Maspin binds to cardiolipin in mitochondria and triggers apoptosis.

A central question in cell biology is how cells respond to stress signals and biochemically regulate apoptosis. One critical pathway involves the change of mitochondrial function and release of cytochrome c to initiate apoptosis. In response to apoptotic stimuli, we found that maspin-a noninhibitory member of the serine protease inhibitor superfamily-translocates from the cytosol to mitochondria and binds to cardiolipin in the inner mitochondrial membrane. Biolayer interferometry assay revealed that recombinant maspin binds cardiolipin with an apparent Kd,of ∼15.8 µM and competes with cytochrome c (apparent Kd of ∼1.31 µM) for binding to cardiolipin-enriched membranes. A hydrophobic, lysine-rich domain in maspin consists of 27 aa, is located at position 268-294, and is responsible for the interaction of this protein with cardiolipin. Depletion of cardiolipin in cells significantly prevents maspin binding to the inner mitochondrial membrane and decreases cytochrome c release and apoptosis. Alteration to maspin's cardiolipin binding domain changes its ability to bind cardiolipin, and tumor cells expressing this mutant have a low frequency of apoptosis. We propose a model of apoptosis in which maspin binds to cardiolipin, displaces cytochrome c from the membrane, and facilitates its release to the cytoplasm.-Mahajan, N., Hoover, B., Rajendram, M., Shi, H. Y., Kawasaki, K., Weibel, D. B., Zhang, M. Maspin binds to cardiolipin in mitochondria and triggers apoptosis.

Supramolecular assembly of multifunctional maspin-mimetic nanostructures as a potent peptide-based angiogenesis inhibitor.

Aberrant angiogenesis plays a large role in pathologies ranging from tumor growth to macular degeneration. Anti-angiogenic proteins have thus come under scrutiny as versatile, potent therapeutics but face problems with purification and tissue retention. We report here on the synthesis of supramolecular nanostructures that mimic the anti-angiogenic activity of maspin, a class II tumor suppressor protein. These maspin-mimetic nanostructures are formed via self-assembly of small peptide amphiphiles containing the g-helix motif of maspin. Using tubulogenesis assays with human umbilical vein endothelial cells, we demonstrate that maspin-mimetic nanostructures show anti-angiogenic activity at concentrations that are significantly lower than those necessary for the g-helix peptide. Furthermore, in vivo assays in the chick chorioallantoic membrane show maspin-mimetic nanostructures to be effective over controls at inhibiting angiogenesis. Thus, the nanostructures investigated here offer an attractive alternative to the use of anti-angiogenic recombinant proteins in the treatment of cancer or other diseases involving abnormal blood vessel formation.

Tumor-suppressive maspin functions as a reactive oxygen species scavenger: importance of cysteine residues.

Maspin is a member of the serine protease inhibitor (serpin) superfamily and displays tumor-suppressing activity by controlling cell migration, proliferation, apoptosis, and adhesion. Here, we provide evidence that maspin acts as a reactive oxygen species (ROS) scavenger through oxidation of three structurally exposed cysteine thiols to sulfenic acid. Ablation of these cysteine residues in maspin resulted in a significant increase in total ROS production in mouse mammary TM40D cells. Also, cells containing a triple-cysteine mutant of maspin showed elevated ERK1/2 activity, a downstream target of ROS, and enhanced proliferation and colony formation. These findings establish a novel mechanism by which maspin utilizes its cysteine thiols to inhibit oxidative stress and cell growth.

Regulation of breast cancer-induced bone lesions by ß-catenin protein signaling.

Breast cancer patients have an extremely high rate of bone metastases. Morphological analyses of the bones in most of the patients have revealed the mixed bone lesions, comprising both osteolytic and osteoblastic elements. ß-Catenin plays a key role in both embryonic skeletogenesis and postnatal bone regeneration. Although this pathway is also involved in many bone malignancy, such as osteosarcoma and prostate cancer-induced bone metastases, its regulation of breast cancer bone metastases remains unknown. Here, we provide evidence that the ß-catenin signaling pathway has a significant impact on the bone lesion phenotype. In this study, we established a novel mouse model of mixed bone lesions using intratibial injection of TM40D-MB cells, a breast cancer cell line that is highly metastatic to bone. We found that both upstream and downstream molecules of the ß-catenin pathway are up-regulated in TM40D-MB cells compared with non-bone metastatic TM40D cells. TM40D-MB cells also have a higher T cell factor (TCF) reporter activity than TM40D cells. Inactivation of ß-catenin in TM40D-MB cells through expression of a dominant negative TCF4 not only increases osteoclast differentiation in a tumor-bone co-culture system and enhances osteolytic bone destruction in mice, but also inhibits osteoblast differentiation. Surprisingly, although tumor cells overexpressing ß-catenin did induce a slight increase of osteoblast differentiation in vitro, these cells display a minimal effect on osteoblastic bone formation in mice. These data collectively demonstrate that ß-catenin acts as an important determinant in mixed bone lesions, especially in controlling osteoblastic effect within tumor-harboring bone environment.

Evidence of post-translational modification of the tumor suppressor maspin under oxidative stress.

Maspin, identified as a 42 kDa unique tumor suppressive serine protease inhibitor (serpin), has multifaceted biological functions by interacting with various target molecules under physiological and pathological conditions including oxidative stress. However, the type of post-translational modification that confers the specific binding affinity of maspin to target molecules, such as glutathione S-transferase (GST), has not been determined. The aim of this study, therefore, is to analyze the molecular heterogeneity of maspin and to identify its modifications in the normal mammary epithelial cell line, MCF-10A, which is known to express the maspin protein abundantly, using electrophoretic analysis. Conventional SDS-PAGE analysis of MCF-10A cell extracts showed that endogenous maspin is composed of both an intact form observed as a 42 kDa band and a smaller form observed as a 36 kDa band. Interestingly, a brief exposure of MCF-10A cells to 10 mM hydrogen peroxide (H2O2) led to the appearance of a novel endogenous maspin form, as demonstrated by non-denaturing PAGE and non-reducing SDS-PAGE. Two-dimensional sequential non-reducing/reducing SDS-PAGE supported that this novel form was generated by intramolecular disulfide-bonded linkage under oxidative stress, and this oxidized maspin form also existed under physiological conditions. Furthermore, a glutathione (GSH) bead pull-down assay revealed that the intramolecular disulfide-bonded maspin lost its binding activity to endogenous GST, indicating that intramolecular disulfide-bonded maspin might have some distinct properties under oxidative stress, although the precise biological significance of this modification remains elusive. In conclusion, we have shown that maspin undertakes different modifications under oxidative stress.

Transcriptional regulation of p21/CIP1 cell cycle inhibitor by PDEF controls cell proliferation and mammary tumor progression.

The Ets family of transcription factors control a myriad of cellular processes and contribute to the underlying genetic loss of cellular homeostasis resulting in cancer. PDEF (prostate-derived Ets factor) has been under investigation for its role in tumor development and progression. However, the role of PDEF in cancer development has been controversial. Some reports link PDEF to tumor promoter, and others show tumor-suppressing functions in various systems under different conditions. So far, there has been no conclusive evidence from in vivo experiments to prove the role of PDEF. We have used both in vitro and in vivo systems to provide a conclusive role of PDEF in the progression process. PDEF-expressing cells block the cell growth rate, and this retardation was reversible when PDEF expression was silenced with PDEF-specific small interfering RNA. When these PDEF-expressing cells were orthotopically implanted into the mouse mammary gland, tumor incidence and growth rate were significantly retarded. Cell cycle analysis revealed that PDEF expression partially blocked cell cycle progression at G(1)/S without an effect on apoptosis. PDEF overexpression resulted in an increase in p21/CIP1 at both the mRNA and protein levels, resulting in decreased Cdk2 activity. Promoter deletion analysis, electrophoresis mobility shift assays, and chromatin immunoprecipitation studies identified the functional Ets DNA binding site at -2118 bp of the p21/CIP1 gene promoter. This site is capable of binding and responding to PDEF. Furthermore, we silenced p21/CIP1 expression in PDEF-overexpressing cells by small interfering RNA. p21-silenced PDEF cells exhibited significantly increased cell growth in vitro and in vivo, demonstrating the p21 regulation by PDEF as a key player. These experiments identified PDEF as a new transcription factor that directly regulates p21/CIP1 expression under non-stressed conditions. This study conclusively proves that PDEF is a breast tumor suppressor for the first time using both in vitro and in vivo systems. PDEF can be further developed as a target for designing therapeutic intervention of breast cancer.

Haploinsufficiency of the maspin tumor suppressor gene leads to hyperplastic lesions in prostate.

Maspin is a key tumor suppressor gene in prostate and breast cancers with diverse biological functions. However, how maspin regulates prostate tumor progression is not fully understood. In this study, we have used maspin heterozygous knockout mice to determine the effect of maspin haploinsufficiency on prostate development and tumor progression. We report that loss of one copy of maspin gene in Mp(+/-) heterozygous knockout mice leads to the development of prostate hyperplastic lesions, and this effect was mediated through decreased level of cyclin-dependent kinase inhibitors p21 and p27. Prostate hyperplastic lesions in Mp(+/-) mice also induced stromal reaction, which occurred in both aged prostate tissues and in neonatal prostates during early ductal morphogenesis. We showed that maspin was also expressed in prostate smooth muscle cells (PSMC), and recombinant maspin increased PSMC cell adhesion but inhibited cell proliferation. We also observed a defective interaction between epithelial cells and basement membrane in the prostate of Mp(+/-) mice, which was accompanied with a changed pattern of matrix deposition and a loss of epithelial cell polarity. Therefore, we have identified a novel property of maspin, which involves the control of the proliferation in prostate epithelial and smooth muscle cells. This is the first report that a partial loss of maspin caused an early developmental defect of the prostate and prostate hyperplastic lesions in mouse.

Maspin controls mammary tumor cell migration through inhibiting Rac1 and Cdc42, but not the RhoA GTPase.

Rac1 and Cdc42 are members of the Rho family of small GTPases that play essential roles in diverse cellular functions, including cell migration. The activities of these Rho family proteins are controlled by growth factor receptor activation and cell-ECM interactions. Here, we show that maspin, a well-documented tumor suppressor gene, also controls cell motility through inhibiting Rac1/Cdc42 activity. Using the GST-PAK and GST-Rho binding protein pull-down assays for GTP-bound Rac1, Cdc42, and RhoA, we showed that treatment of MDA-MB-231 tumor cells with recombinant maspin for a short time period significantly inhibited the activity of Rac1 and Cdc42, but not RhoA. The reactive site loop (RSL) within maspin protein is the functional domain involved in the inhibition. Maspin mutants with the RSL deleted or a point mutation in the RSL region lost their inhibitory activity. We further examined the ability of maspin to inhibit Rac1- and Cdc42-mediated signaling pathways and transcription factors. Treatment of MDA-MB-231 cells with maspin led to the inhibition of JNK kinase activity as assayed by immuno-kinase assays. In addition, the AP-1 transcription activity downstream of JNK kinase pathway was also reduced. Together, we have identified Rac1 and Cdc42 as the downstream targets that mediate the inhibition of mammary tumor cell migration by maspin.

Maspin overexpression modulates tumor cell apoptosis through the regulation of Bcl-2 family proteins.

BACKGROUND: Maspin is a member of serpin family with tumor suppressing activity. Recent studies of maspin in animal models strongly support maspin's role as an inhibitor against the growth of primary tumor sand the process of metastasis. However, the molecular mechanism underlying this inhibition has not been fully elucidated. In this report, we analyze the effect of maspin on tumor cell apoptosis under several stress conditions. METHODS: Stable clones overexpressing maspin are established in the mouse mammary tumor TM40D cells. They are treated with staurosporine, TNF-alpha, and serum starvation. The rates of cell apoptosis are analyzed by TUNEL assay. Inhibitors against caspase 8 and 9 are used in the apoptosis assay. Western blot analysis and ribonuclease protection assay (RPA) are performed to examine the expression of Bcl2 family genes. RESULTS: We report that maspin expressing tumor cells have increased rate of apoptosis when they are treated with staurosporine and serum starvation. The effect is not through extracellular maspin. Maspin-mediated apoptosis is partially blocked by caspase 8 and 9 inhibitors, and is accompanied by changes in the Bcl-2 family proteins. Maspin-expressing tumor cells have a reduced level of anti-apoptotic protein Bcl-2, and an increased level of pro-apoptotic protein Bax. The regulation is not controlled at the transcriptional level but is through selective control of Bcl-2 and Bax protein stability. CONCLUSION: Maspin overexpression modulates tumor cell apoptosis through the regulation of Bcl2 family proteins. Such change results in an increased release of cytochrome c from mitochondria, thus the increased apoptosis in maspin-expressing cells. This evidence strongly suggests that the induction of apoptosis in maspin-overexpressing cells represents a major mechanism by which maspin inhibits breast tumor progression.

Maspin mediates increased tumor cell apoptosis upon induction of the mitochondrial permeability transition.

Maspin is a unique serpin with the ability to suppress certain types of malignant tumors. It is one of the few p53-targeted genes involved in tumor invasion and metastasis. With this in mind, we attempted to study the molecular mechanism behind this tumor suppression. Maspin-expressing mammary tumors are more susceptible to apoptosis in both implanted mammary tumors in vivo, a three-dimensional spheroid culture system, as well as in monolayer cell culture under lowered growth factors. Subcellular fractionation shows that a fraction of maspin (in both TM40D-Mp and mutant maspinDeltaN cells) translocates to the mitochondria. This translocation of maspin to the mitochondria is linked to the opening of the permeability transition pore, which in turn causes the loss of transmembrane potential, thus initiating apoptotic degradation. This translocation is absent in the other mutant, maspinDeltaRSL. It fails to cause any loss of membrane potential and also shows decreased caspase 3 levels, proving that translocation to the mitochondria is a key event for this increase in apoptosis by maspin. Suppression of maspin overexpression by RNA interference desensitizes cells to apoptosis. Our data indicate that maspin inhibits tumor progression through the mitochondrial apoptosis pathway. These findings will be useful for maspin-based therapeutic interventions against breast cancer.

Targeted expression of maspin in tumor vasculatures induces endothelial cell apoptosis.

Angiogenesis, the formation of new blood vessels, is required for normal tissue development and pathological conditions such as tumorigenesis. Most solid tumors can not grow beyond a few millimeters without the recruitment of neovessels since cancer cells require access to blood vessels for nutrients and to escape the local environment and metastasize to other tissue and organ sites. Targeting tumor vessel endothelium therefore should serve as an effective therapy for cancers. Maspin is a serpin that exhibits antiangiogenic properties. In this report, we show that when maspin overexpression is targeted in vivo to endothelial cells, it actively induces endothelial cell apoptosis. Intravascular administration of adenovirus-maspin to mice bearing mammary tumors disrupts tumor-induced angiogenesis. Interestingly, tumor neovessels become leaky after maspin treatment, whereas normal mature vessels are not affected by maspin treatment. We further demonstrate that maspin directly induces endothelial cell apoptosis in vitro, and this effect is maspin specific. The induction of apoptosis is accompanied by changes in the expression of Bcl-2 family genes and is blocked by caspase inhibitors. In addition, the apoptotic effect is mediated by intracellular maspin and is dependent on the RSL region of maspin. Furthermore, we have shown that transient overexpression of Bcl-2 protected the HUVECs from maspin-mediated apoptosis, and the presence of both maspin and Bax accelerated the apoptosis process. These findings demonstrate that neovascular endothelial cells are highly sensitive to maspin level inside the cells. This property can be used for targeted therapy against tumor angiogenesis and metastasis.

Hormonal defect in maspin heterozygous mice reveals a role of progesterone in pubertal ductal development.

Progesterone and PR are mainly thought to affect tertiary ductal side branching and alveologenesis in late stage of mammary gland development. Here, we present evidence that they also play a role in early ductal development. This conclusion derived from our analysis of maspin heterozygous (Mp+/-) mice that showed defective ductal development at puberty. The defect was due to a reduced systemic level of progesterone. We show that treatment of Mp+/- mice with progesterone rescued the defect of ductal development. When both wild-type and Mp+/- mice were ovariectomized at 4 wk of age, treatment with progesterone alone can stimulate their ductal growth. In addition, treatment of wild-type mice with the progesterone inhibitor RU486 slowed ductal development in a dose-dependent manner. To confirm that progesterone receptor (PR) was required for progesterone action in ductal development at pubertal stage, we treated ovariectomized PR-deficient (PRKO) and wild-type mice with progesterone and examined ductal development at 7 wk of age. Whereas wild-type mammary glands displayed abundant ductal growth after progesterone treatment, there was a significant retardation of ductal growth in PRKO mice. Furthermore, we observed reduced ductal development in intact PRKO mice at 7 wk of age compared with that of wild-type mice. However, the defect was rescued at late stage of mammary development in PRKO mice. These data demonstrate that progesterone signaling, which is mediated by PR, plays an important role in early ductal development. In PRKO mice, a compensatory mechanism occurs that rescues the ductal defect at a late stage of mammary development.

Tamoxifen induces the expression of maspin through estrogen receptor-alpha.

Maspin (mammary serine protease inhibitor) is a tumor suppressor gene that plays an important role in inhibiting tumor growth, invasion and metastasis. Maspin expression is down regulated at transcription level in primary and metastatic breast tumor cells. Previous studies on hormonal regulation of maspin prompt us to test whether an estrogen antagonist tamoxifen (TAM) can exert its anti-tumor function by up regulating maspin gene expression. For this purpose, we first tested whether maspin promoter could be activated in normal and several breast tumor cells. We then carried out a series of promoter analysis in which estrogen receptors and TAM were reconstituted in an in vitro cell culture system. Here we report our new finding that tumor suppresser gene maspin is one of the TAM target genes. TAM induces a maspin/luciferase reporter in cell culture and this induction requires the presence of (estrogen receptor alpha) ERalpha but not estrogen receptor-beta (ERbeta). Maspin promoter deletion and mutation analysis showed that the cis element(s) within a region between -90and+87 bp but not the HRE site (-272 bp) was involved in TAM induction of maspin expression. TAM bound ERalpha may directly control maspin gene expression through the interaction with cofactor (s). Analysis using several ERalpha mutants showed that the N-terminal A/B motif (AF-1) was critical for maspin basal level transcription activation. An ERalpha mutant with point mutations at DNA binding domain abolished estrogen induction of an ERE-luciferase reporter but was still active in activating maspin promoter by TAM. LBD-AF2 domain was required for ERalpha-dependent TAM induction. Deletion of LBD-AF2 or a point mutation in the ERalpha LBD-AF2 region (LBDmtL539A) completely abolished the activation of maspin promoter, suggesting that TAM induction of maspin involves the recruitment of cofactor(s) by ERalpha to the maspin promoter region. This finding indicates that one of the pathways for cancer prevention and tumor inhibition by TAM is mediated through the activation of tumor suppressor gene maspin in breast cancer.

Maspin plays an essential role in early embryonic development.

Maspin (Mp) is a member of the serpin family with inhibitory functions against cell migration, metastasis and angiogenesis. To identify its role in embryonic development in vivo, we generated maspin knockout mice by gene targeting. In this study, we showed that homozygous loss of maspin expression was lethal at the peri-implantation stage. Maspin was specifically expressed in the visceral endoderm after implantation; deletion of maspin interfered with the formation of the endodermal cell layer, thereby disrupting the morphogenesis of the epiblast. In vitro, the ICM of the Mp(-/-) blastocysts failed to grow out appropriately. Data from embryoid body formation studies indicated that the Mp(-/-) EBs had a disorganized, endodermal cell mass and lacked a basement membrane layer. We showed that the embryonic ectoderm lineage was lost in the Mp(-/-) EBs, compared with that of the Mp(+/+) EBs. Re-expression of maspin partially rescued the defects observed in the Mp(-/-) EBs, as evidenced by the appearance of ectoderm cells and a layer of endoderm cells surrounding the ectoderm. In addition, a maspin antibody specifically blocked normal EB formation, indicating that maspin controls the process through a cell surface event. Furthermore, we showed that maspin directly increased endodermal cell adhesion to laminin matrix but not to fibronectin. Mp(+/-) endodermal cells grew significantly slower than Mp(+/+) endodermal cells on laminin substrate. We conclude that deletion of maspin affects VE function by reducing cell proliferation and adhesion, thereby controlling early embryonic development.

Inhibition of breast tumor progression by systemic delivery of the maspin gene in a syngeneic tumor model.

Maspin has been shown to possess tumor-suppressing activity against breast tumor growth and metastasis. To test the therapeutic value of the maspin gene (SERPINB5) in breast cancer, we established a syngeneic breast tumor metastasis model. This model involved the implantation of mammary tumor cells orthotopically to mammary gland and allowed tumors to grow within the gland and become metastatic to other organs. The mammary tumor cells were initially isolated from MMTV-polyoma virus middle T transgenic mice and were selected in vitro for high invasiveness. Here, we demonstrate that the mammary tumor cells were highly invasive and metastatic. Overall, 100% of tumor-transplanted mice developed lung metastasis. Using nonviral liposome as a carrier, we delivered SERPINB5 to mice bearing mammary tumors. Our data showed that both primary tumor growth and metastasis were significantly inhibited in this syngeneic metastasis model. Such inhibition is mediated by SERPINB5 transgene through increased apoptosis in SERPINB5-treated tumors. Thus, SERPINB5 can be used in gene therapy against breast tumor growth and metastasis.