Cell Surface Membrane Proteins Research Articles

Low Expression MCEMP1 Promoting Lung Adenocarcinoma Progression by and its Clinical Value.

Lung cancer is a highly prevalent tumor with a lack of biological markers that reflect its progression. Mast cell surface membrane protein 1 (MCEMP1, also known as C19ORF59) has not been reported in lung adenocarcinoma (LUAD). We aimed to investigate the role of MCEMP1 in LUAD. MCEMP1 expression in LUAD was analyzed using The Cancer Genome Atlas (TCGA) data, and conducted univariate and multivariate Cox regression analyses to evaluate the prognostic significance of MCEMP1 expression in TCGA. Tumor Immune Estimation Resource (TIMER) was used for examining the correlation between MCEMP1 expression and immune cell infiltration in LUAD. Furthermore, proliferation, migration, invasion, and colony-forming ability were investigated using LUAD cell lines. MCEMP1 had low expression in LUAD patient tissues and was correlated with lymph node metastasis, differentiation level, and tumor status. The Area under Curve (AUC) value of MCEMP1 for the Receiver Operating Characteristic (ROC) curve analysis was 0.984. The immune infiltration analysis revealed a correlation between MCEMP1 expression and the extent of macrophages and neutrophil infiltration in LUAD. Additionally, MCEMP1 has low expression in clinical samples, MCEMP1 overexpressed in LUAD cells substantially reduced cell growth, migration, and invasion of malignant cells. Low expression MCEMP1 promotes LUAD progression, which provides new insights and a potential biological target for future LUAD therapies.

Glycolysis and tumor progression promoted by the m6A writer VIRMA via m6A-dependent upregulation of STRA6 in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal malignancies, highlighting the urgent need to elucidate the underlying oncogenic mechanisms. VIRMA is a classic isoform of methyltransferases that participates in epigenetic transcriptomic modification in eukaryotic mRNAs. However, the exact roles of VIRMA in PDAC remain unclear. Here, we identified that VIRMA is highly expressed in PDAC, and histone modifications of the promoter may partly account for this dysregulation. Moreover, VIRMA is closely related to glycolysis and poor prognosis in PDAC. We further determined that STRA6 is a direct downstream target of VIRMA in PDAC by RNA sequencing (RNA-seq) and m6A sequencing (m6A-seq). VIRMA is involved in gene expression regulation via 3’ UTR targeting of STRA6 mRNA. Furthermore, the m6A reader IGF2BP2 was shown to critically contribute to the stability of STRA6 mRNA. We describe the role of VIRMA in promoting signaling via the STRA6/STAT3 axis, which results in increased levels of HIF-1α, a key activator of glycolysis. In vivo and in vitro experiments reveal that the VIRMA-STRA6-STAT3-HIF-1α axis plays an instrumental role in glycolysis and tumor progression in PDAC. In conclusion, we demonstrate that VIRMA can increase glycolysis in PDAC by upregulating STRA6, a cell surface membrane protein that stimulates the STAT3 pathway, thereby activating HIF-1α and leading to pancreatic cancer malignancy. Overall, our data strongly suggest that the VIRMA-STRA6-STAT3-HIF-1α axis is a viable therapeutic target in PDAC.

Quantitative Membrane Proteomics for Discovery of Actionable Drug Targets at the Surface of RAS-Driven Human Cancer Cells.

With the advent of promising lung cancer immunotherapies targeting proteins at the cell surface of RAS-driven human cancers, the mass spectrometry (MS)-based surfaceomics remains a feasible strategy for therapeutic target discovery. This chapter describes a protocol for discovery of druggable protein targets at the surface of RAS-driven human cancer cells. This method relies on bottom-up MS-based quantitative surfaceomics that employs in parallel, targeted hydrazide-based cell-surface glycoproteomics and global shotgun membrane proteomics to enable unbiased quantitative profiling of thousands of cell surface membrane proteins. A large-scale molecular map of the KRASG12V surface was attained, resulting in confident detection and quantitation of more than 500 cell surface membrane proteins that were found to be unique or upregulated at the surface of cells harboring the KRASG12V mutant. A multistep bioinformatic progression revealed a subset of unique and/or significantly upregulated proteins as priority drug targets selected for orthogonal cross-validation using immunofluorescence, structured illumination microscopy, and western blotting. Among cross-validated targets, CUB domain containing protein 1 (CDCP1) and basigin (BSG-CD147) were selected as leading targets due to their involvement in cell adhesion and migration, consistent with the KRASG12V malignant phenotype as revealed by scanning electron microscopy and phenotypic cancer cell assays. Follow-up studies confirmed CDCP1 as an actionable therapeutic target, resulting in development of recombinant antibodies capable of killing KRAS-transformed cancer cells in preclinical setting. The present MS-based surfaceomics workflow represents a powerful drug target discovery platform that enables development of innovative immunotherapeutics (e.g., antibody drug conjugate against CDCP1) for attacking oncogenic RAS-driven cancers at the cell surface.

Insulin-like Growth Factor 2-Tagged Aptamer Chimeras (ITACs) Modular Assembly for Targeted and Efficient Degradation of Two Membrane Proteins.

Overexpression of pathogenic membrane proteins drives abnormal proliferation and invasion of tumor cells. Various strategies to durably knockdown membrane proteins with heterobifunctional degraders have been successfully developed, including LYTAC, KineTAC, and AbTAC. However, challenges including complicated synthetic procedures and the inability to simultaneously degrade multiple pathogenic proteins still exist. Herein, we developed insulin-like growth factor 2 (IGF2)-tagged aptamer chimeras (ITACs) that link the cell-surface lysosome-targeting receptor IGF2R and membrane proteins of interest (POIs) based on specific recognition of aptamers to the POIs and high-affinity binding of IGF2 to IGF2R. We demonstrated that ITACs exhibit robust degradation efficiency of various membrane proteins in multiple cell lines. Furthermore, systematic studies revealed that a moderate cell-surface IGF2R level is responsible for the excellent degradation performance of ITACs. Importantly, we further established a modular assembly strategy that allows assembly of one IGF2 with two aptamers with precise stoichiometry (dITACs), enabling cooperative and simultaneous degradation of two membrane proteins. This work provides an efficient and facile target membrane protein degradation platform and will shed light on the treatment of diseases related to the overexpression of membrane proteins.

Lateral interactions between CD276 and CD147 are essential for stemness in breast cancer: a novel insight from proximal proteome analysis

Oncogenic cell-surface membrane proteins contribute to the phenotypic and functional characteristics of cancer stem cells (CSCs). We employed a proximity-labeling proteomic approach to quantitatively analyze the cell-surface membrane proteins in close proximity to CD147 in CSCs. Furthermore, we compared CSCs to non-CSCs to identify CSC-specific cell-surface membrane proteins that are closely interact with CD147 and revealed that lateral interaction between CD147 and CD276 concealed within the lipid raft microdomain in CSCs, confers resistance to docetaxel, a commonly used chemotherapy agent for various cancer types, including metastatic breast cancer. Moreover, we investigated the clinical relevance of CD147 and CD276 co-expression in HER2+ breast cancer (BC) and triple-negative breast cancer patients who underwent chemotherapy. We observed poor disease-free survival and Overall survival rates in patients of CD147 and CD276 (p = 0.04 and 0.08, respectively). Subsequent immunohistochemical analysis in independent cohorts of HER2+ BC support for the association between co-expression of CD147 and CD276 and a poor response to chemotherapy. Collectively, our study suggests that the lateral interaction between CD147 and its proximal partners, such as CD276, may serve as a poor prognostic factor in BC and a predictive marker for the critical phenotypic determinant of BC stemness.

Cell surface profiling of cultured cells by direct hydrazide capture of oxidized glycoproteins

Glycoproteins are a particularly interesting subset of the cellular proteome as a high proportion of proteins present on the extracellular cell surface are glycosylated. These cell surface proteins are ideal targets for biologic drug therapies or for diagnostics tests. Here, we describe a modification of the well-described Cell Surface Capture (CSC) method for the selective isolation and identification of cell surface glycoproteins that contain N-linked carbohydrates. This modification, which we refer to as Direct Cell Surface Capture (D-CSC), is based on oxidation of cell surface glycans on intact cells, followed by direct conjugation of the oxidized oligosaccharides to a solid support using hydrazide chemistry, with no biotinylation step. As a proof-of-principle, we applied D-CSC to the analysis of cell surface membrane proteins of three adherent cancer cell lines (A549, OVCAR3, and U87MG) and compared our results to those published using the well-established Cell Surface Capture (CSC) method, demonstrating comparable selectivity for cell surface proteins.•A method enabling the identification of cell surface proteins from cells in culture is described.•Application of this method to profile the cell surface on three different cancer cell lines is included.

First person – Katherine Paine

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping researchers promote themselves alongside their papers. Katherine Paine is first author on ‘ The phosphatase Glc7 controls the eisosomal response to starvation via post-translational modification of Pil1’, published in JCS. Katherine conducted the research described in this article while a PhD student in Chris MacDonald's lab at University of York, York, UK. She is now a Postdoc in the lab of Simon Wilkinson at Institute of Genetics and Cancer, University of Edinburgh, UK. During her PhD, she was interested in the regulation of cell surface membrane proteins but has now moved into the field of autophagy, where she is working to understand factors involved in regulating this process.

Aptamers Targeting Membrane Proteins for Sensor and Diagnostic Applications.

Many biological processes (physiological or pathological) are relevant to membrane proteins (MPs), which account for almost 30% of the total of human proteins. As such, MPs can serve as predictive molecular biomarkers for disease diagnosis and prognosis. Indeed, cell surface MPs are an important class of attractive targets of the currently prescribed therapeutic drugs and diagnostic molecules used in disease detection. The oligonucleotides known as aptamers can be selected against a particular target with high affinity and selectivity by iterative rounds of in vitro library evolution, known as Systematic Evolution of Ligands by EXponential Enrichment (SELEX). As an alternative to antibodies, aptamers offer unique features like thermal stability, low-cost, reuse, ease of chemical modification, and compatibility with various detection techniques. Particularly, immobilized-aptamer sensing platforms have been under investigation for diagnostics and have demonstrated significant value compared to other analytical techniques. These "aptasensors" can be classified into several types based on their working principle, which are commonly electrochemical, optical, or mass-sensitive. In this review, we review the studies on aptamer-based MP-sensing technologies for diagnostic applications and have included new methodological variations undertaken in recent years.

Comparative site-specific N-glycoproteome analysis reveals aberrant N-glycosylation and gives insights into mannose-6-phosphate pathway in cancer

N-glycosylation is implicated in cancers and aberrant N-glycosylation is recognized as a hallmark of cancer. Here, we mapped and compared the site-specific N-glycoproteomes of colon cancer HCT116 cells and isogenic non-tumorigenic DNMT1/3b double knockout (DKO1) cells using Fbs1-GYR N-glycopeptide enrichment technology and trapped ion mobility spectrometry. Many significant changes in site-specific N-glycosylation were revealed, providing a molecular basis for further elucidation of the role of N-glycosylation in protein function. HCT116 cells display hypersialylation especially in cell surface membrane proteins. Both HCT116 and DKO1 show an abundance of paucimannose and 80% of paucimannose-rich proteins are annotated to reside in exosomes. The most striking N-glycosylation alteration was the degree of mannose-6-phosphate (M6P) modification. N-glycoproteomic analyses revealed that HCT116 displays hyper-M6P modification, which was orthogonally validated by M6P immunodetection. Significant observed differences in N-glycosylation patterns of the major M6P receptor, CI-MPR in HCT116 and DKO1 may contribute to the hyper-M6P phenotype of HCT116 cells. This comparative site-specific N-glycoproteome analysis provides a pool of potential N-glycosylation-related cancer biomarkers, but also gives insights into the M6P pathway in cancer.

Role of phosphatidylserine in the localization of cell surface membrane proteins in yeast.

Phosphatidylserine (PS) is a constituent of the cell membrane, being especially abundant in the cytoplasmic leaflet, and plays important roles in a number of cellular functions, including the formation of cell polarity and intracellular vesicle transport. Several studies in mammalian cells have suggested the role of PS in retrograde membrane traffic through endosomes, but in yeast, where PS is localized primarily at the plasma membrane (PM), the role in intracellular organelles remains unclear. Additionally, it is reported that polarized endocytic site formation is defective in PS-depleted yeast cells, but the role in the endocytic machinery has not been well understood. In this study, to clarify the role of PS in the endocytic pathway, we analyzed the effect of PS depletion on endocytic internalization and post-endocytic transport. We demonstrated that in cell lacking the PS synthase Cho1p (cho1Δ cell), binding and internalization of mating pheromone α-factor into the cell was severely impaired. Interestingly, the processes of endocytosis were mostly unaffected, but protein transport from the trans-Golgi network (TGN) to the PM was defective and localization of cell surface proteins was severely impaired in cho1Δ cells. We also showed that PS accumulated in intracellular compartments in cells lacking Rcy1p and Vps52p, both of which are implicated in endosome-to-PM transport via the TGN, and that the number of Snx4p-residing endosomes was increased in cho1Δ cells. These results suggest that PS plays a crucial role in the transport and localization of cell surface membrane proteins.Key words: phosphatidylserine, endocytosis, recycling, vesicle transport.

Cell Surface Biotinylation Using Furan Cross-Linking Chemistry.

A detailed study of the cellular surfaceome poses major challenges for mass spectrometry analysis. Surface proteins are low abundant compared to intracellular proteins, and their inefficient extraction in aqueous medium leads to their aggregation and precipitation. To tackle such problems, surface biotinylation is frequently used to tag surface proteins with biotin, allowing for their enrichment, leading to a more sensitive mapping of surface proteomes. We here detail a new surface biotinylation protocol based on furan-biotin affinity purification to enrich plasma membrane proteins for proteomics. This protocol involves biotinylation of cell surface membrane proteins on viable cells, followed by affinity enrichment using streptavidin beads, trypsin digestion, peptide cleanup, and LC-MS/MS analysis.

Molecular Mechanism Exploration of Autologous Blood Transfusion with RBC Surface Membrane Protein pMHC/aCD28 Combined with CD8+T Cells to Promote the Proliferation of CD8+T Cells to Inhibit the Malignant Transformation of Liver Cancer.

Autologous blood transfusion is an important blood protection measure. Red blood cells have a certain degree of immunogenicity and their surface membrane proteins CD28 and MHC can participate in the immune response and interact with CD8+ T cells. We build a cell model with a transwell system. The binding characteristics of RBCs and CD8+ T cells were observed with a fluorescent confocal microscope. The content of the inflammatory factor TNF-α and IFN-γ produced was analyzed by ELISA. The proliferation characteristics of CD8+ T cells were analyzed by CFSE staining, and the content of CD3+CD8+ T cells was analyzed by flow cytometry. Cell migration and invasion experiments were used to analyze the malignant metastasis ability of liver cancer cells. The expression of vimentin, E-cadherin, and β-catenin was analyzed by Western blot. We establish a liver cancer model in rats and group them for autologous blood transfusion. The content of CD3+CD8+T cells in the blood of each group of rats was analyzed by flow cytometry. Western blot was used to analyze the expression of vimentin, E-cadherin, and β-catenin in the liver tissues of rats in each group. The red blood cells in the autologous reinfusion blood and CD8+ T cells have an obvious combination. The degree of combination of the two is related to the expression of CD28 and MHC. If CD28 and MHC are expressed at the same time, the combination of the two cells will be high, the proliferation of CD8+ T cells will increase, and the expression of inflammatory factors will also increase, while the expression of the three proteins that are positively correlated with the activity of cancer cells will decrease. If only one of CD28 and MHC is normally expressed, the result is contrary to the situation where both membrane proteins are normally expressed. Our project has proved that autologous infusion of red blood cell surface membrane proteins CD28 and MHC combined with CD8+ T cells can promote the proliferation of CD8+ T cells to inhibit the malignant transformation of liver cancer.

Recycling of cell surface membrane proteins from yeast endosomes is regulated by ubiquitinated Ist1.

Upon internalization, many surface membrane proteins are recycled back to the plasma membrane. Although these endosomal trafficking pathways control surface protein activity, the precise regulatory features and division of labor between interconnected pathways are poorly defined. In yeast, we show recycling back to the surface occurs through distinct pathways. In addition to retrograde recycling pathways via the late Golgi, used by synaptobrevins and driven by cargo ubiquitination, we find nutrient transporter recycling bypasses the Golgi in a pathway driven by cargo deubiquitination. Nutrient transporters rapidly internalize to, and recycle from, endosomes marked by the ESCRT-III associated factor Ist1. This compartment serves as both "early" and "recycling" endosome. We show Ist1 is ubiquitinated and that this is required for proper endosomal recruitment and cargo recycling to the surface. Additionally, the essential ATPase Cdc48 and its adaptor Npl4 are required for recycling, potentially through regulation of ubiquitinated Ist1. This collectively suggests mechanistic features of recycling from endosomes to the plasma membrane are conserved.

NMDA receptor modulation in protracted ethanol withdrawal

Functional dysregulation of the glutamate system during withdrawal from chronic drug exposure is a primary driver of drug craving and relapse. Animal models of psychostimulant use have demonstrated dynamic alterations in NMDA receptor function and expression that contribute to drug seeking behavior. These changes begin as early as 5 days following cessation of drug use are persistently expressed into long term withdrawal (>60d). Evidence of NMDA receptor dependent changes during short term withdrawal (24h) suggest that similar mechanisms may drive drug craving and relapse behaviors following chronic exposure to ethanol. To this end, we investigated NMDA receptor protein and surface expression during protracted withdrawal from chronic intermittent ethanol (CIE) exposure using western blot. We focused on the basolateral amygdala (BLA), as glutamatergic signaling in this region is robustly modulated by short term (24h) withdrawal from CIE (10d, 12hr/day) and regulates anxiety‐like behavior expressed during withdrawal. Adolescent male rats were exposed to repeated cycles of CIE (12hr/day, 4d on/3d off, 3 cycles) and brain tissue was collected during protracted withdrawal. Optimization of experimental procedures is currently being conducted and analysis of total and membrane surface expression will be analyzed by surface biotinylation for NMDAR subunits (GluN1, GluN2B, GluN3). Cell surface biotinylation will be used to isolate cell surface membrane proteins to detect and quantify expression of NMDAR subunits. We expect to identify an increase in membrane surface expression for NMDAR subunits, in agreement with functional data obtained in the laboratory.

A new photolabeling probe for efficient enrichment and deep profiling of cell surface membrane proteome by mass spectrometry

The cell surface membrane proteome is a class of proteins encoded by ∼25% of all protein-coding genes in living organisms and plays a key role in mediating communication between the cells and their surrounding environment. However, most cell surface membrane proteins (CSMPs) are naturally expressed at very low levels compared with intracellular proteins. The difficulties in their purification with high specificity further hinder the understanding of their structure and function. In this study, we developed a new photolabeling probe to achieve efficient tagging and facile enrichment of the CSMPs. The probe is composed of a lipid tail for cell surface localization, a polyethylene glycol (PEG) spacer for increased water solubility, two 4-(N-maleimido)benzophenone (MBP) groups for UV-active tagging of the CSMPs, and a biotin tag for subsequent isolation. Application of this photolabeling probe resulted in the successful enrichment and identification of 3098 annotated CSMPs in HT22 cells with close to 70% selectivity. The proposed photolabeling probe and enrichment strategy were demonstrated to be a powerful method for deep cell surface proteome profiling, representing one of the largest groups of current drug targets.

Paraneoplastic and Other Autoimmune Encephalitides: Antineuronal Antibodies, T Lymphocytes, and Questions of Pathogenesis.

Autoimmune and paraneoplastic encephalitides represent an increasingly recognized cause of devastating human illness as well as an emerging area of neurological injury associated with immune checkpoint inhibitors. Two groups of antibodies have been detected in affected patients. Antibodies in the first group are directed against neuronal cell surface membrane proteins and are exemplified by antibodies directed against the N-methyl-D-aspartate receptor (anti-NMDAR), found in patients with autoimmune encephalitis, and antibodies directed against the leucine-rich glioma-inactivated 1 protein (anti-LGI1), associated with faciobrachial dystonic seizures and limbic encephalitis. Antibodies in this group produce non-lethal neuronal dysfunction, and their associated conditions often respond to treatment. Antibodies in the second group, as exemplified by anti-Yo antibody, found in patients with rapidly progressive cerebellar syndrome, and anti-Hu antibody, associated with encephalomyelitis, react with intracellular neuronal antigens. These antibodies are characteristically found in patients with underlying malignancy, and neurological impairment is the result of neuronal death. Within the last few years, major advances have been made in understanding the pathogenesis of neurological disorders associated with antibodies against neuronal cell surface antigens. In contrast, the events that lead to neuronal death in conditions associated with antibodies directed against intracellular antigens, such as anti-Yo and anti-Hu, remain poorly understood, and the respective roles of antibodies and T lymphocytes in causing neuronal injury have not been defined in an animal model. In this review, we discuss current knowledge of these two groups of antibodies in terms of their discovery, how they arise, the interaction of both types of antibodies with their molecular targets, and the attempts that have been made to reproduce human neuronal injury in tissue culture models and experimental animals. We then discuss the emerging area of autoimmune neuronal injury associated with immune checkpoint inhibitors and the implications of current research for the treatment of affected patients.

Pax5 mediates the transcriptional activation of the CD81 gene

CD81 is an integral membrane protein of the tetraspanin family and forms complexes with a variety of other cell surface membrane proteins. CD81 is involved in cell migration and B cell activation. However, the mechanism of the transcriptional regulation of the CD81 gene remains unclear. Here, we revealed that CD81 transcriptional activation was required for binding of the transcription factor Pax5 at the Pax5-binding sequence (-54)GCGGGAC(-48) located upstream of the transcriptional start site (TSS) of the CD81 gene. The reporter assay showed that the DNA sequence between − 130 and − 39 bp upstream of the TSS of the CD81 gene had promoter activity for CD81 transcription. The DNA sequence between − 130 and − 39 bp upstream of TSS of CD81 harbors two potential Pax5-binding sequences (-87)GCGTGAG(-81) and (-54)GCGGGAC(-48). Reporter, electrophoresis mobility shift, and chromatin immunoprecipitation (ChIP) assays disclosed that Pax5 bound to the (-54)GCGGGAC(-48) in the promoter region of the CD81 gene in order to activate CD81 transcription. Pax5 overexpression increased the expression level of CD81 protein, while the Pax5-knockdown by shRNA decreased CD81 expression. Moreover, we found that the expression level of CD81 was positively correlated with Pax5 expression in human tumor cell lines. Because CD81 was reported to be involved in cell migration, we evaluated the effects of Pax5 overexpression by wound healing and transwell assays. The data showed that overexpression of either Pax5 or CD81 promoted the epithelial cell migration. Thus, our findings provide insights into the transcriptional mechanism of the CD81 gene through transcription factor Pax5.

Fur4-mediated uracil-scavenging to screen for surface protein regulators.

Cell surface membrane proteins perform diverse and critical functions and are spatially and temporally regulated by membrane trafficking pathways. Although perturbations in these pathways underlie many pathologies, our understanding of these pathways at a mechanistic level remains incomplete. Using yeast as a model, we have developed an assay that reports on the surface activity of the uracil permease Fur4 in uracil auxotroph strains grown in the presence of limited uracil. This assay was used to screen a library of haploid deletion strains and identified mutants with both diminished and enhanced comparative growth in restricted uracil media. Factors identified, including various multisubunit complexes, were enriched for membrane trafficking and transcriptional functions, in addition to various uncharacterized genes. Bioinformatic analysis of expression profiles from many strains lacking transcription factors required for efficient uracil‐scavenging validated particular hits from the screen, in addition to implicating essential genes not tested in the screen. Finally, we performed a secondary mating factor secretion screen to functionally categorize factors implicated in uracil‐scavenging.

Targeted Mass Spectrometry-Based Approach for the Determination of Intrinsic Internalization Kinetics of Cell-Surface Membrane Protein Targets.

Successful development of targeted therapeutics aimed at the elimination of diseased cells relies on the target properties and the therapeutics that target them. Currently, target properties have been evaluated through antibody-dependent semiquantitative approaches such as flow cytometry, Western blotting, or microscopy. Since antibodies can alter target properties following binding, antibody-dependent approaches provide at best skewed measurements for target intrinsic properties. To circumvent, here we attempted to develop an antibody-free targeted mass spectrometry-based (ATM) strategy to measure the surface densities and the intrinsic rates (Kint) of CD38 internalization in multiple myeloma cell lines. Using cell-surface biotinylation in conjunction with differential mass tagging to separate inward CD38 molecules from the outbound and nascent ones, the ATM approach revealed diversities in measured CD38 Kint values of 0.239 min-1 S.E. ± 0.076, 0.109 min-1 S.E. ± 0.032, and 0.058 min-1 S.E. ± 0.001 for LP1, NCIH929, and MOLP8 cell lines, respectively. Together with CD38 surface densities, intrinsic Kint values aligned well with the tumor penetration model and supported the outcomes for tumor regression in mouse xenografts upon drug treatment. Additionally, the ATM approach can evaluate molecules with fast Kint as we determined for CTLA4 protein. We believe that the ATM approach has the potential to evaluate diverse cell-surface targets as part of the pharmacological assessment in drug discovery.

Abstract 1989: LSR is a novel prognostic factor by regulating cell proliferation via MEK/ERK pathway in endometrial cancer:Analysis of signal transduction, bioinformatics, and in vitro/in vivo study

Abstract Introduction: Advanced endometrial cancer (EC) still has poor prognosis and requires the development of new therapeutic agents. In ovarian and gastric cancer, we revealed that high expression of lipolysis-stimulated lipoprotein receptor (LSR), a cell-surface membrane protein, was associated with poor prognosis and LSR could be a new therapeutic target. However, in EC, the clinical and cell biological role of LSR is still unclear. We aimed to investigate the functions of LSR in EC. Methods: We evaluated LSR expression by immunohistochemistry and analyzed overall survival (OS) and clinicopathological factors in 228 EC patients. To investigate the mechanism by which LSR affects the prognosis of EC patients, the pathway enrichment analysis and gene ontology analysis were conducted using the publicly available proteogenomic dataset of EC. Cell proliferation was analyzed by WST-8 assay using two LSR-knockdown cell lines (HEC1 and HEC116) developed by transfection of LSR-siRNA, and the activity of several signaling pathways were examined by Western blotting. Results: Patients were divided into two groups based on LSR expression; High (darkly stained cells in ≥25% of the area, n=153) and Low (darkly stained cells in <25% of the area, n=75) groups. 5-year OS rate in High group was significantly lower than Low group (hazard ratio: 3.53, 95% confidence interval: 1.35 - 9.24, p=0.01). Stage III-IV, deep myometrial invasion (≥75% of uterine myometrium), tumor involvement of adnexa or serosa, and distant metastasis were more frequently observed in High group than in Low group (p<0.05, respectively). The pathway analysis demonstrated that genes correlated with high LSR expression were enriched in MAPK signaling pathway. In gene ontology analysis, these genes were enriched in regulation of ERK1/2 and MAPK cascade. In vitro, LSR-knockdown suppressed cell proliferation (p<0.01) and phosphorylation of MEK1/2, ERK1/2, and p90RSK, suggesting that LSR promoted cell proliferation via MEK/ERK pathway. However, LSR did not regulate the other signaling pathways (SAPK/JNK, p38-MAPK, JAK/STAT3, and PI3K/AKT/mTOR pathway). Conclusion: LSR regulated cell proliferation via MEK/ERK pathway, and contributed poor prognosis in EC. LSR may be a new therapeutic target of advanced EC. Citation Format: Yoshikazu Nagase, Kosuke Hiramatsu, Satoshi Nakagawa, Shinya Matsuzaki, Toshihiro Kimura, Satoshi Serada, Yutaka Ueda, Tetsuji Naka, Tadashi Kimura. LSR is a novel prognostic factor by regulating cell proliferation via MEK/ERK pathway in endometrial cancer:Analysis of signal transduction, bioinformatics, and in vitro/in vivo study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1989.