Phosphoprotein Levels Research Articles

Activation of the Soluble Guanylate Cyclase Increases 26S Proteasome Activities and Protects against Proteotoxicity in Cardiomyocytes

BackgroundThe ubiquitin‐proteasome system (UPS) mediates the selective degradation of unwanted and unneeded proteins in the cell, playing crucial roles in protein quality control and diverse other cellular processes. Proteasome malfunction and proteotoxicity are implicated in the genesis of a large subset of heart diseases. Our laboratory has previously discovered that activation of the cGMP‐dependent protein kinase (PKG) primes the proteasome. Here we test the hypothesis that by increasing cGMP and PKG activity, soluble guanylate cyclase (sGC) activation activates the proteasome and thereby protects against proteotoxicity in cardiomyocytes.Methods and ResultsCompared with vehicle control (DMSO), treatment with an sGC activator (cinaciguat) in cultured neonatal rat cardiomyocytes (NRCMs) led to increases in the levels of Ser239‐phosphorylated vasodilator‐stimulated phosphoprotein (VASP) (2.17±0.07 vs. 1.0±0.20, p<0.0001), indicative of PKG activation. We then used adenoviral vectors expressing degron CL1‐fused green fluorescent proteins (GFPu, a proven UPS substrate) or red fluorescent proteins (RFP, a stable protein control) as a reporter system for UPS performance. Compared with DMSO, cinaciguat treatment reduced the GFPu/RFP ratio (0.59±0.06 vs. 1.0±0.18, p=0.0135) as revealed by western blotting, and shortened GFPu half‐life in cultured NRCMs as revealed by cycloheximide chase assays. Cinaciguat treatment decreased Thr41/Ser45‐phosphorylated β‐catenin, an endogenous substrate of the UPS (0.60±0.12 vs. 1.0±0.08, p=0.0014). These data demonstrate that sGC activation enhances UPS performance. Moreover, in‐gel proteasome activity assays followed by western blot analyses reveal that cinaciguat treatment increased the chymotrypsin like activities of 26S proteasomes (1.52±0.17 vs. 1.0±0.09, p=0.0019) without changing 26S proteasome assembly, showing that sGC activation enhances UPS performance via activating 26S proteasomes. We next used NRCMs with adenovirus‐mediated overexpression of a human‐disease‐linked misfolded protein CryABR120Gas a cell model of cardiac proteotoxicity. CryABR120G overexpression‐induced increases in LDH leakage and PKCδ cleavage (both indicative of cell injury/death) were significantly attenuated by cinaciguat treatment (LDH in media: 0.71±0.20 vs.1.44±0.26, p<0.0001); further MTT assays consistently showed cinaciguat promoted NRCMs viability (1.32±0.11 vs. 1.02±0.05, p<0.0001).ConclusionsActivation of sGC with cinaciguat increases PKG activity, activates the 26S proteasome, facilitates UPS functioning, and thereby protects against proteotoxicity in cardiomyocytes.

Inhibition of CHRM1 reverts docetaxel resistance in castration‐resistant prostate cancer

BackgroundChemotherapy remains the major treatment option for castration‐resistant prostate cancer (CRPC). Docetaxel (DTX)‐based chemotherapy provides significant survival benefit in patients with CRPC. However, the clinical utility of DTX is compromised when resistance is encountered. Therefore, a more thorough understanding of mechanisms underlying DTX resistance may potentially improve survival in patients with CRPC. The objective of this study is to investigate the role and mechanism of cholinergic receptor muscarinic 1 (CHRM1) activation upon chemotherapy resistance in PC and the efficacy of the CHRM1 antagonist dicyclomine (Dic) in treating DTX‐resistant PC. The hypothesis is that CHRM1 is essential for developing chemotherapy resistance in PC cells and driving survival and growth of DTX‐resistant PC cells.MethodsThe CHRM1 expression levels and their association with DTX resistance were determined in PC clinical specimens and cultured cells. Human PC cells subjected to stable knockdown or overexpression of CHRM1 were evaluated for their response to DTX by cell proliferation, colony and tumorsphere formation, and TUNEL assays. Synergistic growth inhibition by combination treatment with DTX and Dic was assessed by cell viability and tumorgenicity assays.ResultsCHRM1 expression was elevated under DTX treatment in CRPC 22Rv1 and DU145 cells, which was paralleled by higher CHRM1 expression in these cells after acquiring resistance to DTX (22Rv1DTXR and DU145DTXR). Immunohistochemical analyses revealed higher CHRM1 protein levels in longitudinally collected tumor tissues post‐ versus pre‐chemotherapy from a PC patient cohort. Enforced expression of CHRM1 increased DTX IC50 by up to 5‐fold, along with reduced cell apoptosis marker c‐PARP1 expression, in 22Rv1 and DU145 cells. Conversely, silencing of CHRM1 in 22Rv1DTXR cells decreased cell proliferation, invasion, and colony and tumorsphere formation. CHRM1 inactivation with Dic in combination with DTX resulted in more cell death of both DTX‐sensitive (22Rv1 and DU145) and ‐resistant (22Rv1DTXR, DU145DTXR and PC‐3DTXR) cells compared to either treatment alone. Further, Dic restored DTX sensitivity to suppress the growth of DTX‐resistant cells and xenograft tumors (22Rv1DTXR and PC‐3DTXR). Mechanistically, CHRM1 activation upregulated phosphoprotein levels of ERK, JNK and p38, which all are key players in the MAPK pathway and potential mediators driving chemoresistance in cancer cells, while Dic treatment reduced their phosphorylation levels in 22Rv1DTXR and DU145DTXR cells.ConclusionOur results suggest that CHRM1 contributes to the development of DTX resistance in CRPC, which can be reversed by genetic or pharmacologic inactivation of CHRM1, providing new insights into developing a CHRM1‐targeted therapeutic strategy for overcoming DTX‐based chemotherapy resistance in CRPC.

Exploring causal relationships in proteomic profiles in Cytoscape using the CausalPath App

Introduction: CausalPath compares experimentally measured changes in molecular profiles against curated biological pathways and infers causality between changes in measured features from profiling experiments (e.g., RNA-seq or proteomics from total or phospho-protein levels). Methods: We developed the CausalPath Cytoscape App, an app (i.e., plugin) for visualizing results from the CausalPath method within the Cytoscape Java-based desktop network analysis and visualization platform. Use Cases: Users are given instruction that represents use cases in multiple cancer research areas through the visualization of CausalPath analysis results generated from data by the Clinical Proteomic Tumor Analysis Consortium. Discussion: The CausalPath Cytoscape App visualizes the set of known interactions that are supported by molecular profiling data via the CausalPath method. This integration of CausalPath and Cytoscape benefits users interested in performing secondary analyses (e.g., module detection) on the sub-networks that result from CausalPath analysis by utilizing the many analytical features available in the Cytoscape software ecosystem.

MiR-497-5p promotes osteogenic/odontogenic differentiation of stem cells from the apical papilla by regulation of the TGF-β Smad pathway through Smurf2


 
 
 
 Purpose: To study the influence of miR-497-5p on osteogenic/odontogenic differentiation (OOD) of SCAP, and the signal route involved.
 Methods: Four groups were set up: miR-497-5p overexpression group (OEG), overexpression control OEC), miR-497-5p inhibition group, and inhibition control group. Alkaline phosphatase (ALP) activity was assayed, and calcified nodules measured. Protein expression levels of dentine salivary phosphoprotein (DSPP), collagen type I, ALP, osteoblast-related factors (Runx2, OSX and OPN) were also assayed. The mRNA expression levels of osteogenesis/dentin-related genes were determined.
 Results: ALP activity was significantly higher in miR-497-5p overexpression cells than in control, but was reduced, relative to inhibition control group (p < 0.05). The miR-497-5p OEG had significantly more calcified nodules than OEC (p < 0.05). There were markedly up-regulated protein expressions in cells of miR-497-5p OEG than in OCG. Furthermore, protein expressions of Smad2, Smad3 and Smad4 in cells of miR-497-5p OEG were significantly up-regulated, relative to those in OEC, but wer lower in miR-497- 5p inhibitory cells than in inhibitory cells.
 Conclusion: MiR-497-5p enhances the OOD of SCAP via a mechanism involving TGF-β Smad pathway and Smurf2. Thus, Mir-497-5p may be used as a target for OOD-related drugs.
 
 
 

The effect of two different types of forces on possible root resorption in relation to dentin phosphoprotein levels: a single-blind, split-mouth, randomized controlled trial

BackgroundThe purpose of this 2-arm-parallel split-mouth trial was to evaluate and compare the extent of possible root resorption using dentin phosphoprotein levels in gingival crevicular fluid between controlled continuous and intermittent orthodontic force groups.Materials and methodsA sample of 16 maxillary first premolars from 8 patients requiring bilateral extractions of the upper first premolars as part of their orthodontic treatment were recruited. A buccally directed continuous force of 150 g, reactivated after 28 days, was applied to the upper first premolar on one side for 8 weeks. On the contralateral first premolar, a buccally directed intermittent force (21 days on, 7 days off) of the same magnitude was applied for the same period. Gingival crevicular fluid samples were collected at the beginning of the study, 1st, 3rd, 4th and 5th week, and at the end of the study to quantify and compare dentin phosphoprotein levels in both groups.ResultsDentin phosphoprotein levels showed a higher concentration in the continuous force group than the intermittent force group in week 4 and 8 of sample collection; where the differences were statistically significant (95% CI 0.007–0.14; P < .04) and (95% CI 0.02–0.17; P < .04) respectively. No harm was observed.ConclusionsDentin phosphoprotein was found to be a useful early biomarker to detect and monitor root resorption, showing that the application of an intermittent orthodontic force caused less root resorption than a continuous force.Trial registrationNCT04825665 ClinicalTrials.gov. Registered 1 April 2021—Retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT04825665.

Preclinical development of a bispecific TNF\u03b1/IL-23 neutralising domain antibody as a novel oral treatment for inflammatory bowel disease

Anti-TNFα and anti-IL-23 antibodies are highly effective therapies for Crohn’s disease or ulcerative colitis in a proportion of patients. V56B2 is a novel bispecific domain antibody in which a llama-derived IL-23p19-specific domain antibody, humanised and engineered for intestinal protease resistance, V900, was combined with a previously-described TNFα-specific domain antibody, V565. V56B2 contains a central protease-labile linker to create a single molecule for oral administration. Incubation of V56B2 with trypsin or human faecal supernatant resulted in a complete separation of the V565 and V900 monomers without loss of neutralising potency. Following oral administration of V900 and V565 in mice, high levels of each domain antibody were detected in the faeces, demonstrating stability in the intestinal milieu. In ex vivo cultures of colonic biopsies from IBD patients, treatment with V565 or V900 inhibited tissue phosphoprotein levels and with a combination of the two, inhibition was even greater. These results support further development of V56B2 as an oral therapy for IBD with improved safety and efficacy in a greater proportion of patients as well as greater convenience for patients compared with traditional monoclonal antibody therapies.

Single-cell intracellular epitope and transcript detection reveals signal transduction dynamics

SummaryTo further our understanding of how biochemical information flows through cells upon external stimulation, we require single-cell multi-omics methods that concurrently map changes in (phospho)protein levels across signaling networks and the associated gene expression profiles. Here, we present quantification of RNA and intracellular epitopes by sequencing (QuRIE-seq), a droplet-based platform for single-cell RNA and intra- and extracellular (phospho)protein quantification through sequencing. We applied QuRIE-seq to quantify cell-state changes at both the signaling and the transcriptome level after 2-, 4-, 6-, 60-, and 180-min stimulation of the B cell receptor pathway in Burkitt lymphoma cells. Using the multi-omics factor analysis (MOFA+) framework, we delineated changes in single-cell (phospho)protein and gene expression patterns over multiple timescales and revealed the effect of an inhibitory drug (ibrutinib) on signaling and gene expression landscapes.

Sex Differences and Estradiol Effects in MAPK and Akt Cell Signaling across Subregions of the Hippocampus

Introduction: Rapid effects of estrogens within the hippocampus of rodents are dependent upon cell-signaling cascades, and activation of these cascades by estrogens varies by sex. Whether these pathways are rapidly activated within the dentate gyrus (DG) and CA1 by estrogens across sex and the anatomical longitudinal axis has been overlooked. Methods: Gonadally intact female and male rats were given either vehicle or physiological systemic low (1.1 µg/kg) or high (37.3 µg/kg) doses of 17β-estradiol 30 min prior to tissue collection. To control for the effects of circulating estrogens, an additional group of female rats was ovariectomized (OVX) and administered 17β-estradiol. Brains were extracted, and tissue punches of the CA1 and DG were taken along the longitudinal hippocampal axis (dorsal and ventral) and analyzed for key mitogen-activated protein kinase (MAPK) and protein kinase B (Akt) cascade phosphoproteins. Results: Intact females had higher Akt pathway phosphoproteins (pAkt, pGSK-3β, and pp70S6K) than males in the DG (dorsal and ventral) and lower pERK1/2 in the dorsal DG. Most effects of 17β-estradiol on cell signaling occurred in OVX animals. In OVX animals, 17β-estradiol increased cell signaling of MAPK and Akt phosphoproteins (pERK1/2, pJNK, pAkt, and pGSK-3β) in the CA1 and pERK1/2 and pJNK DG. Discussion/Conclusions: Systemic 17β-estradiol treatment rapidly alters phosphoprotein levels in the hippocampus, dependent on reproductive status, and intact females have greater expression of Akt phosphoproteins than that in intact males in the DG. These findings shed light on underlying mechanisms of sex differences in hippocampal function and response to interventions that affect MAPK or Akt signaling.

Phosphorylation of Ser1452 on BRG1 inhibits the function of the SWI/SNF complex in chromatin activation

BRG1, one of core subunits of the SWI/SNF chromatin remodeling complex, is frequently mutated in cancers. Previously, we reported significant downregulation of the phosphorylation level of BRG1 on Ser1452 (<10%) in cell lines derived from ovarian clear cell carcinoma with frequent recurrence and acquired drug resistance. In this study, we tried to elucidate the roles of BRG1 phosphorylation, using cell lines expressing wild-type, phosphorylation-mimic (brg1-S1452D), or non-phosphorylatable (brg1-S1452A) BRG1. Quantitative proteomic analyses revealed upregulation of proteins and phosphoproteins related to linker histone H1s, histone methylation, and protein ubiquitylation in brg1-S1452D cells, which may coordinately promote the chromatin inactivation and ubiquitin-dependent degradation of target proteins. Consistent with these results, brg1-S1452D cells exhibited an increase in condensed chromatin and polyubiquitylated proteins. In brg1-S1452D cells, we also detected downregulation of various cancer-related proteins (e.g., EGFR and MET) as well as decreased migration, proliferation, and sensitivity to taxanes and oxaliplatin. Together, our results reveal that BRG1 phosphorylation drives tumor malignancy by inhibiting the functions of SWI/SNF complex in chromatin activation, thereby promoting expression of various cancer-related proteins. SignificanceFor the first time we demonstrated that the mutation on Ser1452 phosphorylation site of BRG1, a component of SWI/SNF chromatin remodeling complex, changed protein and phosphoprotein levels of linker histone H1s, binding competitor of histone H1s, and histone methylase/demethylase involved in the heterochromatic histone modifications to promote the chromatin inactivation. In phosphorylation-mimic mutant, significant decrease of various cancer-related proteins as well as migration, proliferation, and sensitivity to specific antitumor agents were detected. Our results reveal that BRG1 phosphorylation drives tumor malignancy by inhibiting the functions of SWI/SNF complex in chromatin activation, thereby promoting expression of various cancer-related proteins.

Enhanced Efficacy of the SYK Inhibitor Entospletinib and Chemotherapy in Infant Acute Lymphoblastic Leukemia

Background: Chemotherapy resistance and subsequent relapse remain a major cause of childhood cancer mortality, particularly for infants (<12 months of age) with KMT2A -rearranged (R) B-cell acute lymphoblastic leukemia (B-ALL), who have extremely poor event-free survival (EFS). Addition of kinase inhibitors to chemotherapy has markedly improved outcomes for patients with some genetic subsets of B-ALL, such as BCR-ABL1 -R (Ph+) ALL. Unfortunately, addition of the FMS-like tyrosine kinase 3 (FLT3) inhibitor lestaurtinib did not improve 3-year EFS for infants with KMT2A -R and FLT3-overexpressing B-ALL in the Children's Oncology Group trial AALL0631 (Brown et al, SIOP 2016), indicating continued need for alternative therapeutic strategies. We recently observed constitutive phosphorylation (p) of spleen tyrosine kinase (SYK) at tyrosine 323 in infant ALL specimens (Figure 1A) and hypothesized that activated signaling is targetable with the selective SYK inhibitor entospletinib (ENTO). We further hypothesized that combining ENTO with chemotherapy would have superior activity in comparison to single-agent treatments.Methods: To test these hypotheses, we established patient-derived xenograft (PDX) models of KMT2A -R (n=7) and non- KMT2A -R (n=4) infant ALL in NSG mice using diagnostic and relapse bone marrow specimens from patients treated on or as per the AALL0631 trial protocol. Total and phosphorylated (p) SYK and other phosphoprotein levels were assessed by Simple WesternTM analysis of splenic lysates from ALL-engrafted PDX mice to identify leukemias with constitutive SYK signaling activation. Infant ALL PDX models were then treated with control mouse chow or chow containing 0.07% ENTO ad libitum, 0.25 mg/kg vincristine (VCR) dosed intraperitoneally once weekly, or a combination of ENTO and VCR for up to 28 days to determine treatment efficacy. Cohorts of additional infant ALL PDX mice were treated for 72 hours to assess plasma levels and pharmacodynamic effects of ENTO administration.Results: Constitutive SYK signaling was observed in several models of de novo KMT2A -R and non- KMT2A -R B-ALL. ENTO treatment of KMT2A -R ALL PDX models ALL3103 with t(9;11) and ALL135 with t(11;19) inhibited leukemic cell burden in vivo versus control animals by flow cytometric and immunohistochemical (IHC) analyses of murine spleens (representative data in Figure 1B). Importantly, combined ENTO and VCR treatment resulted in a greater reduction in ALL cell number, which was confirmed by human CD19+ IHC analysis of murine spleens and bone marrow. ENTO concentrations were maintained throughout study duration with terminal free fraction plasma levels of 61 and 230 nM (ALL3103) and 89 and 214 nM (ALL135) for ENTO and ENTO/VCR treatment arms, respectively. Evaluation of SYK pathway modulation in vivo demonstrated inhibition of pSYKY323 in human ALL cells within murine spleens that correlated with the level of ENTO exposure of treated mice (ALL3103 PDX; Spearman correlation of -0.63, p<0.05). No alterations in total SYK protein were observed. RNA sequencing of ALL3103 PDX tissues showed that ENTO treatment was associated with downregulation of MYC and of cell cycle and DNA damage gene expression signatures.Conclusions: Constitutive activation of SYK signaling occurs in infant KMT2A -R B-ALL. Entospletinib treatment of infant B-ALL PDX models potently inhibited the SYK pathway and decreased leukemic cell burden in vivo . Superior anti-ALL activity was observed with combined ENTO and VCR compared to ENTO alone. Additional studies are ongoing to determine the activity of ENTO in non- KMT2A -R and other ALL PDX models. [Display omitted] DisclosuresYahiaoui:Gilead Sciences, Inc.: Employment, Equity Ownership. Lopez:Gilead Sciences, Inc.: Employment, Equity Ownership. Mikaelian:Gilead Sciences, Inc.: Employment, Equity Ownership. Tannheimer:Gilead Sciences, Inc.: Employment, Equity Ownership. Forslund:Gilead Sciences, Inc.: Employment, Equity Ownership. Tasian:Gilead Sciences, Inc.: Research Funding; Incyte Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Soluble guanylate cyclase activation increases proteasome activities and protects against proteotoxicity in cardiomyocytes

Background The ubiquitin-proteasome system (UPS) mediates the selective degradation of misfolded proteins and most cellular proteins that are normal but no longer needed, thereby playing important roles in diverse cellular processes. Proteasome malfunction and proteotoxicity are implicated in the genesis of a large subset of cardiovascular diseases. We previously discovered that activation of the cGMP-dependent protein kinase (PKG) primes the proteasome. Here we sought to test whether activation of the soluble guanylate cyclase (sGC) could activate the proteasome and improve protein quality control in cardiomyocytes. Methods and Results In cultured neonatal rat ventricular myocytes (NRVMs), treatment with an sGC activator (cinaciguat) led to increases in the levels of Ser239-phosphorylated vasodilator-stimulated phosphoprotein (VASP) in a dose-dependent fashion and increased the activities of 26S proteasomes as assessed with in-gel proteasome activity assays, revealing that sGC activation increases PKG activity and stimulates proteasome activities in cardiomyocytes. Adenoviral vectors expressing degron CL1-fused green fluorescent proteins (GFPu, a proven UPS substrate) or red fluorescent proteins (RFP, a stable protein control) were used as a reporter system for UPS performance. Cinaciguat treatment enhanced the degradation of GFPu in NRVMs. In NRVMs with adenovirus-mediated overexpression of a human-disease-linked misfolded protein HA-tagged CryABR120G, the proteasome-mediated flux of CryABR120G was significantly enhanced by cinaciguat treatment. Consistently, cinaciguat treatment significantly reduced the levels of CryABR120G oligomers, indicating that the cinaciguat treatment promotes proteasomal degradation of the misfolded species of CryABR120G. Furthermore, the LDH leakage and PKCδ cleavage in NRVMs overexpressing CryABR120G were significantly attenuated by cinaciguat treatment. Conclusions Activation of sGC with cinaciguat increases PKG activity, activates the 26S proteasome, facilitates the degradation of misfolded proteins, and thereby protects against proteotoxicity in cardiomyocytes.

Cingulate Cortex Neuroadaptations in a Female Rat Model of Combined Complex Regional Pain Syndrome and Alcohol Neuropathy

Complex Regional Pain Syndrome (CRPS) is an amplified musculoskeletal pain condition that develops following limb injury or immobilization and can persist long after the injury has healed or immobilization has ceased. Although the exact mechanisms underlying the long-lasting nature of the syndrome are unknown, CRPS is associated with central nervous system dysregulation and peripheral hyperalgesia. These symptoms are also observed in alcoholic neuropathy, leading us to hypothesize that these conditions may be pathophysiologically accretive. Additionally, people assigned female at birth appear to be more sensitive to both CRPS and alcoholic neuropathy. To investigate the biobehavioral mechanisms underlying these two conditions, we utilized a model of combined CRPS and alcoholic neuropathy in adult female rats (n=4-5/group). In order to determine the effect of circulating ovarian hormones on these pathologies, half of the animals received an ovariectomy (OVX) at the start of the experiment. To model alcoholic neuropathy, animals were fed either a Leiber-DeCarli alcohol liquid diet or a control diet for 10 weeks. CRPS was modeled in all animals via unilateral hind limb immobilization for 7 days, and mechanical hyperalgesia was measured using the von Frey test 3 days following cast removal. We found that cast immobilization and chronic alcohol both separately and additively decreased mechanical nociceptive thresholds, indicative of hyperalgesia. Interestingly, OVX had no effect on mechanical sensitivity. We next used Western blot analysis to investigate phosphoprotein levels in the cingulate cortex, a limbic region that mediates the affective components of pain. Chronic alcohol increased phosphorylation of glutamatergic receptor subunits GluR1 and NR1 as well as extracellular signal-regulated kinase (ERK) and cAMP-response element binding protein (CREB). Again, we did not observe any OVX effects on protein phosphorylation. Collectively, these findings suggest that chronic alcohol in the context of CRPS facilitates hyperalgesia via hyperexcitability in the cingulate cortex, establishing a novel therapeutic strategy for these conditions.

The miR-155-5p inhibits osteoclast differentiation through targeting CXCR2 in orthodontic root resorption.

Root resorption is an unavoidable side effect of orthodontic tooth movement. The mechanism of root resorption is similar to bone resorption; the odontoclasts share similar characteristics with osteoclasts (OCs). MicroRNAs (miRNAs) such as miR-155-5p play an important role in OC differentiation, but the underlying molecular mechanism of miR-155-5p in this process is not fully understood. We found that the miR-155-5p seed sequences were complementary to a sequence conserved in the 3-untranslated region of CXCR2 mRNA. In this study, we explored the molecular mechanism underlying the effect of miR-155-5p on OC differentiation by targeting CXCR2. In this study, we divided the orthodontic patients into mild, moderate, and severe groups according to the severity of root resorption. The gingival crevicular fluid (GCF) of patients in different groups was collected, and the expression levels of dentin phosphoprotein (DPP) were detected by ELISA, and the expression levels of CXCR2 and miR-155-5p in GCF were detected by real-time quantitative PCR (qRT-PCR). The relationship between miR-155-5p and CXCR2 was verified by double luciferase. We analyzed changes of CXCR2 and miR-155-5p expression after transfection of miR-155-5p mimic and inhibitor into RAW264.7 cells induced by receptor activator of nuclear factor-κB ligand (RANKL) through qRT-PCR and western blotting. The effect of miR-155-5p on OC differentiation was evaluated by tartrate-resistant acid phosphatase (TRAP) staining. QRT-PCR and western blotting were used to analyze expression of the osteoclastic bone resorption-related enzymes carbonic anhydrase 2 (CA II), matrix metalloproteinase-9 (MMP-9), and cathepsin K. To further confirm the direct targeting effect of CXCR2 by miR-155-5p, we blocked CXCR2 using si-CXCR2 in RANKL-induced RAW264.7 cells. Dentin phosphoprotein levels were consistent with the trend of miR-155-5p changes, and the trend of CXCR2 expression was opposite to miR-155-5p changes. miR-155-5p can be directly targeted to act on CXCR2. The expression of miR-155-5p was significantly downregulated in differentiated OCs. MiR-155-5p inhibited OC differentiation, and downregulated CA II, MMP-9, and cathepsin K expression at the protein and mRNA levels. In summary, the results of this study suggested that miR-155-5p inhibited OC differentiation by targeting CXCR2, thus reducing root resorption in orthodontics. MiR-155-5p can be used as an effective target for avoiding or reducing the degree of root resorption in orthodontic treatment.

Comparative phosphoproteomic analysis unravels MAPK1 regulated phosphoproteins in Leishmania donovani

Mitogen Activated Protein Kinase1 (MAPK1) of Leishmania donovani functions as key regulators of various cellular activities, which seem to be imperative for parasite survival, infectivity, drug resistance and post-translational modification of chaperones/co-chaperones. However, very less is known about LdMAPK1 target proteins. With recent advancements in proteomics, we aimed to identify phosphoproteins which were differentially expressed in LdMAPK1 overexpressing (Dd8++/++) and single replacement mutants (Dd8+/) as compared to wild type (Dd8+/+) parasites, utilizing LC-MS/MS approach. An in-depth label-free phospoproteomic analysis revealed that modulation of LdMAPK1 expression significantly modulates expression levels of miscellaneous phosphoproteins which may act as its targets/substrates. Out of 1974 quantified phosphoproteins in parasite, 140 were significantly differentially expressed in MAPK1 overexpressing and single replacement mutants. These differentially expressed phosphoproteins are majorly associated with metabolism, signal transduction, replication, transcription, translation, transporters and cytoskeleton/motor proteins, hence suggested that MAPK1 may act in concert to modulate global biological processes. The study further implicated possible role of LdMAPK1 in regulation and management of stress machinery in parasite through post translational modifications. Precisely, comparative phosphoproteomics study has elucidated significant role of LdMAPK1 in regulating various pathways contributing in parasite biology with relevance to future drug development. SignificanceMAPKinase1, the downstream kinase of MAPK signal transduction pathway, has drawn much attention as potential therapeutic drug target due to their indispensable role in survival and infectivity of Leishmania donovani. However, limited information is available about its downstream effector proteins/signaling networks. Utilizing label free LC-MS/MS analysis, phosphoproteome of LdMAPK1 over-expressing (Dd8++/++) and LdMAPK1 single replacement mutants (Dd8+/−) with wild type (Dd8+/+) parasites was compared and identified 140 LdMAPK1 modulated phosphoproteins, mainly involved in pathways like signal transduction, metabolism, transcriptional, translational, post-translational modification and regulation of heat shock proteins. Interestingly, LdMAPK1 interacts directly with only six phosphoproteins i.e. casein kinase, casein kinase II, HSP83/HSP90, LACK, protein kinase and serine/threonine protein kinase. Thus, the study elucidates significant role of LdMAPK1 in Leishmania biology which may drive drug-discovery efforts against visceral leishmaniasis.

Influence of long-term proteasome inhibition on platelet responsiveness mediated by bortezomib

Introduction: Although platelets contain a full proteasome system, its role in platelet function is not completely understood yet. Since the proteasome system may be involved in time-delayed processes, platelet responsiveness was investigated after long-term, bortezomib-mediated proteasome inhibition.Materials and Methods: Citrate-anticoagulated whole blood was stored with 5 nM and 1 μM bortezomib for 24 h. Consecutively, aggregation was measured by light transmission in platelet-rich-plasma (PRP). Flow cytometry was performed to determine phosphorylation levels of the vasodilator-stimulated phosphoprotein (VASP), fibrinogen binding, PAC1-antibody binding and purinergic receptor expression in PRP, P2Y12 activity or glycoprotein (GP) Ib and IIb expression in whole blood. P2Y1 and P2X1 activities were assessed by calcium flux-induced fluorescence in washed platelets. Using PRP, adherent platelets on fibrinogen-, collagen- and ristocetin-coated surfaces were visualized and quantified by immunostaining.Results: Under bortezomib, VASP phosphorylation was less inducible and nitric oxide-induced inhibition of fibrinogen binding was slightly reduced. Proteasome inhibition did not tamper adenosine diphosphate-mediated aggregation or purinergic receptor expression and activity. Induced expression of activated fibrinogen receptors and fibrinogen binding were not significantly influenced by incubation with bortezomib for 24 h. Aggregation values with threshold agonist concentrations were increased under bortezomib. Despite unchanged GPIb expression, bortezomib-treated platelets showed enhanced adhesion on coated surfaces.Conclusions: In platelets incubated for 24 h, bortezomib mediates a slight attenuation of inhibitory signaling, associated with facilitated platelet aggregation using threshold agonist concentrations and enhanced adhesion on agonist-coated surfaces.

Cell Cycle Regulates Phosphorylation of RUNX1 to Modulate Megakaryocyte-Erythroid Progenitor Fate Specification

Human Megakaryocytic-Erythroid Progenitors (MEP) produce megakaryocytic progenitors (MkP) and erythroid progenitors (ErP). Though some of the players have been identified, the molecular mechanisms underlying the MEP fate decision have not yet been determined. Using a functional single cell CFU-Mk/E assay, and single-cell RNA sequencing (scRNAseq), we have revealed that MEP cell cycling regulates MEP fate decisions with decreased cycling promoting Mk fate commitment and increased cycling promoting E fate commitment (Lu et al, Cell Reports, 2018). Our data point to RUNX1 (aka AML1), already known to be important for both Mk and E maturation, as playing a key role in MEP fate determination. RUNX1 target genes vary significantly between Common Myeloid Progenitors (CMP), MEP, MkP, and ErP. For example, the RUNX1 targets MPL, FLI1, and THBS1 are higher in MkP than in MEP and lowest in CMP and ErP. Analysis of scRNAseq data indicates that 11.8% and 9.3% of differentially (adj.p &amp;lt; 0.05, fold change &amp;gt; 2) expressed genes are predicted RUNX1 targets when comparing MEP to MkP and MEP to ErP, respectively (p=2.9e-8 and 5.6e-16). However, RUNX1 mRNA levels do not change significantly between CMP, MEP, MkP and ErP. We therefore assessed whether total RUNX1 protein levels and post-translational modifications change with Mk and E lineage commitment. Intracellular staining normalized to levels in CMP revealed that total RUNX1 protein is elevated 1.34-fold from CMP to MEP, is highest (1.65-fold) in MkP, and is intermediate in ErP (1.5-fold) consistent with RUNX1 perhaps promoting Mk over E fate. RUNX1 can be phosphorylated by cyclin dependent serine/threonine kinases (CDKs) as well as Src tyrosine kinases. Significant differences in RUNX1 phosphoprotein levels were revealed by intracellular FACS assays for differential phosphorylation of RUNX1. Phosphoserine modified RUNX1 (Ser21, Ser276, and Ser 397) levels, which likely reflect active RUNX1, are highest in MkP (1.28-fold over MEP), and diminished in ErP (-1.32-fold over MEP). To determine whether RUNX1 and phosphor-RUNX1 variants affect MEP commitment, we overexpressed wildtype (WT) RUNX1 or RUNX1-S4D (phospho-mimetic on S249, S277, T273, S276 residues). WT RUNX1 overexpression affected fate specification of primary MEP (p&amp;lt;0.05) with an increase in the Mk-only colony percentage from 24% to 43% at the expense of E-only colonies (from 26% to 16%) with little change in bipotent CFU-Mk/E. RUNX1-S4D more dramatically (p&amp;lt;0.04) increased Mk-only colonies (from 25% to 75%) and decreased E-only (from 25% to &amp;lt; 4%) and Mk/E (from 50% to 21%) colonies. Surprisingly, overexpression of RUNX1-S4D in FACSorted primary human ErP caused them to develop more (p&amp;lt;0.01) Mk/E-colonies (from 12% to 60%) with a dramatic decrease in E-only colonies (from 85% to 28%) suggesting that the E commitment of ErP is reversed (or reprogrammed) by activated RUNX1. In contrast, RUNX1 variants mimicking tyrosine phosphorylation (RUNX1-Y6D) promote E fate specification. Tyrosine phosphorylation on RUNX1 is mediated by Src family kinases (Huang te al, Genes Dev., 2012). We found that Src kinase inhibition results in increasing Mk lineage bias (from 20% to 71%) suggest differential phosphorylation of RUNX1 plays a pivotal role in MEP fate specification. Epigenetic modifications induced by phospho-serine vs phospho-tyrosine mimetics are currently under evaluation. Combining our data on cell cycle control of MEP with RUNX1 reveals that RUNX1 inhibition prevents the Mk bias induced by slowing the cell cycle. For example, the RUNX1 inhibitor Ro5-3335 causes MEP to have an Er bias from 27% to 61% while the mTOR inhibitor Rapamycin (slows MEP cell cycle) induces a Mk bias from 19% to 52%. The combination of Ro5-3335 plus Rapa results in increased Er from 27% to 45% suggest that RUNX1 influences MEP fate specification downstream of the cell cycle machinery. In summary, the phosphorylation status of RUNX1 in primary human hematopoietic progenitors regulates MEP fate commitment. Detailed mechanisms linking the cell cycle machinery to RUNX1 protein levels and function toward Mk vs E specification are being explored. Disclosures No relevant conflicts of interest to declare.

Determining the effects of trastuzumab, cetuximab and afatinib by phosphoprotein, gene expression and phenotypic analysis in gastric cancer cell lines

BackgroundGastric cancer is the fifth most frequently diagnosed cancer and the third leading cause of cancer death worldwide. The molecular mechanisms of action for anti-HER-family drugs in gastric cancer cells are incompletely understood. We compared the molecular effects of trastuzumab and the other HER-family targeting drugs cetuximab and afatinib on phosphoprotein and gene expression level to gain insights into the regulated pathways. Moreover, we intended to identify genes involved in phenotypic effects of anti-HER therapies.MethodsA time-resolved analysis of downstream intracellular kinases following EGF, cetuximab, trastuzumab and afatinib treatment was performed by Luminex analysis in the gastric cancer cell lines Hs746T, MKN1, MKN7 and NCI-N87. The changes in gene expression after treatment of the gastric cancer cell lines with EGF, cetuximab, trastuzumab or afatinib for 4 or 24 h were analyzed by RNA sequencing. Significantly enriched pathways and gene ontology terms were identified by functional enrichment analysis. Furthermore, effects of trastuzumab and afatinib on cell motility and apoptosis were analyzed by time-lapse microscopy and western blot for cleaved caspase 3.ResultsThe Luminex analysis of kinase activity revealed no effects of trastuzumab, while alterations of AKT1, MAPK3, MEK1 and p70S6K1 activations were observed under cetuximab and afatinib treatment. On gene expression level, cetuximab mainly affected the signaling pathways, whereas afatinib had an effect on both signaling and cell cycle pathways. In contrast, trastuzumab had little effects on gene expression. Afatinib reduced average speed in MKN1 and MKN7 cells and induced apoptosis in NCI-N87 cells. Following treatment with afatinib, a list of 14 genes that might be involved in the decrease of cell motility and a list of 44 genes that might have a potential role in induction of apoptosis was suggested. The importance of one of these genes (HBEGF) as regulator of motility was confirmed by knockdown experiments.ConclusionsTaken together, we described the different molecular effects of trastuzumab, cetuximab and afatinib on kinase activity and gene expression. The phenotypic changes following afatinib treatment were reflected by altered biological functions indicated by overrepresentation of gene ontology terms. The importance of identified genes for cell motility was validated in case of HBEGF.

Mechanism of action biomarkers predicting response to AKT inhibition in the I-SPY 2 breast cancer trial

The AKT inhibitor MK2206 (M) was evaluated in I-SPY 2 and graduated in the HER2+, HR−, and HR− HER2+ signatures. We hypothesized that AKT signaling axis proteins/genes may specifically predict response to M and tested 26 phospho-proteins and 10 genes involved in AKT-mTOR-HER signaling; in addition, we tested 9 genes from a previous study in the metastatic setting. One hundred and fifty patients had gene expression data from pretreatment biopsies available for analysis (M: 94, control: 56) and 138 had protein data (M: 87, control: 51). Logistic modeling was used to assess biomarker performance in pre-specified analysis. In general, phospho-protein biomarkers of activity in the AKT-mTOR-HER pathway appeared more predictive of response to M than gene expression or total protein biomarkers in the same pathway; however, the nature of the predictive biomarkers differed in the HER2+ and TN groups. In the HER2+ subset, patients achieving a pCR in M had higher levels of multiple AKT kinase substrate phospho-proteins (e.g., pmTOR, pTSC2). In contrast, in the TN subset responding patients had lower levels of AKT pathway phospho-proteins, such as pAKT, pmTOR, and pTSC2. Pathway mutations did not appear to account for these associations. Additional exploratory whole-transcriptome analysis revealed immune signaling as strongly associated with response to M in the HER2+ subset. While our sample size is small, these results suggest that the measurement of particular AKT kinase substrate phospho-proteins could be predictive of MK2206 efficacy in both HER2+ and TN tumors and that immune signaling may play a role in response in HER2+ patients.

Community Assessment of the Predictability of Cancer Protein and Phosphoprotein Levels from Genomics and Transcriptomics.

Cancer is driven by genomic alterations, but the processes causing this disease are largely performed by proteins. However, proteins are harder and more expensive to measure than genes and transcripts. To catalyze developments of methods to infer protein levels from other omics measurements, we leveraged crowdsourcing via the NCI-CPTAC DREAM proteogenomic challenge. We asked for methods to predict protein and phosphorylation levels from genomic and transcriptomic data in cancer patients. The best performance was achieved by an ensemble of models, including as predictors transcript level of the corresponding genes, interaction between genes, conservation across tumor types, and phosphosite proximity for phosphorylation prediction. Proteins from metabolic pathways and complexes were the best and worst predicted, respectively. The performance of even the best-performing model was modest, suggesting that many proteins are strongly regulated through translational control and degradation. Our results set a reference for the limitations of computational inference in proteogenomics. A record of this paper's transparent peer review process is included in the Supplemental Information.

Temsirolimus combined with etoposide and cyclophosphamide for relapsed/refractory acute lymphoblastic leukemia: Therapeutic advances in Childhood Leukemia Consortium (TACL 2014-001) trial.

10512 Background: PI3K/mTOR signaling, a critical pathway in cell proliferation, metabolism, and apoptosis, is often dysregulated in acute lymphoblastic leukemia (ALL). A phase 1 trial of the mTOR inhibitor temsirolimus combined with etoposide and cyclophosphamide was performed in children with relapsed/refractory (r/r) ALL. Methods: Temsirolimus was administered intravenously (IV) on days 1 and 8 with cyclophosphamide 440 mg/m2 and etoposide 100 mg/m2 IV daily days 1-5. The starting dose level (DL) of temsirolimus was 7.5 mg/m2 (DL1) with escalation to 10 mg/m2 (DL2), 15 mg/m2 (DL3), and 25 mg/m2 (DL4). MRD was performed centrally. PI3K pathway inhibition was measured by phosphoflow cytometry (PFC) analysis of peripheral blood (PB) from treated patients (pts). Results: Sixteen heavily pretreated r/r ALL pts ages 2-19 years with marrow blasts &gt; 25% were enrolled; 15 were evaluable [10 B-ALL/5 T-ALL]. One dose-limiting toxicity (DLT) of grade (Gr) 4 pleural and pericardial effusions with pneumonitis/lung infection leading to Gr 5 cardiorespiratory arrest occurred in a pt treated at DL3. No further DLTs were seen in the DL3 expansion and DL4 cohorts. Gr 3/4 non-hematologic toxicities occurring in ≥ 3 pts included febrile neutropenia, elevated ALT, hypokalemia, mucositis, and tumor lysis syndrome and were independent of dose. Of 15 evaluable pts, 4 (27%; 2 B-ALL/2 T-ALL) had a complete response (CR) after cycle 1, comprised of 1 pt at each DL. Three had MRD &lt; 0.01%. Three pts (20%; 2 B-ALL/1 T-ALL) had partial response (PR). Overall response rate (CR+PR = ORR) was 47%. Pharmacodynamic PFC studies compared phosphoprotein levels pre (day 0) and post treatment (days 3-5) in 9 consenting pts with available PB. All tested pts showed basal activation of PI3K pathway signaling. Dose-dependent inhibition of mTOR targets phosphorylated (p) S6 and/or p4EBP1 was observed in 9/9 and 6/9 pts, respectively, following temsirolimus and chemotherapy treatment. Various patterns of compensatory upregulation of pPI3K, pmTOR, pAkt, and/or pERK was observed. Conclusions: Temsirolimus at 25 mg/m2 combined with salvage etoposide and cyclophosphamide has an acceptable safety profile in high-risk pediatric patients with r/r ALL. Responses were observed at all DLs. mTOR target inhibition was achieved and appeared to correlate with dose level. Future testing of other PI3K/mTOR pathway inhibitors in combination with chemotherapy may be warranted with a goal of further increasing response in r/r ALL. Clinical trial information: NCT01614197.