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. © 2017 Wiley Periodicals, Inc.

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 G(0)/G(1) 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.

Stiff stroma increases breast cancer risk by inducing the oncogene ZNF217.

Women with dense breasts have an increased lifetime risk of malignancy that has been attributed to a higher epithelial density. Quantitative proteomics, collagen analysis, and mechanical measurements in normal tissue revealed that stroma in the high-density breast contains more oriented, fibrillar collagen that is stiffer and correlates with higher epithelial cell density. microRNA (miR) profiling of breast tissue identified miR-203 as a matrix stiffness-repressed transcript that is downregulated by collagen density and reduced in the breast epithelium of women with high mammographic density. Culture studies demonstrated that ZNF217 mediates a matrix stiffness- and collagen density-induced increase in Akt activity and mammary epithelial cell proliferation. Manipulation of the epithelium in a mouse model of mammographic density supported a causal relationship between stromal stiffness, reduced miR-203, higher levels of the murine homolog Zfp217, and increased Akt activity and mammary epithelial proliferation. ZNF217 was also increased in the normal breast epithelium of women with high mammographic density, correlated positively with epithelial proliferation and density, and inversely with miR-203. The findings identify ZNF217 as a potential target toward which preexisting therapies, such as the Akt inhibitor triciribine, could be used as a chemopreventive agent to reduce cancer risk in women with high mammographic density.

Feeling Stress: The Mechanics of Cancer Progression and Aggression.

The tumor microenvironment is a dynamic landscape in which the physical and mechanical properties evolve dramatically throughout cancer progression. These changes are driven by enhanced tumor cell contractility and expansion of the growing tumor mass, as well as through alterations to the material properties of the surrounding extracellular matrix (ECM). Consequently, tumor cells are exposed to a number of different mechanical inputs including cell-cell and cell-ECM tension, compression stress, interstitial fluid pressure and shear stress. Oncogenes engage signaling pathways that are activated in response to mechanical stress, thereby reworking the cell's intrinsic response to exogenous mechanical stimuli, enhancing intracellular tension via elevated actomyosin contraction, and influencing ECM stiffness and tissue morphology. In addition to altering their intracellular tension and remodeling the microenvironment, cells actively respond to these mechanical perturbations phenotypically through modification of gene expression. Herein, we present a description of the physical changes that promote tumor progression and aggression, discuss their interrelationship and highlight emerging therapeutic strategies to alleviate the mechanical stresses driving cancer to malignancy.

New Horizons in Advocacy Engaged Physical Sciences and Oncology Research.

To address cancer as a multifaceted adaptive system, the increasing momentum for cross-disciplinary connectivity between cancer biologists, physical scientists, mathematicians, chemists, biomedical engineers, computer scientists, clinicians, and advocates is fueling the emergence of new scientific frontiers, principles, and opportunities within physical sciences and oncology. In parallel to highlighting the advances, challenges, and acceptance of advocates as credible contributors, we offer recommendations for addressing real world hurdles in advancing equitable partnerships among advocacy stakeholders.

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.

Maspin expression in prostate tumor elicits host anti-tumor immunity.

The goal of the current study is to examine the biological effects of epithelial-specific tumor suppressor maspin on tumor host immune response. Accumulated evidence demonstrates an anti-tumor effect of maspin on tumor growth, invasion and metastasis. The molecular mechanism underlying these biological functions of maspin is thought to be through histone deacetylase inhibition, key to the maintenance of differentiated epithelial phenotype. Since tumor-driven stromal reactivities co-evolve in tumor progression and metastasis, it is not surprising that maspin expression in tumor cells inhibits extracellular matrix degradation, increases fibrosis and blocks hypoxia-induced angiogenesis. Using the athymic nude mouse model capable of supporting the growth and progression of xenogeneic human prostate cancer cells, we further demonstrate that maspin expression in tumor cells elicits neutrophil- and B cells-dependent host tumor immunogenicity. Specifically, mice bearing maspin-expressing tumors exhibited increased systemic and intratumoral neutrophil maturation, activation and antibody-dependent cytotoxicity, and decreased peritumoral lymphangiogenesis. These results reveal a novel biological function of maspin in directing host immunity towards tumor elimination that helps explain the significant reduction of xenograft tumor incidence in vivo and the clinical correlation of maspin with better prognosis of several types of cancer. Taken together, our data raised the possibility for novel maspin-based cancer immunotherapies.

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 Aspartate(346) (D(346)) to Glutamate (E(346)), maspin(D346E), was predominantly nuclear, whereas wild type maspin (maspin(WT)) 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 maspin(D346E) was, at least in part, due to its increased affinity to HDAC1. Maspin(D346E) was also more potent than maspin(WT) as an HDAC inhibitor. Taken together, our evidence demonstrates that D(346) 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.

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 TGFß 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.