Treatment with Low-Dose Interferon- Restores the Balance between Matrix Metalloproteinase-9 and E-Cadherin Expression in Human Transitional Cell Carcinoma of the Bladder

Tumor invasion and metastasis are regulated by the expression of genes such as E-cadherin, which regulates cell adhesion, and matrix metalloproteinase-9 (MMP-9), which alters the integrity of the extracellular matrix. Both up-regulation of MMP-9 and down-regulation of Ecadherin correlate with bladder cancer metastasis. The purpose of this study was first to determine whether an imbalance between MMP-9 and E-cadherin expression correlates with metastasis from human transitional cell carcinoma (TCC) of the bladder after therapy with neoadjuvant chemotherapy and radical cystectomy and then to determine whether treatment of human TCC xenografts growing in nude mice with interferon (IFN)would restore this balance, thereby limiting tumor invasion and metastasis. We used in situ hybridization to evaluate the expression of several metastasis-related genes, including MMP-9 and E-cadherin, in paraffinembedded biopsy specimens from 55 patients with muscle-invasive TCC treated with neoadjuvant methotrexate, vinblastine, doxorubicin, and cisplatin chemotherapy and radical cystectomy. By multivariate analysis, an MMP-9: E-cadherin ratio of >1.8 was an independent prognostic factor for disease progression. In vitro incubation of an IFN-resistant, highly metastatic human TCC cell line, 253J B-V with noncytostatic concentrations of IFNdown-regulated the activity of MMP-9, up-regulated Ecadherin, and inhibited in vitro invasion. 253J B-V cells were implanted into the bladders of athymic nude mice. Systemic therapy with IFN(10,000 units s.c. daily) decreased the expression of MMP-9, increased expression of E-cadherin, reduced tumor volume, and inhibited metastasis. The MMP-9:E-cadherin ratio was 4.5 in untreated controls and 1.1 after IFNtreatment. Moreover, systemic low-dose daily IFNpotentiated the efficacy of paclitaxel. These studies indicate that in addition to its antiproliferative and antiangiogenic effects, IFNlimits tumor invasion by restoring the normal balance between MMP-9 and E-cadherin and enhances the activity of systemic chemotherapy. INTRODUCTION TCC of the bladder is the fifth most common solid malignancy in the United States and is diagnosed in approximately 54,000 patients and results in 12,000 deaths annually (1). The standard treatment for operable invasive bladder cancer is radical cystectomy, whereas systemic chemotherapy is the only viable therapeutic option for patients with distant metastasis (2–5). Although radical cystectomy will cure a substantial fraction of patients with minimally invasive TCC, many patients with deeply muscle-invasive or extravesicular disease treated by radical cystectomy alone die of metastatic TCC (6). For this reason, patients with muscle-invasive TCC at the Kochi Medical School are currently treated uniformly with neoadjuvant M-VAC chemotherapy followed by radical cystectomy. Despite this aggressive approach, some patients still experience disease relapse, and nearly all of these patients die of metastatic disease resistant to conventional chemotherapy. We hypothesize that it is important to identify prognostic markers that predict for disease recurrence after conventional therapy to develop and implement more effective chemotherapeutic strategies that target their expression or activity. Tumor growth and metastasis depend upon the balance of Received 4/10/01; revised 6/18/01; accepted 6/18/01. 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. 1 Supported in part by NIH Grant 67914 and a Cancer Center Core Grant (CA 16672) from the National Cancer Institute. 2 Both authors contributed equally to this article. 3 To whom requests for reprints should addressed, at the Department of Urology, Box 110, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: (713) 7923250; Fax: (713) 792-8747; E-mail: [email protected]. 4 The abbreviations used are: TCC, transitional cell carcinoma; MMP, matrix metalloproteinase; ISH, in situ hybridization; MVD, microvessel density; bFGF, basic fibroblast growth factor; VEGF, vascular endothelial growth factor; IL, interleukin; M-VAC, methotrexate, vinblastine, doxorubicin, and cisplatin; CAT, chloramphenicol acetyltransferase; Act D, actinomycin D. 2840 Vol. 7, 2840–2853, September 2001 Clinical Cancer Research Research. on December 27, 2017. © 2001 American Association for Cancer clincancerres.aacrjournals.org Downloaded from the expression of a number of genes that regulate angiogenesis and invasion (7, 8). The process of invasion is regulated within a complex homeostasis by the expression of enzymes such as the MMPs, which are responsible for the degradation of the extracellular matrix and basal membranes (9, 10) and the adhesion molecule E-cadherin, which promotes homotypic cellular cohesion. The overexpression of the MMPs, especially MMP-9, in tissue, serum, and urine of patients with TCC correlates with disease progression and metastasis (11–21). Likewise, downregulation of E-cadherin is associated with rapid progression (22–26). We reported previously (27) that the expression of the pro-angiogenic factors bFGF and VEGF predicted for disease recurrence of muscle-invasive TCC treated with systemic M-VAC chemotherapy. In the current study, we demonstrate that the ratio of MMP-9:E-cadherin is a stronger independent predictor for recurrence than either VEGF or bFGF. This suggests the hypothesis that therapy that either down-regulates MMP-9 or up-regulates E-cadherin expression may improve the prognosis for these patients. IFNs are cytokines that exert pleiotrophic antitumoral effects; they regulate cell growth and differentiation, inhibit the expression of oncogenes, up-regulate apoptosis, and activate T lymphocytes, natural killer cells, and macrophages (28–31). IFNand directly inhibit the proliferation of tumor cells of different histological origin (32–39) and are endogenous inhibitors of angiogenesis. Recent studies report that IFNdownregulates the in vitro expression of the angiogenesis factors bFGF (34), IL-8 (35, 36), and MMP-2/9 (37–39) and upregulates E-cadherin (40–42). Systemic therapy with IFNinhibited the growth of the human TCC cell line 253J B-V growing within the bladder of athymic nude mice by downregulating the expression of bFGF, resulting in the inhibition of tumor-induced angiogenesis as measured by the reduction in MVD. This antitumor effect was independent of any antiproliferative effects of IFN (43). In this study, we report that systemic therapy of human TCC growing within the bladders of athymic nude mice with IFNdown-regulated the expression of MMP-9, up-regulating E-cadherin expression, and inhibited tumor growth and spontaneous metastasis. IFN therapy also potentiated the efficacy of paclitaxel against established bladder cancers growing within the bladders of athymic nude mice. Thus, virulent TCC is characterized by a high MMP-9:Ecadherin ratio. Systemic low-dose IFN decreases this ratio by down-regulating MMP-9 and up-regulating E-cadherin and, in combination with systemic cytoreductive chemotherapy, provides a novel therapeutic strategy for the treatment of advanced TCC. MATERIALS AND METHODS Surgical Specimens. Formalin-fixed, paraffin-embedded pretherapy biopsy specimens were available from 55 patients with muscle-invasive TCC who underwent neoadjuvant M-VAC chemotherapy (median of three courses) followed by radical cystectomy and pelvic lymph node dissection. All of the patients were treated at the Kochi Medical School (Kochi, Japan) between 1984 and 1998. Initial clinical stage was determined by pathological examination of the transurethral resection in conjunction with the findings of the examination under anesthesia. All of the patients underwent chest X-ray and computed tomographic scans of the abdomen and pelvis, and any patient with lymph node or distant metastasis was excluded from this analysis. The expression of MMP-9 and E-cadherin within viable tumor of each biopsy specimen was determined by ISH. After cystectomy, patients were monitored carefully with periodic measurement of hepatic transaminases and alkaline phosphatase, chest radiography, and abdominal and pelvic computed tomographic scans. Time to recurrence and overall survival were recorded. Cell Lines and Culture Conditions. 253J B-V, a human TCC cell line resistant to the antiproliferative effects of IFN, was grown as a monolayer culture in modified Eagle’s MEM supplemented with IFN-2a (10,000 units/ml), 10% fetal bovine serum, vitamins, sodium pyruvate, L-glutamine, nonessential amino acids, and penicillin-streptomycin (44). In Situ mRNA Hybridization Analysis. For ISH analysis, specific antisense oligonucleotide DNA probes for MMP-9 (45), E-cadherin (46), VEGF, bFGF, and IL-8 (27) were prepared as described previously. These probes were designed to be complementary to the mRNA transcripts based on published reports of the cDNA sequence, as described previously. The specificity of the oligonucleotide sequence was initially determined by a Gene Bank European Molecular Biology Library database search with the use of the Genetics Computer Group sequence analysis program (Genetics Computer Group, Madison, WI) based on the FastA algorithm. These sequences showed 100% homology with the target gene and minimal homology with nonspecific mammalian gene sequences. The specificity of each sequence was also confirmed by Northern blot analysis (47). A poly(dT)20 oligonucleotide was used to verify the integrity and lack of degradation of mRNA in each sample. All of the DNA probes were synthesized with six biotin molecules (hyperbiotinylated) at the 3 end via direct coupling, with the use of standard phosphoramine chemistry (Research Genetics, Huntsville, AL). The lyophilized probes were reconstituted using a stock solution to 1 g/ l in 10 mmol/liter Tris (pH 7.6) and 1 mmol/liter EDTA. Immediately before use, the stock solution was diluted with probe diluent (Research Genetics). In situ mRNA hybridization was performed as described previously (47, 48) with minor modifications. ISH was performed using the Microprobe Manual Staining System (Fisher Scientific, Pittsburgh, PA; Ref. 49). Tissue sections (4 m) of formalin-fixed, paraffin-embedded specimens were mounted on silane-treated ProbeOn slides (Fisher Scientific; Refs. 47, 48). Alternatively, for evaluation of in vitro mRNA expression, cells were grown on dry-sterilized ProbeOn slides fixed in 4% paraformaldehyde in diethyl pyrocarbonate-PBS for 20 min at room temperature. The slides were placed in the Microprobe slide holder and then dewaxed and rehydrated with Autodewaxer and Autoalcohol (Research Genetics) followed by enzymatic digestion with pepsin. Hybridization of the probe was performed for 45 min at 45°C, and the samples were then washed three times with 2 SSC for 2 min at 45°C. The samples were incubated with alkaline phosphataselabeled avidin for 30 min at 45°C, rinsed in 50 mM Tris buffer (pH 7.6), rinsed with alkaline phosphatase enhancer for 1 min, and incubated with fresh chromogen substrate for 15 min at 45°C. If 2841 Clinical Cancer Research Research. on December 27, 2017. © 2001 American Association for Cancer clincancerres.aacrjournals.org Downloaded from necessary, samples were incubated a second time with fresh chromogen to enhance a weak reaction. Complementary mRNA in the sample leads to a red stain in this assay. Control for endogenous alkaline phosphatase included treatment of the sample in the absence of the biotinylated probe and the use of chromogen alone. Four samples of normal urothelium were used as reference material to which all of the tumor samples were referenced (27). Quantification of Color Reaction. Stained sections were examined in a Zeiss photomicroscope (Carl Zeiss, Inc., Thomwood, NY) equipped with a three-chip charged-coupled device color camera (model DXC-960 MD; Sony Corporation, Tokyo, Japan). The images were analyzed using the Optimas image analysis software (version 6.2; Media Cybernetics, Silver Spring, MD). The slides were prescreened by one of us to determine the range in staining intensity of the slides to be analyzed. Images covering the range of staining intensities were captured electronically, a color bar (montage) was created, and a threshold value was set in the red, green, and blue modes of the color camera. All of the subsequent images were quantified based on this threshold. The integrated absorbance of the selected fields was determined by the mean log inverse gray scale values multiplied by the area of the field. The samples were not counterstained, so the absorbance was attributable solely to the product of the ISH reaction. For each section, we determined the absorbance in several 2 2-mm zones located at the periphery of the tumor. Five different fields within each 2 2-mm zone were quantified, and an average value was determined (50). The intensity of staining was recorded as a ratio of the observed intensity:the intensity of the integrated absorbance of poly(dT)20 in the same sections, and this ratio was then normalized by comparison with the integrated absorbance of a reference set of normal urothelium stained simultaneously with the tumor section according to the following equation: 100 (A/B)/(C/D), where A is the absorbance of the gene expression in the tumor specimen, B is the absorbance of poly(dT)20 expression in the tumor specimen, C is the absorbance of the gene expression in the normal urothelium, and D is the absorbance of poly(dT)20 expression in normal urothelium. An example of ISH and relative gene expression of MMP-9 and E-cadherin is shown in Fig. 1. In Vitro mRNA Expression of MMP-9 and E-cadherin after in Vitro Exposure to IFN. We plated 1 10 253J B-V cells onto ProbeOn slides in 150-cm dishes and incubated the cells continuously with increasing doses of IFNfor 96 h. ISH with probes for MMP-9 and E-cadherin was performed as described above, and the relative expression of mRNA of each was quantified. Collagenase Activity. To determine collagenase activity, electrophoresis of serum-free conditioned medium was performed as described previously (51). Cells (5 10) were seeded in six-well plates and grown to 60–70% confluence. The cells were washed with HBSS and grown for 24 h in serum-free medium. The supernatant fluid was collected to determine collagenase activity, and the remaining cells were counted to confirm the cell number. Collected samples were concentrated by centrifugation with MICROCON microconcentrators (Amicon, Inc., Beverly, MA). Thirty l of each sample were electrophoresed with 10 l of loading buffer (10% SDS) on 20% SDSpolyacrylamide gels containing 1 mg/ml gelatin. After electrophoresis, gels were washed in 2.5% Triton X-100 to remove SDS and to allow proteins to renature. Then the gels were immersed in incubation buffer containing 1% Triton X-100, 50 mM Tris (pH 7.5), 5 mM CaCl2, and 1 M ZnCl2 for 24 h at 37°C. The zymograms were stained with 0.1% (w/v) Coomassie Blue R-250 (Sigma Chemical Co.) and destained in 40% methanol10% acetic acid. Identification of a transparent band at Mr 72,000 or 92,000 on the Coomassie Blue background of the slab gel indicated enzymatic activity. The collagenase activity was quantified by densitometry (ImageQuant software program; Molecular Dynamics, Sunnyvale, CA) after normalizing for cell number. To determine whether IFNmediates a decrease in MMP-9 activity, we incubated 253J B-V cells in the presence of different doses (0–100 units/ml) of IFN, and the activity of MMP-9 was determined. CAT Assay. Using the FuGENE 6 protocol (Boehringer Mannheim Corp.), 253J B-V cells were transfected with the basic CAT expression vector with no promoter/enhancer sequences (pCAT-basic) or a control plasmid with SV40 promoter and enhancer (pCAT-control; Promega, Madison, WI). One copy of the full-sequence 570-bp human MMP-9 promoter (a gift of Dr. Seiki Motoharu, Department of Virology and Oncology, Cancer Research Institute, Kanazawa University Kanazawa, Japan) was ligated upstream of the basic CAT expression vector. Cells (5 10/well in a six-well tissue culture dish) were transfected with 2.5 g of the reporter CAT constructs and 2.5 g of a -actin expression plasmid. After 96 h, extracts were prepared from all of the plates, normalized for -actin activity, and assayed for CAT activity (52) as described previously by Hudson et al. (53). The CAT assay was quantified by densitometry of autoradiographs with the use of the ImageQuant software program (Molecular Dynamics) and was evaluated as the ratio of acetylated species:all species. MMP-9 mRNA Half-Life Studies. To determine whether IFNtherapy altered the stability of MMP-9 mRNA, the 253J B-V cells were incubated in IFNfor 96 h, and further transcription in the cells was then blocked by the addition of ActD (Calbiochem-Novabiotechnology, Inc., Lake Placid, NY; final concentration of 5 g/ml). Total RNA was extracted from the cells 0, 1, 2, and 4 h after the addition of ActD, and MMP-9 mRNA expression was determined by Northern blot analysis and compared with the control value at each time point (total RNA extracted from cells before ActD treatment was arbitrarily defined as 100%). The half-life of MMP-9 mRNA was determined by plotting relative MMP-9 mRNA expression levels on a semilogarithmic axis versus time (Cricket Software, Malvern, PA). MMP-9 mRNA half-life was not affected by treatment with IFN(data not shown). Invasion Assay through Matrigel. Polyinylprolidonefree polycarbonate filters (8m pore size; Nuclepore; Becton Dickinson Labware, Franklin Lakes, NJ) were coated with a mixture of basement membrane components (Matrigel; 25 g/ filter) and placed in modified Boyden chambers (54). The cells (5 10) were released from their culture dishes by short exposure to EDTA (1 mmol/liter), centrifuged, resuspended in 0.1% BSA-DMEM, and placed in the upper compartment of the Boyden chamber. Fibroblast-conditioned medium in the lower compartment served as a chemoattractant. After incubation for 6 h at 37°C, the cells on the lower surface of the filter were 2842 IFNRestores Balance between MMP-9 and E-cadherin Research. on December 27, 2017. © 2001 American Association for Cancer clincancerres.aacrjournals.org Downloaded from stained with Diff-Quick (American Scientific Products, McGraw Park, IL) and quantified with a cooled charge coupled device (CCD) Optotronics Tec 470 camera (Optotronics Engineering, Goletha, CA) linked to a computer and digital printer (Sony Corporation). The results were expressed as the average number within a single 200 field in five separate areas on the lower surface of the filter. Animals. Male athymic BALB/c nude mice were obtained from the Animal Production Area of the National Cancer Institute, Frederick Cancer Research Facility (Frederick, MD). The mice were maintained in a laminar airflow cabinet under specific pathogen-free conditions and used at 8 to 12 weeks of age. All of the facilities were approved by the American Association for Accreditation of Laboratory Animal Care in accordance with the current regulations and standards of the United States Department of Agriculture, the Department of Health and Human Services, and the National Institutes of Health. In Vivo Therapy with IFN. Experiments were designed to evaluate the effect of therapy with systemic IFNon the tumorigenic and metastatic potential of 253J B-V cells growing within the bladder of athymic nude mice and the effect of therapy on the relative expression of MMP-9 and E-cadherin. A lower midline incision was made, and viable tumor cells (1 10/0.05 ml) in HBSS were implanted into the bladder wall. The formation of a bulla indicated a satisfactory injection. The bladder was returned to the abdominal cavity, and the abdominal wall was closed with a single layer of metal clips (44). Three days after tumor implantation, the mice were treated with either saline daily or IFNaccording to the following schedule: 70,000 units weekly, 35,000 units twice/week, or 10,000 units daily, administered s.c. for 4 weeks. On or around day 35, mice were anesthetized with sodium pentobarbitol and killed by cervical dislocation, and the presence of tumor within the bladder and at metastatic sites was evaluated. The bladders were then Fig. 1 Examples of ISH. Note the higher expression of MMP-9 (187% compared with 117%) and lower expression of Ecadherin (68% compared with 92%) in the metastatic tumor compared with the nonmetastatic tumor. The ratio of MMP-9:Ecadherin was 2.7 in the metastatic tumor and 1.3 in the nonmetastatic tumor. 2843 Clinical Cancer Research Research. on December 27, 2017. © 2001 American Association for Cancer clincancerres.aacrjournals.org Downloaded from either quickly frozen in liquid nitrogen for mRNA extraction, fixed in 10% buffered formalin, or frozen in OCT compound (Miles Laboratories, Elkhart, IN). We also designed a series of experiments to evaluate combination therapy of established 253J B-V tumors growing within the bladder of athymic nude mice with IFNand paclitaxel. The 253J B-V cells were implanted into the bladder wall as described previously, and therapy commenced 14 days later when the tumors were palpable. Mice were treated with daily saline, daily IFN(10,000 units s.c.), paclitaxel 1 mg/kg weekly, or a combination of IFNand paclitaxel. On or around day 45, when control mice were moribund, mice were anesthetized with sodium pentobarbitol and killed by cervical dislocation, and the presence of tumor within the bladder and at metastatic sites was again evaluated. The bladders were then either fixed in 10% buffered formalin or frozen in OCT compound. Immunohistochemistry. For immunohistochemical analysis, frozen tissue sections (8m thick) were fixed with cold acetone. Tissue sections (5m thick) of formalin-fixed, paraffinembedded specimens were deparaffinized in xylene, rehydrated in graded alcohol, and transferred to PBS. The slides were rinsed twice with PBS, antigen retrieval was performed with pepsin for 12 min, and endogenous peroxidase was blocked by the use of 3% hydrogen peroxide in PBS for 12 min. The samples were washed three times with PBS and incubated for 20 min at room temperature with a protein-blocking solution of PBS (pH 7.5) containing 5% normal horse serum and 1% normal goat serum. Excess blocking solution was drained, and the samples were incubated for 18 h at 4°C with the appropriate dilution (1:100) of rat monoclonal antiCD31 antibody (PharMingen, San Diego, CA; Ref. 55), a 1:100 dilution of mouse monoclonal anti-MMP-9 antibody (Oncogene Research Products, Cambridge, MA) or a 1:200 dilution of anti-Ecadherin antibody (Zymed, San Francisco, CA). The samples were then rinsed four times with PBS and incubated for 60 min at room temperature with the appropriate dilution of the secondary antibody: peroxidase-conjugated goat antimouse IgG1 (Jackson ImmunoResearch Laboratory, Inc., West Grove, PA) or antimouse IgG1 (PharMingen). The slides were rinsed with PBS and incubated for 5 min with diaminobenzidine (Research Genetics). The sections were then washed three times with PBS, counterstained with Gill’s hematoxylin (Biogenex Laboratories, San Ramon, CA), and again washed three times with PBS. The slides were mounted with a mounting medium (Universal Mount; Research Genetics; Ref. 55). Quantification of MVD. MVD was determined by a light microscope after immunostaining of sections with antiCD31 antibodies (56) according to the procedure of Weidner et al. (57). Clusters of stained endothelial cells distinct from adjacent microvessels, tumor cells, or other stromal cells were counted as one microvessel. Microvessels were counted by using a cooled CCD Optotronics Tec 470 camera (Optotronics Engineering), linked to a computer and digital printer (Sony Corporation). The density of microvessels was expressed as the average of the five highest areas identified within a single 200 field. Quantification of Intensity of Immunostaining. The intensity of immunostaining of MMP-9, E-cadherin, bFGF, VEGF, and IL-8 was quantified in three different areas of each sample by an image analyzer using the Optimas software program (Bioscan, Edmonds, WA). Three different areas in each sample were quantified to yield an average measurement of intensity of immunostaining. The results were presented as a number of each cell line compared with the control, which was set to 100. Statistical Analysis. The statistical differences in MVD, the expression of bFGF, VEGF, IL-8, MMP-9, and E-cadherin, and the MMP:E-cadherin ratio were analyzed by the MannWhitney test (58). The median value of each factor was selected as the cutoff point for designating high or low expression. Multivariate analyses were conducted using the Cox proportional hazards model (59). The incidences of tumor and metastasis were statistically analyzed by the 2 test. Statistical significance was defined as P 0.05.