EphA2/EphrinA1: Expression In The Renal Cell Carcinoma And Mechanism Of Action In The Tumor Metastasis

Renal cell carcinoma(RCC) is one kind of malignant tumor from the tubular epithelium of the renal parenchyma,accounting for roughly 80～90% and 2～3%of the renal and adult malignant tumors,respectively.With the improvement of living standards and advance in the medical imaging,early diagnosis and surgical treatment have produced satisfactory results.However, good therapeutic regimens are currently unavailable for the post-operation recurrence and the late stage cases with metastasis.Prediction of the prognosis still depends on the clinical stage and pathological grade,which showed certain limitations.The search of biological markers that may help to predict the degree of the malignance has therefore become a hot topic in the studies of the RCC.Such works may provide new targeting sites and approaches for the biological treatment of the RCC.The EPH(erythropoietin-producing hepatocyte) receptors represent the largest known family of receptor tyrosine kinases.EphA2 is one of the earliest found and expressed in normal endothelial cells.It is also widely over-expressed in various tumor tissues including the mammary cancer, carcinoma of colon,esophageal cancer,prostatic cancer and mesothelial carcinoma,and cell lines.The EphA2 receptor located in the cell junction in normal epitheliums and,after binding with its EphrinA1 ligand under relatively high affinity and the ensuing tyrosine phosphorylation of both with bidirectional signal conduction,regulates the normal cell growth and development through its downstream signal pathway,and simultaneously promotes the degradation of the EphA2 receptor.The location of the EphA2 receptor is aberrant in the malignant tumors:Instead of the cell junction,it is widely distributed in the cell surface which prevented the effective binding with the EphA2 and reduced the degradation of the receptor per se.The EphA2 and EphrinA1 are thus usually over-expressed in many invasive tumors.By affecting the tumor cell lines(PC-3,293,Cos-7) with EphrinA1-Fc,a simulated ligand,investigators recorded rapid and transient actions of EphA2 receptor activation,enhanced phosphorylation,and morphological changes of the cells.So,EphA2 is now under the spotlight in the tumor research.It is reported that over-expression of the EphA2 in the RCC may be associated with the tumor free survival after operation.However,biological changes due to the interaction of the EphA2 and its ligand,and EphA2/AphrinA1 in the RCC are rarely studied in and outside China.It is generally considered tissue type and degree of the tumor may predispose to the outcome and prognosis of the disease.Pathologically,renal clear cell carcinoma is proportionally more in the renal tumors.The current work thus examined the role of the EphA2 and its ligand EphrinA1 in the metastasis of the tumor,by studying the expression of the EphA2 and EphrinA1 in the RCC tissue,clinicopathological features,E-cadherin and microvessel density (MVD).Secondly,the biological effects of the EphA2-EphrinA1 binding and the molecular mechanism of its antitumor action were examined.The latter work was aimed to provide experimental evidence for the expression of the Eph/EphrinA1 in the RCC and its mechanism of action in the metastasis of the RCC.PartⅠ:The effect of EphA2/EphrinA1 in the occurrence of the RCCObjective:To detect the protein expression of EphA2/EphrinA1 in the normal renal tissue and the RCC,explore its role in the occurrence of RCC.Methods:Use the Reversed Transcript-Polymerase Chain Reaction (RT-PCR) and flow cytometry to detect mRNA of the EphA2 and EphrinA1, and the protein expression in 22 cases of RCC and 12 cases of normal renal tissue.Results:1 RT-PCR results showed that in both normal group and RCC group express EphA2/EphrinA1 gene,but the renal group has express significantly higher EphA2/EphrinA1 mRNA expression than the normal group(P＜0.01),there was a significant difference.(1.60±0.80 vs 0.58±0.19;1.37±0.63vs 0.91±0.40,P＜0.01)2 The flow cytometry showed that the expression of the EphA2/EphrinA1 was significantly higher in the RCC group(424.38±43.14 vs 332.44±42.83,P＜0.01;419.44±52.13 vs 325.36±42.33,P＜0.01).3 Correlation analysis showed that in the RCC,EphA2/EphrinA1 protein expression and its mRNA expression were not significantly correlated (r=0.05,P=0.826;r=-0.171,P=0.448).Conclusion:The expression of the EphA2/EphrinA1 gene and protein in the RCC was higher than those in the normal renal tissue.But in the RCC,EphA2/EphrinA1 protein expression and its mRNA expression were not significantly correlated,suggesting that high protein expression is not only associated with the high gene expression,but also with the post-transcriptional regulation and the biological abnormalities of EphA2/EphrinA1.PartⅡ:The role of EphA2/EphrinA1 in the invasion and metastasis of the RCCObjective:To detect the expression of EphA2/EphrinA1 in the RCC and its relationship with the clinicopathological characteristics,E-cadherin and MVD, to explore the role of EphA2/EphrinA1 in the invasion and metastasis of RCC.Methods:Using immunohistochemistry,we detected EphA2/EphrinA1 and E-cadherin expression in 68 cases of RCC and 24 cases of normal renal tissue, also use CD34 antibody to mark microvascular endothelial cells and calculate microvessel density(MVD).Explore the relationship of EphA2/EphrinA1 expression with clinicopathological features and analyze its correlation with E-cadherin,MVD.Results:1 The relationship between clinicopathological features and EphA2/EphrinA1 expression in RCC.Immunohistochemistry results showed that:In the RCC,different grade have different protein expression of EphA2/EphrinA1(x~2=12.611,P=0.002; x~2=8.473,P=0.014),With the increase in grade levels,the protein expression of EphA2/EphrinA1 increase significantly(r=0.431,P=0.000;r=0.355, P=0.003;);stageⅢandⅣhave significantly higher protein expression of EphA2/EphrinA1 than stageⅠandⅡ(Z=-2.975,P=0.003;Z=-2.006,P=0.045); lymph node metastasis group has significantly higher protein expression of EphA2/EphrinA1 than those without lymph node metastasis (Z=-2.320,P=0.02;Z=-2.792,P=0.005);in tumor diameter≥7cm and＜7cm between the two groups,≥45 years and＜45 years old between the two groups, sex between two groups have no significantly difference about the expression of protein(P＞0.05).2 The expression of the E-cadherin in the normal renal tissue and RCC,and its relationship with clinicopathological features.Immunohistochemistry results showed that:positive expression of the E-cadherin protein at the membrane as brown particles.The RCC expressed significantly lower E-cadherin protein than the normal renal tissue (Z=-6.101,P＜0.0001).The expression of E-cadherin has the significant difference(x~2=8.493, P=0.014),and with the increase in grade levels,the expression of E-cadherin decrease significantly(r=-0.320,P=0.008);the expression of E-cadherin in high-stage group and lymph node metastasis group is significantly lower than the low-grade group and without lymph node metastasis group(Z=-2.037, P=0.042;Z=-2.217,P=0.027).In tumor diameter≥7 cm and＜7cm between the two groups,≥45-year-old and＜45 years of age between the two groups, sex between two groups of protein expression were not significantly different (P＞0.05).3 MVD in the normal kidney and RCC and its relationship with the clinicopathological characteristics of the RCC.Immunohistochemistry results showed:CD34 was mainly expressed at the vascular endothelial cell membrane.Microvessel staining was brown,the most densely stained region,e.g."hot spots",was mainly located at the border of tumors,cancer and cancer-intensive areas in the nest CD34 expression was significantly less than the cancer and hyperplasia weeks active region.MVD of tumor tissues was significantly higher than the normal renal tissue(P <0.01).In different grade of the tumor group,MVD have the significant difference(x~2=23.637,P<0.01).With the increase in grade levels,MVD increases significantly(r=0.592,P<0.0001).MVD in the RCC in the group of high-grade,high-stage group,with lymph node metastasis was also significantly higher than low-grade group,low stage and without lymph node metastasis group(Z=3.735,P<0.0001;Z=-3.4,P=0.001;Z=-3.522,P<0.0001);≥45-year-old and <45 years of age between the two groups,sex between two groups of MVD were not significantly difference(P>0.05).4 Correlation of the EphA2,EphrinA1,E-cadherin protein and MVD expression.Correlation analysis showed that in 68 cases of RCC,the protein expression of the EphA2 and EphrinA1 was significantly correlated(r=0.772, P<0.0001),while the protein expression of the E-cadherin was significantly and negatively correlated with that of the EphA2 and EphrinA1(r=-0.378, r=-0.414,P<0.01);EphA2,EphrinA1 expression and MVD were significantly correlated(r=0.555,r=0.485,P<0.01).Conclusion:EphA2/EphrinA1 was highly expressed in tumors with high stage,high grade and with lymph nodes metastasis.EphA2/EphrinA1 was negatively correlated with E-cadherin expression and positively correlated with MVD,indicating that high expression of the EphA2/EphrinA1 protein may predict the degree of malignance of the tumor,possibly through the influence of or the coordination with the E-cadherin and the angiogenesis of microvasculatures.PartⅢ:The anti-cancer effect of the EphrinA1-Fc to the 786-O RCC cells and its molecular mechanism.Objective:Using EphrinA1-Fc to intervene the 786-O renal clear cell carcinoma in vitro study to examine the role and the possible molecular mechanism of the EphrinA-2.Methods:Using the soluble ligand EphrinA1-Fc to intervene the 786-O renal carcinoma cells in vitro to observe the morphological changes in the cells at different time points,by the use of different technologies such as cell counting,Western blotting,immunoprecipitation to temporally examine the phosphorylation of EphA2,ERK1/2 and other changes.Results:1 The changes in the cell morphology after the EphrinA1-Fc intervention:Use soluble ligand EphrinA1-Fc to stimulate renal cancer cells.After 1min, the renal cells exhibited morphological change,shrinking from the normal extended shape of spindle;at 5min,part of the cells appeared to be round;at 30min 90%of the cells were round and loosely attached to the flask;at 40min part of the cells re-extended and adhered to the flask;at 60min 90%of the cells recovered to the pre-stimulation shape.In the control group,there were no detectable morphological changes.2 Impact of the EphrinA1-Fc on the p-EphA2Western blotting results showed that:EphrinA1-Fc at 5min,10min,30min,60min intervention,p-EphA2 expression(p-EphA2/EphA2)was 0.15±0.04, 0.19±0.01,0.21±0.03和0.10±0.00,there is significant difference in different groups(F=9.392,P=0.025).Compared among the different groups,5 min and 60min between the two groups,10min and 30min between two groups were not significantly different(P＞0.05),other groups have the significant difference(P＜0.05).The pre-intervention group and the control group have no expression of the p-EphA2.3 Impact of the EphrinA1-Fc on the phosphorylation of the ERKWestern blotting results showed that:EphrinA1-Fc at 5min,10min,30min,60min intervention,p-ERK expression levels were 0.15±0.07,0.22±0.06,0.26±0.03 and 0.13±0.04.p-ERK expression levels were relative,representing a marked increase in the pre-intervention,there was significant difference (F=4.428,P=0.041).Compared among the different groups,5 min and 30min between the two groups,60min and 30min between two groups were significantly different(P＜0.05).The pre-intervention group and the control group have no expression of the p-ERK. 4 Impact of the EphrinA1-Fc on the cell proliferationAfter repeatedly intervention of the EphrinA1-Fc in the 786-O RCC cell in 12 days,cell growth but not cell morphology showed significant change:the number of cells decreased significantly in the EphrinA1-Fc group[(1.96±0.29)×10~5 vs(3.14±0.49)×10~5,P＜0.01].Conclusion:Addition of the EphrinA1-Fc to the 786-O RCC cell could inhibit cell proliferation and reduce cell adhesion,possibly by affecting the phosphorylation of the intracellular EphA2 and ERK.PartⅣEffects of the EphrinA1-Fe on the nude mice with transplanted human RCCObjective:To investigate the effects of the EphrinA1-Fc on the nude mice with transplanted human RCC and its possible mechanism of action.Methods:the animal model of the transplanted tumor is established by subcutaneous inoculation to nude mice of the human RCC cell 786-O,which is positive for the EphA2 antigen and cultivated in vitro.The growth of the transplanted tumor was observed in both the experimental group(injection of the EphrinA1-Fc in the periphery of the tumor) and the control group (injection of the IgG-Fc in the periphery of the tumor).Expression of EphA2, p-ERK,E-cadherin and MVD was examined by immunohistochemistryResults:1 The establishment of the nude mice model of transplanted human RCC7 weeks after the inoculation of the 786-O cells,nodules about the size of grain were found at the injection site in 8 nude mice,indicating the successful establishing of nude mice model of transplanted human RCC(60%of success rate).2 Effects of the EphrinA1-Fc on the growth of the transplanted RCC in the nude mice8 weeks after the inoculation,the average tumor volume was(382.35±122.04) mm~3;4 weeks after the application of the EphrinA1-Fc,tumor-bearing mice in each group were in generally good condition with no obvious side effects. Time and medical intervention could affect the volume of the transplanted tumor(F=4.085,P=0.012;F=7.469,P=0.034,by ANOVA).Nonparametic test of independent samples showed that,in comparison to that of the control group,the volume of the transplanted tumor was significantly reduced at 4 but not 2 weeks after medical intervention[(115.10±49.16) mm~3 vs(800.00±269.69) mm~3,P=0.021 and(272.65±55.35) mm~3 vs(535.97±216.07) mm~3,P= 0.191],respectively,resulting in a inhibition rate of 80.62%.3 Effects of the EphrinA1-Fc on the EphA2,E-cadherin and MVD in the transplanted RCC in the nude mice.4 weeks after the medical intervention,protein expression of the EphA2 and MVD was lower,and protein expression of the E-cadherin was higher the treatment group than the corresponding value in the control group.Conclusion:RCC 786-O of EphA2 positive was oncogenic with low probability of tumor onset and long time of tumor development;the growth of the tumor could be inhibited by the injection of the EphrinA1-Fc in the periphery of the tumor,its possible mechanism was the up-regulation of the protein expression of the E-cadherin and inhibition of the angiogenesis in the tumor.These findings reciprocally indicated that high expression of the EphA2 and EphrinA1 may contribute to the onset of RCC by promoting cell proliferation and decreasing cell adhesion.Conclusion:1 The expression of EphA2/EphrinA1 in RCC increases significantly.In additional to the up-regulation of the gene expression,the possible mechanism also includes abnormality in the biological aspects of the protein.2 High protein expression of the EphA2/EphrinA1 may predict the malignance of the RCC.and joint examination of the EphA2,EphrinA1 and E-cadherin may be useful for the evaluation of malignancy of RCC and the determination of its metastatic potential.3 EphrinA1-Fc,artificial ligand,was first applied to RCC cell line and nude mice with transplanted RCC,reciprocally indicating that high expression of the EphA2 and EphrinA1 may contribute to the onset of RCC by promoting cell proliferation and increasing cell adhesion.Altered E-cadherin and microvessel growth factor,resulted from the biological abnormality of the EphA2 and EphrinA1,may be one important mechanism enhancing the tumor metastasis.4 The binding of the EphA2 and EphrinA1 may inhibit the proliferation of the tumor by increasing the phosphorylation of the AphA2 and ERK.