Maspin Gene and Chromosome Localization

Human maspin encodes a 375 amino acid protein with a molecular weight of 42 K dalton. By FISH in situ analysis maspin is located at chromosome 18q21.3.¹³ To determine maspin gene structure, we have cloned maspin genomic DNA and sequenced all exon/intron borders as well as partial introns. Our data show that maspin gene contains 7 exons and 6 introns with a total length of 28 kb (Zhang et al, unpublished). It contains a non-coding exon 1 and an unusually long intron 1 (7.4 kb). Further genetic study and computer search of recently published human genome sequence shows that this region has a cluster of serpins belonging to the ovualbumin serpin family (Clan B). These include from the direction of telomere to centromere: PI8, PI10, PAI-2, megsin, SCCA1, SCCA2, headpin, and maspin.^23,24 The gene structure of maspin is different from that of PAI2, SCCA1 and 2 in the same cluster since they all contain 8 exons and 7 introns. In addition, another cluster of serpins within the Clan B family exists in chromosome 6p25. Evidence from genetic study shows that these two clusters may have evolved from both intra- and inter-chromosome duplication.²⁵

The phylogenetic tree shows that maspin is conserved with the ovalbumin family of serpins. The degree of homology at the amino acid level is : 40%, human monocyte-neutrophil elastase inhibitor (ELANH2), 39%, human PI10, 39%, Human PI8, 38%, human PI6, and 35% for human PAI-2.^23,2628 ELANH2 and PI6 are located at chromosome 6p25.²⁹ Other serpins at 18q21.3 such as PI8 are also more homologous to PI9 at 6p25, supporting the hypothesis of interchromosome duplication.

Differential Expression of Maspin in Breast Normal and Tumor Cells

The maspin gene was originally isolated from normal mammary epithelial cells. Northern blot analysis had been used to evaluate the expression pattern of maspin in human cell lines. Maspin was found to be highly expressed in 70N and 76N normal mammary epithelial cells, down-regulated in 21NT and 21PT primary breast tumors, and silent in a series of metastatic tumor cells. The gene was not expressed in cells of non-epithelial origin, such as mammary fibroblast cells, foreskin fibroblast cells, and U937 human monocytic cells. Mouse homolog of maspin was also expressed highly in mouse normal mammary epithelial cells such as NMuMG, HC11. The level of expression was down-regulated in low invasive mouse mammary tumor cells and lost in a highly invasive tumor cell line.

Transcriptional Regulation of Maspin in Breast Cells

To study maspin gene regulation in breast normal and cancer cells, we cloned maspin gene promoter. The cloning of maspin promoter has been quite a challenge because of the presence of a long intron 1. Most lambda genomic clones contain about a 20 kb genomic insert. Thus, using the full length of maspin cDNA as a probe, we were unable to recover any clones containing maspin promoter since the region from exon 2 to the last exon spanned over 20 kb. Finally, a YAC clone containing a cluster of serpins including maspin was used to screen maspin genomic DNA using a 50 mer oligomer as a probe. A positive clone was identified containing a 1.2 kb 5'-flanking region, a 7.4 kb intron 1 and partial exon 2 sequence. Sequencing and primer extension analysis showed that maspin did not have a typical TATA box in its promoter, a common feature for housekeeping or ubiquitously expressed genes. The major transcription start site was located 207 bp from first ATG site and the ATG was located in the second exon.¹⁷

Several important transcription factor binding sites, Ets, Ap1, HRE and p53, are located within the 1 kb promoter region. To identify the responsive elements, promoter deletion and CAT assay were used to identify the activity of maspin in normal mammary epithelial cells (70N), primary tumor cells (21NT), and metastatic tumor cells (MDA-MB231) (Fig.1). As shown in Fig.1, a 1 kb upstream region was sufficient for activating transcription of maspin in normal breast cells. The activity was decreased in 21NT cells, and no detectable CAT activity was found in MDA-MB231 cells. Deletion analysis showed that deletion of an Ets site located at the region from −112 bp to −90 bp, abolished completely maspin activity, suggesting that this Ets was the major positivecis element within 1 kb responsible for up-regulation of maspin in normal mammary epithelial cells. Further analysis confirmed that this Ets site cooperated with a downstream Ap1 site for synergistic activation in normal breast cells; however this cooperative transactivation between Ets and Ap1 was lost in primary breast tumor 21NT cells.

Figure 1

Maspin CAT constructs and CAT assays. On the left: CAT constructs and a schematic representation of maspin promoter with putative transcription factor binding sites. On the right: CAT constructs were transfected into 70N and MDA-MB231 cells. Relative (more...)

Maspin Expression in Prostate Cells

Prostate cancer is the most common cancer in men. The prostate gland depends on androgenic hormones for its growth and development, analogous to the role of mammary hormones in development and morphology changes in the mammary gland.³⁰ The molecular events leading to the development of prostate cancer may be similar to those in breast cancer. Because maspin functions as a tumor suppressor in the mammary gland, we have examined whether maspin could play a similar tumor-suppressing role in the prostate, and more importantly, what is the mechanism underlying gene regulation of maspin in prostate cells.

Northern blot analysis was carried out with RNAs from several human normal prostate and tumor cell lines. Maspin is highly expressed in CF3, CF91, and MLC normal prostate epithelial cells, and down-regulated in LNCaP, PC3, and DU145 prostate tumors. This expression pattern is similar to the findings in the normal mammary epithelial cells and carcinomas, indicating that the down-regulation of maspin expression is a common phenotype of both breast and prostate tumors.^13,17 A 1 kb maspin promoter is active in CF3 normal prostate cells, but little activity is present in LNCaP prostate cancers. As in mammary epithelial cells, the same Ets site is primarily responsible for transcriptional action of maspin in CF3 cells. In addition, we have identified an HRE site located between −297 bp and −265 bp that represses transcriptional activity of maspin. Point mutations at this HRE site abolished the repression completely (see Fig. 2A.). The consensus HRE element has a sequence 5'-GGTACANNNTGT(T/C)CT-3'.³¹ This sequence can be recognized by multiple steroid receptors, such as glucocorticoid receptor, androgen receptor, and progesterone receptor.³² Previous studies have demonstrated that steroid receptors binding to HRE could mediate both transcription activation and repression.^33,34 The HRE site (5'-GTACTCTGATCTCC-3') in the maspin promoter is unique in that its sequence does not share very good homology with the consensus sequence. This maspin HRE sequence acted as a general transcription repressor when it was placed in front of other heterologous promoters. Furthermore, we demonstrated that repression was mediated through AR using electrophoresis mobility shift assay (Fig. 2B). Binding of AR to maspin HRE is specific and can be blocked by AR antibody.³⁵ These data may explain castration-mediated suppression of prostate cancer progression since ablation of androgen by castration may up-regulate maspin in prostate. From the therapeutic point of view, re-expression of maspin in the prostate tumors offers great hope for reversing the tumor phenotypes. Re-expression may be achieved by targeting both activation and repression modes. While it will likely be difficult to restore transcriptional activation of maspin through the Ets site, it may be more feasible to block the repression mediated by the AR binding HRE element. Treating tumors with ligands that block the binding of AR to the HRE or with reagents that compete strongly for binding to AR are possible methods of blocking HRE mediated repression. Our discovery of HRE-mediated repression offers another opportunity to increase the expression of maspin in prostate tumors, which may in turn reduce the progressiveness of prostate cancer.

Figure 2A

HRE element negatively regulates maspin promoter activity. Effect of mutation at HRE site on the promoter activity. CAT constructs were transfected to CF3 cells and activity normalized to pKTCAT control. Values are obtained from at least three repeated (more...)

Figure 2B

HRE element negatively regulates maspin promoter activity. Effect of HRE on the promoter activity of pBLAP1. CAT constructs were transfected to CF3 cells and activity normalized to pBLCAT2 control. Transfected cells were treated with R1881 (50nM) or vehicle (more...)

Regulation of Maspin by Other Transcription Factors and Reagents

A number of groups have contributed to the discovery of new transcription factors and reagents that regulate maspin expression. Notably, a recent report has demonstrated that p53 activates maspin in prostate and breast cancer cells.³⁶ Infection of these cancer cells through adenovirus p53 dramatically induced maspin expression with a rapid kinetics, similar to that of another well-known p53 regulated gene, p21. Inducers of p53 such as DNA-damaging reagents and drugs also induced maspin expression. They further showed that a consensus sequence for p53 binding is located in the maspin promoter and p53 binds to this site in gel shifting assay. A promoter luciferase reporter containing the p53 site was active when cotransfected with wild-type p53 into the prostate cancer cells. This finding is significant since it marks maspin as the first p53 regulated gene involved in cancer invasion and metastasis.

PPAR-γ plays an important role in adipocyte differentiation. A recent study also demonstrated that activation of PPAR-γ by its ligands caused dramatic morphological and nuclear changes that were characteristic of a more differentiated, less malignant state in breast cancer cells. Associated with these changes were the increase of maspin expression and arrest of cell cycle.³⁷ Thus, PPAR-γ-mediated terminal differentiation of cancer cells might result from the increased maspin expression by a mechanism of transcriptional up-regulation. Gamma linolenic acid, an essential fatty acid, has anticancer properties. It was reported that gamma linoneic acid could induce maspin expression very rapidly.³⁸ Cancer cells treated with GLA were shown to have reduced spreading and migration as monitored by video-microscope, and this reduction was reversed if the cells were treated with anti-maspin antibody. Thus, the anticancer effect of GLA was thought to result from the activation of maspin expression. Similarly, maspin was found to be induced by superoxide dismutase (MnSOD), and this induction might explain the tumor-suppressive effect of MnSOD in breast cancer cells.³⁹

While it is relatively easy to identify the transcription factors and their regulatory sites responsible for maspin expression in normal cells, elucidating why certain cancers lose maspin may prove to be a more challenging task. Invasive cancer cells undergo numerous genetic changes such as mutation and chromosome rearrangement; the mechanism for loss of maspin expression in these cells is more complicated than just loss of a single transcription factor such as ETS. Indeed, one recent study has shown that in certain breast cancer cells, methylation of maspin promoter may contribute to the silencing of maspin in breast cancers.⁴⁰

About ten-years ago, Dr. Ruth Sager proposed that tumor suppressor genes could be divided into two categories: class I and class II. Class I genes are mutated or deleted in tumors; examples include Rb and p53, which were identified by virtue of their mutations or deletions. Class II genes are not altered at the DNA level; rather, they affect the phenotype by changes in their expression levels.⁴¹ The focus of expression genetics is to re-express the class II tumor suppressor gene in cancers. Since the maspin gene is not mutated or deleted in tumors, it offers great advantage for one to induce maspin expression in tumors by the reagents identified from the above studies. In the future, some natural products or nontoxic drugs will likely be discovered to accomplish this goal.

Mouse Maspin is Highly Homologous to Human Maspin

Mouse maspin (mMaspin) cDNA was isolated from a mouse mammary gland cDNA library using human maspin cDNA as a probe. The longest cDNA sequence contained 1378 bp, including 69 bp 5' untranslated region, 1128 bp in the coding region, and 181 bp in the 3'-untranslated region. The poly (A) was added 23 bp after the polyadenylation signal site. The deduced amino acid sequence of mMaspin has a 89% identity with its human counterpart. In addition, a major difference in the mRNA lies in the 3' untranslated region. mMaspin cDNA has a short 3'-UTR, compared to the 1.2-kb 3'-UTR in the human maspin cDNA.¹⁶

Tissue Distribution

The maspin tissue expression pattern was examined by Northern blot analysis using RNAs from both human and mouse tissues. The human RNA blots from Clontech, Inc. contained a relatively small collection of tissues. Maspin was found to be abundantly expressed in prostate, thymus, testis, and small intestine.¹⁷ Among mouse tissues, the mammary gland has the highest level of expression. Maspin level varied during mammary gland development, suggesting its expression might be hormonally regulated. mMaspin is also expressed in mouse large intestine, skin, tongue, and stomach, and weakly in other tissues such as lung and thymus.¹⁶ It should be pointed out that Northern blot is not the ideal method to characterize maspin expression pattern. We have found out that some tissues express maspin at such a low level that it can not be detected by Northern blot analysis. One example is ovary, which has very low levels of maspin. However, deletion of one copy of maspin in heterozygous mice is enough to cause a severe defect in ovarian development (Zhang et al, unpublished data). Another feature noticed from Northern blot analysis was that there were multiple maspin transcripts in both human and mouse tissues.^16,17 Alternative splicing may account for the multiplicity. Whether the splicing products play any biological role remains to be identified.

Functional Similarity Between Mouse and Human Maspin

Does mouse maspin function similarly as human maspin? To answer this question, we first compared the changes of maspin expression during mouse and human breast cancer progression.

Northern blot analysis showed that mouse maspin was expressed at a high level in mouse normal mammary epithelial cells (NMuMG, HC11). The level of expression was down-regulated in low invasive mouse mammary tumor cells (CSML0) and lost in a highly invasive tumor cell line (CSML100). The down-regulation of maspin protein was also confirmed by Western blot analysis. These data indicate that mouse maspin, like its human homolog, is also down-regulated during tumor progression.

Recombinant mMaspin proteins were produced in E. coli as a GST-maspin fusion protein. The biological function of recombinant protein was investigated using Boyden chamber assay (Fig. 3). A pair of mouse mammary tumor cells, CSML0 and CSML100 which are well characterized for their invasiveness, were used in both invasion and migration assays.⁴² Normally, about 10% of the low invasive CSML0 cells and 32% of the high invasive CSML100 cells seeded in the upper chamber invade the reconstituted basement membrane. Addition of mMaspin to the cell culture inhibits the invasion of both CSML0 and CSML100 cells. The inhibition by both cell lines increased as the concentration of GST-mMaspin was increased. The highest inhibitions at the concentration of 1.2 mM were about 60% for CSML0 cells (from 10% to 4%) and 62% for CSML100 cells (from 62% to 12.5%). For the motility assay, the recombinant proteins were added at different dosages to CSML0 and CSML100 cell suspensions and incubated with cells for 30 mins at room temperature before seeding. Under control condition, 5.8 % CSML0 cells and 7.2% CSML100 cells move through the polycarbonate membrane. Addition of GST-mMaspin inhibits the motility of both CSML0 and CSML100 cells. The highest inhibitions at the concentration of 1.2 μM were about 78% for CSML0 cells (from 5.9% to 1.25%) and 80% for CSML100 cells (from 7.2% to 1.4%).

Figure 3

Effect of mouse mMaspin protein in motility and invasion by two mouse mammary tumor cell lines, CSML0 and CSML100. Control (untreated cells)and maspin-treated cells at various concentrations were used in the assay. Mutant maspin is GST-mMaspinΔRSL. (more...)

These data strongly suggest that mouse maspin functions like human maspin. Therefore, studying the function of maspin with transgenic and knock-out mice should provide valid insights into its role in the normal human breast and in breast cancers.

Transgenic Maspin Mice and the Role of Maspin in Normal Mammary Gland Development

Why Study the Role of Maspin in Normal Mammary Gland Development?

The mouse mammary gland undergoes a dramatic series of cyclical changes during development (see Fig. 4). In juvenile mice, the mammary gland develops based on a rapid, hormonally-regulated growth and morphogenesis of the epithelial ducts. Mammary ducts penetrate the gland by means of end buds. Upon reaching sexual maturity, the epithelial ducts have filled the whole gland with a tree-like structure. The onset of pregnancy initiates another stage of mammary development in which alveolar epithelial cells rapidly proliferate and differentiate under different regulatory mechanisms. Alveolar cells grow from the ductal skeleton and appear as lobulo-alveolar units by the end of pregnancy. These alveoli are the functional unit of milk production at lactation. Following lactation, the mammary gland undergoes massive remodeling and apoptosis, resulting in involution of the gland and a return to the ductal structure similar to the non-pregnant state.

Figure 4

Mouse normal mammary gland development. Adapted from the website: http://mammary.nih.gov/atlas.

One of the barriers to progress in breast cancer research is lack of the biological study of genes in normal mammary gland development. Since maspin expression is regulated during mammary gland development,⁴³ it is absolutely relevant to examine the role of maspin in this process in order to better understand its involvement in breast cancer.

Establishing Maspin Transgenic Mice

To delineate maspin's function in vivo, we utilized a transgenic mouse system to examine the effect of overexpression of maspin under control of the whey acidic protein (WAP) promoter. The whey acidic protein has been shown to be exclusively expressed in mammary epithelial cells during midpregnancy and lactation.^44,45 Transgenic mice were generated by injecting embryos with the WAP-maspin construct. Screening of founders was performed by Southern blot analysis using DNA isolated from mouse tissues. The expression of both endogenous maspin and the transgene in transgenic mouse mammary glands were examined qualitatively by reverse transcription-PCR. RNA was isolated from the mammary glands of transgenic mice at different stages of development. As expected, the WAP-maspin transgene was expressed during late pregnancy and lactation but was not detectable in virgin mice and glands undergoing involution.

Whole mount and histology of the mammary glands from wild-type and transgenic mice were analyzed. Ductal elongation and branching appeared to be normal in wild-type and transgenic virgin mice. No difference in alveolar structures were present between the transgenic and wild-type animals up to day 10 of pregnancy; however, minor differences did become noticeable at day 15 of pregnancy following the activation of the WAP promoter-driven transgene. These mammary glands exhibited decreased alveolar densities, which was further reduced as compared to controls at day 19 and resembled the morphology of the midpregnant wild-type controls. At high power, the mammary glands from transgenic mice contained not only fewer lobular-alveoli structures, but also the size of each alveolar structure was greatly reduced (Fig. 5). In many cases, the lumens of the alveoli were closed. This defect was due to the expression of the transgene activated by WAP promoter from midpregnancy.

Figure 5

Histological analysis of mammary tissues from the following mice: (A) wild-type at day 15 pregnancy; (B) transgenics at 15-day pregnancy; (C) wild-type at day 19 pregnancy; (D) transgenics at day 19 pregnancy. (E) and (F) (same magnification) were high (more...)

Increased Apoptosis and Proliferation in Midpregnant and Early Lactating Mammary Glands of Transgenic Mice

Since the underdevelopment of the mammary glands in the WAP-maspin transgenic mice could have arisen from either decreased proliferation or increased apoptosis or a combination of the two, TUNEL and PCNA immunohistochemistry assays were carried out utilizing pregnant and early lactating mammary glands from wild-type and transgenic mice. As shown in Table 1, the apoptotic rate was significantly increased in transgenic glands at midpregnancy (2.29±0.26%) as compared to controls (0.91± 0.09%). In contrast, little difference was observed in cell proliferation at day 15 of pregnancy. However, during lactation, the apoptosis and proliferation profiles changed significantly in the transgenic strain. Secretive alveolar cells occupied the majority of the fat pad and there was a low rate of proliferation and apoptosis in samples taken from normal mammary glands. This observation contrasted with the results obtained from the WAP-maspin mice, in which a large percentage of the fat pad was devoid of alveolar cells and an increased rate of proliferation was observed. Both proliferation and apoptosis index decreased quickly as lactation proceeded, and by lactation day 10, very few cells were PCNA-positive and apoptotic in both transgenic and normal mice.

Table 1

The rate of apoptosis in wild-type and transgenic mice.

Maspin Knock-out Mice

A tumor suppressor by definition is a gene, when deleted or inactivated, would result in or facilitate the tumorigenesis of an organism. The two-hit hypothesis by Knudson suggests that loss of heterozygosity in a tumor suppressor gene will render the organism more susceptible to tumorigenesis. This prediction has been convincingly proved by numerous studies. For example, Donehower et al has demonstrated that p53 heterozygous mice can promote cancer formation and that loss of both p53 alleles is not a prerequisite.⁵⁰ To prove maspin does function as a tumor suppressor, we applied gene disruption technique in mice to examine the effect of loss of the maspin function on mammary tumor progression, as well as other mouse development processes. Because of the difficulty to delete a large DNA fragment containing full length of gene, it is a conventional wisdom to target the deletion to a small region of genomic DNA. We have cloned mouse maspin genomic DNAs and designed a vector to selectively knock out the last exon encoding the RSL region and stop codon as well as the entire 3'-UTR. Since 3-UTR is responsible for mRNA stability, it is therefore unlikely that a truncated maspin protein can be made. To this date, we have already generated maspin knock-out mice by the approach described above with help from Dr. Philip Leder at Harvard Medical School and Dr. Francesco DeMayo at the Baylor College of Medicine.

The phenotype of the maspin knock-out mice is surprising. The homozygous mice are embryonic lethal. The existing knock-out mice for serpins and their protease targets are all viable. This demonstrates maspin indeed is a very unique and important serpin. Preliminary data by this laboratory indicate maspin is expressed in early stages of embryonic development (Zhang et al, unpublished data), and lethality demonstrates that maspin plays a critical role. Partial loss of maspin in heterozygotes also displays a phenotype. The heterozygous mothers are less likely to get pregnant and if they do, they deliver small litters of less than 4 pups, which can not be explained by the loss of homozygotes since wildtypes generally produce over 8 pups. Our data indicate that the ovulation efficiency of maspin heterozygotes is greatly reduced compared to control. Histology showed more than 50% of corpus luteum had entrapped oocytes along with hemorrhage in the center of corpus luteum, indicating ovary angiogenesis was affected. Using combined in vitro and in vivo approaches, we find out that maspin indeed plays an important role in the process of lutealization and atresia during ovarian development (Zhang et al, unpublished data). We are currently investigating the effect of loss of heterozygosity on mammary gland development and mammary tumor progression.

Overexpression of Maspin on Tumor Progression in Bitransgenic Mice

To characterize maspin's in vivo activity during tumor progression, we generated transgenic maspin mice exhibiting targeted overexpression of maspin to mammary epithelial cells under the control of mammary specific whey acidic protein (WAP) promoter.⁴³ Overexpression of maspin in normal mammary epithelial cells inhibits mammary gland development and induces apoptosis. Since the primary goal of generating WAP-maspin transgenic mice was to test the protective role of maspin overexpression on mammary tumor progression, we crossed WAP-maspin transgenic mice with a strain of oncogenic WAP-simian virus (SV) 40 T antigen (TAg) mice. WAP-TAg transgenic mice develop mammary tumors with 100% frequency and can be utilized to examine specific mechanisms of tumor progression at both early and late time points.^54,55 The SV40 TAg initiates tumorigenesis through the inactivation of both p53 and the pRb related family of proteins.^56,57 In human breast cancers inactivation of p53 function is found in up to 40% of tumors, and mutations in Rb or related proteins have also been reported.^57,58

Overexpression of Maspin Reduced Tumor Growth and Vessel Density and Increased Apoptosis in Primary Adenocarcinomas

Both maspin overexpression and dominant gain of TAg expression induce mammary apoptosis, likely through different mechanisms. To determine if maspin overexpression could increase the level of TAg-induced apoptosis in mammary epithelial cells, the percentage of cells undergoing apoptosis in WAP-maspin/WAP-TAg double transgenic mice was compared to WAP-TAg single transgenic mice. Maspin overexpression increased the percentage of apoptotic cells from 1.8% in the single WAP-TAg transgenic mice to 5.6% in the WAP-maspin/WAP-TAg double transgenic mice (P< 0.01). In addition, bitransgenic mice exhibited a significant reduction in the number of alveolar-lobular structures and alveolar lumen size when compared to WAP-TAg single transgenic mice. Like WAP-TAg single transgenic mice, none of the WAP-maspin/WAP-TAg double transgenic mice could nurse their offspring beginning with the first litters. These results demonstrate that overexpression of maspin can increase the rate of apoptosis in preneoplastic mammary gland.

We then compared the rate of tumor growth of WAP-TAg and bitransgenic mice (Table 2). Tumors were measured biweekly after they were initially detected, and mice were euthanized when the primary tumor reached 2.5 cm in diameter. The tumor observation time (from the time of first palpable tumor to the time when the tumor reached 2.5 cm in diameter) was used to assess the rate of tumor growth. The presence of maspin overexpression significantly increased the time for a first palpable tumor to reach 2.5 cm in diameter from 36.3 to 49.3 days (p< 0.03).

Table 2

Analysis of primary and lung tumors.

To determine if maspin overexpression has an effect on microvessel density and apoptosis during tumor progression in vivo, microvessel density in adenocarcinomas with and without maspin overexpression were compared. Microvessel density was measured after CD31 staining. In small mammary tumors (tumor size £ 0.6 cm in diameter), the microvessel density was significantly reduced (p< 0.02), while the apoptotic index was significantly increased in the presence of maspin overexpression (p< 0.01).

Maspin as an Angiogenesis Inhibitor

Tumor metastasis is a multi-step process. Malignant tumors need to invade through the basement membrane, stromal tissues, and metastasize to other organs.^59,60 However, for this process to occur, neovascular formation is required.^61,62 This process, termed angiogenesis, provides tumors with nutrients and aids in the removal of metabolic wastes.^63,64 The interaction between endothelial cells and tumor cells also generates a paracrine effect.^65,66

To study the potential anti-angiogenic properties of maspin, the mouse maspin was produced inE. coli as a recombinant GST fusion protein and tested in a variety of angiogenesis assays. Recombinant maspin blocked endothelial cell migration induced by VEGF and bFGF in a dose-dependent manner with an ED₅₀ of 0.2 μM-0.3 mM (Fig. 6). At 1 μM, maspin completely blocked the response of the endothelial cells to both angiogenic inducers, while the GST control was inactive. In vivo, purified maspin effectively inhibited neovascularization. Rat corneas were surgically implanted with non-inflammatory slow release pellets containing maspin with bFGF and examined six or seven days later for ingrowth of vessels. As shown in Fig. 7, maspin completely blocked bFGF-induced neovascularization. To determine if the anti-angiogenic activity of maspin depended on the inhibition of some undefined protease, several mutants were constructed, expressed and tested. The RSL (reactive serpin loop) near the C-terminus of serpin family members is essential for their anti-protease activity. Mutations at the RSL region of other serpins especially at the P1 site abolish serpin activity.⁶⁷ To disrupt this loop in maspin two different mutations were introduced in the RSL region: a C-terminal deletion downstream of P7' residue¹⁶ (maspin DRSL) and a conversion of the critical P1 arginine of the RSL loop to a glutamine (maspin*). A third mutant was constructed in which the first 139 amino acids have been removed but the serpin region left intact (maspinDN). These defective mutants were tested on endothelial cells (Fig. 8). Those with RSL defects retained the ability to inhibit endothelial cell migration and mitogenesis. Protein with mutations in the RSL region also retained the ability to inhibit neovascularization in vivo. The N-terminal deletion, maspinDN, was defective in all assays so it was not possible to determine if a crucial active region had been deleted or if it was not a viable protein.

Figure 6

Effect of maspin on endothelial cell migration. GST-Maspin was tested at a range of concentrations for ability to inhibit endothelial cell migration induced by bFGF (A) or VEGF (B). bFGF, VEGF—indictes migration towards bFGF or VEGF alone; BSA—indicates (more...)

Figure 7

Maspin inhibition of corneal neovascularization. Pellets containing the indicated test substances at 10μM with bFGF (100 ng/ml) were incorporated into Hydron slow release pellets and implanted in rat corneas. After six or seven days animals were (more...)

Figure 8

GST-maspin and its mutants (1μM) were tested for ability to inhibit endothelial cell migration towards bFGF (10 ng/ml). Maspin, GST-maspin fusion protein; Maspin ΔRSL, maspin with a deletion at C-terminus; Maspin*, maspin containing a (more...)

Complete inhibition of endothelial cell migration in vitro was achieved between 0.5 and 1 μM, in the same concentration range where maspin also inhibits tumor cell motility and invasion,¹⁵ but the mechanisms underlying these two maspin activities seem to be different. We and others find that the former requires that the protein have an intact RSL,^16,52 whereas this feature was not essential for the inhibition of angiogenesis.

To determine if the ability of maspin to inhibit angiogenesis plays a role in its well-documented anti-tumor activity, an athymic mouse xenograft model was utilized. LNCaP prostate tumor cells were implanted subcutaneously on the bidorsal back of nude mice and tumor growth and neovascularization were monitored following systemic treatment with exogenous maspin. We found out that maspin-treated tumors contained significantly fewer vessels as determined by CD31 immunostaining than GST treated controls. To determine whether maspin effects on the tumor-induced vasculature were maintained during a more prolonged treatment, the above experiment was replicated with tumors harvested after 7 to 8 weeks. Thirty-two tumor sites were treated with maspin and 37 with GST. When examined at week 8, the growth of 53% of the maspin-treated tumors had been completely inhibited (Fig. 9). The remaining 15 maspin-treated tumors were reduced in size by average 3.43 fold when compared to GST control treated tumors. The effect of maspin was reversible. To examine if the reduced size of maspin-treated tumors coincided with reduced neovascularization, 20 representative tumors from either maspin-treated (10 sites) or GST-treated tumors (10 sites) were used to quantify the density of microvessels after immunostaining with CD31 antibody. The density of vessels in maspin-treated tumors was reduced 2.6 fold in average to that in control tumors and this difference was highly significant (see Fig. 9). We also compared the treated and control tumors of similar size. A reduction of vessel density was also observed in the maspin-treated samples.

Figure 9

Decreased tumor vessels after long-term treatment with exogenous maspin protein. A. Tumors were harvested between 7–8 weeks from GST-treated animals (A, C) and from maspin-treated animals (B, D) and fixed and stained with H & E (A, B) (more...)

The ability to inhibit tumor angiogenesis is only one of several activities associated with the intact maspin protein. Other serpins also have multiple functions and several of them are linked to angiogenesis and tumor growth.^{68 71} PAI-1 is involved in modulating both proteolysis and angiogenesis.¹² PEDF, a known regulator of cell differentiation, is also a very potent anti-angiogenic factor.⁷² Such results indicate that a variety of molecules whose structure places them in the serpin family can be important regulators of natural tumor growth via their influence on neovascularization.