Maspin inhibits cell migration in the absence of protease inhibitory activity

Maspin is a member of the serpin family of protease inhibitors and is a tumor suppressor gene acting at the level of tumor invasion and metastasis. This in vivo activity correlates with the ability of maspin to inhibit cell migration in vitro. This behavior suggests that maspin inhibits matrix-degrading proteases, such as those of the plasminogen activation system, in a similar manner to the serpin PAI-1. However, there is controversy concerning the protease inhibitory activity of maspin. It is devoid of activity against a wide range of proteases, in common with other non-inhibitory serpins, but has recently been reported to inhibit plasminogen activators associated with cells and other biological surfaces (Sheng, S. J., Truong, B., Fredrickson, D., Wu, R. L., Pardee, A. B., and Sager, R. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 499-504; McGowen, R., Biliran, H., Jr., Sager, R., and Sheng, S. (2000) Cancer Res. 60, 4771-4778). We have compared the effects of maspin with those of PAI-1 in a range of situations in which plasminogen activation is potentiated, reflecting the biological context of this proteolytic system: urokinase-type plasminogen activator bound to its receptor on the surface of tumor cells, tissue-type plasminogen activator specifically bound to vascular smooth muscle cells, fibrin, and the prion protein. Maspin was found to have no inhibitory effect in any of these situations, in contrast to the efficient inhibition observed with PAI-1, but nevertheless maspin inhibited the migration of both tumor and vascular smooth muscle cells. We conclude that maspin is a non-inhibitory serpin and that protease inhibition does not account for its activity as a tumor suppressor.     

Dephosphorylation of Tctex2-related dynein light chain by type 2A protein phosphatase

Maspin is a 42kDa tumor suppressor protein that belongs to the serine protease inhibitor (serpin) family. It inhibits cell motility and invasion in vitro, and tumor growth and metastasis in nude mice; however, maspin's molecular mechanism of action has remained elusive. Maspin contains several tyrosine residues and we hypothesized that phosphorylation of maspin could play a role in its biological function. Our study reveals that maspin is phosphorylated on tyrosine moiety(ies) in normal mammary epithelial cells endogenously expressing maspin. In addition, transfection of the maspin gene, using either a stable or inducible system into maspin-deficient breast cancer cell lines, yields a protein product that is phosphorylated on tyrosine residue(s). Furthermore, recombinant maspin protein can be tyrosine-phosphorylated by the kinase domain from the epidermal growth factor receptor in vitro. These novel observations suggest that maspin, which deviates from the classical serpin, may be an important signal transduction molecule in its phosphorylated form.     

p53 regulates the expression of the tumor suppressor gene maspin

Maspin has been shown to inhibit tumor cell invasion and metastasis in breast tumor cells. Maspin expression was detected in normal breast and prostate epithelial cells, whereas tumor cells exhibited reduced or no expression. However, the regulatory mechanism of maspin expression remains unknown. We report here a rapid and robust induction of maspin expression in prostate cancer cells (LNCaP, DU145, and PC3) and breast tumor cells (MCF7) following wild type p53 expression from an adenovirus p53 expression vector (AdWTp53). p53 activates the maspin promoter by binding directly to the p53 consensus-binding site present in the maspin promoter. DNA-damaging agents and cytotoxic drugs induced endogenous maspin expression in cells containing the wild type p53. Maspin expression was refractory to the DNA-damaging agents in cells containing mutant p53. These results, combined with recent studies of the tumor metastasis suppressor gene KAI1 and plasminogen activator inhibitor 1 (PAI1), define a new category of molecular targets of p53 that have the potential to negatively regulate tumor invasion and/or metastasis.     

Biological functions of maspin

Maspin (Mammary Serine Protease Inhibitor) was first reported in 1994 as a serpin with tumor suppressive properties. Maspin was initially isolated through subtractive hybridization and differential display analysis as a 42-kDa protein that is expressed in normal mammary epithelial cells but reduced or absent in breast carcinomas (Zou et al., 1994). Further research led to maspin's characterization as a class II tumor suppressor based on its ability to inhibit cell invasion, promote apoptosis, and inhibit angiogenesis (Sheng et al., 1996; Zhang et al., 2000b; Jiang et al., 2002). Since then, efforts have been made to characterize maspin's tumor suppressive mechanisms. In particular, researchers have studied maspin localization, the regulation of maspin expression, and more recently, maspin protein interactions. By elucidating these mechanisms, researchers are beginning to understand the complex, pleiotropic nature of maspin and the pathways through which maspin exerts its tumor suppressive properties. These new findings not only further enhance our understanding of cancer biology but also provide an avenue to develop maspin's potential as a diagnostic marker for cancer progression, and as a potentially powerful therapeutic agent in the fight against breast cancer.     

Evidence for a direct interaction between the tumor suppressor serpin, maspin, and types I and III collagen.

Maspin (mammary serine protease inhibitor) was originally identified as a tumor suppressor protein in human breast epithelial cells and is a member of the serine proteases inhibitor (serpin) superfamily. It inhibits tumor cell motility and angiogenesis, and although predominantly cytoplasmic, it is also localized to the cell surface. In this study we have investigated the use of the yeast two-hybrid interaction trap to identify novel maspin targets. A target human fibroblast cDNA library was screened, and the alpha-2 chain of type I collagen was identified as a potential interactant. Binding studies with isolated proteins showed interaction between recombinant maspin and types I and III collagen but not other collagen subtypes, a profile strikingly similar to mouse pigment epithelium-derived factor (caspin), which is similarly down-regulated in murine adenocarcinoma tumors and is a potent inhibitor of angiogenesis. Kinetic analysis using an IAsys resonant mirror biosensor determined the dissociation constant of maspin for collagen type I to be 0.63 microm. Further two-hybrid interactions with maspin truncation constructs suggest that collagen binding is localized to amino acids 84-112 of maspin, which aligns with the collagen-binding region of colligin. A direct interaction between exogenous or cell surface maspin and extracellular matrix collagen may contribute to a cell adhesion role in the prevention of tumor cell migration and angiogenesis.     

An emerging role for the nuclear localization of maspin in the suppression of tumor progression and metastasis

Maspin, a member of the serpin family of serine protease inhibitors, was originally identified as a tumor suppressor that is expressed in normal mammary epithelial cells but is reduced or absent in breast carcinomas. Early enthusiasm for maspin as a biomarker for disease progression has been tempered by clinical data that associates maspin with favourable outcomes in some studies and poor prognosis in others. Here, we review all of the published clinical studies for maspin in breast and ovarian cancers and propose that the apparent discordance between clinical reports is a consequence of differential cellular distribution of maspin. Indeed, it was thought that an extracellular pool of maspin possessed tumor suppressor activity, acting by inhibiting migration and increasing cell adhesion. Recent evidence from our group and others indicates, however, that the nuclear localization of maspin in cancer cells is necessary for its tumor suppressor activity. We provide additional data here to demonstrate that nuclear-localized maspin binds to chromatin and is required to effectively prevent cells from metastasizing. Our knowledge of other serpins that localize to the nucleus should help to inform future studies of nuclear maspin. Elucidation of the molecular mechanisms regulating the localization and activities of maspin should pave the way for the development of improved diagnostics and therapies for cancer.     

Modeling human breast cancer metastasis in mice: maspin as a paradigm

Breast cancer is the most common cancer detected in women, accounting for nearly one out of every three cancers diagnosed in the United States. Most cancer patients do not die from the primary tumor but die due to metastasis. Therefore, the study of metastasis is of most importance both to the clinician and patient. In the past, animal models have been used in breast cancer research and mammary gland biology. Our group has also established several animal models to address the function of a novel tumor suppressor gene maspin in breast tumor progression. Maspin was initially isolated from normal mammary epithelial cells. Its expression was down regulated in breast tumors. To test the protective role of maspin overexpression in mammary tumor progression, we crossed maspin overexpression transgenic mice (WAP-maspin) with a strain of oncogenic WAP-SV40 T antigen mice. The bitransgenic mice had reduced tumor growth rate and metastasis. Maspin overexpression increased the rate of apoptosis of both preneoplastic and carcinomatous mammary epithelial cells. Maspin reduced tumor growth through a combination of reduced angiogenesis and increased apoptosis. In a separate animal experiment, maspin overexpressing mammary tumor cells (TM40D) were implanted into the fat pad of syngeneic mice. TM40D tumor cells were very invasive and metastatic. However, both primary tumor growth and metastasis were significantly blocked in TM40D cells that overexpress maspin as a consequence of plasmid or retrovirus infection. These evidences demonstrate that maspin function to inhibit primary tumor growth as well as invasion and metastasis. Elucidating the molecular mechanism of maspin action will shed light on our understanding of breast cancer invasion and metastasis.     

Inhibition of prostate tumor angiogenesis by the tumor suppressor CEACAM1

We have previously shown that CEACAM1, a cell-adhesion molecule, acts as a tumor suppressor in prostate carcinoma. Expression of CEACAM1 in prostate cancer cells suppresses their growth in vivo. However, CEACAM1 has no effect on the growth of prostate cancer cells in vitro. This difference suggests that the antitumor effect of CEACAM1 may be due to inhibition of tumor angiogenesis, perhaps by increased secretion of antiangiogenic molecules from the cells. In this study, we have demonstrated that expression of CEACAM1 in DU145 prostate cancer cells induced the production of a factor or factors that specifically blocked the growth of endothelial but not epithelial cells. Conditioned medium from the CEACAM1-expressing cells but not control luciferase-expressing cells inhibited endothelial cell migration up a gradient of stimulatory vascular endothelial growth factor in vitro and inhibited corneal neovascularization induced by basic fibroblast growth factor in vivo. Moreover, conditioned medium from CEACAM1-expressing cells induced endothelial cell apoptosis in vitro. Only medium conditioned by CEACAM1 mutants that were able to suppress tumor growth in vivo could cause endothelial cell apoptosis. These observations suggest that CEACAM1-mediated tumor suppression in vivo is, at least in part, due to the ability of CEACAM1 to inhibit tumor angiogenesis.     

Maspin and c-erbB-2 expression in correlation with microvessel density in invasive ductal breast cancer

Maspin is a unique member of the serpin family involved in regulation of cell migration, apoptosis and angiogenesis in breast and prostate cancers. In this study maspin expression in comparison with c-erbB-2 (HER2/neu) oncogene expression and microvessel density was investigated. The examined material included specimens of primary invasive ductal breast cancer derived from 69 patients. They were analyzed immunocytochemically to assess maspin and c-erbB-2 expression, as well as microvessel density using endothelium marker CD31. In the studied cancers, maspin expression in cancer cells was detected in more than half of the cases (50.73%). Although statistically insignificant (p=0.27), maspin expression showed decreasing tendency with the increase of tumor grade. C-erbB-2 oncogene expression was observed in 78.26% of the examined cancers. Statistically significant positive correlation was found between c-erbB-2 expression and tumor grade (p<0.005). Analysis of the dependence between maspin and c-erbB-2 expression exhibited statistically significant inverse correlation (p<0.001). Mean microvessel density (MVD) of the studied cancers was 71.64 (SD=19.36). MVD decreased with the increase of maspin expression, whereas in the cases showing c-erbB-2 overexpression MVD was clearly higher. Both correlations were statistically significant (p<0.005). In conclusion, it could be stated that increase in maspin expression is associated with weaker expression of c-erbB-2 oncogene and lower microvessel density, which implies a significant role of maspin in tumor biology. However, the exact mechanism of maspin action (including its potential role in angiogenesis), as well as the assessment of its prognostic significance in breast cancer require further studies.     

Tumor suppressive maspin and epithelial homeostasis

Maspin is a 42-kDa novel serine protease inhibitor (serpin) with multifaceted tumor suppressive activities. To date, the consensus that maspin expression predicts a better prognosis still largely holds for breast, prostate, colon, and oral squamous cancers. Interestingly, however, more detailed analyses revealed a biphasic expression pattern of maspin in early steps of tumorigenicity and re-expression of maspin in dormant cancer metastatic revertants. These data suggest a sensitivity of maspin expression to changes of epithelial microenvironments, and a role of maspin in epithelial homeostasis. Experimental evidence consistently showed that maspin suppresses tumor growth, invasion and metastasis, induces tumor redifferentiation, and enhances tumor cell sensitivity to apoptosis. Maspin protein isolated from biological sources is a monomer, which is present as a secreted, a cytoplasmic, a nuclear, as well as a cell surface-associated protein. Nuclear maspin is associated with better prognoses of cancer. It is further noted that extracellular maspin is sufficient to block tumor induced extracellular matrix degradation, tumor cell motility and invasion, whereas intracellular maspin is responsible for the increased cellular sensitivity to apoptosis. Despite these exciting developments, the mechanistic studies of maspin have proven challenging primarily due to the lack of a prototype molecular model. Although the maspin sequence has overall homologies with other members in the serpin superfamily, it does not behave like a typical serpin, that is, non-inhibitory toward active serine proteases in solution. This novel feature is in line with the X-ray crystallographic evidence. Several recent studies dedicated to finding the maspin partners support a paradigm shift. The current review is intended to summarize these recent findings and discuss a new perspective of maspin in epithelial homeostasis.