The Experts below are selected from a list of 1380 Experts worldwide ranked by ideXlab platform
Mary J.c. Hendrix - One of the best experts on this subject based on the ideXlab platform.
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Oncogenes and Tumor Suppressors Internalization by Multiple Endocytic Pathways and Lysosomal Processing Impact Maspin-Based Therapeutics
2016Co-Authors: Thomas M Bodenstine, Zhila Khalkhali-ellis, Richard E.b. Seftor, Elisabeth A Seftor, Philip A Pemberton, Nicole A Samii, Cesar J Monarrez, Mary J.c. HendrixAbstract:Patients with metastatic disease face high rates of mortality with a paucity of therapeutic options. Protein-based therapeutics provide advantages over traditional chemotherapy through increased specificity, decreased immune impairment, and more direct means of delivery. However, development is often hindered because of insufficient knowledge about protein processing by cells when exogenously applied. This study focuses on recombinant Maspin (rMaspin), a serine protease inhibitor (SERPINB5), which alters invasive properties when directly applied to cancer cells. Previous evidence suggests differences in the effects of rMaspin treatment when compared with endogenous reexpression, with little explanation for these discrepancies. A leading hypothesis is that exogenously applied rMaspin is subject to different regulatory and/or processing mechanisms in cancer cells when compared with endogenous expression. Therefore, a more detailed understanding of the mechanisms of internalization and subcellular trafficking of rMaspin is needed to guide future translational development. We describe the molecular trafficking of rMaspin in cytoplasmic vesicles of the endosomal/lysosomal pathway and characterize its uptake by multiple endocytic mechanisms. Time-lapse laser scanning confocal microscopy shows the uptake, in real time, of dye-labeled rMaspin in cancer cells. This study indicates that cellular processing of rMaspin plays a key role by affecting its biologic activity and highlights the need for new approaches aimed at increasing the availability of rMaspin when used to treat cancer. Implications:Novel characterization of internalization and subcellular trafficking of rMaspin provides new insights for future therapeutic development. Mol Cancer Res; 12(10); 1480–91. 2014 AACR
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internalization by multiple endocytic pathways and lysosomal processing impact Maspin based therapeutics
Molecular Cancer Research, 2014Co-Authors: Thomas M Bodenstine, Richard E.b. Seftor, Zhila Khalkhaliellis, Elisabeth A Seftor, Philip A Pemberton, Nicole A Samii, Cesar J Monarrez, Grace S Chandler, Mary J.c. HendrixAbstract:Patients with metastatic disease face high rates of mortality with a paucity of therapeutic options. Protein-based therapeutics provides advantages over traditional chemotherapy through increased specificity, decreased immune responses and more direct means of delivery. However, development is often hindered due to insufficient knowledge regarding proteolytic processing by cells when exogenously applied. This study focuses on recombinant Maspin (rMaspin), a serine protease inhibitor (SERPINB5), which alters invasive properties when directly applied to cancer cells. Previous evidence suggests differences in the effects of rMaspin treatment when compared to endogenous re-expression, with little explanation for these discrepancies. A leading hypothesis is that exogenously applied rMaspin is subject to different regulatory and/or processing mechanisms in cancer cells when compared to endogenous expression. Therefore, a more detailed understanding of the mechanisms of internalization and subcellular trafficking of rMaspin is needed to guide future translational development. We describe the molecular trafficking of rMaspin in cytoplasmic vesicles of the endosomal/lysosomal pathway and characterize its uptake by multiple endocytic mechanisms. Time-lapse laser scanning confocal microscopy shows the uptake, in real-time, of dye labeled rMaspin in cancer cells. This study indicates that cellular processing of rMaspin plays a key role affecting its biological activity and highlights the need for new approaches aimed at increasing the availability of rMaspin when used to treat cancer.
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Maspin: molecular mechanisms and therapeutic implications
Cancer and Metastasis Reviews, 2012Co-Authors: Thomas M Bodenstine, Zhila Khalkhali-ellis, Richard E.b. Seftor, Elisabeth A Seftor, Philip A Pemberton, Mary J.c. HendrixAbstract:Maspin, a non-inhibitory member of the serine protease inhibitor superfamily, has been characterized as a tumor suppressor gene in multiple cancer types. Among the established anti-tumor effects of Maspin are the inhibition of cancer cell invasion, attachment to extracellular matrices, increased sensitivity to apoptosis, and inhibition of angiogenesis. However, while significant experimental data support the role of Maspin as a tumor suppressor, clinical data regarding the prognostic implications of Maspin expression have led to conflicting results. This highlights the need for a better understanding of the context dependencies of Maspin in normal biology and how these are perturbed in the context of cancer. In this review, we outline the regulation and roles of Maspin in normal and developmental biology while discussing novel evidence and emerging theories related to its functions in cancer. We provide insight into the immense therapeutic potential of Maspin and the challenges related to its successful clinical translation.
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elucidating the function of secreted Maspin inhibiting cathepsin d mediated matrix degradation
Cancer Research, 2007Co-Authors: Zhila Khalkhaliellis, Mary J.c. HendrixAbstract:Cellular interaction with the extracellular milieu plays a significant role in normal biological and pathologic processes. Excessive degradation of basement membrane matrix by proteolytic enzymes is a hallmark of tumor invasion and metastasis, and aspartyl proteinase cathepsin D is implicated as a major contributor to this process. Maspin, a non-inhibitory serpin, plays an important role in mammary gland development and remodeling. Expression of Maspin is decreased in primary tumors and lost in metastatic lesions. Maspin is mostly cytoplasmic and is partially secreted; however, the fate and function of secreted Maspin has remained mostly unexplored. We hypothesized that secreted Maspin is incorporated into the matrix deposited by normal mammary epithelial cells and thus could play a critical role in cathepsin D–mediated matrix degradation and remodeling of mammary tissue. In the absence of Maspin, as is the case with most cancer cells, matrix degradation proceeds unrestricted, thus facilitating the progression to metastasis. To test this, we employed an in vitro model where gels containing both types I and IV collagen were preconditioned with normal mammary epithelial cells to allow the incorporation of secreted Maspin. This conditioned matrix was used to examine cathepsin D–mediated collagen degradation by human breast cancer cell lines. Our results indicate that secretion of Maspin and its deposition into the extracellular milieu play an important role in matrix degradation. In this capacity, Maspin could potentially regulate mammary tissue remodeling occurring under normal and pathologic conditions. In addition, these findings could have a potential effect on future therapeutic intervention strategies for breast cancer. [Cancer Res 2007;67(8):3535–9]
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elucidating the function of secreted Maspin inhibiting cathepsin d mediated matrix degradation
Cancer Research, 2007Co-Authors: Zhila Khalkhaliellis, Mary J.c. HendrixAbstract:Cellular interaction with the extracellular milieu plays a significant role in normal biological and pathologic processes. Excessive degradation of basement membrane matrix by proteolytic enzymes is a hallmark of tumor invasion and metastasis, and aspartyl proteinase cathepsin D is implicated as a major contributor to this process. Maspin, a non-inhibitory serpin, plays an important role in mammary gland development and remodeling. Expression of Maspin is decreased in primary tumors and lost in metastatic lesions. Maspin is mostly cytoplasmic and is partially secreted; however, the fate and function of secreted Maspin has remained mostly unexplored. We hypothesized that secreted Maspin is incorporated into the matrix deposited by normal mammary epithelial cells and thus could play a critical role in cathepsin D-mediated matrix degradation and remodeling of mammary tissue. In the absence of Maspin, as is the case with most cancer cells, matrix degradation proceeds unrestricted, thus facilitating the progression to metastasis. To test this, we employed an in vitro model where gels containing both types I and IV collagen were preconditioned with normal mammary epithelial cells to allow the incorporation of secreted Maspin. This conditioned matrix was used to examine cathepsin D-mediated collagen degradation by human breast cancer cell lines. Our results indicate that secretion of Maspin and its deposition into the extracellular milieu play an important role in matrix degradation. In this capacity, Maspin could potentially regulate mammary tissue remodeling occurring under normal and pathologic conditions. In addition, these findings could have a potential effect on future therapeutic intervention strategies for breast cancer.
Ming Zhang - One of the best experts on this subject based on the ideXlab platform.
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tumor suppressive Maspin functions as a reactive oxygen species scavenger importance of cysteine residues
Journal of Biological Chemistry, 2013Co-Authors: Nitin Mahajan, Thomas J Lukas, Ming ZhangAbstract: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.
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Maspin the molecular bridge between the plasminogen activator system and β1 integrin that facilitates cell adhesion
Journal of Biological Chemistry, 2011Co-Authors: Michael P Endsley, Steven L Gonias, Sally S Twining, Debra J Warejcka, Yong Deng, Ming ZhangAbstract: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.
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Maspin regulates endothelial cell adhesion and migration through an integrin signaling pathway
Journal of Biological Chemistry, 2010Co-Authors: Li Qin, Ming ZhangAbstract:Maspin has been identified as a potent angiogenesis inhibitor. However, the molecular mechanism responsible for its anti-angiogenic property is unclear. In this study, we examined the effect of Maspin on endothelial cell (EC) adhesion and migration in a cell culture system. We found that Maspin was expressed in blood vessels ECs and human umbilical vein endothelial cells (HUVECs). Maspin significantly enhanced HUVEC cell adhesion to various matrix proteins. This effect was dependent on the activation of integrin β1, which subsequently led to distribution pattern changes of vinculin and F-actin. These results indicated that Maspin affects cell adhesion and cytoskeleton reorganization through an integrin signal transduction pathway. Analysis of HUVECs following Maspin treatment revealed increased integrin-linked kinase activities and phosphorylated FAK levels, consistent with increased cell adhesion. Interestingly, when HUVECs were induced to migrate by migration stimulatory factor bFGF, active Rac1 and cdc42 small GTPase levels were decreased dramatically at 30 min following Maspin treatment. Using phosphorylated FAK at Tyr397 as an indicator of focal adhesion disassembly, Maspin-treated HUVECs had elevated FAK phosphorylation compared with the mock treated control. The results were a reduction in focal adhesion disassembly and the retardation in EC migration. This study uncovers a mechanism by which Maspin exerts its effect on EC adhesion and migration through an integrin signal transduction pathway.
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nuclear cytokine activated ikkα controls prostate cancer metastasis by repressing Maspin
Nature, 2007Co-Authors: Junli Luo, Wei Tan, Jill Ricono, Olexandr Korchynskyi, Ming Zhang, Steven L Gonias, David A Cheresh, Michael KarinAbstract:Experiments in a mouse prostate cancer model have identified a signalling pathway that stimulates the formation of metastases. The pathway is initiated when a protein ligand occupies a receptor known as RANK (receptor activator of nuclear factor κB), and is dependent on the activation and nuclear translocation of IKKα (IκB kinase α). Once in the nucleus, activated IKKα represses Maspin gene transcription, whose product is well established as an inhibitor of cell migration and invasion in prostate and breast cancer. RANK may therefore be a general promoter of metastatic behaviour in prostate or mammary carcinoma cells. A previously unknown signalling pathway is shown to enhance the formation of metastases in a mouse model of prostate cancer. This pathway can be activated by RANK ligand that is expressed by inflammatory cells in the tumour and triggers activation of IKKα in the nucleus, where it directly represses the transcription of Maspin, a known metastases suppressor. Inflammation enhances tumour promotion through NF-κB-dependent mechanisms1. NF-κB was also proposed to promote metastatogenesis through epithelial–mesenchymal transition2. Yet a mechanistic link between inflammation and metastasis is missing. We identified a role for IκB kinase α (IKKα), activated by receptor activator of NF-κB (RANK/TNFRSF11A), in mammary epithelial proliferation during pregnancy3. Owing to similarities between mammary and prostate epithelia, we examined IKKα involvement in prostate cancer and its progression. Here we show that a mutation that prevents IKKα activation slows down CaP growth and inhibits metastatogenesis in TRAMP mice, which express SV40 T antigen in the prostate epithelium4. Decreased metastasis correlated with elevated expression of the metastasis suppressor Maspin5, the ablation of which restored metastatic activity. IKKα activation by RANK ligand (RANKL/TNFSF11) inhibits Maspin expression in prostate epithelial cells, whereas repression of Maspin transcription requires nuclear translocation of active IKKα. The amount of active nuclear IKKα in mouse and human prostate cancer correlates with metastatic progression, reduced Maspin expression and infiltration of prostate tumours with RANKL-expressing inflammatory cells. We propose that tumour-infiltrating RANKL-expressing cells lead to nuclear IKKα activation and inhibition of Maspin transcription, thereby promoting the metastatic phenotype.
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Maspin is physically associated with β1 integrin regulating cell adhesion in mammary epithelial cells
The FASEB Journal, 2006Co-Authors: Nathalie Cella, Alejandro Contreras, Khatri Latha, Jeffrey M Rosen, Ming ZhangAbstract:Maspin is a tumor-suppressor serpin (serine protease inhibitor), which inhibits cell invasion and migration. Here, we analyzed Maspin function in cell adhesion in nontransformed mammary epithelial cells and investigated the underlying mechanism involved in this process. We report that Maspin acts in the early steps in the cell adhesion process. Addition of recombinant Maspin rapidly increased MCF-10A cell adhesion to the endogenously deposited matrix, and conversely both an antiMaspin antibody (Ab) and Maspin knockdown by RNA interference resulted in decreased cell adhesion. Mutation analyses revealed that a region of 86 amino acids located between aa 139 and aa 225 was responsible for Maspin effect on adhesion. In addition, we show that Maspin is associated with detergent-insoluble cortical cytoskeleton elements. Collectively, these results suggest that Maspin is part of the supramolecular structure of the adhesion plaque and it modulates cell adhesion via a beta1 integrin-dependent mechanism.
Shijie Sheng - One of the best experts on this subject based on the ideXlab platform.
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Identification of an Intrinsic Determinant Critical for Maspin Subcellular Localization and Function
2016Co-Authors: Sijana H Dzinic, Fulvio Lonardo, Ivory Dean, Er Kaplun, Margarida Bernardo, David Krass, Paul Stemmer, Namhee Shin, Shijie ShengAbstract:Maspin, a multifaceted tumor suppressor, belongs to the serine protease inhibitor superfamily, but only inhibits serine protease-like enzymes such as histone deacetylase 1 (HDAC1). Maspin is specifically expressed in epithelial cells and it is differentially regulated during tumor progression. A new emerging consensus suggests that a shift in Maspin subcellular localization from the nucleus to the cytoplasm stratifies with poor cancer prognosis. In the current study, we employed a rational mutagenesis approach and showed that Maspin reactive center loop (RCL) and its neighboring sequence are critical for Maspin stability. Further, when expressed in multiple tumor cell lines, single point mutation of Aspartate346 (D346) to Glutamate (E346), MaspinD346E, was predominantly nuclear, whereas wild type Maspin (MaspinWT) was both cytoplasmic and nuclear. Evidence from cellular fractionation followed by immunological and proteomic protein identification, combined with the evidence from fluorescent imaging of endogenous proteins, fluorescent protein fusion constructs, as well as bimolecular fluorescence complementation (BiFC) showed that the increased nuclear enrichment of MaspinD346E was, at least in part, due to its increased affinity to HDAC1. MaspinD346E was also more potent than MaspinWT as an HDAC inhibitor. Taken together, our evidence demonstrates that D346 is a critical cis-element in Maspin sequence that determines the molecular context and subcellular localization of Maspin. A mechanistic model derived from our evidence suggests a ne
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Elevated Maspin Expression Is Associated with Better Overall Survival in Esophageal Squamous Cell Carcinoma
2016Co-Authors: Yang Wang, Shijie Sheng, Jianzhi Zhang, Sijana Dzinic, Fang Fang, Qingfeng Zheng, Yue YangAbstract:Tumor suppressor Maspin is a differentially regulated gene in the progression of many types of cancer. While the biological function of Maspin in blocking tumor invasion and metastasis is consistent with the loss of Maspin expression at the late stage of tumor progression, the differential expression and the biological significance of Maspin in early stage of tumor progression appear to be complex and remain to be elucidated. In the current study, we examined the expression of Maspin in 84 esophageal squamous cell carcinoma (ESCC) cases (stages I–III) and 55 non-tumor adjacent esophageal tissue specimens by immunohistochemical (IHC) staining. The correlation of Maspin with clinicopathological parameters was analyzed. Compared to normal esophageal squamous tissue where 80 % (47/55) of the cases expressed Maspin at a low to moderate level, all ESCC specimens (100 % (84/84)) were positive for Maspin expression at a moderate to high level. ESCC with low or moderate Maspin expression had significantly shorter postoperative survival rates compared to those that had high Maspin expression (p,0.001). Since the correlation of Maspin with ESCC histology and the correlation of Maspin with ESCC prognosis seem to be at odds, we further investigated the biological function of Maspin in ESCC using the established ESCC cell lines. The expression of Maspin in five human esophageal squamous cancer cell lines (T12, E450, KYSE150, EC109, and KYSE510) was examined by the Western blot. ESCC cell line KYSE510 that did not express Maspin and was stably transfected by Maspin cDNA or an empty vector. The resulting transfected cells were characterized in vitro. Maspi
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endogenous inhibition of histone deacetylase 1 by tumor suppressive Maspin
Cancer Research, 2006Co-Authors: Shuping Yin, Yonghong Meng, Wael Sakr, Shijie ShengAbstract:Maspin, a noninhibitory serine protease inhibitor, exerts multifaceted tumor-suppressive effects. Maspin expression is associated with better differentiated phenotypes, better cancer prognosis, and better drug sensitivity. Consistently, Maspin also correlates with increased expression of Bax and p21WAF1/CIP1. Interestingly, histone deacetylase 1 (HDAC1), a major HDAC responsible for histone deacetylation, was shown to interact with Maspin in a yeast two-hybrid screening. In this study, we confirmed the Maspin/HDAC1 interaction in human prostate tissues, in prostate cancer cell lines, and with purified Maspin. We produced several lines of evidence that support an inhibitory effect of Maspin on HDAC1 through direct molecular interaction, which was detected in both the nucleus and the cytoplasm. Both endogenously expressed Maspin and purified Maspin inhibited HDAC1. In contrast, small interfering RNA (siRNA) silencing of Maspin in PC3 cells increased HDAC activity. Accordingly, Maspin-transfected DU145 cells exhibited increased expression of HDAC1 target genes Bax, cytokeratin 18 (CK18), and p21WAF1/CIP1, whereas Maspin siRNA decreased CK18 expression in PC3 cells. The Maspin effect on HDAC1 correlated with an increased sensitivity to cytotoxic HDAC inhibitor M344. Interestingly, glutathione S-transferase (GST, another Maspin partner) was detected in the Maspin/HDAC1 complex. Furthermore, a COOH-terminally truncated Maspin mutant, which bound to HDAC1 but not GST, did not increase histone acetylation. Although HDACs, especially the highly expressed HDAC1, are promising therapeutic targets in cancer intervention, our data raise a novel hypothesis that the endogenous inhibitory effect of Maspin on HDAC1 is coupled with glutathione-based protein modification, and provide new leads toward future developments of specific HDAC1-targeting strategies. (Cancer Res 2055; 66(18): 9323-9) (Cancer Res 2006; 66(18): 9323-9)
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tumor suppressive Maspin and epithelial homeostasis
Journal of Cellular Biochemistry, 2006Co-Authors: Jaron Lockett, Shuping Yin, Yonghong Meng, Shijie ShengAbstract: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 tumorigenesity and re-expression of Maspin in dormant cancer metastastic 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. J. Cell. Biochem. 97: 651–660, 2006. © 2005 Wiley-Liss, Inc.
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tumor suppressive Maspin regulates cell response to oxidative stress by direct interaction with glutathione s transferase
The Journal of Urology, 2006Co-Authors: Shuping Yin, Yonghong Meng, Neelima Reddy, Wael Sakr, Russell L Finley, Heng Yang, Shijie ShengAbstract:Abstract 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 Arg340 to Ala (MasR340A) 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 MasR340A, enhanced cellular GST activity. Maspin expression in cancer cell lines also correlated with decreased basal levels of reactive oxygen species (ROS). Furthermore, H2O2 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 H2O2-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.
Zhila Khalkhaliellis - One of the best experts on this subject based on the ideXlab platform.
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internalization by multiple endocytic pathways and lysosomal processing impact Maspin based therapeutics
Molecular Cancer Research, 2014Co-Authors: Thomas M Bodenstine, Richard E.b. Seftor, Zhila Khalkhaliellis, Elisabeth A Seftor, Philip A Pemberton, Nicole A Samii, Cesar J Monarrez, Grace S Chandler, Mary J.c. HendrixAbstract:Patients with metastatic disease face high rates of mortality with a paucity of therapeutic options. Protein-based therapeutics provides advantages over traditional chemotherapy through increased specificity, decreased immune responses and more direct means of delivery. However, development is often hindered due to insufficient knowledge regarding proteolytic processing by cells when exogenously applied. This study focuses on recombinant Maspin (rMaspin), a serine protease inhibitor (SERPINB5), which alters invasive properties when directly applied to cancer cells. Previous evidence suggests differences in the effects of rMaspin treatment when compared to endogenous re-expression, with little explanation for these discrepancies. A leading hypothesis is that exogenously applied rMaspin is subject to different regulatory and/or processing mechanisms in cancer cells when compared to endogenous expression. Therefore, a more detailed understanding of the mechanisms of internalization and subcellular trafficking of rMaspin is needed to guide future translational development. We describe the molecular trafficking of rMaspin in cytoplasmic vesicles of the endosomal/lysosomal pathway and characterize its uptake by multiple endocytic mechanisms. Time-lapse laser scanning confocal microscopy shows the uptake, in real-time, of dye labeled rMaspin in cancer cells. This study indicates that cellular processing of rMaspin plays a key role affecting its biological activity and highlights the need for new approaches aimed at increasing the availability of rMaspin when used to treat cancer.
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elucidating the function of secreted Maspin inhibiting cathepsin d mediated matrix degradation
Cancer Research, 2007Co-Authors: Zhila Khalkhaliellis, Mary J.c. HendrixAbstract:Cellular interaction with the extracellular milieu plays a significant role in normal biological and pathologic processes. Excessive degradation of basement membrane matrix by proteolytic enzymes is a hallmark of tumor invasion and metastasis, and aspartyl proteinase cathepsin D is implicated as a major contributor to this process. Maspin, a non-inhibitory serpin, plays an important role in mammary gland development and remodeling. Expression of Maspin is decreased in primary tumors and lost in metastatic lesions. Maspin is mostly cytoplasmic and is partially secreted; however, the fate and function of secreted Maspin has remained mostly unexplored. We hypothesized that secreted Maspin is incorporated into the matrix deposited by normal mammary epithelial cells and thus could play a critical role in cathepsin D–mediated matrix degradation and remodeling of mammary tissue. In the absence of Maspin, as is the case with most cancer cells, matrix degradation proceeds unrestricted, thus facilitating the progression to metastasis. To test this, we employed an in vitro model where gels containing both types I and IV collagen were preconditioned with normal mammary epithelial cells to allow the incorporation of secreted Maspin. This conditioned matrix was used to examine cathepsin D–mediated collagen degradation by human breast cancer cell lines. Our results indicate that secretion of Maspin and its deposition into the extracellular milieu play an important role in matrix degradation. In this capacity, Maspin could potentially regulate mammary tissue remodeling occurring under normal and pathologic conditions. In addition, these findings could have a potential effect on future therapeutic intervention strategies for breast cancer. [Cancer Res 2007;67(8):3535–9]
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elucidating the function of secreted Maspin inhibiting cathepsin d mediated matrix degradation
Cancer Research, 2007Co-Authors: Zhila Khalkhaliellis, Mary J.c. HendrixAbstract:Cellular interaction with the extracellular milieu plays a significant role in normal biological and pathologic processes. Excessive degradation of basement membrane matrix by proteolytic enzymes is a hallmark of tumor invasion and metastasis, and aspartyl proteinase cathepsin D is implicated as a major contributor to this process. Maspin, a non-inhibitory serpin, plays an important role in mammary gland development and remodeling. Expression of Maspin is decreased in primary tumors and lost in metastatic lesions. Maspin is mostly cytoplasmic and is partially secreted; however, the fate and function of secreted Maspin has remained mostly unexplored. We hypothesized that secreted Maspin is incorporated into the matrix deposited by normal mammary epithelial cells and thus could play a critical role in cathepsin D-mediated matrix degradation and remodeling of mammary tissue. In the absence of Maspin, as is the case with most cancer cells, matrix degradation proceeds unrestricted, thus facilitating the progression to metastasis. To test this, we employed an in vitro model where gels containing both types I and IV collagen were preconditioned with normal mammary epithelial cells to allow the incorporation of secreted Maspin. This conditioned matrix was used to examine cathepsin D-mediated collagen degradation by human breast cancer cell lines. Our results indicate that secretion of Maspin and its deposition into the extracellular milieu play an important role in matrix degradation. In this capacity, Maspin could potentially regulate mammary tissue remodeling occurring under normal and pathologic conditions. In addition, these findings could have a potential effect on future therapeutic intervention strategies for breast cancer.
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Maspin the new frontier
Clinical Cancer Research, 2006Co-Authors: Zhila KhalkhaliellisAbstract:Maspin (mammary serine protease inhibitor) was identified in 1994 by subtractive hybridization analysis of normal mammary tissue and breast cancer cell lines. Subsequently, emerging evidence portrays Maspin as a multifaceted protein, interacting with diverse group of intercellular and extracellular proteins, regulating cell adhesion, motility, apoptosis, and angiogenesis and critically involved in mammary gland development. The tissue-specific expression of Maspin is epigenetically controlled, and aberrant methylation of Maspin promoter is closely associated with Maspin gene silencing. Identification of new tissue sites expressing Maspin and novel Maspin-binding partners has expanded the horizon for Maspin research and promises Maspin-based therapeutic approaches for combating cancer. This perspective briefly outlines the past and present strides in deciphering this unique molecule and speculates on new frontiers in Maspin research and prospects of Maspin as a diagnostic/prognostic indicator in cancer.
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biological functions of Maspin
Journal of Cellular Physiology, 2006Co-Authors: Zhila Khalkhaliellis, Caleb M. Bailey, Elisabeth A Seftor, Mary J.c. HendrixAbstract: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.
Sally S Twining - One of the best experts on this subject based on the ideXlab platform.
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Twining S: Maspin: synthesis by human cornea and regulation of in vitro stromal cell adhesion to extracellular matrix. Invest Ophthalmol Vis Sci 2001
2013Co-Authors: Chatri Ngamkitidechakul, Janice M Burke, William J. O’brien, Sally S TwiningAbstract:PURPOSE. Maspin, a tumor-suppressor protein that regulates cell migration, invasion, and adhesion, is synthesized by many normal epithelial cells, but downregulated in invasive epithelial tumor cells. The purpose of this study was to determine whether cells in the normal human cornea express Maspin and whether Maspin affects corneal stromal cell adhesion to extracellular matrix molecules. METHODS. Maspin expression was analyzed by immunodot blot, Western blot, and RT-PCR analyses in cells obtained directly from human corneas in situ. Maspin protein and mRNA were also studied in primary and passaged cultures of corneal stromal cells using Western blot analysis, RT-PCR, and immunofluorescence microscopy. Maspin cDNA was cloned and sequenced from human corneal epithelial cells and expressed in a yeast system. The recombinant Maspin was used to stud
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Maspin the molecular bridge between the plasminogen activator system and β1 integrin that facilitates cell adhesion
Journal of Biological Chemistry, 2011Co-Authors: Michael P Endsley, Steven L Gonias, Sally S Twining, Debra J Warejcka, Yong Deng, Ming ZhangAbstract: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.
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epigenetic silencing of Maspin expression occurs early in the conversion of keratocytes to fibroblasts
Experimental Eye Research, 2008Co-Authors: Mark Horswill, Debra J Warejcka, M Narayan, Lisa Ann Cirillo, Sally S TwiningAbstract:Maspin, a 42 kDa non-classical serpin (serine protease inhibitor) that controls cell migration and invasion, is mainly expressed by epithelial-derived cells but is also expressed in corneal stromal keratocytes. Upon culture of stromal keratocytes in the presence of FBS, Maspin is down-regulated to nearly undetectable levels by passage two. DNA methylation is one of several processes that controls gene expression during cell differentiation, development, genetic imprinting, and carcinogenesis but has not been studied in corneal stromal cells. The purpose of this study was to determine whether DNA methylation of the Maspin promoter and histone H3 dimethylation is involved in the mechanism of down-regulation of Maspin synthesis in human corneal stromal fibroblasts and myofibroblasts. Human donor corneal stroma cells were immediately placed into serum-free defined medium or cultured in the presence of FBS and passed into serum-free medium or medium containing FBS or FGF-2 to induce the fibroblast phenotype or TGF-beta1 for the myofibroblast phenotype. These cell types are found in wounded corneas. The cells were used to prepare RNA for semi-quantitative or quantitative RT-PCR or to extract protein for Western analysis. In addition, P4 FBS cultured fibroblasts were treated with the DNA demethylating agent, 5-aza-2'-deoxycytidine (5-Aza-dC), and the histone deacetylase inhibitor, trichostatin A (TSA). Cells with and without treatment were harvested and assayed for DNA methylation using sodium bisulfite sequencing. The methylation state of histone H3 associated with the Maspin gene in the P4 fibroblast cells was determined using a ChIP assay. Freshly harvested corneal stromal cells expressed Maspin but upon phenotypic differentiation, Maspin mRNA and protein were dramatically down-regulated. Sodium bisulfite sequencing revealed that the Maspin promoter in the freshly isolated stromal keratocytes was hypomethylated while both the P0 stromal cells and the P1 cells cultured in the presence of serum-free defined medium, FGF-2 and TGF-beta1 were hypermethylated. Down-regulation of Maspin synthesis was also associated with histone H3 dimethylation at lysine 9. Both Maspin mRNA and protein were re-expressed at low levels with 5-Aza-dC but not TSA treatment. Addition of TSA to 5-Aza-dC treated cells did not increase Maspin expression. Treatment with 5-Aza-dC did not significantly alter demethylation of the Maspin promoter but did demethylate histone H3. These results show Maspin promoter hypermethylation and histone methylation occur with down-regulation of Maspin synthesis in corneal stromal cells and suggest regulation of genes upon conversion of keratocytes to wound healing fibroblasts can involve promoter and histone methylation.
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sufficiency of the reactive site loop of Maspin for induction of cell matrix adhesion and inhibition of cell invasion conversion of ovalbumin to a Maspin like molecule
Journal of Biological Chemistry, 2003Co-Authors: Chatri Ngamkitidechakul, Janice M Burke, William J Obrien, Debra J Warejcka, Sally S TwiningAbstract:Maspin, an ov-serpin, inhibits tumor invasion and induces cell adhesion to extracellular matrix molecules. Here, we use Maspin/ovalbumin chimeric proteins and the Maspin reactive site loop (RSL) peptide to characterize the role of the RSL in Maspin-mediated functions. Replacement of the RSL plus the C-terminal region or the RSL alone of Maspin with that of ovalbumin resulted in the loss of the stimulatory effect on adhesion of corneal stromal cells to type I collagen, fibronectin, and laminin and of mammary carcinoma MDA-MB-231 cells to fibronectin. Maspin with ovalbumin as the C-terminal region retained activity, suggesting the Maspin C-terminal polypeptide is not required. An R340Q mutant retained full Maspin activity; however, an R340A mutant lost activity. This indicates the arginine side chain at the putative P1 site forms a hydrogen bond and not an ionic bond. The RSL peptide (P10-P5', amino acids 330-345) alone induced cell-matrix adhesion of mammary carcinoma cells and corneal stromal cells and inhibited invasion of the carcinoma cells. Substitution of the RSL of ovalbumin with that of Maspin converted inactive ovalbumin into a fully active molecule. Maspin bound specifically to the surface of the mammary carcinoma cells with a kd of 367 +/- 67 nM and 32.0 +/- 2.2 x 10(6) binding sites/cell. The Maspin RSL peptide inhibited binding, suggesting the RSL is involved in Maspin binding to cells. Sufficiency of the Maspin RSL for activity suggests the mechanism by which Maspin regulates cell-matrix adhesion and tumor cell invasion does not involve the serpin mechanism of protease inhibition.
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Maspin synthesis by human cornea and regulation of in vitro stromal cell adhesion to extracellular matrix
Investigative Ophthalmology & Visual Science, 2001Co-Authors: Chatri Ngamkitidechakul, Janice M Burke, William J Obrien, Sally S TwiningAbstract:PURPOSE. Maspin, a tumor-suppressor protein that regulates cell migration, invasion, and adhesion, is synthesized by many normal epithelial cells, but downregulated in invasive epithelial tumor cells. The purpose of this study was to determine whether cells in the normal human cornea express Maspin and whether Maspin affects corneal stromal cell adhesion to extracellular matrix molecules. METHODS. Maspin expression was analyzed by immunodot blot, Western blot, and RT-PCR analyses in cells obtained directly from human corneas in situ. Maspin protein and mRNA were also studied in primary and passaged cultures of corneal stromal cells using Western blot analysis, RT-PCR, and immunofluorescence microscopy. Maspin cDNA was cloned and sequenced from human corneal epithelial cells and expressed in a yeast system. The recombinant Maspin was used to study attachment of cultured human corneal stromal cells to extracellular matrices. RESULTS. Maspin mRNA and micromolar amounts of the protein were found in all three layers of the human cornea in situ, including the stroma. Maspin was also detected in primary and first-passage corneal stromal cells, but its expression was downregulated in subsequent passages. Late-passage stromal cells, which did not produce Maspin, responded to exogenous recombinant Maspin as measured by increased cell adhesion not only to fibronectin, similar to mammary gland tumor epithelial cells, but also to type I collagen, type IV collagen, and laminin. CONCLUSIONS. The corneal stromal cell is the first nonepithelial cell type shown to synthesize Maspin. Loss of Maspin expression in late-passage corneal stromal cells in culture and their biological response to exogenous Maspin suggests a role for Maspin on the stromal cells in the cornea. Maspin may function within the cornea to regulate cell adhesion to extracellular matrix molecules and perhaps to regulate the migration of activated fibroblasts during corneal stromal wound healing. (Invest Ophthalmol Vis Sci. 2001;42:3135‐3141)