Adjacent Normal Esophageal Tissues Research Articles

Human leukocyte antigen‐G expression is associated with a poor prognosis in patients with esophageal squamous cell carcinoma

Human leukocyte antigen (HLA)-G inhibits functions of immune component cells and promotes malignant cells evading from antitumor immunity. We investigated the clinical relevance of HLA-G expression in esophageal squamous cell carcinoma (ESCC). In our study, HLA-G expression in 79 primary ESCC lesions and corresponding adjacent normal tissues were analyzed with immunohistochemistry. Soluble HLA-G (sHLA-G) in plasma was detected with enzyme-linked immunosorbent assay (ELISA) in 41 ESCC patients (including 19 case-matched lesions and plasma samples) and in 153 normal healthy controls. HLA-G expression was observed in 65.8% (52/79) of the ESCC lesions but not in adjacent normal esophageal tissues. HLA-G expression was more frequently observed in patients with advanced disease stage (III/IV vs. I/II, p = 0.01). Patients with HLA-G expression had a significantly worse survival, and HLA-G could be an independent prognostic factor. sHLA-G levels in plasma were significantly increased in patients compared to normal controls (median: 152.4 U/ml vs. 8.9 U/ml, p < 0.001). The area under receiver-operating characteristic (ROC) curve for sHLA-G in plasma was 0.992. However, no significant correlation was found between sHLA-G in plasma and clinical parameters studied. In conclusion, our findings indicated that HLA-G expression in ESCC is associated with poor survival and could be a prognostic indicator. Furthermore, increased levels of sHLA-G in plasma might be a useful preoperative biomarker for diagnosis.

Proteomic identification of differentially-expressed proteins in esophageal cancer in three ethnic groups in Xinjiang

The Objective is to identify candidate biomarkers for Squamous cell carcinoma (ESCC) in three ethnics in Xinjiang as well as reveal molecular mechanism. Proteins from 15 pairs of ESCC and matching adjacent normal esophageal tissues (five pairs in each ethnic of Kazakh, Uygur and Han) were separated by 2-DE and differentially proteins were identified by matrix-assisted laser desorption/ionization time-of-flight MS. After identified by Mascot database, some of interesting proteins were confirmed in the other 175 pairs of ESCC by immuno histochemistry. Comparison of patterns revealed 20 proteins significantly changed, of which 12 protein with concordantly increased, such as ACTB protein, COMT protein, Syntaxin binding protein Pyruvate Kinase (PKM2), Cathepsin D, Chromosome 1 open reading frame 8, Heat shock protein 27, Cdc42, Proteosome, LLDBP, Immunoglobulin, TNF receptor associated factor 7; and eight protein spots with concordantly decreased intensity in ESCC, such as Adenylate kinase 1, General transcription factor II H, Smooth muscle protein, Trangelin, Early endosome antigen 1, Annexin A2, Fibrin beta, Tropomyosin. There were a significant difference in protein overexpression of PKM2 (74.9%) and Cathepsin D (85.1%) in ESCC compared to adjacent tissues (P < 0.05) by immunohistochemistry. Further, overexpression of Cathepsin D was obvious difference in Hazakh ESCC (94.7%) than that in Uygur (78.6%) (P < 0.05). Interestingly, the overexpression of Cathepsin D was reversely associated with ESCC differentiation (P < 0.05). Twenty proteins differentially-expressed between ESCC and normal tissues were identified. Cathepsin D and PKM2 were for the first time observed to be dysregulated in Kazakh's ESCC. Moreover, Cathepsin D may play a complicated role both in carcinogenesis and cell-differentiation of ESCC.

Abstract A23: Allele specific regulation of esophageal cancer related gene 2 by miR-1322 in esophageal squamous cell carcinoma

Abstract Background and Aim: TCA3/TCA4 Short Tandem Repeat (STR) polymorphism in 3’ untranslated regions of esophageal cancer related gene 2 (ECRG2) was reported to be associated with esophageal squamous cell carcinoma (ESCC) incidences and the prognosis of ESCC patients. This study is to explore the mechanisms of TCA3 allele as a risk factor and poor prognosis indicator rather than TCA4 allele mainly in the functional relevant of microRNAs regulation in ESCC. Methods: Two genotypes of luciferase reporter plasmids containing ECRG2 3’UTR (TCA3 allele and TCA4 allele respectively) were constructed and expression were compared between the two alleles in three esophageal cancer cell lines: EC9706, EC109 and KYSE150. Bioinformatics predictions of microRNAs binding were performed by software TargetScan5.1, MicroInspector, RNA22 and RNAcofold. Dual-Luciferase reporter assay with or without microRNAs mimics or inhibitors were conducted to verify the effects of predicted microRNAs on expression of different alleles. The different expression of identified microRNAs in cancer tissues and the adjacent normal esophageal tissues from 44 ESCC patients were determined by Real-Time PCR. Results: Dual luciferase reporter assays indicated that expression level of TCA4 allele is 2-4 folds higher than TCA3 allele in all the three esophageal cancer cell lines. Bioinformatics analysis by software TargetScan5.1, MicroInspector, RNA22 and RNAcofold showed 4 potential microRNAs (miR-580, miR-1182, miR-1272 and miR-1322) binding at the TCA polymorphism site of the 3’UTR of ECRG2. Dual luciferase reporter assays indicated that miR-1182 and miR-1272 is not able to regulate either TCA3 or TCA4 allele in all three cell lines, however, miR-1322 and miR-580 dramatically down-regulated TCA3 allele expression (P &amp;lt;0.01) whereas slightly regulated TCA4 allele (P&amp;gt;0.05). The expression of miR-580 were under the detection threshold of real-time PCR analysis in most of the 44 paired tissue samples but the expression of miR-1322 were detectable, and 28 cases (63.6%) had at least 2-fold up regulation in their cancer tissues. No statistical differences were found between metastasis and non-metastasis and in different stage of disease. Conclusions: The differential regulation of TCA3 and TCA4 allele by miR-1322 might explain the difference in cancer risk and prognosis for different genotype carriers. MiR-1322 has an important role in human ESCC by regulating tumor suppressor gene ECRG2 and miR-1322 was up regulated in esophageal cancer, which itself might severs as a tumor promoter and potential marker and therapeutic target for esophageal squamous cell carcinoma. Citation Information: Cancer Prev Res 2010;3(12 Suppl):A23.

Effect of N-cadherin knock-down on invasiveness of esophageal squamous cell carcinoma

e15571 Background: Cell adhesion molecules are of crucial importance in cancer invasion and metastasis. Epithelial to mesenchymal transition, characterized by reduced E-cadherin and increased N-cadherin expression, has been recognized as a feature of aggressive tumors, but the importance of this phenotype has not been settled in esophageal squamous cell carcinoma. Aim: To examine the expressions of N-cadherin and E-cadherin in 62 normal esophageal epithelium specimens, 31 adjacent atypical hyperplasia epithelium specimens and 62 esophageal squamous cell carcinoma specimens, and to investigate the roles of N-cadherin in the invasiveness of esophageal squamous cell carcinoma cell line EC9706 transfected by N-cadherin shRNA.. Methods: PV immunohistochemistry was used to detect the expression pattern of N-cadherin and E-cadherin in 62 normal esophageal epithelium specimens, 31 adjacent atypical hyperplasia epithelium specimens and 62 esophageal squamous cell carcinoma specimens. The invasiveness of esophageal squamous cell carcinoma cell line EC9706 in vitro and in vivo was determined by transwell assay and nude mice experiments after EC9706 was transfected by N-cadherin shRNA. Results: The positive rates of N-cadherin decreased in the sequence of carcinoma, adjacent atypical hyperplasia and normal esophageal tissue, which were 75.8%, 61.3%, 29.0% (P &lt; 0.05), respectively, while those of E-cadherin increased in sequence, which were 40.3%, 71.0% and 95.2% (P &lt; 0.05). The increased expression of N-cadherin and decreased expression of E-cadherin were related to the invasion, differentiation, and lymph node metastasis (P &lt; 0.05). The expression level of N-cadherin decreased in the N- cadherin knocked down cells, and the invasiveness of those cells decreased significantly as well in vitro and in vivo. Conclusions: These results suggest that N-cadherin is an important factor in the invasiveness of esophageal squamous cell carcinoma and N-cadherin may serves as a potential molecular target for biotherapy of esophageal squamous cell carcinoma. No significant financial relationships to disclose.

N-cadherin knock-down decreases invasiveness of esophageal squamous cell carcinoma in vitro

To examine the expressions of N-cadherin and E-cadherin in specimens of 62 normal esophageal epithela, 31 adjacent atypical hyperplastic epithelia and 62 esophageal squamous cell carcinomas (ESCCs), and to investigate the roles of N-cadherin in the invasiveness of ESCC cell line EC9706 transfected by N-cadherin shRNA. PV immunohistochemistry was used to detect the expression pattern of N-cadherin and E-cadherin in specimens of 62 normal esophageal epithelia, 31 adjacent atypical hyperplastic epithelia and 62 ESCCs. The invasiveness of ESCC line EC9706 was determined by transwell assay after EC9706 was transfected by N-cadherin shRNA. The positive rates of N-cadherin decreased in the carcinoma, adjacent atypical hyperplastic and normal esophageal tissues (75.8%, 61.3% and 29.0%, P < 0.05), respectively, while those of E-cadherin increased (40.3%, 71.0% and 95.2%, P < 0.05). The increased expression of N-cadherin and decreased expression of E-cadherin were related to invasion, differentiation, and lymph node metastasis (P < 0.05). The expression level of N-cadherin decreased in the N-cadherin knocked down cells, and the invasiveness of those cells decreased significantly as well. The number of cells which crossed the basement membrane filter decreased from 123.40 +/- 8.23 to 49.60 +/- 6.80 (P < 0.05). E-cadherin and N-cadherin expression is correlated with the invasion and aggravation of ESCC. The down-regulation of N-cadherin lowers the invasiveness of EC9706 cell line.

Proteomic profiling of proteins dysregulted in Chinese esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is one of the leading causes of cancer death in China. In the present study, proteins in tumors and adjacent normal esophageal tissues from 41 patients with ESCC were extracted, and two-dimensional electrophoresis (2-DE) was performed using the pH 3-10 and 4-7 immobilized pH gradient strips. The protein spots expressed differentially between tumors and normal tissues were identified by matrix-assisted laser desorption/ionization and liquid chromatography electrospray/ionization ion trap mass spectrometry. A total of 22 proteins differentially expressed between ESCC and normal esophageal tissues were identified, in which 17 proteins were upregulated and 5 downregulated in tumors. Biological functions of these proteins are related to cell signal transduction, cell proliferation, cell motility, glycolysis, regulation of transcription, oxidative stress processes, and protein folding. Some of the proteins obtained were confirmed by Western blotting and immunohistochemical staining. We showed that high expression of calreticulin and 78-kDa glucose-regulated protein (GRP78) were correlated with poor prognosis by Kaplan-Meier analysis and log rank analysis. Zinc finger protein 410, annexin V, similar to the ubiquitin-conjugating enzyme E2 variant 1 isoform c, mutant hemoglobin beta chain, TPM4-ALK fusion oncoprotein type 2, similar to heat shock congnate 71-kDa protein, GRP78, and pyruvate kinase M2 (M2-PK) were for the first time observed to be dysregulated in human ESCC tissues. The proteins here identified will contribute to the understanding of the tumorigenesis and progression of Chinese ESCC and may potentially provide useful markers for diagnosis or targets for therapeutic intervention and drug development.

Proliferation potential-related protein, an ideal esophageal cancer antigen for immunotherapy, identified using complementary DNA microarray analysis.

To establish effective antitumor immunotherapy for esophageal cancer, we tried to identify an useful target antigen of esophageal cancer. We did cDNA microarray analysis to find a novel candidate antigen, proliferation potential-related protein (PP-RP). We examined cytotoxicity against tumor cells in vitro and in vivo of CTLs specific to PP-RP established from esophageal cancer patients. In 26 esophageal cancer tissues, an average of relative ratio of the expression of the PP-RP mRNA in cancer cells versus adjacent normal esophageal tissues was 396.2. Immunohistochemical analysis revealed that, in 20 of the 22 esophageal cancer tissues, PP-RP protein was strongly expressed only in the cancer cells and not so in normal esophageal epithelial cells. PP-RP protein contains 10 epitopes recognized by HLA-A24-restricted CTLs. These CTLs, generated from HLA-A24-positive esophageal cancer patients, had cytotoxic activity against cancer cell lines positive for both PP-RP and HLA-A24. Furthermore, adoptive transfer of the PP-RP-specific CTL line inhibited the growth of a human esophageal cancer cell line engrafted in nude mice. The expression of PP-RP in esophageal cancer cells was significantly higher than in normal cells, and the CTLs recognizing PP-RP killed tumor cells in vitro and also showed tumor rejection effects in a xenograft model. Therefore, PP-RP may prove to be an ideal tumor antigen useful for diagnosis and immunotherapy for patients with esophageal cancer. cDNA microarray analysis is a useful method to identify ideal tumor-associated antigens.