The podoplanin-CLEC-2 interaction promotes platelet-mediated melanoma pulmonary metastasis.

BACKGROUND: Podoplanin (PDPN) expressed on tumour cells interacts with platelet C-type lectin-like receptor 2 (CLEC-2). This study aimed to investigate the role of the PDPN-platelet CLEC-2 interaction in melanoma pulmonary metastasis. METHODS: Murine melanoma B16-F0 cells, which have two populations that express podoplanin, were sorted by FACS with anti-podoplanin staining to obtain purified PDPN + and PDPN- B16-F0 cells. C57BL/6J mice transplanted with CLEC-2-deficient bone marrow cells were used for in vivo experiments. RESULTS: The in vivo data showed that the number of metastatic lung nodules in WT mice injected with PDPN + cells was significantly higher than that in WT mice injected with PDPN- cells and in WT or CLEC-2 KO mice injected with PDPN- cells. In addition, our results revealed that the platelet Syk-dependent signalling pathway contributed to platelet aggregation and melanoma metastasis. CONCLUSIONS: Our study indicates that the PDPN-CLEC-2 interaction promotes experimental pulmonary metastasis in a mouse melanoma model. Tumour cell-induced platelet aggregation mediated by the interaction between PDPN and CLEC-2 is a key factor in melanoma pulmonary metastasis.

Antibody-mediated targeting of human microglial leukocyte Ig-like receptor B4 attenuates amyloid pathology in a mouse model.

Microglia help limit the progression of Alzheimer's disease (AD) by constraining amyloid-ß (Aß) pathology, effected through a balance of activating and inhibitory intracellular signals delivered by distinct cell surface receptors. Human leukocyte Ig-like receptor B4 (LILRB4) is an inhibitory receptor of the immunoglobulin (Ig) superfamily that is expressed on myeloid cells and recognizes apolipoprotein E (ApoE) among other ligands. Here, we find that LILRB4 is highly expressed in the microglia of patients with AD. Using mice that accumulate Aß and carry a transgene encompassing a portion of the LILR region that includes LILRB4, we corroborated abundant LILRB4 expression in microglia wrapping around Aß plaques. Systemic treatment of these mice with an anti-human LILRB4 monoclonal antibody (mAb) reduced Aß load, mitigated some Aß-related behavioral abnormalities, enhanced microglia activity, and attenuated expression of interferon-induced genes. In vitro binding experiments established that human LILRB4 binds both human and mouse ApoE and that anti-human LILRB4 mAb blocks such interaction. In silico modeling, biochemical, and mutagenesis analyses identified a loop between the two extracellular Ig domains of LILRB4 required for interaction with mouse ApoE and further indicated that anti-LILRB4 mAb may block LILRB4-mApoE by directly binding this loop. Thus, targeting LILRB4 may be a potential therapeutic avenue for AD.

Tumor associated microglia/macrophages utilize GPNMB to promote tumor growth and alter immune cell infiltration in glioma.

Tumor-associated microglia and blood-derived macrophages (TAMs) play a central role in modulating the immune suppressive microenvironment in glioma. Here, we show that GPNMB is predominantly expressed by TAMs in human glioblastoma multiforme and the murine RCAS-PDGFb high grade glioma model. Loss of GPNMB in the in vivo tumor microenvironment results in significantly smaller tumor volumes and generates a pro-inflammatory innate and adaptive immune cell microenvironment. The impact of host-derived GPNMB on tumor growth was confirmed in two distinct murine glioma cell lines in organotypic brain slices from GPNMB-KO and control mice. Using published data bases of human glioma, the elevated levels in TAMs could be confirmed and the GPNMB expression correlated with a poorer survival.

Lipopolysaccharide binding protein resists hepatic oxidative stress by regulating lipid droplet homeostasis.

Oxidative stress-induced lipid accumulation is mediated by lipid droplets (LDs) homeostasis, which sequester vulnerable unsaturated triglycerides into LDs to prevent further peroxidation. Here we identify the upregulation of lipopolysaccharide-binding protein (LBP) and its trafficking through LDs as a mechanism for modulating LD homeostasis in response to oxidative stress. Our results suggest that LBP induces lipid accumulation by controlling lipid-redox homeostasis through its lipid-capture activity, sorting unsaturated triglycerides into LDs. N-acetyl-L-cysteine treatment reduces LBP-mediated triglycerides accumulation by phospholipid/triglycerides competition and Peroxiredoxin 4, a redox state sensor of LBP that regulates the shuttle of LBP from LDs. Furthermore, chronic stress upregulates LBP expression, leading to insulin resistance and obesity. Our findings contribute to the understanding of the role of LBP in regulating LD homeostasis and against cellular peroxidative injury. These insights could inform the development of redox-based therapies for alleviating oxidative stress-induced metabolic dysfunction.

Decorin suppresses tumor lymphangiogenesis: A mechanism to curtail cancer progression.

The complex interplay between malignant cells and the cellular and molecular components of the tumor stroma is a key aspect of cancer growth and development. These tumor-host interactions are often affected by soluble bioactive molecules such as proteoglycans. Decorin, an archetypical small leucine-rich proteoglycan primarily expressed by stromal cells, affects cancer growth in its soluble form by interacting with several receptor tyrosine kinases (RTK). Overall, decorin leads to a context-dependent and protracted cessation of oncogenic RTK activity by attenuating their ability to drive a prosurvival program and to sustain a proangiogenic network. Through an unbiased transcriptomic analysis using deep RNAseq, we identified that decorin down-regulated a cluster of tumor-associated genes involved in lymphatic vessel (LV) development when systemically delivered to mice harboring breast carcinoma allografts. We found that Lyve1 and Podoplanin, two established markers of LVs, were markedly suppressed at both the mRNA and protein levels, and this suppression correlated with a significant reduction in tumor LVs. We further identified that soluble decorin, but not its homologous proteoglycan biglycan, inhibited LV sprouting in an ex vivo 3D model of lymphangiogenesis. Mechanistically, we found that decorin interacted with vascular endothelial growth factor receptor 3 (VEGFR3), the main lymphatic RTK, and its activity was required for the decorin-mediated block of lymphangiogenesis. Finally, we identified that Lyve1 was in part degraded via decorin-evoked autophagy in a nutrient- and energy-independent manner. These findings implicate decorin as a biological factor with antilymphangiogenic activity and provide a potential therapeutic agent for curtailing breast cancer growth and metastasis.

TNF receptor 2 knockout mouse had reduced lung cancer growth and schizophrenia-like behavior through a decrease in TrkB-dependent BDNF level.

The relationship between schizophrenia (SCZ) and cancer development remains controversial. Based on the disease-gene association platform, it has been revealed that tumor necrosis factor receptor (TNFR) could be an important mediatory factor in both cancer and SCZ development. TNF-α also increases the expression of brain-derived neurotrophic factor (BDNF) and tropomyosin receptor kinase B (TrkB) in the development of SCZ and tumor, but the role of TNFR in mediating the association between the two diseases remains unclear. We studied the vital roles of TNFR2 in the progression of tumor and SCZ-like behavior using A549 lung cancer cell xenografted TNFR2 knockout mice. TNFR2 knockout mice showed significantly decreased tumor size and weight as well as schizophrenia-like behaviors compared to wild-type mice. Consistent with the reduced tumor growth and SCZ-like behaviors, the levels of TrkB and BDNF expression were significantly decreased in the lung tumor tissues and pre-frontal cortex of TNFR2 knockout mice. However, intravenous injection of BDNF (160 µg/kg) to TNFR2 knockout mice for 4 weeks increased tumor growth and SCZ-like behaviors as well as TrkB expression. In in vitro study, significantly decreased cell growth and expression of TrkB and BDNF by siTNFR2 transfection were found in A549 lung cancer cells. However, the addition of BDNF (100 ng/ml) into TNFR2 siRNA transfected A549 lung cancer cells recovered cell growth and the expression of TrkB. These results suggest that TNFR2 could be an important factor in mediating the comorbidity between lung tumor growth and SCZ development through increased TrkB-dependent BDNF levels.

The binding of PKCε and MEG2 to STAT3 regulates IL-6-mediated microglial hyperalgesia during inflammatory pain.

Studies have suggested that microglial IL-6 modulates inflammatory pain; however, the exact mechanism of action remains unclear. We therefore hypothesized that PKCε and MEG2 competitively bind to STAT3 and contribute to IL-6-mediated microglial hyperalgesia during inflammatory pain. Freund's complete adjuvant (FCA) and lipopolysaccharide (LPS) were used to induce hyperalgesia model mice and microglial inflammation. Mechanical allodynia was evaluated using von Frey tests in vivo. The interaction among PKCε, MEG2, and STAT3 was determined using ELISA and immunoprecipitation assay in vitro. The PKCε, MEG2, t-STAT3, pSTAT3Tyr705, pSTAT3Ser727, IL-6, GLUT3, and TREM2 were assessed by Western blot. IL-6 promoter activity and IL-6 concentration were examined using dual luciferase assays and ELISA. Overexpression of PKCε and MEG2 promoted and attenuated inflammatory pain, accompanied by an increase and decrease in IL-6 expression, respectively. PKCε displayed a stronger binding ability to STAT3 when competing with MEG2. STAT3Ser727 phosphorylation increased STAT3 interaction with both PKCε and MEG2. Moreover, LPS increased PKCε, MEG2, pSTAT3Tyr705, pSTAT3Ser727, IL-6, and GLUT3 levels and decreased TREM2 during microglia inflammation. IL-6 promoter activity was enhanced or inhibited by PKCε or MEG2 in the presence of STAT3 and LPS stimulation, respectively. In microglia, overexpression of PKCε and/or MEG2 resulted in the elevation of tSTAT3, pSTAT3Tyr705, pSTAT3Ser727, IL-6, and TREM2, and the reduction of GLUT3. PKCε is more potent than MEG2 when competitively binding to STAT3, displaying dual modulatory effects of IL-6 production, thus regulating the GLUT3 and TREM2 in microglia during inflammatory pain sensation.

TREM2 alleviates white matter injury after traumatic brain injury in mice might be mediated by regulation of DHCR24/LXR pathway in microglia.

BACKGROUND: White matter injury (WMI) is an important pathological process after traumatic brain injury (TBI). The correlation between white matter functions and the myeloid cells expressing triggering receptor-2 (TREM2) has been convincingly demonstrated. Moreover, a recent study revealed that microglial sterol metabolism is crucial for early remyelination after demyelinating diseases. However, the potential roles of TREM2 expression and microglial sterol metabolism in WMI after TBI have not yet been explored. METHODS: Controlled cortical injury was induced in both wild-type (WT) and TREM2 depletion (TREM2 KO) mice to simulate clinical TBI. COG1410 was used to upregulate TREM2, while PLX5622 and GSK2033 were used to deplete microglia and inhibit the liver X receptor (LXR), respectively. Immunofluorescence, Luxol fast blue staining, magnetic resonance imaging, transmission electron microscopy, and oil red O staining were employed to assess WMI after TBI. Neurological behaviour tests and electrophysiological recordings were utilized to evaluate cognitive functions following TBI. Microglial cell sorting and transcriptomic sequencing were utilized to identify alterations in microglial sterol metabolism-related genes, while western blot was conducted to validate the findings. RESULTS: TREM2 expressed highest at 3 days post-TBI and was predominantly localized to microglial cells within the white matter. Depletion of TREM2 worsened aberrant neurological behaviours, and this phenomenon was mediated by the exacerbation of WMI, reduced renewal of oligodendrocytes, and impaired phagocytosis ability of microglia after TBI. Subsequently, the upregulation of TREM2 alleviated WMI, promoted oligodendrocyte regeneration, and ultimately facilitated the recovery of neurological behaviours after TBI. Finally, the expression of DHCR24 increased in TREM2 KO mice after TBI. Interestingly, TREM2 inhibited DHCR24 and upregulated members of the LXR pathway. Moreover, LXR inhibition could partially reverse the effects of TREM2 upregulation on electrophysiological activities. CONCLUSIONS: We demonstrate that TREM2 has the potential to alleviate WMI following TBI, possibly through the DHCR24/LXR pathway in microglia.

Conformational changes in the Niemann-Pick type C1 protein NCR1 drive sterol translocation.

The membrane protein Niemann-Pick type C1 (NPC1, named NCR1 in yeast) is central to sterol homeostasis in eukaryotes. Saccharomyces cerevisiae NCR1 is localized to the vacuolar membrane, where it is suggested to carry sterols across the protective glycocalyx and deposit them into the vacuolar membrane. However, documentation of a vacuolar glycocalyx in fungi is lacking, and the mechanism for sterol translocation has remained unclear. Here, we provide evidence supporting the presence of a glycocalyx in isolated S. cerevisiae vacuoles and report four cryo-EM structures of NCR1 in two distinct conformations, named tense and relaxed. These two conformations illustrate the movement of sterols through a tunnel formed by the luminal domains, thus bypassing the barrier presented by the glycocalyx. Based on these structures and on comparison with other members of the Resistance-Nodulation-Division (RND) superfamily, we propose a transport model that links changes in the luminal domains with a cycle of protonation and deprotonation within the transmembrane region of the protein. Our model suggests that NPC proteins work by a generalized RND mechanism where the proton motive force drives conformational changes in the transmembrane domains that are allosterically coupled to luminal/extracellular domains to promote sterol transport.

FLRT2 prevents endothelial cell senescence and vascular aging by regulating the ITGB4/mTORC2/p53 signaling pathway.

The roles of fibronectin leucine-rich transmembrane protein 2 (FLRT2) in physiological and pathological processes are not well known. Here, we identify a potentially novel function of FLRT2 in preventing endothelial cell senescence and vascular aging. We found that FLRT2 expression was lower in cultured senescent endothelial cells as well as in aged rat and human vascular tissues. FLRT2 mediated endothelial cell senescence via the mTOR complex 2, AKT, and p53 signaling pathway in human endothelial cells. We uncovered that FLRT2 directly associated with integrin subunit beta 4 (ITGB4) and thereby promoted ITGB4 phosphorylation, while inhibition of ITGB4 substantially mitigated the induction of senescence triggered by FLRT2 depletion. Importantly, FLRT2 silencing in mice promoted vascular aging, and overexpression of FLRT2 rescued a premature vascular aging phenotype. Therefore, we propose that FLRT2 could be targeted therapeutically to prevent senescence-associated vascular aging.

Structural basis for antiepileptic drugs and botulinum neurotoxin recognition of SV2A.

More than one percent of people have epilepsy worldwide. Levetiracetam (LEV) is a successful new-generation antiepileptic drug (AED), and its derivative, brivaracetam (BRV), shows improved efficacy. Synaptic vesicle glycoprotein 2a (SV2A), a putative membrane transporter in the synaptic vesicles (SVs), has been identified as a target of LEV and BRV. SV2A also serves as a receptor for botulinum neurotoxin (BoNT), which is the most toxic protein and has paradoxically emerged as a potent reagent for therapeutic and cosmetic applications. Nevertheless, no structural analysis on AEDs and BoNT recognition by full-length SV2A has been available. Here we describe the cryo-electron microscopy structures of the full-length SV2A in complex with the BoNT receptor-binding domain, BoNT/A2 HC, and either LEV or BRV. The large fourth luminal domain of SV2A binds to BoNT/A2 HC through protein-protein and protein-glycan interactions. LEV and BRV occupy the putative substrate-binding site in an outward-open conformation. A propyl group in BRV creates additional contacts with SV2A, explaining its higher binding affinity than that of LEV, which was further supported by label-free spectral shift assay. Numerous LEV derivatives have been developed as AEDs and positron emission tomography (PET) tracers for neuroimaging. Our work provides a structural framework for AEDs and BoNT recognition of SV2A and a blueprint for the rational design of additional AEDs and PET tracers.

Cell cycle dependent coordination of surface layer biogenesis in Caulobacter crescentus.

Surface layers (S-layers) are proteinaceous, two-dimensional paracrystalline arrays that constitute a major component of the cell envelope in many prokaryotic species. In this study, we investigated S-layer biogenesis in the bacterial model organism Caulobacter crescentus. Fluorescence microscopy revealed localised incorporation of new S-layer at the poles and mid-cell, consistent with regions of cell growth in the cell cycle. Light microscopy and electron cryotomography investigations of drug-treated bacteria revealed that localised S-layer insertion is retained when cell division is inhibited, but is disrupted upon dysregulation of MreB or lipopolysaccharide. We further uncovered that S-layer biogenesis follows new peptidoglycan synthesis and localises to regions of high cell wall turnover. Finally, correlated cryo-light microscopy and electron cryotomographic analysis of regions of S-layer insertion showed the presence of discontinuities in the hexagonal S-layer lattice, contrasting with other S-layers completed by defined symmetric defects. Our findings present insights into how C. crescentus cells form an ordered S-layer on their surface in coordination with the biogenesis of other cell envelope components.

Regulation of human microglial gene expression and function via RNAase-H active antisense oligonucleotides in vivo in Alzheimer's disease.

BACKGROUND: Microglia play important roles in maintaining brain homeostasis and neurodegeneration. The discovery of genetic variants in genes predominately or exclusively expressed in myeloid cells, such as Apolipoprotein E (APOE) and triggering receptor expressed on myeloid cells 2 (TREM2), as the strongest risk factors for Alzheimer's disease (AD) highlights the importance of microglial biology in the brain. The sequence, structure and function of several microglial proteins are poorly conserved across species, which has hampered the development of strategies aiming to modulate the expression of specific microglial genes. One way to target APOE and TREM2 is to modulate their expression using antisense oligonucleotides (ASOs). METHODS: In this study, we identified, produced, and tested novel, selective and potent ASOs for human APOE and TREM2. We used a combination of in vitro iPSC-microglia models, as well as microglial xenotransplanted mice to provide proof of activity in human microglial in vivo. RESULTS: We proved their efficacy in human iPSC microglia in vitro, as well as their pharmacological activity in vivo in a xenografted microglia model. We demonstrate ASOs targeting human microglia can modify their transcriptional profile and their response to amyloid-ß plaques in vivo in a model of AD. CONCLUSIONS: This study is the first proof-of-concept that human microglial can be modulated using ASOs in a dose-dependent manner to manipulate microglia phenotypes and response to neurodegeneration in vivo.

Dynamic pathological analysis reveals a protective role against skin fibrosis for TREM2-dependent macrophages.

Rationale: Systemic sclerosis (SSc) is a chronic and incurable autoimmune disease with high mortality rates, and skin fibrosis is one of distinguishing hallmarks in the pathogenesis. However, macrophage heterogeneity regulating skin fibrosis remain largely unknown. Methods: We established mouse disease model and performed single-cell RNA-sequencing (scRNA-seq) to resolve the dynamic and heterogenous characteristics of macrophages in skin fibrosis, and the role of TREM2-dependent macrophages in the pathological process was investigated using knockout mice and intraperitoneal transferring TREM2+ macrophages combining with functional assays. Results: We show that TREM2-expressing macrophages (TREM2+ MФs) accumulate in injured skin of mice treated by bleomycin (BLM) and human SSc, and their gene signatures and functional pathways are identified in the course of disease. Genetic ablation of Trem2 in mice globally accelerates and aggravates skin fibrosis, whereas transferring TREM2hi macrophages improves and alleviates skin fibrosis. Amazingly, we found that disease-associated TREM2+ MФs in skin fibrosis exhibit overlapping signatures with fetal skin counterparts in mice and human to maintain skin homeostasis, but each has merits in skin remodeling and development respectively. Conclusion: This study identifies that TREM2 acts as a functional molecule and a major signaling by which macrophage subpopulations play a protective role against fibrosis, and disease-associated TREM2+ MФs in skin fibrosis might undergo a fetal-like reprogramming similar to fetal skin counterparts.

A structure-based designed small molecule depletes hRpn13Pru and a select group of KEN box proteins.

Proteasome subunit hRpn13 is partially proteolyzed in certain cancer cell types to generate hRpn13Pru by degradation of its UCHL5/Uch37-binding DEUBAD domain and retention of an intact proteasome- and ubiquitin-binding Pru domain. By using structure-guided virtual screening, we identify an hRpn13 binder (XL44) and solve its structure ligated to hRpn13 Pru by integrated X-ray crystallography and NMR to reveal its targeting mechanism. Surprisingly, hRpn13Pru is depleted in myeloma cells following treatment with XL44. TMT-MS experiments reveal a select group of off-targets, including PCNA clamp-associated factor PCLAF and ribonucleoside-diphosphate reductase subunit M2 (RRM2), that are similarly depleted by XL44 treatment. XL44 induces hRpn13-dependent apoptosis and also restricts cell viability by a PCLAF-dependent mechanism. A KEN box, but not ubiquitination, is required for XL44-induced depletion of PCLAF. Here, we show that XL44 induces ubiquitin-dependent loss of hRpn13Pru and ubiquitin-independent loss of select KEN box containing proteins.

Endothelial CLEC-1b plays a protective role against cancer hematogenous metastasis.

Metastasis, which is the spread of cancer cells into distant organs, is a critical determinant of prognosis in patients with cancer, and blood vessels are the major route for cancer cells to spread systemically. Extravasation is a critical process for the hematogenous metastasis; however, its underlying molecular mechanisms remain poorly understood. Here, we identified that senescent ECs highly express C-type lectin domain family 1 member B (CLEC-1b), and that endothelial CLEC-1b inhibits the hematogenous metastasis of a certain type of cancer. CLEC-1b expression was enhanced in ECs isolated from aged mice, senescent cultured human ECs, and ECs of aged human. CLEC-1b overexpression in ECs prevented the disruption of endothelial integrity, and inhibited the transendothelial migration of cancer cells expressing podoplanin (PDPN), a ligand for CLEC-1b. Notably, target activation of CLEC-1b in ECs decreased the hematogenous metastasis in the lungs by cancer cells expressing PDPN in mice. Our data reveal the protective role of endothelial CLEC-1b against cancer hematogenous metastasis. Considering the high CLEC-1b expression in senescent ECs, EC senescence may play a beneficial role with respect to the cancer hematogenous metastasis.

Repulsive Sema3E-Plexin-D1 signaling coordinates both axonal extension and steering via activating an autoregulatory factor, Mtss1.

Axon guidance molecules are critical for neuronal pathfinding because they regulate directionality and growth pace during nervous system development. However, the molecular mechanisms coordinating proper axonal extension and turning are poorly understood. Here, metastasis suppressor 1 (Mtss1), a membrane protrusion protein, ensured axonal extension while sensitizing axons to the Semaphorin 3E (Sema3E)-Plexin-D1 repulsive cue. Sema3E-Plexin-D1 signaling enhanced Mtss1 expression in projecting striatonigral neurons. Mtss1 localized to the neurite axonal side and regulated neurite outgrowth in cultured neurons. Mtss1 also aided Plexin-D1 trafficking to the growth cone, where it signaled a repulsive cue to Sema3E. Mtss1 ablation reduced neurite extension and growth cone collapse in cultured neurons. Mtss1-knockout mice exhibited fewer striatonigral projections and irregular axonal routes, and these defects were recapitulated in Plxnd1- or Sema3e-knockout mice. These findings demonstrate that repulsive axon guidance activates an exquisite autoregulatory program coordinating both axonal extension and steering during neuronal pathfinding.

FLRT3 and TGF-ß/SMAD4 signalling: Impacts on apoptosis, autophagy and ion channels in supraventricular tachycardia.

To explore the underlying molecular mechanisms of supraventricular tachycardia (SVT), this study aimed to analyse the complex relationship between FLRT3 and TGF-ß/SMAD4 signalling pathway, which affects Na+ and K+ channels in cardiomyocytes. Bioinformatics analysis was performed on 85 SVT samples and 15 healthy controls to screen overlapping genes from the key module and differentially expressed genes (DEGs). Expression profiling of overlapping genes, coupled with Receiver Operating Characteristic (ROC) curve analyses, identified FLRT3 as a hub gene. In vitro studies utilizing Ang II-stimulated H9C2 cardiomyocytes were undertaken to elucidate the consequences of FLRT3 silencing on cardiomyocyte apoptosis and autophagic processes. Utilizing a combination of techniques such as quantitative reverse-transcription polymerase chain reaction (qRT-PCR), western blotting (WB), flow cytometry, dual-luciferase reporter assays and chromatin immunoprecipitation polymerase chain reaction (ChIP-PCR) assays were conducted to decipher the intricate interactions between FLRT3, the TGF-ß/SMAD4 signalling cascade and ion channel gene expression. Six genes (AADAC, DSC3, FLRT3, SYT4, PRR9 and SERTM1) demonstrated reduced expression in SVT samples, each possessing significant clinical diagnostic potential. In H9C2 cardiomyocytes, FLRT3 silencing mitigated Ang II-induced apoptosis and modulated autophagy. With increasing TGF-ß concentration, there was a dose-responsive decline in FLRT3 and SCN5A expression, while both KCNIP2 and KCND2 expressions were augmented. Moreover, a direct interaction between FLRT3 and SMAD4 was observed, and inhibition of SMAD4 expression resulted in increased FLRT3 expression. Our results demonstrated that the TGF-ß/SMAD4 signalling pathway plays a critical role by regulating FLRT3 expression, with potential implications for ion channel function in SVT.

CD93 regulates breast cancer growth and vasculogenic mimicry through the PI3K/AKT/SP2 signaling pathway activated by integrin ß1.

In women, breast cancer (BC) accounts for 7%-10% of all cancer cases and is one of the most common cancers. To identify a new method for treating BC, the role of CD93 and its underlying mechanism were explored. MDA-MB-231 cells were used in this study and transfected with si-CD93, si-MMRN2, oe-CD93, si-integrin ß1, or oe-SP2 lentivirus. After MDA-MB-231 cells were transfected with si-NC or si-CD93, they were injected into nude mice by subcutaneous injection at a dose of 5 × 106/mouse to construct a BC animal model. The expression of genes and proteins and cell migration, invasion and vasculogenic mimicry were detected by RT‒qPCR, western blot, immunohistochemistry, immunofluorescence, Transwell, and angiogenesis assays. In pathological samples and BC cell lines, CD93 was highly expressed. Functionally, CD93 promoted the proliferation, migration, and vasculogenic mimicry of MDA-MB-231 cells. Moreover, CD93 interacts with MMRN2 and integrin ß1. Knockdown of CD93 and MMRN2 can inhibit the activation of integrin ß1, thereby inhibiting the PI3K/AKT/SP2 signaling pathway and inhibiting BC growth and vasculogenic mimicry. In conclusion, the binding of CD93 to MMRN2 can activate integrin ß1, thereby activating the PI3K/AKT/SP2 signaling pathway and subsequently promoting BC growth and vasculogenic mimicry.