A role of novel serpin maspin in tumor progression: the divergence revealed through efforts to converge

Maspin, a 42 kDa protein, belongs to the serine protease inhibitor (serpin) superfamily and is more closely related to the ovalbumin-like serpin subfamily (ov-serpins). More than a decade after the discovery of the maspin gene, our pursuit of the molecular mechanisms of maspin revealed a significant divergence of maspin from other serpins. This review article summarizes recent advances in the identification of maspin-binding proteins and the potential underlying molecular mechanisms of maspin in tumor progression. Specifically, the molecular interactions of maspin with the cell surface-associated pro-urokinase-type plasminogen activator (pro-uPA) and intracellular histone deacetylase 1 (HDAC1) are highlighted. Our new evidence suggests a new paradigm that maspin acts as a serpin-like molecule to inhibit serine protease-like targets. From an evolution point of view, the uniquely important function of maspin in development and tumor progression is likely due to its ancestral sequence code, and accordingly, its novel "meta"-serpin structure. It is reasonable to hypothesize that the conservation of a serine protease-like catalytic center in many molecules requires the co-existence of endogenous antagonists. The unique inhibitory interaction of maspin with both HDAC1 and pro-uPA might not be substituted by other serpins that have evolved to acquire higher target specificities. Thus, tumor suppressive maspin offers a unique therapeutic opportunity.     

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.     

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.     

HDAC1 inhibition by maspin abrogates epigenetic silencing of glutathione S-transferase pi in prostate carcinoma cells

Both maspin and glutathione S-transferase pi (GSTp) are implicated as tumor suppressors and downregulated in human prostate cancer. It is well established that GSTp downregulation is through DNA methylation-based silencing. We report here that maspin expression in prostate cancer cell line DU145 reversed GSTp DNA methylation, as measured by methylation- specific PCR, MethyLight assay, and bisulfite sequencing. The effect of maspin on GSTp expression was similar to that of the combination of a synthetic histone deacetylase (HDAC) inhibitor and DNA methylation inhibitor 5-aza-2'-deoxycytidine. Maspin expression also led to an increased level of acetylated histone 3, decreased level of methyl transferase, and methyl-CpG-binding domain proteins at the site of demethylated GSTp promoter DNA. Earlier, we have shown that maspin inhibits HDAC1. In PC3 cells, where both maspin and GSTp are expressed at a reduced level, maspin knockdown led to a significant reduction in GSTp expression, whereas dual knockdown of maspin and HDAC1 barely increased the level of GSTp expression. Thus, HDAC1 may play an essential role in cellular response to maspin-mediated GSTp desilencing. Maspin has been shown to increase tumor cell sensitivity to drug-induced apoptosis. Interestingly, GSTp reexpression in the absence of maspin expression perturbation blocked the phosphorylation of histone 2A.X, the induction of hypoxia-induced factor 1α (HIF-1α), and cell death of LNCaP cells under oxidative stress. Because DNA hypermethylation-based silencing may couple with and depend on histone deacetylation, our study suggests that endogenous HDAC inhibition by maspin may prevent pathologic gene silencing in prostate tumor progression.     

G-helix of Maspin Mediates Effects on Cell Migration and Adhesion

Maspin is a member of the serine protease inhibitor (serpin) superfamily that lacks protease inhibitory ability, although displaying tumor metastasis-suppressing activity resulting from its influence on cell migration, invasion, proliferation, apoptosis, and adhesion. The molecular mechanisms of these actions of maspin are as yet undefined. Here, we sought to identify critical functional motifs by the expression of maspin with point mutations at sites potentially involved in protein-protein interactions: the G α-helix (G-helix), an internal salt bridge or the P1 position of the reactive center loop. Our findings indicate that only mutations in the G-helix attenuated inhibition of cell migration by maspin and that this structural element is also involved in the effect of maspin on cell adhesion. The action of maspin on cell migration could be mimicked by a 15-mer G-helix peptide, indicating that the G-helix is both essential and sufficient for this effect. In addition, we provide evidence that the effects of the G-helix of maspin are dependent on β1 integrins. These data reveal that the major extracellular functions associated with the tumor suppressive action of maspin likely involve interactions in which the G-helix plays a key role.     

SMRT‐mediated co‐shuttling enables export of class IIa HDACs independent of their CaM kinase phosphorylation sites

The Class IIa histone deacetylases (HDAC)4 and HDAC5 play a role in neuronal survival and behavioral adaptation in the CNS. Phosphorylation at 2/3 N‐terminal sites promote their nuclear export. We investigated whether non‐canonical signaling routes to Class IIa HDAC export exist because of their association with the co‐repressor Silencing Mediator Of Retinoic And Thyroid Hormone Receptors (SMRT). We found that, while HDAC5 and HDAC4 mutants lacking their N‐terminal phosphorylation sites (HDAC4^MUT, HDAC5^MUT) are constitutively nuclear, co‐expression with SMRT renders them exportable by signals that trigger SMRT export, such as synaptic activity, HDAC inhibition, and Brain Derived Neurotrophic Factor (BDNF) signaling. We found that SMRT's repression domain 3 (RD3) is critical for co‐shuttling of HDAC5^MUT, consistent with the role for this domain in Class IIa HDAC association. In the context of BDNF signaling, we found that HDAC5^WT, which was more cytoplasmic than HDAC5^MUT, accumulated in the nucleus after BDNF treatment. However, co‐expression of SMRT blocked BDNF‐induced HDAC5^WT import in a RD3‐dependent manner. In effect, SMRT‐mediated HDAC5^WT export was opposing the BDNF‐induced HDAC5 nuclear accumulation observed in SMRT's absence. Thus, SMRT's presence may render Class IIa HDACs exportable by a wider range of signals than those which simply promote direct phosphorylation.     

PTEN and p53 are required for hypoxia induced expression of maspin in glioblastoma cells

In response to genotoxic stress, p53 induces the tumor suppressors maspin and PTEN. Here we demonstrate that in response to limited oxygen conditions PTEN and p53 work in tandem to induce maspin in glioblastoma cells. In response to hypoxia a portion of PTEN migrates to the nucleus and complexes with p53, while cytoplasmic PTEN prevents Mdm2 nuclear localization by attenuating Akt signaling. Subcellular distribution of PTEN in the cytoplasm or nucleus protects p53 from inac-tivation and degradation. The presence of nuclear PTEN and p53 coordinates the induction of maspin and p21 (both p53 gene targets) in response to hypoxia. Altering the expression of PTEN and/or p53 attenuated maspin gene induction under hypoxic conditions. Furthermore, implanting U87 (PTEN null) and PTEN reconstituted U87 cells (U87PTEN) in mice we observed by immuno-histochemistry and western blot that Maspin was only detectable in cells with PTEN. The integra-tion of PTEN and p53 into a common pathway for the induction of another tumor suppressor, Maspin, constitutes a tumor suppressor network of PTEN/p53/Mapsin that is operational under limited oxygen conditions.     

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.     

Protein kinase C-related kinase targets nuclear localization signals in a subset of class IIa histone deacetylases

Class IIa histone deacetylases (HDACs) -4, -5, -7 and -9 undergo signal-dependent nuclear export upon phosphorylation of conserved serine residues that are targets for 14-3-3 binding. Little is known of other mechanisms for regulating the subcellular distribution of class IIa HDACs. Using a biochemical purification strategy, we identified protein kinase C-related kinase-2 (PRK2) as an HDAC5-interacting protein. PRK2 and the related kinase, PRK1, phosphorylate HDAC5 at a threonine residue (Thr-292) positioned within the nuclear localization signal (NLS) of the protein. HDAC7 and HDAC9 contain analogous sites that are phosphorylated by PRK, while HDAC4 harbors a non-phosphorylatable alanine residue at this position. We provide evidence to suggest that the unique phospho-acceptor cooperates with the 14-3-3 target sites to impair HDAC nuclear import.

Structured summary

MINT-7710106:HDAC5 (uniprotkb:Q9UQL6) physically interacts (MI:0915) with PRK2 (uniprotkb:Q16513) by pull down (MI:0096)     

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.     

Tumor-suppressive maspin regulates cell response to oxidative stress by direct interaction with glutathione S-transferase

Maspin, a novel serine protease inhibitor, suppresses tumor progression in several cancer models, including an in vivo model for prostate cancer bone metastasis. However, the molecular mechanism of maspin remains illusive, primarily because its molecular targets are unknown. To this end, we used a full-length maspin cDNA bait to screen against both a primary prostate tumor cDNA prey library and a HeLa cDNA prey library by the yeast two-hybrid method. We found that heat shock protein 90, glutathione S-transferase (GST), and heat shock protein 70 interacted with maspin with the highest frequencies. We confirmed the maspin/GST interaction using purified proteins, human epithelial cell lines, and human prostate tissues. A maspin variant that has a point mutation of Arg(340) to Ala (Mas(R340A)) showed a significantly decreased affinity for GST. Although purified maspin had no effect on the activity of purified GST in vitro, intracellular interaction between endogenous maspin and GST correlated with an elevated total GST activity in both MDA-MB-435- and DU145-derived stably transfected cells. Consistently, tumor cells treated with purified wild type maspin, but not Mas(R340A), enhanced cellular GST activity. Maspin expression in cancer cell lines also correlated with decreased basal levels of reactive oxygen species (ROS). Furthermore, H(2)O(2) treatment not only induced GST expression but also increased intracellular maspin/GST interaction, which was inversely correlated with the level of ROS generation. Conversely, maspin knockdown by small interfering RNA increased the basal, as well as H(2)O(2)-induced, ROS generation. Furthermore, the maspin effect on ROS generation was completely abolished by a GST inhibitor, indicating an essential role of GST in maspin-mediated cellular response to oxidative stress. Consistently, oxidative stress-induced vascular endothelial growth factor A expression was significantly inhibited in maspin-expressing cells. Together, our data suggest a new mechanism by which maspin, through its direct interaction with GST, may inhibit oxidative stress-induced ROS generation and vascular endothelial growth factor A induction, thus preventing further adverse effects on tumor genetics and stromal reactivity.     

EspP,an Extracellular Serine Protease from Enterohemorrhagic E. coli,Reduces Coagulation Factor Activities,Reduces Clot Strength,and Promotes Clot Lysis

Background

EspP (E. coli secreted serine protease, large plasmid encoded) is an extracellular serine protease produced by enterohemorrhagic E. coli (EHEC) O157:H7, a causative agent of diarrhea-associated Hemolytic Uremic Syndrome (D+HUS). The mechanism by which EHEC induces D+HUS has not been fully elucidated.

Objectives

We investigated the effects of EspP on clot formation and lysis in human blood.

Methods

Human whole blood and plasma were incubated with EspP^WT at various concentrations and sampled at various time points. Thrombin time (TT), prothrombin time (PT), and activated partial thromboplastin time (aPTT), coagulation factor activities, and thrombelastgraphy (TEG) were measured.

Results and Conclusions

Human whole blood or plasma incubated with EspP^WT was found to have prolonged PT, aPTT, and TT. Furthermore, human whole blood or plasma incubated with EspP^WT had reduced activities of coagulation factors V, VII, VIII, and XII, as well as prothrombin. EspP did not alter the activities of coagulation factors IX, X, or XI. When analyzed by whole blood TEG, EspP decreased the maximum amplitude of the clot, and increased the clot lysis. Our results indicate that EspP alters hemostasis in vitro by decreasing the activities of coagulation factors V, VII, VIII, and XII, and of prothrombin, by reducing the clot strength and accelerating fibrinolysis, and provide further evidence of a functional role for this protease in the virulence of EHEC and the development of D+HUS.     

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.     

Maspin, the molecular bridge between the plasminogen activator system and beta1 integrin that facilitates cell adhesion

Maspin is a non-inhibitory serine protease inhibitor (serpin) that influences many cellular functions including adhesion, migration, and invasion. The underlying molecular mechanisms that facilitate these actions are still being elucidated. In this study we determined the mechanism by which maspin mediates increased MCF10A cell adhesion. Utilizing competition peptides and mutation analyses, we discovered two unique regions (amino acid residues 190-202 and 260-275) involved in facilitating the increased adhesion function of maspin. In addition, we demonstrate that the urokinase-type plasminogen activator (uPA)/uPA receptor (uPAR) complex is required for the localization and adhesion function of maspin. Finally, we showed that maspin, uPAR, and β1 integrin co-immunoprecipitate, suggesting a novel maspin-uPA-uPAR-β1 integrin mega-complex that regulates mammary epithelial cell adhesion.     

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.     

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.     

Maspin is an angiogenesis inhibitor

Maspin, a unique member of the serpin family, is a secreted protein encoded by a class II tumor suppressor gene whose downregulation is associated with the development of breast and prostate cancers. Overexpression of maspin in breast tumor cells limits their growth and metastases in vivo. In this report we demonstrate that maspin is an effective inhibitor of angiogenesis. In vitro, it acted directly on cultured endothelial cells to stop their migration towards basic fibroblast growth factor and vascular endothelial growth factor and to limit mitogenesis and tube formation. In vivo, it blocked neovascularization in the rat cornea pocket model. Maspin derivatives mutated in the serpin reactive site lost their ability to inhibit the migration of fibroblasts, keratinocytes, and breast cancer cells but were still able to block angiogenesis in vitro and in vivo. When maspin was delivered locally to human prostate tumor cells in a xenograft mouse model, it blocked tumor growth and dramatically reduced the density of tumor-associated microvessels. These data suggest that the tumor suppressor activity of maspin may depend in large part on its ability to inhibit angiogenesis and raise the possibility that maspin and similar serpins may be excellent leads for the development of drugs that modulate angiogenesis.