Expressions Of Stem Cell Transcription Factors OCT4, SOX2 And HIWI And Functional Study Of OCT4 In Human Esophageal Squamous Cell Carcinoma

Although much progress has been made in the diagnosis and treatment of cancerover the past decades, evidence strongly indicates that the current treatments usuallyjust hold the tumor at bay and the patients are not truly cured. Successfullyeradicating this disease therefore requires a better understanding of how cancerinitiates and progresses. Cancer stem cells (CSCs) are now thought to play key rolesin cancer genesis and development. Developing approaches to specifically targetmalignant stem cells by applying the principles of stem cell biology might raise hopeto cure this disease.Esophageal carcinoma (EC) is one of the most aggressive neoplasms in the world.At the present, there are more than 480,000 new cases of esophageal carcinoma eachyear in the world and half of which occurs in China. The main type of esophagealcarcinoma in China is squamous cell carcinoma, which is characterized with highmalignance, poor prognosis, fast development, intrinsic resistance to chemotherapyand recurrence.The conventional therapies on tumor often removes the bulk of a tumor masswithout preventing tumor recurrence, suggesting the survival of a subset of cancerstem cells. Cancer stem cells are cancer cells that possess characteristics associatedwith normal stem cells, including the ability of self-renewal and unlimitedproliferation, relative resistance to drugs, radiation, and apoptosis. These cells are tumorigenic and proposed to persist in tumors as a distinct population, causing relapseand metastasis by giving rise to new tumors. Although the cancer stem cells had beenindentified and characterized in the increasing number of cancers, very little is knownabout the esophageal cancer stem cells.The stem cell transcription factors, such as OCT4, SOX2 and HIWI, arenecessary for the maintenance of the ability of self-renewal and pluripotency inembryonic stem cells (ESCs) and primordial germ cells and are down-regulated in alldifferentiated somatic cell types in vitro as well as in vivo. Cancer cells, especially inpoorly differentiated or undifferentiated tumors, have been characterized by manyphenotypic traits similar to undifferentiated embryonic cell. These similarities suggestthe expression of genes determining cell renewal and sternness. Previous studies havedemonstrated that OCT4, SOX2 and HIWI express in many different cancers, such asmammary carcinoma, germinoma, pancreatic carcinoma, hepatocarcinoma, bladdercarcinoma, gastric carcinoma, soft tissue sarcoma, and lung cancer, etc, suggestingthat they play a key role in the genesis and development of cancer.OCT4, the first identified transcription factor only expressed in pluripotent cells,belongs to the POU (Pit-Oct-Unc) transcription factor family. Recent investigationsindicate that OCT4 is involved in controlling not only the maintenance of embryonicstem (ES) cell pluripotency but also the proliferation potential. The signal pathwayOCT4/Tcl1/Akt1 is identified to be involved in ES cell proliferation, which functionsby inhibiting the apoptosis of ES cells.Furthermore, OCT4 is the most informative marker in germ cell tumorsdiagnostics and is expressed in the precursor lesions gonadoblastoma and carcinomain situ, as well as in invasive embryonal carcinoma and seminomatous tumors. Someresearch indicates that OCT4 might play a key role in maintaining the survival ofcancer cells. OCT4 re-expression in epithelial tissues is observed to lead to dysplasticdisorders by inhibiting cellular differentiation in a manner similar to that in embryoniccells. OCT4 has also been reported to be an oncogenic fate determinant. High levelsof OCT4 increase the malignant potential of ES-derived tumors whereas inactivationof OCT4 induces a regression of the malignant component, suggesting that OCT4 might play a critical role in the genesis and development of tumors.There is no report about the expression patterns of OCT4, SOX2 and HIWI inhuman esophageal squamous cell carcinoma as well as the potential functions ofOCT4 on the genesis and development of esophageal squamous cell carcinoma. Thepresent study is to explore the expression patterns of OCT4, SOX2 and HIWI andtheir significance in diagnosis and prognosis in human esophageal squamous cellcarcinoma. Meanwhile, the potential functions of OCT4 on the genesis anddevelopment of esophageal squamous cell carcinoma are also explored in this study.This study includes the following three parts:Partâ… : Expression of Stem cell transcription factors OCT4,SOX2 and HIWI in human esophageal squamous cellcarcinoma and their clinical significanceMethods1. The expressions of OCT4, SOX2 and HIWI in the esophageal squamous cancercell lines KYSE70, KYSE140 and KYSE450 was characterized with the methods ofreal-time PCR, western blotting and immunocytochemistry;2. The expressions of OCT4, SOX2 and HIWI in 153 esophageal squamous cancercases and 10 normal esophageal epithelium was characterized with the methods ofimmunohistochemistry;3. The correlations between the clinicopathological features and the expression ofOCT4, SOX2 and HIWI in the esophageal squamous cancer were analyzed withCrosstabs Analysis, while survival curves regarding to the expression of OCT4, SOX2and HIWI were analyzed using the Kaplan-Meier method;4. Statistical analyses: Bivariate association between ordinal variables was assessedusing Spearman's correlation. For categorical data, Pearson'sχ² test was used. Alltests of statistical significance were two-sided. Survival curves were plotted accordingto the Kaplan-Meier method, and the log-rank test was used to determine significantdifferences among groups (pooled over strata for two groups; pairwise over strata for three groups). Multivariate analysis according to Cox's proportional hazardsregression model adjusted clinicopathological factors. Statistical analyses wereperformed using the SPSS 16.0 package and P＜0.05 was considered as statisticallysignificant difference.Results1. OCT4, SOX2 and HIWI were variably expressed in three esophageal squamouscancer cell lines;2. Immunohistochemically, OCT4 positive immunostaining was observed in thecancer cell nuclei. Among 153 specimens, 105 (68.7%)were negative or weaklypositive for OCT4 staining; 21 (13.7%)were moderately positive and 27 (17.90%)were strong positive;3. Positive immunostaining of SOX2 was also observed in the cancer cell nuclei. Intotal, 98 (64.1%)were either negative or weakly positive for SOX2; 36 (23.5%)weremoderately positive while 19 (12.4%)were strongly positive;4. HIWI immunoreactivity was observed in both nucleus and cytoplasm. Among the153 specimens, 67 (43.8%) were either negative or weakly positive for cytoplasmicimmunostaining; and 86 (56.2%) were strongly positive. For nuclear staining, 104(68.0%) were either negative or weakly positive; 49 (32.0%) were strongly positive.Totally, 46 (30.1%) exhibited negative or weak HIWI expression in both cytoplasmand nucleus, and were classified as negative/weakly positive group; 58 (37.9%) wereonly strongly immunoreactive in cytoplasm, and 21 (13.7%) only stronglyimmunoreactive in nucleus, classified as moderate expression group together; 28(18.3%) showed strong immunoreactivity in both cytoplasm and nucleus, classified asstrongly positive group;5. Crosstabs analysis showed that the expressions of OCT4 and SOX2 weresignificantly correlated (P＜0.001,χ²= 58.248), while there was no correlationbetween OCT4 and HIWI (P=0.515,χ²= 3.263), nor between SOX2 and HIWI (P=0.642,χ²=2.513);6. Higher OCT4 expression was significantly associated with higher histological grade(P＜0.001,χ²=26.706);7. Patients with negative or weakly immunostained OCT4 tumours had an overallbetter survival than patients with moderately stained rumors (P=0.002,χ²=9.462),while patients with moderately immunostained OCT4 tumors were of better survivalthan those with strongly stained tumors (P=0.023,χ²=5.161);8. Higher SOX2 expression was significantly associated with higher histologicalgrade(P＜0.001,χ²=23.058);9. Patients with negative or weakly immunostained SOX2 tumors had an overallbetter survival than patients with moderately stained tumors (P=0.018,χ²=5.579),while patients with moderately immunostained SOX2 tumors had an overall bettersurvival than patients with strongly stained tumors (P=0.01,χ²=6.632);10. The expression level of HIWI in cytoplasm was significantly associated withhistological grade (P=0.011,χ²=9.075) and T stage (P=0.035,χ²=8.589);11. Patients with high cytoplasmic HIWI expression tumors had a poorer overallsurvival than that with low cytoplasmic expression tumors (P＜0.001,χ²=17.05);12. There was no correlation between the expression level of HIWI in nucleus andany clinicopathological features, and the outcome of patients as well;13. Higher expressions of OCT4, SOX2 and HIWI in cytoplasm could be theindependent prognostic factors of survival, and the relative risks of which were 2.625,1.976 and 2.247, respectively.Partâ…¡: The identification and characterization of stem cell-like cellsin human esophageal squamous cancer cell linesMethods1. Hoechest33342 exclusion assay, Aldefluor assay and label retention assay werecarried to identify stem cell-like cancer cells in three human esophageal squamouscancer cell lines KYSE70, KYSE140 and KYSE450, respectively;2. The ability of self-renewal and differentiation of stem cell-like cancer cells wastested by culture-and- reanalysis; 3. Plate colony-forming assay was applied to test the ability of clonegenesis of stemcell-like cancer cells;4. The expression pattern of oct4, sox2, nanog, hiwi, shh, gli1, snai1, twist1, tgf-β1,bcl2, N-cadherin, E-cadherin, egfr, mmp2, mmp9, abcg2, abcb1 in stem cell-likecancer cells was characterized with real-time PCR;5. MTT was applied to test 5-Fu sensitivity of stem cell-like cancer cells;6. Statistical analyses: All quantitative data were present as means±SD ((?)±s).Independent samples t test and one-way ANOVA were adopted to compare the means.Statistical analyses were performed using the SPSS 16.0 package and P＜0.05 wasconsidered as statistically significant difference.Results1. There were SP cells in KYSE70 (0.9±0.17%), but not in KYSE140 andKYSE450;2. There was no Alde⁺ cell KYSE70, KYSE140 and KYSE450;3. There were slow-cycling cells in both KYSE70 (l.l±0.22%) and KYSE450(0.4±0.08%), but not in KYSE140;4. SP cells can differentiate into MP cells while MP cells can not differentiate intoSP cells;5. The colony-forming efficiency of KYSE70 SP cell was 55.33±5.03% while thatof MP cell was 26.00±1.45%;6. The colony-forming efficiency of KYSE70 slow-cycling cell was 45.89±2.52%while that of fast-cycling cell was 18.89±1.02%;7. Compared with in MP cells, the expressions of gli1, oct4, sox2, nanog, mmp2,mmp9, shh, abcg2, bcl2, N-cadherin, egfr, hiwi, twist1 and abcb1 were up-regulatedin SP cells;8. Compared with in fast-cycling cells, the expressions of gli1, oct4, nanog, mmp2,mmp9, Snai1, tgf-β1, abcg2, bcl2, N-cadherin, egfr, sox2, hiwi, shh, twist1 and abcb1were up-regulated in slow-cycling cells;9. The IC50 of 5-Fu in 48h for SP cells was 22.55μg/ml with 19.63～25.77μg/ml as95%CI, while that for MP cells was 11.91μg/ml with 10.02～13.85μg/ml as 95%CI; 10. The IC50 of 5-Fu in 48h for slow-cycling cells was 22.22μg/ml with19.22～25.52μg/ml as 95%CI, while that for fast-cycling cells was 12.79μg/ml with10.84～14.79μg/ml as 95%CI.Partâ…¢: The potential functions of OCT4 on the biological behaviorof human esophageal squamous cancer cell lineMethods1. The cDNA of OCT4 was inserted into pIRESpuro2 to develop eukaryoticexpression recombinant pIRESpuro2-OCT4;2. The esophageal squamous cancer cell line KYSE70 was stably transfected withthe pIRESpuro2-OCT4 and the expression of OCT4 was detected with real-time PCRand western-blot;3. The esophageal squamous cancer cell line KYSE70 was stably transfected withthe commercial OCT4 RNAi vector pLKO.1-OCT4-shRNA and the expression ofOCT4 was detected with real-time PCR and western-blot;4. The effect of OCT4 on the expression levels of sox2, nanog, hiwi, shh, gli1, snai1,twist1, tgf-β1, bcl2, N-cadherin, E-cadherin, egfr, mmp2, mmp9, abcg2 and abcb1were characterized with real-time PCR;5. Plate colony-forming assay was applied to test the function of OCT4 on theability of clonegenesis of KYSE70;6. MTT was applied to test the function of OCT4 on 5-Fu sensitivity of KYSE70;7. The function of OCT4 on the apoptosis of KYSE70 was explored by flowcytometry after double staining with Annexin V-FITC/PI;8. KYSE7-OCT4, KYSE70-pIRESpuro2 and KYSE70-OCT4-shRNA were s.c.inoculated into the right flank area of nude mice with 2×10⁶ cells per mouse and 5mice per group. The tumor size was measured weekly for 5w;9. Statistical analyses: All quantitative data were present as means±SD ((?)±s).Independent samples t test and one-way ANOVA were adopted to compare the means.Statistical analyses were performed using the SPSS 16.0 package and P＜0.05 was considered as statistically significant difference.Results1. The eukaryotic expression recombinant pIRESpuro2-OCT4 was constructedsuccessfully and stably transfected KYSE70, which could increase the OCT4expression level in KYSE70 by 7.43 times;2. After the introduction of commercial OCT4 RNAi vector pLKO.1-OCT4-shRNAinto KYSE70, the expression level of OCT4 in KYSE70 was decreased by 71%;3. After transfected with pIRESpuro2-OCT4, KYSE70 was growing in 3D andcould form cell spheres on ultra-low attachment surface;4. The up-regulation of OCT4 can increase the expression levels of sox2, nanog,mmp9, snai1, abcg2, gli1, egfr, mmp2, twist1, bcl2, shh, tgf-β1, abcb1 and decreasethe E-cadherin expression level;5. The down-regulation of OCT4 can decrease the expression levels of sox2, nanog,mmp9, E-cadherin, snai1, abcg2, gli1, mmp2, twist1, bcl2 and tgf-β1;6. The colony-forming efficiency of KYSE70-pIRESpuro2 was 15.22±2.14%; whilethat of KYSE70-OCT4-shRNA was 9.11±2.01% and that of KYSE70-OCT4 was40.00±2.52%. One-way ANOVA analysis showed there was significant differenceamong three groups (P＜0.001, F= 160.938);7. The IC50 of 5-Fu in 48h for KYSE70-OCT4 was 18.89μg/ml with16.00～21.99μg/ml as 95%CI, while that for KYSE70-pIRESpuro2 was 9.30μg/mlwith 7.44～11.14μg/ml as 95%CI and that for KYSE70-OCT4-shRNA was 5.88μg/mlwith 4.31～7.38μg/ml as 95%CI;8. The apoptosis rate of KYSE70-OCT4 was 3.02±0.75%, while that ofKYSE70-pIRESpuro2 was 3.97±1.03% and that of KYSE70-OCT4-shRNA was25.63±3.57%. The apoptosis rate of KYSE70-OCT4-shRNA was significantly higherthan the other two groups (P＜0.001);9. KYSE70-OCT4 formed invisible tumor on the 7^th day and on the 14^th day, 21^st day, 28^th day and 35^th day, the volumes of tumors were 69.8±10.3mm³,415.4±109.3mm³, 973.4±127.1mm³ and 1713.4±225.1mm³, respectively;KYSE70-pIRESpuro2 formed invisible tumor on the 10^th day and on the eachmeasuring day, the volumes of tumors were 46.6±11.1mm³, 203.4±37.9mm³,532.6±100.4mm³ and 1023.2±149.5mm³, respectively; KYSE70-OCT4-shRNAformed invisible tumor on the 11^th day and on the each measuring day, the volumes oftumors were 41.4±6.19mm³, 122.8±20.1mm³, 259.4±35.9mm³ and 638.4±63.5mm³,respectively. From the 21^st day, the volumes of tumors were significantly differentamong three groups (P＜0.001).Conclusions1. This study is the first report investigating the expression patterns of theembryonic stem cell factors OCT4, SOX2 and HIWI in esophageal squamouscarcinomas and esophageal squamous cancer cell lines;2. The expressions of OCT4 and SOX2 in esophageal squamous carcinoma weresignificantly correlated, while there was no correlation between OCT4 and HIWI norSOX2 and HIWI;3. Higher expression levels of both OCT4 and SOX2 expressions were significantlyassociated with higher histological grade and poorer overall survival of patients;4. Higher expression level of HIWI in cytoplasm was significantly associated withhigher histological grade, T stage and poorer overall survival of patients;5. The expressions of OCT4, SOX2 and HIWI in cytoplasm could be theindependent prognostic factors of survival, respectively;6. There were stem cell-like cancer cells in esophageal squamous cancer cell lines,which were characterized with higher ability of self-renewal, colony-forming, andlower sensitivity for cytotoxic drug; 7. Some stem cell or cancer cell related factors were higher expressed in stemcell-like cancer cells of esophageal squamous cancer cell lines;8. OCT4 played a key role in the morphology, growing pattern, drug sensitivity,apoptosis, abilities of colony-forming and tumorigenesis of esophageal squamous cellcarcinoma.