In transitional cell carcinoma, the most common form of bladder cancer, overexpression of the matrix metalloproteinases MMP-2 and MMP-9 offers prognostic value as markers of disease-specific survival. These molecules have been implicated in metastasis of bladder cancer, but the underlying mechanisms through which they are controlled are poorly defined. In this study, we investigated a role of p38 mitogen-activated protein kinase (MAPK) in this process, using bladder cancer cell lines HTB9 and HTB5 that were derived from different tumor stages. p38 MAPK modulated MMP-2/9 mRNA levels at the levels of transcript stability and MMP-2/9 activity along with invasive capacity. We defined a downstream effector of p38 MAPK, MAPK-activated protein kinase 2 (MAPKAPK2), that was associated with MMP-2/9 activation. Ectopic expression of wild-type or constitutively active forms of MAPKAPK2 increased MMP-2/9 activities and invasive capacity. Conversely, p38 MAPK inhibition blocked the MAPKAPK2-mediated increase in MMP-2/9 activities and the invasive capacity of the cancer cells. Our findings implicate p38 MAPK and MAPKAPK2 in mediating bladder cancer invasion via regulation of MMP-2 and MMP-9 at the level of mRNA stability. Cancer Res; 70(2); 832–41
- Bladder cancer
- p38 MAPK
- mRNA stability
Bladder cancer is a worldwide health problem with an estimated 375,000 cases reported annually. In the United States, an estimated 68,810 new cases and 14,100 deaths from bladder cancer were reported in 2008 alone (http://www.cancer.gov/cancertopics/types/bladder). Transitional cell carcinoma is the most common form of bladder cancer (1). A striking feature of transitional cell carcinoma is the existence of two distinct types of tumors with different clinical features and molecular subtypes. About 75% of the bladder cancer cases were superficial, with relatively benign but recurrent behavior. In contrast, ∼20% of tumors were muscle-invasive at diagnosis, with aggressive behavior (2).
Carcinogenesis of human bladder cancer is a multistep process, and metastasis represents a critical step in carcinogenesis. There are many markers associated with the progression of bladder carcinoma, such as depth of invasion, stage, grade, and multiplicity. Unfortunately, several questions remain unaddressed due to the lack of definitive clinical prognostic markers and targets. Therefore, in bladder cancer, there is a need to identify those molecular targets that may predict which superficial bladder tumors will later progress to become invasive (3). Thus, looking for appropriate molecular targets that regulate bladder cancer metastasis is an important step in identifying and creating newer generation chemotherapeutics with improved efficacy and fewer side effect profiles. Extensive work on the mechanism of tumor invasion and metastasis has identified matrix metalloproteinases (MMP) as the key players in tumor spread. Elevated MMP-2 levels have been suggested to be of independent prognostic value in patients with bladder cancer, however, MMP-9 did not correlate with tumor grade, stage, or overall survival of the patients with bladder cancer (4–6).
Activation of the mitogen-activated protein kinase (MAPK) pathways is a recurrent event in tumorigenesis, and has a potential role in metastasis of tumor cells (7). MAPKs have been shown to be involved in regulating the proteolytic enzymes that degrade the basement membrane, and therefore, have been implicated in the progression and invasion of cancer (8, 9). MAPK-activated protein kinase 2 (MAPKAPK2) and heat shock protein 27 (HSP27), downstream effectors of p38 MAPK are reported to activate MMP-2 and cell invasion in human prostate cancer (10). Interestingly, in bladder cancer cells, there is no direct information on the status of the p38 MAPK pathway, but several events identified in invasive tumors may activate the MAPK and/or the phosphoinositide-3 kinase pathways (2, 11).
Previous studies in our laboratory showed that MAPK pathways are active during the log phase growth of bladder cancer cells (12). In continuation, the present studies were carried out to evaluate the role of p38 MAPK–driven MAPKAPK2 in the regulation and modulation of bladder cancer metastasis in two bladder cell lines HTB9 and HTB5 (derived from grade 2 and 4 bladder cancers, respectively). Our results suggest that p38 MAPK modulates MMP-2 and MMP-9 expression, stability, and activity as well as invasion of bladder cancer cells. Additionally, we found that MAPKAPK2, a downstream effector of p38 MAPK, was associated with invasion as well as MMP-2/9 activities in bladder cancer cell lines. Taken together, our results show, for the first time, that p38 MAPK and MAPKAPK2 regulate the invasion of bladder cancer by modulation of MMP-2 and MMP-9 mRNA stability.
Materials and Methods
Cell lines and reagents
HTB5 and HTB9 cells were purchased from American Type Culture Collection and maintained according to its guidelines. Most of the chemicals as well as SB203580 and actinomycin D were obtained from Sigma. Primary antibodies MAPKAPK2, pp-MAPKAPK2, HSP27, pp-HSP27, p38 MAPK, and pp-p38 MAPK were obtained from Cell Signaling. Plasmids expressing wild-type MAPKAPK2, cDNA3mycMK2WT (MK2WT); dominant-negative kinase-inactive mutant, pcDNA3mycMK2K76R (MK2K76R); and constitutively active mutant, pcDNA3mycMK2T205E317E (MK2EE) were kindly provided by Professor Matthias Gaestel (Institute of Biochemistry, Medical School, Hannover, Germany). MMP-2 luciferase reporter construct was a kind gift from Shaun Marie Sparacio (University of Alabama at Birmingham). MMP-9 proximal promoter constructs were kindly provided by Dina Lev (The University of Texas M.D. Anderson Cancer Center, Houston, TX; ref. 13).
Treatments and transfections
For each experiment, equal numbers of cells were seeded in respective media. In most of the experiments, 10 to 20 μmol/L of SB203580 (stock in DMSO) were used, and the same concentration of DMSO was applied to control cells where indicated. For siRNA transfections, 25 nmol/L of siRNA for p38 MAPK (Ambion, Inc.) were mixed with HiPerFect transfecting reagent (Qiagen) and processed according to the protocol of the manufacturer. For plasmid transfections, 2 to 3 μg of DNA was transfected using Effectene (Qiagen) according to the protocol of the manufacturer. For mRNA stability experiments, cells were treated with actinomycin D (5 μg/mL) in the presence or absence of either SB203580 (20 μmol/L, ∼16 h) or dominant-negative kinase-inactive mutant plasmid (MK2K76R, 2 μg) for different time points, and mRNA levels were measured by relative quantitative reverse transcription-PCR (RT-PCR).
Cell motility and wound healing assays
For persistence migratory directionality assay, confluent cultures (80–90% in serum-free medium) were used (in vitro scratch wound healing assay) and experiments were performed as described (14).
Matrigel invasion assays
In vitro invasion assays were carried out in BD BioCoat Matrigel chambers (Transwell) as described previously (15). Wherever necessary, SB203580 or 100 μg/mL of anti-human MMP-2 and MMP-9 of IgG or mouse IgG were used (16).
Total RNA was extracted using RNEasy mini kit (Qiagen) and was used to prepare cDNA using iScript second-strand cDNA synthesis kit (Bio-Rad). Synthesized cDNA (100 ng) was used for RT-PCR using primers as described in Supplementary Table S1. Transcripts were separated by agarose gel electrophoresis and expression was quantitated by densitometry.
MMP-2 and MMP-9 activities in the conditioned media were determined by zymography as per methods described by Bernhard and Muschel (17).
Cells were transfected with MAPKAPK2 expression plasmids (MK2WT, MK2EE, and MK2K76R; 2 μg of DNA) and 0.5 μg of MMP-2 and MMP-9 luciferase reporter vector along with 10 ng of Renilla luciferase plasmid using Effectene transfection reagent according to the instructions of the manufacturer. Luciferase activity was measured using the Dual luciferase kit (Promega Corporation) with Monolight 2010 Luminometer (Analytical Luminescence Laboratory).
MAPKAPK2 immunocomplex kinase assays
Nonradioactive MAPKAPK2 kinase was assayed using MAPKAP Kinase 2 immunoprecipitation kinase assay kit; Upstate) according to the protocols of the manufacturer. Radioactive MAPKAPK2 assay was performed using the same kit described above except that γ-32P was added to the immunocomplex reaction to measure the phosphotransferase activity.
Measurement of cell proliferation and viability
Western blot analysis
Western blot analysis was performed as described previously (20).
p38 MAPK regulates cell motility in bladder cancer cell lines
Results show that treatment of HTB9 and HTB5 cells with p38 MAPK inhibitor (SB203580) reduced cell motility (Fig. 1A and B). It is important to point out that HTB9 cell motility was more sensitive to p38 MAPK inhibition as compared with HTB5 cells. Similar results were obtained with p38SiRNA treatments (Fig. 1C). Taken together, these results show a critical role for p38 MAPK in the motility of bladder cancer cell lines.
p38 MAPK–dependent cell invasion and gelatinase activity and MMP-2/9 expression and invasion are interdependent events in bladder cancer cell lines
Activated MAPK pathways have been detected in many human tumors, suggesting the possible role of MAPKs in tumor progression and metastasis (21). Results presented in Fig. 2A show that inhibition of p38 MAPK activity with either SB203580 or by p38SiRNA inhibited the invasion of HTB9 bladder cancer cells. MMPs are a family of enzymes whose function primarily relates to degradation of extracellular matrix proteins, and is necessary for cell invasion. Moreover, treatment with SB203580 as well as p38SiRNA strongly inhibited MMP-2 and MMP-9 gelatinolytic activities as well as their promoter activities in the HTB9 cell line (Fig. 2B and C). These treatments, however, did not have a significant effect on cell invasion in the HTB5 cell line (Supplementary Fig. S2E), and HTB5 cells displayed low MMP-9 activity and no MMP-2 activity (Supplementary Fig. S2G). These results suggested that p38 MAPK plays an important role in modulating MMP activity and the invasive phenotype of bladder cancer.
To determine the role of MMPs in bladder cancer invasion, we studied invasion after blocking MMP-2. Results show treatment with an anti–MMP-2 antibody inhibited invasion by 50% to 60% as compared with IgG control (Fig. 2D). Similarly, anti–MMP-9 antibody reduced the invasion of the HTB9 cell line, and had no effect on HTB5 cell invasion (Supplementary Fig. S2F). Taken together, these results indicated that increased expression of MMP-2 and MMP-9 in bladder cancer is associated with increased invasion.
MAPKAPK2 is a downstream effector of p38 MAPK in bladder cancer cells
Results presented in Fig. 3A and B show that treatment of the cells with p38 MAPK–specific inhibitor, SB203580 decreased kinase activity of MAPKAPK2, as determined by reduced phosphorylation of both MAPKAPK2 as well as HSP27 (Fig. 3C). Interestingly, SB203580 treatment increased the phosphorylation of p38 MAPK (data not shown). Indeed, others have previously observed that blocking p38 MAPK activity with another p38 MAPK inhibitor (SB202190) inhibited activity but enhanced p38 MAPK phosphorylation, suggesting a negative feedback regulation of p38 MAPK phosphorylation (22, 23). These results show the requirement of p38 MAPK for HSP27 and MAPKAPK2 in bladder cancer cells.
p38 MAPK regulates MMP-2 and MMP-9 mRNA stability in bladder cancer
To investigate the role of p38 MAPK in the regulation of MMP-2 and MMP-9, we performed RT-PCR in the absence or presence of p38 MAPK inhibitor. Treatment of HTB9 cells with SB203580 completely abolished MMP-9 expression and showed partial effects on MMP-2 expression (Fig. 4A). There were no changes in endogenous tissue inhibitor of MMPs (TIMP1 and TIMP2) after SB203580 treatment at the transcriptional as well as the translational level (Fig. 4A; Supplementary Fig. S4E and F). This modulation of MMP expression with p38 MAPK inhibitor was concomitant with MMP activity obtained by zymogram in HTB9 cell line (Fig. 2B).
We also performed mRNA stability experiments in the presence or absence of either SB203580 or dominant-negative kinase-inactive mutants of MAPKAPK2 plasmid (MK2K76R). Our RT-PCR results indicated that the addition of actinomycin D alone did not influence MMP-2 and MMP-9 mRNA decay (t1/2 = ∼8 and ∼7.2 h, respectively), whereas the addition of SB203580 caused nearly complete decay of MMP-2 (t1/2 = 3.6 h) and MMP-9 mRNA (t1/2 = 5.1 h; Fig. 4B and C; left) in 8 h. A similar result was observed with the inactive mutant of MAPKAPK2 (MK2K76R plasmid; Fig. 4B and C; right). Because MAPKAPK2 is a downstream effector of p38 MAPK, we also performed in vitro MAPKAPK2 kinase assay with actinomycin D to rule out any off-target effects of actinomycin D on MAPKAPK2 activity. Results suggest that actinomycin D does not have any effect on the activity of MAPKAPK2 (Supplementary Fig. S4G). Taken together, these results show that p38 MAPK and associated MAPKAPK2 regulate MMP-2 and MMP-9, at least in part at the posttranscriptional level, by stabilizing mRNA transcripts.
p38 MAPK–driven MAPKAPK2 regulates MMP-2/9 activity and invasion in bladder cancer cell line
The above observations raised the possibility that p38 MAPK regulated invasion and MMP-2/9 activity via regulation of MAPKAPK2. To test this possibility, cells were transfected with either wild-type MAPKAPK2 plasmid (MK2WT), a kinase-inactive dominant-negative mutant (MK2K76R), a constitutive active mutant (MK2EE), or empty vector (VC); and the effects of these treatments on HSP27 phosphorylation and invasion, and MMP-2/9 activity were studied. Results presented in Fig. 5A show that the levels of pMAPKAPK2 and pHSP27 were higher in the constitutive active mutant, MK2EE, compared with other treatments groups. These observations suggest that HSP27 is downstream of MAPKAPK2 in bladder cancer. Furthermore, results presented in Fig. 5B suggest that cells transfected with constitutive active (MK2EE) plasmid showed maximal MMP-2 and MMP-9 luciferase activity, whereas in dominant-negative (MK2K76R) transfected cells, the activity was less than the wild-type (MK2WT) and VC-transfected cells. Interestingly, both wild-type and constitutive active MAPKAPK2 plasmid-transfected cells had reduced MMP-2 and MMP-9 luciferase activities when treated with SB203580 (Fig. 5B). These results were further confirmed by zymograms for MMP-2/9 activity. As seen in Fig. 5C and D, gelatinolytic activity of MMP-2/9 in constitutive active and wild-type MAPKAPK2 plasmids was reduced by SB203580 treatment. Taken together, these results signify the involvement of MAPKAPK2 in p38 MAPK–regulated MMP-2/9 activity in bladder cancer cell lines.
As the activity of MMPs is very much coupled with the invasion/metastasis of the bladder cancer cells (Fig. 2D), we explored the invasive potential of bladder cancer cells expressing MAPKAPK2 with or without SB203580. Results presented in Fig. 6A and B highlight the importance of active MAPKAPK2 in the invasion of HTB9 cells. Cells transfected with kinase-inactive mutant, MK2K76R, showed less invasion compared with wild-type and constitutive active plasmid (Fig. 6A and B). Moreover, treatment with SB203580 also decreased the invasion of bladder cancer cells overexpressing wild-type and constitutively active MAPKAPK2 protein. This was comparable to the invasion of cells overexpressing the kinase-inactive form of MAPKAPK2. These results suggest that the invasive potential of bladder cancer cells is strongly regulated by p38 MAPK via MAPKAPK2 signaling.
p38 MAPK–regulated metastatic potential is not a reflection of inhibition of growth/proliferation of bladder cancer cells
Previous observations indicated that p38 MAPK had an effect on migration and invasion of bladder cancer cell lines. To rule out changes in cell growth or proliferation, we determined the proliferation and viability of bladder cancer cells after treatment with SB203580 or p38SiRNA. Treatment with 10 and 20 μmol/L of SB203580 or with p38 MAPK siRNA for 72 hours caused only ∼25% reduction in cell viability of HTB9 cells (Fig. 6C and D), whereas HTB5 cells were more resistant. The IC50 value for SB203580 over a period of 72 hours was ∼40 μmol/L in these cells. These results indicate that suppression of cell invasion and MMP activity in bladder cancer cells by inhibition of p38 MAPK are distinct from the cytotoxic effects of p38 MAPK inhibition.
Most patients with bladder cancer (∼70%) present superficial disease (Tis, Ta, T1), 20% to 30% present muscle-invasive tumor (grades 2 and 3 bladder cancer), and 5% present clinically evident distant metastases (grade 4 bladder cancer; refs. 24, 25). Transitional cell carcinomas follow the general concept of multistep carcinogenesis and proceeds through two distinct pathways responsible for different transitional cell carcinoma morphologies and aggressiveness. Thus, understanding the molecular biology of the invasive bladder cancer would allow us to develop new strategies to improve prognosis and patient treatment options.
There is a growing body of evidence suggesting the activation of p38 MAPK in the pathogenesis of bladder cancer (2). Preliminary work in our laboratory showed that ERK1/2 and p38 MAPK have differential effects on the growth of invasive bladder cancer cells (12). In the current study, we sought to identify the role of p38 MAPK, a molecular target for invasive cancer cells, in the regulation of metastasis of invasive bladder tumor cell lines, HTB9 and HTB5. We show for the first time, as presented in Figs. 1 and 2, that migration and invasion of bladder cancer cells could be linked to p38 MAPK activity. However, these effects were less prominent in HTB5, which is derived from an advanced bladder cancer (grade 4 tumor). Although the exact reason for this is not known, it can be speculated to be due to differential signaling mechanism in different stages of cancer, and there is great variability in the prognosis of patients with stage II and stage IV bladder tumors (26).
One of the mechanisms by which p38 MAPK may promote tumor cell migration and invasion is by the upregulation of MMPs. Our results showed strong MMP-2 and MMP-9 activity in HTB9 cells, and low MMP-9 activity in HTB5 cells in a basal state (Supplementary Fig. S2F). Furthermore, inhibition of p38 MAPK signaling reduces both MMP-2 and MMP-9 activity (Fig. 2B and C). Therefore, active p38 MAPK signaling may regulate bladder cancer cell migration/invasion by influencing MMP-2/9 activity. Moreover, addition of MMP-2 and MMP-9 antibody inhibited cell invasion, suggesting that invasion in bladder cancer cells is directly dependent on active MMP expression (Fig. 2D). These findings also provide a possible explanation for the highly invasive and aggressive nature of bladder cancer cells in comparison to superficial papillary bladder cancer. Indeed, increased activity of MMP-2 or MMP-9 correlates with increased cell invasion and metastasis in bladder cancer (27, 28).
MMP-2 and MMP-9 are known to induce aggressive invasion and metastasis of different cancer types, and many studies have concentrated on exploring the level of these markers along with their inhibitors (TIMP) in the prognosis of aggressive behavior of various cancers including bladder cancer (29, 30). We observed that the HTB5 cell line expressed only MMP-2, whereas the HTB9 cell line expressed MMP-2 as well as MMP-9. Both the expression of mature MMP protein and its enzyme activity were p38 MAPK–dependent as indicated in Figs. 2 and 4. The reason for the differences in the expression and activity of MMPs between these cell lines is unknown, but one of the reasons might be due to the existence of differential regulatory mechanisms in different stages or grades of cancer. These results highlight the fact that there could be different mechanisms operative in lines derived from different tumor grades, thus putting emphasis on the significance of investigating individual molecular pathways in multiple cell types or tissues.
Imbalance between MMP and TIMP activities seems to be associated with tumor stage, grade, and clinical events (31). Our results show that there was no change in TIMP expression with SB203580 treatment (Fig. 4A; Supplementary Fig. S4E and F). These results suggested that MMP activity could be regulated by p38 MAPK pathway independent of TIMP regulation. Indeed, we observed that p38 MAPK inhibitor and dominant-negative kinase-inactive mutant of MAPKAPK2 significantly reduced the half life of MMP-2 and MMP-9 mRNA. These findings indicate distinct regulation of MMP-2 and MMP-9 transcripts in bladder cancer by p38 MAPK signaling pathway.
MAPKAPK2 is known to be a p38 MAPK downstream signaling protein (32) and is known to be involved in many cellular processes including inflammatory responses, nuclear export, gene expression regulation, and cell proliferation. HSP27 was shown to be one of the substrates of this kinase in vivo (33, 34). Our results show, for the first time, the role of p38 MAPK in MAPKAPK2-dependent invasion and MMP activity in bladder cancer. These results are in agreement with the results of Xu and colleagues (10), who reported the role of MAPKAPK2 and HSP27 in the invasion of prostate cancer cell lines. We found that SB203580 treatment decreases MAPKAPK2 phosphorylation (Fig. 3). Furthermore, the role of MAPKAPK2 in the metastasis or invasion of bladder cancer has not been identified, although a report by Kotlyarov and colleagues (35) regarding the reduction in the migration of MAPKAPK2-deficient mouse embryonic fibroblasts and smooth muscle cells on fibronectin highlight the importance of MAPKAPK2 in the metastasis phenotype of cancer cells. Our study supports the above observation in the bladder cancer cell line, as transfection with a kinase-inactive dominant-negative mutant, MK2K76R, reduced MMP activity and invasion (Figs. 5 and 6). In addition, treatment with SB203580 inhibited gelatinase activity and invasion in cells overexpressing wild-type and constitutive active MAPKAPK2, indicating their importance in the regulation of metastasis of bladder cancer. Taken together, these results imply that p38 MAPK–driven MAPKAPK2 regulates the invasion of bladder cancer cells by stabilizing MMP-2/9 transcripts. Taken together, our results show the essential role of p38 MAPK in regulating an essential component in bladder cancer cell invasion. These results indicate that inhibitors of p38 MAPK pathway might serve as excellent candidates for intervention in invasive bladder cancer.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
The authors gratefully acknowledge Professor Matthias Gaestel for kindly providing us MAPKAPK2 plasmids. We also thank Shaun Marie Sparacio and Dina Lev for kindly providing us MMP-2 and MMP-9 luciferase reporter constructs, respectively.
Grant Support: Department of Surgery and the University of Colorado School of Medicine and Academic Enrichment Funds.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).
- Received August 11, 2009.
- Revision received October 8, 2009.
- Accepted October 23, 2009.
- ©2010 American Association for Cancer Research.