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Cyclooxygenase 2-dependent and independent activation of Akt through casein kinase 2α contributes to human bladder cancer cell survival
© Shimada et al; licensee BioMed Central Ltd. 2011
Received: 7 December 2010
Accepted: 18 May 2011
Published: 18 May 2011
Survival rate for patients presenting muscle invasive bladder cancer is very low, and useful therapeutic target has not been identified yet. In the present study, new COX2 downstream signals involved in urothelial carcinoma cell survival were investigated in vitro and in vivo.
COX2 gene was silenced by siRNA transfection. Orthotopic implantation animal model and transurethral instillation of siRNA with atelocollagen was constructed to examine the effects of COX2 knockdown in vivo. Cell cycle was examined by flowcytoketry. Surgical specimens derived from patients with urinary bladder cancer (all were initially diagnosed cases) were used for immunohistochemical analysis of the indicated protein expression in urothelial carcinoma cells.
Treatment with the COX2 inhibitor or knockdown of COX2 reduced expression of casein kinase (CK) 2 α, a phophorylated Akt and urokinase type plasminogen activator (uPA), resulting in p27 induction, cell cycle arrest at G1 phase and cell growth suppression in human urothelial carcinoma cell lines expressing COX2. Silencing of CK2α exhibited the similar effects. Even in UMUC3 cells lacking the COX2 gene, COX2 inhibition also inhibited cell growth through down-regulation of the CK2α-Akt/uPA axis. The mouse orthotropic bladder cancer model demonstrated that the COX2 inhibitor, meloxicam significantly reduced CK2α, phosphorylated Akt and uPA expression, whereas induced p27 by which growth and invasiveness of bladder cancer cells were strongly inhibited. Immunohistochemically, high expression of COX2, CK2α and phosphorylated form of Akt was found in high-grade, invasive carcinomas as well as carcinoma in situ, but not in low-grade and noninvasive phenotypes.
COX2-dependent and independent activation of CK2α-Akt/uPA signal is mainly involved in urothelial carcinoma cell survival, moreover, not only COX2 but also CK2α could be direct targets of COX2 inhibitors.
Cyclooxygenase (COX) 2 is an inducible enzyme that produces prostaglandins during inflammatory and tumorigenic processes. The biology of COX2 in relation to tumor genesis has been well studied, particularly with regard to colorectal cancer development. Aberrant expression of COX2 as well as deregulation of WNT/β-catenin signaling occurs in the majority of colorectal tumor[2, 3]. Deregulation of COX-2 expression leads to an increased abundance of eicosanoids that affect the hallmarks of cancer. For examples, COX2/prostaglandin E2 signal is thought to protect tumor cells or tumor initiating cells from apoptosis induction by regulating pro- and/or anti-apoptotic molecules[4, 5]. In contrast, studies using in vivo animal models clearly indicated over expression or over activation of COX2 only cannot develop tumors spontaneously: breast cancer or colon cancer cannot be induced in COX2 transgenic mice without a murine mammary tumor virus infection or azoxymethane treatment[6, 7]. Thus, COX2 may be associated with promotion, but not initiation, of several types of human cancer.
Urinary bladder cancer is a common malignancy in industrialized countries including Japan. More than 90% of bladder cancer originates in the urothelial (transitional) cells. Low grade urothelial carcinoma can be usually controlled by intravesical therapy; in contrast, high grade cancer is much difficult to treat. Therefore, identification of target molecules or signals involved in urothelial carcinoma with high malignant potential is needed for successful therapy. COX2 is well known to enhance malignant potential of urothelial carcinoma cells[9, 10], and several types of COX2 inhibitors are clinically used for not only treatment but also prevention of bladder cancer. However, COX2-mediated signals involved in urothelial carcinoma cell survival remain fully undetermined, in addition, the fact that COX2 inhibitors have both COX2 -dependent and -independent cytotoxic effects make us much difficult to understand the biological roles. Casein kinase (CK) 2 is a ubiquitous serine/threonine protein kinase, and its heterotetrameric structure consists of two catalytic subunits (~42 kDa αand 38 kDa α') and two regulatory subunits (~28 kDa β) in α2β2, αα'β2, or α'2β2 configurations. Recent reports demonstrated CK2 is closely associated with tumor progression by phosphorylating a number of kinases [13, 14]. CK2 is widely expressed in various types of malignant tumors[14, 15], and many investigators have focused on CK2 as a therapeutic target. Wang H. et al. showed CK2 down-regulation induced apoptosis in prostate cancer cells that may lead to novel cancer therapies. However, there are no available data on the clinicopathological significance of CK2α in bladder cancer. In the present study, we found that COX2 is an upstream molecule of CK2α leading to Akt activation and urokinase type of plasminogen activator (uPA) induction. Silencing or inhibition of COX2 successfully inhibits CK2α-Akt/uPA axis, resulting in cell cycle arrest and growth suppression of bladder cancer cells in vitro and in vivo. Interestingly, even in urothelial carcinoma cells lacking COX2 gene, COX2 inhibitors reduced expression of CK2α and cell viability. The data suggest both COX2 dependent and independent activation of CK2α-Akt/uPA-mediated promotion and such signals could be targeted by COX2 inhibitors in human bladder cancer.
Bladder cancer cell lines, chemicals and antibodies
Human urothelial carcinoma cell lines UMUC2 and UMUC3 were cultured in RPMI supplemented with 10% fetal bovine serum. The origins of UMUC2 and UMUC3 are urethral tumor (carcinoma in situ) and bladder tumor (invaded carcinoma), respectively. Wild type of p53 is observed in UMUC2 cells, but it is largely deleted in UMUC3 cells. KU7 was derived from human papillary bladder cancer. We generated stable green fluorescent protein (GFP) KU-7 cells as previously described[19, 20]. Antibodies to COX2, p27, Akt and phosphorylated form Akt were purchased from Cell Signaling (Boston, MA, USA); Antibodies to actin and COX1 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); Antibodies to CK2α were supplied by Abcam (Cambridge, UK). COX2 selective inhibitor, CAY10404 or DuP-697 was from Cayman (Ann Arbor, USA). Phosphatidylinositol-3-kinase inhibitor, LY294002, soluble in DMSO was purchased from Cell Signaling Technology Japan (Tokyo, Japan).
Preparation of cell lysates and Western blotting analysis
Cell lysates were resolved in SDS-polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (Millipore, Ltd., MA, USA), which were then blocked in 5% skim milk at room temperature for 1 h. Membranes were incubated with the indicated primary antibody (1:50-1000 dilution according to the manufacture's protocol) for 1 h, and then incubated with horseradish peroxidase-conjugated antimouse or antirabbit IgG (1:5000 dilution) (Amersham Pharmacia Biotech, Boul Morgan Baie-D'Urfe, Canada). We detected peroxidase activity on X-ray films using an enhanced chemiluminescence detection system.
Using the One Step RT-PCR kit (Qiagen), we extracted total RNA using Trizol reagent and subjected it to reverse transcription-PCR (RT-PCR). PCR conditions were 95°C for 30 s, 55 to 58-60°C for 30 s, and 72°C for 1 min through a total of 30 cycles.
The PCR primer sequences for uPA were 5'-TCACCACCAAAATGCTGTGT-3' (sense) and 5'-AGGCCATTCTCTTCCTTGGT-3'(antisense). The primers for COX2 were 5'-GCAATAACGTGAAGGGCTGT-3' (sense) and 5'-CGGGAAGAACTTGCATTGAT-3' (antisense). For glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the primers used were 5'-ACCACAGTCCATGCCATCAC-3' (sense) and 5'-TCCACCACCCTGTTGCTGTA-3' (antisense).
siRNA transfection of COX2/CK2α
For our transfection analyses, 106 cells from each bladder cancer cell line were seeded in 6-cm dish plates and transfected either with 100 nmol/L of control siRNA (Santa Cruz Biotech., CA, USA) or with the siRNA of COX2, CK2α. Transfections were carried out using the Lipofectamine system (Invitrogen, CA, USA) in accordance with the manufacturer's protocol. COX2, CK2α siRNA duplexes, generated with 3'-dTdT overhangs and prepared by Qiagen, were chosen against the DNA target sequences as follows: 5'-AACACCGGATTTTTGACAAG-3' for COX2; 5'-CTGGTCGCTTACATCACTTTA-3' and 5'-TCCATTGAAGCTGAAATGGTA-3'for CK2α (cocktail).
Analysis of uPA activity
uPA activity in culture media was analyzed using an uPA activity assay kit (Chemicon International, Billerica, MA, USA). In accordance to the manufacture's protocol, culture media were collected after indicated treatments or siRNA transfection. Media were concentrated by centrifugation, samples were added into 96-well plate together with positive control, and reacted with several indicated reagents including chromogenic substrates. After incubation at 37°C for 30 min, absorbance was measured on a standard microplate reader at 405 nm. All experiments were done at least thrice in triplicate.
Cell cycle analysis
We performed cell cycle analysis using propidium iodide by flow cytometry as previously described . All experiments were done at least thrice in duplicate.
Orthotropic tumor implantation and intravesical treatment
Animal experiments were approved by the institutional animal care and use committee at Nara Medical University. Eight week-old female nude mice were maintained on a daily 12-h cycle of light and dark and were fed standard diet and water ad libidum. Orthotropic bladder cancer exhibiting muscle invasive phenotype was established by inoculating KU7/GFP cells (5 × 106) into the mouse bladder using transurethral catheter. Seven days after tumor cell inoculations, mice were randomized into control (n = 6) or meloxicam (n = 6), treatment groups. Before the beginning of treatment with meloxicam, we captured in vivo images of tumor masses in the urinary bladder, and identified tumor implantation there were no significant differences of fluorescence intensities between control and drug treatment groups. Meloxicam was fed in the drinking water (3.0 mg/l). Mice were sacrificed 25 d after treatment and bladders were removed, splayed open on filter paper and fixed in 10% neutral buffered formalin. Tumor burden was determined by analyzing images of GFP fluorescence from tumor cells on flat formalin-fixed bladders captured using a macro-imaging station consisting of a SBIG cooled CCD camera model ST-7XME (Santa Barbara Imaging Group Inc., Santa Barbara, CA, USA) mounted onto a dark box. Bladders were embedded in paraffin, step-sectioned, stained with hematoxylin and eosin (H&E) for histological evaluation. Invasion depth means the length from the top to the bottom level of the largest tumor. Results from animal experiments were compiled from data generated from three independent experiments. RNA and protein extractions were performed as follows: Tumor masses expressing GFP was removed under the stereoscopic fluorescence microscopy and the tumors were fragmented by shaver. RNA was extracted by TriReagent (Ambion, Austin, Texas, USA) according to the manufacturers' instructions. Protein was extracted after homogenization using lysis buffer.
A method for the quantification of tumor burden by computer assisted image analysis has been described elsewhere . Briefly, image analysis was performed with ImageJ public domain software available through the National Institutes of Health (Bethesda, MD; available at http://rsb.info.nih.gov/ij/). All images were spatially calibrated for area measurements. Signal strength was recorded as electrons emitted per second of exposure. The area under the curve (AUC) was determined from plot profiles based on fluorescent signal strength and distribution in each individual bladder.
Cell viability assay (MTS assay)
After incubation with the indicated reagents, MTS [(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphonyl)-2H-tetrazolium, inner salt] reagent (Promega, Tokyo, Japan) was added and optical absorbance at 490 nm was measured using a microplate reader. Cell viability was expressed as mean percentages the standard deviations of absorbance before and after treatment with various reagents. All experiments were performed in triplicate.
Tissue samples and immunohistochemistry
Characterization of urothelial carcinomas
Mean Age (years)
72.2(44 to 96)
Data were statistically analyzed using the Student t test or, for nonparametric analysis, the Kruskal-Wallis test. Results were considered significant if P < 0.05.
Inhibition of COX2 suppressed growth of urothelial carcinoma cells
COX2-dependent activation of casein kinase/uPA signal in urothelial carcinoma cells
COX2 affects CK2α-mediated activation of Akt in urothelial carcinoma cells
COX2 inhibitor suppressed COX2- CK2α-Akt-uPA signal independent of COX2
In vivo growth of urothelial carcinoma is suppressed by COX2 inhibitor, Meloxicam through affecting CK2α-Akt-p27 signals
COX2, CK2α and activated Akt are over expressed in human urothelial carcinoma of the urinary bladder
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. 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, 27]. 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, 29], 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, 29, 30], 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.
This research was supported in part by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan (21790392).
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