We demonstrated here for the first time that CK2α-meidated uPA signal through phosphorylated Akt is dependent on COX2 and plays an important role in cell survival of human urothelial carcinoma cells. To date, a number of reports have been accumulated as to the accelerating effects of COX2 on bladder cancer development, but to our knowledge, no investigations have pointed out the importance of CK2α as a downstream molecule. Manna et al. demonstrated the compound 5-(4-methoxyarylimino)-2-N-(3,4-dichlorophenyl)-3-oxo-1,2,4-thiadiazolidine (P(3)-25), which is known to possess anti-bacterial, anti-fungal, anti-tubercular, and local anesthetic activities, reduced phosphorylation of p65 by inhibiting upstream kinases including protein kinase A, CK2α etc resulting in down-regulation of nuclear factor (NF) kappaB-dependent reporter gene, COX2 in cancer cells with constitutive expression of NF kappaB[25
]. Our current data indicated reduced expression of CK2α following COX2 inhibitor or the gene silencing, but phosphorylation of p65 was not observed (data not shown). Whether COX2 interacts with CK2α through NF kappaB-dependent or independent pathway should be estimated using malignant cells. The present study provided COX2/CK2α is an upstream of Akt phosphorylation in urothelial carcinoma cells. CK2 has been previously reported to increase association of Akt to the Heat shock protein (HSP) 90 which maintains stability of phosphorylated Akt at threonine 308 or serine 473 by inhibiting dephosphorylation by protein phosphatase2A[26
]. Because expression of Akt was not affected by knockdown of COX2 or CK2α, CK2α-meditaed stability of Akt-HSP90 might be a main mechanism by which COX2 is closely related to Akt phosphorylation at threonine 308 in urothelial carcinomas. COX2 is fully expressed, but Akt is not constitutively activated, in UMUC2 cells, but it can be activated in response to specific external stimuli (data not shown), therefore, COX2/CK2α may not directly activate Akt but contributes to the maintenance of Akt once phosphorylated. COX2/CK2α and uPA signal actually functioned even in UMUC2 cells lacking activated Akt, therefore, CK2α is able to mediate uPA induction independent of Akt. The interesting data was that COX2 inhibitors down-regulated CK2α-Akt and uPA axis in urothelial carcinoma cells lacking the COX2 gene. Moreover, it is epoch-making that COX2 inhibitor can be used for bladder cancer therapy regardless of COX2 status at least in the cases with functional CK2α-Akt-uPA, even though it should be determined whether all COX2 inhibitors have the same pharmacological action. IC50 values of CAY10404 and DuP-649 for COX1 are 500 μM and 9 μM, respectively. Therefore, there are any possibilities that COX1 contributes to activation of casein kinase 2 alpha as well as COX2 in UMUC3 cells. Previous studies showed the importance of phosphatidyl inositol 3 kinase and Akt pathway in urothelial carcinoma[28
], but there are no clinically available inhibitors of this pathway. Possibly we can substitute COX2 inhibitors in bladder cancer treatment. To make sure we examined the effects of COX2 inhibitors on cell survival in other cell lines used in the current study than UMUC2 and UMUC3. The results showed both CAY10404 and DuP-649 similarly suppressed cell survival of KU-7, UMUC6 and UMUC6R cells at the concentrations more than 25 μM (data not shown).
We found that the COX2 and CK2α-Akt-uPA signal actually function in vivo
using the orthotropic implantation animal model. Oral feeding of COX2 inhibitor could dramatically reduced expression of CK2α, phosphorylated Akt and uPA, resulting in reduction of tumor growth and invasiveness. Unfortunately we have no data to explain the mechanism by which COX2 protein expression in KU7 cells implanted in the urinary bladder was increased in response to treatment with meloxicam. But this reaction is probably due to feedback regulation by inhibition of enzymatic activity of COX2, or due to execution of unidentified molecules or signals by COX2 inhibitors, resulting in transcriptional activation or protein stabilization. It should be further evaluated. Pathological analysis using human bladder cancer specimens clearly indicated that the COX2-CK2α and Akt phosphorylation axis is amplified in human urothelial carcinoma cells, particularly in advanced cases. These results mean that COX2 expression and Akt activation change urothelial carcinoma cells from non-invasive to an invasive phenotype. In addition to COX2/phosphorylated Akt, CK2α induction is essential for the progression to high grade and muscle invasive cancer. In urothelial carcinomas with microinvasion, CK2α signal does not function but Akt is activated, suggesting that Akt can be phosphorylated even in the absence of CK2α, but it is necessary for its sustainable activation and cancer development. We cannot explain why CK2α was not detectable in pT1 tumors, even though COX2 was expressed. Positivity and intensity of COX2 protein did not differ between tumors of pT1 and more than pT2, therefore, COX2 expression level is not essential. There is the possibility that the activity of COX2 or its products, including eicosanoids, might affect the transcription or stabilization of CK2α. Expression of COX2, CK2α or phosphorylated Akt in dysplasia was similar to that in high grade carcinoma in situ (n = 12, intensities of CK2α, COX2 and phosphorylated Akt were 2.2 ± 0.08, 2.0 ± 0.10 and 2.3 ± 0.07, respectively.). Urothelial dysplasia is preclinical cancer and low grade intraepithelial neoplasia, but its malignant potential is almost similar to high grade cancer from immunoprofiles of COX2, CK2α and phosphorylated Akt. CK2α and phosphorylated Akt signal might be useful targets for human bladder cancer treatment. The pathological significance of COX2 or Akt phophorylation has been well investigated independently[10
], and the current study opened the window for the problem, 'how these factors interact for each other'. COX2 affects Akt activation through manipulating CK2α expression, which is mainly involved in bladder cancer development.