Lymphocyte Cytosolic Protein 1 Research Articles

LCP1 correlates with immune infiltration: a prognostic marker for triple-negative breast cancer

ObjectiveTriple-Negative Breast Cancer (TNBC) is known for its aggressiveness and treatment challenges due to the absence of ER, PR, and HER2 receptors. Our work emphasizes the prognostic value of LCP1 (Lymphocyte cytosolic protein 1), which plays a crucial role in cell processes and immune cell activity, to predict outcomes and guide treatments in TNBC.MethodsWe explored LCP1 as a potential biomarker in TNBC and investigated the mRNA and protein expression levels of LCP1. We investigated different databases, including GTEX, TCGA, GEO, cBioPortal and Kaplan-Meier Plotter. Immunohistochemistry on TNBC and benign tumor samples was performed to examine LCP1's relationship with patient clinical characteristics and macrophage markers. We also assessed survival rates, immune cell infiltration, and drug sensitivity related to LCP1 using various bioinformatics tools.ResultsThe results indicated that LCP1 expression was higher in TNBC tissues compared to adjacent normal tissues. However, high expression of LCP1 was significantly associated with favorable survival outcomes in patients with TNBC. Enrichment analysis revealed that genes co-expressed with LCP1 were significantly enriched in various immune processes. LCP1 showed a positive correlation with the infiltration of resting dendritic cells, M1 macrophages, and memory CD4 T cells, and a negative correlation with M2 macrophages. Further analysis suggested a link between high levels of LCP1 and increased survival outcomes in cancer patients receiving immunotherapy.ConclusionLCP1 may serve as a potential diagnostic and prognostic biomarker for TNBC, which was closely associated with immune cell infiltration, particularly M1 and M2 macrophages. Our findings may provide valuable insights into immunotherapeutic strategies for TNBC patients.

Abstract 2040: Targeting MUS81 endonuclease to enhance chemo-immunotherapy response in head and neck squamous cell carcinoma

Abstract Background: In advanced head and neck squamous cell carcinoma (HNSCC), immune checkpoint inhibitors are only effective in 15-20% of cases. Defects in replication stress response represent a promising avenue for enhancing the therapeutic efficacy of immunotherapy through DNA damage and the release of immunostimulatory DNA. MUS81, a DNA structure-specific endonuclease involved in resolving replication stress, is upregulated in many cancer types including HNSCC, and MUS81-associated cytosolic DNA is implicated in triggering cGAS-STING mediated innate immunity. The objectives of this study were to assess the impact of targeting MUS81 on HNSCC cell sensitivity to cisplatin, and investigate its role in enhancing immunotherapy response in HNSCC, through the cell viability and cell cycle analysis, differential gene expression, and DNA release within small extracellular vesicles (exosomes). Methods and Results: We used HNSCC cell lines and patient-derived organoids (PDOs) to investigate the consequences of CRISPR/Cas9-mediated MUS81 knockout (KO). We demonstrated that MUS81 KO increased HNSCC cell sensitivity to cisplatin, inducing a G2/M cell cycle arrest. Transcriptomic analysis of differentially expressed genes (DEG) in MUS81 KO vs control cells highlighted upregulation of ANXA6 (tumor suppressor gene encoding for Annexin A6), MACROH2A2 (involved in the quiescent phenotype acquisition of malignant HNSCC cells) and CDA (encoding for cytidine deaminase, implicated in replication stress). MUS81 KO was also associated with downregulation of LCP1 (encoding for lymphocyte cytosolic protein 1, upregulated in oral cancer and controlling cellular proliferation, invasiveness, and migratory activities). Gene Set Enrichment Analysis (GSEA) of RNA-seq data, revealed positive enrichment in MUS81 KO cells treated with cisplatin in genes involved in the hallmark interferon alpha (Normalized Enrichment Score (NES) 3.03, p-value < 0.001) and interferon gamma (NES 2.83, p- value < 0.001) pathways, indicative of innate and adaptive immune responses. Additionally, the pathway of hallmark inflammatory response was positively enriched (NES 2.22, p < 0.001). In addition, cisplatin treatment in MUS81 KO was associated with notable alterations in the concentration and length of exosomal DNA, with implications for immune response modulation within the tumor microenvironment. Conclusions: Our study underscores the potential significance of MUS81 as a novel therapeutic target in HNSCC. In addition to sensitizing HNSCC cells to chemotherapeutic agents, targeting MUS81 may influence the dynamics of the tumor microenvironment, thereby enhancing the prospects of immunotherapy in this context. Further in-depth investigations are imperative to validate these observations and facilitate the development of innovative therapeutic strategies tailored to targeting MUS81 in HNSCC. Citation Format: Ali Abdulnabi Suwaidan, Anna Pasto, Caitlin McCarthy, Teng Pan, Jinhai Deng, Luigi Dolcetti, Giovanna Alfano, Rami Mustapha, James Monypenny, Martin Forster, Tony Ng. Targeting MUS81 endonuclease to enhance chemo-immunotherapy response in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2040.

Proteomic profiling of formalin-fixed paraffine-embedded tissue reveals key proteins related to lung dysfunction in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) severely affects the lung leading to aberrant deposition of extracellular matrix and parenchymal stiffness with progressive functional derangement. The limited availability of fresh tissues represents one of the major limitations to study the molecular profiling of IPF lung tissue. The primary aim of this study was to explore the proteomic profiling yield of archived formalin-fixed paraffin-embedded (FFPE) specimens of IPF lung tissues. We further determined the protein expression according to respiratory functional decline at the time of biopsy. The total proteins isolated from 11 FFPE samples of IPF patients compared to 3 FFPE samples from a non-fibrotic lung defined as controls, were subjected to label-free quantitative proteomic analysis by liquid chromatography-mass spectrometry (LC-MS/MS) and resulted in the detection of about 400 proteins. After the pairwise comparison between controls and IPF, functional enrichment analysis identified differentially expressed proteins that were involved in extracellular matrix signaling pathways, focal adhesion and transforming growth factor β (TGF-β) signaling pathways strongly associated with IPF onset and progression. Five proteins were significantly over- expressed in the lung of IPF patients with either advanced disease stage (Stage II) or impaired pulmonary function (FVC<75, DLCO<55) compared to controls; these were lymphocyte cytosolic protein 1 (LCP1), peroxiredoxin-2 (PRDX2), transgelin 2 (TAGLN2), lumican (LUM) and mimecan (OGN) that might play a key role in the fibrogenic processes. Our work showed that the analysis of FFPE samples was able to identify key proteins that might be crucial for the IPF pathogenesis. These proteins are correlated with lung carcinogenesis or involved in the immune landscape of lung cancer, thus making possible common mechanisms between lung carcinogenesis and fibrosis progression, two pathological conditions at risk for each other in the real life.

Exploring Corneal Neovascularization: An Integrated Approach Using Transcriptomics and Proteomics in an Alkali Burn Mouse Model.

Corneal neovascularization (CNV) impairs corneal transparency and visual acuity. The study aims to deepen our understanding of the molecules involved in CNV induced by alkali burns, facilitate a better grasp of CNV mechanisms, and uncover potential therapeutic targets. Eighty-four mice were selected for establishing CNV models via alkali burns. On days 3, 7, and 14 after the burns, corneal observations and histological investigations were conducted. An integrated analysis of RNA sequencing (RNA-seq)-based transcriptomics and label-free quantitative proteomics was performed in both normal and burned corneas. Bioinformatics approaches, encompassing Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, were applied to discern differentially expressed genes (DEGs) and crucial signaling pathways. Four potentially CNV-related genes were validated using quantitative real-time PCR (qRT-PCR) and Western blot. Significant CNV was observed on the seventh day. Forty-one genes were differentially expressed in neovascularized corneas, with 15 upregulated and 26 downregulated at both mRNA and protein levels. Bioinformatics analysis revealed that these DEGs participated in diverse biological processes, encompassing retinol and retinoic acid metabolism, neutrophil chemotaxis, and actin filament assembly, along with significant enrichment pathways like cytochrome P450, tyrosine, and phenylalanine metabolism. The upregulation of lymphocyte cytosolic protein 1 (LCP1) and cysteine and glycine-rich protein 2 (CSRP2) genes and the downregulation of transglutaminase 2 (TGM2) and transforming growth factor-beta-induced (TGFBI) genes were confirmed. We analyzed gene expression differences in mouse corneas 7 days after alkali burns, finding 41 genes with altered expression. The exact role of these genes in CNV is not fully understood, but exploring angiogenesis-related molecules offers potential for CNV treatment or prevention.

LCP1 knockdown in monocyte-derived macrophages: mitigating ischemic brain injury and shaping immune cell signaling and metabolism.

Rationale: Ischemic stroke poses a significant health burden with limited treatment options. Lymphocyte Cytosolic Protein 1 (LCP1) facilitates cell migration and immune responses by aiding in actin polymerization, cytoskeletal rearrangements, and phagocytosis. We have demonstrated that the long non-coding RNA (lncRNA) Maclpil silencing in monocyte-derived macrophages (MoDMs) led to LCP1 inhibition, reducing ischemic brain damage. However, the role of LCP1 of MoDMs in ischemic stroke remains unknown. Methods and Results: We investigated the impact of LCP1 on ischemic brain injury and immune cell signaling and metabolism. We found that knockdown of LCP1 in MoDMs demonstrated robust protection against ischemic infarction and improved neurological behaviors in mice. Utilizing the high-dimensional CyTOF technique, we demonstrated that knocking down LCP1 in MoDMs led to a reduction in neuroinflammation and attenuation of lymphopenia, which is linked to immunodepression. It also showed altered immune cell signaling by modulating the phosphorylation levels of key kinases and transcription factors, including p-PLCg2, p-ERK1/2, p-EGFR, p-AKT, and p4E-BP1 as well as transcription factors like p-STAT1, p-STAT3, and p-STAT4. Further bioinformatic analysis indicated that Akt and EGFR are particularly involved in fatty acid metabolism and glycolysis. Indeed, single-cell sequencing analysis confirmed that enrichment of fatty acid and glycolysis metabolism in Lcp1high monocytes/macrophages. Furthermore, Lcp1high cells exhibited enhanced oxidative phosphorylation, chemotaxis, migration, and ATP biosynthesis pathways. In vitro experiments confirmed the role of LCP1 in regulating mitochondrial function and fatty acid uptake. Conclusions: These findings contribute to a deeper understanding of LCP1 in the context of ischemic stroke and provide valuable insights into potential therapeutic strategies targeting LCP1 and metabolic pathways, aiming to attenuating neuroinflammation and lymphopenia.

Inhibition of the Glycolysis Prevents the Cerebral Infarction Progression Through Decreasing the Lactylation Levels of LCP1

Cerebral infarction (CI), also known as ischemic stroke, has a high incidence rate and mortality rate. The purpose of this study was to investigate the potential effect and mechanism of Lymphocyte cytosolic protein 1 (LCP1) in the CI progression. The middle cerebral artery occlusion (MCAO) treated rats and oxygen–glucose deprivation/reoxygenation (OGD/R) stimulated PC12 cells were used to establish CI model in vivo and in vitro. The cell proliferation and apoptosis was determined by CCK-8 assay and flow cytometry, respectively. Immunoprecipitation and western blot was performed to test the lactylation levels of LCP1. The cells were treated with cycloheximide to determined the protein stability of LCP1. The glucose uptake and lactate production was determined with commercial kits. The extracellular acidification rate were evaluated by Seahorse. The results showed that LCP1 was upregulated in the MCAO rats and OGD/R stimulated PC12 cells. LCP1 knockdown dramatically decreased the neurological score, infarct volume and the brain water content of MCAO rats. Besides, LCP1 knockdown promoted the cell viability while decreased the apoptosis rate of the OGD/R stimulated PC12 cells. Additionally, the global lactylation and lactylation levels of LCP1 was prominently enhanced in vivo and in vitro in cerebral infarction. 2-DG treatment prominently decreased it. In conclusion, inhibiting the glycolysis decreased the lactylation levels of LCP1 and resulted in the degradation of LCP1, which eventually relieved the CI progression.

The Role of Lymphocyte Cytosolic Protein 1 in a Mycobacterium Marinum Infection

Mycobacterium tuberculosis is an issue worldwide with multidrug‐resistant forms becoming prevalent due to the lack of effective treatments. The goal of this project is to identify if there are host genes that affect the disease outcome. We decided to study the influence of the Lymphocyte Cytosolic Protein 1 (LCP1) gene in zebrafish larvae infected with mycobacterium marinum. To understand the immune response in the fish we observed the bacterial burden in fish that had LCP1 (heterozygotes) and in fish that did not (knockout mutants). M. marinum was injected into the caudal vein of the zebrafish larvae and the fish were infected. Once the fish were fixed and euthanized, images of the bacterial infection were taken using a fluorescent microscope. The bacteria was tagged with tdTomato, a red fluorescent protein which allows for the bacteria to glow red under the microscope. The images were then analyzed, and the bacterial burden was quantified using ImageJ to count the number of glowing pixels. Following the imaging of the infected larvae, the fish were genotyped. The DNA from the larvae was extracted through a hotSHOT PCR protocol, then LCP1 Exon 2 was amplified through PCR amplification. The PCR product was then cut with the Bsl‐I enzyme to show where the gene was expressed, gel electrophoresis was then run to analyze where the Bsl‐I cut and identify whether the fish was a heterozygote or knockout mutant. We then matched the suitable images of the fish to the genotype and found that fish with LCP1 may have a higher rate of infection. Understanding why fish with LCP1 have a higher bacterial burden could provide an insight to the role LCP1 plays in the human body’s immune response. The future directions of this project are to observe more fish in order to replicate the findings and to test other phenotypes including granuloma size, macrophage motility, and extracellular growth in order to have a better understanding of LCP1's role in the disease outcome. Mycobacterium tuberculosis is an issue worldwide with multidrug‐resistant forms becoming prevalent.

Transcriptome Profiling Analysis Identifies LCP1 as a Contributor for Chidamide Resistance in Gastric Cancer.

Gastric cancer (GC) remains a significant health problem and carries with it substantial morbidity and mortality. Chidamide is a novel and orally administered histone deacetylase (HDAC) inhibitor and has been demonstrated its anti-tumor efficacy on different kinds of hematological and solid tumors. However, the underlying mechanism of chidamide resistance is still poorly characterized. We established chidamide resistant GC cell lines, AGS ChiR and MGC803 ChiR and investigated the toxicologic effects through cell survival, colony formation and flow cytometry assays in vitro, and a subcutaneous xenograft model in vivo. RNA-sequence was then performed to screen chidamide resistance-associated genes between AGS and AGS ChiR cells. The role of Lymphocyte cytosolic protein 1 (LCP1) in chidamide resistance was explored by gain- and loss-of-function analyses. We found that chidamide significantly inhibited cell proliferation and induced the apoptosis in a concentration-dependent manner in wild-type GC cell lines as compared to chidamide resistant cell lines. The transcriptomic profiling, quantitative RT-PCR, and western blot data revealed that LCP1 was upregulated in AGS ChiR cells compared with parental cells. Overexpression of LCP1 conferred and knockdown of LCP1 attenuated the chidamide resistance of GC cells. Epigenetic derepression of LCP1 by chidamide may be a possible reason for the contribution of LCP1 to chidamide resistance. These findings illustrated that LCP1 may play a chidamide resistance role in GC, suggesting that LCP1 could be a potential target for the therapy of GC combined with chidamide.

Lymphocyte Cytosolic Protein 1 I232F Mutation Impairs Granulocytic Proliferation with a G2/M Block in Severe Neutropenia

▪Introduction: As critical effectors of innate immune response, neutrophils migrate from the vasculature into inflamed tissue, where they engage in phagocytosis and clearance of pathogens and apoptotic cells. Migration, phagocytosis, and granule release require multiple well-coordinated events involving remodeling of the actin cytoskeleton. While mutations of some regulators of actin polymerization, such as Wiskott-Aldrich Syndrome gene (WAS) result in Severe Congenital Neutropenia (SCN), the role of actin cytoskeleton in regulating neutrophil ontogeny and functions remain poorly understood. Lymphocyte Cytosolic Protein 1 (LCP1) is an actin bundling protein encoded by LCP1 gene. A 7-year-old girl presented at age of 2 years with chronic severe neutropenia and frequent infections. Next generation sequencing did not reveal a variant in genes known to cause neutropenia. Whole exome sequencing identified a novel LCP1 p.I232F variant of unknown significance, which was not present in either parent. Bone marrow examination revealed a granulocytic hypoplasia with significant dysmorphic giant granulocytes with complex hypersegmentation and increased apoptosis. (Figure 1A) Based on these observations, we hypothesized that missense mutation in LCP1 (I232F) impairs granulocytic proliferation, and causes neutropenia.Aims: To investigate the role of LCP1 I232F, discovered in a child with severe symptomatic neutropenia, in granulopoiesis.Methods: in vitro experiments involving the expression of wild type and mutant LCP1 I232F in murine myeloblast cell (32D cells) and human cervical cancer HeLa cell. We deployed various biologic and functional studies to investigate the role of LCP1 I232F in granulocyte proliferation, differentiation, survival, and function.Results: To determine the pathophysiologic significance of LCP1 I232F, we stably transduced interleukin 3-dependent murine myeloblast 32D and human cervical cancer HeLa cell lines with a doxycycline-inducible lentiviral constructs containing the cDNA for either wild type LCP1 or LCP1 I232F. The mutant LCP1 I232F expressing 32D cells showed impaired proliferation with the appearance of dysplastic granulocytic cells. (Figure 1A, B) However, we did not observe block in differentiation. A cell cycle arrest at G2/M phase occurred only in the LCP1 I232F expressing cells (Figure 1B) and was associated with an upregulation of markers of cell cycle arrest (Cdkn1a and Tp53). LCP1 I232F overexpression did not lead to increased expression of genes involved in apoptosis or the unfolded protein response. The LCP1 I232F cells with dysplastic features were further analyzed by confocal microscopy, which demonstrated increased level of F-actin and enrichment of F-actin and LCP1 at the cell cortex. Flow cytometric evaluation of these cells further confirmed the presence of increased level of F actin. (Figure 1C) Expression of LCP1 I232F impaired cell motility and invasiveness in both 32D and HeLa cells. However, mutant LCP1 expressing 32D cells demonstrated normal oxidative burst upon treatment with N-formyl-Met-Leu-Phe (fMLP) and phorbol myristate acetate (PMA). Confocal imaging and subcellular fractionation revealed diffuse localization of LCP1, but only the mutant form was found in the nucleus. (Figure 1C) In LCP1, two tandem CH domains (CH1-CH2 and CH3-CH4) formed two actin-binding domains (ABD1 and ABD2) that bind to actin to participate in actin bundling. I232F is located at LCP1-CH1 that interacts with actin. To study how the mutation impacts LCP1-actin binding, we constructed a model of LCP1-ABD1 based on the cryo-EM structure of F-actin/LCP1-ABD2 and performed 40 ns of molecular dynamics (MD) simulations of LCP1-ABD1 and LCP1-ABD1 I232F alone to study their dynamical motions. We found that LCP1-ABD1 I232F adopted a more stable open conformation than LCP1-ABD1 to bind effectively with F-actin. The model implies a prolonged association of LCP1 I232F with actin than wild-type LCP1, and the consequence is the increased stabilization of actin bundles.Conclusions: Like the WAS gain-of-function mutations in SCN, LCP1 I232F resulted in severe neutropenia with dysplastic granulocytic precursors, cell cycle arrest, impaired motility, and increased F actin. We also found decreased proliferation of granulocytic progenitor and precursor cells. Our findings suggest that LCP1 is an important gene involved in granulopoiesis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Loss of lymphocyte cytosolic protein 1 (LCP1) induces browning in 3T3-L1 adipocytes via β3-AR and the ERK-independent signaling pathway.

Increased browning of white adipocytes (beiging) is considered a promising therapeutic strategy to fight obesity and its associated metabolic complications. However, the molecular mechanism modulating brown and beige fat-mediated thermogenesis is not fully elucidated. Here, we identified the lymphocyte cytosolic protein 1 (LCP1) as a factor that obstructs fat browning in white adipocytes. LCP1 plays a vital role in non-hematopoietic malignancies, and is also a well-known tumor biomarker; however, evidence regarding its function in adipocytes remains to be elucidated. The current study explores the physiological role of LCP1 in cultured 3T3-L1 white adipocytes, by applying the loss-of-function study using siRNA. Induction of fat browning by LCP1 depletion was evidenced by evaluating the gene and protein expression levels of brown fat-associated markers through real-time qRT-PCR and immunoblot analysis, respectively. We observed that deficiency of LCP1 promotes mitochondrial biogenesis, and significantly enhances expressions of the core brown fat-specific genes (Cd137, Cidea, Cited1, Tbx1, and Tmem26) and proteins (PGC-1α, PRDM16, and UCP1). In addition, deficiency of LCP1 promotes lipid catabolism as well as suppresses adipogenesis and lipogenesis. Loss of LCP1 also ameliorates cellular stress by downregulating JNK and c-JUN in adipocytes, and stimulates apoptosis. A mechanistic study revealed that deficiency of LCP1 induces browning in white adipocytes, independently via β3-AR and the ERK signaling pathway. The current data reveals a previously unknown mechanism of LCP1 in browning of white adipocytes, and highlights the potential of LCP1 as a pharmacotherapeutic target for treating obesity and other metabolic disorders.

Exosomal Transfer of LCP1 Promotes Osteosarcoma Cell Tumorigenesis and Metastasis by Activating the JAK2/STAT3 Signaling Pathway

Increasing evidence indicates that lymphocyte cytosolic protein 1 (LCP1) overexpression contributes to tumor progression; however, its role in osteosarcoma (OS) remains unclear. We aimed to investigate the potential effect of LCP1 in OS and the underlying mechanisms. We first demonstrated that LCP1 is upregulated in OS cell lines and tissues. Then, we found that aberrant expression of LCP1 could induce the proliferation and metastasis of OS cells in vitro and in vivo by destabilizing neuregulin receptor degradation protein-1 (Nrdp1) and subsequently activating the JAK2/STAT3 signaling pathway. When coculturing OS cells with bone marrow-derived mesenchymal stem cells (BMSCs) in vitro, we validated that oncogenic LCP1 in OS was transferred from BMSCs via exosomes. Moreover, microRNA (miR)-135a-5p, a tumor suppressor, was found to interact upstream of LCP1 to counteract the pro-tumorigenesis effects of LCP1 in OS. In conclusion, BMSC-derived exosomal LCP1 promotes OS proliferation and metastasis via the JAK2/STAT3 pathway. Targeting the miR-135a-5p/LCP1 axis may have potential in treating OS.

Lymphocyte cytosolic protein 1 (LCP1) is a novel TRAF3 dysregulation biomarker with potential prognostic value in multiple myeloma

Chromosomal rearrangement involving 14q32 region that results in TNF receptor associated factor 3 (TRAF3) dysfunctional mutation is the most frequent NF-κB pathway mutation in multiple myeloma (MM). Subsequent NF-κB inducing Kinase (NIK) stabilization plays a critical role in alternative NF-κB activation. However, disease progression resulting from TRAF3 dysregulation has not been well understood. In this study, we identified lymphocyte cellular protein 1 (LCP1) as a novel NIK-driven alternative NF-κB target in TRAF3 dysfunctional mutation using RNA-seq, ChIP-seq (RelA/p65 and p52 NF-κB) and other validation methods. LCP1 is exclusively activated in MM cells with TRAF3 loss-of-function mutation. In MM patients, higher LCP1 expression was significantly pronounced in poor prognosis groups such as 4p16 and MAF. CD138 negative MM patient cells showed elevated LCP1 expression and inhibition of LCP1 can sensitize proteasome inhibitor bortezomib in TRAF3 mutant MM cells in vitro. We report that LCP1 is a NIK-driven biomarker in TRAF3 dysfunctional MM and targeting LCP1 can provide a valuable therapeutic intervention in TRAF3 mutated MM.

LCP1 triggers mTORC2/AKT activity and is pharmacologically targeted by enzastaurin in hypereosinophilia.

Hypereosinophilia (HE) is caused by a variety of disorders, ranging from parasite infections to autoimmune diseases and cancer. Only a small proportion of HE cases are clonal malignancies, and one of these, the group of eosinophilia-associated tyrosine kinase fusion-driven neoplasms, is sensitive to tyrosine kinase inhibitors, while most subtypes lack specific treatment. Eosinophil functions are highly dependent on actin polymerization, promoting priming, shape change, and infiltration of inflamed tissues. Therefore, we investigated the role of the actin-binding protein lymphocyte cytosolic protein 1 (LCP1) in malignant and nonmalignant eosinophil differentiation. We use the protein kinase C-β (PKCβ) selective inhibitor enzastaurin (Enza) to dephosphorylate and inactivate LCP1 in FIP1L1-platelet-derived growth factor receptor α (PDGFRA)-positive Eol-1 cells, and this was associated with reduced proliferation, metabolic activity, and colony formation as well as enhanced apoptosis and impaired migration. While Enza did not alter FIP1L1-PDGFRA-induced signal transducer and activator of transcription 3 (STAT3), STAT5, and ERK1/2 phosphorylation, it inhibited STAT1Tyr701 and AKTSer473 (but not AKTThr308 ) phosphorylation, and short hairpin RNA knockdown experiments confirmed that this process was mediated by LCP1 and associated mammalian target of rapamycin complex 2 (mTORC2) activity loss. Homeobox protein HoxB8 immortalized murine bone marrow cells showed impaired eosinophilic differentiation upon Enza treatment or LCP1 knockdown. Furthermore, Enza treatment of primary HE samples reduced eosinophil differentiation and survival. In conclusion, our data show that HE involves active LCP1, which interacts with mTOR and triggers mTORC2 activity, and that the PKCβ inhibitor Enza as well as targeting of LCP1 may provide a novel treatment approach to hypereosinophilic disorders.

Silencing the lncRNA Maclpil in pro-inflammatory macrophages attenuates acute experimental ischemic stroke via LCP1 in mice

Long noncoding RNAs (lncRNA) expression profiles change in the ischemic brain after stroke, but their roles in specific cell types after stroke have not been studied. We tested the hypothesis that lncRNA modulates brain injury by altering macrophage functions. Using RNA deep sequencing, we identified 73 lncRNAs that were differentially expressed in monocyte-derived macrophages (MoDMs) and microglia-derived macrophages (MiDMs) isolated in the ischemic brain three days after stroke. Among these, the lncRNA, GM15628, is highly expressed in pro-inflammatory MoDMs but not in MiDMs, and are functionally related to its neighbor gene, lymphocyte cytosolic protein 1 (LCP1), which plays a role in maintaining cell shape and cell migration. We termed this lncRNA as Macrophage contained LCP1 related pro-inflammatory lncRNA, Maclpil. Using cultured macrophages polarized by LPS, M(LPS), we found that downregulation of Maclpil in M(LPS) decreased pro-inflammatory gene expression while promoting anti-inflammatory gene expression. Maclpil inhibition also reduced the migration and phagocytosis ability of MoDMs by inhibiting LCP1. Furthermore, adoptive transfer of Maclpil silenced M(LPS), reduced ischemic brain infarction, improved behavioral performance and attenuated penetration of MoDMs in the ischemic hemisphere. We conclude that by blocking macrophage, Maclpil protects against acute ischemic stroke by inhibiting neuroinflammation.

Functional analysis of p.Ala253_Leu254insAsn mutation in PLS3 responsible for X-linked osteoporosis.

Mutations in Plastin-3 (PLS3) have been identified as a cause of X-linked osteoporosis. To reveal the molecular mechanism of PLS3 on osteoporosis, we characterized the p.Ala253_Leu254insAsn mutation in PLS3. We first identified Lymphocyte cytosolic protein 1 (LCP1) as a binding partner of PLS3 and the mutation disrupted the interaction between them. We then confirmed the roles of PLS3 and LCP1 in the regulation of intracellular Ca2+ , which was weakened by the mutant PLS3. Moreover, the interaction between PLS3 and LCP1 was enhanced under a low concentration of extracellular Ca2+ . However, the mutation in PLS3 weakened the responsiveness. The reduced regulation on Ca2+ caused by p.Ala253_Leu254insAsn may be the possible molecular mechanism of osteoporosis.

Effects of punctal occlusion on global tear proteins in patients with dry eye

PurposeTo investigate effects of punctal occlusion on global tear protein levels in patients with dry eye. MethodsIn this prospective, longitudinal, single-center study, nonabsorbable punctal plugs were inserted bilaterally into the lower punctum of 30 patients with moderate dry eye. Dry eye symptoms, fluorescein corneal staining, Schirmer I test, tear film break-up time, and safety were assessed in the more severely affected eye. Tear proteins at weeks 1 and 3 were quantified by iTRAQ relative to baseline preocclusion levels. ResultsOf 29 patients who completed the study, 23 (mean age 49.8 years) had sufficient tear samples for analysis. After 3 weeks, punctal occlusion significantly upregulated tear proteins, including glutathione synthase (mean of 1.6-fold, P = 0.01) and interleukin-1 receptor antagonist (1.7-fold, P = 0.032) and downregulated cholinergic receptor (neuronal) alpha-7 (0.79-fold, P = 0.039) and lymphocyte cytosolic protein-1 (0.66-fold, P = 0.012). Clustering analysis of global tear proteins revealed two clear profile changes; the first group of patients (cluster 1, n = 10) had a reduction in the inflammatory proteins (e.g., S100A8) and rise in lacrimal proteins supporting the ocular surface (e.g., lysozyme), whereas the second group (cluster 2, n = 13) had an increase in inflammatory proteins and a decrease in lacrimal proteins. Logistic regression analysis revealed that cluster 1 patients had significantly (P = 0.006) lower Schirmer scores at baseline (mean [standard deviation]: 4.3 [4.3] mm) than cluster 2 (6.8 [2.6] mm). ConclusionsPunctal plugs produced a beneficial pattern of tear protein change in patients with relatively low Schirmer scores within 3 weeks of punctal occlusion.

Evidence for Critical Role of Lymphocyte Cytosolic Protein 1 in Oral Cancer

Lymphocyte cytosolic protein 1 (LCP1), a member of actin-binding protein of the plastin family, has been identified in several malignant tumors of non-hematopoietic sites, such as the colon, prostate, and breast. However, little is known about the roles of LCP1 in oral squamous cell carcinomas (OSCCs). This present study sought to clarify the clinical relevance of LCP1 in OSCCs and investigate possible clinical applications for treating OSCCs by regulating LCP1 expression. We found up-regulation of LCP1in OSCCs compared with normal counterparts using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), immunoblotting, and immunohistochemistry (P < 0.05). We used shRNA models for LCP1 (shLCP1) and enoxacin (ENX), a fluoroquinolone antibiotic drug, as a regulator of LCP1 expression. In addition to the LCP1 knockdown experiments in which shLCP1 cells showed several depressed functions, including cellular proliferation, invasiveness, and migratory activities, ENX-treated cells also had attenuated functions. Consistent with our hypothesis from our in vitro data, LCP1-positive OSCC samples were correlated closely with the primary tumoral size and regional lymph node metastasis. These results suggested that LCP1 is a useful biomarker for determining progression of OSCCs and that ENX might be a new therapeutic agent for treating OSCCs by controlling LCP1 expression.

The serum proteome of nonalcoholic fatty liver disease: A multimodal approach to discovery of biomarkers of nonalcoholic steatohepatitis

Nonalcoholic fatty liver disease (NAFLD) is a common condition affecting up to 25% of the developed world. It is a progressive disease, leading in some to the development of liver cirrhosis. Currently, accurate diagnosis and staging of this condition is only possible with histological examination of a liver biopsy. This gold standard test is neither suitable nor practical for large-scale use as is necessary for a condition as common as NAFLD. The aim of this study is to describe the proteome of human NAFLD using two distinct shotgun proteomic methods, translating the findings into potential biomarkers of NAFLD. Two distinct shotgun proteomic techniques (iTRAQ and label free) were used to describe the proteome of NAFLD. Thereafter, candidate biomarkers were selected for validation by ELISA. Over 550 protein identifications were made in the description of the NAFLD proteome. Several proteins were found to be significantly up/downregulated in nonalcoholic steatohepatitis compared with control, including apolipoprotein E (fold ratio of 1.67), insulin-like growth factor-binding protein 3 (IGFBP3, fold ratio of 1.642), Vitamin D-binding protein (fold ratio of 4.587), and lymphocyte cytosolic protein1 (LCP1, fold ratio of 4.356). ELISA validation of a subset of these proteins confirms the validity of the proteomic studies and suggests possible new biomarkers of NAFLD. Serum markers are able to distinguish between the stages of disease in NAFLD as well as detect the grade of fibrosis. Ultimately, noninvasive serum markers may replace liver biopsy in the investigation of patients with suspected NAFLD.

Lymphocyte cytosolic protein 1 is a chronic lymphocytic leukemia membrane-associated antigen critical to niche homing

AbstractMembrane antigens are critical to the pathogenesis of chronic lymphocytic leukemia (CLL) as they facilitate microenvironment homing, proliferation, and survival. Targeting the CLL membrane and associated signaling patterns is a current focus of therapeutic development. Many tumor membrane targets are simultaneously targeted by humoral immunity, thus forming recognizable immunoglobulin responses. We sought to use this immune response to identify novel membrane-associated targets for CLL. Using a novel strategy, we interrogated CLL membrane-specific autologous immunoglobulin G reactivity. Our analysis unveiled lymphocyte cytosolic protein 1 (LCP1), a lymphocyte-specific target that is highly expressed in CLL. LCP1 plays a critical role in B-cell biology by crosslinking F-actin filaments, thereby solidifying cytoskeletal structures and providing a scaffold for critical signaling pathways. Small interfering RNA knockdown of LCP1 blocked migration toward CXCL12 in transwell assays and to bone marrow in an in vivo xenotransplant model, confirming a role for LCP1 in leukemia migration. Furthermore, we demonstrate that the Bruton's tyrosine kinase inhibitor ibrutinib or the PI3K inhibitor idelalisib block B-cell receptor induced activation of LCP1. Our data demonstrate a novel strategy to identify cancer membrane target antigens using humoral anti-tumor immunity. In addition, we identify LCP1 as a membrane-associated target in CLL with confirmed pathogenic significance. This clinical trial was registered at clinicaltrials.gov; study ID number: OSU-0025 OSU-0156.

Abstract 4925: Lymphocyte cytosolic protein 1 (LCP1) is a membrane-associated molecular target in chronic lymphocytic leukemia and is activated in microenvironment signaling.

Abstract Chronic lymphocytic leukemia (CLL) is an incurable accumulation of malignant B-cells in peripheral circulation, which home to supportive leukemic microenvironments. Critical to the pathogenesis of CLL are membrane-associated antigens since they facilitate leukemic cell signaling, microenvironment homing, proliferation, and survival. Targeting these CLL surface molecules is a current focus of CLL therapeutic development. Rituximab, alemtuzumab, and ofatumumab are commonly used antibody-based therapeutics that proved to be effective at targeting CLL membrane associated antigens. Despite enhanced patient survival, targeting of non-obligate antigens ultimately leads to antibody resistance. In addition, off target expression often produces unacceptable toxicity. Immunosurveilance antigens may provide a key to overcome these obstacles. A number of relevant tumor antigens such as p53, EGFR, WT1, BCR-Abl and ROR1 elicit robust autoimmune responses in cancer patients due to high levels of ectopic expression, high relative immunogenicity and obligate oncogenic signaling that forces expression despite autoimmune recognition. In many cases, autoantibody signatures were realized long after identification of the oncologic target antigens; however, the convergence of these two datasets implies that such signatures could be utilized to identify novel targets. To further explore this possibility, we interrogated anti-tumor humoral reactivity specifically directed against autologous CLL membrane antigens. Immunoreactive leads were identified by mass spectrometry. Potential membrane antigens were further confirmed using immunoblot and ELISA. Our analysis revealed Lymphocyte Cytosolic Protein 1 (LCP1), a membrane associated lymphocyte-specific target that is constitutively expressed on CLL and ectopically expressed in various cancers. Confirmatory assays unveiled robust LCP1-specific IgG autoimmune responses in CLL patients despite a profound absence of reactivity to common viral and vaccine antigens. LCP1 plays a critical role in B-cell biology by cross linking f-actin filaments, thereby solidifying cytoskeletal structures and providing a scaffold for CLL-critical signaling pathways such as PKC. Transwell assays confirmed that LCP1 activity contributed to the migration of CLL cells towards CXCL12. We further utilize a novel xenograft model to demonstrate LCP1 involvement in CLL pathogenesis. We have also characterized LCP1 expression within cancer derived exosomes revealing a previously unknown function of this gene. Our data reveals that LCP1 is a novel membrane associated target antigen in CLL with evidence of differential immune response as compared to common vaccine antigens. We also demonstrate that LCP1 plays a role in CLL microenvironment signaling and progression. Citation Format: Danielle L. Chappell, Jason A. Dubovsky, Leslie A. Andritsos, Joseph M. Flynn, Jeffrey A. Jones, Mike E. Paulaitis, Kitty Agrawal, John C. Byrd, Natarajan Muthusamy. Lymphocyte cytosolic protein 1 (LCP1) is a membrane-associated molecular target in chronic lymphocytic leukemia and is activated in microenvironment signaling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4925. doi:10.1158/1538-7445.AM2013-4925