Protein Phosphorylation In Cells Research Articles

Analyzing Signaling Pathways Using Antibody Arrays.

Cell signaling is comprised of complex networks that regulate homeostasis and human diseases. The analyses of such pathways would improve our understanding of disease pathology and direct drug development. However, it remains a great challenge to study pathways using traditional methods. We developed a high-throughput sandwich-based antibody array technology for the simultaneous detection of multiple targets, capable of identifying the relative expression levels or phosphorylation levels of major signaling pathway proteins. This array-based system features a nitrocellulose membrane or glass slide solid support, spotted with antibodies targeting key proteins of major signaling pathways, including RTK, EGFR, MAPK, AKT, apoptosis, TGFb, JAK/STAT, NFkB, and insulin receptor pathways. We employed these antibody arrays to investigate how the anti-cancer drugs, camptothecin and phorbol 12-myristate 13-acetate (PMA), alter protein phosphorylation in Jurkat and HeLa cells, respectively. Our array data suggest that camptothecin treatment induced DNA double-strand breaks in Jurkat cells and activated the DNA damage pathways ATM and Chk2, which then further induced apoptosis through caspase 3 and PARP. PMA induced the MAPK pathway in HeLa cells through the activation of ERK, CREB, and RSK1. These array results are consistent with previous studies using traditional methods and were validated with Western blotting. Our studies demonstrate that pathway antibody arrays provide a rapid, efficient, and multiplexed approach for profiling phosphorylated proteins.

Detection of protein stoichiometric phosphorylation using Phos-tag SDS-PAGE

Protein phosphorylation plays an important role in many cellular signalings which are relating to many diseases. Therefore, a variety of biochemical techniques has been developed to study protein phosphorylation in cells. Protein phosphorylation has traditionally been detected by radioisotope phosphate labeling of proteins with radioactive ATP. Phosphorylation site-specific antibodies are now available for the analysis of phosphorylation status at target sites. However, these antibodies cannot be used to detect unidentified phosphorylation sites. Recently, the Phos-tag technology has been developed to overcome the disadvantages and limitations of these methods. Phos-tag and its derivatives conjugated to biotin, acrylamide, or agarose, and can capture phosphate monoester dianions bound to serine, threonine, and tyrosine residues, in an amino acid sequence-independent manner. The grouping of the Phos-tag will alter the mobility of protein on the gel depending on the amount of serine, threonine or tyrosine which are phosphorylated. Here, we describe the method to detect the phosphorylation of Pop2 protein, one of the exonucleases in the Ccr4-Not complex regulating the shortening of poly(A) tail of mRNAs using phosphate affinity Phos-tag SDS-PAGE. We observed clear electrophoretic 04 shift bands of Pop2-3XFlag under unstressed conditions. This is the first study which observes Pop2 phosphorylation in normal culture conditions. This study showed the convenience and advantages of Phos-tag SDS-PAGE for research on molecular mechanisms regulating the function of protein.

Altered Phosphorylation of Cytoskeleton Proteins in Peripheral Blood Mononuclear Cells Characterizes Chronic Antibody-Mediated Rejection in Kidney Transplantation.

Chronic antibody-mediated rejection (CAMR) is the major cause of kidney transplant failure. The molecular mechanisms underlying this event are still poorly defined and this lack of knowledge deeply influences the potential therapeutic strategies. The aim of our study was to analyze the phosphoproteome of peripheral blood mononuclear cells (PBMCs), to identify cellular signaling networks differentially activated in CAMR. Phosphoproteins isolated from PBMCs of biopsy proven CAMR, kidney transplant recipients with normal graft function and histology and healthy immunocompetent individuals, have been investigated by proteomic analysis. Phosphoproteomic results were confirmed by Western blot and PBMCs’ confocal microscopy analyses. Overall, 38 PBMCs samples were analyzed. A differential analysis of PBMCs’ phosphoproteomes revealed an increase of lactotransferrin, actin-related protein 2 (ARPC2) and calgranulin-B in antibody-mediated rejection patients, compared to controls. Increased expression of phosphorylated ARPC2 and its correlation to F-actin filaments were confirmed in CAMR patients. Our results are the first evidence of altered cytoskeleton organization in circulating immune cells of CAMR patients. The increased expression of phosphorylated ARPC2 found in the PBMCs of our patients, and its association with derangement of F-actin filaments, might suggest that proteins regulating actin dynamics in immune cells could be involved in the mechanism of CAMR of kidney grafts.

Abstract 4143: Identification of molecular targets for conservative management of early stage, low-grade endometrial cancer through compartment-specific transcriptome profiling

Abstract Understanding tumor microenvironment is critical to improving conservative management (non-surgical) strategies for early stage, low grade endometrial cancer (EC). This remains an unmet clinical need for women who wish to maintain fertility and those who cannot undergo surgery due to medical co-morbidities. Current regimens rely on progestin treatment, but resistance and early relapse require improved therapeutic strategies. We sought to evaluate alterations in gene expression in the epithelial and stromal compartment of EC compared to normal endometrium to identify targets for combination therapies. RNA was extracted from microdissected epithelial and stromal compartments of formalin fixed paraffin embedded early stage, low grade EC and normal endometrium (NE). Comprehensive transcriptome profiling was performed with Affymetrix Clariom D assay and pathway analysis was performed. Immunohistochemistry (IHC) was used for validation. Functional studies of differentially expressed genes were performed in EC cell lines, ECC-1 and Hec1A. Focusing on differentially expressed genes in the epithelial compartment of EC vs NE, multiple activated signaling pathways were identified, including eIF2 signaling, eIF4 and p70S6K signaling, PI3K/AKT/mTOR, and hormone signaling. Top gene expression changes with significantly lower expression in EC included RBPMS (RNA-binding protein with multiple splicing), SORD (sorbitol dehydrogenase), DDX17 (DEAD-box helicase 17), and TPT1 (translationally-controlled tumor protein) gene expression, while MTDH (metadherin) was significantly increased in EC. Differential expression of proteins coded by these genes was confirmed by IHC. Additionally, RBPMS silencing enhanced AKT and S6 protein phosphorylation in Hec1A cells and forced expression of RBPMS in ECC-1 cells attenuated Erk1/2 MAP kinase activation. Expression of RBPMS inhibited cell proliferation and knockdown of RBPMS increased proliferation. Treatment of EC cells with everolimus (mTOR inhibitor) resulted in differential inhibition of proliferation based on RBPMS expression. Communication between tumor and stroma is also being evaluated. While estrogen-regulated changes were prominent in early stage low-grade EC, the discovery of alterations in other signaling pathways suggest additional targets for conservative management of the disease. The identification of signaling nodes shared between hormone-regulated pathways and mTOR signaling provides further justification for the ongoing combination therapy trial (progestin plus everolimus). Additionally, biomarkers from these pathways, such as RBPMS, can be studied to guide the selection of progestin-only therapy versus combination therapy. Citation Format: Qian Zhang, Xiaoping Su, Joseph Celestino, Ying Yuan, Samuel C. Mok, Karen H. Lu, Melinda S. Yates. Identification of molecular targets for conservative management of early stage, low-grade endometrial cancer through compartment-specific transcriptome profiling [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4143.

Abstract 2692: Novel chemotherapy stable subpopulations are conserved across multiple small cell lung carcinoma patient derived xenograft models

Abstract Introduction: Small cell lung cancer (SCLC) is an aggressive neuroendocrine carcinoma of the lung responsible for up to 25% of lung cancer deaths and the 6th leading cause of cancer death. SCLC initially responds well to chemotherapy, but inevitably recurs even after initial complete responses. The etiology of this relapse is likely secondary to tumor heterogeneity and/or chemotherapy resistance subpopulations reconstituting tumor. Characterization of these resistant subpopulations could yield novel therapeutic targets for SCLC treatment. Mass cytometry uses metal labeled antibodies to profile expression and phosphorylation of multiple proteins in a single cell and offers the opportunity to identify new subpopulations as targets for novel therapies in SCLC. Methods: Nude mice with SCLC patient derived xenografts (PDXs) were treated with a single cycle of carboplatin/etoposide or saline injection. PDX samples were stained with a 26-35 marker panel and an intercalator dye to identify nucleated cells. This panel measured phospho-signaling, neuroendocrine, immune, and mesenchymal cell markers, and functional markers including ki67 and cleaved caspase 3. Mouse cells, including leukocytes, were excluded using mouse MHC1 gating and Histone H3 was used to identify nucleated cells Single cell protein expression and phosphorylation was analyzed using viSNE, manual gating, as well as unsupervised clustering approaches with SPADE to identified multiple subpopulations with neuroendocrine and non-neuroendocrine features. Subpopulations were compared across multiple patient derived xenograft (PDX) models with and without chemotherapy treatment. Results: Patient derived Xenograft (PDX) tumors across 3 distinct models including with and without a single cycle of chemotherapy treatment released viable tumor and stromal cells suitable for cryopreservation and mass cytometry. Chemotherapy treated tumors had dramatic changes in subpopulation distribution compared to matched mock treated tumor. This included enrichment in EPCAM+, CD24+, CD44- progenitor like subpopulations. Similar patterns of population shift were observed in multiple models. Of note, chemotherapy stable subpopulations were conserved across 5 different PDX models including SOX2+ and Oct ¾+ tumor populations. These subpopulations sorted similarly in multidimensional space in multiple PDX models suggesting conserved origins. Conclusions: Mass cytometry was able to identify multiple NE and non-neuroendocrine cell populations from SCLC PDXs and characterize their signaling include rare subpopulations with stem like signaling factors of interest. Chemotherapy treated PDX had differential subpopulation distribution with enrichment of progenitor like cells with chemotherapy treatment similar to previous work in mouse genetic SCLC. However, rare conserved chemotherapy stable subpopulations enriched in stem-like signaling factors were identified across 5 PDX models including those with and without chemotherapy treatment. This work raises the possibility that chemotherapy stable subpopulations may contribute to progenitor populations which lead to relapse. Future work in this lab will focus on characterizing the stem like features of these subpopulations and their phenotypic dynamics and signaling. Citation Format: Jonathan M. Lehman, Jeremy Staub, Yong Zou, Jonathan Irish, Pierre Massion. Novel chemotherapy stable subpopulations are conserved across multiple small cell lung carcinoma patient derived xenograft models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2692.

Use of the Polo-like kinase 4 (PLK4) inhibitor centrinone to investigate intracellular signalling networks using SILAC-based phosphoproteomics.

Polo-like kinase 4 (PLK4) is the master regulator of centriole duplication in metazoan organisms. Catalytic activity and protein turnover of PLK4 are tightly coupled in human cells, since changes in PLK4 concentration and catalysis have profound effects on centriole duplication and supernumerary centrosomes, which are associated with aneuploidy and cancer. Recently, PLK4 has been targeted with a variety of small molecule kinase inhibitors exemplified by centrinone, which rapidly induces inhibitory effects on PLK4 and leads to on-target centrosome depletion. Despite this, relatively few PLK4 substrates have been identified unequivocally in human cells, and PLK4 signalling outside centriolar networks remains poorly characterised. We report an unbiased mass spectrometry (MS)-based quantitative analysis of cellular protein phosphorylation in stable PLK4-expressing U2OS human cells exposed to centrinone. PLK4 phosphorylation was itself sensitive to brief exposure to the compound, resulting in PLK4 stabilisation. Analysing asynchronous cell populations, we report hundreds of centrinone-regulated cellular phosphoproteins, including centrosomal and cell cycle proteins and a variety of likely ‘non-canonical’ substrates. Surprisingly, sequence interrogation of ∼300 significantly down-regulated phosphoproteins reveals an extensive network of centrinone-sensitive [Ser/Thr]Pro phosphorylation sequence motifs, which based on our analysis might be either direct or indirect targets of PLK4. In addition, we confirm that NMYC and PTPN12 are PLK4 substrates, both in vitro and in human cells. Our findings suggest that PLK4 catalytic output directly controls the phosphorylation of a diverse set of cellular proteins, including Pro-directed targets that are likely to be important in PLK4-mediated cell signalling.

Dickkopf-3 (DKK3) Signaling in IL-1α-Challenged Chondrocytes: Involvement of the NF-κB Pathway.

Osteoarthritis (OA) is an age-related biomechanical and low-grade inflammometabolic disease of the joints and one of the costliest and disabling forms of arthritis. Studies on matrix-degrading enzymes such as metalloproteases, which are implicated in the increased catabolism of extracellular matrix, are of paramount relevance. DKK3 is a member of DKK family and is best known for its role in cancer. Although there is some information about the participation of DKK3 in cartilage pathophysiology and on metalloproteases regulation, in particular, little is known about DKK3 signaling mechanisms. Thus, the aim of this study is to explore how DKK3 regulates matrix metalloproteinase-13 (MMP-13) expression. Gene, protein expression and protein phosphorylation in primary human chondrocytes and ATDC5 mouse cells were assessed by RT-qPCR and Western blot analysis. Further studies on DKK3 activity were performed by targeting DKK3 gene with a specific siRNA. DKK3 expression was found to be higher in OA human chondrocytes than healthy cells, being its expression decreased in interleukin-1α (IL-1α)-stimulated cells. DKK3 knockdown increased the induction of MMP-13 elicited by IL-1α in human and mouse chondrocytes and after the analysis of different signalling pathways, we observed that NF-κB pathway was involved in the regulation of MMP-13 expression by DKK3. Herein we have demonstrated, for the first time, that DKK3 gene silencing exacerbated NF-κB activation, resulting in an increased IL-1α-driven induction of MMP-13. Our results further confirm that DKK3 may play a protective role in OA by attenuating NF-κB activation and the subsequent production of metalloproteases.

Importance of CNOT8 Deadenylase Subunit in DNA Damage Responses Following Ionizing Radiation (IR).

The Ccr4-Not protein complex (CNOT complex) is a key regulator of gene expression in eukaryotic cells. Ccr4-Not Complex is composed of at least nine conserved subunits in mammalian cells with two main enzymatic activities. CNOT8 is a subunit of the complex with deadenylase activity that interacts transiently with the CNOT6 or CNOT6L subunits. Here, we focused on the role of the human CNOT8 subunit in the DNA damage response (DDR). Cell viability was assessed to measure ATP level using a Cell Titer-Glo Luminescence reagent up to 4 days' post CNOT8 siRNA transfection. In addition, expression level of phosphorylated proteins in signalling pathways were detected by western blotting and immunofluorescence microscopy. CNOT8- depleted Hela cells post- 3 Gy ionizing radiation (IR) treatment were considered as a control. Our results from cell viability assays indicated a significant reduction at 72-hour post CNOT8 siRNA transfection (p= 0.04). Western blot analysis showed slightly alteration in the phosphorylation of DNA damage response (DDR) proteins in CNOT8-depleted HeLa cells following treatment with ionizing radiation (IR). Increased foci formation of γH2AX, RPA, 53BP1, and RAD51 foci was observed after IR in CNOT8-depleted cells compared to the control cells. We conclude that CNOT8 deadenylase subunit is involved in the cellular response to DNA damage.

A Method of Assessment of Human Natural Killer Cell Phenotype and Function in Whole Blood.

The majority of data on human Natural Killer (NK) cell phenotype and function has been generated using cryopreserved peripheral blood mononuclear cells (PBMCs). However, cryopreservation can have adverse effects on PBMCs. In contrast, investigating immune cells in whole blood can reduce the time, volume of blood required, and potential artefacts associated with manipulation of the cells. Whole blood collected from healthy donors and cancer patients was processed by three separate protocols that can be used independently or in parallel to assess extracellular receptors, intracellular signaling protein phosphorylation, and intracellular and extracellular cytokine production in human NK cells. To assess extracellular receptor expression, 200 μL of whole blood was incubated with an extracellular staining (ECS) mix and cells were subsequently fixed and RBCs lysed prior to analysis. The phosphorylation status of signaling proteins was assessed in 500 μL of whole blood following co-incubation with interleukin (IL)-2/12 and an ECS mix for 20 min prior to cell fixation, RBC lysis, and subsequent permeabilization for staining with an intracellular staining (ICS) mix. Cytokine production (IFNγ) was similarly assessed by incubating 1 mL of whole blood with PMA-ionomycin or IL-2/12 prior to incubation with ECS and subsequent ICS antibodies. In addition, plasma was collected from stimulated samples prior to ECS for quantification of secreted IFNγ by ELISA. Results were consistent, despite inherent inter-patient variability. Although we did not investigate an exhaustive list of targets, this approach enabled quantification of representative ECS surface markers including activating (NKG2D and DNAM-1) and inhibitory (NKG2A, PD-1, TIGIT, and TIM-3) receptors, cytokine receptors (CD25, CD122, CD132, and CD212) and ICS markers associated with NK cell activation following stimulation, including signaling protein phosphorylation (p-STAT4, p-STAT5, p-p38 MAPK, p-S6) and IFNγ in both healthy donors and cancer patients. In addition, we compared extracellular receptor expression using whole blood vs. cryopreserved PBMCs and observed a significant difference in the expression of almost all receptors. The methods presented permit a relatively rapid parallel assessment of immune cell receptor expression, signaling protein activity, and cytokine production in a minimal volume of whole blood from both healthy donors and cancer patients.

Control of Platelet CLEC-2-Mediated Activation by Receptor Clustering and Tyrosine Kinase Signaling

Platelets are blood cells responsible for vascular integrity preservation. The activation of platelet receptor C-type lectin-like receptor II-type (CLEC-2) could partially mediate the latter function. Although this receptor is considered to be of importance for hemostasis, the rate-limiting steps of CLEC-2-induced platelet activation are not clear. Here, we aimed to investigate CLEC-2-induced platelet signal transduction using computational modeling in combination with experimental approaches. We developed a stochastic multicompartmental computational model of CLEC-2 signaling. The model described platelet activation beginning with CLEC-2 receptor clustering, followed by Syk and Src family kinase phosphorylation, determined by the cluster size. Active Syk mediated linker adaptor for T cell protein phosphorylation and membrane signalosome formation, which resulted in the activation of Bruton’s tyrosine kinase, phospholipase and phosphoinositide-3-kinase, calcium, and phosphoinositide signaling. The model parameters were assessed from published experimental data. Flow cytometry, total internal reflection fluorescence and confocal microscopy, and western blotting quantification of the protein phosphorylation were used for the assessment of the experimental dynamics of CLEC-2-induced platelet activation. Analysis of the model revealed that the CLEC-2 receptor clustering leading to the membrane-based signalosome formation is a critical element required for the accurate description of the experimental data. Both receptor clustering and signalosome formation are among the rate-limiting steps of CLEC-2-mediated platelet activation. In agreement with these predictions, the CLEC-2-induced platelet activation, but not activation mediated by G-protein-coupled receptors, was strongly dependent on temperature conditions and cholesterol depletion. Besides, the model predicted that CLEC-2-induced platelet activation results in cytosolic calcium spiking, which was confirmed by single-platelet total internal reflection fluorescence microscopy imaging. Our results suggest a refined picture of the platelet signal transduction network associated with CLEC-2. We show that tyrosine kinase activation is not the only rate-limiting step in CLEC-2-induced activation of platelets. Translocation of receptor-agonist complexes to the signaling region and linker adaptor for T cell signalosome formation in this region are limiting CLEC-2-induced activation as well.

Bayesian integrative analysis of epigenomic and transcriptomic data identifies Alzheimer's disease candidate genes and networks.

Biomedical research studies have generated large multi-omic datasets to study complex diseases like Alzheimer’s disease (AD). An important aim of these studies is the identification of candidate genes that demonstrate congruent disease-related alterations across the different data types measured by the study. We developed a new method to detect such candidate genes in large multi-omic case-control studies that measure multiple data types in the same set of samples. The method is based on a gene-centric integrative coefficient quantifying to what degree consistent differences are observed in the different data types. For statistical inference, a Bayesian hierarchical model is used to study the distribution of the integrative coefficient. The model employs a conditional autoregressive prior to integrate a functional gene network and to share information between genes known to be functionally related. We applied the method to an AD dataset consisting of histone acetylation, DNA methylation, and RNA transcription data from human cortical tissue samples of 233 subjects, and we detected 816 genes with consistent differences between persons with AD and controls. The findings were validated in protein data and in RNA transcription data from two independent AD studies. Finally, we found three subnetworks of jointly dysregulated genes within the functional gene network which capture three distinct biological processes: myeloid cell differentiation, protein phosphorylation and synaptic signaling. Further investigation of the myeloid network indicated an upregulation of this network in early stages of AD prior to accumulation of hyperphosphorylated tau and suggested that increased CSF1 transcription in astrocytes may contribute to microglial activation in AD. Thus, we developed a method that integrates multiple data types and external knowledge of gene function to detect candidate genes, applied the method to an AD dataset, and identified several disease-related genes and processes demonstrating the usefulness of the integrative approach.

High glucose induces phosphorylation and oxidation of mitochondrial proteins in renal tubular cells: A proteomics approach

Mitochondrial dysfunction has been thought to play roles in the pathogenesis of diabetic nephropathy (DN). However, precise mechanisms underlying mitochondrial dysfunction in DN remained unclear. Herein, mitochondria were isolated from renal tubular cells after exposure to normal glucose (5.5 mM glucose), high glucose (25 mM glucose), or osmotic control (5.5 mM glucose + 19.5 mM mannitol) for 96 h. Comparative proteomic analysis revealed six differentially expressed proteins among groups that were subsequently identified by tandem mass spectrometry (nanoLC-ESI-ETD MS/MS) and confirmed by Western blotting. Several various types of post-translational modifications (PTMs) were identified in all of these identified proteins. Interestingly, phosphorylation and oxidation were most abundant in mitochondrial proteins whose levels were exclusively increased in high glucose condition. The high glucose-induced increases in phosphorylation and oxidation of mitochondrial proteins were successfully confirmed by various assays including MS/MS analyses. Moreover, high glucose also increased levels of phosphorylated ezrin, intracellular ATP and ROS, all of which could be abolished by a p38 MAPK inhibitor (SB239063), implicating a role of p38 MAPK-mediated phosphorylation in high glucose-induced mitochondrial dysfunction. These data indicate that phosphorylation and oxidation of mitochondrial proteins are, at least in part, involved in mitochondrial dysfunction in renal tubular cells during DN.

Inflammation‐induced Extracellular Vesicles Cause Endothelial Barrier Injury

Extracellular vesicles (EVs) are a heterogenous population of sub‐micron sized vesicles released by cells during physiological or pathological stimulation. Circulating vesicles not only serve as important diagnostic/prognostic biomarkers, but also interact with blood or vascular cells and regulate multiple cellular processes by transferring bioactive cargo. In this study, we characterized EV production and cargo signature in the blood of healthy human subjects and burn patients, as well as in cultured human endothelial cells (ECs) after inflammatory stimulation. We also examined the effects of EVs on endothelial barrier structure and function. The results showed that the plasma level of EVs was increased during sterile inflammation in burn patients, with a significant portion of them derived from leukocytes and the endothelium. Interestingly, the EVs from burn patients expressed higher levels of c‐Src, protein kinase C and focal adhesion kinase, compared to EVs from healthy blood donors. In cultured ECs treated with tumor necrosis factor alpha, there was an increased production of EVs into the culture media. Furthermore, treating endothelial cells with EVs induced myosin light chain and VE‐cadherin phosphorylation and actin stress fiber formation in concomitance with adherens junction discontinuity. Functionally, EV treatment caused an increase in albumin flux across endothelial monolayers in a concentration‐dependent manner. EVs derived from c‐Src knockdown parent ECs displayed an attenuated effect on barrier protein phosphorylation and permeability. Likewise, pharmacological inhibition of Src activity reduced EV‐induced protein phosphorylation and barrier dysfunction in endothelial cells. Together, the data suggest that EVs produced during inflammation carry protein kinases capable of damaging the endothelial barrier.Support or Funding InformationNIH grants HL126646, HL070752 (to SYY), and GM097270. The human blood study was approved by the University of South Florida IRB and supported by the institutional research funds.

Garcinol Alone and in Combination With Cisplatin Affect Cellular Behavior and PI3K/AKT Protein Phosphorylation in Human Ovarian Cancer Cells.

Garcinol is a plant-derived compound that has some physiological benefits to human cells. However, the effect of garcinol on ovarian cancer (OC) cell proliferation and apoptosis is unknown. The current study aimed to examine the effects of garcinol alone and in combination with cisplatin (DDP) on cellular behavior and to explore the expression pattern of PI3K/AKT and nuclear factor-κB (NF-κB) in human OC cells. We found that OVCAR-3 cell viability was decreased after garcinol treatment. Garcinol alone and in combination with DDP significantly inhibited cell proliferation and had a synergistic effect evaluated by CompuSyn software. The cell cycle analysis showed the S phase arrest by garcinol. Furthermore, garcinol alone and in combination with DDP promoted cell apoptosis. The garcinol-induced apoptosis was further confirmed by the detection of cleavage forms of PARP and caspase 3. An increase in proapoptotic factor Bax expression was also found in garcinol-treated cells. Moreover, garcinol significantly decreased the phosphorylation of PI3K and AKT proteins and downregulated the expression of NF-κB. Thus, our data demonstrated that garcinol has the potential to be used as an anticancer agent and may synergize the effect of DDP. These actions are most likely through the regulation of the PI3K/AKT and NF-κB pathways.

Abstract TP276: Identification of Potential Key Genes and Pathways in Ischemic Stroke by Analysis of Human High Throughput Sequencing Data

Background and Purpose: Ischemic stroke (IS) is one of the most common causes of human death worldwide, but the molecular mechanisms of IS are still unclear. To explore the key genes and pathways involved in the pathogenic process of IS, we did a bioinformatics analysis of high throughput sequencing data of IS for the first time, which compared the expression profiles of brain tissues in IS patients and health controls. Methods: The dataset SRP040622 analyzed of expression profiling in 13 samples by deep sequencing in the platform of Illumina HiSeq 2000, including seven cortical ischemic stroke tissues and six control cortex tissues. These data from SRP040622 were downloaded from Sequence Read Archive (SRA) by using Prefech. We adopted fastq-dump, Hisat2, samtools, HTseq and DEseq2 to get the quantification of gene expression from raw data. The differentially expressed genes (DEGs) were identified by limma package, and function enrichment analyses were performed in DAVID website. In addition, the protein-protein interaction (PPI) network was constructed by using STRING and Cytoscape. Results: A total 880 DEGs were identified, including 350 up-regulated genes and 530 down-regulated genes. These DEGs enriched in biological processes(BP) mainly associated with signal transduction, multicellular organism development, positive regulation of cell proliferation, protein phosphorylation, cell-cell adhesion and angiogenesis. And KEGG pathway which DEGS were mainly enriched in is PI3K-Akt signaling pathway, Focal adhesion and Ras signaling pathway. In addition, 34 hub genes were selected by using CytoHubba with degrees >10. Conclusions: Our data reflects that PI3K-Akt signaling pathway, Focal adhesion and Ras signaling pathway may play significant roles in IS, and the hub genes among DEGs(including KDR, ESR1, MMP2, CAV1, PTK2, VWF) may be the potential targets for diagnosis and treatment of IS.

The Role of Neural Precursor Cell Expressed Developmentally Down-Regulated Protein 4.1- Mediated Insulin-Like Growth Factor-1R? Ubiquitination in the Pathogenesis of Alzheimer?s disease

To study the role of Neural precursor cell expressed developmentally down-regulated protein 4.1 on ubiquitination of central Insulin-like growth factor 1β pathway in Alzheimer’s Disease, and to clarify the molecular mechanism of Neural precursor cell expressed developmentally down-regulated protein 4.1 and Insulin-like growth factor 1β for central insulin signalling pathway damage. Mouse hippocampal neurons cells were isolated from Intracerebral haemorrhage mice. 30 μM or 300 μM S-Nitroso-l-cysteine were incubated with hippocampal neurons cells for 4 h. pCMV-Neural precursor cell expressed developmentally down-regulated protein 4.1 or siR- Neural precursor cell expressed developmentally down-regulated protein 4.1 were synthesized and transfected into S-Nitroso-l-cysteine treated hippocampal neurons cells. Cycloheximide and MG132 were incubated with hippocampal neuron cells after S-Nitroso-l-cysteine stimulation. Insulin resistant brain state model were constructed by streptozotocin injection. Western blot were used to test the expression of neural precursor cell expressed developmentally down-regulated protein 4.1, Insulin-like growth factor-1Rβ, Protein kinase B and p- Protein kinase B, Glycogen synthase kinase 3β and p- Glycogen synthase kinase 3β, Tau protein and p-Tau protein in vitro and in vivo. Coimmunoprecipitation were used to analyse Neural precursor cell expressed developmentally downregulated protein 4.1 and Insulin-like growth factor-1Rβ. Neural precursor cell expressed developmentally down-regulated protein 4.1 in the hippocampus of Tg2576 mice increased significantly compared to the control mice (p<0.05), while the expression of Insulin-like growth factor-1Rβ decreased significantly compared to the control mice (p<0.05). S-Nitroso-l-cysteine stimulated hippocampal neuron cells to increase the ubiquitination of Insulin-like growth factor-1Rβ (p<0.05) and decrease the expression of Insulin-like growth factor-1Rβ (p<0.05). Down-regulation of Neural precursor cell expressed developmentally downregulated protein 4.1 can decreased the ubiquitination of Insulin-like growth factor-1Rβ significantly (p<0.05), and rescued the S-Nitroso-l-cysteine inhibition effect on expression of Insulin-like growth factor-1Rβ. Over-expression Neural precursor cell expressed developmentally down-regulated protein 4.1 could increase the ubiquitination level of Insulin-like growth factor-1Rβ (p<0.05). MG132 and cyclohexide both can reverse the degradation of Insulin-like growth factor-1Rβ induced by S-Nitroso-l-cysteine. The phosphorylation degree of Protein kinase B and Glycogen synthase kinase 3β in neurons cells treated with S-Nitroso-l-cysteine decreased significantly (p<0.05), while Tau phosphorylation level in hippocampal neuron cells treated with Insulin-like growth factor-1 increased significantly (p<0.05). S-Nitroso-lcysteine can improve Tau protein phosphorylation (p<0.05) in neurons cells. C1060 site is necessary for its ubiquitination and degradation. Insulin-like growth factor-1Rβ were significantly lower (p<0.05) in the Streptozotocin model while Neural precursor cell expressed developmentally down-regulated protein 4.1 and Aβ were significantly higher (p<0.05) in the Streptozotocin model. p- Protein kinase B/Protein kinase B and p- Glycogen synthase kinase 3β/Glycogen synthase kinase 3β in the Streptozotocin dementia rats were significantly lower (p<0.05), and p-Tau/Tau were significantly increased (p<0.05). Abnormal increasing neural precursor cell expressed developmentally down-regulated protein 4.1 in brain injury tissue induced the rising ubiquitination of Insulin-like growth factor-1Rβ and rising of Aβ amount and Tau protein phosphorylation. C1060 site in Insulin-like growth factor-1Rβ was necessary for the Neural precursor cell expressed developmentally down-regulated protein 4.1 of ubiquitination and degradation of Insulin-like growth factor-1Rβ. In addition, Neural precursor cell expressed developmentally down- regulated protein 4.1 affects Insulin-like growth factor-1 signal pathway in brain, such as inhibiting Protein kinase B and Glycogen synthase kinase 3β phosphorylation, promoting Tau protein phosphorylation, and promoting β-amyloid secretions. The Role of Neural Precursor Cell Expressed Developmentally Down-Regulated Protein 4.1- Mediated Insulin-Like Growth Factor-1Rβ Ubiquitination in the Pathogenesis of Alzheimer’s disease

Comparative Advantages and Limitations of Quantum Dots in Protein Array Applications.

Antibody microarrays have become a powerful tool in multiplexed immunoassay technologies. The advantage of microarray technology is the possibility of rapid analysis of multiple targets in a single sample with a high sensitivity, which makes them ideal for high throughput screening. Usually these microarrays contain biological recognition molecules, such as full-size antibodies, antigen-binding fragments, and single-domain antibodies, and a label for detection. Organic fluorophores are the most popular labels, but they suffer from low sensitivity and instability due to their photodegradation. Here, we describe a protocol for fabricating an antibody microarray with highly fluorescent semiconductor nanocrystals or quantum dots (QDs) as the source of fluorescent signals, which may significantly improve the properties of microarrays, including their sensitivity and specificity. Our approach to analyte detection is based on the use of sandwich approach with streptavidin-biotin to assess and monitor the fluorescence signal instead of direct labeling of samples, which helps improve the reproducibility of results and sensitivity of the microarrays. The antibody microarray developed has been tested for its capacity of detecting DNA-PKcs in glial cell lines and measuring cell protein phosphorylation changes caused by camptothecin-induced DNA damage with different protein kinase inhibitors in HeLa cells.

Anti-Inflammatory Activity of the Active Compounds of Sanguisorbae Radix In Macrophages and in Vivo Toxicity Evaluation in Zebrafish

Sanguisorbae Radix (SR) is the root of the Sanguisorba officinalis L., a plant native to Asian countries and used in traditional medicine. We isolated the active components of SR and investigated their anti-inflammatory potential. Quercetin (QC), (+)-catechin (CC), and gallic acid (GA) were isolated from acetone extracts of SR. To elucidate the molecular mechanism by which these compounds suppress inflammation, we analyzed the transcriptional up-regulation of inflammatory mediators, such as nuclear factor-kappa B (NF-κB) and its target genes, inducible NOS (iNOS), and cyclooxygenase (COX)-2, in lipopolysaccharide (LPS)-stimulated macrophage RAW264.7 cells. Notably, QC, CC, and GA were found to inhibit the production of nitric oxide, tumor necrosis factor-alpha, and prostaglandin in a dose-dependent manner. Western blot results indicate that the compounds decreased the expression of iNOS and COX-2 proteins. Furthermore, the compounds decreased phosphorylation of IKK, IκB, ERK, p-38, and JNK proteins in LPS-induced cells. The results support the notion that QC, CC, and GA can potently inhibit the inflammatory response, with QC showing the highest anti-inflammatory activity. In in vivo toxicity studies in zebrafish (Danio rerio), QC showed no toxicity up to 25 μg/mL. Therefore, QC has non-toxic potential as a skin anti-inflammatory biomaterial.

Abstract GMM-053: ROLE OF LATS AND DYRK1A KINASES IN THE PATHOGENESIS AND TREATMENT OF HGSOC

Abstract Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy, with 75% of women presenting with metastatic disease at the time of diagnosis. High Grade Serous Ovarian Carcinoma (HGSOC) is the most common and aggressive type of epithelial ovarian cancer, with 5-year survival rate of 30% for most patients. Developing of accurate experimental models of HGSOC is necessary to elucidate the disease pathogenesis and to evaluate new treatments. Our study is aimed at determining the roles of the Large Tumor Suppressor 1 and 2 kinases (LATS1/2), and their downstream effector Dual-specificity tyrosine(Y)-Regulated Protein Kinase 1A (DYRK1A), in HGSOC pathogenesis and treatment sensitivity. The Cancer Genome Atlas study revealed copy number losses of the LATS1, LATS2 and DYRK1A genes in 65%, 59% and 38% of HGSOC cases, respectively. Previous studies found that LATS1/2 and DYRK1A mediate a crosstalk between the Hippo tumor suppressor pathway and the G0/G1 cell cycle checkpoint. However, the role of these kinases in HGSOC pathogenesis is not known. We found that loss of both LATS1 and LATS2, or DYRK1A results in increased proliferation of SKOV3 ovarian cancer cells in 2D or 3D cultures, mediated in part by disruption of the DREAM complex that represses cell cycle-regulated genes. Interestingly, we observed upregulation of cyclin D1 and increased phosphorylation of retinoblastoma (RB) family proteins in SKOV3 cells depleted of LATS1/2 or DYRK1A kinases, indicative of aberrant activation of CDK4/6. Similar changes were observed upon depletion of LATS1/2 or DYRK1A using non-transformed human fallopian epithelial cells, suggesting that genetic losses of these kinases could contribute to the HGSOC pathogenesis. Furthermore, we tested whether an aberrant activation of CDK4/6 in SKOV3 cells with low levels of LATS1/2 or DYRK1A could increase their sensitivity to pharmacological CDK4/6 inhibitors such as palbociclib. Using metabolic cell proliferation assays, we found that loss of LATS1/2 or DYRK1A resulted in a significant decrease of palbociclib IC50 in SKOV3 cells. Interestingly, palbociclib treatment increased the DREAM assembly in these cells, resulting in downregulation of DREAM-regulated cell cycle factors such as cyclin A or B-Myb. Our results show that loss of LATS1/2 or DYRK1A could contribute to HGSOC pathogenesis by CDK4/6-mediated disruption of RB family function. Further studies are underway to validate our findings in vivo using orthotopic ovarian tumor xenografts in mice and a bioluminescence imaging approach. In addition, these models will be used for validation of novel therapeutic approaches for HGSOC, such as palbociclib, as well as for the mechanistic studies of the EOC pathogenesis. Citation Format: Fatmata Sesay, Siddharth Saini, Larisa Litovchick. ROLE OF LATS AND DYRK1A KINASES IN THE PATHOGENESIS AND TREATMENT OF HGSOC [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-053.

Oncogenic Mechanisms of CBL E3 Ubiquitin Ligase Mutations in Myeloid Malignancies

CBL encodes an E3 ubiquitin ligase and signaling adaptor that acts downstream of cytokine receptors. Recurrent CBL mutations are found in a variety of myeloid disorders, including 10-15% of chronic myelomonocytic leukemia (CMML) cases, and specifically disrupt the protein's RING domain, which is responsible for E3 ligase activity; adaptor domains of CBL, including the tyrosine kinase-binding domain (TKB), proline-rich region (PRR) and C-terminal phosphotyrosine (pY) residues, remain intact in the context of RING mutations. In prior studies, CBL RING mutations were associated with hyperactivation of signaling pathways that drive cell proliferation. However, the precise mechanism by which CBL mutants act remains incompletely understood. Here we combined functional assays and mass spectrometry (MS) to comprehensively define the phosphoproteome, CBL interactome and molecular mechanism of signaling hyperactivation in a panel of cell lines expressing an allelic series of CBL RING mutants. We identified the SRC family kinase LYN as a key driver of signaling by CBL RING mutants; furthermore, we demonstrated in vitro and in vivo efficacy of LYN inhibition by dasatinib in CBL-mutant cell lines and primary CMML patient samples. We generated cell lines expressing wild-type (WT) or RING-mutant CBL using IL3-dependent mouse 32D cells and GM-CSF-dependent human TF1 cells. Cells expressing CBL RING mutants Y371H, C384Y or R420Q had a proliferative advantage over CBL WT or CBL knockout cells. To determine the role of CBL's adaptor domains in the proliferative advantage conferred by CBL RING mutants, we generated double mutants comprising the C384Y RING mutation in cis with mutations in the TKB domain (G306E), PRR (Δ477-688) or pY residues (Y700/731/774F). The proliferative advantage of cells expressing CBL C384Y was significantly reduced with mutation of the TKB domain, PRR or pY residues, indicating that CBL's adaptor domains are critical for the proliferative advantage of cells expressing RING-mutant CBL. To assess the effects of CBL RING mutation on signaling, we used MS to measure global protein phosphorylation in 32D cells expressing CBL WT or CBL C384Y. Activation of LYN and the PI3 kinase (PI3K) pathway were most significantly increased in cells expressing CBL C384Y compared to CBL WT; western blot confirmed increased phosphorylation of LYN, the PI3K p85 subunit and AKT in cells expressing CBL Y371H, C384Y or R420Q. We next employed immunoprecipitation (IP) followed by MS to characterize the global CBL interactome in 32D cells expressing CBL WT or RING mutants Y371H, C384Y or R420Q. In line with the phosphoproteomic analysis, LYN showed significantly increased binding to CBL RING mutants; the PI3K p85 subunit also showed increased binding to CBL RING mutants. Thus, global proteomic analyses revealed that increased binding of LYN and p85 to CBL RING mutants was directly associated with hyperactivation of LYN and PI3K-AKT signaling pathways. Deletion of CBL's PRR reduced interactions with both LYN and p85, and the CBL-p85 interaction required CBL Y731. Genetic ablation or inhibition of LYN by dasatinib decreased binding of p85 to CBL, suggesting that increased CBL Y731 phosphorylation by LYN enabled the CBL-p85 interaction. Indeed, CBL Y731 phosphorylation and AKT activation were diminished by deletion of CBL's PRR, LYN knockout or LYN inhibition by dasatinib. Altogether, these data demonstrated that enhanced LYN activation in cells expressing RING-mutant CBL drives increased CBL phosphorylation, p85 recruitment and downstream AKT signaling. Given the central role of LYN in signaling by CBL RING mutants, we hypothesized that LYN inhibition by dasatinib would abrogate the hyperproliferation of cells expressing CBL RING mutants. Dasatinib blocked the proliferative advantage of 32D and TF1 cells expressing CBL RING mutants. In addition, dasatinib significantly reduced the number of methylcellulose colonies formed by bone marrow mononuclear cells from 2 patients with CBL-mutated CMML; dasatinib treatment of mice xenografted with the same CMML cells resulted in a substantial decrease in leukemia burden compared to vehicle-treated mice. In summary, we have defined a mechanism by which LYN promotes PI3K-AKT signaling through CBL RING mutants. Our data provide rationale for exploring the therapeutic potential of LYN and/or PI3K-AKT inhibition in patients with CBL-mutated myeloid malignancies. Disclosures Ebert: Celgene: Research Funding; Deerfield: Research Funding.