Visual barcodes for clonal-multiplexing of live microscopy-based assays.

While multiplexing samples using DNA barcoding revolutionized the pace of biomedical discovery, multiplexing of live imaging-based applications has been limited by the number of fluorescent proteins that can be deconvoluted using common microscopy equipment. To address this limitation, we develop visual barcodes that discriminate the clonal identity of single cells by different fluorescent proteins that are targeted to specific subcellular locations. We demonstrate that deconvolution of these barcodes is highly accurate and robust to many cellular perturbations. We then use visual barcodes to generate 'Signalome' cell-lines by mixing 12 clones of different live reporters into a single population, allowing simultaneous monitoring of the activity in 12 branches of signaling, at clonal resolution, over time. Using the 'Signalome' we identify two distinct clusters of signaling pathways that balance growth and proliferation, emphasizing the importance of growth homeostasis as a central organizing principle in cancer signaling. The ability to multiplex samples in live imaging applications, both in vitro and in vivo may allow better high-content characterization of complex biological systems.

Predicting and affecting response to cancer therapy based on pathway-level biomarkers.

Identifying robust, patient-specific, and predictive biomarkers presents a major obstacle in precision oncology. To optimize patient-specific therapeutic strategies, here we couple pathway knowledge with large-scale drug sensitivity, RNAi, and CRISPR-Cas9 screening data from 460 cell lines. Pathway activity levels are found to be strong predictive biomarkers for the essentiality of 15 proteins, including the essentiality of MAD2L1 in breast cancer patients with high BRCA-pathway activity. We also find strong predictive biomarkers for the sensitivity to 31 compounds, including BCL2 and microtubule inhibitors (MTIs). Lastly, we show that Bcl-xL inhibition can modulate the activity of a predictive biomarker pathway and re-sensitize lung cancer cells and tumors to MTI therapy. Overall, our results support the use of pathways in helping to achieve the goal of precision medicine by uncovering dozens of predictive biomarkers.

Astrocyte-specific transcriptome analysis using the ALDH1L1 bacTRAP mouse reveals novel biomarkers of astrogliosis in response to neurotoxicity.

Neurotoxicology is hampered by the inability to predict regional and cellular targets of toxicant-induced damage. Evaluating astrogliosis overcomes this problem because reactive astrocytes highlight the location of toxicant-induced damage. While enhanced expression of glial fibrillary acidic protein is a hallmark of astrogliosis, few other biomarkers have been identified. However, bacterial artificial chromosome - translating ribosome affinity purification (bacTRAP) technology allows for characterization of the actively translating transcriptome of a particular cell type; use of this technology in aldehyde dehydrogenase 1 family member L1 (ALDH1L1) bacTRAP mice can identify genes selectively expressed in astrocytes. The aim of this study was to characterize additional biomarkers of neurotoxicity-induced astrogliosis using ALDH1L1 bacTRAP mice. The known dopaminergic neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP; 12.5 mg/kg s.c.) was used to induce astrogliosis. Striatal tissue was obtained 12, 24, and 48 h following exposure for the isolation of actively translating RNA. Subsequently, MPTP-induced changes in this RNA pool were analyzed by microarray and 184 statistically significant, differentially expressed genes were identified. The dataset was interrogated by gene ontology, pathway, and co-expression network analyses, which identified novel genes, as well as those with known immune and inflammatory functions. Using these analyses, we were directed to several genes associated with reactive astrocytes. Of these, TIMP1 and miR-147 were identified as candidate biomarkers because of their robust increased expression following both MPTP and trimethyl tin exposures. Thus, we have demonstrated that bacTRAP can be used to identify new biomarkers of astrogliosis and aid in the characterization of astrocyte phenotypes induced by toxicant exposures. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. Cover Image for this issue: doi: 10.1111/jnc.14518.

Resistance to paclitaxel is associated with a variant of the gene BCL2 in multiple tumor types.

Paclitaxel, the most commonly used form of chemotherapy, is utilized in curative protocols in different types of cancer. The response to treatment differs among patients. Biological interpretation of a mechanism to explain this personalized response is still unavailable. Since paclitaxel is known to target BCL2 and TUBB1, we used pan-cancer genomic data from hundreds of patients to show that a single-nucleotide variant in the BCL2 sequence can predict a patient's response to paclitaxel. Here, we show a connection between this BCL2 genomic variant, its transcript structure, and protein abundance. We demonstrate these findings in silico, in vitro, in formalin-fixed paraffin-embedded (FFPE) tissue, and in patient lymphocytes. We show that tumors with the specific variant are more resistant to paclitaxel. We also show that tumor and normal cells with the variant express higher levels of BCL2 protein, a phenomenon that we validated in an independent cohort of patients. Our results indicate BCL2 sequence variations as determinants of chemotherapy resistance. The knowledge of individual BCL2 genomic sequences prior to the choice of chemotherapy may improve patient survival. The current work also demonstrates the benefit of community-wide, integrative omics data sources combined with in-lab experimentation and validation sets.

The linker histone H1.0 generates epigenetic and functional intratumor heterogeneity.

Tumors comprise functionally diverse subpopulations of cells with distinct proliferative potential. Here, we show that dynamic epigenetic states defined by the linker histone H1.0 determine which cells within a tumor can sustain the long-term cancer growth. Numerous cancer types exhibit high inter- and intratumor heterogeneity of H1.0, with H1.0 levels correlating with tumor differentiation status, patient survival, and, at the single-cell level, cancer stem cell markers. Silencing of H1.0 promotes maintenance of self-renewing cells by inducing derepression of megabase-sized gene domains harboring downstream effectors of oncogenic pathways. Self-renewing epigenetic states are not stable, and reexpression of H1.0 in subsets of tumor cells establishes transcriptional programs that restrict cancer cells' long-term proliferative potential and drive their differentiation. Our results uncover epigenetic determinants of tumor-maintaining cells.

Autotaxin-Lysophosphatidic Acid Axis Acts Downstream of Apoprotein B Lipoproteins in Endothelial Cells.

OBJECTIVE: As they travel through the blood stream, plasma lipoproteins interact continuously with endothelial cells (ECs). Although the focus of research has mostly been guided by the importance of lipoproteins as risk factors for atherosclerosis, thrombosis, and other cardiovascular diseases, little is known about the mechanisms linking lipoproteins and angiogenesis under physiological conditions, and particularly, during embryonic development. In this work, we performed global mRNA expression profiling of endothelial cells from hypo-, and hyperlipidemic zebrafish embryos with the goal of uncovering novel mediators of lipoprotein signaling in the endothelium. APPROACH AND RESULTS: Microarray analysis was conducted on fluorescence-activated cell sorting-isolated fli1:EGFP(+) ECs from normal, hypo-, and hyperlipidemic zebrafish embryos. We found that opposed levels of apoprotein B lipoproteins result in differential expression of the secreted enzyme autotaxin in ECs, which in turn affects EC sprouting and angiogenesis. We further demonstrate that the effects of autotaxin in vivo are mediated by lysophosphatidic acid (LPA)-a well-known autotaxin activity product-and that LPA and LPA receptors participate as well in the response of ECs to lipoprotein levels. CONCLUSIONS: Our findings provide the first in vivo gene expression profiling of ECs facing different levels of plasma apoprotein B lipoproteins and uncover a novel lipoprotein-autotaxin-LPA axis as regulator of EC behavior. These results highlight new roles for lipoproteins as signaling molecules, which are independent of their canonical function as cholesterol transporters.

hsa-miR-9 controls the mobility behavior of glioblastoma cells via regulation of MAPK14 signaling elements.

BACKGROUND: Glioblastoma Multiforme (GBM) is the most common and lethal primary tumor of the brain. GBM is associated with one of the worst 5-year survival rates among all human cancers, despite much effort in different modes of treatment. RESULTS: Here, we demonstrate specific GBM cancer phenotypes that are governed by modifications to the MAPAKAP network. We then demonstrate a novel regulation mode by which a set of five key factors of the MAPKAP pathway are regulated by the same microRNA, hsa-miR-9.We demonstrate that hsa-miR-9 overexpression leads to MAPKAP signaling inhibition, partially by interfering with the MAPK14/MAPKAP3 complex. Further, hsa-miR-9 overexpression initiates re-arrangement of actin filaments, which leads us to hypothesize a mechanism for the observed phenotypic shift. CONCLUSION: The work presented here exposes novel microRNA features and situates hsa-miR-9 as a therapeutic target, which governs metastasis and thus determines prognosis in GBM through MAPKAP signaling.

Experimental Support for the Ecoimmunity Theory: Distinct Phenotypes of Nonlymphocytic Cells in SCID and Wild-Type Mice.

Immune tolerance toward "self" is critical in multiple immune disorders. While there are several mechanisms to describe the involvement of immune cells in the process, the role of peripheral tissue cells in that context is not yet clear. The theory of ecoimmunity postulates that interactions between immune and tissue cells represent a predator-prey relationship. A lifelong interaction, shaped mainly during early ontogeny, leads to selection of nonimmune cell phenotypes. Normally, therefore, nonimmune cells that evolve alongside an intact immune system would be phenotypically capable of evading immune responses, and cells whose phenotype falls short of satisfying this steady state would expire under hostile immune responses. This view was supported until recently by experimental evidence showing an inferior endurance of severe combined immunodeficiency (SCID)-derived pancreatic islets when engrafted into syngeneic immune-intact wild-type (WT) mice, relative to islets from WT. Here we extend the experimental exploration of ecoimmunity by searching for the presence of the phenotypic changes suggested by the theory. Immune-related phenotypes of islets, spleen, and bone marrow immune cells were determined, as well as SCID and WT nonlymphocytic cells. Islet submass grafting was performed to depict syngeneic graft functionality. Islet cultures were examined under both resting and inflamed conditions for expression of CD40 and major histocompatibility complex (MHC) class I/II and release of interleukin-1α (IL-1α), IL-1ß, IL-6, tumor necrosis factor-α (TNF-α), IL-10, and insulin. Results depict multiple pathways that appear to be related to the sculpting of nonimmune cells by immune cells; 59 SCID islet genes displayed relative expression changes compared with WT islets. SCID cells expressed lower tolerability to inflammation and higher levels of immune-related molecules, including MHC class I. Accordingly, islets exhibited a marked increase in insulin release upon immunocyte depletion, in effect resuming endocrine function that was otherwise suppressed by resident immunocytes. This work provides further support of the ecoimmunity theory and encourages subsequent studies to identify its role in the emergence and treatment of autoimmune pathologies, transplant rejection, and cancer.

Reciprocal Regulation between SIRT6 and miR-122 Controls Liver Metabolism and Predicts Hepatocarcinoma Prognosis.

Mice overexpressing the longevity protein SIRT6 or deficient for the liver's most prevalent microRNA miR-122 display a similar set of phenotypes, including improved lipid profile and protection against damage linked to obesity. Here, we show that miR-122 and SIRT6 negatively regulate each other's expression. SIRT6 downregulates miR-122 by deacetylating H3K56 in the promoter region. MiR-122 binds to three sites on the SIRT6 3' UTR and reduces its levels. The interplay between SIRT6 and miR-122 is manifested in two physiologically relevant ways in the liver. First, they oppositely regulate a similar set of metabolic genes and fatty acid ß-oxidation. Second, in hepatocellular carcinoma patients, loss of a negative correlation between SIRT6 and miR-122 expression is significantly associated with better prognosis. These findings show that SIRT6 and miR-122 negatively regulate each other to control various aspects of liver physiology and SIRT6-miR-122 correlation may serve as a biomarker for hepatocarcinoma prognosis.

MicroRNA regulation of molecular pathways as a generic mechanism and as a core disease phenotype.

The role of microRNAs as key regulators of a wide variety of fundamental cellular processes, such as apoptosis, differentiation, proliferation and cell cycle is increasingly recognized in most aspects of biology and biomedicine. Accretion of results from multiple microRNA studies over multiple pathway networks, led us to hypothesize that microRNAs target molecular pathways. As we show here, this is a network-wide phenomenon. The work presented, uses statistical tools that show how single microRNAs target molecular pathways. We demonstrate that this targeting could not be the result of random associations and cannot be the result of the sheer numeracy of microRNA targets. Furthermore, the strongest evidence for the association microRNA-pathway, is in a demonstration of the way by which these associations are disease-relevant. In our analyses we study ten different types of cancer involving thousands of samples, and show that the identified microRNA-pathway associations demonstrate a clinical affiliation and an ability to stratify patients. The work presented here shows the first evidence for a mechanism of microRNAs-pathway generic regulation. This regulation is tightly associated with clinical phenotype. The presented approach may catalyze targeted treatment through exposure of hidden regulatory mechanisms and a systems-medicine view of clinical observation.

Synaptojanin 2 is a druggable mediator of metastasis and the gene is overexpressed and amplified in breast cancer.

Amplified HER2, which encodes a member of the epidermal growth factor receptor (EGFR) family, is a target of effective therapies against breast cancer. In search for similarly targetable genomic aberrations, we identified copy number gains in SYNJ2, which encodes the 5'-inositol lipid phosphatase synaptojanin 2, as well as overexpression in a small fraction of human breast tumors. Copy gain and overexpression correlated with shorter patient survival and a low abundance of the tumor suppressor microRNA miR-31. SYNJ2 promoted cell migration and invasion in culture and lung metastasis of breast tumor xenografts in mice. Knocking down SYNJ2 impaired the endocytic recycling of EGFR and the formation of cellular lamellipodia and invadopodia. Screening compound libraries identified SYNJ2-specific inhibitors that prevented cell migration but did not affect the related neural protein SYNJ1, suggesting that SYNJ2 is a potentially druggable target to block cancer cell migration.

Shift in GATA3 functions, and GATA3 mutations, control progression and clinical presentation in breast cancer.

INTRODUCTION: GATA binding protein 3 (GATA3) is a regulator of mammary luminal cell differentiation, and an estrogen receptor (ER) associated marker in breast cancer. Tumor suppressor functions of GATA3 have been demonstrated primarily in basal-like breast cancers. Here, we focused on its function in luminal breast cancer, where GATA3 is frequently mutated, and its levels are significantly elevated. METHODS: GATA3 target genes were identified in normal- and luminal cancer- mammary cells by ChIP-seq, followed by examination of the effects of GATA3 expressions and mutations on tumorigenesis-associated genes and processes. Additionally, mutations and expression data of luminal breast cancer patients from The Cancer Genome Atlas were analyzed to characterize genetic signatures associated with GATA3 mutations. RESULTS: We show that some GATA3 effects shift from tumor suppressing to tumor promoting during tumorigenesis, with deregulation of three genes, BCL2, DACH1, THSD4, representing major GATA3-controlled processes in cancer progression. In addition, we identify an altered activity of mutant GATA3, and distinct associated genetic signatures. These signatures depend on the functional domain mutated; and, for a specific subgroup, are shared with basal-like breast cancer patients, who are a clinical group with regard to considerations of mode of treatment. CONCLUSIONS: The GATA3 dependent mechanisms may call for special considerations for proper prognosis and treatment of patients.

Heat acclimation memory: do the kinetics of the deacclimated transcriptome predispose to rapid reacclimation and cytoprotection?

Faster reinduction of heat acclimation (AC) after its decline indicates "AC memory." Our previous results revealed involvement of epigenetic mechanisms of transcriptional regulation. We hypothesized that the decline of AC (DeAC) is a period of "dormant memory" during which many processes are alerted to enable rapid reacclimation (ReAC). Using a genomewide approach we studied the AC, DeAC, and ReAC transcriptomes, to uncover hallmark pathways linked to "molecular memory" in the cardioacclimatome. Fifty rats subjected to heat acclimation [34°C for 2d (AC2d) or 30d (AC30)], DeAC (24°C, 30 days), ReAC (34°C, 2 days), and untreated controls were used. The GeneChip Rat Gene 1.0 ST Array was employed for left ventricular (cardiac) mRNA hybridization. Three independent bioinformatic analyses showed that 1) during AC2d enrichment of DNA impair/repair-linked genes is seen, and this is the molecular on-switch of acclimation; 2) genes activated in AC30 underlie the qualitative physiological adaptations of cardiac performance; 3) particular molecular programs encompassing constitutive upregulation of p38 MAPK, Jak/Stat, and Akt pathways and targets are specifically activated during DeAC and ReAC; and 4) epigenetic markers such as linker histones (histones H1 cluster), associated with nucleosome spacing, transcriptional chromatin modifiers, poly-(ADP-ribose) polymerase-1 (PARP1) linked to chromatin compaction, and microRNAs are only altered during DeAC/ReAC. The latter are newcomers to the AC/DeAC puzzle. We suggest that these transcriptional responses maintain euchromatin and proteostasis and enable faster physiological recovery upon ReAC by rapidly reestablishing the protected acclimated cardiophenotype. We propose that the cardiac AC model can be applied to acclimation processes in general.

PhenoNet: identification of key networks associated with disease phenotype.

MOTIVATION: At the core of transcriptome analyses of cancer is a challenge to detect molecular differences affiliated with disease phenotypes. This approach has led to remarkable progress in identifying molecular signatures and in stratifying patients into clinical groups. Yet, despite this progress, many of the identified signatures are not robust enough to be clinically used and not consistent enough to provide a follow-up on molecular mechanisms. RESULTS: To address these issues, we introduce PhenoNet, a novel algorithm for the identification of pathways and networks associated with different phenotypes. PhenoNet uses two types of input data: gene expression data (RMA, RPKM, FPKM, etc.) and phenotypic information, and integrates these data with curated pathways and protein-protein interaction information. Comprehensive iterations across all possible pathways and subnetworks result in the identification of key pathways or subnetworks that distinguish between the two phenotypes. AVAILABILITY AND IMPLEMENTATION: Matlab code is available upon request. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Reprogrammed and transmissible intestinal microbiota confer diminished susceptibility to induced colitis in TMF-/- mice.

Tata Element Modulatory Factor (TMF/ARA160) is a multifunctional Golgi-associated protein, which accumulates in colonic enterocytes and goblet cells. Mice lacking TMF/ARA160 (TMF(-/-)) produce thick and uniform colonic mucus that resists adherent bacterial colonization and diminishes susceptibility of these mice to induced acute colitis, through a mechanism that is not fully understood. Here, we show that mucus secretion by goblet cells is altered in the colon of TMF(-/-) mice, resulting in the formation of a highly oligomerized colonic gel-forming mucin, MUC2. Microbiome analysis revealed a shift in the microbiota of TMF(-/-) mice leading to predominance of the Firmicutes phylum and a significantly higher abundance of probiotic beneficial bacterial species. Notably, this trait was transmissible, and when cohoused with wild-type animals, TMF(-/-) mice influenced the microbiota and diminished the susceptibility of wild-type mice to chemically induced dextran sulfate sodium colitis. Thus, altered mucus secretion in TMF(-/-) mouse colons is accompanied by a reprogrammed intestinal microbiota, leading to a transmissible reduced sensitivity to induced colitis.

SENP5 mediates breast cancer invasion via a TGFßRI SUMOylation cascade.

Identifying novel mechanisms, which are at the core of breast cancer biology, is of critical importance. Such mechanisms may explain response to treatment, reveal novel targets or drive detection assays. To uncover such novel mechanisms, we used survival analysis on gene expression datasets encompassing 1363 patients. By iterating over the compendia of genes, we screened for their significance as prognosis biomarkers and identified SUMO-specific protease 5 (SENP5) to significantly stratify patients into two survival groups across five unrelated tested datasets. According to these findings, low expression of SENP5 is associated with good prognosis among breast cancer patients. Following these findings, we analyzed SENP5 silencing and show it is followed by inhibition of anchorage-independence growth, proliferation, migration and invasion in breast cancer cell lines. We further show that these changes are conducted via regulation of TGFßRI levels. These data relate to recent reports about the SUMOylation of TGFßRI. Following TGFßRI changes in expression, we show that one of its target genes, MMP9, which plays a key role in degrading the extracellular matrix and contributes to TGFß-induced invasion, is dramatically down regulated upon SENP5 silencing. This is the first report represents SENP5-TGFß-MMP9 cascade and its mechanistic involvement in breast cancer.

MicroRNA-gene association as a prognostic biomarker in cancer exposes disease mechanisms.

The transcriptional networks that regulate gene expression and modifications to this network are at the core of the cancer phenotype. MicroRNAs, a well-studied species of small non-coding RNA molecules, have been shown to have a central role in regulating gene expression as part of this transcriptional network. Further, microRNA deregulation is associated with cancer development and with tumor progression. Glioblastoma Multiform (GBM) is the most common, aggressive and malignant primary tumor of the brain and is associated with one of the worst 5-year survival rates among all human cancers. To study the transcriptional network and its modifications in GBM, we utilized gene expression, microRNA sequencing, whole genome sequencing and clinical data from hundreds of patients from different datasets. Using these data and a novel microRNA-gene association approach we introduce, we have identified unique microRNAs and their associated genes. This unique behavior is composed of the ability of the quantifiable association of the microRNA and the gene expression levels, which we show stratify patients into clinical subgroups of high statistical significance. Importantly, this stratification goes unobserved by other methods and is not affiliated by other subsets or phenotypes within the data. To investigate the robustness of the introduced approach, we demonstrate, in unrelated datasets, robustness of findings. Among the set of identified microRNA-gene associations, we closely study the example of MAF and hsa-miR-330-3p, and show how their co-behavior stratifies patients into prognosis clinical groups and how whole genome sequences tells us more about a specific genomic variation as a possible basis for patient variances. We argue that these identified associations may indicate previously unexplored specific disease control mechanisms and may be used as basis for further study and for possible therapeutic intervention.

IL-27 acts on DCs to suppress the T cell response and autoimmunity by inducing expression of the immunoregulatory molecule CD39.

Dendritic cells (DCs) control the balance between effector T cells and regulatory T cells in vivo. Hence, the study of DCs might identify mechanisms of disease pathogenesis and guide new therapeutic approaches for disorders mediated by the immune system. We found that interleukin 27 (IL-27) signaling in mouse DCs limited the generation of effector cells of the TH1 and TH17 subsets of helper T cells and the development of experimental autoimmune encephalomyelitis (EAE). The effects of IL-27 were mediated at least in part through induction of the immunoregulatory molecule CD39 in DCs. IL-27-induced CD39 decreased the extracellular concentration of ATP and downregulated nucleotide-dependent activation of the NLRP3 inflammasome. Finally, therapeutic vaccination with IL-27-conditioned DCs suppressed established relapsing-remitting EAE. Thus, IL-27 signaling in DCs limited pathogenic T cell responses and the development of autoimmunity.

Altered immune pathway activity under exercise challenge in Gulf War Illness: an exploratory analysis.

Though potentially linked to the basic physiology of stress response we still have no clear understanding of Gulf War Illness (GWI), a debilitating illness presenting with a complex constellation of immune, endocrine and neurological symptoms. Here we compared male GWI (n=20) with healthy veterans (n=22) and subjects with chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) (n=7). Blood was drawn during a Graded eXercise Test (GXT) prior to exercise, at peak effort (VO2 max) and 4-h post exercise. Affymetrix HG U133 plus 2.0 microarray gene expression profiling in peripheral blood mononuclear cells (PBMCs) was used to estimate activation of over 500 documented pathways. This was cast against ELISA-based measurement of 16 cytokines in plasma and flow cytometric assessment of lymphocyte populations and cytotoxicity. A 2-way ANOVA corrected for multiple comparisons (q statistic <0.05) indicated significant increases in neuroendocrine-immune signaling and inflammatory activity in GWI, with decreased apoptotic signaling. Conversely, cell cycle progression and immune signaling were broadly subdued in CFS. Partial correlation networks linking pathways with symptom severity via changes in immune cell abundance, function and signaling were constructed. Central to these were changes in IL-10 and CD2+ cell abundance and their link to two pathway clusters. The first consisted of pathways supporting neuronal development and migration whereas the second was related to androgen-mediated activation of NF-κB. These exploratory results suggest an over-expression of known exercise response mechanisms as well as illness-specific changes that may involve an overlapping stress-potentiated neuro-inflammatory response.