Background
The extracellular microenvironment is an integral and dynamic scaffold that dictates cell function and cell fate for both normal and cancer cells. Cancer invasion and metastasis develop through a series of steps that involve the loss of cell to cell and cell to matrix adhesion, degradation of extracellular matrix and induction of angiogenesis. A number of protease systems, including the matrix metalloproteinases (MMPs) are involved in these steps. MMPs are essential regulators of the tissue microenvironment through their ability to control extracellular matrix (ECM) turnover [
1]. The MMP family is comprised of at least 24 members that can collectively process virtually any component of the ECM [
2], thereby influencing basic cellular processes that underlie cancer development,
e.g., cell dissociation, cell death and cell division.
Based on our previous biomarker studies, we were interested in studying MMP-10, a relatively understudied MMP in cancer biology. MMP-10 (also known as stromelysin 2) is generally limited to epithelial cells [
3] and can target pro-MMP-1, -7, -8, -9, -13, collagen type III, IV, V, gelatin, elastin, fibronectin, proteoglycans and laminin [
4], activities that have been shown to promote tumor cell invasion [
5]. It has been demonstrated that MMP-10 expression is increased in several human tumors of epithelial origin, including gastric cancer [
6,
7], bladder cancer [
8], esophageal cancer [
9], skin cancer [
10] and non-small cell lung cancer (NSCLC) [
11]. These findings suggest that MMP-10 may play an important role in the development and progression of malignant tumors.
In this study, we monitored MMP-10 expression in cohorts of human tumor tissues, and investigated the mechanistic role of this MMP using a panel of in vitro and in vivo studies. We found that MMP-10 expression is positively correlated with an invasive phenotype in both human cervical and bladder cancers. Experimentally, we found that MMP-10 expression is tightly controlled and can be mediated by three-dimensional culture. We show that MMP-10 regulates migration/invasion capability, endothelial cell tube formation, and induces the expression of key angiogenic and metastatic factors (e.g., MMP-9; hypoxia inducible factor-1 alpha, HIF-1α; chemokine (C-X-C motif) receptor 2, CXCR2; and plasminogen activator inhibitor-1, PAI-1). Furthermore, MMP-10 activity causes resistance to apoptosis via both the intrinsic and extrinsic apoptotic pathways. Lastly, we demonstrate that targeting MMP-10 in a human cervical cancer xenograft model with siRNA inhibited angiogenesis and induced apoptosis, resulting in a significant reduction in the growth of xenograft tumors. These results suggest that MMP-10 has distinct, multiple roles in tumor cell-matrix interactions that favor tumor progression.
Methods
Immunohistochemcal (IHC) staining of tissue microarrays
With IRB approval from MD Anderson Cancer Center Orlando, commercial tissue microarrays (TMA) (CR805 and BL2082, BL1002, US Biomax, Inc., Rockville, MD) constructed from clinical samples obtained from a cohort of 80 patients (70 cervical cancers; 67 adenocarcinoma and 3 squamous cell carcinoma and 10 benign cervical tissues) and from a cohort of 258 patients (188 bladder cancers and 70 benign bladder tissues) were examined by immunohistochemical staining. Clinical staging was recorded for cervical cancer using International Federation of Gynecology and Obstetrics (Stages 0-IV) and for bladder cancer using TNM staging (Stage I-IV). Protocol and antibody details are available in Additional file
1.
Cells and reagents
Human cervical cancer cell line HeLa (adenocarcinoma from ATCC, Manassas, VA) and benign human bladder cell line, UROtsa (a generous gift from Dr. Donald Sens at the University of North Dakota School of Medicine, Grand Forks, ND) were available for analysis. HeLa cells were maintained in RPMI 1640 media and UROtsa cells were maintained in DMEM media as previously described [
12]. Primary human umbilical vein endothelial cell (HUVEC, Cambrex) was cultured in EBM-2 basal media supplemented with EGM-2 MV Kit (Lonza) containing 2% FBS. HUVEC cells of passage 6 to 8 were used. To ensure optimal siRNA delivery in xenograft tumors,
in vivo-jetPEI (Polyplus-transfection Inc. NYC, NY), a linear polyethylenimine, was used in conjunction with siRNA [
13].
Gene transfection for stable cell lines
A plasmid with a sequence verified human MMP-10 cDNA cloned within pCMV6-Entry vector and plasmid with vector alone (Origene Technologies) were transfected into HeLa cells using Fugene HD transfection reagent (Roche Diagnostics) to create HeLa-MMP-10OE and HeLaEmpty, respectively. Stable transfectants were selected with 1,200 μg/ml of G418 (Life Technologies, Inc.) for 14 days and subcloned by limiting dilution in 96-well plates. Integration of the transfected gene into chromosome was confirmed by reverse transcriptase-PCR (data not shown). Stable cell lines were maintained in media containing 500 μg/ml of G418 for HeLa clones (HeLa-MMP-10OE and HeLaEmpty).
Transfection of small interfering RNA (siRNA)
UROtsa cells were transfected for 6 hrs with synthesized MMP-10 siRNA (UROtsa-MMP-10
KD-1 and UROtsa-MMP-10
KD-2) or negative control siRNA (UROtsa-MMP-10
Scr) (Life Technologies) using 6 well plates with a 100-pmol of siRNA and 5 μl of lipofectamine 2000 (Life Technology). Forty-eight hrs after transfection, transfected cells were replated for further experiments. MMP-10 siRNA sequences are listed in Additional file
1.
Immunoblotting
Cell lysate and immunoblotting were performed using standard protocols as previously described [
14]. Antibody details are available in Additional file
1.
Quantitative reverse transcriptase-PCR
RNA was extracted from cells using RNeasy mini kit (Qiagen) as per manufacturer’s instructions. Conversion to cDNA was achieved through High Capacity cDNA Reverse Transcription kit (Life Technologies). Quantitative reverse transcriptase (RT)-PCR was carried out using ABI 7300 Real-Time PCR System (Life Technologies) in a 20 μl reaction volume containing 1 μl of the first-strand cDNA, 1 μM of gene-specific TaqMan primer and probe mix. Primer sets can be found in Additional file
1. Relative fold changes in mRNA levels were calculated after normalization to GAPDH using the comparative Ct method [
15].
Zymography
Thirty micrograms of total cell lysate from the parental cervical cancer cell line HeLa and parental urothelial cell line UROtsa as well as 30 μg of total cell lysate from the following clones: HeLa-MMP-10OE, HeLaEmpty, UROtsa-MMP-10KD1&2 and UROtsa-MMP-10Scr, were electrophoresed on 10% SDS-polyacrylamide gels containing 1 mg/ml casein (Sigma, St. Louis, MO) under non-reducing conditions. After electrophoresis and SDS removal, MMP-10 renaturation was achieved by washing the gel for 1 hr in buffer containing 2.5% Triton-X 100, 50 mM tris pH 7.4, 5 mM CaCl2, and 1 μM ZnCl2. Gels were then incubated in a reaction buffer containing 50 mM Tris pH 7.4, 5 mM CaCl2, 1 μM ZnCl2 and 0.02% NaN3 pH 8.0 for 18 hrs at 37°C, followed by staining with Coomassie blue. MMP-10 activity was indicated by the degradation of casein. Gels were photographed using the KODAK Gel Logic 200 Imaging System with Carestream Molecular Imaging Software Standard Edition v5.0.7.24 (Carestream Health, Rochester, NY).
Cell migration and invasion assays
Migration assays were performed in 6 well two-tier invasion chambers (Collaborative Biomedical Products, Bedford, MA, USA), using a protocol similar to that used successfully by our group [
15]. Polycarbonate membranes were coated with 4 mg/ml growth factor reduced Matrigel (BD Biosciences, San Jose, CA) as described for invasion assays, control inserts (migration only) contained no coating. Two separate experimental designs were tested. First, HeLa-MMP-10
OE, HeLa
Empty and UROtsa-MMP-10
KD1&
2, UROtsa-MMP-10
Scr cells were added to each insert at a density of 10
5 cells/ml/well in RPMI media. The lower chamber contained RPMI media with 10% FBS as chemoattractant. After incubation in a humidified incubator in 5% CO
2 at 37°C for 24 hrs, the cells on the top of the polycarbonate membrane were removed. The cells attached to the bottom of the membrane stained for 1 hr with cell viability indicator Calcein AM Fluorscent Dye (BD Biosciences, Franklin Lakes, NJ) and quantified using the FLUOstar OPTIMA at 495 mm excitation and 515 nm emission (BMG LABTECH Inc., Cary, NC).
In additional, HUVEC cells were added to each insert at a density of 105 cells/ml/well in RPMI media. The lower chamber contained conditioned media from HeLa-MMP-10OE, HeLaEmpty, UROtsa-MMP-10KD1&2 and UROtsa-MMP-10Scr. After 24 hrs, the HUVEC cells on the top of the polycarbonate membrane were removed, while the HUVEC cells attached to the bottom of the membrane were stained for 1 hr with cell viability indicator Calcein AM Fluorscent Dye and quantified using the FLUOstar OPTIMA. For the migration and invasion assays, at least three independent experiments consisting of each condition tested in triplicate wells was used to calculate mean ± SD values.
Matrigel (BD Biosciences) was added to 96 well plates (40 μl per well) and allowed to solidify for 30 min at 37°C as previously described [
16]. HUVEC cells were seeded on top of Matrigel in triplicates at a density of 10
4 cells per well in conditioned media from cultured HeLa-MMP-10
OE, HeLa
Empty, UROtsa-MMP-10
KD1&
2 and UROtsa-MMP-10
Scr and allowed to incubate for 6 hrs. Next, images of capillary tube formation were captured using Leica DMIL inverted microscope. The angiogenic activities were quantitatively evaluated by measuring the total tube length of capillary tube in at least four viewed fields per well. At least three independent experiments consisting of each condition tested in triplicate wells was used to calculate mean ± SD values.
RT2Profiler PCR arrays for angiogenesis and metastasis
Cellular RNA from HeLa
Empty and HeLa-MMP-10
OE was recovered and converted to cDNA as described above. RT
2 Profiler ‘human angiogenesis’ PCR arrays, (Catalog # PAHS-024ZA; SABiosciences) and RT
2 Profiler ‘human metastasis’ PCR arrays, (Catalog # PAHS-028ZA; SABiosciences) were analyzed in duplicate according to the manufacturer's instructions (
http://www.sabiosciences.com/pcrarraydataanalysis.php) by quantitative reverse transcriptase (RT)-PCR carried out using ABI 7300 Real-Time PCR system (Life Technologies). The specificity of the SYBR Green assay was confirmed by melting curve analysis. Relative fold changes in mRNA levels were calculated after normalization to housekeeping control gene targets using the comparative Ct method.
Caspase activity assay
HeLa-MMP-10OE and HeLaEmpty cells were lysed in caspase assay buffer containing 50 mM HEPES (pH 7.5), 100 mM NaCl, 2 mM EDTA, 0.1% CHAPS, 10% sucrose and 5 mM DTT. Aliquots of 6 mg of crude cell lysate were incubated with caspase-3 substrate Ac-DEVD-AMC (Pharmingen, San Diego, CA) at 37°C for 30 min. The caspase-3 activity was quantified in a FLUOstar Optima Reader (BMG LABTECH, Ortenberg, Germany) with excitation at 380 nm and emission at 440 nm. At least three independent experiments consisting of each condition tested in triplicate wells was used to calculate mean ± SD values.
Annexin V apoptosis assay
HeLa-MMP-10OE and HeLaEmpty cells were assessed in a LIVE/DEAD Annexin V apoptotic assay (BD Biosciences, Franklin Lakes, NJ) by flow cytometry. Furthermore, resistance to apoptosis was also determined in these same cells by exposing the cells to 1 μM of staurosporine (an agent capable of inducing apoptosis) for 5 hrs. In brief, after 5 hrs of exposure to staurosporine, cells were trypsinized, washed with 1 X PBS, and stained with propidium iodide (PI) in tandem with an APC-conjugated annexinV antibody according to the manufacturer’s instructions (BD Biosciences, San Jose, CA) and quantitated via flow cytometry using the BD FACScaliber with Cell Quest Pro Software (BD Biosciences, San Jose, California) and FlowJo (TreeStar Inc., Ashland, OR). Viable cells were negative for both annexin V and PI, early apoptotic cells exhibit externalization of phosphatidylserine and were annexin V positive but have an intact plasma membrane and were PI negative, while dead and damaged cells which have subsequently lost their membrane integrity were positive for both annexin V and PI.
Mitochondrial membrane potential analysis
Analysis of mitochondrial membrane potential was performed as previously reported by our group [
14]. Briefly, for the analysis of mitochondrial membrane potential (DCm), HeLa-MMP-10
OE and HeLa
Empty cells were seeded at 5 x 10
5 cells in 10 cm tissue culture dishes for 24 hrs. Cells were then treated with 1 μM of staurosporine for 0, 1, 5 and 18 hrs. Next, cells were trypsinized, washed with 1 x PBS, incubated with medium containing JC-1 dye (10 μg/ml) for 20 min at 37°C. Lastly, the cells were washed and resuspended in 1 ml PBS for fluorescent flow cytometry analysis using FACScan flow cytometer (Becton Dickinson) measuring at least 10
4 gated cells. Mitochondrial depolarization is indicated by a decrease in orange/green fluorescence ratio. All mitochondrial membrane potential analyses were performed in triplicate.
Xenograft tumorgenicity
Animal care was in compliance with the recommendations of
The Guide for Care and Use of Laboratory Animals (National Research Council) and approved by our local IACUC at the University of Central Florida and MD Anderson Cancer Center Orlando. First, the subcutaneous tumorigenicity assay was performed in athymic BALB/c (nu/nu) mice, 6 to 8 wks old purchased from Harlan Laboratories (Indianapolis, IN) by inoculating 10
6 HeLa cells as described previously [
17,
18]. After two weeks, mice were divided randomly into four treatment groups (control, human siRNA MMP-10; 10 μg of siRNA with 1.2 μl of
in vivo-jetPEI at a N/P ratio of 6 according to the manufacturer’s protocol, mouse siRNA MMP-10; 10 μg of siRNAs with 1.2 μl of
in vivo-jetPEI at a N/P ratio of 6 and combination of human and mouse siRNA MMP-10; 5 μg of human siRNA and 5 μg of mouse siRNA with 1.2 μl of
in vivo-jetPEI N/P ratio of 6) and treatment was initiated. siRNA therapy was administered intratumorally twice weekly for five weeks. Control mice received 10 μg of non-target siRNA mixed with 1.2 μl of
in vivo-jetPEI N/P ratio of 6 on the same schedule. At least 10 animals were in each group. At the termination of the experiment, mice were sacrificed and tumors resected for IHC analysis.
Immunohistochemical (IHC) analysis of xenograft tumors
Immunohistochemistry was conducted as described in refs. 17 and 18. Microvessel density (MVD) index was calculated as previously described [
19]. Details and antibodies listed in Additional file
1.
Statistical analyses
All data are expressed as mean ± standard deviation (SD). Statistical analyses were conducted using GraphPad Prism 5.0 (GraphPad Software, Inc.). For most comparisons, a 2-tailed unpaired Student t test or Mann-Whitney U test was conducted. The comparison between MMP-10 expression in low-grade, high-grade, low stage and high stage cancer was calculated using Fisher’s exact test. Differences were considered statistically significant at p < 0.05.
Discussion
Our findings reveal that MMP-10 expression can induce the expression of key molecules implicated in angiogenesis, metastasis and apoptosis, major mechanisms involved in the establishment and progression of malignant tumors. Though MMP-10 is well characterized biochemically, compared to other MMPs, little is known about MMP-10 in human cancers. Here, we are the first to report the relative increased expression of MMP-10 in human cervical cancer compared to benign tissue, and that expression correlates with the invasive phenotype of malignant cervical tumors. Given that we have recently demonstrated that MMP-10 expression at the RNA [
20] and protein level [
21,
22] is increased in urine samples obtained from patients with bladder cancer, so there appears be a trend of association between malignancy and MMP-10. Within this study, analysis of 188 tumor specimens confirmed that MMP-10 protein expression was increased in human bladder tumors, and, as in cervical cancer study, a correlation between more aggressive bladder cancers and MMP-10 expression was noted (Additional file
2: Figure S1). Only one small study has previously reported the expression of MMP-10 in human bladder tumors. In that study, Seargent et al., demonstrated that MMP-10 was predominantly present in the epithelial component of tumor tissue, and that bladder cancer had significantly greater levels of MMP-10 than benign bladder specimens. However, the investigators could not demonstrate a difference in MMP-10 expression between low stage and high stage disease, possibly due to the small (n = 60) sample size [
7]. Limited data are available for MMP-10 expression in cervical tissue. However in a small study, Vazquez-Ortiz et al. reported that overexpression of MMP-10 was observed to be consistently overexpressed in invasive cervical cancer biopsies associated by cDNA array. Validation on an independent, large cohort was not performed [
25]. We must stress that HPV status was not reported on the commercial TMA utilized in our study. It would be interesting to investigate whether MMP-10 expression is correlated with disease prognosis in cervical and bladder cancers, as it has been in esophageal carcinoma [
26], current studies to address this are underway.
MMP-10 (also known as stromelysin 2) is generally limited to epithelial cells and its regulation is tightly controlled [
27]. For example, MMP-10 is not present in intact normal skin, but is expressed during cutaneous injury and repair where it is localized to the migrating keratinocytes at the wound edge, alluding to its role as a pro-invasion molecule [
5]. In the HeLa cervical cancer cell line, MMP-10 was not expressed in culture. However, when HeLa cells were grown in a 3D matrix (data not shown), or as a xenograft tumor (Figure
6A), MMP-10 expression was induced. This is similar to the report by Mishra et al., who demonstrated that A549 human lung cancer cells grown in an
ex vivo 3D model expressed significantly more MMP-10 than when grown as a monolayer culture [
28]. Though benign (as defined by inability to form xenograft tumors), the UROtsa cell line has been transformed by SV40 [
29], and thus has a non-functional p53. This could account for the high level of MMP-10 expression, supporting a role for p53 in MMP-10 expression, originally noted by Meyer et al. [
24].
A considerable amount of information has accumulated to suggest that the role of MMPs in cancer is far more complicated than initially presumed. One such role in cancer is related to apoptosis. MMP activity may have proapoptotic or antiapoptotic effects depending on the local cellular production and availability of proteolytically released and activated death-inducing and/or growth and survival factors [
30]. For example, proapoptotic effects can be due to degradation of ECM proteins that serve as ligands for specific classes of integrins. In the absence of the ligand, integrins can no longer trigger signals necessary for epithelial and endothelial survival thus resulting in apoptosis. Alternatively, ECM degradation can result in the release of IGF-1, which could directly promote tumor cell resistance to proapototic signals. In our study, HeLa cells (p53 wild-type) were found to be more resistant to apoptosis when MMP-10 was expressed. This resistance to apoptosis was associated with changes in the expression of key molecules in both the intrinsic (
e.g., reduction in Bax, Bak and cleaved caspase 8 and an increase in Bcl-xl) and extrinsic apoptotic pathway
(e.g., reduction in FasL and cleaved caspase 8).
An interesting co-expression relationship that we were able to confirm was that between MMP-10 and PAI-1. PAI-1 is an important endogenous inhibitor of urokinase-type plasminogen activator. Specifically, PAI-1 normally functions as part of the plasminogen activation (PA) system, which includes the serine protease uPA (urokinase-type plasminogen activator), its receptor uPA-R, tPA (tissue plasminogen activator) and inhibitors PAI-1 and PAI-2 [
31]. PAI-1 expression may be regulated by many intrinsic factors (
e.g., cytokines and growth factors) and extrinsic factors (
e.g., cellular stress) [
32]. Although the canonical function of PAI-1 has been known as an inhibitor of uPA to maintain clot formation [
33] it is now regarded as a pleiotropic factor exerting diverse cellular effects, many related to tumorigenesis; cellular proliferation, migration, invasion, adhesion and angiogenesis. The deregulation of PAI-1 has been correlated to several tumor types (
e.g., breast, colorectal, lung and bladder) and has been associated as a poor prognostic factor [
34‐
40]. Previous studies have reported a positive correlation between MMP-10 and PAI-1 [
41‐
43]. Similar, we report an increase of both MMP-10 and PAI-1 in voided urine samples from subjects with bladder cancer [
21,
22]. Interesting, Wilkins et al. reported that the addition of PAI-1 blocks collagen degradation as well as the conversion of MMP-10 to its catalytically active form. Thus there exist MMP-10/PAI-1/plasmin dependent proteolytic axis that enhances ECM degradation and facilitates angiogenesis and invasion. Further characterization of this axis may have far reaching implications for therapeutic targeting of human malignancies.
Conclusions
In a series of in vitro and in vivo experiments, MMP-10 activity was shown to be pivotal in the tumor growth in a mouse model of cancer, and the expression of this MMP is associated with the upregulation of key molecules related to angiogenesis, metastasis, and apoptosis, creating a milieu favorable to the survival and expansion of malignant lesions. Furthermore, demonstration of a correlation between MMP-10 expression and invasive cervical and bladder cancers supports a role for MMP-10 in human tumor progression. These data suggest that MMP-10 may have value as a novel biomarker and/or provide a molecular target for therapeutic intervention.
Competing interest
S. Goodison and C.J. Rosser are officers in Nonagen Bioscience Corporation. All other authors declare that they have no competing interests.
Authors’ contributions
GZ Perform in vitro and in vivo experiments and analysis. MM Perform in vitro and in vivo experiments and analysis. AL Pathologic review of human and animal tissues. Steve Goodison Study concept and design, drafting of manuscript. CJR Study concept and design, drafting of manuscript, administrative support and funding. All authors read and approved the final manuscript.