Specific Negative Regulator Research Articles

The role of uncertainty and negative feedback loops in the evolution of induced immune defenses.

Organisms use constitutive or induced defenses against pathogens and other external threats. Constitutive defenses are constantly on, whereas induced defenses are activated when needed. Each of these strategies has costs and benefits, which can affect the type of defense that evolves in response to pathogens. In addition, induced defenses are usually regulated by multiple negative feedback mechanisms that prevent overactivation of the immune response. However, it is unclear how negative feedback affects the costs, benefits, and evolution of induced responses. To address this gap, we developed a mechanistic model of the well-characterized Drosophila melanogaster immune signaling network that includes 3 separate mechanisms of negative feedback as a representative of the widespread phenomenon of multilevel regulation of induced responses. We show that, under stochastic fly-bacteria encounters, an induced defense is favored when bacterial encounters are rare or uncertain, but in ways that depend on the bacterial proliferation rate. Our model also predicts that the specific negative regulators that optimize the induced response depend on the bacterial proliferation rate, linking negative feedback mechanisms to the factors that favor induction.

Identifying regulators of aberrant stem cell and differentiation activity in colorectal cancer using a dual endogenous reporter system

Aberrant stem cell-like activity and impaired differentiation are central to the development of colorectal cancer (CRC). To identify functional mediators of these key cellular programs, we engineer a dual endogenous reporter system by genome-editing the SOX9 and KRT20 loci of human CRC cell lines to express fluorescent reporters, broadcasting aberrant stem cell-like and differentiation activity, respectively. By applying a CRISPR screen targeting 78 epigenetic regulators with 542 sgRNAs to this platform, we identify factors that contribute to stem cell-like activity and differentiation in CRC. Perturbation single cell RNA sequencing (Perturb-seq) of validated hits nominate SMARCB1 of the BAF complex (also known as SWI/SNF) as a negative regulator of differentiation across an array of neoplastic colon models. SMARCB1 is a dependency and required for in vivo growth of human CRC models. These studies highlight the utility of biologically designed endogenous reporter platforms to uncover regulators with therapeutic potential.

SPRY4-dependent ERK negative feedback demarcates functional adult stem cells in the male mouse germline†.

Niche-derived growth factors support self-renewal of mouse spermatogonial stem and progenitor cells through ERK MAPK signaling and other pathways. At the same time, dysregulated growth factor-dependent signaling has been associated with loss of stem cell activity and aberrant differentiation. We hypothesized that growth factor signaling through the ERK MAPK pathway in spermatogonial stem cells is tightly regulated within a narrow range through distinct intracellular negative feedback regulators. Evaluation of candidate extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK)-responsive genes known to dampen downstream signaling revealed robust induction of specific negative feedback regulators, including Spry4, in cultured mouse spermatogonial stem cells in response to glial cell line-derived neurotrophic factor or fibroblast growth factor 2. Undifferentiated spermatogonia in vivo exhibited high levels of Spry4 mRNA. Quantitative single-cell analysis of ERK MAPK signaling in spermatogonial stem cell cultures revealed both dynamic signaling patterns in response to growth factors and disruption of such effects when Spry4 was ablated, due to dysregulation of ERK MAPK downstream of RAS. Whereas negative feedback regulator expression decreased during differentiation, loss of Spry4 shifted cell fate toward early differentiation with concomitant loss of stem cell activity. Finally, a mouse Spry4 reporter line revealed that the adult spermatogonial stem cell population in vivo is demarcated by strong Spry4 promoter activity. Collectively, our data suggest that negative feedback-dependent regulation of ERK MAPK is critical for preservation of spermatogonial stem cell fate within the mammalian testis.

MoMaf1 Mediates Vegetative Growth, Conidiogenesis, and Pathogenicity in the Rice Blast Fungus Magnaporthe oryzae.

In eukaryotes, Maf1 is an essential and specific negative regulator of RNA polymerase (Pol) III. Pol III, which synthesizes 5S RNA and transfer RNAs (tRNAs), is suppressed by Maf1 under the conditions of nutrient starvation or environmental stress. Here, we identified M. oryzae MoMaf1, a homolog of ScMaf1 in budding yeast. A heterogeneous complementation assay revealed that MoMaf1 restored growth defects in the ΔScmaf1 mutant under SDS stress. Destruction of MoMAF1 elevated 5S rRNA content and increased sensitivity to cell wall agents. Moreover, the ΔMomaf1 mutant exhibited reduced vegetative growth, conidiogenesis, and pathogenicity. Interestingly, we found that MoMaf1 underwent nuclear-cytoplasmic shuffling, through which MoMaf1 accumulated in nuclei under nutrient deficiency or upon the interaction of M. oryzae with rice. Therefore, this study can help to elucidate the pathogenic molecular mechanism of M. oryzae.

Loss of Adhesion G‐Protein‐Coupled Receptor L2 Expression Impacts Colonic Epithelial Proliferation

BackgroundThe role of adhesion G‐protein‐coupled receptor L2 (ADGRL2, a.k.a. Latrophilin‐2) in human health has been understudied despite discoveries that the receptor is required for both heart development and neural synaptogenesis. This project tests the physiological function of ADGRL2 in the colon where our prior findings that ADGRL2 is lost in human colorectal cancer (CRC), downregulated during hyperproliferative recovery from murine DSS colitis, enriched in stem and progenitor cells, and is inversely correlated with growth arrest. This raises the intriguing possibility that ADGRL2 restricts stem cell expansion and therefore downregulation of ADGRL2 is required for stem‐cell driven regeneration and repair.MethodsThe effect of Adgrl2 on cell proliferation was assessed by histological staining for Ki‐67 antigen or for the thymidine analogue EdU (5‐ethynyl‐2’‐deoxyuridine, administered by intraperitoneal injection 2hr prior to harvest) in intestinal‐epithelial specific Adgrl2 knockout mice (Adgrl2 IE KO). Percent positive cells were quantified per crypt and per crypt region (crypt base, mid crypt, upper crypt) in Image J.Results/ConclusionsAdgrl2 IE KO mice have overall reduced numbers of Ki‐67+ cells (8.2% difference, p = 0.0016) in the colonic epithelium. This seems paradoxical given that we hypothesize a tumor suppressor role for ADGRL2. However, this effect is driven by a pronounced decrease in Ki‐67+ cells in the post‐mitotic upper crypt of Adgrl2 IE KO mice (15.2% difference, p = 0.0121) while percentages of Ki‐67+ cells in the more proliferative mid crypt and crypt base were determined to be roughly equal between groups, suggesting that the observed effect is likely not driven by a reduction in proliferating stem or progenitor cells but is perhaps due to increased cell cycle exit or terminal differentiation with Adgrl2 knockout. Given that Ki‐67 antigen is expressed in all active cell cycle phases, we used EdU labeling of S‐phase proliferating cells to further assess the impact of Adgrl2 loss on colonocyte proliferation. Proliferation is not impaired with loss of Adgrl2 and instead showed a trend towards increased EdU positive cells in the crypt base (6.7% increase, p= 0.0907). As expected, overall number of S‐phase cells in the upper crypt compartment was low for both Adgrl2 KO and WT animals, however a significant decrease in EdU positive cells was still detected in Adgrl2 KO mice (2.98% change, p=0.0500). Based on these data, the loss of Adgrl2 does not decrease proliferative capacity of the stem cell or progenitor niche, but does effect the distribution of active cycling or differentiating cells along the crypt axis, likely by promoting cell cycle exit or terminal differentiation. Further studies will investigate this possibility and the role of Adgrl2 loss on cell renewal in the context of disease. These data support our hypothesis that ADGRL2 modulates epithelial regeneration in the colon, potentially as a stem‐cell specific negative regulator. Mechanistic studies on dysregulated growth and repair in the colon may also illuminate targets to prevent colitis‐associated cancer in IBD patients.

The neural mechanism and transfer effect of creative cognitive reappraisal in negative emotion regulation

<p id=C8>Cognitive reappraisal plays an important role in regulating negative emotions. However, traditional cognitive reappraisal has some issues, such as a lower reconstruction degree and a lower emotion regulation effect. Considering these issues, we propose an effective emotion regulation strategy called creative cognitive reappraisal, which refers to a novel and unique interpretation that is highly creative and appropriate for current emotional stimuli. During the emotion regulation process, an individual suddenly experiences an Aha! moment when combining the interpretation with the stimulus situation. This study found a dramatic shift from negative to positive emotions and a long-lasting regulatory effect. According to imaging results, creative cognitive reappraisal was associated with the formation of a novel association in the hippocampus and positive emotional arousal in the amygdala. <p id=C9>However, the guided reappraisal used in this work was more akin to an understanding of reappraisal interpretation than active emotion regulation, which has lower ecological validity. Furthermore, it is difficult to generate high creative reappraisal on one’s own, making the application of creative cognitive reappraisal challenging. Thus, the goal of this project is to teach participants how to learn the method of creative cognitive reappraisal and then transfer and apply this strategy to specific negative emotion regulation in daily life, i.e., to establish a learn and then use emotion regulation mode that can truly play the role of creative cognitive reappraisal during emotion regulation. Furthermore, because teenagers have more serious mood problems but also have higher creativity, this project intends to apply a creative cognitive reappraisal strategy for emotion regulation to teenagers while also teaching them creative emotion regulation strategies to assist them in learning cognitive transformations and reconstructions. This method can teach teenagers how to use positive emotional regulation strategies to deal with negative life events while also preserving their creativity. <p id=C10>The specific research contents are as follows: 1) exploring the transfer effect of creative cognitive reappraisal in negative emotion regulation of college students and adolescents; 2) investigating changes in brain activation and representation patterns before and after creative cognitive reappraisal transfer; and 3) examining the similarities and differences in the neural mechanisms of creative cognitive reappraisal transfer between adolescents and adults. This project is an extension and expansion of the applicants existing work, and it would establish a theoretical foundation for verifying and promoting creative cognitive reappraisal as a learnable, usable and efficient emotional regulation strategy.

Role of Src‐Dependent Phosphorylation of Mitofusin 2 in Endoplasmic Reticulum‐Mitochondria Tethering

Introduction : Contact sites between the mitochondria and endoplasmic reticulum (ER) are one of the key structures involved in regulating lipid, Ca2+ and reactive oxygen species exchange across the two organelles which is critical for the maintenance of mitochondrial bioenergetics. Recent studies have reported that a major component of the tethering structures between the two organelles is mitofusin 2 (Mfn2), a GTPase associated with fusion of the outer mitochondrial membrane (OMM) as well as the activities of mitophagy and ER stress. While several post-translational modifications (PTMs) that influence the fusion and mitophagy functions of Mfn2 have been identified, it remains unclear whether there are PTMs that can regulate its tethering function. Data from mass spectroscopy reveals that there is basal tyrosine phosphorylation (P-Tyr) of Mfn2, but it is unknown whether Mfn2 is a substrate of mitochondria-localized tyrosine kinases (proline rich tyrosine kinase 2 [Pyk2] and proto-oncogene Src kinase [Src]). Objective : To determine whether P-Tyr of Mfn2 regulates Mfn2 functions. Methods HEK293T cells stably overexpressing shRNA targeted to a specific negative regulator of Src, C-terminal Src kinase (CSK) or a control vector were subjected to several biochemical (Fluorescent western blotting), cell biological (Transmission electron microscopy [TEM], Förster Resonance Energy Transfer [FRET] between cyan-fluorescent protein targeted to the OMM and yellow-fluorescent protein targeted to the ER membrane observed under confocal microscopy) and physiological (mitochondrial calcium [mtCa2+] uptake measurements using a mitochondria-targeted Ca2+ biosensor, mt-RCaMp1h) assays. Results Overexpression of Pyk2 and Src lead to P-Tyr of Mfn2 with Src having greater P-Tyr levels of Mfn2. Generation of a HEK293T cell line with stable knockdown of CSK reveals that CSK knockdown promotes Src activation in the mitochondria and increases P-Tyr of Mfn2 without changing expression levels of Src and Mfn2. CSK knockdown (CSK-KD) cells showed decreased distance between the ER and OMM as assessed by FRET measurement under confocal microscopy. This change was abolished by pretreatment with a Src-specific inhibitor, PP2. TEM images also showed that CSK knockdown decreases the distance between OMM and ER without changing the length of interface. To assess the impact of changes in the physical interaction between ER and mitochondria, mtCa2+ uptake profiles in CSK-KD cells were observed. In CSK-KD cells, mtCa2+ uptake in response to cytosolic Ca2+ elevation induce by Gq-protein coupled receptor stimulation was enhanced with faster kinetics compared to that in control cells. While physical coupling between ER and mitochondria increased, we found that hallmarks of ER stress and mitophagy were not altered in CSK-KD cells compared to control cells. Conclusion : Src-dependent phosphorylation of Mfn2 decreased the distance between the OMM and ER and lead to increased Ca2+ exchange between ER and mitochondria.

COP9 Signalosome Subunit 3 Restricts Neuroinflammatory Responses During Cerebral Ischemia/Reperfusion Injury Through Stabilizing Suppressor of Cytokine Signaling 3 Protein.

BackgroundThe suppressor of cytokine signaling 3 (SOCS3) is a specific negative regulator of signal transducer and activator of transcription 3 (STAT3) signaling, which is predominantly activated to induce neuroinflammatory response in microglia and functions essential roles during cerebral ischemia-reperfusion (I/R) injury. Constitutive photomorphogenesis 9 (COP9) signalosome (CSN) is a signaling platform controlling protein stability by remodeling of cullin-RING ubiquitin ligases, which is recently reported to specifically recognize proteins with SOCS-box domains. However, whether SOCS3 is related to COP9 signalosome in neuroinflammation during cerebral I/R injury is completely unclear.MethodsMice subjected to transient middle cerebral artery occlusion (MCAO) and reperfusion, and BV2 microglia cells treated with oxygen-glucose deprivation and reoxygenation (OGD/R) were used to mimic cerebral I/R injury. Western blot, qRTPCR, immunofluorescence, and co-Immunoprecipitation assays were performed to explore the regulatory mechanism of SOCS3 on neuroinflammation and the relationship of SOCS3 and COP9 signalosome during cerebral I/R injury.ResultsSOCS3 expression is significantly upregulated in microglia during OGD/R treatment, and overexpression of SOCS3 suppresses OGD/R-induced STAT3 activation and inflammatory factor expression. Furthermore, we find that COP9 signalosome subunit 3 (CSN3) interacts with SOCS3 protein to enhance its stability, thereby resulting in restricting OGD/R-induced STAT3 activation and inflammatory response. Moreover, we find that knockdown of CSN3 evidently accelerates STAT3 activation, and aggravates cerebral I/R injury in vivo.ConclusionCSN3 restricts neuroinflammatory responses during cerebral I/R injury through stabilizing SOCS3 protein and indicates that CSN3 a potential therapeutic target for cerebral I/R injury.

RP105 plays a cardioprotective role in myocardial ischemia reperfusion injury by regulating the Toll‑like receptor 2/4 signaling pathways.

The revascularization of blood vessels after myocardial infarction can lead to serious myocardial damage. Previous studies showed that radioprotective 105 kDa protein (RP105) is a specific negative regulator of myocardial ischemia reperfusion injury (MIRI). RP105 can modulate the Toll-like receptor (TLR)2/TLR4 signaling pathways. However, the synergistic effect of TLR2/4 regulated by RP105 during MIRI requires further investigation. To determine this effect, a MIRI model was established in rats in the present study. The expression of RP105 was depleted by transfecting RP105-siRNA and then detected using western blotting. Furthermore, the myocardium tissue was stained with the hematoxylin and eosin staining. Knockdown of RP105 promoted the activity of serum myocardial enzymes during MIRI and increased myocardial infarction. The present results indicated that knockdown of RP105 activated the TLR2/4 signaling pathway by modulating the myeloid differentiation primary response 88 and NF-κB signaling pathways. Furthermore, decreased expression of RP105 promoted myocardial cell apoptosis, which induced the damage of myocardial ischemic reperfusion. The present results suggested both TLR2 and TLR4 as key targets of RP105, thus RP105 may be a promising candidate to facilitate the development of novel therapeutic strategies for MIRI.

ZFYVE1 negatively regulates MDA5- but not RIG-I-mediated innate antiviral response.

The retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), including RIG-I and melanoma differentiation-associated gene 5 (MDA5), sense cytoplasmic viral RNA and initiate innate antiviral responses. How RIG-I and MDA5 are differentially regulated remains enigmatic. In this study, we identified the guanylate-binding protein (GBP) and zinc-finger FYVE domain-containing protein ZFYVE1 as a negative regulator of MDA5- but not RIG-I-mediated innate antiviral responses. ZFYVE1-deficiency promoted MDA5- but not RIG-I-mediated transcription of downstream antiviral genes. Comparing to wild-type mice, Zfyve1-/- mice were significantly protected from lethality induced by encephalomyocarditis virus (EMCV) that is sensed by MDA5, whereas Zfyve1-/- and Zfyve1+/+ mice were comparable to death induced by vesicular stomatitis virus (VSV) that is sensed by RIG-I. Mechanistically, ZFYVE1 interacted with MDA5 but not RIG-I. ZFYVE1 bound to viral RNA and decreased the ligand binding and oligomerization of MDA5. These findings suggest that ZFYVE1 acts as a specific negative regulator of MDA5-mediated innate immune responses by inhibiting its ligand binding and oligomerization.

Adhesion G‐Protein‐Coupled Receptor L2 Expression is Lost in Colorectal Cancer but is Also Associated with Colonic Stem Cells

Colorectal cancer (CRC) is driven by uncontrolled expansion and differentiation of crypt stem cells. We are testing the role of adhesion G‐protein‐coupled receptor L2 (ADGRL2), whose function has yet to be studied in the intestine, as a potential gatekeeper for proliferating colonic stem cells and renewal.MethodsTo assess receptor regulation during disease, mRNA levels were measured in matched normal and tumor specimens from human CRC. We corroborated findings by mining Gene Expression Omnibus (GEO) datasets and examined receptor modulation in response to growth arrest conditions (serum starvation and MAP kinase inhibition) in human CRC cell lines. We further interrogated ADGRL2 function using a stable inducible shRNA knockout line using SW480 cells and compared the gene expression of colonocyte markers in the induced ADGRL2 KO cells versus non‐induced cells. To assess possible inflammatory links, mice were given DSS colitis with 3% DSS in drinking water for 3 days and colons were collected on day 7 in the hyperproliferative recovery phase.ResultsADGRL2 was significantly and selectively reduced in tumors relative to adjacent matched normal tissue (5.14 fold, p &lt; 0.0001). This was confirmed using two GEO datasets, one of which also revealed a pronounced loss in adenomas (3.82 fold, p &lt; 0.0001). ADGRL2 was induced by growth arrest conditions in CRC cell lines, both by serum starvation (2.79 fold, p = 0.0115) and map kinase inhibition (5.59 fold, p = 0.0086). Knockdown in SW480 cells resulted in a loss of the colonic stem cell marker LGR5, while other markers for colonic cell types remained unchanged. Analysis of publicly available microarray data confirmed that ADGRL2 and LGR5 levels are associated in the colonic mucosa. Adgrl2 levels were suppressed in recovery from DSS colitis (1.74 fold, p &lt; 0.0001), suggesting a role in restricting repair mechanisms.ConclusionsDownregulation during recovery from colonic injury, induction by antiproliferative stimuli, and association with LGR5 together suggest that ADGRL2 may have a role in orchestrating epithelial regeneration in the colon, potentially as a stem‐cell specific negative regulator. The substantial loss of ADGRL2 in human CRC adenoma and adenocarcinoma raises the possibility that ADGRL2 functions as a tumor suppressor. Thus, while modulation of ADGRL2 levels may mediate epithelial repair following injury, chronic loss could give way to uncontrollable stem cell expansion and loss of crypt architecture, leading to dysplasia and tumorigenesis.Support or Funding InformationThis work was supported by: NIH R01DK095004 and a Senior Research Award from the Crohn’s and Colitis Foundation (MRF). Support for EB includes The Saban Research Institute Predoctoral Intramural Funding Grant and The Roy E. Thomas Foundation Graduate Scholarship. Support for CM includes an Undergraduate Summer Research Fellowship from the Crohn’s and Colitis Foundation.

Activation of Nogo‐B receptor expression ameliorates type 2 diabetes in mice by improving insulin sensitivity

Reduction of insulin resistance/improvement of insulin sensitivity is the most effective treatment for type 2 diabetes (T2D). We previously reported Nogo‐B receptor (NGBR) is a specific negative regulator for liver X receptor alpha (LXRα)‐dependent hepatic lipogenesis, indicating NGBR may play an important role in hepatic lipid metabolism. However, it remains unknown if NGBR is involved in glucose metabolism and insulin sensitivity. Herein, we report the role of NGBR expression in insulin resistance and T2D development.In vitro, the role of NGBR in insulin sensitivity was analyzed by NGBR knockdown/overexpression in hepatic cell line. The pathophysiological role of NGBR in insulin sensitivity was analyzed in high‐fat diet (HFD)/streptozotocin (STZ)‐induced diabetic mice infected with recombinant adenovirus expressing NGBR by tail vein injection. We found inhibition of NGBR expression by siRNA decreased insulin sensitivity, and attenuated adiponectin‐activated AMPKa phosphorylation in hepatocytes. Reciprocally, NGBR overexpression enhanced insulin response. In vivo, NGBR overexpression was observed in mouse liver after infection of recombinant adenovirus expressing NGBR. Associated with NGBR overexpression, blood glucose was reduced while insulin sensitivity/glucose homeostasis was enhanced. NGBR overexpression also decreased triglyceride and NEFA levels in both liver and serum, and attenuated the injury in pancreatic islets. Furthermore, genes related to ER stress and mitochondrial functions in skeletal muscles and inflammation in adipose tissues were improved by hepatic NGBR overexpression. In conclusion, our study implicates the importance of understanding the role of hepatic NGBR in insulin sensitivity and treatment of T2D.

Inhibition of the histone demethylase, KDM5B, directly induces re-expression of tumor suppressor protein HEXIM1 in cancer cells

BackgroundThe tumor suppressor actions of hexamethylene bis-acetamide (HMBA)-inducible protein 1 (HEXIM1) in the breast, prostate, melanomas, and AML have been reported by our group and others. Increased HEXIM1 expression caused differentiation and inhibited proliferation and metastasis of cancer cells. Historically, HEXIM1 has been experimentally induced with the hybrid polar compound HMBA, but HMBA is a poor clinical candidate due to lack of a known target, poor pharmacological properties, and unfavorable ADMETox characteristics. Thus, HEXIM1 induction is an intriguing therapeutic approach to cancer treatment, but requires better chemical tools than HMBA.MethodsWe identified and verified KDM5B as a target of HEXIM1 inducers using a chemical proteomics approach, biotin–NeutrAvidin pull-down assays, surface plasmon resonance, and molecular docking. The regulation of HEXIM1 by KDM5B and KDM5B inhibitors was assessed using chromatin immunoprecipitation assays, RT-PCR, western blotting, and depletion of KDM5B with shRNAs. The regulation of breast cancer cell phenotype by KDM5B inhibitors was assessed using western blots, differentiation assays, proliferation assays, and a mouse model of breast cancer metastasis. The relative role of HEXIM1 in the action of KDM5B inhibitors was determined by depleting HEXIM1 using shRNAs followed by western blots, differentiation assays, and proliferation assays.ResultsWe have identified a highly druggable target, KDM5B, which is inhibited by small molecule inducers of HEXIM1. RNAi knockdown of KDM5B induced HEXIM1 expression, thus validating the specific negative regulation of tumor suppressor HEXIM1 by the H3K4me3/2 demethylase KDM5B. Known inhibitors of KDM5B were also able to induce HEXIM1 expression, inhibit cell proliferation, induce differentiation, potentiate sensitivity to cancer chemotherapy, and inhibit breast tumor metastasis.ConclusionHMBA and 4a1 induce HEXIM1 expression by inhibiting KDM5B. Upregulation of HEXIM1 expression levels plays a critical role in the inhibition of proliferation of breast cancer cells using KDM5B inhibitors. Based on the novel molecular scaffolds that we identified which more potently induced HEXIM1 expression and data in support that KDM5B is a target of these compounds, we have opened up new lead discovery and optimization directions.

Lgr5 Functions As a Critical Negative Regulator of Wnt/β-Catenin Signaling and Is Essential for B-Lymphopoiesis and Malignant B-Cell Transformation

Introduction: When early B-cell precursors have successfully undergone V(D)J rearrangement of immunoglobulin genes, they receive a strong positive selection signal, which initiates clonal expansion and the first wave of B-lymphopoiesis. Interestingly, this expansion following successful V(D)J recombination is marked by massive up-regulation of Leucine-rich repeat containing G-protein coupled receptor 5 (Lgr5). Results: To test the function of Lgr5 at this particular stage and later in B-cell development, we crossed Lgr5fl/fl mice with Mb1cre and CD21cre mice respectively. While Lgr5 deletion at the time of V(D)J recombination resulted in near complete ablation of the B cell lineage in the bone marrow and periphery, deletion at later stages of B-cell development had no significant impact on B-cell survival and proliferation. Here, we identified Lgr5 as a B-cell specific negative regulator of Wnt/β-catenin signaling, which is essential for normal B-lymphopoiesis but also for initiation of B-cell lineage acute lymphoblastic leukemia (B-ALL). Indeed, in children with B-ALL, higher than median mRNA levels of LGR5 at the time of diagnosis was identified as a predictor of poor clinical outcome (COG P9906, n=207, p=0.008). LGR5 over-expression conferred growth advantage to B-ALL cells, enhanced serial plating capacity and the ability to initiate leukemia in NSG mice. Limiting dilution transplant experiments showed that LGR5 overexpression increased LIC-frequencies by 4-fold. Inducible deletion of Lgr5 in BCR-ABL1- or NRASG12D-transformed B-ALL cell lines led to growth arrest, abolished colony forming capacity and compromised the ability of leukemia cells to initiate fatal disease in NSG recipients. Lgr5 is thought to function as a potentiator of Wnt/β-catenin signaling in epithelial cells. Upon deletion of Lgr5 in B-ALL cells, however, we observed massive accumulation of nuclear β-catenin and increased expression of β-catenin target genes, which suggests that Lgr5 acts as an essential negative regulator of β-catenin in the B-cell context. Phosphoproteomic approaches identified β-catenin to be differentially phosphorylated on S675 upon Lgr5-deletion, which enhances nuclear translocation and transcriptional activity of β-catenin. Deletion of LGR5 in human colon cancer cell lines did not result in β-catenin accumulation, which indicates a lineage specific role of Lgr5 in B-lymphocytes. Studying activating CTNNB1 mutations in 89,325 samples from 23 types of cancer, we found 7,777 activating mutations (8.7%). However, among 2,375 B-cell tumor samples, we did not observe a single activating mutation. Indeed, compared to solid tumors, B-ALL and B-cell lymphomas have very low amounts of nuclear β-catenin. B-ALL cells are extremely sensitive to β-catenin activation, resulting in cell cycle arrest and loss of colony forming ability, which parallels the phenotype observed in the absence of Lgr5. CRISPR-Cas9 mediated deletion of Ctnnb1 in Lgr5 knock-out B-ALL cells strikingly rescued proliferation and colony formation capacity, further corroborating the role of Lgr5 in promoting B-cell leukemogenesis by opposing aberrant activation of β-catenin. Therapeutic implication: Given that Lgr5 is required for leukemia-initiation and marks LICs, we tested an Lgr5-MMAE antibody-drug conjugate (ADC) in the presence and absence of dexamethasone. Combination treatment with dexamethasone not only enforced persistently high surface expression of LGR5, but also potentiated efficacy of by Lgr5-MMAE in B-ALL eradication. Conclusion: Lgr5 is critical for normal B cell development and tumor initiation in B-ALL. Lgr5, restricts the levels of nuclear β-catenin and enables B cell survival through negative regulation of Wnt/β-catenin signaling. Moreover, LGR5 represents a promising target for eradicating leukemia initiating cells. Disclosures No relevant conflicts of interest to declare.

Effect of the Antimicrobial Peptide LL-37 on Gene Expression of Chemokines and 29 Toll-like Receptor-Associated Proteins in Human Gingival Fibroblasts Under Stimulation with Porphyromonas gingivalis Lipopolysaccharide

The antimicrobial peptide LL-37 neutralizes the biological activity of lipopolysaccharide (LPS), while it upregulates the expression of several immune-related genes. We investigated the effect of LL-37 on gene regulation of human gingival fibroblasts (HGFs), stimulated with or without Porphyromonas gingivalis-derived LPS, a ligand for Toll-like receptor (TLR). LL-37 was non-toxic to HGFs up to a concentration of 10 μg/ml. P. gingivalis LPS upregulated the expression of IL8, CXCL10, and CCL2, whereas LL-37 reduced this upregulation. In absence of LPS, LL-37 itself upregulated the expression of IL8 and CCL2. LL-37 increased the expression of P2X7, which was constitutively expressed in HGFs. The P2X7 antagonist A-438079 suppressed the cytotoxicity and upregulatory effect of LL-37 on chemokine response, but not its downregulatory effect on P. gingivalis LPS-induced chemokine response. Whether LL-37 alters the expression of 29 genes that encode TLR-associated proteins, including TLRs, co-receptors, signaling molecules, and negative regulators, in HGFs, under stimulation with LPS, was examined. Among TLRs, P. gingivalis LPS upregulated the level of TLR4, whereas LL-37 reduced it. In co-receptors, LL-37 downregulated the level of CD14. Among signaling molecules, LL-37 augmented the LPS-upregulated expression of IRAK1. Similar effects were observed in the specific negative regulators TNFAIP3, RNF216, TOLLIP, and SIGIRR. Our results suggest that LL-37 exerts cytotoxicity and upregulation of chemokine response via the P2X7 receptor, while it induces downregulation of P. gingivalis LPS-induced chemokine response through alteration in the expression of 7 specific TLR-associated genes: downregulation of TLR4 and CD14 and upregulation of IRAK1, TNFAIP3, RNF216, TOLLIP, and SIGIRR.

Abstract 1718: Down-regulation of p16INK4a by hYSK1 promotes cancer cell migration under hypoxic conditions

Abstract The alteration expression of p16INK4a, a well-known cyclin-dependent kinase inhibitor involved in cancer cell cycle control, is unclear, especially under hypoxic conditions. To evaluate p16INK4a regulation, we performed a protein microarray analysis. Among 1,800 proteins, we identified hYSK1 as a novel protein that interacts with the tumor suppressor p16INK4a. hYSK1, a member of the Ste20 family of serine/threonine protein kinases, promotes cell migration &amp; tumorigenesis and is activated by oxidative stress. However, the molecular mechanisms underlying the oncogenic potential of hYSK1 remain elusive. Here, we report here that hYSK1 interacts with p16INK4a under hypoxic conditions like tumors, where it negatively regulates p16INK4a, enhancing cancer cell migration. Hypoxic stimulation of hYSK1 reduces p16INK4a accumulation through p16 promoter regulation to interact with unphosporylated SP-1 &amp; increases matrix metalloproteinase-2 (MMP-2) expression by activating the MMP-2 promoter associated with cell migration and proliferation.Conversely, knocking down hYSK1 expression activated p16INK4a expression &amp; suppressed MMP-2 expression.Thus, hYSK1 is necessary as a trigger for inactivating p16INK4a &amp; activating MMP-2 during tumor migration, suggesting that hYSK1 is a specific negative regulator of the tumor suppressor p16INK4a may represent a novel molecular target for reactivation of tumor suppressor genes in humans. Citation Format: Mee-Hyun Lee, Jung-Hyun Shim, Zigang Dong, Young-Joon Surh, Bu Young Choi. Down-regulation of p16INK4a by hYSK1 promotes cancer cell migration under hypoxic conditions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1718.

MicroRNA-128 targeting RPN2 inhibits cell proliferation and migration through the Akt-p53-cyclin pathway in colorectal cancer cells.

Colorectal cancer (CRC) is a malignancy with high metastatic rates. The mechanism of miR-128 on the regulation of Ribophorin-II (RPN2) in CRC cells was explored in the present study. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) or western blot analyses were conducted to detect miR-128 and RPN2 levels in tissues and cell lines. AmiR-128 overexpression model was constructed using miR-128 mimic transfection in HT29 CRC cells. Then, cell proliferation was detected using a Cell Counting Kit-8 assay, and the migratory and invasive abilities were measured by Transwell assay. RT-qPCR and western blot analysis were used to detect expression levels of protein kinase-B (Akt)-tumor protein 53 (p53)-cyclin pathway and metastasis-associated factors. In the present study, it was identified that aberrant decreased miR-128 was negatively correlated with RPN2 in CRC tissues. The increased RPN2 levels were significantly associated with poorly-differentiated histology, advanced stages and lymph nodes metastasis in patients with CRC. The survival rate of patients with CRC was also closely associated with RPN2 levels. In HT29 cells, miR-128 upregulation downregulated mRNA and protein levels of RPN2, and significantly inhibited cell proliferative, migratory and invasive abilities. Markedly decreased Akt phosphorylation and cyclin D1 levels and increased p53 levels were detected when cells were transfected with miR-128 mimics. Concurrently, decreased levels of matrix metalloproteinase (MMP)-2, MMP-9 and metastasis-associated protein 1, and increased levels of epithelial-cadherin and tissue inhibitor of metalloproteinases 2, were revealed in miR-128 mimic-transfected cells. Subsequent to screening with miRNA target prediction databases, the specificity of miR-128-targeted RPN2 was validated by a luciferase reporter assay. In conclusion, the results suggested that miR-128 was a specific negative regulator of RPN2, which regulated colorectal cancer cell proliferation and migration by affecting the Akt-p53-cyclin pathway. These data may provide novel evidence for the therapeutic potential of miR-128-based treatments for colorectal cancer.

SUMO2 and SUMO3 redundantly prevent a noncanonical type I interferon response

Detection of nucleic acids by innate immune sensors triggers the production of type I interferons (IFNs). While IFNs are essential for host defense against viral infection, dysregulated production of IFNs underlies numerous autoinflammatory diseases. We have found that the loss of sumoylation results in a potent, spontaneous IFN response. Vertebrates possess three small ubiquitin-like modifiers (SUMOs) that can be conjugated onto target proteins and alter protein function in diverse but still poorly characterized ways. We demonstrate that regulation of IFN by sumoylation is redundantly mediated by both SUMO2 and SUMO3, but not SUMO1, revealing a previously unknown function of SUMO2/3. Remarkably, this IFN response is independent of all known IFN-inducing pathways and does not require either of the canonical IFN-associated transcription factors IRF3 or IRF7. Taken together, our findings demonstrate that SUMO2 and SUMO3 are specific and essential negative regulators of a noncanonical mechanism of IFN induction.

Systems biology analysis of mitogen activated protein kinase inhibitor resistance in malignant melanoma

BackgroundKinase inhibition in the mitogen activated protein kinase (MAPK) pathway is a standard therapy for cancer patients with activating BRAF mutations. However, the anti-tumorigenic effect and clinical benefit are only transient, and tumors are prone to treatment resistance and relapse. To elucidate mechanistic insights into drug resistance, we have established an in vitro cellular model of MAPK inhibitor resistance in malignant melanoma.MethodsThe cellular model evolved in response to clinical dosage of the BRAF inhibitor, vemurafenib, PLX4032. We conducted transcriptomic expression profiling using RNA-Seq and RT-qPCR arrays. Pathways of melanogenesis, MAPK signaling, cell cycle, and metabolism were significantly enriched among the set of differentially expressed genes of vemurafenib-resistant cells vs control. The underlying mechanism of treatment resistance and pathway rewiring was uncovered to be based on non-genomic adaptation and validated in two distinct melanoma models, SK-MEL-28 and A375. Both cell lines have activating BRAF mutations and display metastatic potential.ResultsDownregulation of dual specific phosphatases, tumor suppressors, and negative MAPK regulators reengages mitogenic signaling. Upregulation of growth factors, cytokines, and cognate receptors triggers signaling pathways circumventing BRAF blockage. Further, changes in amino acid and one-carbon metabolism support cellular proliferation despite MAPK inhibitor treatment. In addition, treatment-resistant cells upregulate pigmentation and melanogenesis, pathways which partially overlap with MAPK signaling. Upstream regulator analysis discovered significant perturbation in oncogenic forkhead box and hypoxia inducible factor family transcription factors.ConclusionsThe established cellular models offer mechanistic insight into cellular changes and therapeutic targets under inhibitor resistance in malignant melanoma. At a systems biology level, the MAPK pathway undergoes major rewiring while acquiring inhibitor resistance. The outcome of this transcriptional plasticity is selection for a set of transcriptional master regulators, which circumvent upstream targeted kinases and provide alternative routes of mitogenic activation. A fine-woven network of redundant signals maintains similar effector genes allowing for tumor cell survival and malignant progression in therapy-resistant cancer.

MiR‐330‐5p negatively regulates ovine preadipocyte differentiation by targeting branched‐chain aminotransferase 2

The differentiation of preadipocytes into adipose tissues is tightly regulated by various factors including microRNAs and cytokines. This article aims to study the effect of miR-330-5p on expression of BCAT2 in ovine preadipocytes. Ovine preadipocytes were isolated, and we found that the miR-330-5p expression decreased gradually during the early differentiation of ovine preadipocytes, while BCAT2 expression increased. BCAT2 was identified as a direct target of miR-330-5p, ectopic expression of miR-330-5p could change the expression of both BCAT2 mRNA and protein. Silencing BCAT2 had the same inhibition effects as overexpressing miR-330-5p on the preadipocyte differentiation, but overexpressing BCAT2 had the converse effects. Taken together, we demonstrated that miR-330-5p is a negative regulator of differentiation by targeting BCAT2, and clarified the role of BCAT2 and miR-330-5p during preadipocyte differentiation.