Maspin Re-expression Research Articles

Role of class I histone deacetylases in the regulation of maspin expression in prostate cancer

Maspin repression is frequently observed in prostate cancer; however, the molecular mechanism(s) causing the loss is not completely understood. Here, we demonstrate that inhibition of class I histone deacetylases (HDACs) mediates re-expression of maspin which plays an essential role in suppressing proliferation and migration capability in prostate cancer cells. Human prostate cancer LNCaP and DU145 cells treated with HDAC inhibitors, sodium butyrate, and trichostatin A, resulted in maspin re-expression. Interestingly, an exploration into the molecular mechanisms demonstrates that maspin repression in prostate tumor and human prostate cancer cell lines occurs via epigenetic silencing through an increase in HDAC activity/expression, independent of promoter DNA hypermethylation. Furthermore, transcriptional activation of maspin was accompanied with the suppression of HDAC1 and HDAC8 with significant p53 enrichment at the maspin promoter associated with an increase in histone H3/H4 acetylation. Our results provide evidence of maspin induction as a critical epigenetic event altered by class I HDACs in the restoration of balance to delay proliferation and migration ability of prostate cancer cells.

Re-expression and epigenetic modification of maspin induced apoptosis in MCF-7 cells mediated by myocardin

Breast cancer is the leading cause of cancer death in women worldwide. It is well known that oncogene activation and anti-oncogene inactivation affect the development and progression of breast cancer, but the role of oncogene activation and anti-oncogene inactivation in breast cancer is still not fully understood. We now report that maspin acts as a tumor suppressor gene to induce MCF-7 cell apoptosis. In addition, maspin promoter hypermethylation and histone hypoacetylation lead to silencing of maspin gene expression in MCF-7 cells. Moreover, DNA methyltransferase (DNMT) inhibitor 5-aza-2′-deoxycytidine (5-aza-dc) and/or the histone deacetylase (HDAC) inhibitor Trichostatin A (TSA) strongly up-regulated the expression of maspin in MCF-7 cells. Notably, myocardin can promote the re-expression of maspin in MCF-7 cells. Luciferase assay shows that myocardin activates the transcription of maspin promoter by CArG box. More importantly, 5-aza-dc/TSA and myocardin synergetically enhance re-expression of maspin and augment maspin-mediated apoptosis in MCF-7 cells. Thus, these data reveal the new insight that myocardin meditates apoptosis in breast cancer through affecting maspin re-expression and epigenetic modification to regulate the development of breast cancer, thereby raising the possibility of its use in breast cancer therapy.

Maspin is not required for embryonic development or tumour suppression

Maspin (SERPINB5) is accepted as an important tumour suppressor lost in many cancers. Consistent with a critical role in development or differentiation maspin knockout mice die during early embryogenesis, yet clinical data conflict on the prognostic utility of maspin expression. Here to reconcile these findings we made conditional knockout mice. Surprisingly, maspin knockout embryos develop into overtly normal animals. Contrary to original reports, maspin re-expression does not inhibit tumour growth or metastasis in vivo, or influence cell migration, invasion or survival in vitro. Bioinformatic analyses reveal that maspin is not commonly under-expressed in cancer, and that perturbation of genes near maspin may in fact explain poor survival in certain patient cohorts with low maspin expression.

Abstract 862: Purified recombinant Maspin is processed through the endosomal pathway in breast cancer cells.

Abstract Maspin (Mammary Serine Protease Inhibitor), a gene involved in mammary gland development and morphogenesis, has been shown to act as a tumor suppressor in multiple cancers by inhibiting the aggressive cancer cell phenotype. Recombinant Maspin (rMas) added to cell culture media is internalized by cancer cells and capable of recapitulating many of the effects of endogenously re-expressed Maspin in Maspin-null cancer cells, including the inhibition of cell invasion, migration and induction of angiogenesis. Importantly, these effects are mediated by purified rMas without the need for post-translational modifications prior to application, increasing the future therapeutic potential for utilizing rMas as an adjuvant therapy. However, the therapeutic use of rMas is limited by its rapid clearance from cancer cells, and many of the molecular mechanisms regarding rMas internalization and processing remain to be determined. Building on preliminary data, we hypothesized that rMas is processed by the endosomal pathway. We demonstrate localization of rMas in Rab9 late-endosomes and Lamp-1 positive lysosomes following treatment of 10 μg/ml rMas in BT549, Hs578T and MDA-MB-231 human breast cancer cell lines. rMas internalization was reduced when incubated at 4oC, and increased at temperatures of 20oC and 37oC, with the highest rates of internalization at 37oC, indicative of endocytosis. Furthermore, depletion of K+ decreased rMas internalization in Hs578T and MDA-MB-231, suggestive of clarthrin-mediated endocytosis. Our data show that rMas is capable of acting at the membrane to mediate effects on invasion and migration, but may not be functional once internalized due to rapid degradation by the lysosome. Interestingly, this may provide a plausible explanation for previously reported data in which rMas was not able to reproduce the effects of endogenous Maspin re-expression on cancer cell apoptosis sensitivity, which require cytosolic and mitochondrial mechanisms. Our data provide evidence that strategies which induce endosomal escape of rMas will intensify its anti-tumor effects on cancer cells, and further enhance its potential therapeutic applications. Citation Format: Thomas M. Bodenstine, Richard E. B. Seftor, Elisabeth A. Seftor, Zhila Khalkhali-Ellis, Nicole A. Samii, J. Cesar Monarrez, Philip A. Pemberton, Mary J. C. Hendrix. Purified recombinant Maspin is processed through the endosomal pathway in breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 862. doi:10.1158/1538-7445.AM2013-862

Targeting Serous Epithelial Ovarian Cancer with Designer Zinc Finger Transcription Factors

Ovarian cancer is the leading cause of death among gynecological malignancies. It is detected at late stages when the disease is spread through the abdominal cavity in a condition known as peritoneal carcinomatosis. Thus, there is an urgent need to develop novel therapeutic interventions to target advanced stages of ovarian cancer. Mammary serine protease inhibitor (Maspin) represents an important metastasis suppressor initially identified in breast cancer. Herein we have generated a sequence-specific zinc finger artificial transcription factor (ATF) to up-regulate the Maspin promoter in aggressive ovarian cancer cell lines and to interrogate the therapeutic potential of Maspin in ovarian cancer. We found that although Maspin was expressed in some primary ovarian tumors, the promoter was epigenetically silenced in cell lines derived from ascites. Transduction of the ATF in MOVCAR 5009 cells derived from ascitic cultures of a TgMISIIR-TAg mouse model of ovarian cancer resulted in tumor cell growth inhibition, impaired cell invasion, and severe disruption of actin cytoskeleton. Systemic delivery of lipid-protamine-RNA nanoparticles encapsulating a chemically modified ATF mRNA resulted in inhibition of ovarian cancer cell growth in nude mice accompanied with Maspin re-expression in the treated tumors. Gene expression microarrays of ATF-transduced cells revealed an exceptional specificity for the Maspin promoter. These analyses identified novel targets co-regulated with Maspin in human short-term cultures derived from ascites, such as TSPAN12, that could mediate the anti-metastatic phenotype of the ATF. Our work outlined the first targeted, non-viral delivery of ATFs into tumors with potential clinical applications for metastatic ovarian cancers.

Abstract 486A: Biological characterization of recombinant maspin in breast cancer cells

Abstract Mammary serine protease inhibitor (maspin) is a non-inhibitory member of the serine protease inhibitor (serpin) superfamily and is involved in mammary gland development. Loss of maspin expression is commonly observed during breast cancer progression while re-expression of maspin confers tumor suppressive capabilities. Cellular localization of maspin within the cytosol, nucleus, and association in complexes at the plasma membrane confer different properties in cancer cells adding to the complexity of maspin function. The recombinant form of maspin (rMas) when added to growth media of maspin-null cancer cells recapitulates many of the effects of exogenous maspin re-expression including inhibition of invasion through 3D matrices. These effects highlight the potential of rMas for therapeutic development as a novel adjuvant therapy. The aim of this study was to characterize the uptake, processing and distribution of wild-type and mutated forms of rMas in cancer cells. Using the MDA-MB-231, BT549 and Hs578T breast cancer cell lines we demonstrate that rMas is directly internalized by cancer cells and degraded rapidly. Time-lapse confocal microscopy using Alexa Fluor 594 labeled rMas and nuclear/cytoplasmic extraction of rMas following cellular uptake demonstrate that rMas is predominantly localized and processed in the cytosol. Wild-type rMas inhibited cancer cell invasion through collagen based matrices and this effect was independent of nuclear localization. Interestingly, site-directed mutagenesis of rMas to induce reported polymorphisms (Ile to Val 66, Pro to Ser 176, Leu to Val 187, Ile to Val 319) abrogated the ability of rMas to inhibit invasion. These preliminary experiments contribute to the characterization of rMas following internalization by cancer cells and aid in the therapeutic development of rMas as a potential therapy. Additionally, mutational data from this study support growing evidence that mutations in the maspin gene alter functionality in vitro, and may correlate to clinical outcomes in patients in which maspin is expressed in tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 486A. doi:1538-7445.AM2012-486A

Mammary serine protease inhibitor inhibits epithelial growth factor‐induced epithelial‐mesenchymal transition of esophageal carcinoma cells

By using proteomic technology, the authors previously observed the substantial down-regulation of mammary serine protease inhibitor (maspin) in esophageal squamous cell carcinoma and metastases. In the current study, they examined the effects of maspin re-expression in a maspin-null esophageal cancer cell line EC109 and also investigated the underlying mechanism. A cell line with stable maspin expression was established. An epithelial growth factor (EGF)-induced epithelial-mesenchymal transition (EMT) model was used to mimic some aspects of the metastatic process in vitro. The effects of maspin reintroduction on EGF-induced EMT and cell growth characteristics were evaluated. Comparative proteomic analysis of transfected cells versus parental cells was then performed to explore the potential mechanism. The introduction of maspin into EC109 cells was able to inhibit EGF-induced EMT and altered cell growth characteristics, including the serum dependence, proliferative response to EGF stimulation, and colony formation ability in soft agar, indicating a conversion from a malignant phenotype to a benign phenotype. Proteomic analysis revealed a significant down-regulation of a group of glycolytic enzymes in maspin-transfected cells. In addition, maspin-transfected cells expressed much lower levels of hypoxia-inducible factor 1alpha than parental cells or empty vector transfected cells. Maspin exhibited a metastasis-suppressive effect, which may be a consequence of the reversal of the malignant phenotype of EC109 cells. The switch of cellular metabolic phenotype to low glycolysis by the gain of maspin function may play a key role in the process. This finding provides additional evidence of the tumor metastasis-suppressive activity of maspin and may indicate a new direction for future studies of the mechanism of maspin.

Clinical relevance of maspin expression in bladder cancer

Transitional cell carcinoma (TCC) of the bladder is a solid tumor that induces angiogenesis to maintain nutrition and oxygenation of tumor cells. Maspin, a serpin with tumor suppressing activity, has recently been identified as an inhibitor of angiogenesis. This study examined the impact of maspin expression in the growth pattern of TCC of the bladder. Maspin was identified in a panel of normal tissues, in several bladder carcinoma cell lines, and 51 patient samples of TCC of the bladder. Expression was detected by RT-PCR and immunohistochemistry. Furthermore, the level of maspin was correlated to the growth rate of bladder tumor cell lines in vitro and in vivo. Maspin expression was found in high quantities in normal urothelium. Maspin expression was preserved in superficial bladder cancers but was significantly diminished in invasive carcinomas. Within the group of invasive TCCs, maspin expression was inversely correlated to the patient prognosis. Furthermore, low maspin expression level was coupled to an increased tumor cell growth in vivo. Down-regulation of maspin expression seems to be a specific event in the progression of invasive bladder carcinoma. Maspin might be a useful marker to determine the prognosis of invasive TCC. Furthermore, maspin re-expression might become a therapeutic option in the treatment of invasive, metastatic TCC.

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.

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.

Expression and regulation of tumor suppressor gene maspin in human bladder cancer

Maspin is a member of serine protease inhibitor family with tumor suppressing activity for breast and prostate cancers, acting at the level of tumor invasion and metastasis. However, there have been no published data regarding the role of maspin in human bladder cancer. We evaluated maspin expression in 65 series of bladder cancer samples (22 transurethral resection (TUR) and 43 radical cystectomy) and studied the regulatory mechanism of maspin gene activation in bladder cancer cells. Maspin expression was immunohistochemically detected in four (18.2%) patients with TUR and 22 (51.2%) patients with radical cystectomy whereas no expression was observed in normal transitional cells located at tumor-free area in bladder. The maspin expression was significantly correlated with the development of muscle invasive bladder cancer ( P=0.00008). Using a luciferase reporter system, maspin promoter activity was induced in the maspin-positive bladder cancer cell lines as well as maspin-negative RT4 cells. Furthermore, treatment with the DNA methyltransferase inhibitor, 5-aza-2′ deoxycytidine, and histone deacetylase inhibitor, trichostatin A, led to re-expression of maspin in RT4 cells. Our results indicate that maspin may contribute to bladder cancer development and that DNA methylation and histone deacetylation may be important for regulating maspin gene activation in bladder cancer cells.

Expression and Regulation of Tumor Suppressor Gene Maspin in Breast Cancer

Maspin (mammary serpin) is an inhibitor of serine proteases with tumor suppressor activity in breast cancer. Maspin was originally identified by subtractive hybridization in normal breast epithelial cells, but its expression decreased during tumor progression. The loss of maspin gene expression with increasing malignancy is by transcriptional regulation. Maspin is known to be involved in invasion and metastasis, interact with the p53 tumor suppressor pathway, and act as an inhibitor of angiogenesis. The immunohistochemical analysis and reverse-transcription polymerase chain reaction of maspin in normal human breast tissue and breast carcinoma indicated a stepwise reduction of maspin expression during the progression from ductal carcinoma in situ to invasive carcinoma to lymph node metastasis. The lack of maspin expression in breast cancer seems to be associated with a short disease-free survival and supports maspin's function as an indicator for tumor aggressiveness and metastatic potential. New studies on the gene regulation of maspin provide evidence for promising potential of possible re-expression of maspin in tumor cells. The function of maspin as a tumor suppressor gene involved in tumor invasion, metastasis, and angiogenesis may not be limited to breast cancer.

Hypermethylation and histone deacetylation lead to silencing of the maspin gene in human breast cancer

Maspin is a member of the serine protease inhibitor family with tumor suppressing activity in breast cancer. Maspin expression was found in normal breast epithelial cells, but was frequently decreased in breast cancer cells and lost in metastatic cells. In this study, we examined the regulatory mechanism of maspin expression and described the re-activation of maspin expression in a series of maspin-negative breast cancer cell lines. All of the 6 maspin-negative breast cancer cells showed induction of maspin promoter activity in a promoter reporter assay. In addition, the treatment of 5-aza-2 ′ deoxycytidine, trichostatin A or a combination of both led to the re-expression of maspin in a series of maspin-negative breast cancer cell lines. These findings indicate that DNA methylation and/or histone deacetylation are/is partially responsible for the silencing of maspin gene expression in breast cancer cells. The re-expression of maspin by pharmacological intervention potentially offers a promising new target as a therapeutic option in breast cancer.

Maspin, neuer Protease-Inhibitor mit Tumorsuppressor-Aktivität beim Brustkrebs - Maspin, Novel Protease Inhibitor with Tumor Suppressing Activity in Breast Cancer -

Maspin (mammary serpin) ist ein neuer Serin-Protease-Inhibitor mit Tumorsuppressor-Aktivität beim Brustkrebs. Das entsprechende Maspin-Gen wurde ursprünglich durch subtraktive Hybridisierung identifiziert und konnte auf dem Chromosom 18 lokalisiert werden. Maspin zeigt starke Expression in normalen Brustepithelzellen bei überwiegender Down-Regulation in Brustkrebszellen. Maspins Expression sinkt kontinuierlich mit steigendem Malignitätsgrad von normalem Brustepithel über duktales Carcinoma in situ und invasive Karzinome bis hin zu Lymphknotenmetastasen und peripheren Metastasen. In-vitro-und In-vivo-Untersuchungen ergaben eine hemmende Wirkung von Maspin auf Tumorzellinvasion und Metastasierung. Die unterschiedliche Expression von Maspin in normalem Brustepithel und Brusttumoren beruht auf regulatorischen Veränderungen auf Transkriptionsebene. Damit unterscheidet sich Maspin von den klassischen Tumorsuppressor-Genen (z. B. P53, Retinoblastom) und kann der sogenannten Klasse II Tumorsuppressor-Genen zugerechnet werden. Die Tumorsuppressor-Gene der Klasse II besitzen eine besondere klinische Relevanz, da eine therapeutische Intervention im Sinne einer Re-Expression des Maspin-Gens durch medikamentöse (Gen-)Therapien möglich ist.

Maspin-A Novel Protease Inhibitor with Tumor-suppressing Activity in Breast Cancer

Maspin (mammary serpin) is a novel serine protease inhibitor related to the serpin family with a tumor-suppressing function in breast cancer. Maspin was originally identified from normal mammary epithelium by subtractive hybridization and might function as a class II tumor-suppressor gene. Maspin's decreased expression with increased level of malignancy and its loss in metastatic cells is regulated at the transcriptional level. Cytosin methylation and heterochromatinization in the promoter region might account for this down-regulation of maspin. Transfection of tumor cells with maspin cDNA inhibits invasion and motility and decreases tumor growth and metastatic ability in nude mice. Maspin interacts with the p53 tumor-suppressor pathway and function as an inhibitor of angiogenesis in vitro and in vivo. The progressive loss of expression of maspin during tumor progression makes this new protein an interesting diagnostic and prognostic marker. The re-expression of maspin by pharmacological intervention potentially offers a promising approach as a therapeutic option in breast cancer therapy.