Maspin Overexpression Research Articles

Role of cytoplasmic localization of maspin in promoting cell invasion in breast cancer with aggressive phenotype

Mammary serine protease inhibitor (maspin) is a tumor suppressor gene that is downregulated during carcinogenesis and breast cancer progression. While the nuclear localization of maspin is essential for tumor suppression, we previously reported that the cytoplasmic localization of maspin was significantly correlated with poor prognosis in breast cancer patients. To understand the mechanisms that underlie oncogenic role of cytoplasmic maspin, we studied its biological function in breast cancer cell lines. Subcellular localization of maspin in MDA-MB-231 breast cancer cells was mainly detected in the cytoplasm, whereas in MCF10A mammary epithelial cells, maspin was present in both cytoplasm and nucleus. In MDA-MB-231 cells, maspin overexpression promoted cell proliferation and cell invasion, whereas maspin downregulation resulted in the opposite effect. Further, we observed that SRGN protein levels were increased in MDA-MB-231 cells stably overexpressing maspin. Finally, maspin overexpression in MDA-MB-231 cells resulted in the N-cadherin and epithelial mesenchymal transition (EMT)-related transcription factors upregulation, and TGFβ signaling pathway activation. These results suggested that cytoplasmic maspin enhances the invasive and metastatic potential in breast cancer cells with aggressive phenotype by inducing EMT via SRGN/TGFβ axis. This study demonstrated a novel biological function of cytoplasmic maspin in progression of breast cancer cells with an aggressive phenotype.

Zinc oxide nanoparticles enhance expression of maspin in human breast cancer cells.

Toxic and apoptotic impacts of zinc oxide nanoparticle (ZNP) on different cancer cells have been reported. Maspin (a mammary serine protease inhibitor) as a tumor suppressor gene can inhibit tumor growth and metastasis. The expression of maspin is modulated by p53, Bcl-2 family genes, and estrogen receptor α (ER-α). This study aimed to assess the ZNP effects on maspin expression in MCF-7 cells (a breast cancer cell). Experimental groups (ZNP5, ZNP10, and ZNP20) received 5, 10, and 20μM/mL ZNP for 48h, respectively. 17-β-estradiol (E2) was used to evaluate the role of ER-α in the anticancer impact of ZNP. Cell viability, Annexin V, migration assay, gene expression, and western blotting methods were applied to evaluate ZNP effects on the MCF-7 cells. ZNP at the concentrations of 10 and 20μM/mL could significantly decrease the viability and migration rate, and significantly increase apoptosis percentage in the MCF-7 cells. ZNP significantly enhanced mRNA expression and protein level of maspin in MCF-7 cells in a concentration-dependent way. ZNP concentration-dependently elevated mRNA expression and protein level of p53 and Bax while reduced the expression of Bcl-2 and ER-α. E2 promoted cancer cell growth by enhancing survival and migration rates. E2 treatment reduced mRNA expression and protein level of maspin and p53, and elevated Bcl-2 expression. ZNP considerably changed these events induced by E2 in the MCF-7 cells. It is concluded that the maspin overexpression is one of the toxic mechanisms of the ZNP on the ER-α-positive breast cancer cells, and can suppress the migration of these cells.

Maspin as a Prognostic Marker for Early Stage Colorectal Cancer With Microsatellite Instability.

Colorectal cancers are among the most common cancers and a leading cause of cancer death. In our pursuit to discover molecular markers for better characterization and precision theranostics of these cancers, we first conducted global deep proteome analyses and identified maspin (serpin B5, peptidase inhibitor 5) as an upregulated protein in tumor tissue. We then validated its expression in a large cohort of 743 patients with colorectal cancers of all stages and found that both cytoplasmic and nuclear expression varied widely between different patients. Comparison with clinicopathological features revealed that maspin expression levels correlate significantly only with mismatch repair (MMR) status but not with other features. To elucidate the prognostic significance of maspin, we analyzed two outcome-annotated cohorts, one of 572 early stage cancer patients and another of 93 late stage cancer patients. Kaplan–Meier survival, univariate, and multivariate analyses revealed that maspin overexpression predicts longer overall and disease-free survival for early stage microsatellite instability (MSI) subtype colorectal cancer, but there is no correlation with survival for patients with early stage cancer of the microsatellite stability (MSS) subtype or late stage cancer. Our study identifies maspin expression as an independent prognostic marker for risk stratification of early stage MSI subtype colorectal cancer and may provide guidance for improved therapeutic management.

Maspin suppresses growth, proliferation and invasion in cutaneous squamous cell carcinoma cells.

Cutaneous squamous cell carcinoma (cSCC) is a common malignant tumor. Mammary serine protease inhibitor (Maspin), a member of serpin family, has been reported as a tumor suppressor in various carcinomas. In this study, we detected the expression level of Maspin in cSCC tissues by real-time PCR and western blotting, and found that Maspin was downregulated in the cSCC tissues compared with the adjacent normal tissues. Moreover, Maspin was stably overexpressed in A431 cells, and CCK-8 assay, colony formation assay, Transwell assay, Hoechst staining and western blotting were carried out to detect the growth, proliferation, invasion, cell cycle and apoptosis of A431 cells. The results revealed that overexpression of Maspin inhibited growth, proliferation, invasion and cell cycle G1/S/G2 transition and enhanced apoptosis of A431 cells. The pro-apoptotic protein cleaved caspase-3, poly(ADP-ribose) polymerase (PARP) and Bax increased, and the anti-apoptotic protein Bcl-2 decreased after Maspin overexpression. Therefore, we demonstrated that Maspin suppressed growth, proliferation and invasion by delaying cell cycle transition and promoting apoptosis in cSCC cells, which may provide new insights for the clinical diagnosis and therapy of cSCC.

Maspin enhances cisplatin chemosensitivity in bladder cancer T24 and 5637 cells and correlates with prognosis of muscle-invasive bladder cancer patients receiving cisplatin based neoadjuvant chemotherapy.

BackgroundMaspin, a non-inhibitory member of the serine protease inhibitor superfamily, has been characterized as a tumor suppressor gene in multiple cancer types. Chemotherapeutic insensitivity is one of major obstacles to effectively treating muscle invasive bladder cancer (MIBC). This study was conducted to investigate the role and probable mechanism of Maspin enhancing cisplatin chemosensitivity of bladder cancer in vitro and MIBC patients.MethodsMaspin expression was quantified by qRT-PCR in two MIBC cell lines (T24 and 5637). After successful established Maspin overexpression model by lipidosome transfection, MTT and cell apoptosis assay were used to assess the MIBC’s cisplatin sensitivity. Western blot method was used to test PI3K/ AKT/mTOR signal passway and apoptosis related molecules Caspase3 and Bcl-2. Additionally, we evaluated Maspin expression and prognosis in 62 MIBC cases who underwent cisplatin based neoadjuvant chemotherapy (NACT) using immunohistochemistry.ResultUpregulate Maspin expression could enhance the chemosensitivity induced by cisplatin in T24 and 5637 cell lines. The cell viability, cloning ability and IC50 were reduced while apoptosis rate was upregulated when cells were transfected Maspin. Phospho(p)-AKT, PI3K, mTOR, and Bcl-2 expression were significantly decreased, whereas Caspase3 was greatly increased in the Maspin group. In the clinic study, there was significant correlation between Maspin expression and overall survival (OS) and progression-free survival (PFS) rate in MIBC patients who received cisplatin based NACT.ConclusionMaspin could enhance cisplatin chemosensitivity in T24 and 5637 cell lines. Its expression correlated with prognosis of MIBC patients who received cisplatin based neoadjuvant chemotherapy.

Maspin influences response to doxorubicin by changing the tumor microenvironment organization

Altered degradation and deposition of extracellular matrix are hallmarks of tumor progression and response to therapy. From a microarray supervised analysis on a dataset of chemotherapy-treated breast carcinoma patients, maspin, a member of the serpin protease inhibitor family, has been the foremost variable identified in non-responsive versus responsive tumors. Accordingly, in a series of 52 human breast carcinomas, we detected high maspin expression in tumors that progressed under doxorubicin (DXR)-based chemotherapy. Our analysis of the role of maspin in response to chemotherapy in human MCF7 and MDAMB231 breast and SKOV3 ovarian carcinoma cells transfected to overexpress maspin and injected into mice showed that maspin overexpression led to DXR resistance through the maspin-induced collagen-enriched microenvironment and that an anti-maspin neutralizing monoclonal antibody reversed the collagen-dependent DXR resistance. Impaired diffusion and decreased DXR activity were also found in tumors derived from Matrigel-embedded cells, where abundant collagen fibers characterize the tumor matrix. Conversely, liposome-based DXR reached maspin-overexpressing tumor cells despite the abundant extracellular matrix and was more efficient in reducing tumor growth. Our results identify maspin-induced accumulation of collagen fibers as a cause of disease progression under DXR chemotherapy for breast cancer. Use of a more hydrophilic DXR formulation or of a maspin inhibitor in combination with chemotherapy holds the promise of more consistent responses to maspin-overexpressing tumors and dense-matrix tumors in general.

Tumor Suppressor Maspin as a Rheostat in HDAC Regulation to Achieve the Fine-Tuning of Epithelial Homeostasis

Maspin, a class II tumor suppressor, is often downregulated during tumor progression and its depletion from the nucleus is associated with poor prognosis. Recently, we reported that reintroduction of maspin is sufficient for redifferentiation of prostate cancer cells to epithelial phenotype, a reversal of epithelial-to-mesenchymal transition. We have linked this effect of maspin with its ability to directly inhibit HDAC1, thereby influencing the acetylation state of transcription factors and other proteins. Maspin overexpression leads to changes in the expression level of a large number of proteins and these changes are often microenvironment specific. In this review, we summarize the epigenetic effects of maspin and provide comprehensive bioinformatic analysis of microarray-derived gene expression changes caused by maspin in different microenvironments. The analysis was performed on multiple levels, including identification of statistically enriched gene ontology groups, detection of overreprepresented transcription factors binding sites in promoters of differentially expressed genes, followed by searching for key nodes of regulatory networks controlling these transcription factors. The results are consistent with our hypothesis that maspin serves as an endogenous regulator of HDAC activity and suggest that the effect of maspin is primarily mediated by TGFβ, β-catenin/E-cadherin pathways, and network key nodes such as Abl kinase, p62, IL1, and caspases 6 and 8.

Comparative proteomic analysis of the function and network mechanisms of MASPIN in human lung cells

MASPIN, which is also known as Serpin B5, is a novel tumor suppressor. Emerging evidence suggests that MASPIN acts as a multifaceted protein in various types of cancer, including prostate, breast and pancreatic cancer. It interacts with diverse groups of intercellular and extracellular proteins, regulating cell adhesion, motility, apoptosis and angiogenesis, and is involved in mammary gland development. As MASPIN is a multifunctional factor in cancer pathways, its function remains poorly illuminated. In this study, we compared the protein profiles of LC5 cell lines with MASPIN overexpression and knockdown using comparative two-dimensional gel electrophoresis. The differences in protein expression, visualized as differences in spots, were identified by time-of-flight (TOF)/TOF mass spectometry. Significant differences were observed between overexpressing and knocked down cells, including eight spots that were unique and sixteen spots that were up- or down-regulated by more than 4-fold. Six genes, including Sdccag8, Ldoc1, SCAI, SDCCAG3, CT62 and NEDD9 were unique in MASPIN-expressing cell lines, but absent in knock-out cell lines, in which most of them play a significant role in the invasion of cancer cells. Moreover, the Brms1 and CAGE1 genes were identified as being uniquely expressed in knocked down cell lines, which were associated with the development and progression of tumors. The data from this study shed some light on the function, as well as the general network mechanisms of MASPIN in lung cancer.

Maspin expression in gastrointestinal stromal tumors

BackgroundTo investigate the role of maspin expression in the progression of gastrointestinal stromal tumors, and its value as a prognostic indicator.MethodsIn the study 54 patients with GIST diagnosis were included in Uludag University of Faculty of Medicine, Department of Pathology between 1997-2007. The expression of maspin in 54 cases of gastrointestinal stromal tumor was detected by immunohistochemistry and compared with the clinicopathologic tumor parameters.ResultsThe positive expression rates for maspin in the GISTs were 66,6% (36 of 54 cases). Maspin overexpression was detected in 9 of 29 high risk tumors (31%) and was significantly higher in very low/low (78.6%) and intermediate-risk tumors (63.6%) than high-risk tumors.ConclusionsMaspin expression might be an important factor in tumor progression and patient prognosis in GIST. In the future, larger series may be studied to examine the prognostic significance of maspin in GISTs and, of course, maspin expression may be studied in different mesenchymal tumors.

Maspin overexpression correlates with positive response to primary chemotherapy in ovarian cancer patients

Objective Maspin is a member of the serine protease inhibitor superfamily. Experimental studies revealed that maspin suppresses tumor growth, angiogenesis, invasion and metastasis. We examined maspin expression in human ovarian tumors and relation between maspin expression and clinicopathological features as well as the role of maspin in predicting clinical outcome in patients with ovarian cancer. Methods Tissue samples consisted of 42 benign tumors, 10 borderline (LMP) tumors, 76 ovarian carcinomas, 8 Krukenberg tumors and 32 normal tissues. Immunoblot analysis was performed to evaluate the relative expression of maspin/β-actin. Results Relative maspin level was significantly higher in patients with LMP tumors (median 0.74) and early stages ovarian cancers (median 0.46) when compared with healthy tissues (median 0.03), those with benign (median 0.23) and metastatic tumors (median 0.22). Overexpression of maspin was found to correlate with the early stage of the disease ( p = 0.001), non-serous subtype of ovarian cancer ( p = 0.03) and positive response to chemotherapy ( p = 0.02). A statistically significant longer PFS was seen in women with high as compared with low expression of maspin ( p = 0.03). Conclusions Maspin is upregulated in borderline tumors and the early stages of ovarian carcinoma and then significantly downregulated with malignant transformation. High expression may paradoxically promote the invasion and metastasis of ovarian carcinomas. Our study revealed that maspin expression could play an important role in predicting the results of treatment of ovarian cancer patients.

Maspin overexpression correlates with increased expression of vascular endothelial growth factors A, C, and D in human ovarian carcinoma

The vascular endothelial growth factor (VEGF) family, including VEGFA, VEGFC, and VEGFD, plays an essential role in the angiogenesis of both pathologic and nonpathologic conditions. Maspin belongs to the serpin superfamily and has been identified as a tumor suppressor because it inhibits motility, invasion, and angiogenesis. Few studies have compared maspin with VEGF in ovarian carcinoma. Therefore, we investigated the expression and correlation of maspin, VEGFA, VEGFC, and VEGFD with the tumorigenesis of the ovary and clinicopathologic variables. Using immunohistochemistry, we examined maspin, VEGFA, VEGFC, and VEGFD expression in 60 ovarian carcinoma tissues (35 serous papillary carcinomas, 18 endometrioid carcinomas, and 7 primary ovarian mucinous carcinomas). Staining of cells was scored as +2 if more than 50% of the cells were positive, as +1 if less than 50% of the cells were positive, and as negative if none of the cells stained positive. Overexpression of maspin, VEGFC, and VEGFD was significantly associated with high tumor grade ( P<.001, P=.004, P<.001, respectively), clinical stage ( P=.002, .01, and .001, respectively), the presence of ascites ( P<.001, P=.03, and P=.001, respectively), and the presence of metastatic lymph nodes ( P=.002, P<.001, and P<.001, respectively). Maspin was correlated with VEGFA ( P=.01), VEGFC ( P<.001), and VEGFD ( P<.001). The VEGFA score was positively correlated with high tumor grade ( P=.04), lymphovascular space invasion (LVSI) ( P<.001), International Federation of Gynecology and Obstetrics (FIGO) stage ( P=.009), maspin, VEGFC ( P=.003), and VEGFD ( P=.003), but it was not correlated with the presence of ascites and metastatic lymph nodes. Maspin, VEGFC, and VEGFD are expressed in ovarian tumors with a poor prognostic parameters, and seem to play a role in ovarian cancer angiogenesis, progression, and lymph node metastases. Our results indicate that in contrast to most other carcinomas, maspin expression is directly associated with the biological aggressiveness of ovarian carcinoma. These results may offer new insights regarding the role of maspin in ovarian cancer and might also affect the diagnosis and treatment strategies.

Maspin Expression in Oral Squamous Cell Carcinoma

Maspin (mammary serine protease inhibitor) is a member of the serpin superfamily of protease inhibitors and it has a role as a tumor suppressor. Maspin has been reported to be important in processes relevant to tumor growth and metastasis such as cell invasion, angiogenesis, and apoptosis. A high expression of maspin was correlated with better rates of survival and absence of nodal metastases in head and neck squamous cell carcinoma. In contrast, some studies have shown that maspin overexpression is correlated with a poor prognosis in pancreatic and ovarian cancers and in lung adenocarcinoma. The aim of this study was an immunohistochemical evaluation of the maspin expression in oral squamous cell carcinoma and thus 89 patients were evaluated. Maspin expression in oral squamous cell carcinoma was significantly associated with the tumor differentiation grade (chi test: P = 0.0318) and the lymph node status (chi test: P < 0.005), but not with the tumor stage (chi test: P = 0.666). Metastatic involvement of lymph nodes was observed more frequently in maspin-negative cases than in tumors with more than 5% of positive cells (P = 0.0024). The present results confirm that maspin expression predicts a better prognosis in oral squamous cell carcinoma and that maspin probably plays a role in tumor progression.

Prognostic significance of maspin in pancreatic ductal adenocarcinoma: tissue microarray analysis of 223 surgically resected cases

Maspin (SERPINB5), a serine proteinase inhibitor, was first identified as a potential tumor suppressor on the basis of its differential expression between normal mammary epithelial cells and human breast carcinoma cell lines. Recent studies have shown that maspin might be a prognostic tumor marker. Pancreatic ductal adenocarcinoma acquires maspin expression through hypomethylation of the maspin promoter. However, no study has investigated the prognostic significance of maspin expression in pancreatic ductal adenocarcinomas. In this study, we investigated maspin protein expression in a large series of 223 surgically resected pancreatic ductal adenocarcinomas using immunohistochemical staining and high throughput tissue microarrays. Maspin expression was correlated with postoperative survival and other clinicopathologic factors. Maspin was detected in 209 of these 223 (94% cases) pancreatic ductal adenocarcinomas including 39 (18% cases) focal (5–50% tumor cells) and 170 (76% cases) diffuse (>50% tumor cells). Fourteen (or 6% cases) pancreatic ductal adenocarcinomas did not show maspin expression by immunohistochemical staining (<5% tumor cells). Normal ductal epithelium is not labeled with maspin. Overexpression of maspin in pancreatic ductal adenocarcinoma is associated with worse postoperative survival especially in patients whose tumors exhibit diffuse expression of maspin. After adjusting other clinicopathologic factors, maspin expression remains to be an independent adverse prognosticator for postoperative survival. Maspin expression is not associated with patient age, gender, tumor size, tumor pathologic stage, lymph node status, and vascular invasion or perineural invasion. Nuclear labeling of maspin is associated with better tumor differentiation although this staining pattern is not associated with a better prognosis. In addition, maspin overexpression is also observed in 48% low-grade (grades 1a and 1b) pancreatic intraepithelial neoplasias (PanINs) and 78% high-grade (grades 2 and 3) PanINs, suggesting that maspin upregulation occurs early during the multi-step progression model of pancreatic ductal adenocarcinoma.

Doxazosin‐derived DZ‐3 Compound Enhances Apoptotic Ability of Maspin‐sensitized Prostate Cancer Cells

The quinazoline-based α1-adrenoceptor antagonist, doxazosin, has previously been shown to induce apoptosis in both benign and malignant prostate cancer cells via the death receptor-mediated pathway. Pharmacologic exploitation of doxazosin structure led the development of a series novel potential antitumor agents for prostate cancer treatment. In this study, we investigate the effect of a newly-derived quinazoline-based compound DZ-3, on human prostate caner cell growth using the maspin-overexpressing clone of human prostate cancer cells DU-145 as a model. Maspin, a mammary serine protease inhibitor, has both tumor suppressive and antiangiogenic activity targeting tumor cell motility, invasion, and metastasis. Using the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay, we determined cell viability in DU-145 parental and DU-145 maspin cells after treatment with DZ-3. Exposure to DZ-3 results in apoptotic cell death in both cell lines, however maspin expressing cells exhibited an enhanced sensitivity to DZ-3-induced apoptosis. Time course analysis of DZ-3 treatment was subsequently performed different time in both cell lines and Western blotting was conducted to determine caspase activation. The results revealed that Maspin overexpression in prostate cancer cells resulted in an enhanced temporal activation in caspase-8 vs the parental DU-145 control cells. This study provides initial evidence on the ability the novel doxazosin-derived DZ-3 to induce caspase-8mediated apoptosis in human prostate cancer cells.

Simultaneous evaluation of maspin and CXCR4 in patients with breast cancer

Aim: To study simultaneously the actions of maspin and CXCR4, which share several similar pathways in cancer, including apoptosis and angiogenesis. Methods: Our material consisted of 151 invasive breast carcinomas...

Maspin sensitizes prostate cancer cells to doxazosin-induced apoptosis.

Maspin is a mammary serine protease inhibitor or serpin with tumor suppressive and antiangiogenic activity that inhibits tumor motility, invasion and metastasis, at least by its actions on cell membrane and extracellular matrix (ECM) proteins. Previous studies documented that the quinazoline-derived alpha1-adrenoceptor antagonist doxazosin affects the attachment and migration of prostate cancer cells. In this study, we investigated the effect of maspin overexpression on the apoptotic/antiadhesion response of prostate cancer cells to doxazosin. The response of maspin-overexpressing clones of human prostate cancer cells DU-145 to doxazosin was evaluated by determining cell viability, apoptosis and cell proliferation on the basis of the trypan blue exclusion assay/methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay, Hoechst staining and caspase-3 activation, and [(3)H]thymidine incorporation assay. Vascular endothelial growth factor (VEGF), transforming growth factor betaRII (TGFbetaRII), Smad4 (a TGFbeta intracellular effector) and bax expression was evaluated at the mRNA and protein level using reverse transcriptase-polymerase chain reaction and Western blotting, respectively. The effect of doxazosin on cell attachment of maspin-expressing prostate cancer cells was evaluated on collagen- and fibronectin-coated plates. Cell migration was assessed using the wounding assay. In response to tumor necrosis factor-related apoptosis-inducing ligand, DU-145-maspin expressing cells undergo apoptosis, via poly(ADP-ribose) polymerasecleavage and caspase-3 activation. DU-145-maspin cells exhibited higher sensitivity to doxazosin and an earlier temporal activation of caspase-3. The number of apoptotic cells detected in response to doxazosin was significantly higher compared to the neo control (P<0.0001). Doxazosin resulted in dramatic downregulation of the 189 isoform of VEGF in maspin transfectants, while a fivefold induction of Smad4 mRNA expression was detected in those cells after 24 h of treatment. Maspin overexpression in prostate cancer cells resulted in an increased ability to attach to ECM-coated plates, and doxazosin treatment considerably antagonized this effect by decreasing the attachment potential to collagen and fibronectin. The present study supports the ability of maspin to enhance the apoptotic threshold of prostate cancer cells to the quinazoline-based alpha1-adrenoceptor antagonist doxazosin. These findings may have therapeutic significance in the development of antiangiogenic targeting by doxazosin and derivative agents for advanced prostate cancer.

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

BackgroundMaspin 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.MethodsStable 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.ResultsWe 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.ConclusionMaspin 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.

Cell-Type-Specific Repression of the Maspin Gene Is Disrupted Frequently by Demethylation at the Promoter Region in Gastric Intestinal Metaplasia and Cancer Cells

Maspin, a serine protease inhibitor, was originally reported as a tumor suppressor gene in breast and prostatic cancers. We examined maspin expression and/or the allele-specific methylation status in four gastric cancer cell lines, as well as normal, metaplastic, and cancerous epithelia obtained from 50 gastric cancer patients. Three gastric cancer cell lines exhibiting maspin overexpression showed hypomethylation at either both alleles or a haploid allele. Only one cell line (GCIY) was maspin-negative but maspin expression was reactivated after treatment with a demethylating agent, 5-aza-2'-deoxycytidine. Dense and diffuse immunoreactivity for maspin was observed in 40 (80%) of 50 gastric cancers and all gastric normal epithelia (GNE) with intestinal metaplasia (IM), but not in GNE without IM. We further analyzed the allele-specific methylation status in 10 of 50 cases subjected to immunohistochemistry by the crypt isolation technique followed by a bisulfite genome sequencing method. The maspin gene promoter region of all GNE without IM was hypermethylated on both alleles whereas those with IM frequently represented the haploid type of hypomethylation status. In six of seven gastric cancers in which crypt isolation was possible, demethylation frequently occurred and extended to both alleles. Maspin mRNA was amplified from GNE with IM and cancerous crypts but not from GNE without IM. These results suggest that demethylation at the maspin gene promoter disrupts the cell-type-specific gene repression in both GNE and gastric cancer.