The Expression Of EphA2, EphrinA1, ER, PR,ER-β And PR In Esophageal Squamous Cell Carcinoma And Their Relation With Clinicopathological Feature And Clinical Prognosis

Among the PTKs, the largest family is Eph.Fourteen Eph receptors and eight cphrins ligands have been identified to date. EphA2 was a number of Eph subfamily sifted from human horny cDNA library by Linsberg in 1990, it was the first discovered gene with tyrosine kinase active in Eph subfamily. EphrinAl was originally cloned from human umbilical vein endothelial cells as a factor induced by tumor necrosis factor a(TNF-a).Signal conducting regulated by Eph receptor and EphrinAl participating in the formation of many tissues and organs, Including thenervous system signal transmitting, angiogenesis ,the regulation of cell adhesion among cells, And involving in the development of tumor.EphA2 is found at low levels on adult epithelial cells and is frequently overexpressed in a large number of cancer cells, including breast cancer, aggressive melanomas,prostate cancer. Recent research reported: The overexpression of EphA2 is related to histological grade,number of lymph node metastases ,and the poor prognosis. Suggesting that its potential prognostic value in tumor treatment, where very limited number of study is available in literature about EphA2 and EphrinAl in esophageal squamous cell carcinoma.ER and PR exist in sex-hormone-related tumors(including breast cancer ,endometrial carcinoma),And they have guiding significance in endocrine treatment and the prognosis judgment .Sex hormone receptors reported surely exist in alimentary canal tumors recently, Endocrine treatment made definite curative effect in some cases. During the research of the expression and changing of EphA2 and ER influenced each other. ER-B is the second type of estrogen receptor which was found in Mouse s subject animal and human body recently, Its gene and chromosome location had been defined, But there are not reports related to the expression of ER-6 in ESCC tissues.Esophageal cancer is one of the most frequently developed cancers in the world, and is a common cancer among the Chinese. The annual rate varies from 3/105 to 64/105,depending upon genetic vulnerability, diet and environmental factors. Although surgery is a curative treatment for early stage esophageal cancer. It is still a limited clinical option since most patients present with advanced disease. The survivalrate at 5 years was reported to be about 10%, and recent advancements in both surgical and adjuvant therapies have improved the 5-year survival rate to be about 40%,a fact far from satisfactory. Therefore, determination of prognostic factors in esophageal cancer is important to commence proper treatment and to increase survival. At present, The co-expression of EphA2, EphrinAl, ER, PR , ER-8 and PR in ESCC have not been reported inside and outside country. So it will be have important theoretic and practical value to understand their expression state and sigificance.Tissue micoarray and immunohistochemistry were used to assess the protein expressions of EphA2, EphrinAl, ER, ER-6 and PR in tumors from 173 patients with esophageal squamous cell carcinoma(ESCC). Paraffin sections from 20 cases were used for Laser Capture Microdissection and processed for RT-PCR detection of EphA2 and EphrinAl mRNA in ESCC. Immunohistochemistry , Western blot and RT-PCR were used to research the orientation and equalization of EphA2, EphrinAl, ER, ER-fiand PR in ESCC lines Kyse-70, Kyse-140, Kyse-450 and HET-lA.Then discussed the relation to the clinicopa- thological features such as: number of lymph node metastases, clinical stage, tumor size, histological grade, survival , In order to clarify the clinicopathological significance in the occurring, procession and prognostic judgment of ESCC, Our research can be divided into there parts.Part One: The research of the protein expression of EphA2, EphrinAl, ER, PR , ER-fl in ESCC and their relation to clinicopathological features and prognosis factors.Methods:1.Using Tissue micoarray to establish the micoarray lineup of 173 UICC( I -IV stage )ESCC tissues and 19 normal esophageal tissues.2.Using Immunohistochemistry to assess the protein expression of EphA2, EphrinAl, ER, ER-8 and PR in tumors form 173 ESCCs and in 19 normal esophageal tissues. Then discussing the relation to clinicopathological feature and prognostic factors.3.The SPSS version 11.0 Statistical package (SPSS.Inc,Chicago,USA)was used for the statistical analysis. We also used X2 test(chi-square), t test(student t), ANVOA, related test and univariate analysis values of <0.05 were considered statistically significant.Results:l.Among the 19 normal esophageal epithelial cells, 6 (31.6%)as grade 1 for EphA2 and 13(68.4%) were negative, For EphrinAl protein expression,l(5.3%) as grade 2,6 (31.5%)as grade l,and 12(63.2%) were negative. Through Immunohistochemical straining for three times, All the ER, PR , ER-13 results are negative in normal esophageal squamous epithelial tissues.2.Among the 173 ESCC cases, 38(22.0%) as grade 3 for EphA2, 58(33.5%) as grade 2 and 44(25.3%) as grade 1, 33(19.1%) were negative, total positive rate 80.9%. For EphrinAl protein expression,28(16.2%) as grade 3,76(43.9%) as grade 2, 42(24.3%) as grade 1 and 27(15.6%) were negative, total positive rate 84.4%. The intensity of EphA2 and EphrinAl in esophageal squamous cells were stronger thanthose in the normal esophageal epithelia.(P<0.001)3.Through Immunohistochemical straining for three times, All the results of ER were negative. Positive ER-B and PR immunostraining was observed in the nucleus. Among the 173 ESCCs cases ,45(26.0%) as grade 3 for ER-B,47(27.2%) as grade 2.25(14.5%) as grade 1 and 56(32.4%) were negative, total positive rate 67.6%. For PR protein expression, 11(6.4%) as grade 3,32(18.5%) as grade 2,47(27.2%) as grade 1 and 83(48.0%) were negative, total positive rate52.0%.4.EphA2 protein expressions were not associated with age, histological grade, tumor location, tumor size and clinical stage; But gender (P<0.05) and lymph node metastastases (P<0.001). EphrinAl protein expressions were not associated with age, gender, tumor location, histological grade, tumor location, tumor size, lymph node metastases, but significantly correlated with clinical stage(P<0.05).5.ER-B protein expression were not associated with histological grade, age, tumor location, tumor size, clinical stage and lymph node metastases, but correlated with gender(P<0.05).PR protein expressions were not associated with age, gender, tumor size, clinical stage, lymph node metastases, But corrected with histological grade(P<0.001).6.Statistical analysis suggests the stronger EphA2 and EphrinAl immunostraining are, The shorter survival for the ESCC are. There are significant differences of EphA2 protein form negative to grade 3(all P<0.05). There are significant differences of EphA2 and EphrinAl protein expression form differ survival time(^3 years, ^5 years, >5 years, all P<0.01).7.EphA2 and EphrinAl protein expressions co-localized in the same tumor areas and vascular endothelial cells, and their expression were significantly associated.(P<0.01) Their expression distribution are fundamentally unanimous.S.Univariate analysis suggest that the overall survival of ESCCs shows no apparent difference between ER-B and PR protein expression form negative to grade 3.9.Statistical analysis indicated that the exprssion of EphA2 and EphrinAl were significantly associated with the positive expression of ER-B in ESCQall P<0.05),the expression of EphA2 and EphrinAl may have an inverse proportion to the expression of ER-B(all P<0.05).The expression of EphrinAl was significantly associated with the positive expression of PR in ESCC. (P<0.05) The positive expression of EphrinAl also had an inverse proportion to the expression of PR.10.In Cox multivariate analysis, EphA2 protein expression was associated with the survival.(P<0.01) The lymph node metastases(P<0.05) and clinical stage (P<0.01) were also significantly associated with survival.Part Two: Apply LCM to study the mRNA expression of EphA2 and EphrinAl in ESCC Methods:l.The selected 40 cases form the 173 ESCC paraffin samples, Among which there were 20 cases confirmed to have various protein expression of EphA2 by immunohistochemistry .There were also 20 cases confirmed to have different protein expression of EphrinAl.LCM was used to choose and cut minor tumor areas formevery scction(about 200 tumor cells),Then put them onto the LCM CapSure cap to extract RNA.2.Using RT-PCR to analyze the expression of EphA2 and EphrinAl, comparing the relation between mRNA and its protein expression.3.Statistical analysis :A11 the data were analyzed by the SPSS version 11.0 statistical package (SPSS,Inc,Chicago,USA).We also used X2 test(chi-square), t test(student t), ANVOA, related test and univariate analysis. P values of <0.05 were considered statistically significant. Results:l.All the 20 ESCC specimens, which expressed different protein expression of EphA2 mRNA expression. The mRNA expression levels of negative expressing protein (0.27±0.11) were obviously lower than those of 16 cases expressing 1-3 grade protein(grade 1: 1.13±0.33; grade 2: 1.67±0.27 grade 3: 1.40+0.31; each compare with grade 0: all p<0.05). Among the 16 cases expressing protein form grade 1 to 3, There were no significant difference in their mRNA expression levels. (p>0.05) . Indicating that mRNA expression did not fully correspond to its protein expression in ESCC.2.All the 20 ESCC specimens, which expressed different EphrinAl protein expression found EphrinAl mRNA expression. The 4 case mRNA expression levels of negative expressing protein (0.66+0.22) were obviously lower than those of 16 cases expressing 1-3 grade protein( grade 1: 1.68+0.26; grade 2: 2.16±0.64; grade 3: 1.81 ±0.57; each compare with grade 0: all p<0.05). Among the 16 cases expressingprotein form grade 1 to 3,There were no significant difference in their mRNA expression levels of EphrinAl. (p>0.05) Indicating that mRNA expression did not fully correspond to its protein expression in ESCC.Part Three: The expression of EphA2, EphrinAl, ER, ER-B and PR in the Kyse-70, Kyse-140, Kyse-450 and HET-1A cell lines in ESCCMethods:1.Constructing the embedded paraffin block of the Kyse-70, Kyse-140, Kyse-450 andHET-lAinESCC.2.Checking the protein expression of EphA2, EphrinAl ,ER>ER-B and PR of the Kyse-70, Kyse-140, Kyse-450 and HET-1A cell lines in ESCC by Immunohistochemistry.3.Using Western blot to analyze the protein expression of EphA2 and EphrinAl in the Kyse-70, Kyse-140, Kyse-450 and HET-lAcell lines of ESCC.4.Western blot was used to detect the protein expression that EphrinAl-Fc gomphosls body stimulated and derivated in the Kyse-70, Kyse-140, Kyse-450 and HET-1A cell lines of ESCC.5.Using RT-PCR to analyze the expression of EphA2 and EphrinAlmRNA in the Kyse-70, Kyse-140, Kyse-450 and HET-1A cell lines of ESCC. Then comparing the relativity between mRNA and its protein expression.Results:1.Imuunohistochemistry was used to access the protein of EphA2 in ESCC celllines. It was strong positive of grade 3 in Kyse-70,Kyse-450 and HET-lA;And weak positive of grade 1, in kyse-140 positive immunostraining was observed in the cytoplasm. For EphrinAl, It was weak positive of grade 1 inKyse-70,Kyse-140 and HET-lA,andit was positive of grade 2 in Kyse-450. Positive immunostraining was also observed in the cytoplasm.2.Through Immunohistochemical straining for three times, all the results of ER were negative. For the expression of ER-B,It was weak positive for grade 1 in Kyse-70;And positive for grade 2 in Kyse-140, Kyse- 450;Strong positive for grade 3 in HET-lA;For the expression of PR, It was positive for grade 2 in Kyse-70, Kyse-140, Kyse-450 ;And weak positive for grade 1 in HET-lA.Positive immunostraining was observed in the nucleus.3.The result of Western bolt showed: All the protein expression of EphA2 and EphrinAl for all levels were observed in Kyse-70, Kyse-140, Kyse-450 and HET-IA cell lines of ESCC. Their expression levels were similar with the results of immunohistochemistry for EphA2 and EphrinAl.4.Western blot was used to detect the protein expression that EphrinAl-Fc gomphosls body stimulated and derivated in the Kyse-70, Kyse-140, Kyse-450 and HET-IA cell lines of ESCC: There are stronger protein sediments in the cell lines of Kyse-140, Kyse-450 and HET-IA ,But there is not obvious change in Kyse-70 cell lines.5.RT-PCR was used to analyze the mRNA expression of EphA2 and EphrinAl in the Kyse-70, Kyse-140, Kyse-450 and HET-IA cell lines of ESCC: There are positiveexpression of all levels for both in all lines, The results was not fully correspond to their protein expression (immunohistochemistry and Western blot)Conclusion:1.Tissue micoarray was used to analyze the co-protein-expression of EphA2, EphrinAl, ER, ER-B and PR in ESCC for the first time,LCM was used for the RT-PCR monitoring of the mRNA level of EphA2 and EphrinAl to the paraffin samples of ESCC.2.The protein overexpression of EphA2 and EphrinAl are correlated with the unfavorable survival of ESCC, Indicating their potential prognosis value and the hopeful of becoming a new marker to the prognosis evaluation of ESCC. The overexpression of EphA2 is associated with lymph node metastases, indicating the possibility that it may be participate in metastasting mechanism of ESCC.3.EphA2 and EphrinAl proteins highly expressed overlapping patterns in both tumor and endothelial in ESCC. Suggesting that EphA2 and EphrinAl may participate in angiogenesis and the possibility of EphA2 and EphrinAl of becoming the new target of ESCC gene therapy.4.Finding the expression difference of ER-B and EphA2 in ESCC for the first time, Indicating the possibility why the prognosis of the female ESCC are better than that of males.5.Finding the fact that EphA2 and EphrinAl are negative regulated by ER-B , EphrinAl is negative regulated by PR,But ER-B and PR protein are not correlated with the survival of ESCC.6.Making sure that mRNA expression of EphA2 and EphrinAl did not fully correspond to their protein expression in ESCC tissues and the cell lines of ESCC indicating there is obstacle in regulation of translation level and the protein gathering and degradation.7.EphA2 and EphrinAl protein are overexpressed in ESCC, Their mRNA expression and protein levels are positively correlated, there is conducting obstruction below EphrinAl-EphA2 phosphorylation in ESCC low-differentiation cell line Kyse-70,Suggesting that both may play a cooperated role in the biological behavior of ESCC.8. Finding the fact that ER are negative expressed in ESCC tissues and the cell lines of ESCC for the first time, Indicating that the expression of ER may lack its expression or the antigenic determinant may have structural aberrance.