Ivory Dean

A NOVEL GERANYLGERANYL TRANSFERASE INHIBITOR IN COMBINATION WITH LOVASTATIN INHIBITS PROLIFERATION AND INDUCES AUTOPHAGY IN STS-26T MPNST CELLS

Prenylation inhibitors have gained increasing attention as potential therapeutics for cancer. Initial work focused on inhibitors of farnesylation, but more recently geranylgeranyl transferase inhibitors (GGTIs) have begun to be evaluated for their potential antitumor activity in vitro and in vivo. In this study, we have developed a nonpeptidomimetic GGTI, termed GGTI-2Z [(5-nitrofuran-2-yl)methyl-(2Z,6E,10E)-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraenyl 4-chlorobutyl(methyl)phosphoramidate], which in combination with lovastatin inhibits geranylgeranyl transferase I (GGTase I) and GGTase II/RabGGTase, without affecting farnesylation. The combination treatment results in a G0/G1 arrest and synergistic inhibition of proliferation of cultured STS-26T malignant peripheral nerve sheath tumor cells. We also show that the antiproliferative activity of drugs in combination occurs in the context of autophagy. The combination treatment also induces autophagy in the MCF10.DCIS model of human breast ductal carcinoma in situ and in 1c1c7 murine hepatoma cells, where it also reduces proliferation. At the same time, there is no detectable toxicity in normal immortalized Schwann cells. These studies establish GGTI-2Z as a novel geranylgeranyl pyrophosphate derivative that may work through a new mechanism involving the induction of autophagy and, in combination with lovastatin, may serve as a valuable paradigm for developing more effective strategies in this class of antitumor therapeutics.

Maspin reprograms the gene expression profile of prostate carcinoma cells for differentiation.

Maspin is an epithelial-specific tumor suppressor gene. Previous data suggest that maspin expression may redirect poorly differentiated tumor cells to better differentiated phenotypes. Further, maspin is the first and only endogenous polypeptide inhibitor of histone deacetylase 1 (HDAC1) identified thus far. In the current study, to address what central program of tumor cell redifferentiation is regulated by maspin and how tumor microenvironments further define the effects of maspin, we conducted a systematic and extensive comparison of prostate tumor cells grown in 2-dimensional culture, in 3-dimensional collagen I culture, and as in vivo bone tumors. We showed that maspin was sufficient to drive prostate tumor cells through a spectrum of temporally and spatially polarized cellular processes of redifferentiation, a reversal of epithelial-to-mesenchymal transition (EMT). Genes commonly regulated by maspin were a small subset of HDAC target genes that are closely associated with epithelial differentiation and TGFβ signaling. These results suggest that a specific endogenous HDAC inhibitor may regulate one functionally related subset of HDAC target genes, although additional maspin-induced changes of gene expression may result from tumor interaction with its specific microenvironments. Currently, EMT is recognized as a critical step in tumor progression. To this end, our current study uncovered a link between maspin and a specific mechanism of prostate epithelial differentiation that can reverse EMT.

Identification of an Intrinsic Determinant Critical for Maspin Subcellular Localization and Function

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 new window of opportunity for the development of maspin-based biologically competent HDAC inhibitors for cancer treatment.

The secretion and biological function of tumor suppressor maspin as an exosome cargo protein

Maspin is an epithelial-specific tumor suppressor shown to exert its biological effects as an intracellular, cell membrane-associated, and secreted free molecule. A recent study suggests that upon DNA-damaging g-irradiation, tumor cells can secrete maspin as an exosome-associated protein. To date, the biological significance of exosomal secretion of maspin is unknown. The current study aims at addressing whether maspin is spontaneously secreted as an exosomal protein to regulate tumor/stromal interactions. We prepared exosomes along with cell extracts and vesicle-depleted conditioned media (VDCM) from normal epithelial (CRL2221, MCF-10A and BEAS-2B) and cancer (LNCaP, PC3 and SUM149) cell lines. Atomic force microscopy and dynamic light scattering analysis revealed similar size distribution patterns and surface zeta potentials between the normal cells-derived and tumor cells-derived exosomes. Electron microscopy revealed that maspin was encapsulated by the exosomal membrane as a cargo protein. While western blotting revealed that the level of exosomal maspin from tumor cell lines was disproportionally lower relative to the levels of corresponding intracellular and VDCM maspin, as compared to that from normal cell lines, maspin knockdown in MCF-10A cells led to maspin-devoid exosomes, which exhibited significantly reduced suppressive effects on the chemotaxis activity of recipient NIH3T3 fibroblast cells. These data are the first to demonstrate the potential of maspin delivered by exosomes to block tumor-induced stromal response, and support the clinical application of exosomal maspin in cancer diagnosis and treatment.

Maspin Expression in Prostate Tumor Cells Averts Stemness and Stratifies Drug Sensitivity

Future curative cancer chemotherapies have to overcome tumor cell heterogeneity and plasticity. To test the hypothesis that the tumor suppressor maspin may reduce microenvironment-dependent prostate tumor cell plasticity and thereby modulate drug sensitivity, we established a new schematic combination of two-dimensional (2D), three-dimensional (3D), and suspension cultures to enrich prostate cancer cell subpopulations with distinct differentiation potentials. We report here that depending on the level of maspin expression, tumor cells in suspension and 3D collagen I manifest the phenotypes of stem-like and dormant tumor cell populations, respectively. In suspension, the surviving maspin-expressing tumor cells lost the self-renewal capacity, underwent senescence, lost the ability to dedifferentiate in vitro, and failed to generate tumors in vivo. Maspin-nonexpressing tumor cells that survived the suspension culture in compact tumorspheres displayed a higher level of stem cell marker expression, maintained the self-renewal capacity, formed tumorspheres in 3D matrices in vitro, and were tumorigenic in vivo. The drug sensitivities of the distinct cell subpopulations depend on the drug target and the differentiation state of the cells. In 2D, docetaxel, MS275, and salinomycin were all cytotoxic. In suspension, while MS275 and salinomycin were toxic, docetaxel showed no effect. Interestingly, cells adapted to 3D collagen I were only responsive to salinomycin. Maspin expression correlated with higher sensitivity to MS275 in both 2D and suspension and to salinomycin in 2D and 3D collagen I. Our data suggest that maspin reduces prostate tumor cell plasticity and enhances tumor sensitivity to salinomycin, which may hold promise in overcoming tumor cell heterogeneity and plasticity.

The Opportunity of Precision Medicine for Breast Cancer With Context-Sensitive Tumor Suppressor Maspin

To improve the precision of molecular diagnosis and to develop and guide targeted therapies of breast cancer, it is essential to determine the mechanisms that underlie the specific tumor phenotypes. To this end, the application of a snapshot of gene expression profile for breast cancer diagnosis and prognosis is fundamentally challenged since the tissue‐based data are derived from heterogonous cell types and are not likely to reflect the dynamics of context‐dependent tumor progression and drug sensitivity. The intricate network of epithelial differentiation program can be concertedly controlled by tumor suppressor maspin, a homologue of clade B serine protease inhibitors (serpin), through its multifaceted molecular interactions in multiple subcellular localizations. Unlike most other serpins that are expressed in multiple cell types, maspin is epithelial specific and has distinct roles in luminal and myoepithelial cells. Endogenously expressed maspin has been found in the nucleus and cytoplasm, and detected on the surface of cell membrane. It is also secreted free and as an exosomal cargo protein. Research in the field has led to the identification of the maspin targets and maspin‐associated molecules, as well as the structural determinants of its suppressive functions. The current review discusses the possibility for maspin to serve as a cell type‐specific and context‐sensitive marker to improve the precision of breast cancer diagnosis and prognosis. These advancements further suggest a new window of opportunity for designing novel maspin‐based chemotherapeutic agents with improved anti‐cancer potency. J. Cell. Biochem. 118: 1639–1647, 2017.

Towards Curative Cancer Therapy with Maspin: A Unique Window of Opportunity to Target Cancer Dormancy

Tumor dormancy is considered to be the last frontier in the battle to cure cancer. Although experimental evidence and clinical studies led to some consensus regarding the phenotypical characteristics of tumor dormancy, the underlying biological controls remain elusive. As a result, in the absence of dormancy-targeted therapeutic strategies, cancer drug resistance and recurrence are a certainty in a matter of time. In this review, we discuss a novel opportunity to target prostate tumor dormancy based on the expression of tumor suppressor maspin, an epithelial-specific endogenous inhibitor of histone deacetylase 1 (HDAC1).

Abstract 5067: Human prostate epithelial cells secrete the tumor suppressor maspin as a soluble and exosome-associated protein .

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Maspin, an epithelial-specific member of the serine protease inhibitor superfamily, inhibits tumor invasion and metastasis. Overall maspin expression is downregulated or lost in invasive and metastatic carcinomas. Maspin subcellular distribution among the nuclear, cytoplasmic and extracellular compartments is differentially regulated during tumor progression. Extracellular maspin has been shown to inhibit tumor cell invasion and motility in vitro. Also, recombinant maspin was shown to inhibit endothelial cell tube formation in vitro and tumor-angiogenesis in vivo. To date, the mechanisms governing maspin trafficking remain unclear. In the current study, we aim to investigate the mechanism and biological significance of maspin secretion. Normal and tumor cells known to secrete maspin, were treated with or without drugs targeting classical or non-classical secretory pathways. The CM were collected and analyzed for soluble and exosome-associated proteins. We also utilized atomic force microscopy to characterize the physical characteristics of exosomes. We found maspin as a soluble protein in the CM. Maspin secretion was not inhibited by the drug treatments. In fact, maspin secretion increased under drug treatment targeting the classical secretory pathway. Furthermore, maspin was detected in the exosomes. Under drug treatment, the overall increase in maspin secretion was accompanied by an increase in detection of maspin in the exosomes. Based on the drug treatment-induced changes in maspins secretion patterns, we speculate that maspin is secreted via a non-classical pathway. In addition, we provide the first evidence that maspin is secreted inclusively as both a free and an exosome-encapsulated molecule in normal and tumor cells. The existence of extracellular maspin as a free and an exosome-associated molecule implies a dual role for maspin in the tumor microenvironment. These novel findings are currently under investigation since the tumor suppressive activity of exosome-associated maspin may be of particular significance in cell-cell communication, and tumor suppressive activity of free maspin may be associated with extracellular matrix remodeling. Citation Format: Ivory S. Dean, Sijana Dzinic, Alexander Kaplun, Yi Zou, Guangzhao Mao, Shijie Sheng. Human prostate epithelial cells secrete the tumor suppressor maspin as a soluble and exosome-associated protein . [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 5067. doi:10.1158/1538-7445.AM2013-5067

Abstract 5203: Identification of an intrinsic determinant critical for maspin subcellular localization and function.

Abstract: Maspin is a novel serine protease inhibitor (serpin) that does not act as a classical serine protease inhibitor. Our lab demonstrated that maspin directly inhibits the serine protease-like enzyme, histone deacetylase 1 (HDAC1), which is a major deacetylase up-regulated in many types of cancers. To our knowledge, maspin is the only endogenous polypeptide inhibitor of HDAC1 identified thus far. Interestingly, the differential regulation of maspin during tumor progression is characterized by changes in overall expression and subcellular distribution. In particular, a significant shift in maspin subcellular localization from the nucleus to the cytosol has been observed at the transition from benign epithelial cells to precancerous or low grade carcinoma cells. Accumulated clinical evidence suggests that nuclear retention of maspin is correlated with better overall patient survival. We propose that hypothesized that the configuration and conformation of maspin coded by its primary sequence, i.e. cis elements are the key determinants for where maspin may be retained in a given molecular context. We noted that at the C-terminal end of the RCL sequence is an Aspartate 346 (D 346 ) which is also unique among all maspin homologs and orthologs. In the current study, using the mutagenesis approach, we aimed to determine whether D 346 acts as the cis element essential for maspin subcellular localization and function. We showed that in comparison to the maspin WT , which is distributed in both cytosol and nuclei of cancer cells, maspin D346E is predominantly localized in the nuclei of cancer cells. In addition, maspin D346E had an increased affinity towards HDAC1 and it was more effective inhibitor of HDAC1 as compared to the maspin WT . These findings led to a novel molecular model that explains how maspin may be disregulated during tumor progression resulting in loss of its tumor suppressive potential. Citation Format: Sijana H. Dzinic, Alexander Kaplun, Xiaohua Li, Margarida M. Bernardo, Ivory Dean, Fulvio Lonardo, Shijie Sheng. Identification of an intrinsic determinant critical for maspin subcellular localization and function. [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 5203. doi:10.1158/1538-7445.AM2013-5203

Abstract 3359: Maspin induces MET in prostate cancer cells cultured in 3D-collagen I

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The aggressiveness of carcinoma is typically associated with a significant epithelial-to-mesenchymal phenotypical shift (EMT). While several pathways have been identified as the driving force for EMT, mechanisms that epithelial cells depend on to prevent or reverse EMT are largely unknown. To this end, the tumor suppressive maspin inhibits tumor cell motility, invasion, pericellular plasminogen activation and extracellular matrix (ECM) degradation, and blocks the prostate tumor-induced bone remodeling. Furthermore, a general correlation between maspin expression and better differentiated epithelial phenotypes has been noted, with established cell lines, in an in vivo model for prostate tumor bone metastasis and in human prostate tissue specimens. The objective of the current study is to test whether maspin is sufficient to drive the program of mesenchymal-to-epithelial transition (MET). We showed that stably transfected DU145 that express maspin (M7) formed spheroid structures of polarized cells with a hollow lumen in 3D collagen I culture. The M7 acini in 3D collagen were encircled with reorganized fibrillar collagen, while the Neo control cells degraded collagen fibrils. The spheroid formation was abolished when M7 cells were transfected with maspin shRNA. Under the same condition, no redifferentiation of DU145 cells, with or without maspin, occurred in 3D Matrigel. To examine what other cellular and molecular changes are associated with the maspin-led tumor cell redifferentiation, we performed immunofluorescent staining and found that maspin correlated with highly organized β1-integrin localized on the periphery of the M7 spheroids. We confirmed the secretion of maspin protein by prostate epithelial cells. To test whether the effect of maspin on prostate epithelial cell differentiation depends on uPA or uPAR, we performed the 3D collagen spheroid formation assay in the presence of specific neutralizing antibodies. We found that only the neutralizing antibodies of either uPA or uPAR led to a slight increase of the size of the spheroids. While uPA and uPAR may be targeted as a part of the biological activities of maspin, our results suggest that the effect of maspin more than just blocks the uPA/uPAR cascade. Indeed, mRNA microarray, with cDNA derived from Neo and M7 cells grown in 2D cultures, 3D culture in collagen I and bone tumors in the Scid-Hu model, identified 32 commonly up-regulated genes and commonly down-regulated genes upon maspin expression. These epigenetic changes, along with other evidence, support the hypothesis that maspin induces MET in prostate tumor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3359. doi:10.1158/1538-7445.AM2011-3359