The Experimental Study Of EphA2 Expression And Vasculogenic Mimicry In Transitional Cell Carcinoma Of Bladder

Background and objective: bladder carcinoma is the most common malignancy of urinary system, Approximately 90% of the urinary bladder cancers are transitional cell carcinomas that typically occur either as superficial (low-grade) or muscle-invasive (intermediate to high-grade) carcinoma. Superficial tumors can be surgically resected; however, local recurrence is common and 20% to 30% of recurrent lesions progress to higher grade or stage. Muscle-invasive cancers have the greatest propensity to metastasize and are fatal in～50% of the patients. Although the most reliable prognostic factors for recurrence and progression are tumor stage and grade. It is still necessary to search better prognostic markers, to improve clinical management of bladder carcinoma patients.The Eph family of kinases constitutes the largest group of transmembrane receptor tyrosine kinases (RTKs). EphA2 is primarily found in adult human epithelial cells. Lower level of EphA2 expression in epithelial cells are observed, while high levels of EphA2 expression have been reported in different human tumor tissues and cell lines, including breast cancer, metastatic melanoma, prostate cancer, colon cancer, and esophagealcancer, specially with higher expression in some aggressive tumors. Increasing evidence indicates that EphA2 overexpression is a common event during the progression of cancer. The study on tumor-based models suggests potential roles for EphA2 in the regulation of cell growth, survival, migration, and angiogenesis. EphA2 has been recently identified as an important mediator of vasculogenie mimicry in vitro. The patterned microcirculation characteristic of vasculogenic mimicry was first described in uveal (intraocular) melanoma. The de novo generation of vascular channels by aggressive and metastatic tumor cells is not strictly a vasculogenic event, therefore the name "vasculogenic mimicry" is assigned to the process by which aggressive tumor cells generate non-endothelial cell-lined charnnels delimited by extracelhlar matrix. Recent researchs discover that other malignant tumors also can generate the characteristic of vasculogenic mimicry. Tumors with vaseulogenic mimicry have a high-grade malignancy, early blood metastasis and poor clinical prognosis. Bissell has further noted that vaseulogenic mimicry poses challenges to the practice of surgical pathology and provides opportunities for the development of new imaging techniques and cancer treatment strategies.Although the researches about VM and EphA2 expression in tumor have been sporadically reported, the related study on bladder transitional cell carcinoma was seldom reported. We used Immunohistochemistry, Western blotting and immunoprecipitation to detect the expression of EphA2, EphA2-P and EphA2 mRNA in bladder transitional cell carcinoma and normal mucous membrane of urinary bladder; to investigate the relations between EphA2 and bladder transitional cell carcinoma biological characteristics;to explore the mechanism about EphA2 expression, at the same time, the possible vasculogenic mimicry(VM) presence in transitional cell carcinoma of bladder(TCCB) was investigated to explain the clinical significance of VM in bladder cancer. The relationship between Epha2 and VM, MVD was analysised.Experiment part one: Expression and molecular regulation of EphA2 in transitional cell carcinoma of bladder.Purpose: to study the relations between EphA2 and TCCB biological characteristics; to explore the molecular mechanism regulating EphA2 expression.Methods: Western blotting and Immunoprecipitation were used to detect the protein expression of EphA2 and EphA2-P in 20 cases of TCCB and 10 cases of normal bladder. The expression of EphA2 mRNA in these cases was determined by RT-PCR.Results: The EphA2 protein and EphA2 mRNA in TCCB were signigicantly higher expressed than those in normal mucous membrane of urinary bladder(P＜0.001). The EphA2 protein was highly expressed in poorly differentiated group than that in well differentiated group, the EphA2-P expression in normal mucous membrane of urinary bladder was signigicantly higher than that in TCCB(P＜0.001). The expression of EphA2 mRNA was not correlated with pathological grade of TCCB(P=0.295).Experiment part two: Vasculogenic mimicry and expression of EphA2 protein in transitional cell carcinoma of bladder.Purpose: to search whether vasculogenic mimicry(VM) exsits in TCCB, to explore the importment biological significances of VM and EphA2 in TCCB, to analyze the relations between EphA2 and VM, MVD in TCCB.Methods: the results demonstrated that endothelium-lined vessels were stained positively for CD31 and PAS and were in the majority of the tumor microvasculature.PAS-positive pattern of VM were found in 8 BTCC specimens among 85 TCCB specimens,the 8 specimens belong toⅡ～Ⅲgrade and T2～T4 stage, 6 of them with lymphatic metastasis, the positive rate of EphA2 in TCCB and normal bladder group was 89.1% and 40% ,and was significantly different between the two groups(P＜0.01).VM correlate with the expression level of EphA2(P＜0.01).the MVD was significantly different between the EphA2 positive group and the EphA2 negative group in TCCB(P＜0.01), the MVD among the EphA2 positive groups was not different signigicantly (P＞0.05). VM and the expression of EphA2 correlate with clinic stage, histological grading and lymphatic metastasis(P＜0.01).Conclusion: our results suggest that VM exists in some moderately /poorly differentiated TCCB; EphA2 correlates with VM and angiogenesis in TCCB; VM and EphA2 will be some good markers to monitor the malignant degree and some novel targets for therapeutic intervention against TCCB. The inconformity in expression of EphA2 mRNA and protein in TCCB indicate that the posttranscriptional mechanism may play an important role in the reglation of EphA2.