Current advances of nitric oxide in cancer and anticancer therapeutics

Nitric oxide (NO) is a short-lived, ubiquitous signaling molecule that affects numerous critical functions in the body. There are markedly conflicting findings in the literature regarding the bimodal effects of NO in carcinogenesis and tumor progression, which has important consequences for treatment. Several preclinical and clinical studies have suggested that both pro- and antitumorigenic effects of NO depend on multiple aspects, including, but not limited to, tissue of generation, the level of production, the oxidative/reductive (redox) environment in which this radical is generated, the presence or absence of NO transduction elements, and the tumor microenvironment. Generally, there are four major categories of NO-based anticancer therapies: NO donors, phosphodiesterase inhibitors (PDE-i), soluble guanylyl cyclase (sGC) activators, and immunomodulators. Of these, NO donors are well studied, well characterized, and also the most promising. In this study, we review the current knowledge in this area, with an emphasis placed on the role of NO as an anticancer therapy and dysregulated molecular interactions during the evolution of cancer, highlighting the strategies that may aid in the targeting of cancer

Modular Evolution of DNA-Binding Preference of a Tbrain Transcription Factor Provides a Mechanism for Modifying Gene Regulatory Networks

Gene regulatory networks (GRNs) describe the progression of transcriptional states that take a single-celled zygote to a multicellular organism. It is well documented that GRNs can evolve extensively through mutations to cis-regulatory modules (CRMs). Transcription factor proteins that bind these CRMs may also evolve to produce novelty. Coding changes are considered to be rarer, however, because transcription factors are multifunctional and hence are more constrained to evolve in ways that will not produce widespread detrimental effects. Recent technological advances have unearthed a surprising variation in DNA-binding abilities, such that individual transcription factors may recognize both a preferred primary motif and an additional secondary motif. This provides a source of modularity in function. Here, we demonstrate that orthologous transcription factors can also evolve a changed preference for a secondary binding motif, thereby offering an unexplored mechanism for GRN evolution. Using protein-binding microarray, surface plasmon resonance, and in vivo reporter assays, we demonstrate an important difference in DNA-binding preference between Tbrain protein orthologs in two species of echinoderms, the sea star, Patiria miniata, and the sea urchin, Strongylocentrotus purpuratus. Although both orthologs recognize the same primary motif, only the sea star Tbr also has a secondary binding motif. Our in vivo assays demonstrate that this difference may allow for greater evolutionary change in timing of regulatory control. This uncovers a layer of transcription factor binding divergence that could exist for many pairs of orthologs. We hypothesize that this divergence provides modularity that allows orthologous transcription factors to evolve novel roles in GRNs through modification of binding to secondary sites

Survey of variation in human transcription factors reveals prevalent DNA binding changes

Published in final edited form as: Science. 2016 Mar 25; 351(6280): 1450–1454. Published online 2016 Mar 24. doi: 10.1126/science.aad2257Sequencing of exomes and genomes has revealed abundant genetic variation affecting the coding sequences of human transcription factors (TFs), but the consequences of such variation remain largely unexplored. We developed a computational, structure-based approach to evaluate TF variants for their impact on DNA binding activity and used universal protein-binding microarrays to assay sequence-specific DNA binding activity across 41 reference and 117 variant alleles found in individuals of diverse ancestries and families with Mendelian diseases. We found 77 variants in 28 genes that affect DNA binding affinity or specificity and identified thousands of rare alleles likely to alter the DNA binding activity of human sequence-specific TFs. Our results suggest that most individuals have unique repertoires of TF DNA binding activities, which may contribute to phenotypic variation.National Institutes of Health; NHGRI R01 HG003985; P50 HG004233; A*STAR National Science Scholarship; National Science Foundatio

Highly parallel assays of tissue-specific enhancers in whole Drosophila embryos

Transcriptional enhancers are a primary mechanism by which tissue-specific gene expression is achieved. Despite the importance of these regulatory elements in development, responses to environmental stresses, and disease, testing enhancer activity in animals remains tedious, with a minority of enhancers having been characterized. Here, we have developed ‘enhancer-FACS-Seq’ (eFS) technology for highly parallel identification of active, tissue-specific enhancers in Drosophila embryos. Analysis of enhancers identified by eFS to be active in mesodermal tissues revealed enriched DNA binding site motifs of known and putative, novel mesodermal transcription factors (TFs). Naïve Bayes classifiers using TF binding site motifs accurately predicted mesodermal enhancer activity. Application of eFS to other cell types and organisms should accelerate the cataloging of enhancers and understanding how transcriptional regulation is encoded within them

Using a structural and logics systems approach to infer bHLH–DNA binding specificity determinants

Numerous efforts are underway to determine gene regulatory networks that describe physical relationships between transcription factors (TFs) and their target DNA sequences. Members of paralogous TF families typically recognize similar DNA sequences. Knowledge of the molecular determinants of protein–DNA recognition by paralogous TFs is of central importance for understanding how small differences in DNA specificities can dictate target gene selection. Previously, we determined the in vitro DNA binding specificities of 19 Caenorhabditis elegans basic helix-loop-helix (bHLH) dimers using protein binding microarrays. These TFs bind E-box (CANNTG) and E-box-like sequences. Here, we combine these data with logics, bHLH–DNA co-crystal structures and computational modeling to infer which bHLH monomer can interact with which CAN E-box half-site and we identify a critical residue in the protein that dictates this specificity. Validation experiments using mutant bHLH proteins provide support for our inferences. Our study provides insights into the mechanisms of DNA recognition by bHLH dimers as well as a blueprint for system-level studies of the DNA binding determinants of other TF families in different model organisms and humans.National Institute of General Medical Sciences (U.S.) (DK068429)National Institute of General Medical Sciences (U.S.) (HG003985)European Union (PROSPECTS HEALTH-F4-2008-201648

Notch and MAML-1 Complexation Do Not Detectably Alter the DNA Binding Specificity of the Transcription Factor CSL

Canonical Notch signaling is initiated when ligand binding induces proteolytic release of the intracellular part of Notch (ICN) from the cell membrane. ICN then travels into the nucleus where it drives the assembly of a transcriptional activation complex containing the DNA-binding transcription factor CSL, ICN, and a specialized co-activator of the Mastermind family. A consensus DNA binding site motif for the CSL protein was previously defined using selection-based methods, but whether subsequent association of Notch and Mastermind-like proteins affects the DNA binding preferences of CSL has not previously been examined.Here, we utilized protein-binding microarrays (PBMs) to compare the binding site preferences of isolated CSL with the preferred binding sites of CSL when bound to the CSL-binding domains of all four different human Notch receptors. Measurements were taken both in the absence and in the presence of Mastermind-like-1 (MAML1). Our data show no detectable difference in the DNA binding site preferences of CSL before and after loading of Notch and MAML1 proteins.These findings support the conclusion that accrual of Notch and MAML1 promote transcriptional activation without dramatically altering the preferred sites of DNA binding, and illustrate the potential of PBMs to analyze the binding site preferences of multiprotein-DNA complexes

Role of nitric oxide-based immunotherapy in augmenting prostate cancer progression by targeting androgen receptor heterogeneity

e17537 Background: A significant proportion of men with Prostate Cancer (PCa) develop castration resistant prostate cancer (CRPC) and do not respond to hormonal agents that decrease androgens. In trying to understand the causes of androgen resistance that develop in CRPC, it is considered most relevant to study the role of Androgen receptor (AR) in the development and progression of PCa from androgen dependent to androgen independent state. Recent studies have highlighted the significance of tumor microenvironment (TME) in regulation of PCa progression in addition to AR. A key molecule in the regulation of TME interactions is nitric oxide (NO). We have shown in our recent study, the critical association of NO with the TME in CRPC. However, the effects of NO to modulate the progression of PCa to CRPC with respect to AR still remains largely unexplored. Methods: 22RV1, LNCaP, LNCaP APIPC (cells expressing no AR), and LNCaP shAR/pATK (cells expressing low AR), cells were used for the study. Cell proliferation was first assessed by MTT assay. The castrated SCID mice were grafted with 22RV1 cells and were treated with GSNO at the dosage of 10mg/kg/day IP. After treatment, animals were humanely sacrificing. Tumor RNA and proteins were analysed for markers that are important for PCa progression using qPCR, western blot and cytokine antibody array. Animal experiments were carried out in compliance with the IACUC of University of Miami. GraphPad Prism (GraphPad Software) was used for statistical analysis. Results: In addition to reducing the tumor burden, the expression of anti-inflammatory (M2) macrophages (CD206 and Arginase1) is decreased and that of the pro-inflammatory (M1) macrophage (iNOS) is increased in mice which received increased NO levels. Furthermore, to study the effects of NO on progression of PCa from androgen dependent to androgen independent stage, we characterized the LNCAP cell models with differential extent of AR knockdown (LNCaP, LNCaP shAR/pATK and LNCaP APIPC ) for the effects of increased NO levels. Results showed that NO had significant impact on cell proliferation on androgen dependent PCa cells however the effects were negligible in cells expressing low or no AR, suggesting that effects of NO on PCa cell proliferation are AR dependent. Conclusions: Our results suggest that during PCa progression, NO suppresses TAMs to target the TME in an AR dependent manner. Further studies are undergoing to establish the impacts of NO in PCa progression

Mesenchymal Stem Cells: Characterization, Properties and Therapeutic Potential

Mesenchymal stem cells are one of the most widely studied cell types with promising applications in regenerative medicine, thanks to their abundant supply, ease of isolation, unique differentiation potential, immunosuppressive phenotype, and stimulation of endogenous repair mechanisms. Diverse sources of MSCs include embryonic and extraembryonic lineages and adult tissues, such as bone, fat, and cartilage, each intricately regulated by specific signaling pathways. MSCs are at the forefront of stem cell therapy, where they can be utilized as whole-cell, cell-free, or in combination with other cells to produce meaningful therapeutic outcomes for the treatment of cardiovascular, neurological, pulmonary, liver, and kidney disease. Here, we review the underlying biology and regenerative potential of MSCs

Abstract 137: Ghrhr is a Cell-surface Marker of Human Pluripotent Stem Cell-derived Cardiomyogenic Precursors

Introduction: A major roadblock for generating human pluripotent stem cell (hPSCs) derivatives highly enriched in cardiomyogenic precursors (CPCs), has been the lack of CPC-specific cell surface markers. Hypothesis: Based on observations that adult CPCs are responsive to growth hormone-releasing hormone (GHRH) signaling, we hypothesized that the GHRH receptor (GHRHR) is a specific cell-surface marker for hPSC-derived CPCs. Methods: We performed temporal analysis of GHRHR expression in an in-vitro model of human cardiogenesis using induced hPSCs (hiPSCs) and SOX10::GFP embryonic hPSCs (hESCs) ; and mouse ( in-vivo ) cardiogenesis in wild-type (WT), MEF2c-AHF-Cre, Wnt1-Cre2 and cKit-CreERT2/+ reporter mice. Results: Gene expression and confocal immunofluorescence analyses during chemically-defined, stage-specific, cardiac lineage differentiation indicated that GHRHR is not expressed in undifferentiated hiPSCs or during specification into primitive streak-like Brachyury + or Mesp1 + precardiac cells; but is induced in cardiogenic mesoderm-like cells, at the stage of commitment into NKX2.5 + and/or ISL1 + CPCs ( p =0.001) and persists in Troponin-T + cardiomyocytes. Similarly, experiments modeling cardiac neural crest (CNC) with SOX10::GFP hESCs indicated that GHRHR is not expressed by GFP + CNCs but is induced following differentiation into NKX2.5 + and/or ISL1 + derivatives. Importantly, stimulation with 1μm recombinant GHRH during days 5-7 of hiPSCs differentiation increased NKX2.5 expression 2.5-fold, an effect that was abolished by exposure to 1μM Somatostatin, a GHRH antagonist ( p =0.0009). Last, in vivo analyses in WT , MEF2c-AHF-Cre, Wnt1-Cre2 and cKit-CreERT2/+ reporter embryonic and postnatal hearts corroborated that GHRHR specifically marks NKX2.5 + mesoderm- and CNC-lineage descendants in vivo, whereas GHRHR is not expressed by Wnt1-Cre2 and cKit-CreERT2/+ CNCs descendants that are Nkx2.5 – . Conclusions: Together these findings indicate that GHRHR is universally expressed by NKX2.5 + /ISL1 + CPCs and cardiomyocytes of both mesoderm and CNC origin. Therefore, GHRHR appears to be a valuable cell-surface marker for the selection and enrichment of CPCs from hPSCs for biomedical and regenerative medicine applications