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Cancer Research 67, 4956-4964, May 15, 2007. doi: 10.1158/0008-5472.CAN-07-0380
© 2007 American Association for Cancer Research
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Experimental Therapeutics, Molecular Targets, and Chemical Biology

MMP-9 Short Interfering RNA Induced Senescence Resulting in Inhibition of Medulloblastoma Growth via p16INK4a and Mitogen-Activated Protein Kinase Pathway

Jasti S. Rao1,3, Praveen Bhoopathi1, Chandramu Chetty1, Meena Gujrati2 and Sajani S. Lakka1

Departments of 1 Cancer Biology and Pharmacology, 2 Pathology, and 3 Neurosurgery, University of Illinois College of Medicine at Peoria, Peoria, Illinois

Requests for reprints: Sajani S. Lakka, Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, One Illini Drive, Peoria, IL 61605. Phone: 309-671-3445; Fax: 309-671-3442; E-mail: slakka{at}uic.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The involvement of matrix metalloproteinases (MMP) has been suggested in cellular mechanisms leading to medulloblastoma, the most common malignant brain tumor in children. A significant association of the expression levels of MMP-9 with survival and M stage suggests that patients with medulloblastoma metastatic disease at diagnosis may benefit from the anti-MMP therapy. Here, we have evaluated the tumorigenicity of medulloblastoma cells after infection with an adenovirus containing a 21-bp short interfering RNA sequence of the human MMP-9 gene (Ad-MMP-9). Infection of Daoy medulloblastoma cells with Ad-MMP-9 reduced MMP-9 activity and protein levels compared with parental and Ad-SV controls. Ad-MMP-9 decreased the number of viable Daoy cells in a concentration-dependent manner. Fluorescence-activated cell sorting analysis indicated that Ad-MMP-9 infection caused a dose-dependent cell cycle arrest in the G0-G1 phase. Ad-MMP-9–induced cell cycle arrest seems to be mediated by the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway and the cell cycle inhibitor p16INK4a and is phenotypically indistinguishable from senescence. Ad-MMP-9 treatment inhibited medulloblastoma tumor growth in an intracranial model and was mediated by up-regulation of p16 expression. These studies validate the usefulness of targeting MMP-9 and provide a novel perspective in the treatment of medulloblastoma. [Cancer Res 2007;67(10):4956–64]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Medulloblastoma is an embryonic brain tumor that arises within the external germinal layer of the cerebellum. There have been many recent advances in the treatment of medulloblastoma, including improved surgical resection techniques, radiation, and chemotherapy (1, 2). The prognosis for patients with these tumors remains variable and is relatively poor in infants and adult patients with metastatic disease. The traditional treatments are also toxic and can lead to long-term disabilities (3). Therefore, development of novel therapeutic approaches, such as those aimed at targeting tumor cell invasion and metastasis, is greatly needed.

The underlying molecular mechanisms of brain tumor invasiveness have been found to be closely related to the proteolytic degradation of the extracellular matrix. The extracellular matrix of the brain is mainly comprised of proteins such as fibronectin, laminin, vitronectin, thrombospondin, tenascin, heparin sulfate proteoglycan, and collagen IV (4). Several proteases are thought to be involved in the degradation of extracellular matrix components, including matrix metalloproteinases (MMP). In addition to extracellular matrix degradation, MMPs are also capable of releasing growth factors and/or inactive complexes, cleaving growth factor receptors, and activating growth factors excreted as pre-proenzymes, such as transforming growth factor {alpha} and ß, macrophage-colony stimulating factor, insulin-like growth factor, and fibroblast growth factor receptor-1 (5–8). Finally, some MMPs have been shown to play a crucial role in tumor invasion. Among the MMPs thought to be involved in cancer, attention has been focused on MMP-2 and MMP-9, which specifically degrade the main structural component of basement membranes (i.e., type IV collagen). Studies on regional distribution of MMPs in medulloblastoma tumors indicated that MMP-2 and MMP-9 were strongly expressed (9–11). A number of studies have linked elevated MMP-2 and MMP-9 expression to increased metastasis and advanced tumor stage (12, 13). Notably, MMP-9 is not expressed in normal adult tissues but is expressed in invasive tumors and represents a key protein in brain tumor progression. MMP-9, MT1-MMP, and MT2-MMP are often and strongly expressed in classic and desmoplastic medulloblastomas and correlates with prognosis in classic medulloblastomas (14).

RNA interference (RNAi) has been shown to be an effective method for inhibiting the expression of a specific gene in human cells via targeting with short duplex RNA (short interfering RNA or siRNA). Antitumor activities have been attained in vivo through siRNA knockdown of several components for tumor cell growth, metastasis, angiogenesis, and chemoresistance (15). Here, we assessed the potential of RNAi-mediated MMP-9 gene knockdown in medulloblastoma cells. In the present study, we constructed a replication-deficient recombinant adenovirus (Ad-MMP-9) to efficiently deliver MMP-9 siRNA targeted to the MMP-9 gene, thereby down-regulating MMP-9 expression in a medulloblastoma cell line. Our results show that the down-regulation of MMP-9 has a therapeutic effect in inhibiting medulloblastoma cell growth and invasion in vitro and in vivo. We also show that Ad-MMP-9 promotes p16INK4a-mediated senescence and cell cycle arrest in the Daoy medulloblastoma cancer cell line. These results provide insight about the underlying mechanisms of the antitumorigenic effects of Ad-MMP-9.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell cultures. We used the Daoy medulloblastoma cell line, which was derived from a tumor in the posterior fossa of a 4-year-old boy (ATCC #HTB 186). Daoy cells were cultured in advanced-MEM supplemented with 5% fetal bovine serum, 2 mmol/L L-glutamine, 2 mmol/L sodium pyruvate, 100 units/mL penicillin, and 100 µg/mL streptomycin. Cells were maintained in a humidified atmosphere containing 5% CO2 at 37°C.

Extracellular matrix components and antibodies. Laminin, fibronectin, and vitronectin were obtained from Sigma. Antibodies for MMP-9, p16, p21, extracellular signal-regulated kinase (ERK), phosphorylated ERK (pERK), and pRb were obtained from Santa Cruz Biotechnology; cyclin-dependent kinase 2 (cdk2), cdk4, cyclin D2, and cyclin E were from Biomeda; {alpha}vß3, {alpha}5ß1, and ß4 were from Chemicon.

Adenovirus construction. We constructed two adenoviruses: one carrying siRNA targeting the MMP-9 gene (Ad-MMP-9) and the other carrying a scrambled sequence of the MMP-9 siRNA (Ad-SV). Oligonucleotides were designed using an adenoviral pSuppressor kit (Imgenex) inserted into the suppressor vector under the control of the modified polymerase II promoter as per manufacturer's instructions. This plasmid was cotransfected with the pAd vector backbone in 293 cells. Adenovirus generation, amplification, and titer were done according to previously described procedures (16). Briefly, viral particles were purified using a cesium chloride density gradient. Viral titers were assessed using the plaque-forming test (PFU) and counting infectious virus particles.

Adenoviral infection. Infection with recombinant viruses was accomplished by exposing cells to adenovirus in serum-free cell culture medium for 30 min followed by addition of serum-containing medium. We used green fluorescent protein–expressing recombinant adenovirus (Ad-SV/GFP) as a control when determining transfection efficiency.

Gelatin zymography. Gelatin zymography was done as described previously (17). For tumor-conditioned medium, DAOY cells were grown in six-well tissue culture plates and infected with mock (PBS), 100 multiplicities of infection (MOI) of Ad-SV, or 25 to 100 MOI of Ad-MMP-9. After a 36-h incubation period, cells were washed with PBS and cultured overnight in serum-free DMEM/F-12 medium. The total protein concentration of the conditioned media was estimated using bicinchoninic acid (BCA) reagent (Pierce). Equal amounts of protein from various treatments were used to determine gelatinase activity.

Western blotting. Western blot analysis was done as described previously (17). Briefly, Daoy cells were grown in 100-mm plates and infected with mock, 100 MOI of Ad-SV, or various MOI of Ad-MMP-9 and incubated for 48 h at 37°C. Cell lysates were prepared in radioimmunoprecipitation assay buffer, and protein concentrations were measured using bicinchoninic acid (BCA) protein assay regents (Pierce). For electrophoresis, 30 to 40 µg of protein in 6x sample buffer were loaded to each well of a SDS-PAGE gel. The blot was blocked and probed overnight with primary antibodies for either MMP-9, ERK, pERK, p21, p16, pRb, Cdk2, Cdk4, cyclin D, and cyclin E at 4°C and detected with horseradish peroxidase using enhanced chemiluminescence.

Reverse transcription-PCR. Daoy cells were infected as above, and after 36 h at 37°C, total RNA was extracted as described by Chomczynski and Sacchi (18). PCR was done using an reverse transcription-PCR (RT-PCR) kit (Invitrogen): 35 cycles of denaturation at 94°C for 1 min, annealing at 67°C for 30 s, and extension at 72°C for 90 s. The expected PCR products were visualized using ethidium bromide after resolving on 2% agarose gels. RT-PCR for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was done to normalize input RNA. We used the following primers: sense, 5'-TGGACGATGCCTGCAACGTG-3' and antisense, 5'-GTCGTGCGTGTCCAAAGGCA-3' (MMP-9); sense, 5'-TGAAGGTCGGAGTCAACGGATTTGGT-3' and antisense, 5'-CATGTGGGCCATGAGGTCCACCAC-3' (GAPDH).

Cell proliferation assays. Cell growth rate was determined using a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (R&D Systems) as a measurement of mitochondrial metabolic activity. Cells were infected with mock, 100 MOI of Ad-SV, or various doses of Ad-MMP-9 and incubated at 37°C. After 0 to 72 h, MTT reagent was added, cells incubated for 1 h at 37°C, and the rate of absorbance of formazan (a dye produced by live cells) was measured with a microplate reader at A550.

Flow cytometry. Daoy cells were plated overnight in 100-mm tissue culture plates and infected for 36 h as described above. We used fluorescence-activated cell sorting (FACS) analysis that uses propidium iodide staining of nuclear DNA to characterize cell cycle phase (19). Briefly, cells were harvested by trypsinization and stained with propidium iodide (2 mg/mL) in 4 mmol/L sodium citrate containing 3% (w/v) Triton X-100 and RNase A (0.1 mg/mL; Sigma). Suspensions of 2 x 106 cells were analyzed by FACSCalibur System (Becton Dickinson Bioscience). The percentages of cells in the various phases of the cell cycle were assessed using Cell Quest software (Becton Dickinson Bioscience). For integrin analysis, cells were incubated with monoclonal anti-integrin antibodies {alpha}vß3 and {alpha}5ß1, ß4, or control mouse IgG in 0.5% bovine serum albumin (BSA) for 30 min on ice. After two PBS washes, cells were incubated with FITC-conjugated secondary antibodies in 0.5% BSA for 30 min on ice. Cells were again washed, and cell surface integrins were determined using a flow cytometer. All experiments were done in triplicate.

Cell adhesion assay. We used the established crystal violet colorimetric method to determine cell adhesion (20). Briefly, 48-well tissue culture plates were coated with purified extracellular matrix proteins including laminin (10 µg/mL), fibronectin (10 µg/mL), or vitronectin (5 µg/mL) for 18 h at 4°C. Wells were covered with BSA (10 mg/mL; fraction V; Sigma) in Ca2+- and Mg2+-free PBS for 1 h. BSA was removed, and the wells were washed with PBS. Daoy cells were infected with mock, 100 MOI of Ad-SV, or the indicated MOI of Ad-MMP-9 and incubated for 24 h. Cells were harvested by trypsinization, washed, and suspended in serum-free medium containing 0.1% BSA at 1 x 106 cells per mL, then plated onto cell culture plates coated with extracellular matrix and incubated at 37°C for 2 h, and attached cells were stained with 0.1% crystal violet (Sigma) at room temperature for 25 min. Cell adhesion was quantified by counting the average number of cells per five microscopic fields. All experiments were done in triplicate, and the data represent the average of three independent experiments. The Student's t test was used to compare treatment groups with control cells, with P < 0.05 considered significant.

Senescence-associated ß-galactosidase activity. Senescence-associated ß-galactosidase (SA-ß-gal) activity was determined using a SA-ß-gal staining kit from Cell Signaling (Chemicon International). Briefly, Daoy cells (2 x 103 per well) were plated in eight-well chamber slides and infected with Ad-MMP-9 as described earlier. After 36 h, we used standard light microscopy to identify senescent cells, which were blue stained. Five fields were evaluated for each well with three wells per condition (x40 magnification).

Cell invasion assay. To gauge the invasive capacity of the tumor cells, we used Transwell cell culture chambers (Corning Costar) as described previously (17). Briefly, Daoy cells were infected with mock, 100 MOI of Ad-SV, or various doses of Ad-MMP-9 for 48 h. Viable cells (1 x 106) from each treatment were allowed to invade through polycarbonate filters (12-µm pore size) coated with Matrigel. The migrating cells on the reverse side of the filter were photographed and counted. Five different fields per filter were analyzed, and all experiments were done in triplicate.

Intracranial tumor model. Daoy cells (1 x 105) were stereotactically implanted as described elsewhere with minor modifications (21). Two weeks after tumor cell implantation, the animals were randomized into three groups (six animals per group). Each mouse received three intratumoral injections on the 14th, 15th, and 16th day: group 1 received PBS (6 µL; n = 6), group 2 received 5 x 107 PFU of Ad-SV virus in 6 µL of PBS, and group 3 received 5 x 107 PFU of Ad-MMP-9 in 6 µL of PBS. Animals losing ≥20% of body weight or having trouble ambulating, feeding, or grooming were sacrificed. Animals were monitored for 180 days when we arbitrarily terminated the experiment. Mouse brains were fixed in 10% formalin and embedded in paraffin. Tissue sections (4 µm thick) were obtained from the paraffin blocks and stained with H&E using standard histologic techniques. Sections were also subjected to immunostaining with antibodies for either MMP-9 or p16. Protein expression was detected using 3,3-diaminobenzidine solution (Sigma). Sections were counterstained with hematoxylin, and negative control slides were obtained by nonspecific IgG. Sections were washed and mounted with anti-fade mounting solution (Invitrogen) and analyzed with an inverted microscope.

Statistical analyses. All data are expressed as mean ± SD. Statistical analysis was done using the Student's t test or a one-way ANOVA. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ad-MMP-9 infection decreased MMP-9 activity and expression. We constructed a recombinant adenovirus encoding siRNA targeted to MMP-9 and then tested its effect on MMP-9 expression and activity. We determined MMP-9 activity, protein and mRNA levels in Daoy medulloblastoma cells infected with various doses of Ad-MMP-9. Gelatin zymography of conditioned media showed that Ad-MMP-9 infection inhibited MMP-9 activity in a dose-dependent manner (Fig. 1A ). There was no significant change in MMP-2 expression indicating that this inhibition was specific to MMP-9. Western blot analysis of conditioned media using anti-MMP-9 antibodies showed that Ad-MMP-9 infection decreased MMP-9 protein expression levels in a dose-dependent manner compared with mock and scrambled vector controls (Fig. 1B). To determine whether decreased production of MMP-9 was caused by gene transcription, we examined MMP-9 transcripts using RT-PCR. As shown in Fig. 1C, the levels of transcripts of MMP-9 in Ad-MMP-9–infected cells were significantly lower compared with cells infected with mock and Ad-SV.


Figure 1
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  		Figure 1. Effect of Ad-MMP-9 on Daoy cells. Daoy cells were infected with mock, 100 MOI of Ad-SV, or the indicated doses of Ad-MMP-9 for 36 h, and then the conditioned medium was collected. A, zymographic analysis for MMP-9 activity in the conditioned medium. B, Western blot analysis of MMP-9 protein expression in the conditioned medium. C, RT-PCR. Total RNA was extracted as per standard protocols, and cDNA was synthesized. The PCR reaction was set up using first-stand cDNA as the template for MMP-9. GAPDH served as a loading control. Data from a typical experiment done in triplicate. D, MTT proliferation assay was carried out for Daoy cells infected with mock, 100 MOI of Ad-SV, or the indicated doses of Ad-MMP-9. MTT activities were measured at 550 nm in triplicate at 24, 36, 48, and 72 h. Proliferation curve of average values from a typical experiment. The experiment was repeated thrice.

 
Ad-MMP-9 infection inhibited cell proliferation in Daoy cells. Daoy medulloblastoma cells were infected with various MOI of Ad-MMP-9 for 72 h. Ad-MMP-9 infection led to a dose-dependent decrease in cell proliferation, as shown in Fig. 1D. A 75% to 80 % inhibition of cell proliferation occurred at an MOI of 50 at 72-h time points. This decrease reached almost 90% when Daoy cells were infected with 100 MOI of Ad-MMP-9 compared with mock and Ad-SV controls at 72 h.

Ad-MMP-9 infection blocks medulloblastoma invasion. Matrigel-coated Transwell chambers were used in a standard test to study whether infection by Ad-MMP-9 suppressed the invasive capacity of Daoy cells. The results in Fig. 2A show that Ad-MMP-9 infection for 48 h inhibited the number of Daoy cells invaded through the Matrigel in a dose-dependent manner compared with the mock and Ad-SV controls. Quantification of the invaded cells indicated that 24% and 39% less cells invaded in 10 and 25 MOI Ad-MMP-9–infected Daoy cells than that of the controls. Infection with 50 and 100 MOI of Ad-MMP-9 resulted in a more significant effect on invasion with 59% and 78% inhibition compared with the mock-infected controls (Fig. 2B).


Figure 2
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  		Figure 2. Effect of Ad-MMP-9 on the invasive capacity of Daoy cells through Matrigel. Daoy cells were infected with mock, 100 MOI of Ad-SV, or the indicated doses of Ad-MMP-9 and incubated for 48 h. Cells were then allowed to invade Transwell inserts containing 12-µm pore polycarbonate membranes precoated with Matrigel for 24 h at 37°C. Cells were then fixed and stained with Hema-3. A, cells that had migrated to the lower side of the membrane were photographed under a light microscope at x20 magnification. B, percentages of invading cells were quantified by counting five fields in each condition. Data from a typical experiment done in triplicate.

 
Ad-MMP-9 affects medulloblastoma cell adhesion to extracellular matrix protein–coated plates. To investigate the effect of MMP-9 inhibition on cell-matrix adhesion, we examined the effect of Ad-MMP-9 infection on Daoy cell attachment onto various extracellular matrix components. MMP-9 inhibition was associated with an overall increase in adhesion to extracellular matrices, an effect that was most evident when cells were allowed to adhere to fibronectin (Fig. 3A ). Stimulation of adhesion was dose dependent in all the matrices, and Ad-MMP-9 infection caused a more prominent increase in adhesion on fibronectin compared with the other matrices and controls (Fig. 3B).


Figure 3
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  		Figure 3. Effect of Ad-MMP-9 on Daoy cell adhesion to various matrices. Daoy cells were infected with mock, 100 MOI of Ad-SV, or the indicated doses of Ad-MMP-9 for 24 h. A, adhesion assays were done on vitronectin, fibronectin, and laminin. B, percentage of mock control adhesion to vitronectin. Columns, mean of three experiments; bars, SD. All experiments were done in triplicate. P < 0.05 is significant. C, FACS analysis of integrin expression. Daoy cells were washed in blocking solution and incubated with monoclonal anti-integrin antibodies {alpha}vß3, {alpha}5ß1, and ß4 for 30 min. Integrin expression was determined using a FITC-conjugated secondary antibody. Data from a representative experiment repeated thrice with similar results.

 
Ad-MMP-9 affects expression of {alpha}vß3, {alpha}5ß1, and ß4 integrins. Because cell-matrix interactions are critically determined by integrins, we hypothesized that MMP-9 inhibition could modulate integrin expression. Expression of {alpha}vß3, {alpha}5ß1, and ß4 levels were not significantly different between the mock and Ad-SV control groups. However, the expression of these integrins in Ad-MMP-9–infected Daoy cells was markedly increased in a dose-dependent manner (Fig. 3C).

Ad-MMP-9 infection arrests cells in G0-G1 phase. It is known from previous studies with tumor cells that synthetic MMP inhibitors may induce cell cycle arrest (22, 23). Therefore, we hypothesized that Ad-MMP-9 could cause similar effects. FACS analysis for nuclear DNA content by propidium iodide staining showed that cell growth was arrested in the G0-G1 cell cycle phase. Figure 4A shows that 52% to 54% of Daoy cells were in G0-G1, 15% to 14% cells were in S phase, and 18% to 21% were in G2-M phase in mock- and Ad-SV–treated cells. In contrast, Daoy cells infected with 100 MOI of Ad-MMP-9 remained to a high extent, about 73% in the G0-G1 phase. In addition, the number of cells in the G2-M (mitotic/dividing) phase was significantly decreased, thereby resulting in limited cell cycle progression, which in turn translates into a marked decrease in proliferation.


Figure 4
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  		Figure 4. Effect of Ad-MMP-9 on cell cycle and cell cycle regulatory proteins. A, cell cycle analysis was done using flow cytometry as detailed in Materials and Methods. The labeled cells were analyzed using a FACSCalibur System. Percentage of cells in G0-G1, S, and G2-M phases were calculated using Cell Quest software. Data from a typical experiment repeated thrice. B, immunoblot of cell lysates corresponding to Daoy cells infected with mock, 100 MOI of Ad-SV, or the indicated MOI of Ad-MMP-9. Total protein lysates were analyzed for the levels of p21, p16, cyclin D2, cyclin E, cdk2, cdk4, pRb, and E2F-1. As detailed in Materials and Methods, the cells were infected with mock, 100 MOI of Ad-SV, or various doses of Ad-MMP-9, and total cell lysates were prepared for immunoblot analysis. Data from a representative experiment repeated thrice.

 
Ad-MMP-9 infection induces senescence in Daoy cells. Cellular senescence is characterized by accumulation of lysosomal enzymes. Cells that were treated with Ad-MMP-9 were subsequently growth arrested acquired the enlarged and flattened morphology characteristic of cellular senescence. To examine the relative levels of cellular senescence, we assessed the levels of SA-ßGal, a well-known marker for cellular senescence (24). As shown in Fig. 5A , senescence, as indicated by ß-gal staining (blue color), increased in a dose-dependent manner with Ad-MMP-9 infection. We did not observe senescent cells in mock- and Ad-SV–treated cells.


Figure 5
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  		Figure 5. A, photographs of Daoy cells infected with Ad-MMP-9 stained for ß-gal (pH 6.0) activity. B, effect of Ad-MMP-9 treatment on protein expression of ERK1/2 and pERK levels. Simultaneous treatment of Ad-MMP-9 infection and Dn-ERK mutant showed inhibition of ERK and p16 expression (C) and senescence marker SA-ß-gal (D). Data from experiments repeated in triplicate.

 
Ad-MMP-9 infection increases p16 and p21 protein expression. To further characterize the nature of the cell cycle arrest caused by Ad-MMP-9 infection, we looked at the expression of several cell cycle regulatory proteins in Ad-MMP-9–infected Daoy cells (Fig. 4B). The changes that distinguish cellular senescence from quiescence are thought to include up-regulation of the cell cycle inhibitors p16INK4a and p21CIP (25, 26). We observed a significant induction of p21 with Ad-MMP-9 infection. The expression of p16 protein was at nearly undetectable levels in the mock- and Ad-SV–infected cells but increased dramatically with Ad-MMP-9 infection. The expression of p16 increased in a dose-dependent manner as cells reached senescence. These results, along with the accumulation of SA-ß-gal, strongly suggest that the cell cycle arrest induced by Ad-MMP-9 is indeed senescence. Using immunoblot analysis, we also assessed the effect of Ad-MMP-9 infection on the protein expression of the cyclins and cdks, which are known to be regulated by p21. Ad-MMP-9 treatment of the cells resulted in a dose-dependent decrease in protein expression of cyclin D2 and cyclin E as well as cdk2 and cdk4.

Ad-MMP-9 inhibits protein expression of pRb and E2F1. Down-regulation of cdk2/4 has been shown to be associated with a decrease in the expression of retinoblastoma (pRb) tumor suppressor protein, a key regulator of the G1-S phase transition in the cell cycle (27). Therefore, we next examined the effect of Ad-MMP-9 on the protein expression of pRb. Immunoblot data revealed that Ad-MMP-9 treatment of cells resulted in a significant decrease in the protein expression of pRb (Fig. 4B). Because pRb controls cell cycle by binding to and inhibiting E2F transcription factors, we assessed the protein expression of E2F transcription factors. As shown in Fig. 4B, Ad-MMP-9 treatment of cells resulted in a dose-dependent decrease in E2F transcription factors.

Ad-MMP-9–induced senescence is mediated by the ERK/mitogen-activated protein kinase pathway. The ERK pathway was shown to be responsible for senescence (28). We found that both ERK and pERK levels were increased in Daoy cells infected with Ad-MMP-9 compared with the controls (Fig. 5B). To further validate the role of ERK1/2 in Ad-MMP-9–induced cell cycle arrest leading to senescence, we silenced ERK1/2 phosphorylation by transiently transfecting Daoy cells with a dominant-negative mutant of ERK (Dn-ERK) before Ad-MMP-9 infection. We observed that Ad-MMP-9 did not induce ERK1/2 expression in this condition (Fig. 5C). To determine if p16 up-regulation was mediated by ERK activation, we stripped the blot and tested for p16 expression. Figure 5C shows that Dn-ERK transfection reduced p16 expression, thereby suggesting that p16 expression in Ad-MMP-9–treated cells is mediated by ERK. Furthermore, transfection with a Dn-ERK led to a decrease of about 50% in the number of SA-ß-Gal–positive cells compared with the levels observed without infection with Dn-ERK (Fig. 5D).

Ad-MMP-9 treatment causes loss of tumorigenicity in nude mice. To directly evaluate the role of Ad-MMP-9 on tumor formation in vivo, we injected Daoy cells into nude mice and treated the pre-formed tumors with intratumoral injections of Ad-MMP-9. Mice treated with mock (PBS) and Ad-SV developed tumors, were symptomatic within 4 weeks, and were sacrificed. In striking contrast, Ad-MMP-9–injected mice survived for 6 months, at which point the animals were sacrificed, and their brains were examined for tumor growth. Histologic examination of the paraffin-embedded tissue sections of the brains from mice that received mock and Ad-SV showed large tumors in the cerebellum. However, H&E staining did not reveal any tumor cells in four of six mice treated with Ad-MMP-9 (Fig. 6A ). Very small tumors were observed in the other two Ad-MMP-9–treated animals.


Figure 6
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  		Figure 6. Tumorigenicity in vivo. Daoy cells were implanted intracranially in nude mice and treated with intratumoral injections of mock, Ad-SV, and Ad-MMP-9 as described in Materials and Methods. A, H&E staining of brain sections showing neoplastic growth (inset, x20). Immunohistochemical analysis of MMP-9 expression (B) and p16 (C) expression in brain sections as described in Materials and Methods. D, proposed schematic model for Ad-MMP-9–mediated cell cycle dysregulation and induction of senescence.

 
To determine whether MMP-9 was expressed in vivo, brain sections were stained with a monoclonal antibody for human MMP-9. Figure 6B indicates that brain sections from mock- and Ad-SV–treated mice showed intense expression of MMP-9. In contrast, MMP-9 expression was not detectable in Ad-MMP-9–treated mice brain sections. Based on the in vitro experiments, we decided to assess p16 expression in brain sections of the mice. We observed a remarkable increase in p16 expression in brain sections from mice that received Ad-MMP-9 treatment. However, we barely found any signal in the control brains sections from the mock- and Ad-SV–treated mice brain sections (Fig. 6C).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A number of studies have shown that the introduction of siRNA into mammalian and human cells causes specific and effective suppression of the corresponding mRNA molecules (29, 30). Therapeutic application of siRNA technology requires an efficient gene delivery system for transduction of siRNA into target cells. Adenoviral vectors have been shown to efficiently transduce genes into many types of cells. The present study describes the profound effects of an adenovirus carrying siRNA against MMP-9 on medulloblastoma tumor growth in vitro and in vivo.

In this study, we have shown that adenovirally transduced siRNA against MMP-9 (Ad-MMP-9) exerted a significant effect on inhibiting MMP-9 mRNA expression using RT-PCR and MMP-9 protein levels by Western blotting. Furthermore, the results show that Ad-MMP-9 infection causes cell cycle arrest in the G0-G1 phase, senescence in Daoy cells in vivo, and inhibits tumor growth in an intracranial model. Cell cycle arrest occurs as a function of cdk inhibitors. In regard to this observation, our results definitively show that MMP-9 inhibition induces the expression of CDK inhibitors p21 and p16 in a dose-dependent manner. The induction of these CDK inhibitors has been implicated in the initiation and maintenance of cellular senescence (31, 32). p21 inhibits cyclin-dependent protein kinase (33) and the proliferating cell nuclear antigen–dependent DNA replication (34), causing G1 arrest of cell cycle. Expression of the p21 gene suppressed growth of human brain, lung, and colon tumor cells in culture (35).

Cellular senescence is strongly implicated as an important mechanism of tumor suppression, and the ability of p53 and p16 to mediate cell cycle arrest is central to their activity in blocking tumor development (36). We show that Ad-MMP-9 infection causes a dramatic increase in p16 expression. In addition, p16 is a specific inhibitor of the cyclin D1–dependent kinases cdk4 and cdk6. As expected, the levels of cdk4 and cdk2 were decreased. Normally, in the absence of p16, cyclin D1/cdk4 and cyclin D1/cdk6 complexes phosphorylate and inactivate the Rb protein, permitting E2F-dependent transcription of genes encoding proteins to initiate chromosome replication and ultimately another round of cell division (37). In this study, we show that Ad-MMP-9 infection decreased phosphorylated Rb levels, thereby activating it, which in turn resulted in cell cycle arrest. Deregulated activity of the D-type cdk4 and cdk6 is widely observed in various tumor cells, illustrating their importance in controlling cell cycle (38). We also show that the brain sections of mice that received Ad-MMP-9 treatment show remarkable levels of p16 expression, which was completely absent in the brain sections of control and Ad-SV–treated mice, suggesting p16-mediated inhibition of tumor growth. Our results confirm earlier studies indicating the involvement of the p16-Rb pathway in medulloblastomas (39).

We next investigated the signaling mechanism mediating senescence upon Ad-MMP-9 infection. Cells acquire increased adhesion to the extracellular matrix while losing cell-cell contacts during the process of senescence. Ad-MMP-9 infection of Daoy cells caused increased adhesion on various extracellular matrix proteins and increased expression of {alpha}vß3, {alpha}5ß1, and ß4 integrins. Several studies have suggested a link between integrin-mediated signaling and the Ras/mitogen-activated protein kinase (MAPK) pathway. Specifically, adhesion of cells to extracellular matrix components, such as fibronectin, induces activation, tyrosine phosphorylation, and nuclear translocation of MAPKs through an integrin-dependent mechanism (40), thereby suggesting that integrin-matrix interactions activate a MAPK cascade. Interestingly, the ability of oncogenic ras to induce premature senescence depends on the Raf/MAPK kinase (MEK)/ERK pathway that mediates cell proliferation (28). We found that Ad-MMP-9 infection elicited ERK activation. Of the three matrices studied, cell adhesion to fibronectin is greatly increased with Ad-MMP-9 infection, with a concomitant increase in its receptor {alpha}5ß1. p16INK4a was found to sensitize cells to detachment-induced apoptosis by increased transcription of integrin {alpha}5ß1 (41). The biological and molecular basis of the promotion of adhesion in our study remains to be elucidated. Although we found an increase in integrin expression with MMP-9 inhibition, further work is required to determine whether the observed changes in integrin expression are causing senescence-mediated growth arrest. One possibility is that the cytoskeleton or some other pathway is activated by MMP-9 inhibition. This would explain the increase in adhesion on several matrices. Cytoskeletal reorganization was a key player in induction of anoikis in breast cancer cells (42). In addition, cytoskeletal-disrupting drugs, such as LatA, induced apoptosis in normal MCF10A cells (43).

Next, our results indicate that the ERK pathway plays an important role in the premature senescence that resulted from Ad-MMP-9 infection in Daoy cells. Studies have shown that sustained ERK activation is required to pass the G1 restriction point (44), and that ERK regulates cyclin D1 expression during mid-G1 (45). However, under certain circumstances, the Ras/Raf/MEK/ERK cascade lead to cell cycle arrest instead of proliferation. Ras-induced cell cycle arrest is driven through ERK-mediated up-regulation of p53 and p16INK4a activity (28). Ad-MMP-9 infection in Daoy cells caused an increase in ERK and pERK levels in a dose-dependent manner. Our studies also show that transfection with a Dn-ERK construct before Ad-MMP-9 infection inhibited Ad-MMP-9–mediated senescence. We also show here that induction of p16 expression with Ad-MMP-9 infection is also inhibited by transfection with Dn-ERK. The ERK/MAPK is known to regulate transcription factors, thereby controlling gene expression (46). In fact, p16INK4a expression can be directly regulated by transcription factors of the ETS family, which in turn are regulated by ERK (47). Based on the outcome of this study and as shown in the scheme in Fig. 6D, we suggest that MMP-9 inhibition induces activation of ERK1/2, which leads to induction of cyclin kinase inhibitor p27/KIP1 and p16 and, in turn, inhibits cell cycle regulatory molecules resulting in G1 arrest and senescence. Down-regulation of cdk4/6 inhibits pRb and inhibits protein expression of the E2F family, thereby leading to senescence.

Induction of cell cycle arrest and apoptosis represent an established method to treat malignant disorders (48). The influence of synthetic MMP inhibitors on cell cycle and apoptosis is well documented. The MMP inhibitor batimastat (BB-94) was shown to enhance IFN-induced apoptosis in mice with ovarian cancer (22) and to block ovarian cancer cells in the G0-G1 phase of cell cycle (23). Another MMP inhibitor (AG3340) promoted apoptosis in human prostate and colon carcinoma models (49). GM-6001, a nonspecific MMP inhibitor, was shown to induce apoptosis in smooth muscle cells (50).

In conclusion, our studies provide the first evidence that MMP-9 inhibition causes ERK-mediated p16 expression, resulting in cell cycle arrest in Daoy cells in vitro and in vivo. These findings show that MMP-9 may play an important role in inhibiting medulloblastoma invasion and tumor growth and identifies MMP-9 as a promising target for adenoviral-mediated, siRNA-based therapy in medulloblastoma.


   Acknowledgments
 
Grant support: National Cancer Institute grants CA 75557, CA 92393, CA 95058, and CA 116708; National Institute of Neurological Disorders and Stroke grants NS47699 and NS57529; Caterpillar, Inc.; OSF Saint Francis, Inc., Peoria, IL (J.S. Rao); and Children's Miracle Network (S.S. Lakka).

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.

We thank Noorjehan Ali for technical assistance in animal experiments, Shellee Abraham for preparing the manuscript, and Diana Meister and Sushma Jasti for reviewing the manuscript.

Received 1/30/07. Revised 3/ 7/07. Accepted 3/16/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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