Sijana H. Dzinic

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.

The mouse prostate: a basic anatomical and histological guideline

Despite substantial similarities in embryological, cellular and molecular biology features, human and mouse prostates differ in organ morphology and tissue architecture. Thus, a clear understanding of the anatomy and histology of the mouse prostate is essential for the identification of urogenital phenotypes in genetically engineered mice, as well as for the study of the etiology, development, and treatment of human prostatic diseases for which mouse models are used. The purpose of this manuscript is to provide a brief guide for the dissection of the mouse prostate and the identification of its different lobes and histology, to both basic researchers and medical pathologists who are unfamiliar with mouse tissues.

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.

Elevated Maspin Expression Is Associated with Better Overall Survival in Esophageal Squamous Cell Carcinoma (ESCC)

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. Maspin expression significantly inhibited cell proliferation, motility and matrigel invasion. Taken together, our data suggest that the transient up-regulation of maspin in the early development of ESCC may be a defense mechanism against further transition towards more malignant phenotypes, ultimately slowing down ESCC tumor progression.

Maspin expression patterns differ in the invasive versus lepidic growth pattern of pulmonary adenocarcinoma

To test whether changes in the subcellular localization of maspin parallel morphological progression in pulmonary adenocarcinoma, we compared its expression between lepidic and invasive growth patterns.

An essential role of maspin in embryogenesis and tumor suppression

Maspin (SerpinB5) is an epithelial-specific tumor suppressor gene product that displays context-dependent cellular functions. Maspin-deficient mouse models created to date have not definitively established maspin functions critical for cancer suppression. In this study, we generated a mouse strain in which exon 4 of the Maspin gene was deleted, confirming its essential role in development but also enabling a breeding scheme to bypass embryonic lethality. Phenotypic characterization of this viable strain established that maspin deficiency was associated with a reduction in maximum body weight and a variety of context-dependent epithelial abnormalities. Specifically, maspin-deficient mice exhibited pulmonary adenocarcinoma, myoepithelial hyperplasia of the mammary gland, hyperplasia of luminal cells of dorsolateral and anterior prostate, and atrophy of luminal cells of ventral prostate and stratum spinosum of epidermis. These cancer phenotypes were accompanied by increased inflammatory stroma. These mice also displayed the autoimmune disorder alopecia aerate. Overall, our findings defined context-specific tumor suppressor roles for maspin in a clinically relevant model to study maspin functions in cancer and other pathologies. Cancer Res; 77(4); 886-96. ©2017 AACR.

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.

Bacterial chemotaxis differences in Escherichia coli isolated from different hosts

The mechanisms mediating the association between Escherichia coli and specific hosts are unknown. This study investigates the hypothesis that the host-specific associations of E. coli strains are mediated in part by differences in chemotaxis. To test this hypothesis, chemotactic responses of E. coli strains isolated from different host groups (carnivores, herbivores, and omnivores) were tested with various attractants. In low-density agar chemotaxis assays, the average motility of E. coli in response to aspartate, serine, and ribose among the different groups was not significantly different; however, strains from carnivores responded significantly more to aspartate, relative to their responses to serine, in comparison with strains from herbivores, which responded equally or better to serine than to aspartate. The relatively greater chemotactic response of strains from carnivores to aspartate than to serine was confirmed in a subset of strains by capillary chemotaxis assay. Differences in responses to serine and aspartate were not due to growth differences, as determined by comparison of 24 h growth curves with glycerol, aspartate, and serine carbon sources. The differences in chemotactic behavior of E. coli strains isolated from herbivores and carnivores support the hypothesis that host-specific associations of E. coli strains are mediated in part by differences in chemotactic behavior.

Variable length tandem repeat polyglutamine sequences in the flexible tether region of the Tsr chemotaxis receptor of Escherichia coli

Methyl-accepting chemotaxis proteins (MCPs) are receptors that play an important role in bacterial chemotaxis. Methylation of Tsr, the MCP that mediates chemotaxis towards serine in Escherichia coli, is thought to be facilitated by binding of the methyltransferase to a flexible tether region at the C-terminal end of Tsr. This study analysed natural length variants of the tether that occur in E. coli due to genetic instability in tandem repeat DNA sequences that code for glutaminyl (Q) residues, creating polyQ sequences of variable lengths in the tether region. The tsr gene of E. coli K-12 (strain MG1655) codes for 4Q at the beginning of its 35 aa tether region. The tether varies in length from 35 to 47 residues among pathogenic and non-pathogenic strains of Escherichia, Shigella spp., Salmonella, Yersinia and Photorhabdus. Among previous sequences, Escherichia and Shigella mostly have 4Q and 7Q variants, and one strain (E. coli HS) has 10Q. In E. coli isolated from 50 humans and 75 animals (dogs, cats, horses, birds, etc.), polyQ up to 13Q (44 aa tether) were identified (6 strains); relative frequencies were 7Q ( approximately 77 % of the total) >4Q (14 %) >13Q (5 %) >10Q (4 %). Phylogenetic analysis revealed that E. coli strains with 10Q or 13Q largely fell within two clusters. Serine chemotaxis was not significantly different among 7Q, 10Q and 13Q strains, and was comparable to chemotaxis in the frequently studied K-12 strain. These results are consistent with models indicating that polyQ sequences from 7Q to 13Q are flexible, and that longer tethers, within this range, would not change the precision of adaptation mediated by methylation. Studies of this naturally variable polyQ region in E. coli may also have relevance to mechanisms that mediate polyQ instability in human genetic diseases.