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• W1965172023 abstract "In this study, we explored the possibility of SOX17 promoter region methylation as an esophageal cancer detection marker, the regulation of SOX17 expression, and the function of SOX17 in the WNT signaling pathway in esophageal cancer. Eight esophageal cancer cell lines, 9 normal esophageal mucosa samples, 60 cases of dysplasia, and 169 cancer tissue samples were included. Methylation-specific PCR, semiquantitative reverse transcription–PCR, immunohistochemistry, luciferase reporter assay, colony formation, and Western blot analysis were used to analyze methylation and function of SOX17 in esophageal cancer. MicroRNA-related detection methods were performed to evaluate microRNA regulation of SOX17. SOX17 methylation was found in progression tendency with 0% of normal mucosa, 39% of grade 1 dysplasia, 48% of grades 2 and 3 dysplasia, and 65% of primary cancer. SOX17 methylation is related to esophageal cancer patients' history of alcohol use and may induce β-catenin expression and redistribution. Loss of SOX17 expression is correlated to promoter region hypermethylation, and re-expression was activated by 5-aza-2′-deoxycytidine treatment in esophageal cancer cell lines. Restoration of SOX17 expression suppresses TCF/β-catenin–dependent transcription and colony formation. MicroRNA 141 was also found to down-regulate SOX17 expression and activate the WNT signal pathway. SOX17 is frequently methylated in esophageal cancer and in a progression tendency during esophageal carcinogenesis. Loss of SOX17 removes the normal inhibition of WNT signaling and promotes esophageal tumorigenesis. In this study, we explored the possibility of SOX17 promoter region methylation as an esophageal cancer detection marker, the regulation of SOX17 expression, and the function of SOX17 in the WNT signaling pathway in esophageal cancer. Eight esophageal cancer cell lines, 9 normal esophageal mucosa samples, 60 cases of dysplasia, and 169 cancer tissue samples were included. Methylation-specific PCR, semiquantitative reverse transcription–PCR, immunohistochemistry, luciferase reporter assay, colony formation, and Western blot analysis were used to analyze methylation and function of SOX17 in esophageal cancer. MicroRNA-related detection methods were performed to evaluate microRNA regulation of SOX17. SOX17 methylation was found in progression tendency with 0% of normal mucosa, 39% of grade 1 dysplasia, 48% of grades 2 and 3 dysplasia, and 65% of primary cancer. SOX17 methylation is related to esophageal cancer patients' history of alcohol use and may induce β-catenin expression and redistribution. Loss of SOX17 expression is correlated to promoter region hypermethylation, and re-expression was activated by 5-aza-2′-deoxycytidine treatment in esophageal cancer cell lines. Restoration of SOX17 expression suppresses TCF/β-catenin–dependent transcription and colony formation. MicroRNA 141 was also found to down-regulate SOX17 expression and activate the WNT signal pathway. SOX17 is frequently methylated in esophageal cancer and in a progression tendency during esophageal carcinogenesis. Loss of SOX17 removes the normal inhibition of WNT signaling and promotes esophageal tumorigenesis. Esophageal cancer is the eighth most common malignant tumor and the sixth most fatal disease all over the world.1Jemal A. Siegel R. Ward E. Hao Y. Xu J. Thun M.J. Cancer statistics, 2009.CA Cancer J Clin. 2009; 59: 225-249Crossref PubMed Scopus (9886) Google Scholar, 2Kamangar F. Dores G.M. Anderson W.F. Patterns of cancer incidence, mortality, and prevalence across five continents: defining priorities to reduce cancer disparities in different geographic regions of the world.J Clin Oncol. 2006; 24: 2137-2150Crossref PubMed Scopus (3034) Google Scholar, 3Pohl H. Welch H.G. The role of overdiagnosis and reclassification in the marked increase of esophageal adenocarcinoma incidence.J Natl Cancer Inst. 2005; 97: 142-146Crossref PubMed Scopus (1084) Google Scholar Although multiple genetic and epigenetic changes have been associated with esophageal cancer, its underlying molecular mechanism remains incompletely understood.4Guo M. House M.G. Suzuki H. Ye Y. Brock M.V. Lu F. Liu Z. Rustgi A.K. Herman J.G. Epigenetic silencing of CDX2 is a feature of squamous esophageal cancer.Int J Cancer. 2007; 121: 1219-1226Crossref PubMed Scopus (34) Google Scholar, 5Guo M. Liu S. Lu F. Gefitinib-sensitizing mutations in esophageal carcinoma.N Engl J Med. 2006; 354: 2193-2194Crossref PubMed Scopus (43) Google Scholar Carcinogenesis is a multifactorial process with the accumulation of genetic and epigenetic changes involving oncogenes and tumor suppressor genes. Methylation patterns are different in esophageal adenocarcinoma and squamous cell carcinoma. APC is frequently methylated in human esophageal adenocarcinoma, and CDX2 is a feature of human esophageal squamous cell carcinoma.4Guo M. House M.G. Suzuki H. Ye Y. Brock M.V. Lu F. Liu Z. Rustgi A.K. Herman J.G. Epigenetic silencing of CDX2 is a feature of squamous esophageal cancer.Int J Cancer. 2007; 121: 1219-1226Crossref PubMed Scopus (34) Google Scholar, 6Guo M. Ren J. House M.G. Qi Y. Brock M.V. Herman J.G. Accumulation of promoter methylation suggests epigenetic progression in squamous cell carcinoma of the esophagus.Clin Cancer Res. 2006; 12: 4515-4522Crossref PubMed Scopus (104) Google Scholar, 7Brock M.V. Gou M. Akiyama Y. Muller A. Wu T.T. Montgomery E. Deasel M. Germonpre P. Rubinson L. Heitmiller R.F. Yang S.C. Forastiere A.A. Baylin S.B. Herman J.G. Prognostic importance of promoter hypermethylation of multiple genes in esophageal adenocarcinoma.Clin Cancer Res. 2003; 9: 2912-2919PubMed Google Scholar Increasing evidence has shown that constitutive aberrant activation of the WNT/β-catenin signaling pathway is associated with human cancers, including esophageal tumorigenesis.8Kudo J. Nishiwaki T. Haruki N. Ishiguro H. Shibata Y. Terashita Y. Sugiura H. Shinoda N. Kimura M. Kuwabara Y. Fujii Y. Aberrant nuclear localization of beta-catenin without genetic alterations in beta-catenin or Axin genes in esophageal cancer.World J Surg Oncol. 2007; 5: 21Crossref PubMed Scopus (27) Google Scholar, 9Salahshor S. Naidoo R. Serra S. Shih W. Tsao M.S. Chetty R. Woodgett J.R. Frequent accumulation of nuclear E-cadherin and alterations in the Wnt signaling pathway in esophageal squamous cell carcinomas.Mod Pathol. 2008; 21: 271-281Crossref PubMed Scopus (60) Google Scholar SRY-box containing gene 17 (SOX17) is a member of the SRY-related high-mobility group (HMG)-box transcription factor superfamily. SOX17 is structurally related to T-cell transcription factor (TCF)/lymphoid enhancer factor (LEF), binding to target DNA sequence 5′-(A/T)(A/T)CAA(A/T)G-3′ and promoting degradation of β-catenin/TCF via a GSK3β-independent mechanism in the WNT signaling pathway.10Zhang W. Glockner S.C. Guo M. Machida E.O. Wang D.H. Easwaran H. Van Neste L. Herman J.G. Schuebel K.E. Watkins D.N. Ahuja N. Baylin S.B. Epigenetic inactivation of the canonical Wnt antagonist SRY-box containing gene 17 in colorectal cancer.Cancer Res. 2008; 68: 2764-2772Crossref PubMed Scopus (154) Google Scholar SOX17 was reported to be frequently methylated in human cancers, but the function and epigenetic changes in esophageal cancer and early lesions were not studied before.10Zhang W. Glockner S.C. Guo M. Machida E.O. Wang D.H. Easwaran H. Van Neste L. Herman J.G. Schuebel K.E. Watkins D.N. Ahuja N. Baylin S.B. Epigenetic inactivation of the canonical Wnt antagonist SRY-box containing gene 17 in colorectal cancer.Cancer Res. 2008; 68: 2764-2772Crossref PubMed Scopus (154) Google Scholar, 11Jia Y. Yang Y. Liu S. Herman J.G. Lu F. Guo M. SOX17 antagonizes WNT/beta-catenin signaling pathway in hepatocellular carcinoma.Epigenetics. 2010; 5: 743-749Crossref PubMed Scopus (109) Google Scholar A total of 169 cases of primary esophageal squamous cell cancer were studied in this study, including 127 male cases and 42 female cases. Snap-frozen, fresh tissue samples were collected by surgical resection. All samples were classified by TNM (Union for International Cancer Control, 2009) staging, with 5 cases of stage I, 110 cases of stage II, 53 cases of stage III, and 1 case of stage IV. Sixty of these cases had alcohol and smoking use records, and 121 cases had differentiation, tumor size, or metastasis description. Thirty-nine cases of low-grade dysplasia, 12 cases of intermediate-grade dysplasia, and 9 cases of high-grade dysplasia were collected as paraffin-embedded samples. Samples from 9 cases of normal esophageal epithelia were collected by endoscopy biopsy with snap-frozen tissue. Matched adjacent tissue was available in 39 cases, with paraffin blocks for 169 primary cancer samples. All samples were collected under the guidelines approved by the Chinese PLA General Hospital's institutional review board and with written informed consent from patients. All esophageal cancer cell lines were previously established from primary esophageal cancer, and maintained in 90% RPMI 1640 (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum.4Guo M. House M.G. Suzuki H. Ye Y. Brock M.V. Lu F. Liu Z. Rustgi A.K. Herman J.G. Epigenetic silencing of CDX2 is a feature of squamous esophageal cancer.Int J Cancer. 2007; 121: 1219-1226Crossref PubMed Scopus (34) Google Scholar, 12Guo M. House M.G. Akiyama Y. Qi Y. Capagna D. Harmon J. Baylin S.B. Brock M.V. Herman J.G. Hypermethylation of the GATA gene family in esophageal cancer.Int J Cancer. 2006; 119: 2078-2083Crossref PubMed Scopus (57) Google Scholar Esophageal cancer cell lines (SKGT4, YSE2, KYSE30, KYSE70, KYSE140, KYSE150, KYSE180, and KYSE450) were split to low density (30% confluence) 12 hours before treatment. Cells were treated with 5-aza-2′-deoxycytidine (DAC; Sigma, St. Louis, MO) at a concentration of 2 μmol/L. Growth medium, conditioned with DAC at 2 μmol/L, was exchanged every 24 hours for a total of 96 hours of treatment. Total RNA was isolated by Trizol reagent (Life Technologies, Gaithersburg, MD). First-strand cDNA was synthesized according to the manufacturer instruction's (Invitrogen). PCR amplification of SOX17 was performed using primers: 5′-GGCTGGCGCAGCAGAATC-3′ (forward) and 5′-AGCCCTGCTCGGGGAACT-3′ (reverse). Amplified products were analyzed on 1.5% agarose gels. A total of 32 cycles of amplification were performed for each of the reverse transcription–PCR (RT-PCR) experiments. As an internal control, GAPDH was amplified with 25 cycles to ensure cDNA quality and quantity for each RT-PCR. DNA was prepared by the proteinase K method. The bisulfite treatment was performed as described in a previous report.13Herman J.G. Graff J.R. Myohanen S. Nelkin B.D. Baylin S.B. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands.Proc Natl Acad Sci U S A. 1996; 93: 9821-9826Crossref PubMed Scopus (5225) Google Scholar Methylation-specific PCR (MSP) and nested MSP were performed as described previously.6Guo M. Ren J. House M.G. Qi Y. Brock M.V. Herman J.G. Accumulation of promoter methylation suggests epigenetic progression in squamous cell carcinoma of the esophagus.Clin Cancer Res. 2006; 12: 4515-4522Crossref PubMed Scopus (104) Google Scholar MSP primers were designed according to genomic sequences flanking presumed translation start sites and are as follows: 5′-GGGGCGTTCGTAGTGTTATTAGGTC-3′ (M-forward); 5′-AAACACTAAAATACCCCGAAAACTACG-3′ (M-reverse); 5′-TTAGGGGTGTTTGTAGTGTTATTAGGTT-3′ (U-forward) and 5′-TAAAACACTAAAATACCCCAAAAACTACA-3′ (U-reverse). The external primer sequences of nested MSP were as follows: 5′-AGAAAAGGTTTYGYGGTTTAGG-3′ (nest-forward) and 5′-TAAAAAATAAAACACTAAAATACCC-3′ (nest-reverse). Bisulfite-treated DNA was subjected to PCR using bisulfite sequencing (BSSQ) primers flanking the targeted MSP regions. Sequencing primers were designed as follows: 5′-GTTTTATTGGTTATATTTGTGTAG-3′ (BSSQ-forward) and 5′-ACTACTCATAACRCTCCAAAC-3′ (BSSQ-reverse). Bisulfite sequencing was performed as described in a previous report.11Jia Y. Yang Y. Liu S. Herman J.G. Lu F. Guo M. SOX17 antagonizes WNT/beta-catenin signaling pathway in hepatocellular carcinoma.Epigenetics. 2010; 5: 743-749Crossref PubMed Scopus (109) Google Scholar β-Catenin antibody (ZSGB Biotech, Beijing, China) and SOX17 antibody (OriGene Technologies, Rockville, MD) were used in this study. Immunohistochemistry was performed as previously described.11Jia Y. Yang Y. Liu S. Herman J.G. Lu F. Guo M. SOX17 antagonizes WNT/beta-catenin signaling pathway in hepatocellular carcinoma.Epigenetics. 2010; 5: 743-749Crossref PubMed Scopus (109) Google Scholar The χ2 test was used to analyze the correlation between SOX17 methylation and location of β-catenin and SOX17; P < 0.05 was considered statistically significant. Full-length SOX17 cDNA was cloned as described previously.10Zhang W. Glockner S.C. Guo M. Machida E.O. Wang D.H. Easwaran H. Van Neste L. Herman J.G. Schuebel K.E. Watkins D.N. Ahuja N. Baylin S.B. Epigenetic inactivation of the canonical Wnt antagonist SRY-box containing gene 17 in colorectal cancer.Cancer Res. 2008; 68: 2764-2772Crossref PubMed Scopus (154) Google Scholar To generate microRNA (miRNA) expression vector, a genomic fragment of Homo sapiens miRNA precursor was amplified and cloned into pcDNA3.0 (Invitrogen). SOX17 3′-untranslated region (3′-UTR) was also generated by PCR with primers designed depending on bioinformatics websites. The PCR product was subcloned into pGL3 control vector with XbaI and NdeI sites (Promega) immediately downstream of the stop codon of the luciferase reporter gene. Mutant construct of 3′-UTR of SOX17 mRNA was generated by PCR to disrupt the microRNA 141 (miR-141) binding site. KYSE140 cells were grown in six-well culture plates 24 hours before transfection. Cells were transfected with empty control vector or SOX17 expression construct according to the manufacturer's instructions (Roche Applied Science, Penzberg, Germany). Colony formation assay was performed as reported before.11Jia Y. Yang Y. Liu S. Herman J.G. Lu F. Guo M. SOX17 antagonizes WNT/beta-catenin signaling pathway in hepatocellular carcinoma.Epigenetics. 2010; 5: 743-749Crossref PubMed Scopus (109) Google Scholar KYSE140 cells were seeded at 5 × 104 cells/well in 24-well culture plates 24 hours before transfection. To examine transcriptional activity driven by β-catenin/TCF, KYSE140 cells were transfected with 200 ng/well pGL3-OT (TCF/LEF-responsive reporter),14Shih I.M. Yu J. He T.C. Vogelstein B. Kinzler K.W. The beta-catenin binding domain of adenomatous polyposis coli is sufficient for tumor suppression.Cancer Res. 2000; 60: 1671-1676PubMed Google Scholar 30 ng/well pRL-TK control vector (Promega) as an internal control reporter, and 600 ng/well β-catenin construct. Basal transcriptional activity in KYSE140 cells was tested only by transfection with empty vectors. KYSE140 cells were then transfected with 100 ng/well pGL3-OT, 10 ng/well pRL-TK, 150 ng/well β-catenin constructs, and 200 ng/well SOX17 constructs (wild type and mutant). Thereafter, 100 ng/well pGL3-OT, 10 ng/well pRL-TK, 100 ng/well β-catenin expression vector, and increased amounts of pcDNA3.1/V5-SOX17-His B wild-type, mutant (30 ng, 90 ng, and 120 ng, SOX17 constructs 50–414, 135–414, and 1–353, respectively), or empty vector were transfected into KYSE140 cells with FuGENE 6 (Roche Applied Science). pGL3-SOX17-3′-UTR vector, which contains the 3′-UTR of the SOX17 gene and the firefly luciferase reporter, was cotransfected at 125 ng/well with 1 ng/well pRL-CMV control vector (Promega) and different miRNA expression vectors (pcDNA3.0-miRNA-26a, −26b, −141, −151, −200a, −329, and −362) or empty vector, respectively, at 375 ng/well using FuGENE 6 (Roche Applied Science); 1 ng/well pRL-CMV vector control (Promega) and 125 ng/well wild-type pGL3-SOX17-3′-UTR or 125 ng/well pGL3-SOX17-3′-UTR mutant were cotransfected with 375 ng/well pcDNA3.0-miRNA-141 in KYSE140 cells by FuGENE 6 (Roche Applied Science). Relative luciferase activity was measured according to the manufacturer's instructions (GloMax luminometer; Promega) 48 hours after transfection. KYSE140 cells were transfected with SOX17 constructs (wild type and mutant). YSE2 cells were transfected with miRNA mimics or miRNA control. Forty-eight hours later, transfected cells were harvested for Western blotting according to the manufacturer's instructions. For miRNA detection, total RNA was polyadenylated by poly(A) polymerase according to the manufacturer's protocol (Ambion, Austin, TX). Reverse transcription was performed using 1 μg of poly(A)-tailed total RNA and 1 μg of reverse transcription primer (5′-GCGAGCACAGAATTAATACGACTCACTATAGG(T)18VN-3′) with 1 μL ImPro-II Reverse Transcriptase (Promega) according to the manufacturer's protocol. Quantitative PCR was performed as described in the method of Quantitect SYBR Green PCR Kit (Qiagen, Hilden, Germany) with Mx3000p (Stratagene, La Jolla, CA). GenEX software, version 4.10 (TATAA Biocenter, Munich, Germany) was used to analyze real-time PCR results. Statistical analysis was performed using the χ2 test, and P < 0.05 was considered statistically significant. SOX17 expression level was determined using semiquantitative RT-PCR in esophageal cancer cell lines. SOX17 was expressed in the YSE2 cell line and weakly in the KYSE70, KYSE180, and KYSE450 cell lines. However, no expression was detected in the SKGT4, KYSE30, KYSE140, and KYSE150 cell lines. SOX17 gene promoter region was completely methylated in the SKGT4, KYSE30, KYSE140, and KYSE150 cell lines, which did not express this gene. Partial methylation and weak expression were detected in KYSE70, KYSE180, and KYSE450 cells. SOX17 is unmethylated and strongly expressed in the YSE2 cell line (Figure 1, A and B). The SOX17 promoter region CpG island distribution and methylation detection location are shown in Figure 1C, transcription start site is located in the CpG island. Two sets of primers were designed encompassing the transcription start site to perform MSP and BSSQ. The representative BSSQ of SOX17 was performed in the SKGT4, KYSE140, YSE2, and KYSE70 cell lines. Consistent with MSP results, BSSQ of 10 individual clones from SKGT4 and KYSE140 revealed dense methylation of CpGs within the promoter region. The mixed methylated and unmethylated results by bisulfite sequencing in KYSE70 may represent both methylated and unmethylated alleles, or both methylated and unmethylated clonal subpopulations within cultured cells. Result from YSE2 shows unmethylation status of CpG in the promoter region of SOX17. To further validate whether SOX17 expression is regulated by promoter region methylation, the effect of DAC on SOX17 expression was determined using semiquantitative RT-PCR in esophageal cancer cell lines. DAC is a DNA methylation transferase inhibitor.15Qin T. Jelinek J. Si J. Shu J. Issa J.P. Mechanisms of resistance to 5-aza-2′-deoxycytidine in human cancer cell lines.Blood. 2009; 113: 659-667Crossref PubMed Scopus (198) Google Scholar, 16Griffiths E.A. Gore S.D. DNA methyltransferase and histone deacetylase inhibitors in the treatment of myelodysplastic syndromes.Semin Hematol. 2008; 45: 23-30Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar After DAC treatment for 96 hours, SOX17 was re-expressed in the SKGT4, KYSE30, KYSE140, and KYSE150 cell lines. Increased expression of SOX17 was also detected in the KYSE70, KYSE180, and KYSE450 cell lines, which were partially methylated and had reduced expression compared to YSE2 (Figure 1A). These results suggest that SOX17 expression is silenced by promoter region methylation in esophageal cancer. To explore whether SOX17 is methylated during esophageal carcinogenesis, we examined 9 cases of normal esophageal mucosa, 60 cases of different grades of dysplasia, and 169 cases of primary esophageal cancer. A total of 109 of 169 (65%) cases of primary esophageal cancer were methylated (Figure 2A), and this appears to be a neoplasia-specific alteration, since all 9 cases of normal esophageal mucosa were unmethylated (Figure 2B). There was slightly more frequent methylation of SOX17 in late (stage III and IV) versus early stage esophageal cancer. To determine the place in carcinogenesis that methylation of SOX17 occurs, we examined dysplastic lesions. Twenty-five of 60 (42%) cases were methylated in esophageal dysplasia (Figure 2C), more frequently in grades 2 and 3 dysplasia (48%) than in grade 1 (39%), suggesting that methylation of SOX17 is associated with the progression of esophageal cancer and is a frequent and early event. In the 169 cases of esophageal cancer, 127 patients were male and 42 patients were female. The ratio of male/female is similar to the ratios observed in other high-incidence areas in the world. It was reported that alcohol is a major cause of squamous cell carcinoma in the United States and Europe.17Eslick G.D. Epidemiology of esophageal cancer.Gastroenterol Clin North Am. 2009; 38: 17-25Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 18Glickman J.N. Section II: pathology and pathologic staging of esophageal cancer.Semin Thorac Cardiovasc Surg. 2003; 15: 167-179Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 19Freedman N.D. Abnet C.C. Leitzmann M.F. Mouw T. Subar A.F. Hollenbeck A.R. Schatzkin A. A prospective study of tobacco, alcohol, and the risk of esophageal and gastric cancer subtypes.Am J Epidemiol. 2007; 165: 1424-1433Crossref PubMed Scopus (320) Google Scholar SOX17 methylation is significantly correlated with alcohol history in 60 patients with alcohol and smoking records in this study (χ2 test, P = 0.0057). No significant difference was found when grouped by differentiation, tumor size, or metastasis for the 121 cases with detailed descriptions. There were no significant differences among age, sex, smoking history, and TNM stages for SOX17 methylation (Table 1). As shown in Figure 2D, the SOX17 methylation rate increases with the progression of esophageal carcinogenesis from normal esophageal epithelia, grade 1, grade 2, and grade 3 dysplasia to advanced cancer (χ2 test, P = 0.0002). These results suggest that SOX17 gene promoter region methylation is a potential early detection marker of esophageal cancer.Table 1Clinicopathological Characteristics and SOX17 Methylation Status of 169 Patients with Esophageal CancerClinical parameterNo.SOX17 methylation statusP value⁎P values are obtained by the χ2 test. Statistical significance is indicated by P < 0.05.MethylatedUnmethylatedn = 109 (64.5%)n = 60 (35.5%)Age (years) <502216 (72.7%)6 (27.3%)0.3870 ≥5014793 (63.3%)54 (36.7%)Sex Male12785 (66.9%)42 (33.1%)0.2506 Female4224 (57.1%)18 (42.9%)Alcohol abuse†In 169 patients, 60 cases had alcohol and smoking records. Negative3118 (58.1%)13 (41.9%)0.0057 Positive2926 (89.7%)3 (10.3%)Smoking†In 169 patients, 60 cases had alcohol and smoking records. Negative3321 (63.6%)12 (36.4%)0.0604 Positive2723 (85.2%)4 (14.8%)Tumor size (cm)‡In 169 patients, 121 cases had pathological descriptions of tumor size, metastasis, and differentiation. <58662 (72.1%)24 (27.9%)0.6985 ≥53524 (68.6%)11 (31.4%)Differentiation‡In 169 patients, 121 cases had pathological descriptions of tumor size, metastasis, and differentiation. Poorly3727 (73.0%)10 (27.0%)0.3247 Moderately7450 (67.6%)24 (32.4%) Well109 (90.0%)1 (10.0%)Tumor stage I54 (80%)1 (20%)0.6387 II11068 (61.8%)42 (38.2%) III5336 (67.9%)17 (32.1%) IV11 (100%)0 (0%)Metastasis‡In 169 patients, 121 cases had pathological descriptions of tumor size, metastasis, and differentiation. Negative7451 (68.9%)23 (31.1%)0.5117 Positive4735 (74.5%)12 (25.5%)SOX17 methylation is significantly correlated with alcohol history in 60 cases with alcohol and smoking records in this study (χ2 test, P = 0.0057). P values are obtained by the χ2 test. Statistical significance is indicated by P < 0.05.† In 169 patients, 60 cases had alcohol and smoking records.‡ In 169 patients, 121 cases had pathological descriptions of tumor size, metastasis, and differentiation. Open table in a new tab SOX17 methylation is significantly correlated with alcohol history in 60 cases with alcohol and smoking records in this study (χ2 test, P = 0.0057). β-Catenin is reported to be widely distributed in cellular membrane, cytoplasm, and nucleus. Transcription of the WNT signaling pathway downstream genes may be activated by stabilized β-catenin accumulation in the cytoplasm and translocation to the nucleus. To explore the possible interaction of SOX17 and β-catenin, IHC was performed on 39 pairs of available paraffin samples (esophageal cancer and adjacent tissue). SOX17 methylation status was also already analyzed in these 9 cases of esophageal cancer. Thirty-three samples were methylated, and 6 samples were unmethylated. In 39 samples of adjacent tissue, 7 samples were methylated and 32 samples were unmethylated. Nuclear SOX17 staining was found in 28 samples of unmethylated adjacent esophageal tissue, but only 3 cases of esophageal cancer showed positive SOX17 nuclear staining (Figure 3A). Nuclear SOX17 staining inversely correlated to SOX17 methylation (χ2 test, P = 0.0359). In 33 samples of esophageal cancer, 10 cases positive for methylated SOX17 nuclear β-catenin were found, and cytoplasmic β-catenin staining was found in 26 cases (χ2 test, P = 0.0105) (Figure 3B). In six cases of SOX17 unmethylated esophageal cancer, β-catenin staining was different: two cases were cell membrane positive, three cases had cytoplasmic staining, and two cases were negative for staining. The results indicate SOX17 methylation is correlated with β-catenin expression in the nucleus and cytoplasm in esophageal cancer. β-Catenin was predominantly expressed in membrane, but no expression was found in the nucleus for the unmethylated adjacent noncancerous tissues (Figure 3A). SOX17 and nuclear β-catenin expression were significantly different in the matched esophageal cancer and adjacent tissues (χ2 test, P = 0.00001, P = 0.0011, respectively) (Figure 3B). SOX17 was mainly expressed in unmethylated adjacent tissues, whereas nuclear and cytoplasmic β-catenin was mainly expressed in methylated esophageal cancer tissues. These results indicate that the WNT/β-catenin signaling pathway may play an important role in esophageal cancer. To evaluate the inhibitory effect of SOX17 on clonogenicity of esophageal cancer cells, colony formation was assayed in KYSE140 cells. Re-expression of SOX17 was confirmed by Western blot after transfection of wild-type SOX17 expression vector. Colony formation was inhibited by restoration of SOX17 expression (Figure 4), suggesting that SOX17 may serve as a potential tumor suppressor during esophageal cancer development. In this study, the TCF/LEF luciferase reporter system was used to explore SOX17's effect on WNT/β-catenin and downstream genes. Relative luciferase activity was increased sharply in KYSE140 cells transfected with β-catenin expression vector compared with control cells that were transfected with empty control vector (Figure 5A). A schematic diagram of different structural deletions on the SOX17 gene is shown in Figure 5B. In the luciferase reporter assay, pGL3-OT vector is a TCF/LEF-responsive reporter containing three consensus TCF binding sites, and pRL-TK vector is used as system control.14Shih I.M. Yu J. He T.C. Vogelstein B. Kinzler K.W. The beta-catenin binding domain of adenomatous polyposis coli is sufficient for tumor suppression.Cancer Res. 2000; 60: 1671-1676PubMed Google Scholar, 20Korinek V. Barker N. Morin P.J. van Wichen D. de Weger R. Kinzler K.W. Vogelstein B. Clevers H. Constitutive transcriptional activation by a beta-catenin-Tcf complex in APC-/- colon carcinoma.Science. 1997; 275: 1784-1787Crossref PubMed Scopus (2927) Google Scholar After transfection with pGL3-OT, pRL-TK, β-catenin, and SOX17 expression vector or its different deletion segments, SOX17 fragment 135–414 with deletion in the HMG domain, showed no suppressive effect on β-catenin/TCF-LEF transcriptional activity. By contrast, the results of using other deletion segments, including SOX17 fragment 50–414 and fragment 1–353, were similar to wild-type SOX17 (Figure 5C). To further explore this inhibition of the regulatory effect of SOX17 HMG, increasing amounts of mutant or wild-type SOX17 vectors were transfected into KYSE140 cells separately. In groups that were transfected with SOX17 constructs with a HMG region fragment, the downstream transcriptional activity of β-catenin/TCF-LEF was suppressed in a dose-dependent manner (Figure 5D). Furthermore, Western blotting was performed to test the expression of downstream genes such as cyclinD1. CyclinD1 expression was detected in KYSE140 cells transfected with the SOX17 construct including fragment 135–414; however, expression of cyclinD1 was decreased in other groups (Figure 5E). Increasing evidence indicates that miRNAs play important roles during human cancer development. MicroRNAs negatively regulate gene expression by targeting mRNA 3′-UTR and degrading mRNA or repressing translation.21Bartel D.P. MicroRNAs: genomics, biogenesis, mechanism, and function.Cell. 2004; 116: 281-297Abstract Full Text Full Text PDF PubMed Scopus (29453) Google Scholar To clarify whether SOX17 expression is regulated by miRNAs, the TargetScan and miRBase bioinformatics databases were used.22Lewis B.P. Shih I.H. Jones-Rhoades M.W. Bartel D.P. Burge C.B. Prediction of mammalian microRNA targets.Cell. 2003; 115: 787-798Abstract Full Text Full Text PDF PubMed Scopus (4215) Google Scholar As predicted by these databases, the 3′-UTR of SOX17 harbors potential binding sites of several miRNAs, including miR-26a, miR-141, and miR-200a. Luciferase reporter assay was used to screen candidate miRNAs targeting on 3′-UTR of SOX17 mRNA in the KYSE140 cell line. Relative luciferase activity was sharply decreased in miR-141–, miR-26a–, and miR-200a–transfected cells in comparison with the control group (Figure 6A). To further validate the inhibitory effect of these miRNAs, Western blotting was used in this study. Expression of SOX17 was down-regulated by miR-141. Furthermore, MYC (alias c-myc) and cyclinD1, downstream genes of the WNT signaling pathway, were simultaneously up-regulated by miR-141 indirectly. By contrast, the expression levels of SOX17 and downstream genes (cyclinD1 and MYC) were not different between miR-26a, miR-200a, and control groups (Figure 6B). The putative binding sites of SOX17 3′-UTR were analyzed by TargetScan. Only one miR-141 binding site was discovered in the 3′-UTR of SOX17 mRNA. The binding sites are also conserved among H. sapiens, Mus musculus, Rattus norvegicus, and Oryctolagus cuniculus (Figure 6C). To validate the interaction of miR-141 and the 3′-UTR of SOX17 mRNA, the 3′-UTR of SOX17 mutant was generated by PCR (Figure 6C). Fifty-five percent of relative luciferase activity was reduced in miR-141–transfected cells, in comparison with the control group. No significant changes was found in the 3′-UTR of the SOX17 mutant group related to miR-141 (Figure 6D). These results indicate that miR-141 may effectively interact with the 3′-UTR of SOX17 mRNA, but no effective interaction was found in the mutant group. To explore the relationship of miR-141 and SOX17 expression, miR-141 expression was detected by real-time PCR in eight esophageal cancer cell lines. SOX17 expression in these cell lines was described as above. The results indicated miR-141 and SOX17 expression were inversely correlated in these cell lines (Figure 6E). miR-141 expression level was higher in the KYSE30, KYSE140, KYSE150, and SKGT4 cell lines, but was lower in the KYSE70, KYSE180, and KYSE450 cell lines. The expression level of miR-141 was very low in the YSE2 cell line. SOX17 was expressed in the YSE2 cell line and weakly in the KYSE70, KYSE180, and KYSE450 cell lines. No expression was detected in the KYSE30, KYSE140, KYSE150, and SKGT4 cell lines. These studies suggest that miR-141 may be involved in the silencing of SOX17. All the above results demonstrate that SOX17 expression may be regulated by both DNA methylation and miR-141 in esophageal cancer cell lines. In this study, 8 cancer cell lines, 9 normal esophageal mucosa, 60 dysplasia, and 169 cases of esophageal primary cancer were analyzed for SOX17 methylation. SOX17 methylation was correlated to loss of gene expression in esophageal cancer cell lines, and SOX17 expression was restored in SKGT4, KYSE30, KYSE140, and KYSE150 cells after DAC treatment (Figure 1). These results indicate that SOX17 expression is regulated by promoter region methylation. Bisulfite sequencing was performed to confirm the MSP results and further to analyze the distribution and density of methylation in the promoter region in esophageal cancer cell lines. The results indicate that the selected location and primer sequences are proper. SOX17 is frequently methylated in human primary esophageal cancer (65%) and dysplasia (42%), but not in normal esophageal mucosa. As shown in Figure 2, SOX17 methylation is in progression tendency during esophageal cancer carcinogenesis from normal epithelia, different grades of dysplasia, and advanced cancer. The above results indicate that SOX17 methylation may serve as an esophageal cancer early detection marker. Immunohistochemistry results hint that SOX17 expression is regulated by promoter region methylation, and further affects β-catenin expression and distribution. These results suggest that SOX17 plays an important role in the WNT signaling pathway in esophageal cancer. As shown in Table 1, SOX17 methylation is related to alcohol history in esophageal cancer. This suggests that alcohol may cause SOX17 methylation and further lead to esophageal cancer. Reduction of alcohol consumption may help prevent esophageal cancer by hindering SOX17 gene methylation. The canonical WNT signaling pathway is involved in many biological processes, including embryogenesis and carcinogenesis.23Klaus A. Birchmeier W. Wnt signalling and its impact on development and cancer.Nat Rev Cancer. 2008; 8: 387-398Crossref PubMed Scopus (1251) Google Scholar, 24Clevers H. Wnt/beta-catenin signaling in development and disease.Cell. 2006; 127: 469-480Abstract Full Text Full Text PDF PubMed Scopus (4491) Google Scholar It was reported that free cytoplasmic β-catenin enters the nucleus, forms a complex with members of the TCF/LEF transcription factor family, and then up-regulates target gene expression.25Sinner D. Rankin S. Lee M. Zorn A.M. Sox17 and beta-catenin cooperate to regulate the transcription of endodermal genes.Development. 2004; 131: 3069-3080Crossref PubMed Scopus (241) Google Scholar, 26Sinner D. Kordich J.J. Spence J.R. Opoka R. Rankin S. Lin S.C. Jonatan D. Zorn A.M. Wells J.M. Sox17 and Sox4 differentially regulate beta-catenin/T-cell factor activity and proliferation of colon carcinoma cells.Mol Cell Biol. 2007; 27: 7802-7815Crossref PubMed Scopus (256) Google Scholar The activated WNT/β-catenin signaling pathway may induce MYC, cyclinD1, and other downstream genes expression and, finally, promote cell proliferation and cause carcinogenesis. The SOX gene family plays different roles during development and carcinogenesis. Nuclear protein SOX17 may inhibit the WNT signaling pathway by degradation of the β-catenin/TCF complex. Inactivation of SOX17 may promote the WNT/β-catenin signaling pathway and lead to carcinogenesis. The SOX17 promoter region was frequently methylated, with silenced expression, in esophageal cancer cell lines. Restoration of SOX17 expression reduced colony formation and inhibited β-catenin/TCF–dependent transcriptional activity in KYSE140 cells. As shown in Figure 5, loss of the HMG box of SOX17 may promote β-catenin and its downstream gene transcription. These results suggested that SOX17 is an important negative regulator of the canonical WNT signaling pathway in esophageal cancer. Reversion of SOX17 expression may inhibit esophageal carcinogenesis. The above results lead us to consider further studies of targeting therapy in esophageal cancer by demethylation of SOX17 in the future. MicroRNAs are endogenous, 18- to 25-nucleotide, non-coding RNAs that mainly serve as regulators of gene expression by binding to the 3′-untranslated region of targeting mRNAs. The miR-141/200 family was reported to take part in maintenance of epithelial phenotype, and loss of its expression may lead to epithelial to mesenchymal transition.27Burk U. Schubert J. Wellner U. Schmalhofer O. Vincan E. Spaderna S. Brabletz T. A reciprocal repression between ZEB1 and members of the miR-200 family promotes EMT and invasion in cancer cells.EMBO Rep. 2008; 9: 582-589Crossref PubMed Scopus (1422) Google Scholar Recent publications indicate that miR-141 may stimulate cell proliferation.28Hyun S. Lee J.H. Jin H. Nam J. Namkoong B. Lee G. Chung J. Kim V.N. Conserved microRNA miR-8/miR-200 and its target USH/FOG2 control growth by regulating PI3K.Cell. 2009; 139: 1096-1108Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar A high level of miR-141 in plasma is associated with a short survival time in colorectal cancer patients.29Cheng H. Zhang L. Cogdell D.E. Zheng H. Schetter A.J. Nykter M. Harris C.C. Chen K. Hamilton S.R. Zhang W. Circulating plasma MiR-141 is a novel biomarker for metastatic colon cancer and predicts poor prognosis.PLoS One. 2011; 6: e17745Crossref PubMed Scopus (377) Google Scholar As shown in Figure 6, our screening results demonstrate that miR-141, miR-200a, and miR-26a may interact with the 3′-UTR of SOX17 mRNA, but only miR-141 was found to effectively down-regulate SOX17 expression on further study. Up-regulation of MYC and cyclinD1 were also induced indirectly by miR-141 in this study; and the expression of miR-141 was inversely correlated with SOX17. All the above results indicate that SOX17, the WNT signal pathway gene, is regulated by both promoter region methylation and miR-141 in esophageal cancer. In conclusion, SOX17 is frequently methylated in esophageal cancer. The progression tendency of SOX17 methylation during esophageal carcinogenesis indicates it may serve as an esophageal cancer early detection marker. SOX17 methylation is significantly correlated with alcohol history. SOX17 promoter region methylation may induce β-catenin up-regulation and redistribution. SOX17 expression was regulated by both promoter region methylation and miR-141 in esophageal cancer. SOX17 is probably a tumor suppressor and involved in the WNT signaling pathway." @default.
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• W1965172023 title "Inhibition of SOX17 by MicroRNA 141 and Methylation Activates the WNT Signaling Pathway in Esophageal Cancer" @default.
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