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[Cancer Research 61, 2365-2370, March 15, 2001]
© 2001 American Association for Cancer Research

Advances in Brief

Stimulation of Mammary Tumorigenesis by Systemic Tissue Inhibitor of Matrix Metalloproteinase 4 Gene Delivery1

Yangfu Jiang, Mingsheng Wang, Mahmut Y. Çeliker, Yiliang E. Liu, Qing Xiang Amy Sang, Itzhak D. Goldberg and Y. Eric Shi2

Department of Radiation Oncology, Long Island Jewish Medical Center, The Long Island Campus for Albert Einstein College of Medicine, New Hyde Park, New York 11040 [Y. J., M. W., M. Y. C., Y. E. L., I. D. G., Y. E. S.], and Department of Chemistry and Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida 32306 [Q-X. A. S.]


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
Tissue inhibitors of matrix metalloproteinase (TIMPs) are multifunctional proteins with both matrix metalloproteinase (MMP) inhibitory effects and growth-regulatory activity. TIMPs inhibit MMP activity, suggesting a use for cancer gene therapy. However, here we report that systemic administration of human TIMP-4 by electroporation-mediated i.m. injection of naked TIMP-4 DNA stimulates tumorigenesis of human breast cancer cells in nude mice. Consistent with tumor stimulation, TIMP-4 up-regulates Bcl-2 and Bcl-XL protein. TIMP-4 also inhibits apoptosis in human breast cancer cells in vitro and mammary tumors in vivo. A synthetic MMP inhibitor BB-94 did not have such antiapoptotic effect. Analysis of TIMP-4 expression in human mammary specimens indicates that TIMP-4 protein is increased in mammary carcinoma cells compared with normal mammary epithelial cells. These data indicate an antiapoptotic activity in breast cancer cells and a tumor-stimulating effect of TIMP-4 when administrated systemically.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
Overproduction and unrestrained activity of MMPs3 have been linked to malignant conversion of tumor cells. Augmented MMP activity is associated with the metastatic phenotype of carcinomas (1, 2, 3, 4) . Decreased production of TIMP could also result in greater MMP activity, leading to increased invasive potential of cancer cells (5 , 6) . In fact, tumor invasion and metastasis can be inhibited by up-regulation of TIMP expression in tumor cells (7, 8, 9, 10, 11, 12, 13) . Alternatively, down-regulations of TIMP-1 and TIMP-2 have been reported to contribute significantly to the tumorigenic and invasive potentials (14, 15, 16) . In addition to inhibiting tumor cell invasion and metastasis, overexpression of TIMPs in tumor cells also inhibits primary tumor growth (8, 9, 10, 11, 12, 13) .

TIMPs have been shown to be multifunctional factors. In addition to their anti-MMP activity, TIMPs also regulate cell growth. The stimulatory effect on cell growth was initially recognized when TIMP-1 and TIMP-2 were identified having erythroid-potentiating activities (17 , 18) . It is now clear that TIMP-1 and TIMP-2 are also mitogenic for nonerythroid cells, including normal keratinocytes, fibroblasts, lung adenocarcinoma cells, and melanoma cells (19, 20, 21) . In addition, recent evidence indicates that the TIMP family is involved in apoptosis. Whereas it has been demonstrated that TIMP-1 and TIMP-2 have antiapoptotic effects in mammary epithelial cells and lymphocytes (22, 23, 24) , TIMP-3 induces apoptosis (25) . The role of TIMP-4 on apoptosis has not been identified.

Although the inhibitory effect of TIMP on tumor growth and metastasis was achieved by local expression of the TIMP genes in tumor cells, most MMPs and TIMPs are not expressed in genetically altered cancer cells but rather synthesized and secreted by adjacent stromal fibroblasts (26, 27, 28) . Potential therapeutic applications of TIMPs for cancer treatment is limited by the lack of a method for systemic administration of TIMPs, which can reach distant tumor locations and by the lack of systemic assessment of the net effect between their tumor-suppressing MMP inhibitory effect and the cell survival pro-tumor activity. An imbalance between MMPs and TIMPs in favor of enzymatic inhibition might be important in inhibiting tumor angiogenesis and malignant progression. It was reported that i.p. injection of recombinant TIMP-1 inhibited the invasion of murine melanoma cells and reduced lung colonization, whereas recombinant TIMP-1 had no significant effect on the tumor growth (29 , 30) . Our previous study indicated that TIMP-4 inhibited tumor growth and metastasis when it was transfected into human breast cancer cells (13) . In the current study, we investigated the effect of systemic TIMP-4 gene delivery on mammary tumorigenesis. Unexpectedly, we demonstrated for the first time that systemic delivery of TIMP-4 by i.m. administration of naked TIMP-4 DNA significantly stimulated mammary tumorigenesis in vivo.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Preparation of Recombinant Human TIMP-4 Protein and Anti-TIMP-4 Antibody.
rhTIMP-4 protein and anti-TIMP-4 antibody were prepared as described previously (31) . Briefly, rhTIMP-4 was expressed in baculovirus-infected insect cells and purified to homogeneity by a combination of cation exchange, hydrophobic, and size-exclusion chromatography. The purified protein migrated as a single Mr 23,000 band in SDS-PAGE and in Western blot (31) . A peptide sequence corresponding to amino acids 61–74 of human TIMP-4 was synthesized to immunize New Zeland rabbits. Antibodies were separated from serum by ion exchange and purified with TIMP-4 peptide affinity column (43) .

Immunohistochemistry and TUNEL Assay.
Paraffin-embedded tissues were used to examine TIMP-4 protein expression. Briefly, after deparaffinization and rehydration, endogenous peroxidase activity was inactivated by incubating slides in 0.3% H2O2 in methanol for 15 min and digested with 0.1% trypsin for 20 min. Nonspecific antibody binding was blocked with 10% BSA in PBS for 30 min at room temperature prior to incubation with the affinity-purified specific anti-TIMP-4 antibody (0.2 µg/ml) for 1 h. To assess nonspecific background staining in negative control slides, anti-TIMP-4 antibody (0.2 µg/ml) was preincubated with recombinant TIMP-4 protein (1 µg/ml). Sections were incubated with biotin-conjugated secondary antirabbit antibodies (Dako Corp., Carpinteria, Pleasanton, CA). Slides were incubated with LSAB regents (Dako Corp.), following the manufacturer’s instructions for the colorimetric detection of TIMP-4 staining. The two-step PCNA detection (Antibody Diagnostic Inc., Pleasanton, CA) and in situ apoptotic TUNEL (Roche Diagnostics, Germany) assay were performed according to the manufacturers’ instructions. The proliferative index (PCNA) and the apoptotic index (TUNEL) were evaluated by the percentage of cells scored under a light microscope at 200-fold magnification.

In Situ Hybridization.
Deparaffinized and acid-treated sections (5-µm thick) were treated with proteinase K, prehybridized, and hybridized overnight with digoxigenin-labeled antisense transcripts from a TIMP-4 cDNA insert. The TIMP-4 antisense probe is a 550-bp fragment from nucleic acids 130 to 683. The full-length TIMP-4 cDNA was cut by KPNI and SmaI; the 550-bp inset was subcloned into Bluescript II plasmid, and the resulting plasmid was named as Bluescript TIMP4-B. The 550-bp antisense probe was generated by SamI digestion of Bluescript TIMP4-B plasmid, followed by T7 polymerase. After hybridization, RNase treatment and three stringent washes were carried out, sections were incubated with mouse antidigoxigenin antibodies (Boehringer), followed by incubation with biotin-conjugated secondary rabbit antimouse antibodies (Dako). The colorimetric detection was performed by a standard indirect streptavidin-biotin immunoreaction method using Universal LSAB kit (Dako) according to the manufacturer’s instructions.

i.m. Injection of Plasmid and Electroporation.
Fifty µl (150 µg) of TIMP-4 plasmid DNA (TIMP-4 cDNA in pCI-neo mammalian expression vector; Promega Corp., Madison, WI) or empty vector (the parental plasmid) as a control was injected into the bilateral tibialis anterior muscles of 6-week-old of female nude mice using a disposable insulin syringe with a 25-gauge needle. For electroporation, a pair of electrode needles was inserted into the muscle with a 5-mm gap within the DNA injection sites, and electric pulses were delivered using an electric pulse generator Electro Square Porator ECM 830 (Genetronics, Inc., San Diego, CA). Three pulses of 150 V each were delivered to the injection site at a rate of one pulse/second, each pulse lasting for 50 ms. Then, three pulses of the opposite polarity were applied.

Tumor Growth in Athymic Nude Mice.
Nude mouse tumorigenic assay was performed as we described previously (13) . Briefly, parental MDA-MB-435 cells were grown to 80–90% of confluence in 150-cm2 dishes and were harvested by incubation with 5 mM EDTA in PBS. EDTA was neutralized with medium containing serum. The cells were washed twice with serum-free medium, counted, and resuspended in serum-free Iscove’s modified medium at a concentration of 5 x 106 cells/ml. Approximately 0.5 x 106 cells (0.1 ml) were injected into 5–6-week-old female athymic nude mice (Frederick Cancer Research and Development Center, Frederick, MD). Each animal received two injections, one on each side, in the mammary fat pads between the first and second nipples. The animals were ear tagged. Primary tumor growth was assessed by measuring the volume of each tumor at weekly intervals. Tumor size was determined at intervals by three-dimensional measurements (mm) using a caliper.

Cell Death Assay.
Human breast cancer MD-MBA-435 cells were maintained in monolayer culture in Iscove’s modified medium containing 5% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. The effect of recombinant human TIMP-4 protein on cell death in response to Adriamycin was assessed using the 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt assay (Roche Molecular Biochemicals). Briefly, cells were cultured in 96-well plates at 3000 cells/well. Cells were incubated with or without 80 nM recombinant human TIMP-4 protein (29) . After 24 h, cells were treated with 0.5 µM of Adriamycin. Forty-eight h later, cells were incubated at 37°C with 100 µl of 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt labeling mixture for 4 h; absorbance was measured with ELISA plate reader at 490 nm.

Immunoblot Analysis.
Equal amounts of protein were subjected to 12% SDS-PAGE and transferred to Hybond-C membrane (Amersham, Arlington Heights, IL). Antibodies against bcl-2, bcl-XL, bax, Fas, and Fas ligand were purchased from Oncogene Research Products. Western blot analyses were carried out using the appropriate antibody as noted in the figure legends, and protein bands were visualized with horseradish peroxidase-conjugated anti-IgG antibody and enhanced chemiluminescence according to the manufacturer’s recommendations (Amersham).


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
Expression of TIMP-4 in Human Breast Samples.
To assess the biological relevance of TIMP-4 on mammary tumors, we first analyzed TIMP-4 protein expression in human mammary specimens including both normal glands and malignant carcinomas. A total of 40 human breast cancer specimens were analyzed. A strong TIMP-4 staining (Fig. 1A)Citation was observed in 9 of 10 breast carcinomas. We also selected 15 human breast sections that contain both infiltrated breast cancer cells and residual normal breast epithelial cells to assess TIMP-4 protein expression in normal versus cancer cells in the same tissue section. Whereas minimal TIMP-4 staining was observed in residual normal lobular and ductal breast epithelial cells, a significant TIMP-4 staining was observed in the infiltrating malignant breast cancer cells (Fig. 1, C and D)Citation .



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  		Fig. 1. Expression of TIMP-4 protein and mRNA in human mammary gland. Cells stained brown indicate TIMP-4 expression. All sections were counterstained lightly with hematoxylin for viewing negatively stained epithelial and stromal cells. Sections in A–E are immunohistochemical staining, and section F is in situ hybridization. A, strong TIMP-4 protein staining in a mammary ductal carcinoma in situ. B, as a negative control, no signal of TIMP-4 protein was detected in the same sample when the antibody was preincubated with TIMP-4 protein. C, strong positive TIMP-4 staining in infiltrated malignant breast cancer cells (arrows) in comparison with much weaker TIMP-4 staining in surrounding residual normal mammary lobule epithelial cells. D, relatively high levels of TIMP-4 staining in malignant breast cancer cells (arrowhead) compared with surrounding residual normal mammary ductal epithelial cells (arrow). E, strong positive staining of TIMP-4 protein in malignant epithelial cells; in contrast, nondetectable or much less TIMP-4 staining was observed in stromal cells surrounding ductal carcinoma in situ. F, in the same slide as E, TIMP-4 mRNA was detected in stromal cells surrounding the ductal carcinoma in situ but not in malignant epithelial cells. The section was also hybridized with the sense probe, and no detectable background staining was observed at the same conditions for the antisense probe.

 
To localize the cellular source of the TIMP-4 expression in mammary gland, we performed in situ hybridization analysis. Fourteen of 15 in situ hybridization analyses showed that TIMP-4 mRNA was expressed in stromal cells but not in epithelial cells. Although TIMP-4 mRNA was detected in the stromal fibroblasts surrounding the carcinoma (Fig. 1F)Citation , in the same sample, the TIMP-4 protein was primarily localized on the malignant breast epithelial cells (Fig. 1E)Citation . The expression of TIMP-4 mRNA in stroma is consistent with the inability to detect the TIMP-4 mRNA in most breast cancer cell lines by Northern blot (32) .

Systemic Tumor Stimulating Effect of i.m. Administered TIMP-4 Gene.
Because TIMP-4 is synthesized in mammary stromal cells, secreted, and accumulated on cancer cells, local expression of TIMP-4 in cancer cells by gene transfection may not mimic the in vivo stromal-epithelial interactions. To examine the functions of increased TIMP-4 protein expression on mammary tumorigenesis and to study the effect of systemic administration of TIMP-4 on tumor growth, we undertook a gene therapy approach through i.m. administration of TIMP-4 expression plasmid. On the basis of published data on i.m. delivery of DNA plasmid (33) , we selected two doses (50 and 150 µg) of TIMP-4 expression plasmid for i.m. injection, followed by electroporation. Sera were collected prior to the injection and at 4, 8, 13, and 21 days after the injection. TIMP-4 protein levels were determined by Western blot analysis. For the dose of 50 µg of plasmid, serum levels of TIMP-4 were slightly increased over control at day 4 (data not shown). However, at the dose of 150 µg of plasmid, serum levels of TIMP-4 were significantly increased. As seen in Fig. 2ACitation , although there was a minimal TIMP-4 protein in the plasma prior to the injection, when 150 µg of plasmid was administrated, the amount of TIMP-4 was increased 3-fold at 4 days after the injection. A significant amount TIMP-4 protein was observed at 8 and 13 days with a 19- and 29-fold increase over control, respectively.



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  		Fig. 2. Effects of systemic administration of the TIMP-4 gene on mammary tumor growth and incidence. A, serum-derived TIMP-4 protein following i.m. injection of the plasmid. Ten-µl aliquots of sera collected before and at 4, 8, 13, and 21 days after a single i.m. injection of 150 µg of TIMP-4 expression plasmid were subjected to Western blot analysis with anti-TIMP-4 antibody. The figure represents a time course of serum TIMP-4 levels in one mouse. Similar TIMP-4 levels were observed in all five mice tested for each time point. B, time course of TIMP-4-stimulated mammary tumorigenesis. The data were plotted based on experiment 1 in Table 1Citation . The number of cells injected was 5 x 105. C and D, accumulation of TIMP-4 on tumor xenograft sections. Tumor sections collected from nine control and nine TIMP-4-injected mice were subject to immunohistochemical analysis with TIMP-4 antibody. Bars, SD. Whereas there was a minimal TIMP-4 protein on tumor xenograft from control mice (C), tumor xenograft from TIMP-4 plasmid-injected mice showed strong TIMP-4 immunoreactivity (D). E, tumor incidences. MDA-MB-435 cells were injected at 2 x 105 cells/injection. TIMP-4 administration was described in Table 1Citation . Tumor incidences were analyzed in control and TIMP-4 injected mice. Control group, nine control mice were injected with 2 x 105 cells/injection. TIMP-4 group, eight TIMP-4-treated mice were injected with 2 x 105 cells/injection. For both groups, each mouse received two injections.

 
We injected either TIMP-4 plasmid or control plasmid into i.m. sites of nude mice 5 days before inoculation of MDA-MB-435 breast cancer cells and every 7 days thereafter. Three independent experiments were done to confirm reproducibility, and the data from these experiments are summarized in Table 1Citation . When 5 x 105 of cells were injected, tumors developed after a lag phase of 5–7 days. There was no significant difference in tumor incidence among control and TIMP-4 injected mice. However, the tumor volume of TIMP-4-injected mice was significantly larger than that of empty vector injected mice. Fig. 2BCitation shows a representative of experiment 1. The size of tumors in TIMP-4-injected mice were 283, 327, 253, and 171% of that in control mice at days 7, 10, 15, and 21, respectively. Consistent with this data, a strong immunohistochemical staining of TIMP-4 was observed in breast cancer xenografts from TIMP-4-injected mice (Fig. 2D)Citation , whereas very weak TIMP-4 signal was detectable in xenografts from control mice (Fig. 2C)Citation .


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  		Table 1 Effects of systemic administration of TIMP-4 plasmid on mammary tumorigenesis

 
In a separate experiment, we tested whether systemic administration of the TIMP-4 gene could increase the tumor incidence when the number of injected tumor cells was reduced. When the number of injected MDA-MB-435 cells was reduced from 5 x 105 to 2 x 105, the tumor incidences was greatly reduced from 80% (13 of 16) to 5% (1 of 18) at week 1 and from 90% (15 of 16) to 40% (7 of 18) at week 2, respectively. However, when TIMP-4 was preadministered to the mice inoculated with 2 x 105 tumor cells, a significant increase in tumor incidence and a decrease in tumor latency were observed. As shown in Fig. 2ECitation , the tumor incidences were increased from 5% in control mice to 44% (7 of 16) in TIMP-4-treated mice at week 1 and from 40% in control mice to 88% (14 of 16) in treated mice at week 2, respectively (p< 0.01).

TIMP-4 Enhanced Survival of Breast Cancer Cells in Vitro and Breast Tumor Xenograft in Vivo.
To study whether TIMP-4-mediated stimulation of tumorigenesis is mediated by its antiapoptotic effect or growth stimulation, we selected MDA-MB-435 and MDA-MB-453 cells for testing the direct effect of exogenously added recombinant human TIMP-4 on cell survival in response to Adriamycin treatment. Dose dependence experiments (0.1–1 µM) showed that Adriamycin induced 8% of cell death at the dose of 0.1 µM and >95% of cell death at the dose of 1 µM (data not shown). As shown in Fig. 3Citation , in the absence of TIMP-4, 19 and 25% of MDA-MB-435 and MDA-MB-453 cells remained viable after 48 h treatment with 0.5 µM of Adriamycin, respectively. However, in the presence of TIMP-4 (80 nM), cell survival increased to 61% for MDA-MB-435 cells and 65% for MDA-MB-453 cells, respectively. Synthetic MMP inhibitor BB-94 (5 µM) had no significant effect on Adriamycin-induced apoptosis (data not shown). To determine whether cell proliferation may be affected by TIMP-4, both MDA-MB- 435 and MDA-MB-453 cells were treated with 80 nM TIMP-4 for 2 days, followed by [3H]thymidine labeling for 6 h. The incorporation of [3H]thymidine was measured by standard scintillation counting. TIMP-4 had no significant effect on the proliferation of MDA-MB-435 and MDA-MB-453 cells.



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  		Fig. 3. Effects of recombinant TIMP-4 protein on Adriamycin-induced apoptosis. MDA-MB-453 (A) and MDA-MB-435 (B) cells were grown in 96-well plates. Each treatment group consisted of 10 wells. Cells were pretreated with or without 80 nM rhTIMP-4 for 24 h and then treated with 0.5 µM Adriamycin for 12 h. Cells were harvested 2 days after Adriamycin treatment. Survivals of untreated cells (control) were taken as 100%, and all other survival ratios were normalized to the control. Data represent the means of 10 cultures; bars, SD.

 
The effect of systemic administration of the TIMP-4 gene on apoptosis was investigated in a MDA-MB-435 human breast carcinoma xenograft model. Because the most possible antiapoptotic effects of TIMP-4 in vivo are present during the early phase of tumor onset and growth, mice bearing established tumors from both control and TIMP-4-injected mice were sacrificed at day 7 after tumor cell injection. PCI-neo-TIMP-4 plasmid or empty vector as control was injected 5 days before tumor cell inoculation and 2 days after tumor cell injection. As shown in Fig. 4ACitation , the tumor sizes increased significantly in TIMP-4-treated mice versus control mice. Immunohistochemical analyses of tumors showed that there was no significant difference in proliferative index between control and TIMP-4-treated mice (Fig. 4B)Citation . TUNEL assay revealed that apoptotic cells in tumor decreased 3-fold in TIMP-4-treated mice (Fig. 4C)Citation . Thus, TIMP-4 protects breast cancer cells from apoptosis both in vitro and in vivo.



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  		Fig. 4. Effects of systemic administration of TIMP-4 on apoptosis and proliferation of breast cancer xenograft. A, tumor size from control and TIMP-4-treated mice at 7 days. i.m. injection of control, and TIMP-4 plasmid and tumor cell injections were described in Table 1Citation . The tumor sizes were much bigger in the TIMP-4-treated mice than in control mice. B, histological sections of tumors from TIMP-4-treated or control human breast carcinomas were analyzed for proliferation (PCNA). There was no significant difference in the proliferative index of tumor cells in treated versus untreated tumors. Bars, SD. C, histological sections of tumors were also analyzed for apoptosis (TUNEL assay). The apoptotic index of the tumor cells decreased in the TIMP-4-treated mice (P < 0.01).

 
Effect of TIMP-4 on Apoptotic Proteins.
To further investigate the role of TIMP-4 in the regulation of apoptosis in breast cancer cells, we investigated Bcl-2, Bcl-XL, Bax, Fas, and Fas ligand protein expression in control TIMP-4-435 versus rhTIMP-4-treated cells by Western blot. Whereas Bax, Fas, and Fas ligand proteins were not detectable in these cells, both Bcl-2 and Bcl-XL protein expression was significantly increased in 100 nM rhTIMP-4-treated cells (Fig. 5)Citation . In the absence of Adriamycin, rhTIMP-4 stimulated Bcl-XL expression by 3.5-fold and Bcl-2 expression by 1.5-fold. In the presence of Adriamycin, >4-fold stimulation in rhTIMP-4-treated cells versus control cells was observed for Bcl-XL; Bcl-2 expression in rhTIMP-4-treated cells was 3-fold more than that in untreated cells. Bcl-2 and Bcl-XL protein expression were also increased in TIMP-4-transfected MDA-MB-435 cells (data not shown).



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  		Fig. 5. Analyses of the bcl-2 and bcl-XL protein expression. Both control MDA-MB-435 cells and rhTIMP-4-treated cells were treated with 1 µM Adriamycin for 6 h. Total cellular proteins were isolated before and after Adriamycin treatment and normalized, and 40 µg of total protein were subjected to Western blots. Bcl-2 protein was detected as a Mr 28,000 band by mouse anti-bcl-2 monoclonal antibody. Bcl- XL protein was detected as a Mr 30,000 band by rabbit polyclonal antibody. Expression of ß-actin was used as a control for protein loading.

 

   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
The blocking of MMP activity by TIMPs might be important in inhibiting tumorigenesis and subsequent malignant progression. However, some TIMP members also have a growth-stimulating and antiapoptotic effect, which may promote tumorigenesis. These conflicting facts prompted us to investigate the role of systemic expression of TIMP-4 on mammary tumorigenesis. The significance of the current study is 4-fold: (a) more TIMP-4 protein accumulated in mammary cancer cells versus normal cells in clinical specimens; (b) TIMP-4 stimulates Bcl-2 and Bcl-XL protein expression and protects breast tumor cells in vitro and mammary tumor xenografts in vivo from apoptosis; (c) electroporation-mediated i.m. delivery of naked TIMP DNA can provide a sustained high level of TIMP-4 protein in serum; and (d) results in a significant stimulation of the growth of breast cancer cells.

The observation of increased TIMP-4 protein in malignant cancer cells versus normal cells in human mammary specimens prompted us to investigate the role of TIMP-4 on mammary tumorigenesis. In contrast to the previous approach of local expression of TIMP-4 by transfection of cancer cells with the gene (13) , we undertook a systemic gene therapy approach of i.m. injection of naked TIMP-4 DNA. This approach is based on two rationales: (a) local expression of TIMP in cancer cells may not mimic stromal-epithelial interaction in vivo, because TIMP-4 mRNA is expressed in stromal cells but not in malignant epithelial cells; and (b) delivery of heterologous genes into skeletal muscles has been shown to be able to provide sustained production of proteins (33) . Our data show that TIMP-4 protein can be systemically produced in muscle cells by a single injection of naked DNA, followed by electroporation. The production of TIMP-4 and its release into the circulation were sustained for at least 2 weeks. Furthermore, a significant amount of TIMP-4 protein was accumulated on tumor xenografts. Unexpectedly, systemic administration of TIMP-4 resulted in a significant stimulation of primary mammary tumorigenesis.

It is intriguing that systemic delivery of TIMP-4 via i.m. injection of DNA results in an stimulatory effect on mammary tumorigenesis, which is in contrast to the previously demonstrated antitumor and antimetastatic effect of TIMPs (7, 8, 9, 10, 11, 12, 13) . The TIMP-4-mediated, tumor-stimulating effect can be viewed from two perspectives; TIMP-4 has both anti-MMP activity, which favors the tumor-suppressing effect, and growth-stimulating and antiapoptotic activity, which may favor the pro-tumor effect. It should be noted that the previously established dominant antitumor effect of TIMP is primarily based on local expression of the TIMP gene in cancer cells but not by systemic evaluation of TIMP function. The difference of local versus systemic effects of TIMP-4 on mammary tumorigenesis may be attributable to a different dominancy between anti-MMP and antiapoptotic activities of TIMP-4. The dominancy between anti-MMP and antiapoptotic effect on tumor growth may depend on the amount of bioavailable TIMP-4 protein in the tumor microenvironment. In this regard, the higher level of TIMP-4 may have a tumor-suppressing effect because of its dominant anti-MMP function, whereas a lower level of TIMP-4 may favor tumor growth because of its antiapoptotic activity. When a gene transfection approach is taken, it usually results in a selection of the most highly TIMP-expressing clones. The overexpression of TIMP-4 locally in every breast cancer cell would generate abundant inhibitory proteins and create a microenvironment in the tumor-stromal interface where the pro-tumor MMP activity is blocked. In contrast, in the i.m. gene therapy approach, TIMP-4 protein has to cross a vast amount of extracellular matrix proteins and circulation before reaching the target tumor cells. Therefore, the amount of TIMP-4 bioavailable to the tumor cells may be much lower than that from locally expressed TIMP-4 in transfected cells. In addition, when the TIMP-4 gene was administrated i.m., the anti-MMP function of circulating TIMP-4 may be neutralized in part by circulating MMPs, and therefore, the balance was shifted in favor of its antiapoptotic activity when it reached to the tumor site.

Regulation of apoptosis by TIMPs has been reported. Whereas TIMP-3 induces apoptosis, TIMP-1 and TIMP-2 have an antiapoptotic effect. As for the mammary gland, TIMP-1 inhibits apoptosis of human breast epithelial cells in vitro (23) and rescues mammary epithelial cell from apoptosis in transgenic mice (24) . Consistent with this antiapoptotic effect of TIMP-1 on the mammary gland, we reported here a similar apoptosis-inhibiting effect of TIMP-4 on human breast cancer cells as well as on mammary tumor xenografts. On the apoptotic pathways, TIMP-1, which is up-regulated by Bcl-2, seems to function downstream of Bcl-2 (23) . TIMP-4 stimulates Bcl-2 and Bcl-XL expression, which may contribute to the inhibition of apoptosis. By using a TIMP-4 affinity column, we have identified a putative TIMP-4 binding protein on the cell surface (data not shown). We are currently in the process of cloning this putative TIMP-4 binding protein and to study the mechanism of TIMP-4-mediated regulation of apoptosis.

MMPs and TIMPs also play a complex role in regulating angiogenesis. On one hand, MMP may trigger an angiogenic switch. On the other hand, it can convert matrix proteins into angiogenic inhibitors. Systemic inhibition of tumor growth by administration of the endostatin gene has been demonstrated previously (33) . Because endostatin is converted from type 18 collagen, which may be mediated by MMPs (34) , we wondered whether TIMP-4-induced tumor stimulation is mediated by reduction of endostatin levels as result of inhibition of MMPs. Levels of mouse endostatin in sera isolated from tumor-bearing control mice and TIMP-4-injected mice were determined using a commercially available ELISA kit. This assay showed that TIMP-4-induced tumor stimulation was not associated with a decrease in endostatin levels but rather a slight increase in endostatin level (data not shown). The increased endostatin expression in TIMP-4-injected mice may be attributable to the larger tumor volume of the TIMP-4-injected mice compared with that of control mice.

Although TIMPs have been shown to inhibit tumor invasion and metastasis, the data presented here demonstrated a higher level of TIMP-4 expression in breast carcinoma cells than in normal breast epithelial cells. The increased TIMP expression was demonstrated in a variety of different tumors. TIMP-1 expression is often associated with poor prognosis in many human solid tumors, including metastatic breast cancer (35, 36, 37, 38) , colorectal cancer (39) , gastric carcinoma (40) , lymphoma (41) , and non-small cell lung carcinoma (42) . It has been demonstrated that the outcome of patients with breast cancer is more closely related to the presence of TIMP-2 in the peritumoral stroma than to the corresponding MMPs (35, 36, 37) . It is not easy to understand why the elevated content of TIMP expression is associated with malignant cancer cells. One explanation is that the increased expression of TIMPs may be reciprocally related to the increased expression of MMPs during the tumor-mediated degradation of extracellular matrix. Therefore, elevated level of TIMP in the invasive breast carcinomas may represent one of the subsequent acute host responses to the remodeling stimuli in an attempt to regulate the local tissue degradation. Alternatively, the high level of TIMP expression in breast cancer may favor the proposed MMP-independent growth regulatory and apoptotic regulatory functions. Our evaluation of biological effect of TIMP-4 on stimulation of mammary tumorigenesis provides the rationale for unexpected results of these clinical studies. Our data indicated that the antiapoptotic effect of TIMP-4 plays a key role in TIMP-4 mediated tumor stimulation. Therefore, systemic administration of full-length TIMP protein or DNA clinically to block MMP may not exert the tumor-suppressing effect expected. In this regard, the potential cancer treatment by using truncated forms of TIMP, which lacks antiapoptotic function, warrants further investigation. These results provide a new conceptual basis for design of strategies that use MMP inhibitors as a probe for proteinase function in cancer gene therapy. Such an approach will be useful for evaluating the functions of other members of TIMP and MMP families.


   FOOTNOTES
 
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 CA68064-01 and United States Department of the Army Breast Cancer Research Program Grant DAMD17-96-1-6261. Back

2 To whom requests for reprints should be addressed, at Department of Radiation Oncology, Long Island Jewish Medical Center, New Hyde Park, NY. Phone: (718) 470-3086; Fax: (718) 962-6675; E-mail: shi{at}lij.edu. Back

3 The abbreviations used are: MMP, matrix metalloproteinase; TIMP, tissue inhibitors of MMP; rhTIMP, recombinant human; TUNEL, terminal deoxynucleotidyltransferase-mediated nick end labeling; PCNA, proliferating cell nuclear antigen. Back

Received 10/30/00. Accepted 1/30/01.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

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