Lymphocyte-specific Protein 1 Research Articles

Lymphocyte-Specific Protein 1 Regulates Expression and Stability of Endothelial Nitric Oxide Synthase.

Nitric oxide (NO), synthesized by endothelial nitric oxide synthase (eNOS), plays a critical role in blood pressure regulation. Genome-wide association studies have identified genetic susceptibility loci for hypertension in human lymphocyte-specific protein 1 (LSP1) gene. LSP1 is recognized as modulator of leukocyte extravasation, and endothelial permeability, however, the role of LSP1 in regulation of NO signaling within endothelial cells (ECs) remains unknown. The present study investigated the role of LSP1 in the regulation of eNOS expression and activity utilizing human macrovascular ECs in vitro and LSP1 knockout (KO) mice. In ECs, specific CRISPR-Cas9 genomic editing deleted LSP1 and caused downregulation of eNOS expression. LSP1 gain-of-function through adenovirus-mediated gene transfer was associated with enhanced expression of eNOS. Co-immunoprecipitation and confocal fluorescence microscopy revealed that eNOS and LSP1 formed a protein complex under basal conditions in ECs. Furthermore, LSP1 deficiency in mice promoted significant upregulation and instability of eNOS. Utilizing a mass-spectrometry-based bottom-up proteomics approach, we identified novel truncated forms of eNOS in immunoprecipitates from LSP1 KO aortae. Our experimental data suggest an important role of endothelial LSP1 in regulation of eNOS expression and activity within human ECs and murine vascular tissues.

Tim-3 Is Differentially Expressed during Cell Activation and Interacts with the LSP-1 Protein in Human Macrophages.

T-cell Immunoglobulin and Mucin Domain 3 (TIM-3) is an immune checkpoint receptor known to regulate T-cell activation and has been targeted for immunotherapy in cancer and other diseases. However, its expression and function in other cell types, such as macrophages, are poorly understood. This study investigated TIM-3 expression in human macrophages polarized to M1 (stimulated with IFN-γ and LPS) and M2 (stimulated with IL-4 and IL-13) phenotypes using an in vitro model. Our results show that M1 macrophages have a lower frequency of TIM-3+ cells compared to M2 macrophages at 48 and 72 hr poststimulation. Additionally, we observed differential levels of soluble ADAM 10, an enzyme responsible for TIM-3 release, in the supernatants of M1 and M2 macrophages at 72 hr. We also found that the TIM-3 intracellular tail might associate with lymphocyte-specific protein 1 (LSP-1), a protein implicated in cell motility and podosome formation. These findings enhance our understanding of TIM-3 function in myeloid cells such as macrophages and may inform the development of immunotherapies with reduced immune-related adverse effects.

Dawn-to-dusk dry fasting induces anti-atherosclerotic, anti-inflammatory, and anti-tumorigenic proteome in peripheral blood mononuclear cells in subjects with metabolic syndrome

BackgroundMetabolic syndrome characterized by abdominal obesity, high blood pressure, elevated fasting glucose and triglyceride levels and low high-density lipoprotein cholesterol level is associated with pro-inflammatory state, increased risk for atherosclerosis, and multiple cancers. Our previous results on subjects with metabolic syndrome showed that 4-week dawn-to-dusk (sunset) dry fasting resulted in significant changes in the serum proteome and improvement in several metabolic risk factors. Peripheral blood mononuclear cells (PBMC) proteomics is a powerful tool that can provide mechanistic insights into how dawn-to-dusk dry fasting affects protein expression in metabolic pathways at cellular level. In this study, we determined whether dawn-to-dusk dry fasting would induce favorable changes in PBMC proteome in subjects with metabolic syndrome, similar to the changes induced by dawn-to-dusk dry fasting in the same subjects' serum proteome. MethodsWe conducted a prospective study on subjects with metabolic syndrome and collected blood specimens before 4-week dawn-to-dusk dry fasting, at the end of 4-week dawn-to-dusk dry fasting, and one week after 4-week dawn-to-dusk dry fasting. We performed untargeted proteomics using nano ultra-high performance liquid chromatography-tandem mass spectrometry to assess the impact of 4-week dawn-to-dusk dry fasting on PBMC proteome. ResultsThere were 14 subjects with metabolic syndrome with a mean age of 59 who fasted from dawn to dusk (strict dry fasting without any liquid or food intake) for more than 14 h daily for 29 days. The quantitative proteome analysis showed that apolipoprotein B (APOB) gene protein products (GP) levels were downregulated and had the most statistical significance of the observed difference at the end of 4-week dawn-to-dusk dry fasting (P = 0.008) and one week after 4-week dawn-to-dusk dry fasting (P = 0.0004) compared with the levels before 4-week dawn-to-dusk dry fasting. The comparison between GP levels before and at the end of 4-week dawn-to-dusk dry fasting showed an alteration in the expression of genes associated with lipid and atherosclerosis pathway (P = 6.014e-4) and C-type lectin receptor signaling pathway (P = 1.064e-5). The genes that were differentially expressed in the lipid and atherosclerosis pathway were APOB (P = 0.008), CD36 (P = 0.040), CALM1, CALM2, CALM3 (P = 0.015), and HSPA8 (P = 0.047). One of the differentially expressed genes in the C-type lectin receptor signaling pathway was lymphocyte-specific protein 1 (LSP1), which showed an average of 19-fold increase at the end of 4-week dawn-to-dusk dry fasting compared with the GP levels before fasting (P = 0.004). Several GPs associated with tumor-suppressor effect (TUBB4B, LSP1, ACTR3B) were upregulated, and GPs associated with tumor-promoter effect (CD36, CALM1, CALM2, CALM3, FLOT2, PPIF) were downregulated at the end of 4-week dawn-to-dusk dry fasting or one week after 4-week dawn-to-dusk dry fasting compared with the GP levels before 4-week dawn-to-dusk dry fasting. ConclusionBased on our results, we conclude that in subjects with metabolic syndrome, 4-week dawn-to-dusk dry fasting induced anti-atherosclerotic, anti-inflammatory, and anti-tumorigenic PMBC proteome. Randomized, controlled clinical trials are needed to further investigate the effect of dawn-to-dusk dry fasting on subjects with chronic metabolic diseases and metabolic syndrome-induced cancers.

Cyclosporine A Modulates LSP1 Protein Levels in Human B Cells to Attenuate B Cell Migration at Low O2 Levels.

Coordinated migration of B cells within and between secondary lymphoid tissues is required for robust antibody responses to infection or vaccination. Secondary lymphoid tissues normally expose B cells to a low O (hypoxic) environment. Recently, we have shown that human B cell migration is modulated by an O-dependent molecular switch, centrally controlled by the hypoxia-induced (transcription) factor-1 (HIF1A), which can be disrupted by the immunosuppressive calcineurin inhibitor, cyclosporine A (CyA). However, the mechanisms by which low O environments attenuate B cell migration remain poorly defined. Proteomics analysis has linked CXCR4 chemokine receptor signaling to cytoskeletal rearrangement. We now hypothesize that the pathways linking the O sensing molecular switch to chemokine receptor signaling and cytoskeletal rearrangement would likely contain phosphorylation events, which are typically missed in traditional transcriptomic and/or proteomic analyses. Hence, we have performed a comprehensive phosphoproteomics analysis of human B cells treated with CyA after engagement of the chemokine receptor CXCR4 with CXCL12. Statistical analysis of the separate and synergistic effects of CyA and CXCL12 revealed 116 proteins whose abundance is driven by a synergistic interaction between CyA and CXCL12. Further, we used our previously described algorithm BONITA to reveal a critical role for Lymphocyte Specific Protein 1 (LSP1) in cytoskeletal rearrangement. LSP1 is known to modulate neutrophil migration. Validating these modeling results, we show experimentally that LSP1 levels in B cells increase with low O exposure, and CyA treatment results in decreased LSP1 protein levels. This correlates with the increased chemotactic activity observed after CyA treatment. Lastly, we directly link LSP1 levels to chemotactic capacity, as shRNA knock-down of LSP1 results in significantly increased B cell chemotaxis at low O levels. These results directly link CyA to LSP1-dependent cytoskeletal regulation, demonstrating a previously unrecognized mechanism by which CyA modulates human B cell migration. Data are available via ProteomeXchange with identifier PXD036167.

Finasteride induces Epigenetic Modulation of LSP1: A Gene implicated in Neutrophil Actin Dysfunction disease

Lymphocyte Specific Protein 1 (LSP1) gene translates F‐actin protein expressed in leucocytes such as neutrophils, lymphocytes, macrophages, and endothelium. It controls neutrophil motility, adhesion to fibrinogen matrix proteins, and transendothelial migration. Overexpression or increased level of Lymphocyte Specific Protein 1 (LSP1) is implicated in a rare immunologic disorder Neutrophil Actin Dysfunction characterized by spontaneous onset of recurrent infections and inflammation of oral cavity, skin, and respiratory tract. In this experiment aimed at studying the methylation profile and genes expression following finasteride treatment, we cultured human Leydig cells in the presence of 0.5 μM finasteride for 14 days and performed whole‐genome DNA methylation analysis using the NimbleGen Human DNA Methylation 3×720K Promoter Plus CpG Island Array and Ingenuity Pathway Analysis. Results identified several genes that were differentially methylated following finasteride treatment. Lymphocyte Specific Protein 1 (LSP1) gene was downregulated through methylation. We therefore deduced that finasteride could be a gateway to the treatment of Neutrophil Actin Dysfunction disease through epigenetic modification and decreased expression of LSP1 gene.

Lymphocyte-Specific Protein-1 Suppresses Xenobiotic-Induced Constitutive Androstane Receptor and Subsequent Yes-Associated Protein–Activated Hepatocyte Proliferation

Activation of constitutive androstane receptor (CAR) transcription factor by xenobiotics promotes hepatocellular proliferation, promotes hypertrophy without liver injury, and induces drug metabolism genes. Previous work demonstrated that lymphocyte-specific protein-1 (LSP1), an F-actin binding protein and gene involved in human hepatocellular carcinoma, suppresses hepatocellular proliferation after partial hepatectomy. The current study investigated the role of LSP1 in liver enlargement induced by chemical mitogens, a regenerative process independent of tissue loss. 1,4-Bis [2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP), a direct CAR ligand and strong chemical mitogen, was administered to global Lsp1 knockout and hepatocyte-specific Lsp1 transgenic (TG) mice and measured cell proliferation, hypertrophy, and expression of CAR-dependent drug metabolism genes. TG livers displayed a significant decrease in Ki-67 labeling and liver/body weight ratios compared with wild type on day 2. Surprisingly, this was reversed by day 5, due to hepatocyte hypertrophy. There was no difference in CAR-regulated drug metabolism genes between wild type and TG. TG livers displayed increased Yes-associated protein (YAP) phosphorylation, decreased nuclear YAP, and direct interaction between LSP1 and YAP, suggesting LSP1 suppresses TCPOBOP-driven hepatocellular proliferation, but not hepatocyte volume, through YAP. Conversely, loss of LSP1 led to increased hepatocellular proliferation on days 2, 5, and 7. LSP1 selectively suppresses CAR-induced hepatocellular proliferation, but not drug metabolism, through the interaction of LSP1 with YAP, supporting the role of LSP1 as a selective growth suppressor.

Identification of Diagnostic Signatures and Immune Cell Infiltration Characteristics in Rheumatoid Arthritis by Integrating Bioinformatic Analysis and Machine-Learning Strategies.

BackgroundRheumatoid arthritis (RA) refers to an autoimmune rheumatic disease that imposes a huge burden on patients and society. Early RA diagnosis is critical to preventing disease progression and selecting optimal therapeutic strategies more effectively. In the present study, the aim was at examining RA’s diagnostic signatures and the effect of immune cell infiltration in this pathology.MethodsGene Expression Omnibus (GEO) database provided three datasets of gene expressions. Firstly, this study adopted R software for identifying differentially expressed genes (DEGs) and conducting functional correlation analyses. Subsequently, we integrated bioinformatic analysis and machine-learning strategies for screening and determining RA’s diagnostic signatures and further verify by qRT-PCR. The diagnostic values were assessed through receiver operating characteristic (ROC) curves. Moreover, this study employed cell-type identification by estimating relative subsets of RNA transcript (CIBERSORT) website for assessing the inflammatory state of RA, and an investigation was conducted on the relationship of diagnostic signatures and infiltrating immune cells.ResultsOn the whole, 54 robust DEGs received the recognition. Lymphocyte-specific protein 1 (LSP1), Granulysin (GNLY), and Mesenchymal homobox 2 (MEOX2) (AUC = 0.955) were regarded as RA’s diagnostic markers and showed their statistically significant difference by qRT-PCR. As indicated from the immune cell infiltration analysis, resting NK cells, neutrophils, activated NK cells, T cells CD8, memory B cells, and M0 macrophages may be involved in the development of RA. Additionally, all diagnostic signatures might be different degrees of correlation with immune cells.ConclusionsIn conclusion, LSP1, GNLY, and MEOX2 are likely to be available in terms of diagnosing and treating RA, and the infiltration of immune cells mentioned above may critically impact RA development and occurrence.

Genetic basis and identification of candidate genes for wooden breast and white striping in commercial broiler chickens

Wooden breast (WB) and white striping (WS) are highly prevalent and economically damaging muscle disorders of modern commercial broiler chickens characterized respectively by palpable firmness and fatty white striations running parallel to the muscle fiber. High feed efficiency and rapid growth, especially of the breast muscle, are believed to contribute to development of such muscle defects; however, their etiology remains poorly understood. To gain insight into the genetic basis of these myopathies, a genome-wide association study was conducted using a commercial crossbred broiler population (n = 1193). Heritability was estimated at 0.5 for WB and WS with high genetic correlation between them (0.88). GWAS revealed 28 quantitative trait loci (QTL) on five chromosomes for WB and 6 QTL on one chromosome for WS, with the majority of QTL for both myopathies located in a ~ 8 Mb region of chromosome 5. This region has highly conserved synteny with a portion of human chromosome 11 containing a cluster of imprinted genes associated with growth and metabolic disorders such as type 2 diabetes and Beckwith-Wiedemann syndrome. Candidate genes include potassium voltage-gated channel subfamily Q member 1 (KCNQ1), involved in insulin secretion and cardiac electrical activity, lymphocyte-specific protein 1 (LSP1), involved in inflammation and immune response.

MiR-920 and LSP1 co-regulate the growth and migration of glioblastoma cells by modulation of JAK2/STAT5 pathway.

This study probes the function and mechanism of lymphocyte-specific protein 1 (LSP1) in glioblastoma pathogenesis. According to the data acquired from TCGA, Oncomine and GEO databases, the expression and prognostic value of LSP1 and miR-920 in glioblastoma patients were analyzed. The expression levels of LSP1 in U251 and A172 cell lines were analyzed by qRT-PCR and western blotting. CCK8, colony formation and transwell assays were utilized to test glioblastoma cell malignant abilities. Furthermore, the associations between LSP1 and miR-920 were indentified by bioinformatics analysis and rescue assays. Moreover, the protein expression levels of p-JAK2, JAK2, p-STAT5 and STAT5, as the hallmark of JAK/STAT5 signaling, were detected by western blotting. The observations showed that LSP1 was highly augmented in glioblastoma samples. Additionally, up-regulation of LSP1 was associated with a unfavorable prognosis in glioblastoma patients. Biological experiments revealed that depletion of LSP1 significantly suppressed the proliferation, invasion and migration of U251 and A172 cells. MiR-920, as an upstream regulator of LSP1, negatively modulated LSP1 expression and promoted U251 cells malignant behaviors after miR-920 inhibitor treatment. However, together knockdown LSP1 and miR-920 inhibited these effects. Moreover, the expression levels of p-JAK2 and p-STAT5 were increased or decreased in U251 cells after transfection of miR-920 inhibitor or si-LPS1. Taken together, miR-920 might blocked the malignant development of glioblastoma cells, which is possibly realized by targeting LSP1 and modulation of JAK/STAT5 pathway. These findings implied that miR-920/LSP1 was a potential therapeutic target for glioblastoma treatment.

Lymphocyte‐specific protein 1 (LSP1) regulates bone marrow stromal cell antigen 2 (BST‐2)‐mediated intracellular trafficking of HIV‐1 in dendritic cells

Human immunodeficiency virus type 1 (HIV-1) subverts intracellular trafficking pathways to avoid its degradation and elimination, thereby enhancing its survival and spread. The molecular mechanisms involved in intracellular transport of HIV-1 are not yet fully defined. We demonstrate that the actin-binding protein lymphocyte-specific protein 1 (LSP1) interacts with the interferon-inducible protein bone marrow stromal antigen 2 (BST-2) in dendritic cells (DCs) to facilitate both endocytosis of surface-bound HIV-1 and the formation of early endosomes. Analysis of the molecular interaction between LSP1 and BST-2 reveals that the N terminus of LSP1 interacts with BST-2. Overall, we identify a novel mechanism of intracellular trafficking of HIV-1 in DCs centering on the LSP1/BST-2 complex. We also show that the HIV-1 accessory protein Vpu subverts this pathway by inducing proteasomal degradation of LSP1, augmenting cell-cell transmission of HIV-1.

Lymphocyte Specific Protein‐1 suppresses TCPOBOP induced hepatocellular proliferation

Lymphocyte specific protein‐1 (LSP‐1), an f‐actin binding protein expressed in hematopoietic cells as well as hepatocytes, is the gene with the most copy number variations detected in human HCC. Our previous work has demonstrated that LSP‐1 suppresses hepatocellular proliferation following partial hepatectomy as well as in primary hepatocyte cultures. LSP‐1 acts as a scaffold for the Ras/MEK/ERK MAP kinase pathway and along with kinase suppressor of Ras (KSR) inhibits ERK phosphorylation and thus its activation preventing downstream signaling to induce cellular proliferation and migration. To determine if LSP‐1 functions to attenuate hepatocellular growth in models which induce proliferation without loss of tissue or liver injury, we aimed to explore if LSP‐1 can modulate TCPOBOP induced hepatocellular proliferation and hepatomegaly. TCPOBOP (1,4‐Bis [2‐(3,5‐Dichloropyridyloxy)] benzene), a constitutive androstane receptor (CAR) agonist, has been shown to induce hepatocellular hyperplasia through a CAR dependent mechanism. Since LSP‐1 has been shown to suppress hepatocyte proliferation during liver regeneration following partial hepatectomy and in culture, we hypothesized that LSP‐1 will attenuate TCPOBOP driven hepatocellular proliferation. In this study, we administered TCPOBOP to both a global LSP1 knockout mouse model as well as a hepatocyte specific LSP‐1 transgenic (TG) mouse model. Livers were harvested at various time point after TCPOBOP gavage and proliferation was measured using Ki67 and PCNA immunohistochemistry as well as liver to body weight ratios. Increased LSP‐1 expression in the transgenic livers caused decreased Ki67 staining on day 2 after TCPOBOP administration as well as no measurable change in liver to body weight ratios as compared to baseline. However, by day 5, the TG liver to body weight ratios increased above those of the WT but the percentage of Ki67 positive hepatocytes was decreased in the TG as compared to the wild type controls. Loss of LSP‐1 expression led to a statistically significant difference in the liver to body weight ratios between the KO and WT mice on day 2 after TCPOBOP administration as well as increased hepatocellular proliferation on both days 2 and 5 after gavage in the KO livers. CAR target genes and downstream signaling pathways that are modulated by LSP‐1 expression will be analyzed.ConclusionLSP‐1 attenuates TCPOBOP induced hepatocellular proliferation and loss of expression leads to increased hyperplasia supporting the role of LSP‐1 as a growth suppressor.Support or Funding InformationSupported by the Department of Defense grant CA160119, the University of Pittsburgh Menten Endowment, and the Cleveland Foundation Morningstar Fund.

Multi-omic profiling reveals associations between the gut mucosal microbiome, the metabolome, and host DNA methylation associated gene expression in patients with colorectal cancer

BackgroundThe human gut microbiome plays a critical role in the carcinogenesis of colorectal cancer (CRC). However, a comprehensive analysis of the interaction between the host and microbiome is still lacking.ResultsWe found correlations between the change in abundance of microbial taxa, butyrate-related colonic metabolites, and methylation-associated host gene expression in colonic tumour mucosa tissues compared with the adjacent normal mucosa tissues. The increase of genus Fusobacterium abundance was correlated with a decrease in the level of 4-hydroxybutyric acid (4-HB) and expression of immune-related peptidase inhibitor 16 (PI16), Fc Receptor Like A (FCRLA) and Lymphocyte Specific Protein 1 (LSP1). The decrease in the abundance of another potentially 4-HB-associated genus, Prevotella 2, was also found to be correlated with the down-regulated expression of metallothionein 1 M (MT1M). Additionally, the increase of glutamic acid-related family Halomonadaceae was correlated with the decreased expression of reelin (RELN). The decreased abundance of genus Paeniclostridium and genus Enterococcus were correlated with increased lactic acid level, and were also linked to the expression change of Phospholipase C Beta 1 (PLCB1) and Immunoglobulin Superfamily Member 9 (IGSF9) respectively. Interestingly, 4-HB, glutamic acid and lactic acid are all butyrate precursors, which may modify gene expression by epigenetic regulation such as DNA methylation.ConclusionsOur study identified associations between previously reported CRC-related microbial taxa, butyrate-related metabolites and DNA methylation-associated gene expression in tumour and normal colonic mucosa tissues from CRC patients, which uncovered a possible mechanism of the role of microbiome in the carcinogenesis of CRC. In addition, these findings offer insight into potential new biomarkers, therapeutic and/or prevention strategies for CRC.

Elevated lymphocyte specific protein 1 expression is involved in the regulation of leukocyte migration and immunosuppressive microenvironment in glioblastoma.

Immune cell infiltration mediates therapeutic response to immune therapies. The investigation on the genes regulating leukocyte migration may help us to understand the mechanisms regulating immune cell infiltration in tumor microenvironment. Here, we collected the data from Chinese Glioma Genome Atlas (CGGA) and The Cancer Genome Atlas (TCGA) to analyze the expression of leukocyte migration related genes in glioblastoma (GBM). Lymphocyte specific protein 1 (LSP1) was identified as the only gene in this family which not only has an elevated expression, but also serve as an independent predictive factor for progressive malignancy in glioma. We further confirmed these results in clinical glioma samples by quantitative PCR, immunofluorescence, immunohistochemistry, and western blot. Moreover, LSP1 expression was closely related to the response to radio- and chemotherapy in GBM, and positively correlated with immunosuppressive cell populations, including M2 macrophages, neutrophil, and regulatory T cell. Additionally, elevated LSP-1 expression enhanced the expression of immunosuppression related genes like programmed cell death 1 (PD1) and leukocyte associated immunoglobulin like receptor 1 (LAIR1) in macrophages. LSP1 also promoted the migration of macrophages. Together, our study suggests a novel role of LSP1 contributing to immunosuppressive microenvironment in GBM and serving as a potential therapeutic target for it.

Identification and analysis of pathogenic nsSNPs in human LSP1 gene.

LSP1 (Lymphocyte-specific protein 1) protein plays an important role in neutrophil motility, fibrinogen matrix proteins adhesion, and trans-endothelial migration. Variation in the LSP1 gene is associated with leukemia and lymphomas in tumor cells of Hodgkin's disease and breast cancer. Despite extensive study on the human LSP1, a comprehensive analysis on the Single Nucleotide Polymorphism (SNPs) of the gene is not available. Therefore, it is of interest to identify, collect, store and analyze the SNPs of the LSP1 gene in relation to several known diseases. Hence, the SNP data (398 rsids) from dbSNP database was downloaded and mapped to the genomic coordinate of "NM_002339.2" transcript expressed by LSP1 (P33241). There were 300 nsSNPs with missense mutation in the dataset. Tools such as SIFT, PROVEAN, Condel, and PolyPhen-2 were further used to identify 29 highly deleterious or damaging on synonymous SNP (nsSNPs) for LSP1. These high confident damaging nsSNPs were further analyzed for disease association using SNPs and GO tool. SNPs of the gene such as nsSNPs C283R, G234R, Y328D and H325P showed disease association with high prevalence.

A novel scoring system for acute myeloid leukemia risk assessment based on the expression levels of six genes.

Acute myeloid leukemia (AML) is the most common type of acute leukemia and is a heterogeneous clonal disorder. At present, the pathogenesis of AML and potential methods to effectively prevent AML have become areas of interest in research. In the present study, two messenger ribonucleic acid sequencing datasets of patients with AML were downloaded from the Cancer Genome Atlas and Gene Expression Omnibus databases. The differentially expressed genes (DEGs) of the poor and good prognosis groups were screened using the Linear Models for Microarray Data package, and the prognosis-related genes were screened using univariate Cox regression analysis. A total of 206 significant DEGs were identified. Following univariate and multivariate Cox regression analysis, 14 genes significantly associated with prognosis were screened and six of these genes, including triggering receptor expressed on myeloid cells 2 (TREML2), cysteine-glutamate transporter (SLC7A11), NACHT, LRR, and PYD domains-containing protein 2 (NLRP2), DNA damage-inducible transcript 4 protein (DDIT4), lymphocyte‑specific protein 1 (LSP1) and C-type lectin domain family 11 member A (CLEC11A), were used to construct model equations for risk assessment. The prognostic scoring system was used to evaluate risk for each patient, and the results showed that patients in the low-risk group had a longer survival time, compared with those in the high-risk group (P=9.59e-06 for the training dataset and P=0.00543 for the validation dataset). A total of eight main Kyoto Encyclopedia of Genes and Genomes pathways were identified, the top three of which were hematopoietic cell lineage, focal adhesion, and regulation of actin cytoskeleton. Taken together, the results showed that the scoring system established in the present study was credible and that the six genes were identified, which were significantly associated with the risk assessment of AML, offer potential as prognostic biomarkers. These findings may provide clues for further clarifying the pathogenesis of AML.

Genetic Breast Cancer Susceptibility Variants and Prognosis in the Prospectively Randomized SUCCESS A Study.

Large-scale genotyping studies have identified over 70 single nucleotide polymorphisms (SNPs) associated with breast cancer (BC) risk. However, knowledge regarding genetic risk factors associated with the prognosis is limited. The aim of this study was therefore to investigate the prognostic effect of nine known breast cancer risk SNPs. BC patients (n = 1687) randomly sampled in an adjuvant, randomized phase III trial (SUCCESS A study) were genotyped for nine BC risk SNPs: rs17468277 (CASP8) , rs2981582 (FGFR2) , rs13281615(8q24), rs3817198 (LSP1) , rs889312 (MAP3K1) , rs3803662 (TOX3) , rs13387042(2q35), rs4973768 (SLC4A7) , rs6504950 (COX11) . Cox proportional hazards models were used to test the SNPs' association with overall survival (OS) and progression-free survival (PFS). Additional analyses were carried out for molecular subgroups. rs3817198 in LSP1 (lymphocyte-specific protein 1) was the only SNP that significantly influenced OS (p = 0.01) and PFS (p < 0.01) in the likelihood ratio test comparing the genetic survival model with the clinical survival model. In the molecular subgroups, triple-negative patients with two minor alleles in rs3817198 had a much better prognosis relative to OS (adjusted HR 0.03; 95% CI 0.002 - 0.279) and PFS (HR 0.09; 95% CI 0.02 - 0.36) than patients with the common alleles. The same effect on PFS was shown for patients with luminal A tumors (HR 0.19; 95% CI 0.05 - 0.84), whereas patients with luminal B tumors had a poorer PFS with two minor alleles (HR 2.13; 95% CI 1.02 - 4.40). The variant in rs3817198 has a prognostic effect particularly in the subgroup of patients with triple-negative BC, suggesting a possible link with immunomodulation and BC.

Abstract 237: The Role of Lymphocyte Specific Protein-1 in the Phenotypic Switch of Smooth Muscle Cells After Arterial Injury

Background: After vascular injury, vascular smooth muscle cells (SMCs) switch from a differentiated contractile state to synthetic de-differentiated phenotype which contributes to the pathophysiology of restenosis. Experimental data generated by our lab indicate that TGF-β downregulates contractile proteins and stimulates migration. To understand how TGF-β promotes SMC phenotypic switch in injured arteries, we performed an Affymetrix Array analysis and identified Lymphocyte Specific Protein-1 (LSP1) among other upregulated genes. LSP1 is known to play a role in neutrophil extravasation, however the role of LSP1 within SMCs is unknown. We hypothesize that LSP1 contributes to SMC pathophysiological behavior through changes in cell architecture and migration in-vivo and in-vitro. Methods and Results: After carotid artery angioplasty, male Sprague-Dawley rats were sacrificed at 3, 7, and 14 days after injury for immunohistochemistry. Immunofluorescence staining revealed a unique upregulation of LSP1 within the neointima, media, and adventitia at 7 and 14 days, but not at 3 days after injury. Confocal images revealed that the LSP1 positive cells minimally express α-SMA (Pierson’s Coefficient, r=.017). Additional characterization experiments using immune cell markers CD3 and CD45 show no co-localization with LSP1 positive cells. To mimic the in-vivo neointimal cells and vascular injury induced de-differentiation in-vitro , rat A10 cells were treated with solvent or PDGF-bb (10 ng/mL). Quantitative RT-PCR demonstrated an upregulation of LSP1 mRNA after 24 hrs of PDGF-BB stimulation. Using Western Blotting, we confirm an upregulation of LSP1 protein after 48 hrs of PDGF-BB stimulation. Lastly, we performed nuclear and cytoplasmic fractionation followed by Western Blotting which demonstrated that LSP1 is remained within cytoplasmic fraction of the A10 cell after treatment with PDGF-BB. Conclusion: These results demonstrate that LSP1 is increased in-vivo after balloon injury, and in-vitro after PDGF-BB stimulation. Experiments to characterize the identity of these LSP1 cells in-vivo are in process, with future in-vitro experiments to focus on the role of LSP1 phosphorylation as a part of cytoskeletal remodeling and cellular migration.

Lymphocyte-specific protein 1 inhibits the growth of hepatocellular carcinoma by suppressing ERK1/2 phosphorylation.

Lymphocyte‐specific protein 1 (LSP1) has been reported to regulate cell biology in several human cancers including lymphoma and breast cancer. However, the functions of LSP1 in human hepatocellular carcinoma (HCC) are still unknown. In this study, we found that LSP1 expression was downregulated in HCC tissues and cell lines, and lower LSP1 expression was correlated with poor clinicopathological features including large tumor size, high Edmondson–Steiner grading and advanced tumor–node–metastasis (TNM) stage. Additionally, we demonstrated that patients with high LSP1 expression had significantly better overall survival and disease‐free survival. Moreover, LSP1 was found to be an independent factor for predicting the prognosis of HCC patients. In vitro and in vivo assays showed that overexpressing LSP1 inhibited HCC growth by inducing both apoptosis and growth arrest. Mechanistically, we found that expression of phosphorylated extracellular regulated protein kinases 1 and 2 (ERK1/2) was downregulated after LSP1 overexpression, indicating LSP1 could suppress HCC growth by inhibiting the ERK pathway in HCC cells. Taken together, these results indicate that LSP1 may serve as a prognostic marker and a potential therapeutic target in human HCC.

Temporal regulation of Lsp1 O-GlcNAcylation and phosphorylation during apoptosis of activated B cells.

Crosslinking of B-cell receptor (BCR) sets off an apoptosis programme, but the underlying pathways remain obscure. Here we decipher the molecular mechanisms bridging B-cell activation and apoptosis mediated by post-translational modification (PTM). We find that O-GlcNAcase inhibition enhances B-cell activation and apoptosis induced by BCR crosslinking. This proteome-scale analysis of the functional interplay between protein O-GlcNAcylation and phosphorylation in stimulated mouse primary B cells identifies 313 O-GlcNAcylation-dependent phosphosites on 224 phosphoproteins. Among these phosphoproteins, temporal regulation of the O-GlcNAcylation and phosphorylation of lymphocyte-specific protein-1 (Lsp1) is a key switch that triggers apoptosis in activated B cells. O-GlcNAcylation at S209 of Lsp1 is a prerequisite for the recruitment of its kinase, PKC-β1, to induce S243 phosphorylation, leading to ERK activation and downregulation of BCL-2 and BCL-xL. Thus, we demonstrate the critical PTM interplay of Lsp1 that transmits signals for initiating apoptosis after BCR ligation.

Proteome changes in auricular lymph nodes and serum after dermal sensitization to toluene diisocyanate in mice

Some reactive chemicals, such as diisocyanates, are capable of initiating an allergic response, which can lead to occupational asthma after a latency period. Clinical symptoms such as cough, wheezing, and dyspnea occur only late, making it difficult to intervene at an early stage. So far, most studies using proteomics in lung research have focused on comparisons of healthy versus diseased subjects. Here, using 2D-DIGE, we explored proteome changes in the local draining lymph nodes and serum of mice dermally sensitized once or twice with toluene-2,4-diisocyanate (TDI) before asthma is induced. In the lymph nodes, we found 38 and 58 differentially expressed proteins after one and two treatments, respectively, between TDI-treated and vehicle-treated mice. In serum, seven and 16 differentially expressed proteins were detected after one and two treatments, respectively. We identified 80-85% of the differentially expressed proteins by MS. Among them, lymphocyte-specific protein-1, coronin 1a, and hemopexin were verified by Western blotting or ELISA in an independent group of mice. This study revealed alterations in the proteomes early during sensitization in a mouse model before the onset of chemical-induced asthma. If validated in humans, these changes could lead to earlier diagnosis of TDI-exposed workers.