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Molecular Cancer Research 4:803-810 (2006)
© 2006 American Association for Cancer Research

Angiogenesis, Metastasis, and the Cellular Microenvironment

Wild-type p53 Inhibits Nuclear Factor-{kappa}B–Induced Matrix Metalloproteinase-9 Promoter Activation: Implications for Soft Tissue Sarcoma Growth and Metastasis

Jue Liu1, Maocheng Zhan1, Jonathan A.F. Hannay1, Parimal Das1, Svetlana V. Bolshakov2, Dhanashankren Kotilingam2, Dihua Yu1, Alexander F. Lazar3, Raphael E. Pollock1 and Dina Lev2

Departments of 1 Surgical Oncology, 2 Cancer Biology, and 3 Pathology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas

Requests for reprints: Dina Lev, Department of Cancer Biology, Unit 173, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-792-1637; Fax: 713-794-0722. E-mail: dlev{at}mdanderson.org


   Abstract
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 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Human soft tissue sarcoma (STS) is a highly lethal malignancy in which control of metastasis determines survival. Little is known about the molecular determinants of STS dissemination. Here, we show that human STS express high levels of matrix metalloproteinase-9 (MMP-9) and that MMP-9 expression levels correlate with sequence analysis–defined p53 mutational status. Reintroduction of wild-type p53 (wtp53) into mutant p53 STS cell lines decreased MMP-9 mRNA and protein levels, decreased zymography-assessed MMP-9 proteolytic activity, and decreased tumor cell invasiveness. Reintroduction of wtp53 into STS xenografts decreased tumor growth and MMP-9 protein expression. Luciferase reporter studies showed that reintroduction of wtp53 into mutant p53 STS cells decreased MMP-9 promoter activity. Deletion constructs of the MMP-9 promoter identified a region containing a p53-responsive element that lacked a p53 consensus binding site but did contain a nuclear factor-{kappa}B (NF-{kappa}B) site. Mutating this NF-{kappa}B binding site eliminated the wtp53-repressive effect. Electrophoretic mobility shift assays confirmed decreased NF-{kappa}B binding in STS cells in the presence of wtp53. Our findings suggest a role for MMP-9 in STS progression and expand the role of p53 in molecular control of STS growth and metastasis. Therapeutic interventions in human STS targeting MMP-9 activity directly or via reintroduction of wtp53 merit further investigation. (Mol Cancer Res 2006;4(11):803–10)


   Introduction
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 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 References
 
Soft tissue sarcoma (STS) are mesenchymal-origin malignancies that include >50 histologic subtypes, occur anywhere in the body, and have a predilection for hematogenous pulmonary dissemination (1). STS overall survival is only 50% at 5 years, and pulmonary metastases cause 80% of sarcoma-specific deaths. Chemotherapy to treat STS dissemination has disappointing 40% to 50% response rates (2). Therapies targeting molecular determinants of STS metastasis are needed if survival is to be improved.

A potential target is the matrix metalloproteinase (MMP) family, consisting of numerous proteases separable by substrate specificity, inhibitor, and extracellular membrane binding efficiency. MMPs are implicated in invasion and angiogenesis (3). MMP-9 (gelatinase B), the focus of this study, preferentially degrades denatured collagens and native collagen type IV, a major component of extracellular matrix and basement membranes whose disruption is central to tumor invasion and metastasis. Tumor MMP-9 also exerts early control of the angiogenic switch necessary in primary tumor progression (4). Degradation of extracellular matrix facilitates endothelial cell migration and subsequent proliferation. Experimentally, overexpression of MMP-9 contributes to pulmonary metastasis (5-7), whereas inhibiting MMP-9 expression blocks metastasis (8-10).

Given invasive and pulmonary metastatic STS propensities, it is surprising that MMP-9 in STS has received scant attention. Increased STS expression of MMP-2 and MMP-9 (with concomitant decreased expression of their cognate inhibitors tissue inhibitor of metalloproteinase-2 and tissue inhibitor of metalloproteinase-1) correlates with overall survival (11). Abrogating MMP-9 activity by transfecting anti-MMP-9 small interfering RNA resulted in decreased Ewing's sarcoma spread on extracellular matrix–coated surfaces (12).

Regulation of MMP-9 expression and activity is multifactorial yet incompletely understood (7); MMP-9 regulation in STS has apparently not been examined. Because p53 mutations are prevalent in STS and other malignancies, it is intriguing that possible p53 regulation of MMP-9 activity has also received little consideration.

Our studies show that MMP-9 expression levels in human STS correlate with both metastasis and p53 mutational status. Reintroduction of wild-type p53 (wtp53) into mutant p53 (mutp53) STS favorably alters MMP-9 tumor biology by down-regulation of the MMP-9 promoter, mediated via decreased nuclear factor-{kappa}B (NF-{kappa}B) activity, a potentially effective anti-MMP-9 therapeutic strategy. To have an impact, such approaches should target early-stage STS patients in whom MMP-9 activity has an evolving role in facilitating primary tumor angiogenesis and subsequent metastasis.


   Results
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 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 References
 
Immunohistochemistry was done on 68 randomly selected STS primary, recurrent, and metastatic specimens of various histologic subtypes arising in different anatomic locations. MMP-9 expression was detected at varying intensities (no expression, n = 11; low intensity, n = 21; high intensity, n = 38; Fig. 1 ). MMP-9 expression was observed in stromal cells as well as in tumor cells (Fig. 1). Out of this initial cohort of specimens, a more homogenous group of tumors, including only the primary-extremity STS belonging to the three most common histologic subtypes (malignant fibrous histiocytoma, synovial sarcoma, and leiomyosarcoma; n = 13) was selected for further MMP-9 analysis: three were negative (score 0), six were low to moderate (score 1), and four were strongly positive (score 2). All STS exhibiting cytoplasmic granular MMP-9 immunoreactivity (scores 1 and 2) were deemed elevated expressers; this was observed in 10 of 13 (77%) primary STS (Table 1 ). Nine of these 10 (90%) MMP-9–expressing STS primary tumors subsequently metastasized, whereas no metastases developed in the three primary STS nonexpressers (P = 0.0017; Table 1).


Figure 1
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  		FIGURE 1. Immunohistochemistry for MMP-9 confirms expression in a broad spectrum of human STS. MMP-9 expression is observed in stromal cells as well as in tumor cells. Arrows, normal stromal fibroblasts and endothelial cells expressing MMP-9. Malignant fibrous histiocytoma (MFH; magnification, x100), leiomyosarcoma (magnification, x200), synovial sarcoma (magnification, x200), and fibrosarcoma (magnification, x400).

 

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  		Table 1. Association of Elevated MMP-9 Expression with Clinicopathologic Variables in STS

 
Because p53 mutational status correlates with STS metastatic behavior (13), we sequenced tumor genomic DNA for p53 gene mutation status in the 13 STS primary tumors, demonstrating p53 mutations in 7 of 13 (54%). All mutp53 primary STS metastasized (seven of seven), whereas only two of six without p53 mutation metastasized (P = 0.021). Furthermore, all mutp53 STS expressed MMP-9 (seven of seven), whereas only three of those without p53 mutation exhibited MMP-9, all at low to intermediate levels (P = 0.05; Table 1). Taken together, these results suggest that MMP-9 is highly expressed in STS and that primary STS with metastatic capacity is characterized by both high MMP-9 expression and also p53 mutation.

We considered possible regulatory relationships between mutp53 status and MMP-9 expression/function. SKLMS1 human leiomyosarcoma cells have p53 core-binding point mutation at codon 245. Previously, we stably transfected SKLMS1 cells with wtp53 (designated SK-p53) and temperature-sensitive 143Ala-p53 (SKAla-10, SKAla-14, and SKAla-21), demonstrating that restoration of wtp53 suppresses the malignant phenotype in vitro and in vivo (14). Immunohistochemistry of severe combined immunodeficient mouse xenografts generated from these cell lines showed strongly decreased MMP-9 expression and induction of p21 in wtp53 cells compared with controls (Fig. 2A ). Immunohistochemistry done on severe combined immunodeficient mouse SKLMS1 xenografts intratumorally injected with either Adp53 or AdLacZ showed increased p21 expression as well as much lower MMP-9 expression with Adp53 compared with AdLacZ treatment (Fig. 2B). These in vivo results collectively suggest that wtp53 might help regulate MMP-9 expression in human STS.


Figure 2
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  		FIGURE 2. Immunohistochemical staining of MMP-9 expression in STS xenografts. A. SKLMS1 parental mutp53 cells and SKp53-1 wtp53 stable transfectants. B. SKLMS1 xenografts intratumorally injected with Adp53 or AdLacZ. Increased p21 expression confirms wtp53 function (original magnification, x200).

 
The in vitro effect of mutp53 status on STS invasiveness was examined in SKAla temperature-sensitive p53 mutant cell lines. SKAla cells grown at 32°C express p53 with wild-type function (permissive condition), whereas SKAla grown at 37°C or greater express mutp53 (restrictive condition; ref. 14). SKAla transfectants grown at 32°C or 38°C, vector control SKNeo, and parental SKLMS1 cells were studied in a chemoinvasion assay. Markedly less chemoinvasion was observed when SKAla-10, SKAla-14, and SKAla-21 cells were cultured at 32°C compared with 38°C (Fig. 3A ), whereas only a slight nonsignificant difference was seen between parental or neo control cells grown at 32°C or 38°C or SKAla transfectants grown at 38°C, implying that decreased chemoinvasion was associated with wtp53 function rather than temperature shifts per se. In contrast, a migration assay revealed no difference in motility between these cells or in the different temperatures (Fig. 3B). These results suggested that the reexpression of wtp53 affects the degradative ability of SKLMS1 cells, but not their motile capabilities. In another set of experiments, we have treated SKAla-14 at the mutp53-permissive temperature (38°C) with 20 nmol/L of a selective MMP-9 inhibitor and observed a significant reduction (P < 0.05) in cell invasion with no impact on cell migration (Fig. 3C), similar to the results obtained by reexpressing wtp53, suggesting that MMP-9 plays an important role in STS invasion and that it is possible that the effects of wtp53 on STS invasive properties are mediated, at least partially, through MMP-9 inhibition. It is, however, imperative to state that the more pronounced inhibition in invasion, observed after wtp53 reexpression, suggests that multiple molecular mechanisms, and not solely MMP-9 inhibition, are affected by wtp53.


Figure 3
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  		FIGURE 3. Reconstitution of wild-type p53 suppresses STS invasion in vitro. A. Chemoinvasive activity was determined by counting the number of cells per high-powered field (x200) that had migrated to the lower side of the filters (see text for details). The difference in the invasion rates between 32°C and 38°C was analyzed using a two-tailed Student's t test (P < 0.05). B. Chemotaxis assays using 30 µg/mL laminin as a chemoattractant, showing no difference in migration of STS cell after reexpression of wtp53 or due to change of temperature. C. SKAla-14 cells incubated at 38°C (mutp53-permissive temperature) with 20 nmol/L of an MMP-9 inhibitor exhibited significant decrease in invasion (P < 0.05) but no change in migration rate.

 
Taking into account our previous results, suggesting a possible interplay between p53 status and MMP-9 expression, we next questioned whether the decreased invasion observed with wtp53 reexpression could be secondary to decrease in MMP-9 expression and activity. To further explore wtp53 chemoinvasion inhibition, MMP-9 activity after wtp53 reintroduction was assessed. A slight decrease in MMP-9 activity measured by zymography was observed in control SKLMS1 and SKneo cells conditioned medium when shifted to 32°C, suggesting that the lower temperature affects the activity of MMP-9 to some extent (Fig. 4A ). However, a much more pronounced reduction of MMP-9 activity was detected in conditioned medium of 32°C versus 38°C SKAla transfectants (Fig. 4A). Similarly, Adp53-infected SKLMS1 cells exhibited a significant reduction in MMP-9 activity, whereas only a slight reduction was observed in AdLacZ-infected SKLMS1 cells (Fig. 4B). Taken together, these data suggest that wtp53 inhibits MMP-9 activity in STS cells in vitro.


Figure 4
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  		FIGURE 4. Reconstitution of wtp53 suppresses MMP-9 enzymatic activity, secreted protein levels, and mRNA levels. A. Zymography shows significantly decreased MMP-9 activity in SKAla cells manifesting wtp53 at 32°C compared with mutp53 at 38°C. However, only a slight reduction in MMP-9 activity is seen in parental SKLMS1 or control SKNeo cells at 32°C. B. Decreased MMP-9 activity was observed in SKLMS1 cells treated with Adp53 for 48 hours and analyzed as above. C. Western blot. The concentrated conditioned medium collected for the zymography assay in A was subjected to Western blot analysis. MMP-9 protein levels were lower in medium from SKAla cells at 32°C than at 38°C. Total cell protein lysates were also subjected to Western blot analysis: p21 induction in the SKAla cells at 32°C was used as a surrogate for wtp53 reexpression; no change was observed in JAG-1 protein level used as control, suggesting that the change seen in the MMP-9 level secondary to reexpression of wtp53 is specific. D. Reverse transcription-PCR confirms lower MMP-9 mRNA levels in SKAla cells incubated at 32°C than at 38°C. ß-Actin served as an internal control.

 
Next, we considered the mechanism underlying wtp53 inhibition of MMP-9 activity, beginning with MMP-9 protein and mRNA expression. SKAla cells had lower MMP-9 protein levels at 32°C than at 38°C (Fig. 4C); Western blots, using the total cell lysates extracted during the collection of the conditioned medium used for MMP-9 level evaluations, showed the induction of p21 in the SKAla cells at 32°C as a surrogate for wtp53 reexpression. Moreover, no change in JAG-1 protein level was used as a control to verify that the change observed in the MMP-9 level secondary to reexpression of wtp53 is specific (Fig. 4C). Changes in MMP-9 mRNA expression levels found using reverse transcription-PCR were similar to those found for the MMP-9 protein (Fig. 4D). In contrast, only slight or no differences in either protein or mRNA levels were seen in parental SKLMS1 or vector control SKNeo cells grown at either temperature.

The decreased MMP-9 mRNA observed in 32°C SKAla cells suggested that wtp53 might exert at least partial transcriptional repression of MMP-9 gene expression. To test this hypothesis, we first examined the effect of wtp53 on MMP-9 promoter activity. The –674 to +3 bp MMP-9 promoter fragment (relative to the transcription start site) was subcloned into the firefly luciferase reporter gene plasmid pGL3 to generate pMMP-9-674Luc. SKLMS1 cells preinfected with incremental doses of Adp53 or AdLacZ were transiently transfected with the pMMP-9-674Luc reporter construct, and luciferase expression was assayed 48 hours later. Compared with control SKLMS1 and AdLacZ-infected cells, Adp53-infected SKLMS1 showed significant inhibition of MMP-9 promoter activity, up to 40% (±6) promoter inhibition at 500 plaque-forming units of Adp53 (two-tailed Student's t test; P < 0.05; Fig. 5A ). An increased p21 promoter-driven luciferase expression in the presence of wtp53 verified the activity of the wtp53 plasmid used for transfection (Fig. 5B). These results suggest that wtp53 transcriptionally regulates MMP-9 expression. Although not further explored in the present study, the relatively modest differences in luciferase activity observed point to the possibility that additional wtp53 regulatory mechanisms might be affecting MMP-9 level and activity in our cells.


Figure 5
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  		FIGURE 5. A. Luciferase reporter assay. wtp53 suppresses transcriptional activity from pMMP-9-674Luc in a dose-dependent manner in SKLMS1 cells pretreated with Adp53 but not mock or AdLacZ. Representative of three independent experiments. B. Luciferase reporter assay. A genetic element between –674 and –328 is required for wtp53 to exert suppression of MMP-9 transcriptional activity. Point mutations in the –607 to –595 NF-{kappa}B binding site abolish wtp53 ability to suppress transcriptional activity from the MMP-9 promoter. Control p21 promoter constructs confirm wtp53-transactivating function. The results represent three independent experiments. C. EMSA. NF-{kappa}B binding to its consensus sequence is attenuated in nuclear extracts from SKLMS1 cells pretreated with Adp53. NE, nuclear extract.

 
To localize the promoter region responsible for wtp53-mediated suppression of MMP-9 transcription, we generated a MMP-9 promoter deletion construct from pMMP-9-674Luc (Fig. 5B) and repeated transient transfection reporter gene assays in SKLMS1 cells preinfected with Adp53 or AdLacZ. The promoter activity for both the control mutp53- and the wtp53-infected cells decreased when the MMP-9 promoter was truncated to –328, indicating that the –674 to –328 region contains elements that activate its expression (Fig. 5B). However, the wtp53 inhibitory effect was attenuated from >40% to <15% (Fig. 5B) in the shorter construct, indicating that the deleted fragment contains a p53-responsive element. As expected, the promoterless luciferase pGL3-basic vector lacked promoter activity and was not repressed by wtp53.

Sequence analysis revealed that the identified wtp53-responsive region contains a consensus NF-{kappa}B binding site (5'-ggaattcccc-3') at positions –604 to –595. To further explore the role of this site in wtp53-mediated MMP-9 transcriptional repression, the putative NF-{kappa}B site was mutated to AAaaAATTTc, and the full-length MMP-9 promoter (–674/+3) containing this mutation was subcloned into pGL3 and transfected into STS cells. Reporter assays using this NF-{kappa}B–mutated construct showed that the difference in MMP-9 promoter–driven luciferase expression observed after reintroduction of wtp53 was now abolished (Fig. 5B). NF-{kappa}B binding at this site was previously shown to be critical for both basal MMP-9 promoter activation and also induction of MMP-9 by growth factors and cytokines (15, 16). Taken together, our data suggests that NF-{kappa}B–induced basal MMP-9 promoter activity is inhibited by wtp53. Although not previously shown in the context of the STS MMP-9 promoter, an inverse relationship between wtp53 and NF-{kappa}B activity has been identified in other tumor systems (17). We further investigated whether the wtp53-induced decrease in NF-{kappa}B–mediated MMP-9 promoter activity could be due to decreased NF-{kappa}B expression and/or DNA binding. Western blotting did not show decreased NF-{kappa}B (p50 and p65) protein levels in nuclear protein extracts after Adp53 transfection of SKLMS1 cells compared with parental SKLMS1 or AdLacZ transfectants (data not shown). Electrophoretic mobility shift assays (EMSA) using 32P-labeled NF-{kappa}B consensus site oligonucleotides confirmed NF-{kappa}B DNA binding in SKLMS1 nuclear extracts (Fig. 5C and data not shown). Anti-NF-{kappa}B antibodies "supershifted" the protein-oligonucleotide complex, and radiolabeled probe signal was competitively abolished by excess unlabeled oligonucleotides. EMSA signal intensity was decreased in nuclear extracts from Adp53-treated SKLMS1 cells but not in extracts from AdLacZ-treated cells (Fig. 5C). EMSAs done with probes specific for the MMP-9 NF-{kappa}B –604 to –595 region showed similar findings (data not shown). Taken together, these data indicate that wtp53 represses MMP-9 transcription via inhibition of NF-{kappa}B activation of the MMP-9 promoter.


   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 References
 
We show that MMP-9 is expressed in both primary and metastatic STS, that MMP-9 expression in primary STS is associated with subsequent development of metastases, and that p53 mutation in primary STS correlates with both MMP-9 overexpression and subsequent metastasis. Restoration of wtp53 into mutp53 STS inhibits invasion in vitro and decreases MMP-9 expression in vitro and in vivo, at least partially due to MMP-9 transcriptional repression.

Multiple genetic mutations underlie STS proliferation and dissemination. p53 alterations are the most frequently identified derangement in STS, and contribute to metastasis-promoting behaviors, including loss of cell cycle control (14), enhanced angiogenesis (18), and STS chemoresistance (19). p53 may regulate some MMPs (20); mutp53 activates synovial cell MMP-1 and MMP-13, processes inhibited by wtp53 (21). wtp53 inhibits MMP-2 in melanoma cells (22). In contrast, wtp53 has been shown to activate the MMP-2 promoter (23). Before our study, a regulatory wtp53 inhibition of MMP-9 expression has apparently not been shown.

Which cis-DNA element is responsible for the wtp53 inhibition of MMP-9 promoter activity? Sequence analysis of the MMP-9 promoter revealed that there is no conservative p53 DNA-binding sequence therein; however, recent studies show that wtp53 can transcriptionally regulate downstream target genes through conservative binding site–independent pathways, such as Sp1 (19). The 5' flanking sequence of the MMP-9 promoter contains putative binding sites within the first 670 bp for activator protein-1, NF-{kappa}B, Sp1, and Ets transcription factors, which have been implicated in the induction of MMP-9 gene expression by tumor necrosis factor, Src, and Ras (15, 24). Our experiments identified the NF-{kappa}B binding site as the cis-element responsible for the repression of the MMP-9 promoter by wtp53. NF-{kappa}B transcription factor is a dimer of proteins (p50/p105 or NF-{kappa}B1, p52/p100 or NF-{kappa}B2, p65 or RelA, and c-Rel and RelB) that is retained in the cytoplasm by I{kappa}B family inhibitors until cells receive an appropriate stimulus. In response to growth factors, cytokines, hormones, or other agents, I{kappa}B phosphorylation and degradation is induced, resulting in liberation and nuclear translocation of NF-{kappa}B. In cell nuclei, NF-{kappa}B regulates expression of genes controlling differentiation, apoptosis, cell growth, migration, and inflammation. NF-{kappa}B participate in the basal activity of the MMP-9 promoter as well as its induction by growth factors and cytokines (16, 25). Tumor-suppressor genes, such as PTEN, have been shown to decrease MMP-9 expression by inhibiting NF-{kappa}B promoter binding activity in vascular smooth muscle (26). wtp53 plays a role in NF-{kappa}B expression and activity: wtp53 transfection into human colon cancer cells resulted in suppressed NF-{kappa}B protein levels and decreased constitutive NF-{kappa}B activity, resulting in enhanced apoptosis (27). Our results concur with these findings, showing decreased NF-{kappa}B promoter-binding activity in mutp53 STS cells with wtp53 reintroduction, and are apparently the first demonstration of an interaction between wtp53 and NF-{kappa}B in regulating MMP-9 activity in STS or any other malignancy. The recent demonstration that transcriptionally active mutp53 expressed in H1299 lung carcinoma cell lines induces expression of ~100 cell growth and survival genes, including NF-{kappa}B, suggests a gain of function for p53 mutations in NF-{kappa}B regulation (28). Treatment of these cells with small interfering RNA specific for NF-{kappa}B caused enhanced chemosensitivity to etoposide. However, the complexity of p53/NF-{kappa}B interplay is suggested because contradictory findings have also been reported. Induction of wtp53 in Saos2 osteosarcoma cells has been observed to cause activation of NF-{kappa}B that correlated with wtp53 apoptosis-inducing abilities, whereas loss of NF-{kappa}B activity decreased wtp53-induced apoptosis (28); further study is needed.

Our findings suggest that increased MMP-9 expression/activity is associated with decreased survival in STS, suggesting a rationale for anti-MMP-9–targeted therapy in STS patient subsets. Although MMP inhibitors have yet to be tested in STS, several have been evaluated as antiangiogenic or antimetastatic agents in clinical trials for advanced pancreatic, gastric, prostate, and lung cancer (29-32). Collectively, the results of these trials have been disappointing with no apparent efficacy but increased concomitant morbidity. It is possible that MMP inhibitors have little effect on advanced-stage tumors, but might have impact if administered in earlier-stage tumors before their deployment of multiple metastasis-facilitating molecular derangements. The results of the present study also suggest that wtp53 restorative therapy might help inhibit MMP-9 function in human STS. Adding p53/MMP-9 abrogating therapies to standard chemotherapy might possibly facilitate control of STS metastasis, thereby improving outcomes in this devastating malignancy.


   Materials and Methods
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 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
References
 
This Health Insurance Portability and Accountability Act–compliant study was approved by the institutional review board at The University of Texas M.D. Anderson Cancer Center, which also granted a waiver of informed consent for the use of patient tissue samples.

Cell Lines and Constructs
SKLMS1 cells (human leiomyosarcoma; p53 codon 245 mutation) were obtained from American Type Culture Collection (Rockville, MD) and cultured in DMEM/Ham's F-12 with 10% fetal bovine serum (complete culture medium; Life Technologies, Inc., Grand Island, NY). SKp53; SKAla-10, SKAla-14, and SKAla-21 temperature-sensitive p53; and SKneo control cell lines were generated as previously reported (14). Adp53 and AdLacZ adenoviruses were produced and titered by the Vector Core Laboratory at M.D. Anderson Cancer Center.

Antibodies
Anti-MMP-9: Ab-3 monoclonal antibody (Oncogene Research Products, Boston, MA); anti-p21: C-19 polyclonal; anti-p50: C-19 polyclonal; and anti-p65: C-20 polyclonal (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Anti-ß-actin: monoclonal (Sigma-Aldrich, St. Louis, MO). Anti-p53: DO1 monoclonal (Santa Cruz Biotechnology Inc., CA). Anti-Jagged-1 (Santa Cruz Biotechnology). Horseradish peroxidase–conjugated secondary antibodies were detected by ECL chemiluminesence (Amersham Biosciences, Plc., United Kingdom). IRdye680- and IRdye800-conjugated secondary antibodies (Molecular Probes, Eugene, OR) were detected using Odyssey Imaging (LICOR Biosciences, Lincoln, NE).

Tissue Specimens
Human STS samples were randomly selected from M.D. Anderson Sarcoma Tissue Bank and formalin fixed before paraffin block sectioning at 5 µmol/L for immunohistochemical staining. Genomic DNA was extracted from tumor and autologous normal tissue using a QIAamp DNA minikit according to instructions of the manufacturer (QIAGEN Sciences, Germantown, MA). Mock-, AdLacZ-, and Adp53-treated SKLMS1 severe combined immunodeficient mouse xenografts were harvested as previously published (33).

Immunohistochemistry
Immunohistochemistry was done as previously described (33). Briefly, paraffin sections were dewaxed and rehydrated before antigen retrieval in 0.01 mol/L sodium citrate buffer (pH 6). Endogenous peroxidase activity was quenched with 0.6% hydrogen peroxide before blocking with horse serum. Immunohistochemistry was done with MMP-9, and p21 was diluted 1:200 in PBS containing 0.1% sodium azide and 0.5% bovine serum albumin. Biotinylated secondary antibodies were applied at 1:200 before ABC peroxidase system application (Vectastain ABComplex; Vector Laboratories, Inc., Burlingame, CA), 3,3'-diaminobenzidine color development (Sigma Chemical Co., St. Louis, MO), and Mayer's hematoxylin counterstaining. MMP-9 expression was evaluated by two independent reviewers counting 20 representative fields.

Sequencing of p53 in STS Samples
Genomic DNA was isolated from pathologist-confirmed tumor tissue. PCR amplification of all coding exons and intron-exon boundaries (exons 2-11) was done with primers and conditions as previously described (34). DNA sequencing was done in the DNA core facility at M.D. Anderson Cancer Center.

In vitro Chemoinvasion and Migration Assays
In vitro invasiveness was assayed as previously described (35). Chemoinvasion was measured using 24-well BioCoat Matrigel invasion chambers with 8 µmol/L pore size polycarbonate filters coated with Matrigel (Becton Dickinson Labware, Bedford, MA). Lower compartments contained 0.6 mL laminin (Becton Dickinson Labware) as a chemoattractant. Cells were seeded in the upper compartments and incubated for 72 hours at 38°C or 32°C in a humidified atmosphere of 95% air and 5% CO2. After incubation, filters were fixed and stained with Giemsa (Fisher Scientific, Orangeburg, NY). Cells on the upper surface of the filters were removed by wiping with a cotton swab, and chemoinvasive activity was determined by counting the number of cells per high-power field (x200) that had migrated to the lower side of the filter. Migrant cells were counted in at least three high-power fields per filter. Each sample was assayed in triplicate, and the assays were done twice. The chemotaxis assay was done essentially as described previously (36) using BioCoat cell culture inserts and polycarbonate filters with 8 µmol/L pores (Becton Dickinson Labware). Lower compartments of the insert contained 0.6 mL laminin at 30 µg/mL in DMEM/Ham's F-12 as a chemoattractant or DMEM/Ham's F-12 alone as a negative control. Cells were seeded in the upper compartment and incubated for 6 hours at 37°C in a humidified atmosphere of 95% air and 5% CO2 before fixation, staining, and counting as described above for the chemoinvasion assay. All of the assays were done in duplicate and repeated thrice. Both assays were repeated using SKAla-14 incubated at 38°C with or without 20 nmol/L of a selective MMP-9 inhibitor (Calbiochem, San Diego, CA; a kind gift from Dr. Dougles Boyd, M.D. Anderson Cancer Center).

Zymography Assay
This assay was done as previously described (36). Cells (5 x 105) were seeded onto 100-mm tissue culture plates in serum-containing medium and cultured overnight. Monolayers were then washed and incubated in serum-free medium for 30 hours. Conditioned medium supernatant was concentrated (10-fold) using Amicon spin columns (Amicon, Beverly, MA) before protein quantitation, and subjected to 10% SDS-PAGE (with gel containing 1.5% gelatin) without reducing agents. After electrophoresis, the gel was washed thrice with wash buffer; incubated at 37°C for 16 to 24 hours in 0.2% Triton X-100, 50 mmol/L NaCl, 10 mmol/L CaCl, 50 mmol/L Tris-HCl (pH 7.5), and 1 µmol/L ZnCl containing 0.05% sodium azide; and then stained by Coomassie brilliant blue and destained with 10% acetic acid.

Western Blot Analysis
Western blotting was done by standard methods. Briefly, the conditioned medium (as used in the zymography assay) or equimolar amounts of nuclear extract proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes. Membranes were then blocked and blotted with anti-MMP-9, or anti-p50 and anti-p65 (antibodies as above), and visualized with ECL or IR-conjugated secondary antibodies.

Reverse Transcription-PCR
Reverse transcription-PCR was done as previously described (37). Briefly, total RNA was isolated from cultured cells using TRIzol reagent (Life Technologies) per instructions of the manufacturer. After denaturation at 65°C for 5 minutes, 2 µg RNA was added to 20 µL reverse transcriptase mixture [10 µg/mL oligo(dT), 1x reverse transcriptase buffer; Promega, Madison, WI], four deoxynucleosides (each 0.5 mmol/L), 1 unit/µL RNasein (Promega), and 10 units of avian myeloblastosis virus reverse transcriptase (Promega). RNA was reverse transcribed at 42°C for 60 minutes, and 2 µL of the products were used as templates for multiplex PCR containing both target MMP-9 and ß-actin primers for normalization. Primers used were MMP-9 (PCR product size, 120 bp), 5'-GAGGTTCGACGTGAAGGCGCAGATG-3' and 5'-CATAGGTCACGTAGCCCACTTGGTC-3'; ß-actin (product size, 621 bp), 5'-ACACTGTGCCCATCTACGAGG-3' and 5'-AGGGGCCGGACTCGTCATACT-3'. The PCR reaction mixture contained 50 ng genomic DNA; 1.5 mmol/L MgCl2; 0.2 mmol/L dATP, dTTP, dGTP, and dCTP; primers (each 0.5 µmol/L); and 1x Q solution (Qiagen, Valencia, CA). PCR consisted of 30 cycles of denaturation for 1 minute at 95°C, annealing for 1 minute at 62°C, and an extension for 40 seconds at 72°C. PCR cycles were terminated by an extension at 72°C for 7 minutes and products were resolved on a 2% agarose gel.

MMP-9 Promoter Deletion and Mutation Constructs
MMP-9 proximal promoter sequences (from –674 and –328, to +3 bp of the transcription start site—numbered according to Genbank locus NT_011362.9, GI:51475129) were kindly provided by Dr. Douglas Boyd (M.D. Anderson Cancer Center) and cloned into pGL3. PCR-based substitution mutations (capitalized) were incorporated into the –604 to –595 bp NF-{kappa}B consensus site to generate sequence 5'-AAaaAATTTc-3'.

Reporter Gene Assays
Transfections were done in six-well cluster plates using Fugene 6 transfection reagent (Roche Diagnostics GmbH, Denzburg, Germany) per instructions of the manufacturer, with 100 ng of the MMP-9-Luc–based luciferase reporter per well and 20 ng pRL/cytomegalovirus control per well for normalization. SKLMS1 cells were pretreated with wtp53- or lacZ-expressing adenoviruses (500 plaque-forming units per cell) or mock-infected for 48 hours before transfection. The total amount of DNA transfected per well was 120 ng. Lysates were prepared 48 hours posttransfection and luciferase activity was measured using Promega dual luciferase assay kit per instructions of the manufacturer (Promega). The p53 response element–containing region of the p21 promoter was subcloned from WWP-LUC (38) into pGL3 and used as a positive control for wtp53 activity. All of the reporter assays were done in duplicates and repeated thrice.

EMSA
Nuclear extracts were prepared from SKLMS1 cells in cold nuclear extraction buffer. Binding reaction was initiated by adding 5 µg nuclear extract to binding buffer and 1 µg poly(deoxyinosinic-deoxycytidylic acid) from Amersham Biosciences (Piscataway, NJ), 3 x 105 cpm 32P-labeled target double-stranded oligonucleotide, and 1% NP40 (total volume 20 µL), and incubated for 30 minutes at 37°C. The reaction was terminated by adding 4 µL of 6x DNA loading dye and then placing samples on ice before 5% native PAGE. The dried gel was autoradiographed. For supershifts, 1 µg antibody was added to the nuclear extract and incubated at room temperature for 30 minutes before being added to the binding reaction. NF-{kappa}B consensus site probe sequence: 5'-AGTTGAGGGGACTTTCCCAGGC-3'; MMP-9 NF-{kappa}B –604 to –595 containing probe sequence: 5'-GACAGGGGTTGCCCCAGTGGAATTCCCCAGCCTTGCCTAGCA-3'. EMSA was repeated thrice.

Statistical Analysis
The correlations of MMP-9, p53 mutation, and clinicopathologic variables were assessed using Fisher's exact test. Differences in invasion rate were analyzed using a two-tailed Student's t test. Significance was P ≤ 0.05.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Grant support: M.D. Anderson Cancer Center Core grant P30-CA16672 from the National Cancer Institute, and NIH grant RO1 CA 67802 (R.E. Pollock) and CA 60488 (D. Yu).

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.

Received 7/ 7/06; revised 9/21/06; accepted 9/25/06.


   References
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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