Biomarker LEPRE1 induces pelitinib-specific drug responsiveness by regulating ABCG2 expression and tumor transition states in human leukemia and lung cancer.

Biomarkers for treatment sensitivity or drug resistance used in precision medicine include prognostic and predictive molecules, critical factors in selecting appropriate treatment protocols and improving survival rates. However, identification of accurate biomarkers remain challenging due to the high risk of false-positive findings and lack of functional validation results for each biomarker. Here, we discovered a mechanical correlation between leucine proline-enriched proteoglycan 1 (LEPRE1) and pelitinib drug sensitivity using in silico statistical methods and confirmed the correlation in acute myeloid leukemia (AML) and A549 lung cancer cells. We determined that high LEPRE1 levels induce protein kinase B activation, overexpression of ATP-binding cassette superfamily G member 2 (ABCG2) and E-cadherin, and cell colonization, resulting in a cancer stem cell-like phenotype. Sensitivity to pelitinib increases in LEPRE1-overexpressing cells due to the reversing effect of ABCG2 upregulation. LEPRE1 silencing induces pelitinib resistance and promotes epithelial-to-mesenchymal transition through actin rearrangement via a series of Src/ERK/cofilin cascades. The in silico results identified a mechanistic relationship between LEPRE1 and pelitinib drug sensitivity, confirmed in two cancer types. This study demonstrates the potential of LEPRE1 as a biomarker in cancer through in-silico prediction and in vitro experiments supporting the clinical development of personalized medicine strategies based on bioinformatics findings.

The Role of Podoplanin in Skin Diseases.

Podoplanin is a sialomucin-like type I transmembrane receptor glycoprotein that is expressed specifically in lymphatic vessels, sebaceous glands, and hair follicles in normal skin. However, under pathological conditions podoplanin expression is upregulated in various cells, such as keratinocytes, fibroblasts, tumor cells, and inflammatory cells, and plays pivotal roles in different diseases. In psoriasis, podoplanin expression is induced in basal keratinocytes via the JAK-STAT pathway and contributes toward epidermal hyperproliferation. Podoplanin expression on keratinocytes can also promote IL-17 secretion from lymphocytes, promoting chronic inflammation. During wound healing, the podoplanin/CLEC-2 interaction between keratinocytes and platelets regulates re-epithelialization at the wound edge. In skin cancers, podoplanin expresses on tumor cells and promotes their migration and epithelial-mesenchymal transition, thereby accelerating invasion and metastasis. Podoplanin is also expressed in normal peritumoral cells, such as cancer-associated fibroblasts in melanoma and keratinocytes in extramammary Paget's disease, which promote tumor progression and predict aggressive behavior and poor prognosis. This review provides an overview of our current understanding of the mechanisms via which podoplanin mediates these pathological skin conditions.

CD38-mediated Inhibition of Bruton's Tyrosine Kinase in Macrophages Prevents Endotoxemic Lung Injury.

TLR4 signaling via endotoxemia in macrophages promotes macrophage transition to the inflammatory phenotype through NLRP3 inflammasome activation. This transition event has the potential to trigger acute lung injury (ALI). However, relatively little is known about the regulation of NLRP3 and its role in the pathogenesis of ALI. Here we interrogated the signaling pathway activated by CD38, an ectoenzyme expressed in macrophages, in preventing ALI through suppressing NLRP3 activation. Wild-type and Cd38-knockout (Cd38-/-) mice were used to assess inflammatory lung injury, and isolated macrophages were used to delineate underlying TLR4 signaling pathway. We showed that CD38 suppressed TLR4 signaling in macrophages by inhibiting Bruton's tyrosine kinase (Btk) through the recruitment of Src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2) and resulting in the dephosphorylation of activated Btk. Cd38-/- mice show enhanced lung polymorphonuclear leukocyte extravasation and severe lung injury. LPS- or polymicrobial sepsis-induced mortality in Cd38-/- mice were markedly augmented compared with wild types. CD38 in macrophages functioned by inhibiting Btk activation through activation of SHP2 and resulting dephosphorylation of Btk, and thereby preventing activation of downstream targets NF-κB and NLRP3. Cd38-/- macrophages displayed markedly increased activation of Btk, NF-κB, and NLRP3, whereas in vivo administration of the Btk inhibitor ibrutinib (a Food and Drug Administration-approved drug) prevented augmented TLR4-induced inflammatory lung injury seen in Cd38-/- mice. Our findings together show upregulation of CD38 activity and inhibition of Btk activation downstream of TLR4 activation as potential strategies to prevent endotoxemic ALI.

Platelet Phagocytosis via P-selectin Glycoprotein Ligand 1 and Accumulation of Microparticles in Systemic Sclerosis.

OBJECTIVE: It is unclear why activated platelets and platelet-derived microparticles (MPs) accumulate in the blood of patients with systemic sclerosis (SSc). This study was undertaken to investigate whether defective phagocytosis might contribute to MP accumulation in the blood of patients with SSc. METHODS: Blood samples were obtained from a total of 81 subjects, including 25 patients with SSc and 26 patients with stable coronary artery disease (CAD). Thirty sex- and age-matched healthy volunteers served as controls. Studies were also conducted in NSG mice, in which the tail vein of the mice was injected with MPs, and samples of the lung parenchyma were obtained for analysis of the pulmonary microvasculature. Tissue samples from human subjects and from mice were assessed by flow cytometry and immunochemical analyses for determination of platelet-neutrophil interactions, phagocytosis, levels and distribution of P-selectin, P-selectin glycoprotein ligand 1 (PSGL-1), and HMGB1 on platelets and MPs, and concentration of byproducts of neutrophil extracellular trap (NET) generation/catabolism. RESULTS: Activated P-selectin+ platelets and platelet-derived HMGB1+ MPs accumulated in the blood of SSc patients but not in the blood of healthy controls. Patients with CAD, a vasculopathy independent of systemic inflammation, had fewer P-selectin+ platelets and a negligible number of MPs. The expression of the receptor for P-selectin, PSGL-1, in neutrophils from SSc patients was significantly decreased, raising the possibility that phagocytes in SSc do not recognize activated platelets, leading to a failure of phagocytosis and continued neutrophil release of MPs. As evidence of this process, activated platelets were not detected in the neutrophils from SSc patients, whereas they were consistently present in the neutrophils from patients with CAD. HMGB1+ MPs elicited generation of NETs, which were only detected in the plasma of SSc patients. In mice, P-selectin-PSGL-1 interaction resulted in platelet phagocytosis in vitro and influenced the ability of MPs to elicit NETs, endothelial activation, and migration of leukocytes through the pulmonary microvasculature. CONCLUSION: The clearance of activated platelets via PSGL-1 limits the undesirable effects of MP-elicited neutrophil activation. This balance is disrupted in patients with SSc. Its reconstitution might curb vascular inflammation and prevent fibrosis.

The Role of TRAPγ/SSR3 in Preproinsulin Translocation Into the Endoplasmic Reticulum.

In the endoplasmic reticulum (ER), the translocation-associated protein complex (TRAP), also called signal sequence receptor (SSR), includes four integral membrane proteins TRAPα/SSR1, TRAPß/SSR2, and TRAPδ/SSR4 with the bulk of their extramembranous portions primarily in the ER lumen, whereas the extramembranous portion of TRAPγ/SSR3 is primarily cytosolic. Individually diminished expression of either TRAPα/SSR1, TRAPß/SSR2, or TRAPδ/SSR4 mRNA is known in each case to lower TRAPα/SSR1 protein levels, leading to impaired proinsulin biosynthesis, whereas forced expression of TRAPα/SSR1 at least partially suppresses the proinsulin biosynthetic defect. Here, we report that diminished TRAPγ/SSR3 expression in pancreatic ß-cells leaves TRAPα/SSR1 levels unaffected while nevertheless inhibiting cotranslational and posttranslational translocation of preproinsulin into the ER. Crucially, acute exposure to high glucose leads to a rapid upregulation of both TRAPγ/SSR3 and proinsulin protein without change in the respective mRNA levels, as observed in cultured rodent ß-cell lines and confirmed in human islets. Strikingly, pancreatic ß-cells with suppressed TRAPγ/SSR3 expression are blocked in glucose-dependent upregulation of proinsulin (or insulin) biosynthesis. Most remarkably, overexpression of TRAPγ/SSR3 in control ß-cells raises proinsulin levels, even without boosting extracellular glucose. The data suggest the possibility that TRAPγ/SSR3 may fulfill a rate-limiting function in preproinsulin translocation across the ER membrane for proinsulin biosynthesis.

Role of podoplanin and Kupffer cells in liver injury after ischemia-reperfusion in mice.

AIM: To investigate the relationship between the intrahepatic expression of podoplanin (PDPN) and Kupffer cells (KCs) in ischemia-reperfusion (I/R) liver damage. METHODS: C57Bl/6 mice were injected with 200 µl of clodronate liposomes (macrophage depletion; MDP group) to deplete KCs or control liposomes (control group) via the ophthalmic vein plexus 24 h prior to ischemia. Animals were subjected to 90 min of partial hepatic ischemia (70%), followed by reperfusion, and were then killed at designated time points. Serum and liver tissues were harvested for further analyses. RESULTS: Serum ALT levels, mortality rates, and the percentage of necrotic area in liver sections were significantly higher in the MDP group than in the control group. PDPN was expressed in the lymphatic epithelium, interlobular bile duct epithelium, and in some hepatocytes in each group. Its expression in hepatocytes was down-regulated in the MDP group. The accumulation of platelets in the sinusoid was reduced 6 h after I/R in the MDP group. Tissue HGF and IGF-1 levels decreased in the MDP group. CONCLUSIONS: These results suggest that KCs play a key role in the activation of platelets through direct contact with PDPN-positive hepatocytes in I/R livers.

Leukocyte Membrane Enzymes Play the Cell Adhesion Game.

For a long time, proteins with enzymatic activity have not been usually considered to carry out other functions different from catalyzing chemical reactions within or outside the cell. Nevertheless, in the last few years several reports have uncovered the participation of numerous enzymes in other processes, placing them in the category of moonlighting proteins. Some moonlighting enzymes have been shown to participate in complex processes such as cell adhesion. Cell adhesion plays a physiological role in multiple processes: it enables cells to establish close contact with one another, allowing communication; it is a key step during cell migration; it is also involved in tightly binding neighboring cells in tissues, etc. Importantly, cell adhesion is also of great importance in pathophysiological scenarios like migration and metastasis establishment of cancer cells. Cell adhesion is strictly regulated through numerous switches: proteins, glycoproteins and other components of the cell membrane. Recently, several cell membrane enzymes have been reported to participate in distinct steps of the cell adhesion process. Here, we review a variety of examples of membrane bound enzymes participating in adhesion of immune cells.

TREM2-independent oligodendrocyte, astrocyte, and T cell responses to tau and amyloid pathology in mouse models of Alzheimer disease.

Non-neuronal responses in neurodegenerative disease have received increasing attention as important contributors to disease pathogenesis and progression. Here we utilize single-cell RNA sequencing to broadly profile 13 cell types in three different mouse models of Alzheimer disease (AD), capturing the effects of tau-only, amyloid-only, or combined tau-amyloid pathology. We highlight microglia, oligodendrocyte, astrocyte, and T cell responses and compare them across these models. Notably, we identify two distinct transcriptional states for oligodendrocytes emerging differentially across disease models, and we determine their spatial distribution. Furthermore, we explore the impact of Trem2 deletion in the context of combined pathology. Trem2 knockout mice exhibit severely blunted microglial responses to combined tau and amyloid pathology, but responses from non-microglial cell types (oligodendrocytes, astrocytes, and T cells) are relatively unchanged. These results delineate core transcriptional states that are engaged in response to AD pathology, and how they are influenced by a key AD risk gene, Trem2.

A Genetic Variation of Lipopolysaccharide Binding Protein Affects the Inflammatory Response and Is Associated with Improved Outcome during Sepsis.

LPS binding protein (LBP) is an important innate sensor of microbial cell wall structures. Frequent functionally relevant mutations exist and have been linked to influence susceptibility to and course of bacterial infections. We examined functional properties of a single nucleotide polymorphism resulting in an exchange of phenylalanine to leucine at position 436 of LBP (rs2232618) and compared the frequent variant of the molecule with the rare one in ligand binding experiments. We then stimulated RAW cells with bacterial ligands in the presence of serum obtained from individuals with different LBP genotypes. We, furthermore, determined the potential effects of structural changes in the molecule by in silico modeling. Finally, we analyzed 363 surgical patients for this genetic variant and examined incidence and course of sepsis following surgery. We found that binding of LBP to bacterial ligands was reduced, and stimulation of RAW cells resulted in an increased release of TNF when adding serum from individuals carrying the F436L variant as compared with normal LBP. In silico analysis revealed structural changes of LBP, potentially explaining some of the effects observed for the LBP variant. Finally, patients carrying the F436L variant were found to be similarly susceptible for sepsis. However, we observed a more favorable course of severe infections in this cohort. Our findings reveal new insights into LPS recognition and the subsequent activation of the innate immune system brought about by LBP. The identification of a genetic variant of LBP influencing the course of sepsis may help to stratify individuals at risk and thus reduce clinical complications of patients.

The Collagen Receptor uPARAP in Malignant Mesothelioma: A Potential Diagnostic Marker and Therapeutic Target.

Malignant mesothelioma (MM) is a highly aggressive cancer with limited therapeutic options. We have previously shown that the endocytic collagen receptor, uPARAP, is upregulated in certain cancers and can be therapeutically targeted. Public RNA expression data display uPARAP overexpression in MM. Thus, to evaluate its potential use in diagnostics and therapy, we quantified uPARAP expression by immunohistochemical H-score in formalin-fixed paraffin-embedded bioptic/surgical human tissue samples and tissue microarrays. We detected pronounced upregulation of uPARAP in the three main MM subtypes compared to non-malignant reactive mesothelial proliferations, with higher expression in sarcomatoid and biphasic than in epithelioid MM. The upregulation appeared to be independent of patients' asbestos exposure and unaffected after chemotherapy. Using immunoblotting, we demonstrated high expression of uPARAP in MM cell lines and no expression in a non-malignant mesothelial cell line. Moreover, we showed the specific internalization of an anti-uPARAP monoclonal antibody by the MM cell lines using flow cytometry-based assays and confocal microscopy. Finally, we demonstrated the sensitivity of these cells towards sub-nanomolar concentrations of an antibody-drug conjugate formed with the uPARAP-directed antibody and a potent cytotoxin that led to efficient, uPARAP-specific eradication of the MM cells. Further studies on patient cohorts and functional preclinical models will fully reveal whether uPARAP could be exploited in diagnostics and therapeutic targeting of MM.

Enhanced pro-BDNF-p75NTR pathway activity in denervated skeletal muscle.

AIMS: Brain derived neurotrophic factor (BDNF) and the related receptors TrkB and p75NTR are expressed in skeletal muscle, yet their functions remain to be fully understood. Skeletal muscle denervation, which occurs in spinal injury, peripheral neuropathies, and aging, negatively affects muscle mass and function. In this study, we wanted to understand the role of BDNF, TrkB, and p75NTR in denervation-induced adverse effects on skeletal muscle. MAIN METHODS: Mice with unilateral sciatic denervation were used. Protein levels of pro- and mature BDNF, TrkB, p75NTR, activations of their downstream signaling pathways, and inflammation in the control and denervated muscle were measured with Western blot and tissue staining. Treatment with a p75NTR inhibitor and BDNF skeletal muscle specific knockout in mice were used to examine the role of p75NTR and pro-BDNF. KEY FINDINGS: In denervated muscle, pro-BDNF and p75NTR were significantly upregulated, and JNK and NF-kB, two major downstream signaling pathways of p75NTR, were activated, along with muscle atrophy and inflammation. Inhibition of p75NTR using LM11A-31 significantly reduced JNK activation and inflammatory cytokines in the denervated muscle. Moreover, skeletal muscle specific knockout of BDNF reduced pro-BDNF level, JNK activation and inflammation in the denervated muscle. SIGNIFICANCE: These results reveal for the first time that the upregulation of pro-BDNF and activation of p75NTR pathway are involved in denervation-induced inflammation in skeletal muscle. The results suggest that inhibition of pro-BDNF-p75NTR pathway can be a new target to treat skeletal muscle inflammation.

The conserved fertility factor SPACA4/Bouncer has divergent modes of action in vertebrate fertilization.

Fertilization is the fundamental process that initiates the development of a new individual in all sexually reproducing species. Despite its importance, our understanding of the molecular players that govern mammalian sperm-egg interaction is incomplete, partly because many of the essential factors found in nonmammalian species do not have obvious mammalian homologs. We have recently identified the lymphocyte antigen-6 (Ly6)/urokinase-type plasminogen activator receptor (uPAR) protein Bouncer as an essential fertilization factor in zebrafish [S. Herberg, K. R. Gert, A. Schleiffer, A. Pauli, Science 361, 1029-1033 (2018)]. Here, we show that Bouncer's homolog in mammals, Sperm Acrosome Associated 4 (SPACA4), is also required for efficient fertilization in mice. In contrast to fish, in which Bouncer is expressed specifically in the egg, SPACA4 is expressed exclusively in the sperm. Male knockout mice are severely subfertile, and sperm lacking SPACA4 fail to fertilize wild-type eggs in vitro. Interestingly, removal of the zona pellucida rescues the fertilization defect of Spaca4-deficient sperm in vitro, indicating that SPACA4 is not required for the interaction of sperm and the oolemma but rather of sperm and the zona pellucida. Our work identifies SPACA4 as an important sperm protein necessary for zona pellucida penetration during mammalian fertilization.

Reduced TREM2 activation in microglia of patients with Alzheimer's disease.

Loss-of-function variants of triggering receptor expressed on myeloid cells 2 (TREM2) increase the risk of developing Alzheimer's disease (AD). The mechanism through which TREM2 contributes to the disease (TREM2 activation vs inactivation) is largely unknown. Here, we analyzed changes in a gene set downstream of TREM2 to determine whether TREM2 signaling is modified by AD progression. We generated an anti-human TREM2 agonistic antibody and defined TREM2 activation in terms of the downstream expression changes induced by this antibody in microglia developed from human induced pluripotent stem cells (iPSC). Differentially expressed genes (DEGs) following TREM2 activation were compared with the gene set extracted from microglial single nuclear RNA sequencing data of patients with AD, using gene set enrichment analysis. We isolated an anti-TREM2-specific agonistic antibody, Hyb87, from anti-human TREM2 antibodies generated using binding and agonism assays, which helped us identify 300 upregulated and 251 downregulated DEGs. Pathway enrichment analysis suggested that TREM2 activation may be associated with Th2-related pathways. TREM2 activation was lower in AD microglia than in microglia from healthy subjects or patients with mild cognitive impairment. TREM2 activation also showed a significant negative correlation with disease progression. Pathway enrichment analysis of DEGs controlled by TREM2 activity indicated that TREM2 activation in AD may lead to anti-apoptotic signaling, immune response, and cytoskeletal changes in the microglia. We showed that TREM2 activation decreases with AD progression, in support of a protective role of TREM2 activation in AD. In addition, the agonistic anti-TREM2 antibody can be used to identify TREM2 activation state in AD microglia.

Characterization of nuclear pore complex targeting domains in Pom152 in Saccharomyces cerevisiae.

Pom152 is a transmembrane protein within the nuclear pore complex (NPC) of fungi that is important for NPC assembly and structure. Pom152 is comprised of a short amino-terminal region that remains on the cytosolic side of the nuclear envelope (NE) and interacts with NPC proteins, a transmembrane domain, and a large, glycosylated carboxy-terminal domain within the NE lumen. Here we show that the N-terminal 200 amino acids of Pom152 that include only the amino-terminal and transmembrane regions are sufficient for localization to the NPC. Full-length, glycosylation-deficient, and truncated Pom152-GFP chimeras expressed in cells containing endogenous Pom152 localize to both NPCs and cortical endoplasmic reticulum (ER). Expression of Pom152-GFP fusions in pom152Δ cells results in detectable localization at only the NE by full-length and amino-terminal Pom152-GFP fusions, but continued retention at both the NE and ER for a chimera lacking just the carboxy-terminal 377 amino acids. Neither deletion of Pom152 nor its carboxy-terminal glycosylation sites altered the nuclear protein export rate of an Msn5/Kap142 protein cargo. These data narrow the Pom152 region sufficient for NPC localization and provide evidence that alterations in other domains may impact Pom152 targeting or affinity for the NPC.

Epithelial membrane protein 3 regulates lung cancer stem cells via the TGF­ß signaling pathway.

Epithelial membrane protein 3 (EMP3) is a transmembrane glycoprotein that contains a peripheral myelin protein 22 domain. EMP3 first received attention as a tumor suppressor, but accumulating evidence has since suggested that it may exhibit a tumor­promoting function. Nonetheless, the biological function of EMP3 remains largely unclear with regards to its role in cancer. Herein, it was shown that EMP3 expression is upregulated in non­small cell lung cancer (NSCLC) cells overexpressing aldehyde dehydrogenase 1 (ALDH1). EMP3 was shown to be involved in cell proliferation, the formation of cancer stem cells (CSCs) and in epithelial­mesenchymal transition (EMT). The ability to resist irradiation, one of the characteristics of CSCs, decreased when the EMP3 mRNA expression was knocked down using small interfering RNA. In addition, when EMP3 knockdown reduced the migratory ability of cells, a characteristic of EMT. Additionally, it was shown that the TGF­ß/Smad signaling axis was a target of EMP3. EMP3 was found to interact with TGF­ß receptor type 2 (TGFBR2) upon TGF­ß stimulation in lung CSCs (LCSC). As a result, binding of EMP3­TGFBR2 regulates TGF­ß/Smad signaling activation and consequently affects CSCs and EMT. Kaplan­Meier analysis results confirmed that patients with high expression of EMP3 had poor survival rates. Taken together, these findings showed that EMP3 may be a potential target for management of LCSCs with high expression of ALDH1, and that EMP3 is involved in TGF­ß/Smad signaling activation where it promotes acquisition of cancerous properties in tumors.

P-Selectin Glycoprotein Ligand 1: A Potential HIV-1 Therapeutic Target.

Antiretroviral therapy (ART), which is a life-long therapeutic option, remains the only currently effective clinical method to treat HIV-1 infection. However, ART may be toxic to vital organs including the liver, brain, heart, and kidneys, and may result in systemic complications. In this context, to consider HIV-1 restriction factors from the innate immune system to explore novel HIV therapeutics is likely to be a promising investigative strategy. In light of this, P-selectin glycoprotein ligand 1 (PSGL-1) has recently become the object of close scrutiny as a recognized cell adhesion molecule, and has become a major focus of academic study, as researchers believe that PSGL-1 may represent a novel area of interest in the research inquiry into the field of immune checkpoint inhibition. In this article, we review PSGL-1's structure and functions during infection and/or inflammation. We also outline a comprehensive review of its role and potential therapeutic utility during HIV-1 infection as published in contemporary academic literature.

Transcriptomic Analysis and C-Terminal Epitope Tagging Reveal Differential Processing and Signaling of Endogenous TLR3 and TLR7.

Toll-like receptor (TLR) signaling is critical for defense against pathogenic infection, as well as for modulating tissue development. Activation of different TLRs triggers common inflammatory responses such as cytokine induction. Here, we reveal differential impacts of TLR3 and TLR7 signaling on transcriptomic profiles in bone marrow-derived macrophages (BMDMs). Apart from self-regulation, TLR3, but not TLR7, induced expression of other TLRs, suggesting that TLR3 activation globally enhances innate immunity. Moreover, we observed diverse influences of TLR3 and TLR7 signaling on genes involved in methylation, caspase and autophagy pathways. We compared endogenous TLR3 and TLR7 by using CRISPR/Cas9 technology to knock in a dual Myc-HA tag at the 3' ends of mouse Tlr3 and Tlr7. Using anti-HA antibodies to detect endogenous tagged TLR3 and TLR7, we found that both TLRs display differential tissue expression and posttranslational modifications. C-terminal tagging did not impair TLR3 activity. However, it disrupted the interaction between TLR7 and myeloid differentiation primary response 88 (MYD88), the Tir domain-containing adaptor of TLR7, which blocked its downstream signaling necessary to trigger cytokine and chemokine expression. Our study demonstrates different properties for TLR3 and TLR7, and also provides useful mouse models for further investigation of these two RNA-sensing TLRs.

Glycoprotein non-metastatic melanoma protein B functions with growth factor signaling to induce tumorigenesis through its serine phosphorylation.

Breast cancer is the most common cancer among women. Glycoprotein non-metastatic melanoma protein B (GPNMB), a type I transmembrane protein that is highly expressed in many cancers, including breast cancer, has been shown to be a prognostic factor. We previously reported that GPNMB overexpression confers tumorigenic potential, as evidenced by invasive tumor growth in vivo, sphere formation, and cellular migration and invasion to non-tumorigenic mammary epithelial cells. In this study, we focused on the serine (S) residue in the intracellular domain of GPNMB (S530 in human isoform b and S546 in mouse), which is predicted to be a phosphorylation site. To investigate the roles of this serine residue, we made an antibody specific for S530-phosphorylated human GPNMB and a point mutant in which S530 is replaced by an alanine (A) residue, GPNMB(SA). Established GPNMB(SA) overexpressing cells showed a significant reduction in sphere formation in vitro and tumor growth in vivo as a result of decreased stemness-related gene expression compared to that in GPNMB(WT)-expressing cells. In addition, GPNMB(SA) impaired GPNMB-mediated cellular migration. Furthermore, we found that tyrosine kinase receptor signaling triggered by epidermal growth factor or fibroblast growth factor 2 induces the serine phosphorylation of GPNMB through activation of downstream oncoproteins RAS and RAF.

FLRT2 and FLRT3 Cooperate in Maintaining the Tangential Migratory Streams of Cortical Interneurons during Development.

Neuron migration is a hallmark of nervous system development that allows gathering of neurons from different origins for assembling of functional neuronal circuits. Cortical inhibitory interneurons arise in the ventral telencephalon and migrate tangentially forming three transient migratory streams in the cortex before reaching the final laminar destination. Although migration defects lead to the disruption of inhibitory circuits and are linked to aspects of psychiatric disorders such as autism and schizophrenia, the molecular mechanisms controlling cortical interneuron development and final layer positioning are incompletely understood. Here, we show that mouse embryos with a double deletion of FLRT2 and FLRT3 genes encoding cell adhesion molecules exhibit an abnormal distribution of interneurons within the streams during development, which in turn, affect the layering of somatostatin+ interneurons postnatally. Mechanistically, FLRT2 and FLRT3 proteins act in a noncell-autonomous manner, possibly through a repulsive mechanism. In support of such a conclusion, double knockouts deficient in the repulsive receptors for FLRTs, Unc5B and Unc5D, also display interneuron defects during development, similar to the FLRT2/FLRT3 mutants. Moreover, FLRT proteins are chemorepellent ligands for developing interneurons in vitro, an effect that is in part dependent on FLRT-Unc5 interaction. Together, we propose that FLRTs act through Unc5 receptors to control cortical interneuron distribution in a mechanism that involves cell repulsion.SIGNIFICANCE STATEMENT Disruption of inhibitory cortical circuits is responsible for some aspects of psychiatric disorders such as schizophrenia or autism. These defects include interneuron migration during development. A crucial step during this process is the formation of three transient migratory streams within the developing cortex that determine the timing of interneuron final positioning and the formation of functional cortical circuits in the adult. We report that FLRT proteins are required for the proper distribution of interneurons within the cortical migratory streams and for the final laminar allocation in the postnatal cortex. These results expand the multifunctional role of FLRTs during nervous system development in addition to the role of FLRTs in axon guidance and the migration of excitatory cortical neurons.

The Multifunctional Role of EMP3 in the Regulation of Membrane Receptors Associated with IDH-Wild-Type Glioblastoma.

Epithelial membrane protein 3 (EMP3) is a tetraspan membrane protein overexpressed in isocitrate dehydrogenase-wild-type (IDH-wt) glioblastoma (GBM). Several studies reported high EMP3 levels as a poor prognostic factor in GBM patients. Experimental findings based on glioma and non-glioma models have demonstrated the role of EMP3 in the regulation of several membrane proteins known to drive IDH-wt GBM. In this review, we summarize what is currently known about EMP3 biology. We discuss the regulatory effects that EMP3 exerts on a variety of oncogenic receptors and discuss how these mechanisms may relate to IDH-wt GBM. Lastly, we enumerate the open questions towards EMP3 function in IDH-wt GBM.