ABSTRACT
BACKGROUND & AIMS: Pancreatic ductal adenocarcinomas (PDACs) are characterized by fibrosis and an abundance of cancer-associated fibroblasts (CAFs). We investigated strategies to disrupt interactions among CAFs, the immune system, and cancer cells, focusing on adhesion molecule CDH11, which has been associated with other fibrotic disorders and is expressed by activated fibroblasts. METHODS: We compared levels of CDH11 messenger RNA in human pancreatitis and pancreatic cancer tissues and cells with normal pancreas, and measured levels of CDH11 protein in human and mouse pancreatic lesions and normal tissues. We crossed p48-Cre;LSL-KrasG12D/+;LSL-Trp53R172H/+ (KPC) mice with CDH11-knockout mice and measured survival times of offspring. Pancreata were collected and analyzed by histology, immunohistochemistry, and (single-cell) RNA sequencing; RNA and proteins were identified by imaging mass cytometry. Some mice were given injections of PD1 antibody or gemcitabine and survival was monitored. Pancreatic cancer cells from KPC mice were subcutaneously injected into Cdh11+/+ and Cdh11-/- mice and tumor growth was monitored. Pancreatic cancer cells (mT3) from KPC mice (C57BL/6), were subcutaneously injected into Cdh11+/+ (C57BL/6J) mice and mice were given injections of antibody against CDH11, gemcitabine, or small molecule inhibitor of CDH11 (SD133) and tumor growth was monitored. RESULTS: Levels of CDH11 messenger RNA and protein were significantly higher in CAFs than in pancreatic cancer epithelial cells, human or mouse pancreatic cancer cell lines, or immune cells. KPC/Cdh11+/- and KPC/Cdh11-/- mice survived significantly longer than KPC/Cdh11+/+ mice. Markers of stromal activation entirely surrounded pancreatic intraepithelial neoplasias in KPC/Cdh11+/+ mice and incompletely in KPC/Cdh11+/- and KPC/Cdh11-/- mice, whose lesions also contained fewer FOXP3+ cells in the tumor center. Compared with pancreatic tumors in KPC/Cdh11+/+ mice, tumors of KPC/Cdh11+/- mice had increased markers of antigen processing and presentation; more lymphocytes and associated cytokines; decreased extracellular matrix components; and reductions in markers and cytokines associated with immunosuppression. Administration of the PD1 antibody did not prolong survival of KPC mice with 0, 1, or 2 alleles of Cdh11. Gemcitabine extended survival of KPC/Cdh11+/- and KPC/Cdh11-/- mice only or reduced subcutaneous tumor growth in mT3 engrafted Cdh11+/+ mice when given in combination with the CDH11 antibody. A small molecule inhibitor of CDH11 reduced growth of pre-established mT3 subcutaneous tumors only if T and B cells were present in mice. CONCLUSIONS: Knockout or inhibition of CDH11, which is expressed by CAFs in the pancreatic tumor stroma, reduces growth of pancreatic tumors, increases their response to gemcitabine, and significantly extends survival of mice. CDH11 promotes immunosuppression and extracellular matrix deposition, and might be developed as a therapeutic target for pancreatic cancer.
Subject(s)
Cadherins/metabolism , Cancer-Associated Fibroblasts/metabolism , Carcinoma, Pancreatic Ductal/immunology , Deoxycytidine/analogs & derivatives , Pancreatic Neoplasms/immunology , Animals , Cadherins/antagonists & inhibitors , Cadherins/genetics , Cancer-Associated Fibroblasts/immunology , Carcinoma, Pancreatic Ductal/drug therapy , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/surgery , Deoxycytidine/pharmacology , Deoxycytidine/therapeutic use , Disease Models, Animal , Disease Progression , Drug Resistance, Neoplasm/genetics , Drug Resistance, Neoplasm/immunology , Extracellular Matrix/immunology , Extracellular Matrix/pathology , Female , Gene Expression Regulation, Neoplastic , Humans , Metallothionein 3 , Mice , Mice, Knockout , Pancreas/cytology , Pancreas/immunology , Pancreas/pathology , Pancreas/surgery , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/surgery , Pancreaticoduodenectomy , Tumor Escape/drug effects , Tumor Escape/genetics , Tumor Escape/immunology , Tumor Microenvironment/genetics , Tumor Microenvironment/immunology , GemcitabineABSTRACT
microRNAs are thought to regulate tumor progression and invasion via direct interaction with target genes within cells. Here the microRNA17/20 cluster is shown to govern cellular migration and invasion of nearby cells via heterotypic secreted signals. microRNA17/20 abundance is reduced in highly invasive breast cancer cell lines and node-positive breast cancer specimens. Cell-conditioned medium from microRNA17/20-overexpressing noninvasive breast cancer cell MCF7 was sufficient to inhibit MDA-MB-231 cell migration and invasion through inhibiting secretion of a subset of cytokines, and suppressing plasminogen activation via inhibition of the secreted plasminogen activators (cytokeratin 8 and alpha-enolase). microRNA17/20 directly repressed IL-8 by targeting its 3' UTR, and inhibited cytokeratin 8 via the cell cycle control protein cyclin D1. At variance with prior studies, these results demonstrated a unique mechanism of how the altered microRNA17/20 expression regulates cellular secretion and tumor microenvironment to control migration and invasion of neighboring cells in breast cancer. These findings not only reveal an antiinvasive function of miR-17/20 in breast cancer, but also identify a heterotypic secreted signal that mediates the microRNA regulation of tumor metastasis.
Subject(s)
Breast Neoplasms/genetics , Breast Neoplasms/pathology , MicroRNAs/genetics , Signal Transduction , 3' Untranslated Regions , Animals , Breast Neoplasms/metabolism , Cell Line, Tumor , Cell Movement , Enzyme Activation , Gene Expression Regulation, Neoplastic , Humans , Interleukin-8/genetics , Interleukin-8/metabolism , Neoplasm Invasiveness , Neoplasm Metastasis , Plasminogen/metabolism , Protein BindingABSTRACT
Progression of invasive carcinoma involves the deregulation of molecular signaling pathways that results in the acquisition of oncogenic phenotypes. Functional enrichment analysis allows for the identification of deregulated pathways from omics scale expression data. Given the importance of post-transcriptional regulatory mechanisms on protein expression and function, identification of deregulated pathways on the basis of protein expression data is likely to provide new insights. In this study, we have developed methods for label-based mass spectrometry in a large number of samples and applied these methods toward identification and quantification of protein expression in samples of infiltrating ductal carcinoma, benign breast growths, and normal adjacent tissue. We identified 265 proteins with differential expression patterns in infiltrating ductal carcinoma relative to benign growths or normal breast tissue. Analysis of the differentially expressed proteins indicated the deregulation of signaling pathways related to proliferation, invasion and metastasis, and immune response. Our approach provides complementary information to gene expression microarray data and identifies a number of deregulated molecular signaling pathways indicative of breast cancer progression that may enable more accurate, biologically relevant diagnoses and provide a stepping stone to personalized treatment.
Subject(s)
Breast Neoplasms/metabolism , Carcinoma, Ductal, Breast/metabolism , Proteome/analysis , Proteomics/methods , Tumor Microenvironment , Aged , Breast Neoplasms/chemistry , Breast Neoplasms/pathology , Carcinoma, Ductal, Breast/chemistry , Carcinoma, Ductal, Breast/pathology , Chromatography, Liquid , Cluster Analysis , Electrophoresis, Gel, Two-Dimensional , Female , Gene Expression Regulation, Neoplastic , Humans , Middle Aged , Proteome/metabolism , Signal Transduction , Tandem Mass SpectrometryABSTRACT
Mass cytometry, or cytometry by time-of-flight (the basis for Fluidigm® CyTOF® technology), is a system for single-cell detection using antibodies tagged with metal probes. Without the need for compensation, the highly parametric Helios™ mass cytometer has a detection range of 135 distinct mass channels (75-209 Da). Optimized for mass cytometry, the Maxpar® Direct™ Immune Profiling Assay™ is a dry, metal-tagged antibody cocktail for immunophenotyping 37 immune cell populations found in human peripheral blood in a single tube. The Maxpar Direct Assay utilizes 31 mass channels for marker detection and live/dead viability staining, with at least 14 additional marker channels available from the Fluidigm catalog for flexible custom panel design. Here, we describe a workflow combining the assay with additional surface and intracellular cytokine antibodies for peripheral blood mononuclear cell (PBMC) staining using lanthanide-, bismuth-, and cadmium-tagged antibodies.
Subject(s)
Cytokines/analysis , Antibodies , Biomarkers , Flow Cytometry , Humans , Immunophenotyping , Intracellular Space , Leukocytes, Mononuclear/immunology , Staining and Labeling , WorkflowABSTRACT
Activation of the inflammasome generates the pro-inflammatory cytokines interleukin-1 beta and -18, which are important mediators of inflammation. Abnormal activation of the inflammasome leads to many inflammatory diseases, including gout, silicosis, neurodegeneration, and genetically inherited periodic fever syndromes. Therefore, identification of small molecule inhibitors that target the inflammasome is an important step toward developing effective therapeutics for the treatment of inflammation. Here, we show that the herbal NF-kappaB inhibitory compound parthenolide inhibits the activity of multiple inflammasomes in macrophages by directly inhibiting the protease activity of caspase-1. Additional investigations of other NF-kappaB inhibitors revealed that the synthetic I kappaB kinase-beta inhibitor Bay 11-7082 and structurally related vinyl sulfone compounds selectively inhibit NLRP3 inflammasome activity in macrophages independent of their inhibitory effect on NF-kappaB activity. In vitro assays of the effect of parthenolide and Bay 11-7082 on the ATPase activity of NLRP3 demonstrated that both compounds inhibit the ATPase activity of NLRP3, suggesting that the inhibitory effect of these compounds on inflammasome activity could be mediated in part through their effect on the ATPase activity of NLRP3. Our results thus elucidate the molecular mechanism for the therapeutic anti-inflammatory activity of parthenolide and identify vinyl sulfones as a new class of potential therapeutics that target the NLRP3 inflammasome.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Inflammation/drug therapy , Nitriles/pharmacology , Sesquiterpenes/pharmacology , Sulfones/pharmacology , Animals , Bone Marrow Cells/metabolism , Caspase 1/metabolism , Cell Death , Humans , Immunoblotting , L-Lactate Dehydrogenase/metabolism , Macrophages/metabolism , Mice , NF-kappa B/metabolism , Sulfones/chemistryABSTRACT
The activity of the p53 gene product is regulated by a plethora of posttranslational modifications. An open question is whether such posttranslational changes act redundantly or dependently upon one another. We show that a functional interference between specific acetylated and phosphorylated residues of p53 influences cell fate. Acetylation of lysine 320 (K320) prevents phosphorylation of crucial serines in the NH(2)-terminal region of p53; only allows activation of genes containing high-affinity p53 binding sites, such as p21/WAF; and promotes cell survival after DNA damage. In contrast, acetylation of K373 leads to hyperphosphorylation of p53 NH(2)-terminal residues and enhances the interaction with promoters for which p53 possesses low DNA binding affinity, such as those contained in proapoptotic genes, leading to cell death. Further, acetylation of each of these two lysine clusters differentially regulates the interaction of p53 with coactivators and corepressors and produces distinct gene-expression profiles. By analogy with the "histone code" hypothesis, we propose that the multiple biological activities of p53 are orchestrated and deciphered by different "p53 cassettes," each containing combination patterns of posttranslational modifications and protein-protein interactions.
Subject(s)
Cell Cycle/genetics , Gene Expression Regulation/genetics , Protein Processing, Post-Translational/genetics , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Acetylation , Amino Acid Sequence/physiology , Apoptosis/genetics , Binding Sites/genetics , Cell Line, Tumor , Cell Survival/genetics , Cell Transformation, Neoplastic/genetics , Genes, cdc/physiology , Humans , Lysine/metabolism , Phosphorylation , Promoter Regions, Genetic/genetics , Protein Binding/genetics , Protein Structure, Tertiary/physiology , Regulatory Elements, Transcriptional/genetics , Repressor Proteins/genetics , Repressor Proteins/metabolism , Tumor Suppressor Protein p53/chemistryABSTRACT
Recently, we reported that human breast cancer-associated fibroblasts show functional inactivation of the retinoblastoma (RB) tumor suppressor and down-regulation of caveolin-1 (Cav-1) protein expression. However, it remains unknown whether loss of Cav-1 is sufficient to confer functional RB inactivation in mammary fibroblasts. To establish a direct cause-and-effect relationship, mammary stromal fibroblasts (MSFs) were prepared from Cav-1(-/-) null mice and subjected to phenotypic analysis. Here, we provide evidence that Cav-1(-/-) MSFs share many characteristics with human cancer-associated fibroblasts. The Cav-1(-/-) MSF transcriptome significantly overlaps with human cancer-associated fibroblasts; both show a nearly identical profile of RB/E2F-regulated genes that are up-regulated, which is consistent with RB inactivation. This Cav-1(-/-) MSF gene signature is predictive of poor clinical outcome in breast cancer patients treated with tamoxifen. Consistent with these findings, Cav-1(-/-) MSFs show RB hyperphosphorylation and the up-regulation of estrogen receptor co-activator genes. We also evaluated the paracrine effects of "conditioned media" prepared from Cav-1(-/-) MSFs on wild-type mammary epithelia. Our results indicate that Cav-1(-/-) MSF "conditioned media" is sufficient to induce an epithelial-mesenchymal transition, indicative of an invasive phenotype. Proteomic analysis of this "conditioned media" reveals increased levels of proliferative/angiogenic growth factors. Consistent with these findings, Cav-1(-/-) MSFs are able to undergo endothelial-like transdifferentiation. Thus, these results have important implications for understanding the role of cancer-associated fibroblasts and RB inactivation in promoting tumor angiogenesis.
Subject(s)
Breast Neoplasms/genetics , Breast Neoplasms/pathology , Caveolin 1/deficiency , Caveolin 1/genetics , Fibroblasts/pathology , Stromal Cells/pathology , Blotting, Western , Breast/cytology , Breast/physiology , Breast Neoplasms/mortality , Cell Culture Techniques , Cell Division , Disease Progression , Disease-Free Survival , Epithelial Cells/cytology , Epithelial Cells/pathology , Epithelial Cells/physiology , Female , Fibroblasts/cytology , Fibroblasts/physiology , Humans , Polymerase Chain Reaction , RNA, Messenger/genetics , RNA, Neoplasm/genetics , Stromal Cells/cytology , Stromal Cells/physiology , Survival AnalysisABSTRACT
Here, we show that functional loss of a single gene is sufficient to confer constitutive milk protein production and protection against mammary tumor formation. Caveolin-3 (Cav-3), a muscle-specific caveolin-related gene, is highly expressed in muscle cells. We demonstrate that Cav-3 is also expressed in myoepithelial cells within the mammary gland. To determine whether genetic ablation of Cav-3 expression affects adult mammary gland development, we studied the phenotype(s) of Cav-3(-/-)-null mice. Interestingly, Cav-3(-/-) virgin mammary glands developed lobulo-alveolar hyperplasia, akin to the changes normally observed during pregnancy and lactation. Genome-wide expression profiling revealed up-regulation of gene transcripts associated with pregnancy/lactation, mammary stem cells, and human breast cancers, consistent with a constitutive lactogenic phenotype. Expression levels of three key transcriptional regulators of lactation, namely Elf5, Stat5a, and c-Myc, were also significantly elevated. Experiments with pregnant mice directly showed that Cav-3(-/-) mice underwent precocious lactation. Finally, using orthotopic tumor cell implantation, we demonstrated that virgin Cav-3(-/-) mice were dramatically protected against mammary tumor formation. Thus, Cav-3(-/-) mice are a novel preclinical model to study the protective effects of a lactogenic microenvironment on mammary tumor onset and progression. Our current studies have broad implications for using the lactogenic microenvironment as a paradigm to discover new therapies for the prevention and/or treatment of human breast cancers.
Subject(s)
Caveolin 3/genetics , Caveolin 3/metabolism , Gene Expression , Lactation/physiology , Mammary Neoplasms, Experimental/genetics , Animals , Cell Movement/physiology , Female , Gene Expression Profiling , Humans , Immunohistochemistry , In Situ Nick-End Labeling , Male , Mammary Glands, Animal/metabolism , Mammary Neoplasms, Experimental/metabolism , Mice , Mice, Mutant Strains , Milk, Human/metabolism , Oligonucleotide Array Sequence Analysis , Phenotype , Polymerase Chain Reaction , PregnancyABSTRACT
Cyclin D1 is overexpressed in human tumors, correlating with cellular metastasis, and is induced by activating Rho GTPases. Herein, cyclin D1-deficient mouse embryo fibroblasts (MEFs) exhibited increased adhesion and decreased motility compared with wild-type MEFs. Retroviral transduction of cyclin D1 reversed these phenotypes. Mutational analysis of cyclin D1 demonstrated that its effects on cellular adhesion and migration were independent of the pRb and p160 coactivator binding domains. Genomewide expression arrays identified a subset of genes regulated by cyclin D1, including Rho-activated kinase II (ROCKII) and thrombospondin 1 (TSP-1). cyclin D1(-/-) cells showed increased Rho GTP and ROCKII activity and signaling, with increased phosphorylation of LIM kinase, cofilin (Ser3), and myosin light chain 2 (Thr18/Ser19). Cyclin D1 repressed ROCKII and TSP-1 expression, and the migratory defect of cyclin D1(-/-) cells was reversed by ROCK inhibition or TSP-1 immunoneutralizing antibodies. cyclin E knockin to the cyclin D1(-/-) MEFs rescued the DNA synthesis defect of cyclin D1(-/-) MEFs but did not rescue either the migration defect or the abundance of ROCKII. Cyclin D1 promotes cellular motility through inhibiting ROCK signaling and repressing the metastasis suppressor TSP-1.
Subject(s)
Cell Movement , Cyclin D1/metabolism , Gene Expression Regulation , Protein Serine-Threonine Kinases/metabolism , Signal Transduction , Thrombospondin 1/antagonists & inhibitors , Animals , Cell Adhesion , Cells, Cultured , Cyclin D1/chemistry , Cyclin D1/deficiency , Cyclin-Dependent Kinase Inhibitor p16/metabolism , Cyclin-Dependent Kinases/metabolism , DNA/biosynthesis , Fibroblasts/cytology , Humans , Intracellular Signaling Peptides and Proteins , Mice , Mutation/genetics , Protein Structure, Tertiary , RNA, Messenger/genetics , RNA, Messenger/metabolism , Stress Fibers/metabolism , Thrombospondin 1/genetics , Thrombospondin 1/metabolism , rho-Associated KinasesABSTRACT
The NAD-dependent histone deacetylase Sir2 plays a key role in connecting cellular metabolism with gene silencing and aging. The androgen receptor (AR) is a ligand-regulated modular nuclear receptor governing prostate cancer cellular proliferation, differentiation, and apoptosis in response to androgens, including dihydrotestosterone (DHT). Here, SIRT1 antagonists induce endogenous AR expression and enhance DHT-mediated AR expression. SIRT1 binds and deacetylates the AR at a conserved lysine motif. Human SIRT1 (hSIRT1) repression of DHT-induced AR signaling requires the NAD-dependent catalytic function of hSIRT1 and the AR lysine residues deacetylated by SIRT1. SIRT1 inhibited coactivator-induced interactions between the AR amino and carboxyl termini. DHT-induced prostate cancer cellular contact-independent growth is also blocked by SIRT1, providing a direct functional link between the AR, which is a critical determinant of progression of human prostate cancer, and the sirtuins.
Subject(s)
Dihydrotestosterone/metabolism , Gene Expression Regulation , Prostatic Neoplasms , Receptors, Androgen/metabolism , Sirtuins/metabolism , Acetylation , Amino Acid Sequence , Animals , Cell Cycle Proteins/metabolism , Cell Line , Cell Proliferation , Genes, Reporter , Histone Acetyltransferases/metabolism , Humans , Lysine/metabolism , Male , Models, Molecular , Molecular Sequence Data , Peptides/genetics , Peptides/metabolism , Receptors, Androgen/genetics , Signal Transduction/physiology , Sirtuin 1 , Sirtuins/genetics , Transcription Factors/metabolism , Transcription, Genetic , p300-CBP Transcription FactorsABSTRACT
The cyclin D1 gene encodes a regulatory subunit of the holoenzyme that phosphorylates and inactivates the pRb tumor suppressor to promote nuclear DNA synthesis. cyclin D1 is overexpressed in human breast cancers and is sufficient for the development of murine mammary tumors. Herein, cyclin D1 is shown to perform a novel function, inhibiting mitochondrial function and size. Mitochondrial activity was enhanced by genetic deletion or antisense or small interfering RNA to cyclin D1. Global gene expression profiling and functional analysis of mammary epithelial cell-targeted cyclin D1 antisense transgenics demonstrated that cyclin D1 inhibits mitochondrial activity and aerobic glycolysis in vivo. Reciprocal regulation of these genes was observed in cyclin D1-induced mammary tumors. Cyclin D1 thus integrates nuclear DNA synthesis and mitochondrial function.
Subject(s)
Cyclin D1/metabolism , Mitochondria/metabolism , Animals , Base Sequence , Cyclin D1/deficiency , Cyclin D1/genetics , DNA/genetics , Female , Gene Expression Profiling , Glycolysis , Hexokinase/genetics , Hexokinase/metabolism , Humans , Lipogenesis/genetics , Mammary Glands, Animal/metabolism , Mammary Neoplasms, Experimental/genetics , Mammary Neoplasms, Experimental/metabolism , Mice , Mice, Knockout , Mice, Transgenic , Mitochondria/genetics , Models, Biological , Multigene Family , Oligonucleotide Array Sequence Analysis , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/geneticsABSTRACT
We have developed a fully three-dimensional (3D) model of calcium signaling in epithelial cells based on a set of reaction diffusion equations that are solved on a large-scale finite-element code in three dimensions. We have explicitly included the cellular compartments including the cell nucleus, cytoplasm, and gap junctions. The model allows for buffering of free Ca2+, calcium-induced calcium release, and the explicit inclusion of mobile buffers. To make quantitative comparisons to experimental results, we used fluorescence microscopy images of cells to generate an accurate mesh describing cell morphology. We found that Ca2+ wave propagation through the tissue is a function of both initial conditions used to start the wave and various geometrical parameters that affect propagation such as gap junction density and distribution, and the presence of nuclei. The exogenous dyes used in experimental imaging also affect wave propagation.
Subject(s)
Calcium Signaling/physiology , Epithelial Cells/physiology , Extracellular Fluid/physiology , Image Processing, Computer-Assisted/methods , Models, Biological , Animals , Calcium/metabolism , Cell Line , Cell Membrane/chemistry , Cell Membrane/physiology , Dogs , Epithelial Cells/chemistry , Extracellular Fluid/chemistry , Gap Junctions/chemistry , Gap Junctions/physiology , Nuclear Envelope/chemistry , Nuclear Envelope/physiologyABSTRACT
Time-Of-Flight Mass Spectrometry (TOF-SIMS) was used to determine elemental and biomolecular ions from isolated protein samples. We identified a set of 23 mass-to-charge ratio (m/z) peaks that represent signatures for distinguishing biological samples. The 23 peaks were identified by Singular Value Decomposition (SVD) and Canonical Analysis (CA) to find the underlying structure in the complex mass-spectra data sets. From this modified data, SVD was used to identify sets of m/z peaks, and we used these patterns from the TOF-SIMS data to predict the biological source from which individual mass spectra were generated. The signatures were validated using an additional data set different from the initial training set used to identify the signatures. We present a simple method to identify multiple variables required for sample classification based on mass spectra that avoids overfit. This is important in a variety of studies using mass spectrometry, including the ability to identify proteins in complex mixtures and for the identification of new biomarkers.
Subject(s)
Biological Products/analysis , Spectrometry, Mass, Secondary Ion/methods , Proteins/analysisABSTRACT
BACKGROUND: Recent technological advances in high-throughput data collection allow for experimental study of increasingly complex systems on the scale of the whole cellular genome and proteome. Gene network models are needed to interpret the resulting large and complex data sets. Rationally designed perturbations (e.g., gene knock-outs) can be used to iteratively refine hypothetical models, suggesting an approach for high-throughput biological system analysis. We introduce an approach to gene network modeling based on a scalable linear variant of fuzzy logic: a framework with greater resolution than Boolean logic models, but which, while still semi-quantitative, does not require the precise parameter measurement needed for chemical kinetics-based modeling. RESULTS: We demonstrated our approach with exhaustive search for fuzzy gene interaction models that best fit transcription measurements by microarray of twelve selected genes regulating the yeast cell cycle. Applying an efficient, universally applicable data normalization and fuzzification scheme, the search converged to a small number of models that individually predict experimental data within an error tolerance. Because only gene transcription levels are used to develop the models, they include both direct and indirect regulation of genes. CONCLUSION: Biological relationships in the best-fitting fuzzy gene network models successfully recover direct and indirect interactions predicted from previous knowledge to result in transcriptional correlation. Fuzzy models fit on one yeast cell cycle data set robustly predict another experimental data set for the same system. Linear fuzzy gene networks and exhaustive rule search are the first steps towards a framework for an integrated modeling and experiment approach to high-throughput "reverse engineering" of complex biological systems.
Subject(s)
Artificial Intelligence , Fuzzy Logic , Models, Genetic , Oligonucleotide Array Sequence Analysis/statistics & numerical data , Cell Cycle/genetics , Computational Biology/statistics & numerical data , Linear Models , Saccharomyces cerevisiae/geneticsABSTRACT
Improved clinical management of prostate cancer has been impeded by an inadequate understanding of molecular genetic elements governing tumor progression. Gene signatures have provided improved prognostic indicators of human prostate cancer. The TGF-ß/BMP-SMAD4 signaling pathway, which induces epithelial-mesenchymal transition (EMT), is known to constrain prostate cancer progression induced by Pten deletion. Herein, cyclin D1 inactivation reduced cellular proliferation in the murine prostate in vivo and in isogenic oncogene-transformed prostate cancer cell lines. The in vivo cyclin D1-mediated molecular signature predicted poor outcome of recurrence-free survival for patients with prostate cancer (K-means HR, 3.75, P = 0.02) and demonstrated that endogenous cyclin D1 restrains TGF-ß, Snail, Twist, and Goosecoid signaling. Endogenous cyclin D1 enhanced Wnt and ES cell gene expression and expanded a prostate stem cell population. In chromatin immunoprecipitation sequencing, cyclin D1 occupied genes governing stem cell expansion and induced their transcription. The coordination of EMT restraining and stem cell expanding gene expression by cyclin D1 in the prostate may contribute to its strong prognostic value for poor outcome in biochemical-free recurrence in human prostate cancer.
Subject(s)
Cyclin D1/physiology , Epithelial-Mesenchymal Transition , Gene Expression Regulation, Neoplastic , Prostatic Neoplasms/metabolism , Animals , Cell Line, Tumor , Cell Proliferation , Disease Progression , Disease-Free Survival , Gene Deletion , Humans , Male , Mice , Mice, Transgenic , Oligonucleotide Array Sequence Analysis , PTEN Phosphohydrolase/metabolism , Prognosis , Recurrence , Signal Transduction , Treatment OutcomeABSTRACT
Breast cancer is a leading form of cancer in the world. The Drosophila Dac gene was cloned as an inhibitor of the hyperactive epidermal growth factor (EGFR), ellipse. Herein, endogenous DACH1 co-localized with p53 in a nuclear, extranucleolar compartment and bound to p53 in human breast cancer cell lines, p53 and DACH1 bound common genes in Chip-Seq. Full inhibition of breast cancer contact-independent growth by DACH1 required p53. The p53 breast cancer mutants R248Q and R273H, evaded DACH1 binding. DACH1 phosphorylation at serine residue (S439) inhibited p53 binding and phosphorylation at p53 amino-terminal sites (S15, S20) enhanced DACH1 binding. DACH1 binding to p53 was inhibited by NAD-dependent deacetylation via DACH1 K628. DACH1 repressed p21CIP1 and induced RAD51, an association found in basal breast cancer. DACH1 inhibits breast cancer cellular growth in an NAD and p53-dependent manner through direct protein-protein association.
Subject(s)
Breast Neoplasms/metabolism , Eye Proteins/metabolism , Transcription Factors/metabolism , Tumor Suppressor Protein p53/metabolism , Acetylation , Amino Acid Sequence , Apoptosis/physiology , Binding Sites , Breast Neoplasms/genetics , Cell Cycle Checkpoints/genetics , Cell Cycle Checkpoints/physiology , Cell Differentiation/physiology , Cell Line, Tumor , Chromatin/genetics , Chromatin/metabolism , DNA Damage , Eye Proteins/genetics , Female , Gene Expression , HEK293 Cells , Humans , Mutation , Promoter Regions, Genetic , Protein Binding , Sequence Homology, Amino Acid , Signal Transduction , Transcription Factors/genetics , Transfection , Tumor Suppressor Protein p53/geneticsABSTRACT
Identification of regulatory molecules in signaling pathways is critical for understanding cellular behavior. Given the complexity of the transcriptional gene network, the relationship between molecular expression and phenotype is difficult to determine using reductionist experimental methods. Computational models provide the means to characterize regulatory mechanisms and predict phenotype in the context of gene networks. Integrating gene expression data with phenotypic data in transcriptional network models enables systematic identification of critical molecules in a biological network. We developed an approach based on fuzzy logic to model cell budding in Saccharomyces cerevisiae using time series expression microarray data of the cell cycle. Cell budding is a phenotype of viable cells undergoing division. Predicted interactions between gene expression and phenotype reflected known biological relationships. Dynamic simulation analysis reproduced the behavior of the yeast cell cycle and accurately identified genes and interactions which are essential for cell viability.
ABSTRACT
Molecular biomarkers of early stage breast cancer may improve the sensitivity and specificity of diagnosis. Plasma biomarkers have additional value in that they can be monitored with minimal invasiveness. Plasma biomarker discovery by genome-wide proteomic methods is impeded by the wide dynamic range of protein abundance and the heterogeneity of protein expression in healthy and disease populations which requires the analysis of a large number of samples. We addressed these issues through the development of a novel protocol that couples a combinatorial peptide ligand library protein enrichment strategy with isobaric label-based 2D LC-MS/MS for the identification of candidate biomarkers in high throughput. Plasma was collected from patients with stage I breast cancer or benign breast lesions. Low abundance proteins were enriched using a bead-based combinatorial library of hexapeptides. This resulted in the identification of 397 proteins, 22% of which are novel plasma proteins. Twenty-three differentially expressed plasma proteins were identified, demonstrating the effectiveness of the described protocol and defining a set of candidate biomarkers to be validated in independent samples. This work can be used as the basis for the design of properly powered investigations of plasma protein expression for biomarker discovery in larger cohorts of patients with complex disease.
Subject(s)
Biomarkers, Tumor/blood , Breast Neoplasms/chemistry , Breast Neoplasms/blood , Breast Neoplasms/pathology , Chromatography, Liquid/methods , Combinatorial Chemistry Techniques/methods , Early Diagnosis , Female , Humans , Middle Aged , Peptide Library , Tandem Mass Spectrometry/methodsABSTRACT
Cyclin D1 belongs to a family of proteins that regulate progression through the G1-S phase of the cell cycle by binding to cyclin-dependent kinase (cdk)-4 to phosphorylate the retinoblastoma protein and release E2F transcription factors for progression through cell cycle. Several cancers, including breast, colon, and prostate, overexpress the cyclin D1 gene. However, the correlation of cyclin D1 overexpression with E2F target gene regulation or of cdk-dependent cyclin D1 activity with tumor development has not been identified. This suggests that the role of cyclin D1 in oncogenesis may be independent of its function as a cell cycle regulator. One such function is the role of cyclin D1 in cell adhesion and motility. Filamin A (FLNa), a member of the actin-binding filamin protein family, regulates signaling events involved in cell motility and invasion. FLNa has also been associated with a variety of cancers including lung cancer, prostate cancer, melanoma, human bladder cancer, and neuroblastoma. We hypothesized that elevated cyclin D1 facilitates motility in the invasive MDA-MB-231 breast cancer cell line. We show that MDA-MB-231 motility is affected by disturbing cyclin D1 levels or cyclin D1-cdk4/6 kinase activity. Using mass spectrometry, we find that cyclin D1 and FLNa coimmunoprecipitate and that lower levels of cyclin D1 are associated with decreased phosphorylation of FLNa at Ser2152 and Ser1459. We also identify many proteins related to cytoskeletal function, biomolecular synthesis, organelle biogenesis, and calcium regulation whose levels of expression change concomitant with decreased cell motility induced by decreased cyclin D1 and cyclin D1-cdk4/6 activities.
Subject(s)
Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Movement/physiology , Contractile Proteins/metabolism , Cyclin D1/metabolism , Cyclin-Dependent Kinase 4/metabolism , Microfilament Proteins/metabolism , Amino Acid Sequence , Cell Line, Tumor , Cyclin-Dependent Kinase 6/metabolism , Filamins , Humans , Molecular Sequence Data , Neoplasm Invasiveness , Phosphoproteins/metabolismABSTRACT
The role of mammary epithelial cell (MEC) NF-κB in tumor progression in vivo is unknown, as murine NF-κB components and kinases either are required for murine survival or interfere with normal mammary gland development. As NF-κB inhibitors block both tumor-associated macrophages (TAM) and MEC NF-κB, the importance of MEC NF-κB to tumor progression in vivo remained to be determined. Herein, an MEC-targeted inducible transgenic inhibitor of NF-κB (IκBαSR) was developed in ErbB2 mammary oncomice. Inducible suppression of NF-κB in the adult mammary epithelium delayed the onset and number of new tumors. Within similar sized breast tumors, TAM and tumor neoangiogenesis was reduced. Coculture experiments demonstrated MEC NF-κB enhanced TAM recruitment. Genome-wide expression and proteomic analysis showed that IκBαSR inhibited tumor stem cell pathways. IκBαSR inhibited breast tumor stem cell markers in transgenic tumors, reduced stem cell expansion in vitro, and repressed expression of Nanog and Sox2 in vivo and in vitro. MEC NF-κB contributes to mammary tumorigenesis. As we show that NF-κB contributes to expansion of breast tumor stem cells and heterotypic signals that enhance TAM and vasculogenesis, these processes may contribute to NF-κB-dependent mammary tumorigenesis.