Συγκριτική μελέτη της έκφρασης των κινασών RHO στον καρκίνο της ουροδόχου κύστης

Abstract

Urinary bladder cancer (BC) is the most common malignancy of the urinary tract, responsible for significant mortality and morbidity worldwide. In Europe, an estimate of 105.000 new cases of bladder cancer are diagnosed annually, while approximately 20.000 patients die from bladder cancer each year. Nearly all bladder cancers are carcinomas, arising from the urothelium. At presentation, 75-85% of tumours are restricted to the mucosa, or invade the lamina propria mucosae. The remainder present with invasion of the muscular layer of the bladder wall or extend to perivesical tissue, adjacent organs and even the pelvic wall. The behaviour of urothelial cell carcinoma (UCC) is highly diverse and defined by two separate, but related processes: tumour recurrence and progression. In specific, more than 60% of the superficial tumours will recur at least once and progress to less differentiated or invasive neoplasms in a significant percentage of patients. In current clinical practice the most useful prognostic parameters are tumour grade, stage, size, prior recurrence rate and the synchronous presence of carcinoma in situ (CIS). However, neither recurrence nor progression can be predicted accurately and a better understanding of the natural history of UCC is expected upon the elucidation its molecular mechanisms. Rho kinases comprise a major subgroup of the Ras superfamily that mediate housekeeping aspects of cell biology. In particular, Rho kinases play a fundamental role in the reorganization of actin and microtubule cytoskeleton. Therefore, Rho proteins govern a variety of cytoskeletondependent processes including alterations in cell shape, polarity, adhesion, cell motility, as well as cell to cell and cell to matrix interactions. Furthermore, Rho proteins appear to be involved in gene transcription by activating Serum Response Factor (SRF) and in the regulation of cell cycle progression, growth and apoptosis. Rho members affect the process of tumourigenesis either by over-expression of some members of the family with oncogenic activity or by down-modulation of other members with suggested tumour suppressor activity. Moreover, since Rho GTPases regulate actomyosin-based contractility and extracellular matrix remodelling, they may be involved in the transmigration of cells, thereby facilitating local invasiveness and metastasis. In the present study we investigated the expression of RhoA, RhoB, RhoC, Rac1 and Cdc42 kinases in UCC of the urinary bladder. Additionally, we determined the expression profile of 107 Rho-associated genes, including GTPases, GDIs, GAPs and GEFs. The expression was investigated using microarrays, qPCR and Western blotting in 77 UCC specimens compared to paired normal urothelium. Computational analysis was also performed on Gene Expression Omnibus datasets in order to further elucidate whether differences in the expression of Rho kinases occur among xviii superficial and invasive tumours. Further microarray analysis was carried out for the expression profiling of the Rho-associated genes. Finally, the correlation of the mRNA and protein levels of the genes of interest was also examined in UCC and normal tissue, using the Spearman rank correlation test According to our results RhoB RNA and protein levels were significantly lower in UCC, suggesting a tumour-suppressor role. On the contrary, RNA of RhoC and protein levels of RhoA, RhoC and Cdc42, respectively, were significantly higher in UCC compared to normal urothelium. High Cdc42 RNA levels correlated with worse overall survival, whereas high RhoB RNA levels correlated both with better overall and cancer-specific survival. Computational analysis verified the expression profile of Rho kinases among superficial UCC, muscle-invasive UCC and normal urothelium. Our findings in conjunction with the results of other investigators indicate that overexpression of either RhoA and/or RhoC is involved in tumourigenesis irrespective of its origin. In addition, over-expression of RhoA appears to be one of the events leading to more aggressive UCCs, characterized by higher histological grade and more extensive local invasion. RhoB in BC plays a tumour suppressor role, opposing the positive functions of RhoA and RhoC. Regarding its role in other malignancies a review of literature yielded contradictory results. These data suggest different Rho kinases play different roles in tumourigenesis and one Rho kinase may have different roles in different tumour development. Rac1 and Cdc42 seem to cross activate RhoA and/or RhoC in UCC and may contribute to tumour formation and progression. More importantly, preliminary evidence suggests that Cdc42 silencing leads to growth suppression in human bladder-cancer cells. In conclusion, the present study showed that the RNA and protein levels of RhoC and the protein levels of RhoA and Cdc42 were significantly higher in UCC compared to normal tissue. RhoB RNA and protein levels exhibited an inverse expression profile, supporting its role as a tumour suppressor gene. Elucidating the regulatory mechanisms that modulate Rho-mediated signaling pathways in tumours may therefore provide novel targets for small molecule therapeutic agents against cancer