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. Author manuscript; available in PMC: 2010 Dec 1.
Published in final edited form as: Exp Mol Pathol. 2009 Sep 9;87(3):184–188. doi: 10.1016/j.yexmp.2009.09.002

Thrombospondin-1 (TSP-1) Up-regulates Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) Production in Human Tumor Cells: Exploring the Functional Significance in Tumor Cell Invasion

Anitha S John ^, Xioulong Hu *, Vicki L Rothman, George P Tuszynski *
PMCID: PMC2783349  NIHMSID: NIHMS150758  PMID: 19747478

Abstract

Thrombospondin-1 (TSP-1), a matrix-bound adhesive glycoprotein, has been shown to modulate tumor progression. We previously demonstrated that TSP-1 up-regulates matrix metalloproteinases MMP-2 and MMP-9. Our studies suggested that the balance between MMPs and tissue inhibitors of metalloproteinases (TIMPs) is a key determinant in tumor cell invasion. We now report that TSP-1 up-regulates TIMP-1 expression in both human breast and prostate cancer cell lines. The effect of TSP-1 on TIMP-1 expression was examined in human breast adenocarcinoma cell lines (MDA-MB-231) and human prostate cancer cell lines (PC3-NI and PC3-ML) treated with exogenous TSP-1. TIMP-1 expression was also examined in TSP-1 stably transfected breast cancer cell line (MDA-MB-435). Northern and western blot analysis revealed TIMP-1 mRNA and TIMP-1 protein expression increased with increasing concentrations of TSP-1. This effect was inhibited by antibodies against the type I repeat domain of TSP-1 further suggesting that TSP-1 mediates TIMP-1 secretion. Inhibition of TSP-1 induced TIMP-1 levels increased tumor cell invasion. We conclude that TSP-1 is involved in influencing the critical balance between MMPs and their inhibitors, maintaining the controlled degradation of the extracellular matrix needed to support metastasis and our results may provide an explanation for the divergent activities reported for TSP-1 in tumor progression.

Keywords: Thrombospondin-1, TIMP-1, Tumor Cell Metastasis, Tumor Cell Invasion

Introduction

The process of metastasis involves the survival of the primary tumor through a series of steps including: tumor cell invasion, extravasation from the blood or lymphatic circulation, and finally tumor cell colonization and angiogenesis to form the metastatic lesion (Stetler-Stevenson et al., 1993). Once a tumor cell is formed, it relies on the body’s extracellular matrix (ECM) to survive and grow. ECM proteins such as laminin, collagen, and thrombospondin-1 (TSP-1) have been shown to stimulate tumor cells to secrete proteolytic enzymes needed for tumor cell metastasis (Haas et al., 1998; Song et al., 1997; Tuszynski et al., 1987).

TSP-1 is a 450 kDa trimeric glycoprotein involved in a variety of processes including cell adhesion, cell migration, and angiogenesis (Qian and Tuszynski, 1996). Each monomer of TSP-1 is composed of repeating homologous amino acid sequences, with specific receptors responsible for the numerous cellular processes involving TSP-1 (Lawler, 1986; Lawler et al., 1978). The complexity of the TSP-1 molecule and the many receptors makes the study of TSP-1 difficult, often leading to conflicting results especially in the fields of angiogenesis and tumor progression.

One of the mechanisms by which TSP-1 influences tumor cell invasion and metastasis is through the regulation of several proteolytic enzyme families, including the metalloproteinases (MMPs). MMPs are used by tumor cells to invade the basement membrane and its underlying connective tissue, providing a mechanism for tumor cells to enter the small blood vessels and lymphatics (Parsons et al., 1997). Our laboratory was also the first to identify that TSP-1 is capable of stimulating MMP-9 production in bovine aortic endothelial cells and in pancreatic tumor cells, stimulating TSP-1 mediated tumor progression and endothelial tube formation (Qian et al., 1997).

Critical to the function of MMPs is the balance of the enzyme to its physiologic inhibitor, tissue inhibitor of metalloproteinase (TIMPs). The TIMPs are comprised of a family of four members, TIMP-1, TIMP-2, TIMP-3, and TIMP-4. A balance of inhibitor to proteolytic enzyme is needed to achieve tumor cell invasion as unregulated enzyme action has been shown to actually inhibit tumor cell invasion in vivo (Qian et al., 1997).

In this study, we report the finding that TSP-1 stimulates the expression of TIMP-1 in both breast and prostate carcinoma cell lines. We hypothesize that the control of net proteolysis of the ECM by TSP-1 is through both up-regulation of MMP-9 and its inhibitor TIMP-1 leading to a controlled proteolytic system.

Materials and methods

Materials

All reagents, unless specified otherwise, were reagent grade and purchased from Sigma Chemical Co. (St. Louis, MO). Tissue culture supplies were purchased from Fisher Scientific (Malvern, PA). Reagents for sodium dodecyl sulfate-polyacylamide gel electrophoresis (SDS-PAGE) were purchased from Bio-Rad Laboratories (Richmond, CA). Laminin, type IV collagen and fibronectin were purchased from Collaborative Research (Bedford, MA). Rabbit anti-human TIMP-1 and mouse anti-human TIMP-1 were purchased from Triple Point (Forest Grove, OR) and Oncogene Science (Cambridge, MA), respectively. Horseradish peroxidase-conjugated anti-rabbit and anti-mouse IgG were purchased from Boehringer Mannheim (Indianapolis, IN). Goat polyclonal anti-human TSP-1 IgG and rabbit polyclonal CSVTCG antibody were raised in our laboratory. Type I repeat peptides and irrelevant peptides were purchased from Peptidogenic (Livermore, California).

Boyden Chamber Invasion Assay

Breast tumor cell invasion was measured using the modified Boyden chamber. Polycarbonate filters, 8 μm pore size (Millicell, Millipore Corporation, Bedford, MA), were coated with 100 μg Type IV collagen (1 mg/ml 60% EtOH) and dried overnight at 25°C. Blind-well Boyden chambers were filled with 700 μl of serum-free media containing 0.1% BSA in the lower compartment, and the coated filters were mounted in the chamber. Approximately 50,000 cells (tested to be greater that 95% viable) suspended in 300 μl of the same media were placed in the upper chamber of the apparatus and allowed to settle onto the collagen-coated membrane. Neutralizing antibodies as well as peptides were placed in the upper chamber. After an incubation period of 3-6 h at 37°C, the cells on the upper surface of the filter were removed with a cotton swab. The filters were fixed in 3% glutaraldehyde solution and stained with 0.5% crystal violet solution. Invasive cells adhering to the under-surface of the filter were counted using a phase contrast microscope (400 X). The data were expressed as the summation of the number of invasive tumor cells in five representative fields.

Cell Culture and Treatment

The human breast adenocarcinoma cell line MDA-MB-231 was purchased from the American Type Culture Collection (CRL 10317, Rockville, MD). The human prostate cancer cell lines, PC3-NI and PC3-ML, were kindly provided by Dr. Mark Sterns, Drexel School of Medicine, Philadelphia, PA. The TSP-1 transfected breast adenocarcinoma cell line, MDA-MB-435, was kindly provide by Dr. David Roberts, National Cancer Institute, Bethesda, MD. The origin of the MDA-MB-235 cell line has been in question with some studies suggesting that the line was identical to a M14 melanoma line, however recent published data is consistent with both M14 and MDA-MB-235 cell lines being of breast cancer origin (Chambers, 2009). The lines obtained from Dr. Roberts include three lines: a vector control (TH5), a high TSP-1 producer (TH26), and a COOH-terminally truncated TSP-1 producer (TH50). These cells were transfected with the pCMVBamNeo vector. All cells were grown at 37°C and 5% CO2 in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 50 U/ml of penicillin, 50 μg/ml of streptomycin, and 50 μg/ml of gentamicin sulfate (Sigma Chemical Co). The TSP-1 transfected cells were cultured with media supplemented with 50 μg/ml G418 antibiotic to maintain the transformed phenotype. Cells were cultured in 6-well plates for TIMP-1 analysis or T75 flasks for RNA isolation. Cells were grown to 85% confluence and were washed and incubated in serum-free medium containing 0.1% BSA. Different concentrations of TSP-1 (20-60 μg/ml) and/or 10 μg/ml of antibody IgG, conrol IgG or peptides were added. After 48-72 hours of culture, the conditioned medium was collected, clarified by centrifugation, and assayed by enzyme-linked immunoadsorbant assay (ELISA) and Western blot analysis for TIMP-1. The amount of conditioned media analyzed was corrected for the total number of cells in each well so that TIMP-1 secretion was compared from the same amount of cells from each treatment group. Cell viability after treatments was assessed using trypan blue exclusion.

ELISA Analysis

Conditioned media from cells were analyzed for TIMP-1 levels using commercial TIMP-1 ELISA kits (Oncogene Science, Cambridge MA). Statistical Analysis was performed using GraphPad Prizm software, San Diego CA.

Northern Blot Analysis

Total RNA was isolated from tumor cells by Rneasy Total RNA Kits (QIAGEN Inc. Chatsworth, CA) following the manufacturer’s direction. 15 μg of total RNA was electrophoresed on 1% agarose/formaldehyde gels and blotted onto nitrocellulose paper. The paper was hybridized with the appropriate 32P-labeled cDNA probes and exposed for autoradiography at −80° C with an intensifying screen. After recording the results, the same paper was re-probed with a beta-actin cDNA probe and exposed at −80° C. The cDNAs were radiolabeled with 32P by the random primer labeling method using the Stratagene labeling kit (Stratagene, La Jolla, CA).

Thrombospondin-1 Purification

TSP-1 was purified from Ca+2 ionophore A23187-activated platelets in our laboratory as previously described (Tuszynski et al., 1985). Purity was assessed by SDS-PAGE using Coomassie blue or silver staining. All TSP-1 used was further purified to remove all bound TGF-β1 according to the procedure of Murphy-Ullrich et al (Murphy-Ullrich et al., 1992). TGF-β1 levels was monitored by human TGF-β1 ELISA kits (Quantikine, R&D Systems, Minneapolis, MN).

Western Blotting

Concentrated conditioned media was fractionated on 8-12% SDS-PAGE and then transferred to a nitrocellulose membrane using a Pharmacia Phast gel electrophoresis system. Nonspecific sites of the membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBS-T) for 1h. The immunoblots were incubated with primary antibodies diluted in TBS-T for 1 h at a concentration of 1 μg/ml. After washing, the immunoblots were incubated in TBS-T with horseradish peroxidase-conjugated secondary antibodies for 1 h at a concentration of 0.1 μg/ml. The immunoreactivity was detected using enhanced chemoluminesence (ECL) system (Amersham, Arlington Heights, IL).

Results

TSP-1 treatment stimulates TIMP-1 production in a dose- dependent manner

MDA-MB-231, a ductal breast adenocarcinoma cell line, and PC3-NI and PC3-ML, a non-invasive and a highly metastatic prostate cancer cell line, respectively, were treated with exogenous TSP-1 at various concentrations. After a 48 hour incubation time, conditioned media from these cell lines were examined by Western blot analysis. TSP-1 treatment resulted in a dose-dependent increase in TIMP-1 secretion by both the MDA-MB-231 and PC3 cell lines (Fig. 1). With the PC3-ML cell line, however, TIMP-1 production increased with a TSP-1 concentration of 20 μg/ml but then reached a plateau at higher concentrations. TIMP-2 production was not affected by TSP-1 treatment in any of the three cell lines (data not shown).

Fig. 1.

Fig. 1

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TSP-1 up-regulates TIMP-1 production in human tumor cells in a dose-dependant manner. MDA-MB-231 breast cancer cells, PC3-NI, non-invasive human prostate cancer cells and PC3-ML, metastatic human prostate cancer cells were plated in 6 well plates and were allowed to grow to 85 % confluency. Cells were then were treated with TSP-1 at the indicated concentrations in serum-free media containing 0.1% BSA . Cells treated with 100ng/ml of PMA were used as a positive control and those treated with bis-Tris-Phosphate buffer (BTP) were the vehicle buffer control for TSP-1 treatment. Conditioned media were harvested after 72 hours and analyzed by western blot or ELISA for TIMP-1 levels. The amount of conditioned media analyzed was corrected for the total number of cells in each well so that TIMP-1 secretion was compared from the same amount of cells from each treatment group. Experiments were repeated three times and the results of a representative experiment are shown in the figure.

Conditioned media from the cell lines treated as above were also analyzed by a commercial TIMP-1 ELISA. Analysis revealed a dose-dependent increase of TIMP-1 production in response to TSP-1 treatment (Fig. 2A and 2B). Both MDA-MB-231 cells and PC3-NI cells showed a two-fold increase in TIMP-1 production with TSP-1 treatment of 60μg/ml. In contrast to the MB-231 cells and the PC3-NI cells, the PC3-ML cells did not exhibit a significant TIMP-1 up-regulation in response to increasing doses of TSP-1. These data are consistent with the Western blot analysis of PC3-ML cells which showed that TIMP-1 secretion plateaued at 20 μg/ml. In all of the experiments, TSP-1 treated samples were compared to bis-tris-phosphate (BTP) buffer treated controls.

Fig. 2.

Fig. 2

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Quantitative analysis of TSP-1 induced TIMP-1 production of human cancer cells through ELISA analysis. Conditioned media from cells treated with TSP-1 as described in figure legend were analyzed for TIMP-1 levels using an ELISA kit from Oncogene Science of Siemens Healthcare Diagnostics Inc., Cambridge, MA. A: MDA-MB-231 conditioned media. B: PC3-NI and PC3-ML conditioned media. TIMP-1 levels were normalized to 106 cells and are presented as percent of control. Each data point is the average of three replicates and the error bars represent standard deviation. The two curves in panel B are statistically different (p<0.05). Statistical analysis was performed using GraphPad Prizm software, San Diego CA.

TSP-1 treatment stimulates TIMP-1 mRNA production

The MDA-MB-231 cell line was treated with media containing TSP-1 at a concentration of 40 μg/ml. Total RNA was collected at time points of 3 hours, 6 hours, 12 hours, and 24 hours of TSP-1 treatment. Cells were also treated with phorbol 12-myristate 13-acetate (PMA) at a concentration of 100 ng/ml and used as a positive control. The results reveal a 0.9 kB band signifying TIMP-1 message with a marked up-regulation at the 12 hour time point (Fig. 3). The message remained up-regulated at the 24 hour time point as well. TSP-1 induced TIMP-1 expression was not detectable at the protein level until the cells had been exposed to TSP-1 for 24 hours. A 2.4 kB β-actin probe was used as an internal control for equal loading.

Fig. 3.

Fig. 3

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Time dependant effect of exogenous TSP-1 treatment on TIMP-1 mRNA in human breast cancer cells, MDA-MB-231. Cells were treated with either 40 μg/ml of TSP-1 in serum –free media or 100 ng/ml PMA and RNA isolated as described in Material and methods. Lane 1: PMA for 12 hours. Lane 2: BTP buffer for 24 hours. Lanes 3-5: TSP-1 treatment for 6hours, 12hours, and 6-24 hours as noted. A 2.4 kB β-actin probe was used as an equal loading control. Experiments were repeated three times and the results of a representative experiment are shown in the figure.

Regions of TSP-1 responsible for TIMP-1 production

To confirm specificity of TSP-1 induced TIMP-1 production, MDA-MB-231 cells were treated with 10 μg/ml polyclonal TSP-1 antibody IgG and concurrently with 40 μg/ ml exogenous TSP-1. Treatment with the goat TSP-1 antibody IgG resulted in inhibition of TSP-1 induced TIMP-1 production (Fig. 4A). In contrast, goat non-immune IgG (10 μg/ml) had no effect on TIMP-1 production. The experiment was repeated with a polyconal rabbit antibody IgG (10 μg/ml) against the type I repeat peptide CSVTG of TSP-1 to determine if this particular domain is involved in TIMP-1 regulation. This polyclonal antibody was also capable of inhibiting TSP-1 induced TIMP-1 production (Fig. 4B). The peptide CSVTCG from the TSP-1 type I repeats of TSP-1 was also tested as a competitive inhibitor of TSP-1. MDA-MB-231 cells were treated with either buffer, 40 μg/ml TSP-1 alone, 40 μg/ml TSP-1 plus 10 μg/ml CSVTCG peptide, or 40 μg/ml TSP-1 plus 10 μg/ml of an irrelevant control peptide (Fig. 4C). The type I peptide was capable of inhibiting TIMP-1 production to a level lower than that of the buffer control or irrelevant peptide control.

Fig. 4.

Fig. 4

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Anti-TSP-1 antibody and type 1 repeat peptide (CSVTCG) inhibits TIMP-1 production in human breast cancer cells. In all panels, cells were incubated in serum-free media with TSP-1 alone (40 μg/ml) or with TSP-1 (40 μg/ml) and (10 μg/ml) of the indicated antibodies or peptides for 48 hours. Conditioned media was then collected and analyzed by Western blot analysis using a rabbit polyclonal anti-TIMP-1 antibody. A. Effect of a polyclonal goat anti-TSP-1 antibody on TIMP-1 production. B. Effect of polyclonal rabbit antibody against CSVTCG present in the type I repeat domain of TSP-1. C. Effect of the CSVTCG peptide (from the type 1 repeat of TSP-1) on TIMP-1 production. The last lane in all experiments was conditioned media of HT-1080 cells treated with PMA (positive control). Experiments were repeated three times and the results of a representative experiment are shown in the figure.

TIMP-1 production was also analyzed in several TSP-1 stably transfected MD-MBA-435 breast adenocarcinoma cell lines engineered to produce TSP-1 at varying levels as reviewed in the methods. Cells were serum starved and the 72 hour conditioned media analyzed for TIMP-1 production by Western Blot analysis (Fig. 5). The results show that the high TSP-1 producer (TH26) secreted a higher level of TIMP-1 than the vector control (TH5). The COOH-terminally truncated TSP-1 producer (TH50) secreted TIMP-1 at concentrations similar to that of the TH5 cell line implying that several TSP-1 domains may be involved in TIMP-1 regulation.

Fig. 5.

Fig. 5

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TIMP-1 expression in TSP-1 stably transfected MDA-MB-435 cells as assessed by western blot analysis. Cells were grown in serum-free media for 72 hours and the conditioned media measured for TIMP-1expression by western blot analysis. Lane #1: conditioned media from HT-1080 cells treated with PMA. Lane #2: vector control cell line (TH5). Lane #3: high TSP-1 producers (TH26). Lane #4: the COOH-terminal truncated TSP-1 producers (TH50). Experiments were repeated three times and the results of a representative experiment are shown in the figure.

Functional significance of TSP-1 induced TIMP-1 production on tumor cell invasion

Because TSP-1 mediated TIMP-1 up-regulation is not detectable until after 24 hours after exposure to TSP-1, Boyden chamber invasion assays were done with MB-231 cells pretreated with either BTP buffer or 40 μg/ml TSP-1 for 48 hours. Cells were placed in the upper chamber either alone or with 10 μg/ml TIMP-1 neutralizing antibody or 10 μg/ml IgG control and were allowed to incubate for five hours. Filters were then fixed in 3% gluteraldehyde and stained in 0.5% crystal violet solution. Invasion was stimulated in the TSP-1 containing wells as compared to the BTP buffer treated control samples (Fig. 6). Furthermore, when anti-TIMP-1 antibody was added to the cells, invasion was increased by more than 25% as compared to TSP-1 alone or TSP-1 and control IgG treated cells.

Fig. 6.

Fig. 6

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TSP-1 inhibits breast cancer cell invasion through TIMP-1 production. MDAMB-231 cells were pretreated with either BTP (buffer control) or TSP-1 (40 μg/ml) for 36 hours. Cells were harvested and placed in the top chamber of the Boyden chamber assay with or without the neutralizing antibodies as indicated. Cells were treated with antibody or control IgG at a concentration of 10 μg/ml. No TSP-1 or BTP buffer was added to the bottom chamber in this experiment. Bottom chambers contained serum-free media with 0.1% BSA. Cells were incubated for 5 hours at 37° C, fixed and then stained. Invasive cells adhering to the bottom of the chamber were counted using a phase contrast microscope (400X). The data were expressed as the summation of the number of invasive tumor cells in five representative fields. Experiments were repeated three times and the results of a representative experiment are shown in the figure.

Discussion

TSP-1 is a multifunctional protein involved in many aspects of tumor progression including cell adhesion, cell motility, and tumor angiogenesis (Staniszewska et al., 2007; Taraboletti et al., 1987; Varani et al., 1986). The involvement of TSP-1 in tumor progression has often been shown to be contradictory as to whether TSP-1 serves as a promoter or inhibitor of tumor cell progression and angiogenesis. Certainly, there is data to support both viewpoints. We have shown previously in our laboratory that TSP-1 mediates tumor cell invasion (Albo and Tuszynski, 2004). There is, however, a great deal of evidence that TSP-1 inhibits tumor progression. For example, a study examining breast cancer cells revealed an inverse correlation with TSP-1 expression and metastatic potential (Zabrenetzky et al., 1994). These results have also been observed for melanoma cells and also transformed endothelial cells (Sheibani and Frazier, 1995).

In this study, we show that TSP-1 is capable of stimulating TIMP-1 production in a time and dose-dependent manner, beginning at 12 hours. Modulating TIMP-1 expression may be one mechanism TSP-1 uses to regulate tumor progression and angiogenesis and may be one explanation for the often contradictory results seen with TSP-1 and tumor progression. TSP-1 has been shown to stimulate endothelial tube formation at a concentration of 5 μg/ml but inhibits tube formation at 15 μg/ml (Qian et al., 1997). While these observations were correlated with TSP-1 induced MMP-9 production, TIMP-1 production may have also played a role in TSP-1’s biphasic mechanism. TIMP-1 expression was enhanced by a 10 μg/ml dose of TSP-1. Lower doses, however, had no effect. As TIMP-1 has been shown to be a potent inhibitor of angiogenesis, TSP-1 induced TIMP-1 production at a 15 μg/ml concentration may have been the stimulus for decreased endothelial tube formation.

Further substantiating this theory, the modified Boyden chamber invasion assay results show TSP-1 induced TIMP-1 production decreases invasion. Again, this ability of TSP-1 to up-regulate TIMP-1 production could play a role in why TSP-1 has been shown to have conflicting roles in tumor cell angiogenesis and invasion.

The TSP-1 transfected MDA-MB-435 cells when examined in vivo reveal that the high TSP-1 producers, the TH26 cell line, show decreased tumorigenesis and metastasis in vivo when compared to the vector control, the TH5 cell line (Weinstat-Saslow et al., 1994). This data correlates with the TIMP-1 expression patterns of these cell lines with the TH26 cells expressing at least four times as much TIMP-1 as the TH5 cells. All of the transfected cell lines produce minimal amounts of the metalloproteinases, MMP-2 and MMP-9, when analyzed by gelatin zymography (data not shown). The TIMP-1 expression, therefore, may be one factor that contributes to the decreased tumor growth and decreased angiogenesis observed for the TH26 cells when tested in vivo.

To establish specificity, a polyclonal TSP-1 antibody was used to try to inhibit TIMP-1 production. The TSP-1 specific antibody blocked TSP-1 mediated TIMP-1 expression, returning expression back to that of the buffer treated cells. To further localize the domain of TSP-1 responsible for TIMP-1 up-regulation, an antibody directed against the type I repeat sequence (CSVTCG) of TSP-1 was used to try to inhibit TIMP-1 up-regulation. Similar to the TSP-1 polyclonal antibody, the anti-type 1 repeat antibody also was able to inhibit TIMP-1 up-regulation. This implies that the type I repeat may play a role in TIMP-1 regulation. Specifically, the CSVTCG peptide, present in two of the type I repeats, was able to inhibit TIMP-1 production while an irrelevant peptide ASVTAR had no effect. This adhesive domain of TSP-1 has also been shown to be involved in MMP-9 regulation as well . Other groups have shown that this domain of TSP-1 is not only capable of inhibiting tumor progression but also angiogenesis as assessed through a rat cornea assay (Good et al., 1990). The CSVTCG peptide binds angiocidin, a unique tumor associated protein, originally isolated in our laboratory, which has been shown to have anti-angiogenic and anti-tumorigenic properties (Sabherwal et al., 2006; Tuszynski et al., 1993). This protein was originally thought to be a receptor for the type 1 repeat domain of TSP-1, but has also been found in the serum of patients with hepatocellular carcinoma (Sabherwal et al., 2007).

An additional domain that may be responsible for TIMP-1 production is the carboxyl terminal domain. Examining expression of TIMP-1 in the TH50 cell line, the carboxyl terminal truncated TSP-1 expressors, revealed a decrease in TIMP-1 production. The TH50 appeared to show similar expression with the TH5 cells. In vivo, these cells display a more metastatic phenotype than the TH26 cell line, supporting the idea that TIMP-1 suppresses metastasis (Weinstat-Saslow et al., 1994). The carboxyl terminal domain is also involved in tumor cell adhesion and has two known receptors (Qian and Tuszynski, 1996). Although this domain does seem to be one of the critical cell adhesive domains required for tumor cell invasion, there are no reports of this region being able to mediate protease activity. Additional studies regarding the function of this domain and its receptors is warranted.

The role of TSP-1 in tumor progression is a complicated one. TSP-1 is not only capable of stimulating proteolytic enzymes involved in tumor invasion but also their corresponding inhibitors. The critical balance of enzyme to inhibitor needed for successful invasion might partially explain the varied results observed with TSP-1’s involvement in tumor progression and metastasis as observed by the dose and time dependent effects of TSP-1 on MMP-9 and TIMP-1. Its role in cancer has yet to be clearly defined, but is an exciting area of research both towards the understanding of the physiology of tumor progression and the development of cancer therapeutics.

Acknowledgments

This work was supported in part by NIH grant R01 CA88931.

Abbreviations

ECM

extracellular matrix

MMP

metalloproteinase

SDS-PAGE

sodium dodecyl sulfate-polyacylamide gel electrophoresis

(TIMP)

tissue inhibitor of metalloproteinase

TSP-1

thrombospondin-1

SDS-PAGE

sodium dodecyl sulfate-polyacylamide gel electrophoresis

Footnotes

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