Plexin Research Articles

Semaphorin Receptors Antagonize Wnt Signaling Through Beta-Catenin Degradation.

Precise control of morphogen signaling levels is essential for proper development. An outstanding question is: what mechanisms ensure proper morphogen activity and correct cellular responses? Previous work has identified Semaphorin (SEMA) receptors, Neuropilins (NRPs) and Plexins (PLXNs), as positive regulators of the Hedgehog (HH) signaling pathway. Here, we provide evidence that NRPs and PLXNs antagonize Wnt signaling in both fibroblasts and epithelial cells. Further, Nrp1/2 deletion in fibroblasts results in elevated baseline Wnt pathway activity and increased maximal responses to Wnt stimulation. Notably, and in contrast to HH signaling, SEMA receptor-mediated Wnt antagonism is independent of primary cilia. Mechanistically, PLXNs and NRPs act downstream of Dishevelled (DVL) to destabilize β-catenin (CTNNB1) in a proteosome-dependent manner. Further, NRPs, but not PLXNs, act in a GSK3b/CK1-dependent fashion to antagonize Wnt signaling, suggesting distinct repressive mechanisms for these SEMA receptors. Overall, this study identifies SEMA receptors as novel Wnt pathway antagonists that may also play larger roles integrating signals from multiple inputs.

Expression of semaphorin-3A in the joint and role in osteoarthritis.

Osteoarthritis (OA) is characterised by the deterioration of cartilage in the joints and pain. We hypothesise that semaphorin-3A (sema-3A), a chemorepellent for sensory nerves, plays a role in joint degradation and pain. We used the mechanical joint loading (MJL) model of OA to investigate sema-3A expression in the joint and examine its association with the development of OA and pain. We also analyse its effect on chondrocyte differentiation using the ATDC5 cell line. We demonstrate that sema-3A is present in most tissues in the healthy joint and its expression increases in highly innervated tissues, such as cruciate ligaments, synovial lining and subchondral bone, in loaded compared to nonloaded control joints. In contrast, sema-3A expression in cartilage was decreased in the severe OA induced by the application of high loads. There was a significant increase in circulating sema-3A, 6 weeks after MJL compared to the nonloaded mice. mRNA for sema-3A and its receptor Plexin A1 were upregulated in the dorsal root ganglia of mice submitted to MJL. These increases were supressed by zoledronate, an inhibitor of bone pain. Sema-3A was expressed at all stages of Chondrocyte maturation and, when added exogenously, stimulated expression of markers of chondrocyte differentiation. This indicates that sema-3A could affect joint tissues distinctively during the development of OA. In highly innervated joint tissues, sema-3A could control innervation and/or induce pain-associated neuronal changes. In cartilage, sema-3A could favour its degeneration by modifying chondrocyte differentiation.

Single-Cell Transcriptional Response of the Placenta to the Ablation of Caveolin-1: Insights into the Adaptive Regulation of Brain-Placental Axis in Mice.

Caveolin-1 (Cav1) is a major plasma membrane protein that plays important functions in cellular metabolism, proliferation, and senescence. Mice lacking Cav1 show abnormal gene expression in the fetal brain. Though evidence for placental influence on brain development is emerging, whether the ablation of Cav1 affects the regulation of the brain-placental axis remains unexamined. The current study tests the hypothesis that gene expression changes in specific cells of the placenta and the fetal brain are linked to the deregulation of the brain-placental axis in Cav1-null mice. By performing single-nuclei RNA sequencing (snRNA-seq) analyses, we show that the abundance of the extravillious trophoblast (EVT) and stromal cells, but not the cytotrophoblast (CTB) or syncytiotrophoblast (STB), are significantly impacted due to Cav1 ablation in mice. Interestingly, specific genes related to brain development and neurogenesis were significantly differentially expressed in trophoblast cells due to Cav1 deletion. Comparison of single-cell gene expression between the placenta and the fetal brain further showed that specific genes such as plexin A1 (Plxna1), phosphatase and actin regulator 1 (Phactr1) and amyloid precursor-like protein 2 (Aplp2) were differentially expressed between the EVT and STB cells of the placenta, and also, between the radial glia and ependymal cells of the fetal brain. Bulk RNA-seq analysis of the whole placenta and the fetal brain further identified genes differentially expressed in a similar manner between the placenta and the fetal brain due to the absence of Cav1. The deconvolution of reference cell types from the bulk RNA-seq data further showed that the loss of Cav1 impacted the abundance of EVT cells relative to the stromal cells in the placenta, and that of the glia cells relative to the neuronal cells in the fetal brain. Together, the results of this study suggest that the ablation of Cav1 causes deregulated gene expression in specific cell types of the placenta and the fetal brain in mice.

Transcriptome Analysis of the Spleen Provides Insight into the Immune Regulation of GCRV Resistance in Grass Carp (Ctenopharyngodon idella).

Grass carp (Ctenopharyngodon idella) is one of the most economically important fish in China, and its production is commonly lost due to GCRV infection. To understand the molecular mechanism of GCRV resistance in grass carp, we compared the spleen transcriptome of the GCRV-resistant and susceptible individuals under GCRV infection (Res-Sus) and the GCRV-resistant individuals under different conditions of injection with GCRV and PBS (Res-Ctl). A total of 87.56GB of clean data were obtained from 12 transcriptomic libraries of spleen tissues. A total of 379 DEGs (156 upregulated genes and 223 downregulated genes) were identified in the comparison group Res-Ctl. A total of 1207 DEGs (633 upregulated genes and 574 downregulated genes) were identified in the comparison group Res-Sus. And 54 DEGs were shared including immune-related genes of stc2 (stanniocalcin 2), plxna1 (plexin A1), ifnα (interferon alpha), cxcl 11 (C-X-C motif chemokine ligand 11), ngfr (nerve growth factor receptor), mx (MX dynamin-like GTPase), crim1 (cysteine-rich transmembrane BMP regulator 1), plxnb2 (plexin B2), and slit2 (slit guidance ligand 2). KEGG pathway analysis revealed significant differences in the expression of genes mainly involved in immune system and signal transduction, including antigen processing and presentation, Toll-like receptor signaling pathway, natural killer cell-mediated cytotoxicity, and Hippo signaling pathway. This study investigates the immune mechanism of the resistance to GCRV infection in grass carp and provides useful information for the development of methods to control the spread of the GCRV infection.

RGC-32 facilitates reactive astrocytosis by modulating the expression of axonal guidance molecules

Abstract Response Gene to Complement (RGC)-32 modulates TGF-β-induced extracellular matrix secretion and the ability of astrocytes to undergo reactive changes in vivo during experimental autoimmune encephalomyelitis (EAE). However, the molecular pathways underlying these effects are still not well understood. In this study, we investigated how lack of RGC-32 affects the transcriptomic profile and the expression of axonal guidance molecules (AGM) in astrocytes during EAE. We performed next-generation RNA sequencing on brain neonatal astrocytes isolated from wild type (WT) and RGC-32 knock-out (KO) mice, either unstimulated or stimulated with TGF-β. Results were then validated by using Real-Time PCR. Spinal cords from WT and RGC-32 KO mice with EAE (at days 0 and 14) were stained by immunohistochemistry for the astrocyte marker GFAP, AGM, and for nuclear factor IA (NFIA), a gliogenic factor and transcriptional regulator of AGM. Lack of RGC-32 had a significant impact on the transcriptomic programs normally associated with brain development whose re-expression is usually seen in reactive astrocytes. Connectivity analysis revealed that genes coding for AGM were particularly affected. We found lower transcript levels of ephrin receptor A type 7 (Epha7), plexin A1 and Slit guidance ligand 2 in RGC-32 KO astrocytes. Moreover, our results showed that NFIA and EPHA7 are expressed by astrocytes during EAE. We found a lower number of astrocytes expressing EPHA7 and NFIA in RGC-32 KO mice with acute EAE when compared with WT mice. These results suggest that RGC-32 might facilitate reactive astrogliosis during acute EAE through regulating the expression of AGM and NFIA. Veterans Administration Merit Award I01BX001458 (to HR)

Upregulated Genes in Contact Co-culture of Osteoblasts and Sympathetic Neuronal Cells

Many works have been done on the interaction between neurons and osteoblasts. However, the cells used in our previous study were derived from mouse and rat, and it is impossible to exclude the possibility of cross-reactivity of the mouse and rat probes Osteoblasts cell line labeled with red fluorescent protein and sympathetic neuronal cell line labeled with green fluorescent protein were generated. A DNA microarray analysis was conducted by using these cell line in co-culture system. It was filtered for genes with a ratio more than 2.0-fold in the biological process category by the search term “axon” and cellular component by the search term “membrane”. Eight genes, i.e., neuropilin 1, growth associated protein 43, dopamine receptor D2, plexin A2, disabled 1, ephrin receptor A7, ephrin B2, and fibronectin leucine rich transmembrane protein 3 were found. The molecules expressed on the plasma membrane of osteoblasts were upregulated in contact co-culture with sympathetic neuronal cells. It was filtered for genes with a ratio more than 2.0-fold in the biological process category by the search term “osteoblast” and cellular component by the search term “membrane”. Four genes, i.e., gap junction protein, alpha 1, collagen, type VI, alpha 1 and bone morphogenetic protein 4 were found. The molecules expressed on the plasma membrane of neurons were upregulated in co-culture with osteoblasts. These molecules may be candidates for factors that promote osteoblast differentiation and neural differentiation.

Semaphorin 6D–expressing mesenchymal cells regulate IL-10 production by ILC2s in the lung

Group 2 innate lymphoid cells (ILC2s) have been implicated in both physiologic tissue remodeling and allergic pathology, yet the niche signaling required for ILC2 properties is poorly understood. Here, we show that an axonal guidance cue semaphorin 6D (Sema6D) plays critical roles in the maintenance of IL-10-producing ILC2s. Sema6d -/- mice exhibit a severe steady-state reduction in ILC2s in peripheral sites such as the lung, visceral adipose tissue, and mesentery. Interestingly, loss of Sema6D results in suppressed alarmin-driven type 2 cytokine production but increased IL-10 production by lung ILC2s both in vitro and in vivo. Consequently, Sema6d -/- mice are resistant to the development of allergic lung inflammation. We further found that lung mesenchymal cells highly express Sema6D, and that niche-derived Sema6D is responsible for these phenotypes through plexin A1. Collectively, these findings suggest that niche-derived Sema6D is implicated in physiological and pathological characteristics of ILC2s.

Efficacy of an Anti-Semaphorin 3A Neutralizing Antibody in a Male Experimental Retinal Vein Occlusion Mouse Model.

PurposeSemaphorin 3A (Sema3A) is a promising therapeutic target for macular edema in age-related macular degeneration, diabetic retinopathy, and retinal vein occlusion (RVO). Anti-vascular endothelial growth factors (anti-VEGFs) are the current standard of care for many retinal diseases. This study investigated the Sema3A neutralizing antibody BI-X and/or anti-VEGF therapy (aflibercept) in an RVO mouse model. Treatment efficacy was examined and grouped by timing subsequent to the RVO mouse model induction: efficacy against the onset of intraretinal edema 1 day postinduction and protective effects at 7 days postinduction.MethodsWe examined the changes in expression of Sema3A in the retina of an RVO mouse model. In addition, changes in expression of tumor necrosis factor (TNF)-α and semaphorin-related proteins (neuropilin-1 and plexin A1) in the retina upon treatment were analyzed by Western blotting. The effects of BI-X and/or aflibercept were evaluated using measures of retinal edema, blood flow, and thinning of the inner nuclear layer.ResultsInduction of vein occlusion in the RVO mouse model significantly increased Sema3A expression in the retina, particularly in the inner nuclear layer. BI-X was effective as a monotherapy and in combination with anti-VEGF therapy, demonstrating a beneficial effect on intraretinal edema and retinal blood flow. Moreover, in the RVO mouse model, BI-X monotherapy normalized the changes in expression of TNF-α and semaphorin-related proteins.ConclusionsThese findings support targeting Sema3A to treat intraretinal edema and retinal ischemia.

RUVBL1 promotes enzalutamide resistance of prostate tumors through the PLXNA1-CRAF-MAPK pathway.

Although enzalutamide improves the overall survival of patients with metastatic prostate cancers, enzalutamide resistance (ENZR) will be inevitably developed. Emerging evidence support that alternative oncogenic pathways may bypass the androgen receptor (AR) signaling to promote ENZR progression, however, the underpinning mechanisms remain poorly defined. Here, we report that the expression of RuvB like AAA ATPase 1 (RUVBL1) is upregulated in ENZR cells and xenograft models and prostate tumors in patients. Enzalutamide increases RUVBL1 accumulation in the cytoplasm, which in turn enhances the recruitment of CRAF proto-oncogene serine/threonine kinase protein to plexin A1 (PLXNA1) and the subsequent activation of the downstream MAPK pathway. Co-overexpression of RUVBL1 and PLXNA1 defines a subgroup of prostate cancer (PCa) patients with a poor prognosis. Furthermore, pharmacological inhibition of RUVBL1 by CB-6644 suppresses ENZR cell proliferation and xenograft growth and allows re-sensitization of ENZR cells and xenografts to enzalutamide, indicating that RUVBL1 may act to substitute the AR signaling to promote cancer cell survival and ENZR development. Together, these findings may lead to the identification of RUVBL1 as a potential therapeutic target for ENZR tumors.

Transcriptomic Profiling Revealed Plexin A2 Downregulation With Migration and Invasion Alteration in Dacarbazine-Treated Primary Melanoma Cells.

Melanoma is highly heterogeneous type of malignant neoplasm that is responsible for the majority of deaths among other types of skin cancer. In the present study, we screened a list of differentially expressed genes in two primary, drug-naïve melanoma cell lines derived from patients with melanoma following treatment of the cells with the chemotherapeutic agent dacarbazine. The aim was to determine the transcriptomic profiles and associated alterations in the cell phenotype. We found the vascular endothelial growth factor A/vascular endothelial growth factor receptor 2, phosphoinositide 3-kinase/protein kinase B and focal adhesion signaling pathways to be top altered after dacarbazine treatment. In addition, we observed the expression levels of genes associated with tumor dissemination, integrin β8 and matrix metalloproteinase-1, to be diminished in both cell lines studied, the results of which were confirmed by reverse transcription-quantitative polymerase chain reaction. By contrast, plexin A2 expression was found to be upregulated in K2303 cells, where reduced migration and invasion were also observed, following dacarbazine treatment. Plexin A2 downregulation was associated with the promotion of migrative and invasive capacities in B0404 melanoma cells. Since plexin A2 is semaphorin co-receptor that is involved in focal adhesion and cell migration regulation, the present study suggested that plexin A2 may be implicated in the dacarbazine-mediated phenotypic shift of melanoma cells. We propose that the signature of cancer cell invasiveness can be revealed by using a combination of transcriptomic and functional approaches, which should be applied in the development of personalized therapeutic strategies for each patient with melanoma.

Luteolin Stimulates Proliferation and Inhibits Late Differentiation of Primary Rat Calvarial Osteoblast Induced by High-dose Dexamethasone via Sema3A /NRP1/Pleixin A1.

Although Semaphorin 3A (Sema3A)/ Neuropilin-1(NRP1)/Plexin A1 is one of the important targets in bone metabolism, few studies are performed on this target in the glucocorticoids- induced osteoporosis. Luteolin is a chemical component of Honeysuckle and it has various bioactivities. The effect of Luteolin on the glucocorticoids-induced osteoporosis (primary osteoblasts model) remain unknown. The aim of this study was to investigate the action of Sema3A/ NRP1/Plexin A1 in Luteolin- induced osteoprotection against high-dose Dexamethasone. The effect of Luteolin on the proliferation, late differentiation, and apoptosis of primary osteoblasts model of glucocorticoids-induced osteoporosis in vitro as well as the expression of Sema3A/ NRP1/Plexin A1 are investigated by using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-Htetrazolium bromide, Cell Counting Kit-8, reverse transcription-quantitative real-time polymerase chain reaction, western bolting and so on. Suckling SD rats' calvarial osteoblasts were isolated and identified. The glucocorticoidsinduced primary osteoporosis cell model was established by 100 μM Dexamethasone in 48 h (P<0.01). Luteolin promotes osteoblasts either in physiological condition (without Dexamethasone) or pathological condition (with Dexamethasone) at 1 μM concentration for 48h (P<0.01). Luteolin partly reverses down-regulated expression of proliferation markers such as proliferating cell nuclear and CyclinD1. (P<0.01) Similarly, Luteolin partly reverses up-regulated expression of apoptosis markers such as Bax/B-cell lymphoma-2. (P<0.01) The expression of mRNA and protein of Sema3A/ NRP1/Plexin A1 decreased in model one which significantly increased in Luteolin protecting one. (P<0.05) Interestingly, the late differentiation marker such as osteocalcin and collagenase Ⅰ sharply decreased in Luteolin protecting group compared with model one. (P<0.01). The thesis concludes that Luteolin promoted the proliferation of osteoblast and inhibited its apoptosis and late differentiation in this glucocorticoids-induced primary osteoporosis cell model. This function may be related to the expression of up-regulated Sema3A/NRP1/Plexin A1. Therefore, Luteolin may be a potential medicine for the glucocorticoids-induced osteoporosis.

Perturbation of semaphorin and VEGF signaling in ACDMPV lungs due to FOXF1 deficiency

BackgroundAlveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a rare lethal congenital lung disorder in neonates characterized by severe progressive respiratory failure and refractory pulmonary hypertension, resulting from underdevelopment of the peripheral pulmonary tree. Causative heterozygous single nucleotide variants (SNVs) or copy-number variant (CNV) deletions involving FOXF1 or its distant lung-specific enhancer on chromosome 16q24.1 have been identified in 80–90% of ACDMPV patients. FOXF1 maps closely to and regulates the oppositely oriented FENDRR, with which it also shares regulatory elements.MethodsTo better understand the transcriptional networks downstream of FOXF1 that are relevant for lung organogenesis, using RNA-seq, we have examined lung transcriptomes in 12 histopathologically verified ACDMPV patients with or without pathogenic variants in the FOXF1 locus and analyzed gene expression profile in FENDRR-depleted fetal lung fibroblasts, IMR-90.ResultsRNA-seq analyses in ACDMPV neonates revealed changes in the expression of several genes, including semaphorins (SEMAs), neuropilin 1 (NRP1), and plexins (PLXNs), essential for both epithelial branching and vascular patterning. In addition, we have found deregulation of the vascular endothelial growth factor (VEGF) signaling that also controls pulmonary vasculogenesis and a lung-specific endothelial gene TMEM100 known to be essential in vascular morphogenesis. Interestingly, we have observed a substantial difference in gene expression profiles between the ACDMPV samples with different types of FOXF1 defect. Moreover, partial overlap between transcriptome profiles of ACDMPV lungs with FOXF1 SNVs and FENDRR-depleted IMR-90 cells suggests contribution of FENDRR to ACDMPV etiology.ConclusionsOur transcriptomic data imply potential crosstalk between several lung developmental pathways, including interactions between FOXF1-SHH and SEMA-NRP or VEGF/VEGFR2 signaling, and provide further insight into complexity of lung organogenesis in humans.

Interactions between semaphorins and plexin–neuropilin receptor complexes in the membranes of live cells

Signaling of semaphorin ligands via their plexin–neuropilin receptors is involved in tissue patterning in the developing embryo. These proteins play roles in cell migration and adhesion but are also important in disease etiology, including in cancer angiogenesis and metastasis. While some structures of the soluble domains of these receptors have been determined, the conformations of the full-length receptor complexes are just beginning to be elucidated, especially within the context of the plasma membrane. Pulsed-interleaved excitation fluorescence cross-correlation spectroscopy allows direct insight into the formation of protein–protein interactions in the membranes of live cells. Here, we investigated the homodimerization of neuropilin-1 (Nrp1), plexin A2, plexin A4, and plexin D1 using pulsed-interleaved excitation fluorescence cross-correlation spectroscopy. Consistent with previous studies, we found that Nrp1, plexin A2, and plexin A4 are present as dimers in the absence of exogenous ligand. Plexin D1, on the other hand, was monomeric under similar conditions, which had not been previously reported. We also found that plexin A2 and A4 assemble into a heteromeric complex. Stimulation with semaphorin 3A or semaphorin 3C neither disrupts nor enhances the dimerization of the receptors when expressed alone, suggesting that activation involves a conformational change rather than a shift in the monomer–dimer equilibrium. However, upon stimulation with semaphorin 3C, plexin D1 and Nrp1 form a heteromeric complex. This analysis of interactions provides a complementary approach to the existing structural and biochemical data that will aid in the development of new therapeutic strategies to target these receptors in cancer.

Abstract 3161: The perlecan-semaphorin3A-plexinA1-neuropilin-1 Complex: Toggle control of prostate cancer cycles of dormancy and reactivation

Abstract Introduction: Metastatic prostate cancer (PCa) undergoes cycles of dormancy and reactivation, resulting in secondary metastasis, and leading to lethal disease. PCa preferentially metastasizes to bone where it can remain dormant for years after initial colonization. Previous work showed different growth and dormancy phenotypes in PCa tumors injected subcutaneously and intrafemorally, though the exact source of the dormancy-inducing factors remains undefined. We identified a novel receptor complex, the Perlecan-Semaphorin3A-PlexinA1-Neuropilin-1 (PSPN) complex, that is modulated by context-specific endothelial cell populations. The overall goal of this project is to identify the role of endothelial cells in stabilizing and silencing metastatic behavior via the PSPN complex, it's downstream pathways, and it's turnover by matrix metalloproteinase 7/Matrilysin (MMP-7). Methods: Live/dead assays coupled with immunolabeling and molecular approaches were used to determine the molecular impact of secreted endothelial cell factors on PCa signaling and behavior through the PSPN Complex in 2.5D and 3D microtumor cultures. Reverse phase protein arrays (RPPA) provided an unbiased means to assess the downstream molecular events in 3D co-cultures of PCa and microvascular cells. Results: Intact perlecan (P) induced clustering via engagement of PSPN complex. Destruction of complex components by MMP-7 activated growth and migration. FOXM1 was activated downstream of PSPN complex cleavage and resulting destabilization, consistent with escape from dormancy. Secreted factors from endothelial cells induced dormancy and reduced FOXM1 activation. Immunolocalization data showed internalization of plexin A1 with immunostaining both at the PCa cell surface and perinuclear, consistent with dynamic turnover associated with activation of signaling after loss of the PSPN complex. Conclusion: Intact PSPN complex at the PCa cell surface favors a dormant state that is reversed by MMP7 cleavage. In the cohesive state (PSPN complex intact) FOXM1 is “off”, inhibiting the transcription of growth and metastasis promoting factors. Context-specific endothelial cell populations secrete PSPN complex stabilizing proteins, silence downstream signaling, and result in a dormant phenotype. We propose that the PSPN complex at metastatic sites such as bone acts as a molecular toggle that can maintain dormancy until the switch is “flipped” by MMP-7 allowing reactivation, dyscohesion and favoring growth and metastasis. Citation Format: Tristen V. Tellman, Lindsey K. Sablatura, Lissette A. Cruz, Sue-Hwa Lin, Leland W. Chung, Mary C. Farach-Carson. The perlecan-semaphorin3A-plexinA1-neuropilin-1 Complex: Toggle control of prostate cancer cycles of dormancy and reactivation [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 3161.

A prognostic model based on seven immune-related genes predicts the overall survival of patients with hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is a disease with a high incidence and a poor prognosis. Growing amounts of evidence have shown that the immune system plays a critical role in the biological processes of HCC such as progression, recurrence, and metastasis, and some have discussed using it as a weapon against a variety of cancers. However, the impact of immune-related genes (IRGs) on the prognosis of HCC remains unclear.MethodsBased on The Cancer Gene Atlas (TCGA) and Immunology Database and Analysis Portal (ImmPort) datasets, we integrated the ribonucleic acid (RNA) sequencing profiles of 424 HCC patients with IRGs to calculate immune-related differentially expressed genes (DEGs). Survival analysis was used to establish a prognostic model of survival- and immune-related DEGs. Based on genomic and clinicopathological data, we constructed a nomogram to predict the prognosis of HCC patients. Gene set enrichment analysis further clarified the signalling pathways of the high-risk and low-risk groups constructed based on the IRGs in HCC. Next, we evaluated the correlation between the risk score and the infiltration of immune cells, and finally, we validated the prognostic performance of this model in the GSE14520 dataset.ResultsA total of 100 immune-related DEGs were significantly associated with the clinical outcomes of patients with HCC. We performed univariate and multivariate least absolute shrinkage and selection operator (Lasso) regression analyses on these genes to construct a prognostic model of seven IRGs (Fatty Acid Binding Protein 6 (FABP6), Microtubule-Associated Protein Tau (MAPT), Baculoviral IAP Repeat Containing 5 (BIRC5), Plexin-A1 (PLXNA1), Secreted Phosphoprotein 1 (SPP1), Stanniocalcin 2 (STC2) and Chondroitin Sulfate Proteoglycan 5 (CSPG5)), which showed better prognostic performance than the tumour/node/metastasis (TNM) staging system. Moreover, we constructed a regulatory network related to transcription factors (TFs) that further unravelled the regulatory mechanisms of these genes. According to the median value of the risk score, the entire TCGA cohort was divided into high-risk and low-risk groups, and the low-risk group had a better overall survival (OS) rate. To predict the OS rate of HCC, we established a gene- and clinical factor-related nomogram. The receiver operating characteristic (ROC) curve, concordance index (C-index) and calibration curve showed that this model had moderate accuracy. The correlation analysis between the risk score and the infiltration of six common types of immune cells showed that the model could reflect the state of the immune microenvironment in HCC tumours.ConclusionOur IRG prognostic model was shown to have value in the monitoring, treatment, and prognostic assessment of HCC patients and could be used as a survival prediction tool in the near future.

Cdk5 and GSK3\u03b2 inhibit fast endophilin-mediated endocytosis

Endocytosis mediates the cellular uptake of micronutrients and cell surface proteins. Fast Endophilin-mediated endocytosis, FEME, is not constitutively active but triggered upon receptor activation. High levels of growth factors induce spontaneous FEME, which can be suppressed upon serum starvation. This suggested a role for protein kinases in this growth factor receptor-mediated regulation. Using chemical and genetic inhibition, we find that Cdk5 and GSK3β are negative regulators of FEME. They antagonize the binding of Endophilin to Dynamin-1 and to CRMP4, a Plexin A1 adaptor. This control is required for proper axon elongation, branching and growth cone formation in hippocampal neurons. The kinases also block the recruitment of Dynein onto FEME carriers by Bin1. As GSK3β binds to Endophilin, it imposes a local regulation of FEME. Thus, Cdk5 and GSK3β are key regulators of FEME, licensing cells for rapid uptake by the pathway only when their activity is low.

PLXNA2 and LRRC40 as candidate genes in autism spectrum disorder.

Autism spectrum disorder (ASD) is a neurodevelopmental disability with high heritability yet the genetic etiology remains elusive. Therefore, it is necessary to elucidate new genotype-phenotype relationships for ASD to improve both the etiological knowledge and diagnosis. In this work, a copy-number variant and whole-exome sequencing analysis were performed in an ASD patient with a complex neurobehavioral phenotype with epilepsy and attention deficit hyperactivity disorder. We identified rare recessive single nucleotide variants in the two genes, PLXNA2 encoding Plexin A2 that participates in neurodevelopment, and LRRC40, which encodes Leucine-rich repeat containing protein 40, a protein of unknown function. PLXNA2 showed the heterozygous missense variants c.614G>A (p.Arg205Gln) and c.4904G>A (p.Arg1635Gln) while LRRC40 presented the homozygous missense variant c.1461G>T (p.Leu487Phe). In silico analysis predicted that these variants could be pathogenic. We studied PLXNA2 and LRRC40 mRNA and proteins in fibroblasts from the patient and controls. We observed a significant PlxnA2 subcellular delocalization and very low levels of LRRC40 in the patient. Moreover, we found a novel interaction between PlxnA2 and LRRC40 suggesting that participate in a common neural pathway. This interaction was significant decreased in the patient's fibroblasts. In conclusion, our results identified PLXNA2 and LRRC40 genes as candidates in ASD providing novel clues for the pathogenesis. Further attention to these genes is warranted in genetic studies of patients with neurodevelopmental disorders, particularly ASD. LAY SUMMARY: Genomics is improving the knowledge and diagnosis of patients with autism spectrum disorder (ASD) yet the genetic etiology remains elusive. Here, using genomic analysis together with experimental functional studies, we identified in an ASD complex patient the PLXNA2 and LRRC40 recessive genes as ASD candidates. Furthermore, we found that the proteins of these genes interact in a common neural network. Therefore, more attention to these genes is warranted in genetic studies of patients with neurodevelopmental disorders, particularly ASD.

Semaphorin 3A mediated brain tumor stem cell proliferation and invasion in EGFRviii mutant gliomas

BackgroundGlioblastoma multiforme (GBM) is the most common primary brain tumor in adults, with a median survival of approximately 15 months. Semaphorin 3A (Sema3A), known for its axon guidance and antiangiogenic properties, has been implicated in GBM growth. We hypothesized that Sema3A directly inhibits brain tumor stem cell (BTSC) proliferation and drives invasion via Neuropilin 1 (Nrp1) and Plexin A1 (PlxnA1) receptors.MethodsGBM BTSC cell lines were assayed by immunostaining and PCR for levels of Semaphorin 3A (Sema3A) and its receptors Nrp1 and PlxnA1. Quantitative BrdU, cell cycle and propidium iodide labeling assays were performed following exogenous Sema3A treatment. Quantitative functional 2-D and 3-D invasion assays along with shRNA lentiviral knockdown of Nrp1 and PlxnA1 are also shown. In vivo flank studies comparing tumor growth of knockdown versus control BTSCs were performed. Statistics were performed using GraphPad Prism v7.ResultsImmunostaining and PCR analysis revealed that BTSCs highly express Sema3A and its receptors Nrp1 and PlxnA1, with expression of Nrp1 in the CD133 positive BTSCs, and absence in differentiated tumor cells. Treatment with exogenous Sema3A in quantitative BrdU, cell cycle, and propidium iodide labeling assays demonstrated that Sema3A significantly inhibited BTSC proliferation without inducing cell death. Quantitative functional 2-D and 3-D invasion assays showed that treatment with Sema3A resulted in increased invasion. Using shRNA lentiviruses, knockdown of either NRP1 or PlxnA1 receptors abrogated Sema3A antiproliferative and pro-invasive effects. Interestingly, loss of the receptors mimicked Sema3A effects, inhibiting BTSC proliferation and driving invasion. Furthermore, in vivo studies comparing tumor growth of knockdown and control infected BTSCs implanted into the flanks of nude mice confirmed the decrease in proliferation with receptor KD.ConclusionsThese findings demonstrate the importance of Sema3A signaling in GBM BTSC proliferation and invasion, and its potential as a therapeutic target.

Abstract 1785: Domain-specific antibodies to Neuropilin 2 implicate VEGF-C and not Semaphorin 3F in breast cancer stem cell function

Abstract Introduction: There is a strong body of evidence indicating that the expression of Neuropilin-2 (NRP2) is enriched in breast cancer stem cells (CSCs) and that NRP2 signaling is critical for breast CSC function and resistance development. For this reason, the rationale for targeting NRP2 as a therapeutic strategy is compelling and timely. A major limitation that has hampered the development of this therapy, however, has been the lack of availability of high-quality anti-human NRP2 monoclonal antibodies (mAbs) that block NRP2 signalling. aTyr Pharma has generated a panel of high-quality, anti-human NRP2 mAbs that have the potential to be developed for the clinical management of breast cancer. A significant advance made by aTyr is that through specific domain reactivity, they have demonstrated differential effects on ligand blocking, receptor homo and heterodimerization and functional activity. Importantly a subset of such antibodies show differential activity in the mammosphere assay of triple negative breast cancer. Results: Flow cytometry was used to assess the specificity of the in-house anti-NRP2 mAbs to NRP2 using A549 wild type versus NRP2 knockout clonal cells. The in-house anti-NRP2 mAbs bound to A549 wild-type cells while showing little or no binding to the NRP2 knockout clonal cells, and exhibited significantly superior specificity, and sensitivity compared to existing commercially available antibodies. Displacement studies demonstrated that the tested anti-NRP2 mAbs showed different capabilities in blocking of VEGF-C or SEMA-3F binding to Expi293-hNRP2 cells, and were categorized as blockers (&amp;gt;90% inhibition), partial blockers (30-90% inhibition), or non-blockers (no obvious inhibition). To further extend the assessment of the biological activity of the anti-NRP2 antibodies, their activity was assessed in a receptor dimerization assay. Vectors encoding a split luciferase pBiT1.1 and pBiT2.1, and a cell permeable substrate, were obtained from Promega corporation. The complete extracellular domain and transmembrane helices of NRP2, FLT4 (VEGFR3), KDR (VEGFR2) and plexin A1 (PLXNA1) were cloned into the vectors and screened for optimal orientation, following established methods. Tested antibodies were able to impair respective VEGF and SEMA3 induced dimerization of receptor pairs NRP2/FLT4, NRP2/KDR and NRP2/PLXNA1. Surprisingly select antibodies show extremely specific and non-obvious functional differentiation. Direct functional assessment of a subset of these antibodies on breast CSCs revealed that the VEGF blocking, but not the SEMA3 blocking anti-NRP2 amAbs had the ability to inhibit serial passage mammosphere formation, an indicator of self-renewal potential. Conclusions: A series of domain specific antibodies to NRP2 have been developed and characterized. These antibodies show differential binding to specific domains of NRP2, can inhibit either VEGF-C or Sema3F binding to NRP2, and differentially effect receptor dimerization. The use of these antibodies enabled us to implicate VEGF-C/NRP2 signaling but not SEMA-3F/NRP2 signaling in the function of breast CSCs. Citation Format: Leslie A. Nangle, Luke Burman, Hira L. Goel, Zhiwen Xu, Kristina Hamel, Nathaniel Bloom, Arthur M. Mercurio. Domain-specific antibodies to Neuropilin 2 implicate VEGF-C and not Semaphorin 3F in breast cancer stem cell function [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1785.

Abstract 2650: Perlecan-Semaphorin 3A-Plexin A1-Neuropilin-1 complex: Flipping the switch controlling metastatic behavior of prostate cancer cells

Abstract Introduction: For prostate cancer (PCa), the major site of metastasis is the bone, where disseminated PCa cells can remain dormant for years after colonization. The events that end dormancy and trigger recurrence in the skeletal system, leading to a transition from indolent to lethal widely metastatic disease, are poorly understood. The last seven Ig repeats of perlecan/HSPG2 domain IV (domain IV-3) function as an extracellular scaffold that can anchor the Perlecan-Semaphorin 3A-Plexin A1-Neuropilin-1 (PSPN) signaling complex. Perlecan interacts with semaphorin 3A (Sema3A) sequestered on PCa cells to modulate cell-cell and cell-matrix adhesion decisions. Secreted Sema3A functions as a neuropilin-1 ligand, but a plexin is required to transduce an intracellular signal. We are investigating the means by which the PSPN complex functions as a molecular switch that controls dormancy and reactivation of PCa cells in the perlecan-rich bone niche. Methods: Biochemical, immunolabeling and molecular approaches were used to identify the components of the PSPN complex and demonstrate their functions. Susceptibility of PSPN complex components to MMP7/Matrilysin was evaluated by SDS-PAGE. Endocytosis and cellular localization of complex components was determined by immunolocalization. Dormancy induced by IV-3 engagement of SPN was evaluated using pp38 and pRb. FOXM1 activation was monitored downstream of the PSPN complex to demonstrate dormancy/cohesion or dyscohesion/invasion. Results: Intact perlecan and domain IV-3 induce clustering via engagement of PSPN complex. Plexin knockdown reduced the cohesive phenotype in PCa cells. Destruction of complex components by the matrix metalloproteinase MMP-7/Matrilysin was demonstrated. FOXM1 was activated downstream of MMP-7-activated PSPN complex cleavage, consistent with escape from dormancy. Perlecan IV-3 induced dormancy and reduced FOXM1 activation. Immunolocalization data showed internalization of plexin A1 with immunostaining both at the PCa cell surface and perinuclear, consistent with dynamic turnover associated with activation of signaling after loss of the PSPN complex. Conclusion: Intact PSPN complex at the PCa cell surface favors a dormant state that is reversed by MMP-7 cleavage. In the cohesive state (PSPN complex intact) FOXM1 is “off”, inhibiting the transcription of growth and metastasis promoting factors. We propose that the PSPN complex at metastatic sites such as bone acts as a molecular switch that can maintain dormancy until the switch is “flipped” by MMP-7 allowing reactivation, dyscohesion and favoring growth and metastasis. Ongoing work is investigating the role of plexin endocytosis in signal activation. Citation Format: Tristen V. Tellman, Lissette A. Cruz, Lindsey K. Sablatura, Brian J. Grindel, Leland W. Chung, Mary C. Farach-Carson. Perlecan-Semaphorin 3A-Plexin A1-Neuropilin-1 complex: Flipping the switch controlling metastatic behavior of prostate cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2650.