide. As an effective and easily performed method, microscopy-based Lauren classification has been widely accepted by gastrointestinal surgeons and pathologists for GC subtyping, but molecular characteristics of different Lauren subtypes were poorly revealed. METHODS: GSE62254 was used as a derivation cohort, and GSE15459 was used as a validation cohort. The difference between diffuse and intestinal GC on the gene expression level was measured. Gene ontology (GO) enrichment analysis was performed for both subgroups. Hierarchical clustering and heatmap exhibition were also performed. Kaplan-Meier plot and Cox proportional hazards model were used to evaluate survival grouped by the given genes or hierarchical clusters. RESULTS: A total of 4598 genes were found differentially expressed between diffuse and intestinal GC. Immunity- and cell adhesion–related GOs were enriched for diffuse GC, whereas DNA repair– and cell cycle–related GOs were enriched for intestinal GC. We proposed a 40-gene signature (χ2 = 30.71, P < .001) that exhibits better discrimination for prognosis than Lauren classification (χ2 = 12.11, P = .002). FRZB [RR (95% CI) = 1.824 (1.115-2.986), P = .017] and EFEMP1 [RR (95% CI) = 1.537 (0.969-2.437), P = .067] were identified as independent prognostic factors only in diffuse GC but not in intestinal GC patients. KRT23 [RR (95% CI) = 1.616 (0.938-2.785), P = .083] was identified as an independent prognostic factor only in intestinal GC patients but not in diffuse GC patients. Similar results were achieved in the validation cohort. CONCLUSION: We found that GCs with different Lauren classifications had different molecular characteristics and identified FRZB, EFEMP1, and KRT23 as subtype-specific prognostic factors for GC patients.

Regulation of Desmocollin3 Expression by Promoter Hypermethylation is Associated with Advanced Esophageal Adenocarcinomas

BACKGROUND: Desmocollin3 (DSC3) is a member of the cadherin superfamily of calcium-dependent cell adhesion molecules and plays an important role in tumor invasion and metastasis. In this study, we investigated the epigenetic mechanism that regulates DSC3 expression in esophageal adenocarcinomas (EACs). METHODS: Expression of DSC3 was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). The promoter DNA methylation level of DSC3 was examined using quantitative bisulfite pyrosequencing. RESULTS: The qRT-PCR analysis demonstrated significant down-regulation of the DSC3 mRNA levels in human EAC cell lines and tissue samples (P<.001). In addition, the EAC cell lines and tumor samples have aberrant promoter hypermethylation as compared to normal esophageal samples (P<.001). DSC3 promoter hypermethylation (>10% methylation level) was detected in 97.5% (39/40) of EAC samples whereas none of the normal tissue samples showed hypermethylation (P<.0001). There was a significant inverse correlation between promoter DNA methylation levels and mRNA expression folds for DSC3 (coefficient r=-0.685, P<.0001). Treatment of FLO-1 and SKGT4 EAC cells with 5-Aza-deoxytidine led to a significant reduction in the promoter DNA methylation levels with restoration of the DSC3 expression, suggesting that promoter DNA methylation is a key epigenetic mechanism regulating DSC3 expression. High DSC3 promoter DNA methylation levels were significantly correlated with advanced tumor stage (P<.001) and lymph node metastasis (P<.001). CONCLUSION: Taken together, our results demonstrate that epigenetic silencing of DSC3 is a frequent finding in EAC that is possibly associated with advanced stages.

Abnormal Localization and Tumor Suppressor Function of Epithelial Tissue-Specific Transcription Factor ESE3 in Esophageal Squamous Cell Carcinoma

Esophageal cancer is one of the most common malignant cancers worldwide. The molecular mechanism of esophageal squamous cell carcinoma (ESCC) is still poorly understood. ESE3 is a member of the Ets transcription family, which is only expressed in epithelial tissues and acts as a tumor suppressor gene in prostate cancer. Our study aim was to confirm whether ESE3 is involved in the carcinogenesis of ESCC. Immunohistochemical analysis revealed that ESE3 was mainly located in cell nuclei of normal tissues and the cytoplasm in ESCC tissues. Immunofluorescence and western blot analyses of the normal esophageal cell line HEEpiC and ESCC cell lines EC9706 TE-1, KYSE150, and KYSE410 confirmed these results. pEGFP-ESE3 and pcDNA3.1-V5/HisA-ESE3 plasmids were constructed for overexpression of ESE3 in EC9706 and KYSE150 cells. The stably transfected cells showed restoration of the nuclear localization of ESE3. EC9706 cells with re-localization of ESE3 to the nucleus showed inhibition of proliferation, colony formation, migration, and invasion. To explore the possible mechanism of the differences in localization of ESE3 in normal esophageal cells and ESCC cells, ESCC cell lines were treated with the nuclear export inhibitor leptomycin B, transcription inhibitor actinomycin D, PKC inhibitor sphinganine, P38 MAPK inhibitor SB202190, and CK II inhibitor TBCA. These reagents were chosen according to the well-known mechanisms of protein translocation. However, the localization of ESE3 was unchanged after these treatments. The sequence of ESE3 cDNA in ESCC cells was identical to the standard sequence of ESE3 in the NCBI Genebank database, indicating that there was no mutation in the coding region of ESE3 in ESCC. Taken together, our study suggests that ESE3 plays an important role in the carcinogenesis of ESCC through changes in subcellular localization and may act as a tumor suppressor gene in ESCC, although the mechanisms require further study.

The motility of esophageal sphincters during liquid and solid bolus swallows: a multicenter normative value study of high-resolution manometry in China.

It is gradually accepted that solid bolus swallow needs to be added to the procedure of manometry. The motility differences in the upper esophageal sphincter (UES) and lower esophageal sphincter (LES) were not well described. Sierra Scientific Instruments solid‐state high‐resolution manometry (HRM) system, the most popular HRM system in China, lacks the Chinese normative values for both liquid and solid bolus swallow parameters.

RGD conjugated, Cy5.5 labeled polyamidoamine dendrimers for targeted near-infrared fluorescence imaging of esophageal squamous cell carcinoma

The early detection of esophageal squamous cell carcinoma (ESCC), one of the most common human neoplasms, is of great importance in improving prognosis. However, there is a lack of screening methods with high sensitivity for early diagnosis of ESCC. In this study, we developed a αvβ3 integrin targeted near-infrared (NIR) fluorescence nanoprobe by conjugation of NIR dye Cy5.5 and tumor vasculature targeted cyclic RGDfK peptide onto a polyamidoamine (PAMAM) dendrimer (RGD–PEG–PAMAM–Cy5.5). The applicability of the as-prepared targeted nanoprobe for NIR fluorescence imaging of 4-NQO induced large and tiny esophageal neoplasms in mice was examined. It was demonstrated that the NIR fluorescence imaging with our targeted nanoprobe enables the identification of ESCC at an early stage.

Factors associated with gastric adenocarcinoma and dysplasia in patients with chronic gastritis: a population-based study

Objective Gastric cancer (GC) is one of the leading causes of death in China and other Asian countries. Recently, gastric endoscopy has become the main approach for GC screening, but the identification of high-risk individuals remains a challenge in GC screening programs. Methods There were 7,302 patients with chronic gastritis involved in this study. Endoscopic examinations were performed, and their demographic characteristics and lifestyle data were collected. Each possible associated factor of GC/premalignant and precursor lesions was evaluated by univariate and multivariate logistic regressions. Nomograms were used for visualization of those models, and receiver operating characteristic (ROC) curve analysis was used to present the predictive accuracy. Results We detected 8 (0.11%) gastric adenocarcinomas, 17 (0.23%) dysplasia cases, 14 (0.19%) hyperplasia cases, 52 (0.71%) intestinal metaplasia cases, 217 (2.97%) inflammatory lesions, 141 (1.93%) gastric ulcers, 10 (0.14%) atrophic gastritis cases, 1,365 (18.69%) erosive gastritis cases, and 5,957 (81.58%) superficial gastritis cases in 7,302 patients. The age (P<0.001), gender (P=0.086), labor intensity (P=0.018) and leek food intake (P=0.143) were identified as independent predictive factors of GC/premalignant lesions possibility. The corresponding nomogram exhibited an area under the curve (AUC) [95% confidence interval (95% CI)] of 0.82 (0.74–0.89) for the modeling group and 0.80 (0.75–0.85) for the validation group. The age (P=0.002), gender (P=0.024), smoking (P=0.002) and leek food intake (P=0.039) were independent predictive factors of precursor lesions possibility. The corresponding nomogram exhibited an AUC (95% CI) of 0.62 (0.60–0.65) for the modeling group and 0.61 (0.59–0.63) for the validation group. Conclusions We identified several potential associated factors and provided a preclinical nomogram with the potential to predict the possibility of GC/premalignant and precursor lesions.

Loss of sonic hedgehog gene leads to muscle development disorder and megaesophagus in mice

Sonic hedgehog (Shh) is crucial for organogenesis in the foregut. This study investigated the function of Shh at the late‐gestational stage; during which, the esophagus continues to differentiate. We established cytokeratin 14 (CK14)‐Cre;Shhfl/fl mice in which the down‐regulation of Shh in the epithelium occurred at approximately the same time as esophageal muscle conversion. Hematoxylin and eosin and immunohistochemical staining, with antibodies against keratin 14, Shh, patched 1 (Ptch1), Gli1, proliferating cell nuclear antigen (PCNA), α‐smooth muscle actin (αSMA), high‐molecular‐weight caldesmon (hCD), myogenin, paired box 7 (Pax7), β3‐tubulin, and protein gene product 9.5 (PGP9.5), was performed to detect specific tissue dysplasia. Organ culture was conducted in vitro, and total mRNA was extracted to determine the transcriptional dysregulation. The esophagus of CK14‐Cre; Shhfl/fl mice developed into an independent tube with an obvious dilatation at postnatal d 0.5. The number of cell layers and the expression of PCNA were decreased in mutant mice, compared with those in wild‐type mice. The expression of hCD declined progressively in the middle, distal, and lower esophageal sphincter levels of the mutant esophagus from embryonic d 17.5, compared with the expression in wild‐type littermates. Pax7 accumulation and myogenin reduction in mutant mice indicated that esophageal skeletal‐myoblast progression was blocked. RNA sequencing analysis revealed a significant down‐regulation of genes involved in proliferation and muscular motivation in CK14‐Cre;Shhfl/fl mice. Thus, loss of Shh at the late‐ gestational stage leads to megaesophagus with reduced proliferation and a muscle development disorder in mice. mice.—Jia, X., Min, L., Zhu, S., Zhang, S., Huang, X. Loss of sonic hedgehog gene leads to muscle development disorder and megaesophagus in mice. FASEB J. 32, 5703–5715 (2018). www.fasebj.org

Cadherin Expression Shift Could Well Distinguish Esophageal Squamous Cell Carcinoma from Non- Cancerous Esophageal Tissues

Background: Biomarkers for esophageal squamous cell carcinoma (ESCC) identification with high sensitivity are not well established. Since abnormal expression of cadherins has been widely reported in cancer, we explored its feasibility as an ESCC biomarker. Methods: Expression of E-cadherin and N-cadherin were detected in 150 esophageal tissues by immunohistochemistry. Staining strength and percentage in different subcellular structures of each specimen were evaluated by 2 independent pathologists. A logistic regression-based classifier derived from E-cadherin and N-cadherin staining was generated. Results: E-cadherin exhibited decreased membrane expression in ESCC, while N-cadherin exhibited decreased expression in the nucleus but elevated expression in the cytoplasm. Both E-cadherin and N-cadherin could distinguish ESCC tissues from non-cancerous tissues (area under the curve (AUC) = 0.748, 0.801, respectively). E-cadherin and N-cadherin staining scores could be merged into a cadherin (CDH) logistic index, which showed better discrimination (AUC = 0.909) than E-cadherin or N-cadherin alone. Further investigation indicated that the CDH logistic index was significantly correlated with tumor size and differentiation in ESCC. Conclusion: Both E-cadherin and N-cadherin had a strong expression shift in ESCC compared with non-cancerous tissues. The CDH logistic index, a parameter integrating the expression data of both cadherins, could be used as a marker with high sensitivity and specificity in the identification of ESCC.

Downregulation of NONO induces apoptosis, suppressing growth and invasion in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies in China, and is associated with high morbidity and mortality. However, the molecular mechanisms that control ESCC tumorigenicity and metastasis remain unclear. Here, we report that the RNA splicing factor, NONO, is an important regulator of ESCC growth, apoptosis and invasion. NONO protein levels were dramatically upregulated in ESCC when compared with that in adjacent benign esophageal squamous epithelium. Particularly, NONO expression was statistically higher in tumors with greater tumor invasion depth. Using multiple ESCC cell models, we further showed that NONO depletion using siRNA significantly inhibited proliferation, invasion, and promoted apoptosis of ESCC cells. In addition we found that knockdown of NONO could reduce protein levels of phosphorylated Akt and Erk1/2. Our findings suggest that NONO plays a potent role in multiple biological aspects of ESCC through activation of the Akt and Erk1/2 signaling pathways. Taken together, our findings suggest that NONO might play an important role in promoting tumorigenesis of ESCC. It may provide a promising approach to prevent the progress of ESCC.

Chronic stress and intestinal permeability: Lubiprostone regulates glucocorticoid receptor-mediated changes in colon epithelial tight junction proteins, barrier function, and visceral pain in the rodent and human

Chronic psychological stress is associated with increased intestinal epithelial permeability and visceral hyperalgesia. Lubiprostone, an agonist for chloride channel‐2, promotes secretion and accelerates restoration of injury‐induced epithelial barrier dysfunction. The mechanisms underlying how lubiprostone regulates colon epithelial barrier function and visceral hyperalgesia in chronic stress remain unknown.