Membrane Receptor Tyrosine Kinases Research Articles

Comprehensive analysis of Epha10 as a predictor of clinical prognosis and immune checkpoint therapy efficacy in non-small cell lung cancer

The EphA family belongs to a large group of membrane receptor tyrosine kinases. Emerging evidence indicates that the EphA family participates in tumour occurrence and progression. Nonetheless, the expression patterns and prognostic values of the nine EphAs in non-small cell lung cancer (NSCLC) have rarely been studied before. In the current study, we comprehensively analysed the expression and prognostic role of EphA family members by different means. The Cancer Genome Atlas and Gene Expression Profiling Interactive Analysis databases were used to investigate the expression of EphAs in NSCLC. The cBioPortal database was applied to analyse the prognostic values and genetic mutations of EphAs.We discovered that the expression of EphA10 was significantly higher in NSCLC tissues than in adjacent noncancerous tissues, and survival analyses showed that a higher level of EphA10 predicted poor prognosis. Further exploration into the role of EphA10 by ESTIMATE, CIBERSORT, and ssGSEA analysis found that it was also related to immune infiltration and higher expression of targets of ICI targets. In conclusion, this study revealed that among the EphA family members, EphA10 played an oncogenic role and was a promising biomarker for poor prognosis and better immunotherapy response in NSCLC.

GAS6/TAM Axis as Therapeutic Target in Liver Diseases.

TAM (TYRO3, AXL, and MERTK) protein tyrosine kinase membrane receptors and their vitamin K-dependent ligands GAS6 and protein S (PROS) are well-known players in tumor biology and autoimmune diseases. In contrast, TAM regulation of fibrogenesis and the inflammation mechanisms underlying metabolic dysfunction-associated steatohepatitis (MASH), cirrhosis, and, ultimately, liver cancer has recently been revealed. GAS6 and PROS binding to phosphatidylserine exposed in outer membranes of apoptotic cells links TAMs, particularly MERTK, with hepatocellular damage. In addition, AXL and MERTK regulate the development of liver fibrosis and inflammation in chronic liver diseases. Acute hepatic injury is also mediated by the TAM system, as recent data regarding acetaminophen toxicity and acute-on-chronic liver failure have uncovered. Soluble TAM-related proteins, mainly released from activated macrophages and hepatic stellate cells after hepatic deterioration, are proposed as early serum markers for disease progression. In conclusion, the TAM system is becoming an interesting pharmacological target in liver pathology and a focus of future biomedical research in this field.

GM1 structural requirements to mediate neuronal functions.

Since the 1980s, it has been known that the administration of ganglioside GM1 to cultured cells induced or enhanced neuronal differentiation. GM1 mechanism of action relies on its direct interaction and subsequent activation of the membrane tyrosine kinase receptor, TrkA, which naturally serves as NGF receptor. This process is mediated by the sole oligosaccharide portion of GM1, the pentasaccharide β-Gal-(1-3)-β-GalNAc-(1-4)-[α-Neu5Ac-(2-3)]-β-Gal-(1-4)-β-Glc.Here we detailed the minimum structural requirements of the oligosaccharide portion of GM1 for mediating the TrkA dependent neuritogenic processing. By in vitro and in silico biochemical approaches, we demonstrated that the minimal portion of GM1 required for the TrkA activation is the inner core of the ganglioside's oligosaccharide β-Gal-(1-3)-β-GalNAc-(1-4)-[α-Neu5Ac-(2-3)]-β-Gal. The addition of a sialic acid residue at position 3 of the outer galactose of the GM1 oligosaccharide, which forms the oligosaccharide of GD1a, prevented the interaction with TrkA and the resulting neuritogenesis. On the contrary, the addition of a fucose residue at position 2 of the outer galactose, forming the Fucosyl-GM1 oligosaccharide, did not prevent the TrkA-mediated neuritogenesis.

Nuclear translocation of Axl contributes to the malignancy of oral cancer cells

Background/purposeDysregulation of receptor tyrosine kinases is implicated in cancer development. This study aimed to investigate the nuclear translocation of Axl, a membrane protein and receptor tyrosine kinase in cancer malignancy. Materials and methodsWe examined Axl's entry into the cell nucleus and validated it with the nuclear export inhibitor leptomycin. Transfection experiments with mutated nuclear localization signals were conducted to assess the impact of reduced nuclear Axl levels on cancer cell malignancy. Additionally, we evaluated the effects of decreased nuclear Axl on sensitivity to radiation and cisplatin, a chemotherapeutic drug. ResultsIn the present study, we observed nuclear translocation of Axl in cancer cells. Reducing nuclear Axl levels led to a decrease in cancer cell malignancy. This nuclear translocation was further validated using a nuclear export inhibitor, leptomycin. Additionally, transfection experiments with mutated nuclear localization signals confirmed the functional significance of Axl's nuclear localization. Notably, decreased nuclear Axl levels also increased the sensitivity of cancer cells to radiation and cisplatin treatment. ConclusionThis study suggests that Axl's nuclear translocation plays a significant role in cancer malignancy. Targeting Axl's nuclear localization could offer a potential strategy to inhibit cancer progression and improve the efficacy of radiation and chemotherapy treatments.

Screening and Analysis of Possible Drugs Binding to PDGFRα: A Molecular Modeling Study.

The platelet-derived growth factor receptor (PDGFR) is a membrane tyrosine kinase receptor involved in several metabolic pathways, not only physiological but also pathological, as in tumor progression, immune-mediated diseases, and viral diseases. Considering this macromolecule as a druggable target for modulation/inhibition of these conditions, the aim of this work was to find new ligands or new information to design novel effective drugs. We performed an initial interaction screening with the human intracellular PDGFRα of about 7200 drugs and natural compounds contained in 5 independent databases/libraries implemented in the MTiOpenScreen web server. After the selection of 27 compounds, a structural analysis of the obtained complexes was performed. Three-dimensional quantitative structure-activity relationship (3D-QSAR) and absorption, distribution, metabolism, excretion, and toxicity (ADMET) analyses were also performed to understand the physicochemical properties of identified compounds to increase affinity and selectivity for PDGFRα. Among these 27 compounds, the drugs Bafetinib, Radotinib, Flumatinib, and Imatinib showed higher affinity for this tyrosine kinase receptor, lying in the nanomolar order, while the natural products included in this group, such as curcumin, luteolin, and epigallocatechin gallate (EGCG), showed sub-micromolar affinities. Although experimental studies are mandatory to fully understand the mechanisms behind PDGFRα inhibitors, the structural information obtained through this study could provide useful insight into the future development of more effective and targeted treatments for PDGFRα-related diseases, such as cancer and fibrosis.

Activated EGFR and PDGFR internalize in separate vesicles and downstream AKT and ERK1/2 signaling are differentially impacted by cholesterol depletion

The interplay between membrane subregions and receptor tyrosine kinases (RTK) will influence signaling in both normal and pathological RTK conditions. In this study, epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor β (PDGFR-β) internalizations were investigated by immunofluorescent microscopy following simultaneous treatment with EGF and PDGF-BB. We found that the two receptors utilize separate routes of internalization, which merges in a common perinuclear endosomal compartment after 45 min of stimulation. This is further strengthened when contrasting the recruitment of either EGFR or PDGFR-β to either clathrin or caveolin-1: PDGFR-β dissociates from caveolin-1 upon stimulation, and engages clathrin, whilst an increased recruitment of EGFR, to both clathrin and caveolin-1, was observed upon EGF stimulation. The association between EGFR and caveolin-1 is supported by the observation that EGFR was localized in lipid raft associated fractions, whereas PDGFR-β was not. We also found that disruption of lipid rafts using MβCD led to an increased EGFR dimerization and phosphorylation in response to ligand, as well as a dramatic decrease in AKT- and a smaller but robust decrease in ERK1/2 phosphorylation. This suggest that lipid rafts may be important to effectively connect the EGFR with downstream proteins to facilitate signaling. Our data implies that cholesterol depletion of the plasma membrane affect the signaling of EGFR and PDGFRβ differently.

Abstract 4039: RET overexpression is frequent in lung neuroendocrine tumors (NET) and associates with response to RET tyrosine kinase inhibitors (RET TKIs) in NET cell lines

Abstract Background: RET (rearranged during transfection) is a proto-oncogene encoding for a tyrosine kinase membrane receptor that promotes protein synthesis and cell proliferation. A small percentage of human tumors harbor RET fusions or mutations as oncogenic drivers and RET-TKIs have been developed. Two of them, selpercatinib and pralsetinib, have recently been approved. However, their effect on tumors overexpressing wild-type RET (wt-RET) has not been investigated. A large-scale mRNA analysis performed by our group revealed that ~2% of lung tumors present high expression of wt-RET in absence of fusions. Interestingly, ~60% of high wt-RET tumors presented neuroendocrine characteristics. Methods: Twenty large cell neuroendocrine (LCNEC) and 19 small cell (SCLC) lung carcinomas from two additional institutions were analyzed by nCounter® to validate the previous results. For in vitro experiments, a panel of cell lines derived from lung NETs (DMS-53, NCI-H82, NCI-H727) was used, together with a lung adenocarcinoma cell line (LC2/ad, a positive control harboring the CDCC6-RET translocation). RET fusions and mRNA expression were analyzed by nCounter®, RET protein expression by Western blotting and immunohistochemistry (IHC) and RET mutations by next generation sequencing. The inhibitory effects of selpercatinib and pralsetinib were analyzed in vitro by MTT in 2D and spheroid/3D cultures. Finally, in vivo experiments implanting tumor cells into athymic nude mice are ongoing. Results: Among the 20 LCNECs and 19 SCLCs analyzed, 3 (15%) and 3 (15%) presented very high levels of wt-RET mRNA by nCounter, respectively. Fusions were absent in all cases. The NCI-H82 and NCI-H727 NET cell lines showed very low RET mRNA and protein expression by nCounter, Western blotting and IHC. In contrast, DMS-53 cells presented levels of RET mRNA and protein higher than the observed in the RET-fusion positive LC2/ad. In 2D models, the three NET cell lines tested showed moderate resistance to pralsetinib and selpercatinib, with IC50s of 1.5 - 9.7 µM for pralsetinib and 4.6 - 6.1 µM for selpercatinib; while the RET-fusion positive LC2/ad cells showed an IC50 in the nM range, as expected. In 3D cultures, DMS-53 was found to be sensitive to RET TKIs, with IC50s of and 0.3 µM for praseltinib and 2.5 µM for selpercatinib. In contrast, spheroids of the two RET low cell lines showed complete resistance to both drugs (IC50>10 µM). Experiments in animal models are currently ongoing, final results will be presented at the meeting. Conclusions: Overexpression of RET is frequent in lung tumors with neuroendocrine features (LCNEC, SCLC). Selpercatinib and Pralsetinib show antitumor activity in lung NETs cells expressing high levels of wt-RET. Citation Format: Jaume Roca-Arias, Jordi Bertran-Alamillo, Ruth Román, Cristina Aguado, Laura López, Leticia Ferro-Leal, Beatriz Garcia-Pelaez, Rodrigo de Oliveira Cavagna, Silvia Teixeira, Rafael Rosell, Rui Manuel Reis, Miguel A. Molina-Vila, Ivana Sullivan. RET overexpression is frequent in lung neuroendocrine tumors (NET) and associates with response to RET tyrosine kinase inhibitors (RET TKIs) in NET cell lines. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4039.

Insulin receptor-inspired soluble insulin binder

The insulin receptor (IR) is a 320 kDa membrane receptor tyrosine kinase mediating the pleiotropic actions of insulin, leading to phosphorylation of several intracellular substrates including serine/threonine-protein kinase (AKT1), and IR autophosphorylation. Structural details of the IR have been recently revealed. A high-binding insulin site, L1 (Kd =2 nM), consists of two distant domains in the primary sequence of the IR. Our design simplified the L1 binding site and transformed it into a soluble insulin binder (sIB). The sIB, a 17 kDa protein, binds insulin with 38 nM affinity. The sIB competes with IR for insulin and reduces by more than 50% phosphorylation of AKT1 in HEK 293 T cells, with similar effects on IR autophosphorylation. The sIB represents a new tool for research of insulin binding and signaling properties.

Molecular and functional interaction of single pass ephrin receptor tyrosine kinase, EphA2 in lipid nanodisc using solution NMR.

Eph receptor, the largest subfamily of single pass trans membrane receptor tyrosine kinase family play crucial role as a regulator during embryonic development, cell maturation, and adulthood. EphA2, a member of the Eph family receptor tyrosine kinases (RTK) is a key regulator of tumorigenesis and cancer progression. EphA2 can be activated canonically by ephrin ligand which normally is a repulsive signal OR non-canonical (unliganded EphA2) promoting cell migration in several cancer types. Here we focus to dissect the effects of the sterile α motif (SAM) domain together with the phosphorylation on intracellular region (ICR) interactions as well as the transmembrane construct of EphA2 in solution to understand the signaling mechanism at molecular level. Our study of the ICR shows that the sterile α motif (SAM) domain acts as a regulator impeding kinase domains interaction and conversely increasing the kinase activity upon deletion of SAM domain. While the presence of the SAM domain hinders oligomerization of phosphorylated ICR, it appears that such protein-protein interactions are required for kinase activity regulation. Mutation studies of the kinase domain, SAM domain and the linker region between kinase domain and SAM domain give insight into its regulatory role for EphA2 activity. More importantly, we have recently optimized truncated construct with single Fibronectin-III, FN-III (Ecto)-Transmembrane (TM)-Juxta-Membrane, JM (ICR) in presence of DDM with well dispersed peaks in TROSY-HQSC indicating well folded protein. We will incorporate this truncated construct into MSP lipid nanodisc for NMR studies and biophysical characterization. In addition to the ICR regulation via SAM domain, we will have in-depth perspective on the configurational coupling of membrane-proximal transmembrane domains to each other.

Tyrosine kinase receptor RON activates MAPK/RSK/CREB signal pathway to enhance CXCR4 expression and promote cell migration and invasion in bladder cancer.

Bladder cancer (BC) is one of the most lethal malignancies worldwide. The poor survival may be due to a high proportion of tumor metastasis. RON and CXCR4 are the key regulators of cell motility in BC, while the relationship between RON and CXCR4 remains elusive. In the present study, immunohistochemistry analysis of BC and adjacent normal tissues found that higher RON expression was positively correlated with CXCR4 expression. Inhibiting and replenishing RON level were used to regulate CXCR4 expression, observing the effects on migration and invasion of BC cells. Overexpression of RON reversed the inhibited cell migration and invasion following siCXCR4 treatment. Conversely, overexpression of CXCR4 restored the inhibition of cell migration and invasion caused by shRON. The activation of RON-MAPK/RSK/CREB pathway was demonstrated in BC cells under MSP treatment. Dual luciferase and CHIP assay showed that p-CREB targeted CXCR4 by binding to its CRE sequence. RON knockdown suppressed BC tumor growth in xenograft mouse tumors, accompanied by reduced expression of CXCR4. In conclusion, our data adds evidence that RON, a membrane tyrosine kinase receptor, promotes BC migration and invasion not only by itself, but also by activating MAPK/RSK/CREB signaling pathway to enhance CXCR4 expression.

Discovery of Raf Family Is a Milestone in Deciphering the Ras-Mediated Intracellular Signaling Pathway.

The Ras-Raf-MEK-ERK signaling pathway, the first well-established MAPK pathway, plays essential roles in cell proliferation, survival, differentiation and development. It is activated in over 40% of human cancers owing to mutations of Ras, membrane receptor tyrosine kinases and other oncogenes. The Raf family consists of three isoforms, A-Raf, B-Raf and C-Raf. Since the first discovery of a truncated mutant of C-Raf as a transforming oncogene carried by a murine retrovirus, forty years of extensive studies have provided a wealth of information on the mechanisms underlying the activation, regulation and biological functions of the Raf family. However, the mechanisms by which activation of A-Raf and C-Raf is accomplished are still not completely understood. In contrast, B-Raf can be easily activated by binding of Ras-GTP, followed by cis-autophosphorylation of the activation loop, which accounts for the fact that this isoform is frequently mutated in many cancers, especially melanoma. The identification of oncogenic B-Raf mutations has led to accelerated drug development that targets Raf signaling in cancer. However, the effort has not proved as effective as anticipated, inasmuch as the mechanism of Raf activation involves multiple steps, factors and phosphorylation of different sites, as well as complex interactions between Raf isoforms. In this review, we will focus on the physiological complexity of the regulation of Raf kinases and their connection to the ERK phosphorylation cascade and then discuss the role of Raf in tumorigenesis and the clinical application of Raf inhibitors in the treatment of cancer.

ADAM10 Site-Dependent Biology: Keeping Control of a Pervasive Protease.

Enzymes, once considered static molecular machines acting in defined spatial patterns and sites of action, move to different intra- and extracellular locations, changing their function. This topological regulation revealed a close cross-talk between proteases and signaling events involving post-translational modifications, membrane tyrosine kinase receptors and G-protein coupled receptors, motor proteins shuttling cargos in intracellular vesicles, and small-molecule messengers. Here, we highlight recent advances in our knowledge of regulation and function of A Disintegrin And Metalloproteinase (ADAM) endopeptidases at specific subcellular sites, or in multimolecular complexes, with a special focus on ADAM10, and tumor necrosis factor-α convertase (TACE/ADAM17), since these two enzymes belong to the same family, share selected substrates and bioactivity. We will discuss some examples of ADAM10 activity modulated by changing partners and subcellular compartmentalization, with the underlying hypothesis that restraining protease activity by spatial segregation is a complex and powerful regulatory tool.

Phage-Displayed Peptides for Targeting Tyrosine Kinase Membrane Receptors in Cancer Therapy.

Receptor tyrosine kinases (RTKs) regulate critical physiological processes, such as cell growth, survival, motility, and metabolism. Abnormal activation of RTKs and relative downstream signaling is implicated in cancer pathogenesis. Phage display allows the rapid selection of peptide ligands of membrane receptors. These peptides can target in vitro and in vivo tumor cells and represent a novel therapeutic approach for cancer therapy. Further, they are more convenient compared to antibodies, being less expensive and non-immunogenic. In this review, we describe the state-of-the-art of phage display for development of peptide ligands of tyrosine kinase membrane receptors and discuss their potential applications for tumor-targeted therapy.

English

BACKGROUND Human epidermal growth factor receptor-2 HER2 / neu, is a trans membrane tyrosine kinase receptor of epidermal growth factor receptor (EGFR) family and is involved in the pathogenesis of urinary bladder cancer. In this study we attempted to evaluate the HER2 / neu expression in urothelial carcinoma of bladder and its association with tumour grading. METHODS This was a cross sectional study with a sample size of 75. Routine 4 micrometre thick sections of formalin fixed paraffin embedded tissue blocks stained with haematoxylin & eosin were reviewed. Tumour grade was determined by using the World Health Organization (WHO) / International Society of Urological Pathologist criteria (ISUP). Immunohistochemistry was done by using HER2 / neu monoclonal antibody and its expression were observed. The membrane staining intensity and pattern were studied and scored. RESULTS In our study 75 cases of urothelial carcinoma were studied, of which 49 cases were papillary urothelial carcinoma low grade, 26 cases were papillary urothelial carcinoma high grade. Among these, 19 cases were infiltrating urothelial carcinoma. HER2 / neu positivity were observed in 27 (36 %) cases and overexpression in 8 (10 %) cases. Low grade urothelial carcinoma showed HER2 / neu positivity in 11 (22 %) cases and overexpression in 1 (2 %) case. High grade urothelial carcinoma showed HER2 / neu positivity in 16 (64 %) cases, among which 7 (28 %) cases showed overexpression. HER2 / neu positivity was seen in 13 (68 %) cases of infiltrating urothelial carcinoma with 4 (21 %) cases showing overexpression. A statistically significant difference in HER2 / neu expression was noted in high grade and invasive urothelial carcinoma compared to low grade and non-invasive urothelial carcinoma. CONCLUSIONS Urothelial carcinomas show overexpression of HER2 / neu and this over expression increases with increasing grade of tumour and muscle invasiveness. KEYWORDS Urothelial Carcinoma, HER2 / neu, Overexpression, Tumour Grade, Trastuzumab

Transcriptional and Post-Translational Control Mechanisms for EphB4 Expression in Physiology and Cancer Disease.

EphB4 is a membrane tyrosine kinase receptor involved in a number of physiologic and pathologic conditions, cancer being the one drawing the highest attention. In mammalians, EphB4 regulates blood vessel growth and differentiation. EphB4 also mediates a number of cancer promoting effects including angiogenesis, invasion, and metastasis. Its effects have been linked to both EphrinB2-dependent and to EphrinB2-independent mechanisms. In spite of its emerging role in physiology and disease, the regulation of EphB4 cellular expression has been poorly described until recently. The present mini-review summarizes the latest findings on EphB4 regulation at the transcriptional and post-transcriptional level and discusses the biologic implications of such novel findings. Perspective on the scenarios opened by such findings and the open questions in EphB4 research is also provided.

PI3K/AKT/mTOR Signaling Pathway in Breast Cancer: From Molecular Landscape to Clinical Aspects.

Breast cancer is a serious health problem worldwide, representing the second cause of death through malignancies among women in developed countries. Population, endogenous and exogenous hormones, and physiological, genetic and breast-related factors are involved in breast cancer pathogenesis. The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) is a signaling pathway involved in cell proliferation, survival, invasion, migration, apoptosis, glucose metabolism and DNA repair. In breast tumors, PIK3CA somatic mutations have been reported, located in exon 9 and exon 20. Up to 40% of PIK3CA mutations are estrogen receptor (ER) positive and human epidermal growth factor receptor 2 (HER2) -negative in primary and metastatic breast cancer. HER2 is overexpressed in 20–30% of breast cancers. HER1, HER2, HER3 and HER4 are membrane receptor tyrosine kinases involved in HER signaling to which various ligands can be attached, leading to PI3K/AKT activation. Currently, clinical studies evaluate inhibitors of the PI3K/AKT/mTOR axis. The main purpose of this review is to present general aspects of breast cancer, the components of the AKT signaling pathway, the factors that activate this protein kinase B, PI3K/AKT-breast cancer mutations, PI3K/AKT/mTOR-inhibitors, and the relationship between everolimus, temsirolimus and endocrine therapy.

Working together for the family: determination of HER oncogene co-amplifications in breast cancer.

HER2 is a well-studied tyrosine kinase (TK) membrane receptor which functions as a therapeutic target in invasive ductal breast carcinomas (IDC). The standard of care for the treatment of HER2-positive breast is the antibody trastuzumab. Despite specific treatment unfortunately, 20% of primary and 70% of metastatic HER2 tumors develop resistance. HER2 belongs to a gene family, with four members (HER1-4) and these members could be involved in resistance to anti-HER2 therapies. In this study we designed a probemix to detect the amplification of the four HER oncogenes in a single reaction. In addition, we developed a protocol based on the combination of MLPA with ddPCR to detect the tumor proportion of co-amplified HERs. On 111 IDC, the HER2 MLPA results were validated by FISH (Adjusted r2 = 0,91, p < 0,0001), CISH (Adjusted r2 = 0,938, p < 0,0001) and IHC (Adjusted r2 = 0,31, p < 0,0001). HER1-4 MLPA results were validated by RT-qPCR assays (Spearman Rank test p < 0,05). Of the 111 samples, 26% presented at least one HER amplified, of which 23% showed co-amplifications with other HERs. The percentage of cells with HER2 co-amplified varied among the tumors (from 2–72,6%). Independent in-silico findings show that the outcome of HER2+ patients is conditioned by the status of HER3 and HER4. Our results encourage further studies to investigate the relationship with patient’s response to single or combined treatment. The approach could serve as proof of principle for other tumors in which the HER oncogenes are involved.

Abstract B02: Ephrin receptor A10 promotes PD-L1 expression for breast cancer immune evasion

Abstract Programmed death-ligand 1(PD-L1) is an immune checkpoint protein and its engagement with programmed cell death protein-1 (PD-1) receptor on T cells activates co-inhibitory signaling to induce cancer immune evasion. PD-L1 or PD-1 blockade has demonstrated encouraging clinical outcomes in several advanced cancer treatment. Elucidating the regulatory mechanisms of PD-L1 is critical to improve the efficiency of PD-L1-targeting immunotherapy. Recent studies indicated that contact-dependent pathways downregulate anticancer immunity, highlighting the importance of cell contact-induced signaling for cancer immune escape. In this study, we show that tumor cell contact promotes PD-L1 expression and reduces immune surveillance via membrane receptor tyrosine kinase ephrin receptor A10 (EphA10), which is only expressed in normal testis tissue and mediates cell contact-dependent juxtacrine signaling. Abolishing the expression of EphA10 enhances T-cell-mediated antitumor immunity in 4T1 mouse model. A positive correlation between EphA10 and PD-L1 expression is observed in human breast cancer tissues. Overall, our findings indicate that cell contact-mediated juxtacrine signaling increases PD-L1 expression, suggesting that tumor cells avoid immune surveillance via this mechanism and blockade of EphA10 may be a new immunotherapy strategy for breast cancer patients. Citation Format: Li-Chuan Chan, Jong-Ho Cha, Wen-Hao Yang, Weiya Xia, Heng-Huan Lee, Ying-Nai Wang, Jennifer L. Hsu, Guoxin Ren, Mien-Chie Hung. Ephrin receptor A10 promotes PD-L1 expression for breast cancer immune evasion [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B02.

Multiplexing Angiogenic Receptor Quantification via Quantum Dots.

Clinical and biomedical research seeks single-cell quantification to better understand their roles in a complex, multicell environment. Recently, quantification of vascular endothelial growth factor receptors (VEGFRs) provided important insights into endothelial cell characteristics and response in tumor microenvironments. However, existing technologies for quantifying plasma membrane receptor tyrosine kinases (RTKs) lack multiplexing capabilities, limiting detailed characterization. Here, we use the unique spectral properties of quantum dots (Qdots) to optimize and dually quantify VEGFR1 and VEGFR2 on human umbilical vein endothelial cells (HUVECs). To enable this quantification, we reduce nonspecific binding between Qdot-conjugated antibodies and cells via buffer optimization. Second, we identify optimal labeling conditions by examining Qdot-conjugated antibody binding to five receptors: VEGFRs (VEGFR1 and VEGFR2), their coreceptor neuropilin1 (NRP1), and platelet-derived growth factor receptor (PDGFRα and PDGFRβ). We establish that 800-20 000 is the dynamic range where accurate Qdot-enabled quantification can be achieved. Through these optimizations, we demonstrate measurement of 1 100 VEGFR1 and 6 900 VEGFR2 per HUVEC. We induce ∼90% upregulation of VEGFR1 and ∼30% downregulation of VEGFR2 concentration via 24 h VEGF-A165 treatment. We observe no change in VEGFR1 or VEGFR2 concentration with 24 h VEGF-B167 treatment. We further apply Qdots to analyze HUVEC heterogeneity and observe that 24 h VEGF-A165 treatment induces a ∼15% decrease in VEGFR2 heterogeneity, but little to no change in VEGFR1 heterogeneity. We observe that VEGF-B167 induces little to no change in either VEGFR1 or VEGFR2 heterogeneity. Overall, we demonstrate experimental and analytical strategies for quantifying two or more RTKs at single-level using Qdots, which will help provide new insights into biological systems.

Endocytosis and Transport of Growth Factor Receptors in Peripheral Axon Regeneration: Novel Lessons from Neurons Expressing Lysine-Deficient FGF Receptor Type 1 in vitro.

ABSTRACTIn the course of peripheral nerve regeneration, axons encounter different extracellular growth factors secreted by non‐neuronal cells at the injury site and retrogradely transported after binding to neuronal membrane receptor tyrosine kinases. The present study reviews the role of receptor transport in peripheral axon outgrowth and provides novel data on trafficking of fibroblast growth factor receptor type 1 (FGFR1). Differences in receptor transport are determined by different numbers of lysine residues acting as ubiquitination sites in the intracellular receptor domain. We previously demonstrated that overexpression of mutant FGFR1‐25R (25 out of 29 intracellular lysines replaced with arginine) results in enhanced receptor recycling as compared to wild‐type FGFR1 followed by strong stimulation of elongative axon growth in vitro. Here, the effects of lysine‐deficient FGFR1 (FGFR1‐29R lacking all 29 cytoplasmic lysine residues) or of only 15 lysine mutations (FGFR1‐15R) on axon outgrowth and concomitant changes in signal pathway activation were investigated by immunocytochemistry and morphometry of cultured primary neurons. Overexpression of FGFR1‐15R in adult sensory neurons resulted in enhanced receptor recycling, which was accompanied by increased axon elongation without stimulating axon branching. By contrast, FGFR1‐29R was neither endocytosed nor axon outgrowth affected. Although overexpression of FGFR1‐15R or FGFR1‐25Ra strongly promoted elongation, we did not detect increased signal pathway activation (ERK, AKT, PLC, or STAT3) in neurons expressing mutant FGFR1 as compared with wild‐type neurons raising the possibility that other signaling pathways or signaling independent mechanisms may be involved in the axon outgrowth effects of recycled FGF receptors. Anat Rec, 302:1268–1275, 2019. © 2019 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.