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Experimental Therapeutics, Molecular Targets, and Chemical Biology |
1 Departments of Medicine and 2 Biochemistry, Dartmouth Medical School, Hanover, New Hampshire and 3 Norris Cotton Cancer Center, Dartmouth Hitchcock Medical Center, Lebanon, New Hampshire
Requests for reprints: Constance E. Brinckerhoff, Dartmouth Hitchcock Medical Center, Norris Cotton Cancer Center, Room 602, Rubin Building, 1 Medical Center Drive, Lebanon, NH 03756. Phone: 603-653-9957; Fax: 603-653-9952; E-mail: brinckerhoff{at}dartmouth.edu.
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Introduction |
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Type I collagen is a major component of the ECM and it is degraded by enzymes called matrix metalloproteinases (MMP). MMPs with enzyme activity directed against type I collagen include the interstitial collagenases MMP-1 and MMP-13 and the membrane-bound MMP-14 (MT1-MMP; ref. 1). In normal physiology, basal expression of most MMPs, including the interstitial collagenases, is low; however, high expression has been linked to several pathologies, including cancer cell invasiveness. In clinical studies, increased MMP-1 expression has been associated with the incidence or invasiveness of several cancers: colorectal, esophageal, pancreatic, gastric, breast, and malignant melanoma (5–10). Furthermore, elevated MMP-1 expression in atypical ductal hyperplastic tissues may serve as a marker for predicting which patients will develop invasive breast cancer (11).
To begin to understand the contributions of MMP-1 to tumor growth and progression, MMP-1 has been overexpressed in various experimental systems. In a differentiated epithelial cell line (Madin-Darby canine kidney cells), overexpressing proMMP-1 did not confer an invasive phenotype in an in vitro peritoneal invasion model or in a three-dimensional collagen matrix, perhaps because these cells did not express the proteinases needed to fully activate the latent enzyme (3, 12–14). However, when MMP-1 was overexpressed in the skin of transgenic mice, it increased their susceptibility to skin carcinogenesis (15).
An alternative to overexpression has been blocking MMP-1 expression. Durko et al. (16) developed a 777-nucleotide antisense RNA molecule capable of specifically silencing MMP-1 expression and showed that blocking this expression in melanoma cells blocked in vitro invasion of a collagen matrix. These findings were extended to chondrosarcoma cells using the same antisense RNA plasmid, and more recently, the experiments with chondrosarcoma cells were repeated using transiently transfected short interfering RNA (siRNA) molecules (17, 18). Both approaches resulted in a substantial reduction in MMP-1 protein levels, which was accompanied by a significant decrease in the ability of the cells to invade through a type I collagen gel.
A plasmid-based system that stably expresses short hairpin RNA (shRNA) molecules, which silence gene expression by RNA interference (19–21), has been developed, thereby providing a mechanism for long-term gene silencing. In this study, we developed a shRNA sequence which, when stably integrated into cellular DNA, selectively targets MMP-1 expression. In MDA-231 breast cancer cells that have constitutively high levels of MMP-1, this shRNA molecule specifically blocks MMP-1 expression and MMP-1-mediated destruction of collagen in an in vitro assay and the MMP-1 shRNA molecule significantly inhibits the growth of breast cancer cells in nude mice.
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Materials and Methods |
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Matrix metalloproteinase-1 and enhanced green fluorescent protein expression plasmids. The pCMV-MMP-1 expression construct was prepared with NotI and SalI restriction enzymes (Invitrogen, Carlsbad, CA), used to excise the complete MMP-1 cDNA from pSP64-MMP-1 (ATCC) and to digest the pCMV-Tag4c vector (Stratagene, La Jolla, CA). The purified digestion products and the MMP-1 cDNA fragment were ligated into the pCMV-Tag 4c vector to create the pCMV-MMP-1 vector. The enhanced green fluorescent protein expression vector (peGFP) is commercially available from BD Biosciences (Franklin Lakes, NJ).
Short hairpin RNA expression plasmids. Circular pSuper vectors (pSuper and pSUPER.Retro.Neo.GFP, Oligoengine) were linearized with BglII and HindIII restriction enzymes (Invitrogen) and dephosphorylated with calf intestinal phosphatase (Life Technologies, Inc., Gaithersburg, MD). DNA oligonucleotides (see below) specifically designed for use with the pSuper plasmids and containing either MMP-1 shRNA sequence or scrambled shRNA sequence were annealed to create double-stranded oligos to be cloned into the pSuper vectors. Annealed oligonucleotides were phosphorylated with T4 polynucleotide kinase (Promega, Madison, WI), ligated to the pSUPER plasmids, and then transformed into bacteria. The newly created pSuper-MMP-1 shRNA, pSuper-retro-MMP-1 shRNA, and pSuper-retro-scrambled shRNA vectors were prepared from individual bacterial colonies. Correct orientation and location of the oligonucleotide cloning were confirmed by sequencing the plasmids with T3 and T7 primers (T3, 5'-AATTAACCCTCACTAAAGGG-3'; T7, 5'-TAATACGACTCACTATAGGG-3') and the Big Dye sequencing reagent (Applied Biosystems, Foster City, CA). MMP-1 shRNA oligonucleotides (MMP-1 sense, 5'-GATCCCCACCAGATGCTGAAACCCTGTTCAAGAGACAGGGTTTCAGCATCTGGTTTTTTGGAAA-3'; MMP-1 antisense, 5'-AGCTTTTCCAAAAAACCAGATGCTGAAACCCTGTCTCTTGAACAGGGTTTCAGCATCTGGTGGG-3'; patent pending) contained a region specific to bases 234 to 252 of MMP-1 mRNA (bold), a hairpin loop region (italic), and 5' and 3' linker sequences for subcloning into the BglII and HindIII sites of the pSUPER vectors. The scrambled shRNA oligonucleotides contained identical hairpin loop and linker sequence but contained a sequence of DNA that was not complementary to any human gene (AAGTGGAGGGACGTATGCA).
Transfection of NIH 3T3 and MDA-231 cells. NIH 3T3 cells were transiently transfected with Lipofectamine 2000 (Invitrogen) along with 0.5 µg peGFP, 0.5 µg pCMV-MMP-1, and 2 µg pSUPER-MMP-1 shRNA or pSuper with no insert. Transfection efficiency was monitored using eGFP signal. Total cellular RNA was harvested 48 hours after transfection and analyzed for MMP-1 mRNA by real-time reverse transcription-PCR (RT-PCR; see below). MDA-231 cells were stably transfected in triplicate with Lipofectamine 2000 (Invitrogen) and 2 µg of the pSUPER.Retro.Neo.GFP plasmid containing the MMP-1 shRNA, the scrambled sequence, or the empty vector. Stable cell lines were selected by growth in the presence of 1 mg/mL G418 (Stratagene) and individual cell lines were isolated with cloning discs (PGC Scientific, Frederick, MD).
RNA isolation and real-time reverse transcription-PCR. MDA-231 stable cell lines were cultured in serum-free medium for 24 hours, RNA was harvested using the RNeasy RNA isolation kit (Qiagen, Valencia, CA), and DNA contamination was removed with RNase-free DNase (Qiagen). For real-time RT-PCR, reverse transcription was done using protocols and reagents from Applied Biosystems TaqMan reverse transcription reagent kit. Briefly, 2 µg of DNase treated RNA were reverse transcribed in a 20-µL reaction containing 5.5 mmol/L MgCl2, 500 µmol/L each deoxynucleotide triphosphate (dNTP), 2.5 µmol/L oligo d(T)16, 0.4 units/µL RNase inhibitor, and 1.25 units/µL Multiscribe reverse transcriptase. The reactions were incubated at 25°C for 5 minutes, 48° for 30 minutes, then 95°C for 5 minutes.
Real-time PCR was done with reagents and protocols from the Applied Biosystems SYBR Green master mix kit. Five microliters of each reverse transcription reaction were used to amplify MMP cDNA in triplicate real-time PCR reactions and 2 µL of each reverse transcription reaction were used to amplify glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or eGFP cDNA in duplicate reactions. For quantitative comparisons between PCR assays, standard curves were generated with every assay (see standards outlined below). The PCRs contained 200 nmol/L of each primer and were incubated on an M.J. Research Opticon (Waltham, MA) real-time detection thermal cycler at 95°C for 10 minutes, followed by 50 PCR cycles of 95° for 15 seconds, and 60°C for 1 minute, and plate read. The PCR cycles were followed by a SYBR Green melting curve from 55°C to 90°C. MMP mRNA levels are reported as copies of the target gene per picogram of GAPDH or eGFP.
Real-time PCR primer sequences. The MMP-2 and MMP-9 primer sequences were generous gifts from Dr. Ian Clark (University of East Anglia, Norwich, United Kingdom). The remaining primers were designed using Oligo primer analysis software (Molecular Biology Insights, Cascade, CO). Forward and reverse real-time PCR primers for MMP-1, MMP-2, MMP-9, MMP-14 (MT1-MMP), GAPDH, and peGFP were MMP-1 forward 5'-AGCTAGCTCAGGATGACATTGATG-3', MMP-1 reverse 5'-GCCGATGGGCTGGACAG-3'; MMP-2 forward 5'-TGGCGATGGATACCCCTTT-3', MMP-2 reverse 5'-TTCTCCCAAGGTCCATAGCTCAT-3'; MMP-9 forward 5'-CCTGGGCAGATTCCAAACCT-3', MMP-9 reverse 5'-GCAAGTCTTCCGAGTAGTTTTGGAT-3'; MMP-13 forward 5'-TGGCATTGCTGACATCATGA-3', MMP-13 reverse 5'-GCCAGAGGGCCCATCAA-3'; MMP-14 forward 5'-CCCCGAAGCCTGGCTACA-3', MMP-14 reverse 5'-GCATCAGCTTTGCCTGTTACT-3'; GAPDH forward 5'-CGACAGTCAGCCGCATCTT-3', GAPDH reverse 5'-CCCCATGGTGTCTGAGCG-3'; and eGFP forward 5'-TATCATGGCCGACAAGCAGAAGAAC-3', eGFP reverse 5'-TTTGCTCAGGGCGGACTGGGTGCTC-3'.
Real-time PCR standards. The real-time standards for MMP-1, MMP-2, MMP-9, GAPDH, and peGFP were plasmids containing either a portion or all of the cDNA of the target gene. The standard for MMP-14 was a PCR fragment generated with the real-time primers. Plasmid dilutions were serial log dilutions from 1 ng to 10 fg. The MMP-14 standards were diluted from 10 pg to 1 fg. Numbers for converting picograms to copies of mRNA were 360,000 copies/pg MMP-1, 480,000 copies/pg MMP-2, 340,000 copies/pg MMP-9, 270,000 copies/pg MMP-13, and 10 x 106 copies/pg MMP-14 (MT1-MMP).
IFN response reverse transcription-PCR assay. Three micrograms of RNA pooled from MDA-231 cells containing the empty vector or the MMP-1 shRNA were reverse transcribed with Moloney murine leukemia virus (MMLV) reverse transcriptase (Invitrogen) according to the instructions of the manufacturer. Briefly, 3 µg of RNA, 99 ng of random hexamers, and 10 nmol of dNTP were combined to a total volume of 12 µL. The mixture was then incubated at 65°C for 5 minutes and cooled to 4°C. Four microliters of 5x reverse strand buffer, 2 µL of 0.1 mol/L DTT, and 1 µL of RNase inhibitor A (40 units/µL) were added to the reaction and incubated at 37°C for 2 minutes. One microliter of MMLV reverse transcriptase was added to the reaction, which was incubated at 25°C for 10 minutes, 37°C for 50 minutes, and 70°C for 15 minutes. Expression of 11 IFN-responsive genes and GAPDH was estimated using semiquantitative PCR and the human IFN response MultiGene-12 RT-PCR Profiling Kit (#PH-009B) from Superarray (Frederick, MD).
ELISA for matrix metalloproteinase-1 protein. MMP-1 protein in serum-free medium of stably transfected MDA-231 cells was quantified with the human MMP-1 biotrak ELISA system for latent and active enzyme (Amersham, Piscataway, NJ). Cells were grown in six-well culture dishes with 1 mL of serum-free medium for 24 hours and 100 µL of a 1:30 dilution of the medium were used for the ELISA. Cells were lysed with 350 µL of passive lysis buffer (Promega) and total protein was determined with 70 µL in a Bradford assay (Bio-Rad, Hercules, CA). Nanograms of MMP-1 protein from the ELISA were normalized to micrograms of total protein.
Western blotting. MDA-231 cells were cultured in 1 mL serum-free DMEM/F12 medium in six-well plates in the presence or absence of a collagen gel, with or without aprotinin (50 units/mL; Sigma, St. Louis, MO). After 24 hours, medium was precipitated with cold 10% trichloroacetic acid and resuspended in 50 µL SDS loading buffer (Promega). Following SDS-PAGE and transfer to a polyvinylidene difluoride membrane (Immobilon-P, Millipore, Billerica, MA), the membrane was incubated in a 5% milk, TBS solution with 0.1% Tween 20 (milk TBST) for 1 hour. The membrane was probed with a MMP-1 polyclonal antibody (Chemicon, Temecula, CA) diluted 1:5,000 in milk TBST overnight at 4°C, washed thrice with TBST, and a 1:2,000 dilution of goat anti-rabbit horseradish peroxidase (HRP)–conjugated antibody (Cell Signaling, Beverly, MA) in milk TBST was added for 60 minutes. The blot was washed thrice with TBST and HRP activity was detected with the Western Lightning chemiluminescence reagent (Perkin-Elmer, Boston, MA).
Cell proliferation assay. The proliferation rate of the stably transfected MDA-231 cell lines at 24 and 48 hours was determined with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (ATCC). The assay was optimized to be sure signal obtained was within the linear range for the reagent. For each cell line, the optimum cell count was 10,000 cells per well of a 96-well tissue culture dish.
In vitro collagen gel destruction assay. Collagen preparations were carried out on ice to prevent premature jelling. A solution of purified type I bovine collagen (Cohesion Technologies, Palo Alto, CA) was neutralized with a sterile 10x PBS (pH 7.4) and 0.1 mol/L NaOH. Neutralized collagen (2 mg/mL) was mixed with an equivalent volume of cells suspended in serum-free DMEM to yield a final concentration of 2 x 105 cells/mL in 1 mg/mL fibrillar collagen, and 1 mL of the collagen/cell mixture was added to each well of a six-well plate. After the collagen jelled (
1 hour), 1 mL of serum-free medium was added on top of the gel. For some experiments, 25 µmol/L Ilomastat (Chemicon), 50 units/mL aprotinin (Sigma), 1 µg/mL neutralizing MMP-1 antibody (MMP-1 Ab-5, Oncogene, Cambridge, MA), 1 µg/mL monoclonal FLAG antibody (Sigma), or 1% DMSO was added to the collagen before it jelled (1 µg antibody/mL collagen). After 36 hours at 37°C, the medium was removed and weighed. The weight of medium added at the start of the 37°C incubation (1 g) was subtracted from the total medium weight to reveal the amount liberated by collagen destruction.
Tumorigenesis studies in mice. Pooled MDA-231 stable cell lines containing the empty vector or the MMP-1 shRNA (1 x 106 cells/100 µL HBSS) were injected into the fourth inguinal fat pad of 51 female, 6-week-old nude mice (nu/nu, Charles River, Wilmington, MA) using 28 gauge x 1/2 in. single-use insulin needles. Mice were examined weekly until tumors were grossly apparent; then they were examined several times a week. Tumors were measured twice with Vernier calipers and tumor volume was calculated using the formula (4/3)
r3. When the two measurements differed, the smaller radius measurement was squared and multiplied by the largest radius measurement. This number was then substituted for the r3 portion of the formula.
Statistical methods. Student's t test was used for calculating the statistical significance of measurements of MMP levels, collagen destruction, cell proliferation, and tumor size. Fisher's exact test was used to determine whether there was a correlation between tumor incidence and the presence of the MMP-1 shRNA.
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Results |
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Matrix metalloproteinase-1 short hairpin RNA efficacy: blocking ectopic gene expression by transient transfections. To test the efficacy of the selected shRNA sequence, we used two pSUPER plasmids: one with the MMP-1 shRNA sequence to block MMP-1 gene expression and one with no shRNA insert. NIH 3T3 mouse embryonic fibroblasts were used because they do not contain a homologue to MMP-1 and because they are easily transfectable. MMP-1 mRNA production was supplied to the NIH 3T3 cells by a plasmid producing the mRNA from a CMV promoter (pCMV-MMP-1-tag) and transfection efficiency was tracked by cotransfecting with peGFP. The eGFP mRNA also served as a control gene to normalize the MMP-1 mRNA levels. Forty-eight hours after transfection, MMP-1 mRNA levels of cells transfected with the MMP-1 shRNA vector were 73% lower than the MMP-1 levels of cells transfected with the empty vector (P = 0.015; Fig. 1). These findings indicated that the MMP-1 shRNA reduced MMP-1 mRNA levels; consequently, the shRNA oligonucleotides were cloned into the stably integrating pSUPER-retro plasmid to create MDA-231 cell lines.
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After neomycin selection and isolation of individual stable cell lines, MMP-1 mRNA and protein levels were determined using quantitative RT-PCR and ELISA. The ELISA measured total (active and zymogen) MMP-1 protein. Three cell lines producing MMP-1 shRNA had a >90% reduction in MMP-1 mRNA and protein levels compared with the cell lines containing the empty vector (Table 1).
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50%) increase in expression in the MMP-1 shRNA cell lines when compared with the empty vector cell lines and three genes (SCYB10/IP-10, MxB, and OAS1) with a modest (35-50%) decrease in expression (Fig. 2). Overall, there is no consistent up-regulation of IFN-responsive genes to suggest that the MMP-1 shRNA induced an IFN response.
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1 mL (0.8 ± 0.06 g) of medium was liberated from the gels containing the MDA-231 cells with the empty vector (Fig. 3B). This volume was comparable to the volume of the gel at the beginning of the experiments (1 mL), indicating that these cells had completely degraded the collagen matrix. Conversely, no medium (–0.01 ± 0.03 g) was liberated from collagen containing MDA-231 cells producing the MMP-1 shRNA. The slight decrease in the volume of medium recovered may be due to evaporation during the incubation period. These results show that MMP-1 produced by cells containing the empty vector can destroy a collagen gel and that shRNA-mediated reduction of MMP-1 expression blocked the collagenolytic activity of the cells.
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1 mL of medium (0.8 ± 0.05 g; Fig. 3B). The three MMPs primarily responsible for degrading type 1 collagen are MMP-1, MMP-13, and the membrane-bound MT1-MMP (1). MMP-1 and MMP-13 are secreted as proenzymes and require stepwise cleavage of the propeptide (often by serine proteases) before the enzymes become active (3). MT1-MMP is embedded in the cell membrane as an active enzyme. Therefore, to distinguish between MT1-MMP–mediated destruction of the collagen versus that mediated by MMP-1 and MMP-13, the serine protease inhibitor aprotinin (50 units/mL collagen) was embedded in collagen with MDA-231 cells containing the empty vector. After 36 hours, no medium had been liberated from collagen containing cells and aprotinin (0.01 ± 0.1 g; Fig. 3B), implying that MMP-1 or even MMP-13 (despite its low level of expression; Table 2) was responsible for the collagenolytic activity of the MDA-231 cells. Western blot analysis of the culture medium confirmed that aprotinin blocked activation of MMP-1. We found no detectable MMP-1 protein in the medium of MDA-231 cells producing the MMP-1 shRNA (Fig. 3C, lane 1), confirming the ELISA and quantitative RT-PCR findings. However, a band representing cleaved and activated MMP-1 was visible when cells containing the empty vector were grown on plastic (Fig. 3C, lane 2). When these cells were embedded in collagen, there were two MMP-1 protein bands representing the 54 kDa zymogen and the smaller activated form of the enzyme (Fig. 3C, lane 3). Finally, medium from aprotinin-treated cells contained only the higher molecular weight zymogen band (Fig. 3C, lane 4). The presence of more MMP-1 protein in the medium of cells grown in collagen agrees with our findings that embedding MDA-231 cells in a collagen matrix stimulates a 2- to 3-fold increase in MMP-1 expression (data not shown). Cleavage of the propeptide domain of the 54-kDa MMP-1 zymogen produces a 44-kDa active form; however, smaller 24-kDa MMP-1 proteins represent active MMP-1 (26, 27). The Western blot confirms that MDA-231 cells produce proteinases that activate proMMP-1 (28) and that aprotinin can block this activation.
Finally, to definitively prove that the collagenolytic activity of the MDA-231 cells is due to MMP-1, MMP-1-neutralizing antibody was embedded in the collagen along with MDA-231 cells containing the empty vector. A monoclonal antibody against the FLAG epitope was used as the negative control. At 24 hours, there was a noticeable difference in the thickness of the collagen gels (data not shown). After 40 hours, the weight of medium liberated from gels containing FLAG antibody and cells was comparable to that of the medium liberated from wells containing cells alone (0.78 ± 0.03 g) whereas very little medium was liberated from collagen gels containing the MMP-1 antibody and cells (0.14 ± 0.04 g; Fig. 3B). The small volume of medium liberated from collagen by MDA-231 cells incubated with the MMP-1-neutralizing antibody may be attributed to the limited effectiveness of the MMP-1-neutralizing antibody in blocking MMP-1 activity for a long period of time. Indeed, by 50 hours, the collagen was completely destroyed (data not shown), indicating that MMP-1 production by these cells could eventually overwhelm the neutralizing antibody. Conversely, when the MDA-231 cell lines producing MMP-1 shRNA were incubated in collagen for as long as 5 days, there was no collagen destruction (data not shown). These results confirm that the MDA-231 cell–mediated destruction of collagen is a result of their MMP-1 expression and supports the hypothesis that blocking MMP-1 expression with a stably integrated MMP-1 shRNA vector blocks the ability of MDA-231 cells to degrade a type 1 collagen matrix.
In vivo tumorigenesis. We next determined whether the shRNA-mediated reduction of MMP-1 in MDA-231 cells would affect the growth of the cells in an in vivo model of breast tumor growth. Two separate groups of nude mice were injected in the fourth inguinal mammary fat pad with MDA-231 cells containing the empty vector or the MMP-1 shRNA. At week 6, tumors began to appear and at week 11, the mice were sacrificed (Fig. 4). The frequency of tumor formation in mice injected with the empty vector cell lines (57%, n = 23) was not significantly greater than that in mice injected with MMP-1 shRNA–producing cells (36%, n = 28; P = 0.11, Fisher's exact test; Table 4). However, at the time of sacrifice, the average tumor size in mice injected with cells containing the empty vector (1,217 ± 334 mm3, mean ± SE; n = 13) was significantly greater (P = 0.027, Student's t test) than the average tumor size in mice injected with cells producing the MMP-1 shRNA (272 ± 117 mm3, mean ± SE; n = 10; Fig. 4A). Furthermore, exponential curve fits of the average weekly tumor sizes generated by the Excel program reveal that the tumor cells producing MMP-1 were growing at a faster rate (tumor volume = 2.4874e0.5431week, R2 = 0.9412) than tumor cells not producing MMP-1 (tumor volume = 21.802e0.1892week, R2 = 0.2883; Fig. 4B). Examination at autopsy revealed no macroscopic metastases to any organs in the peritoneal cavity, the lungs, or the brain (data not shown). Furthermore, histologic slides of these organs revealed no microscopic metastases (data not shown). These data suggest that MMP-1 is not necessary for tumor formation but that it contributes to the growth of primary tumors in this mouse model.
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Discussion |
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MMP-1 activity may be required to break down the fibrous stroma within the mammary fat pad, removing a physical barrier to growth; however, degradation of ECM molecules does more than facilitate cell mobility by removing a physical barrier. The ECM contains growth factors that are liberated by breakdown of matrix proteins. MMP-1, in particular, is capable of cleaving the proteoglycan perlecan, which can release basic fibroblast growth factor (29). MMP-1 can also cleave insulin-like growth factor (IGF) binding proteins that are present in the ECM, thereby increasing the availability of IGF and increasing cell proliferation (30). Other MMP-1 substrates within the ECM include proteins that modulate immune responses, such as monocyte chemoattractant proteins (31). When MMP-1 cleaves monocyte chemoattractant protein, it produces a CC chemokine receptor antagonist which may block phagocytic cell recruitment to the site of tumor cell injection. Although nude mice are athymic, they have some components of their immune system, including monocyte/macrophages. Thus, MMP-1 expression may have contributed to tumor growth in these mice through mechanisms beyond the traditional collagen destruction model of tumor invasion.
Our findings are similar to those describing decreased collagenolytic activity of chondrosarcoma cells transiently transfected with MMP-1 siRNA molecules (18). Transient transfection of three siRNA molecules targeted at MMP-1 resulted in a 50% to 80% inhibition of MMP-1 between 48 and 72 hours after transfections with a recovery to pretransfection levels by 120 hours. By using a stably integrating plasmid to express our shRNA molecule, we obtained a >90% reduction in MMP-1 expression that persisted in the cells for several months (8 months to date). The persistent inhibition effected by stably integrating the shRNA vector into cellular DNA may account for the increased effectiveness of our shRNA molecule compared with transient transfection.
The specificity of the MMP-1 shRNA for its target gene agrees with previously published reports on the specificity of RNA interference molecules (23, 32). It is unclear why the cells lines with the scrambled shRNA vector did not grow because the sequence used did not show homology to any human gene. RNA interference molecules can induce IFN responses in a sequence-specific and cell type–specific manner (25, 33). Therefore, the scrambled shRNA may have triggered an IFN response in the MDA-231 cells. Indeed, our recent findings indicate that the same scrambled shRNA sequence induced IFN-responsive genes in melanoma cell lines (data not shown). siRNAs may cause changes in untargeted proteins in mammalian cells; however, the mechanism is unknown (34, 35). One possibility is that transiently transfected siRNAs engage toll-like receptors on the target cells (36). shRNA stably expressed from an integrated vector circumvents this potential difficulty. Additionally, to minimize the possibility of off-target effects, we selected one shRNA sequence that showed efficacy in reducing the levels of both an ectopically and endogenously expressed gene (Fig. 1; Table 1). Furthermore, we measured closely related genes to verify the specificity of the shRNA and found no consistent differences in expression. The effects of blocking MMP-1 expression on cancer cell invasion and tumor growth described here are consistent with published findings on blocking MMP expression in vitro and in vivo (17, 18, 37).
A recent report describes the ability of RNA interference targeted to two other proteinases, MMP-9 and cathepsin B, to suppress tumor cell invasion, tumor growth, and angiogenesis in a glioblastoma model (37). Similarly, RNA interference blocked MT1-MMP expression in fibrosarcoma and gastric cancer cells, resulting in decreased in vitro invasion (38). Our results show that MMP-1 is another protease that can affect the growth of tumors formed by cancer cells introduced into an animal model. Furthermore, another recent article hints at the importance of MMP-1 expression in the development of invasive breast cancer and suggests that MMP-1 expression can be used as a predictive marker for the development of this disease in patients with atypical ductal hyperplasia (11). Our findings confirm that siRNA/shRNA-mediated suppression of proteinases represents a powerful new approach to understanding the role of these enzymes in cancer and provides a potential avenue for new antiproteinase cancer therapies.
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Acknowledgments |
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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 the biostatistics center at the Norris Cotton Cancer Center and the pathology department at Dartmouth Hitchcock Medical Center for support services.
Received 7/12/05. Revised 9/14/05. Accepted 9/23/05.
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 Z. Hassani, J.-C. Francois, G. Alfama, G. M. Dubois, M. Paris, C. Giovannangeli, and B. A. Demeneix A hybrid CMV-H1 construct improves efficiency of PEI-delivered shRNA in the mouse brain Nucleic Acids Res., May 14, 2007; 35(9): e65 - e65. [Abstract] [Full Text] [PDF]
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Y. Mitsui, S. Mochizuki, T. Kodama, M. Shimoda, T. Ohtsuka, T. Shiomi, M. Chijiiwa, T. Ikeda, M. Kitajima, and Y. Okada ADAM28 Is Overexpressed in Human Breast Carcinomas: Implications for Carcinoma Cell Proliferation through Cleavage of Insulin-like Growth Factor Binding Protein-3. Cancer Res., October 15, 2006; 66(20): 9913 - 9920. [Abstract] [Full Text] [PDF]
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) formed larger tumors (1,217 ± 334 mm3, average ± SE; n = 13) than MDA-231 cells containing the MMP-1 shRNA (+; 272 + 117 mm3, average ± SE; n = 10; P = 0.027, Student's t test). Superficial tumor measurements were used to calculate the tumor volumes [(4/3)