Overexpression Of CD9 Research Articles

Bioinformatics investigation of the prognostic value and mechanistic role of CD9 in glioma

In recent years, CD9 has been extensively studied as a potential biomarker for cancer. However, the biological role of CD9 in gliomas remains unclear. This study investigates the function of CD9 in gliomas and its molecular mechanisms. Utilizing pan-cancer analysis with TCGA, CGGA, and GEO databases, differential expression of CD9 was observed in 11 tumor types within the TCGA cohort, and it was associated with patient survival rates. Analysis of the CGGA glioma database revealed that patients with high CD9 expression had lower survival rates. The area under the ROC curve (AUC) for GSE16011 was greater than 0.7, indicating a high discriminative ability. Through gene set enrichment analysis (GSEA), immune-related analysis, and CD9 mutation detection, CD9 was found to have the strongest correlation with neutrophil involvement (cor = 0.30, P < 0.05), and the high CD9 expression group exhibited higher rejection responses and TIDE scores, suggesting a lower likelihood of successful immunotherapy. The high CD9 expression group was more sensitive to 81 drugs, indicating potential therapeutic effects for gliomas. Furthermore, overexpression of CD9 in gliomas may be associated with gene mutations. Down-regulation or up-regulation of CD9 expression in the glioblastoma cell line LN229 showed that CD9 could positively regulate the migratory ability of LN229 cells. Further, several marker genes, such as VEGFR-2, TGF-β1, CASP1 and PI3K, were down regulated in CD9 knockdown cell lines and up regulated in CD9 overexpression cell lines, compared with control cell line. This study preliminarily explores the role of CD9 in gliomas and its prognostic value, providing new insights for personalized treatment strategies in glioma therapy.

A Novel Rat Model of Venous Hypertensive Myelopathy Produced by Arteriovenous Bypass Plus Venous Stenosis.

Venous hypertensive myelopathy (VHM), mainly induced by the spinal dural arteriovenous fistula, is a congestive spinal cord injury that currently has no appropriate animal model available in preclinical research. Sprague Dawley rats (280-320 g) were used. The rats were divided into 3 groups: (1) Group 1, which underwent renal artery-dorsal spinal venous bypass (AVB group); (2) Group 2, which underwent renal artery-dorsal spinal venous bypass and drainage vein stenosis (AVB/VS group); and (3) Control group, with T13 dorsal vein ligation. The success of the model was assessed using Doppler ultrasound and 7.0-T magnetic resonance imaging. Transmission electron microscopy, histochemistry, proteomics, and western blot analysis were used to evaluate ultrastructural, pathological, and molecular features in the spinal cord and cerebrospinal fluid (CSF). The success rate of the arteriovenous bypass was 100% at 5 days and 83% at 2 weeks. The locomotor assessment showed decreased lower extremity strength in the AVB/VS group ( P = .0067), whereas unremarkable changes were found in the AVB and Control groups. Histochemical staining suggested a 2-fold expansion of the dorsal spinal vein in the AVB/VS group, which was lower than that in the AVB group ( P < .05); however, the former displayed greater myelin and neuronal damage ( P < .05) and slight dilatation of the central canal ( P > .05). Proteomics analysis revealed that the complement and coagulation cascade pathways were upregulated in the CSF of AVB/VS rats, whereas the C3 level was elevated both in the CSF and bilateral spinal cord. Furthermore, overexpression of C3, ITGB2, and CD9 in the spinal cord was confirmed by immunoblotting. These findings suggest that the AVB/VS model can effectively mimic the clinical and molecular characteristics of VHM. Furthermore, they suggest that impaired deep intramedullary venous drainage is the key reason for the VHM.

High expression of CD9 and Epidermal Growth Factor Receptor promotes the development of tongue cancer.

Tongue cancer is distinguished by aggressive behavior, a high risk of recurrence, lymph, and distant metastases. Hypoxia-Induced Factor 1 α functions as a CD9 transcription factor. CD9 is a transmembrane protein that may be found on the cell membrane. It can modulate the expression of the Epidermal Growth Factor Receptor (EGFR) pathway. ELISA was used to measure serum CD9, p-EGFR, and p-Akt levels in 70 tongue cancer patients and 35 healthy controls. RT-PCR was used to analyze the gene expression of the related genes. The gene as well as protein expression of CD9, EGFR/p-EGFR, and Akt/p-Akt was significantly higher in case subjects when compared with the controls. The expression of CD9 was higher in case subjects who were smokers/alcoholics when to control subjects who were smokers/alcoholics. Overexpression of CD9 due to hypoxic conditions leads to the activation of EGFR-signaling pathway resulting in cancer progression, resistance to chemotherapy. Hence, CD9 could be a potential target to suppress cancer progression.

The Tetraspanin CD9 Shapes Glucocorticoid Sensitivity in Pediatric B-Precursor Acute Lymphoblastic Leukemia

Background: De novo or acquired resistance to glucocorticoids (GCs), the common therapeutic regimens for remission-induction in pediatric B-precursor acute lymphoblastic leukemia, confronts treatment success and is a strong predictor for disease relapse. Discovery of predictive markers and underlying mechanisms of GC resistance could lead to new treatment and prognostic modalities. Aims: This study aims: (1) to demonstrate the association between CD9 expression and GC sensitivity in childhood BCP-ALL; (2) to dissect the underlying mechanisms driving GC resistance; and (3) to discover the possible therapeutic modalities for restoring GC susceptibility. Methods: Ex vivo drug sensitivity profiling to prednisone (Pred) and dexamethasone (Dex) was performed on 6 BCP-ALL cell lines and 18 patient samples with differential CD9 expression. Association of CD9 expression with patient responses to Pred prophase was analyzed in a pediatric BCP-ALL cohort (n=182). Functional impact of CD9 on GC susceptibility was dissected by gain- and loss-of-function experiments in BCP-ALL cells. Mechanistic basis of CD9-driven GC response was investigated through genomics, transcriptomics, and proteomics. Reversal of GC resistance by pathway-targeted agents were explored on BCP-ALL cells and xenograft models. Results: Distinctive differences in the drug sensitivity pattern were found between CD9- and CD9+ BCP-ALL cell lines, with the former showing resistance to GCs but not other chemotherapeutic agents. Concurrently, CD9- primary lymphoblasts exhibited resistance to Dex (IC50s: 2,761 vs. 31 nM) and Pred (IC50s: 8,185 vs. 268 nM) compared with CD9+ samples (positivity defined by the presence of ≥ 20% CD9+ blasts). More Pred poor responders (defined by the presence of ≥ 1×10 9/L circulating lymphoblasts on day 8) were found in CD9- than CD9+ patients (19.4% vs. 6.2%, P=0.018). Subgroup analyses revealed more poor responders with CD9- phenotype were enriched in patients with older age (60% vs. 3.7%, P=0.008), male gender (21.7% vs. 6.7%, P=0.045), higher white blood cell count (66.7% vs. 15.4%, P=0.004), and in those with BCR-ABL1 translocation (100% vs. 12.5%, P=0.024), or not otherwise cytogenetically specified BCP-ALL (30.8% vs. 5.8%, P=0.019). Univariate and multivariate analyses further confirmed CD9 negativity as an independent predictor for GC resistance (OR=5.1, P=0.009). Overexpression of CD9 in CD9- cells (SEM, KOPN-8) by lentiviral transduction significantly enhanced GC sensitivity by 2.8-62.3-fold ( P&amp;lt;0.024), whereas CD9 knockout in CD9+ cells (697) using CRISPR-Cas9 decreased GC susceptibility ( P&amp;lt;0.003). No significant differences in mRNA or protein expression of the GC receptor NR3C1 were identified between CD9+ and CD9- cells, as well as its isoforms or mutants. The cytoplasm-to-nucleus translocation of GC receptor was consistent among cell lines regardless of CD9 expression status, but CD9 was physically interacted with NR3C1. Upon GC stimulation, the presence of CD9 increased the number of DEGs (110 vs. 82 DEGs) and magnitude of GC-responsive genes exclusively detected in the background of CD9 (1.3-18.6-fold; e.g. AKAP13, STAT5A, EZR). CD9- cells showed constitutive activation of the MAPK pathway and could be preferentially restored for GC susceptibility by the MEK1/2 inhibitor trametinib in vitro. BCP-ALL xenograft modeling revealed that, compared to single agent Dex treatment, combination with trametinib significantly reduced the leukemic load in animal transplanted with CD9- SEM but not CD9+ BV-173 cell lines. Ex vivo drug testing further showed that the resistance to GCs in some CD9+ cases could be partially reversed by combination of trametinib and the JAK inhibitor ruxolitinib due to the activation of STAT5. Conclusions: This study provides a definitive linkage of CD9 with GC sensitivity in childhood BCP-ALL mediated through a NR3C1-independent mechanism. CD9- cases could be prone to trametinib combination therapy, whereas CD9+ cases might need a third drug such as ruxolitinib to counteract GC resistance.

Epigenetic Silencing of CD9 Shapes a New Mechanism to Render Differentiation Block and Immune Evasion in Pediatric Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy characterized by clonal expansion of myeloid progenitor cells with impaired terminal differentiation. Despite intensive treatment regimes, the clinical outcomes remain suboptimal. Emerging evidence suggest myeloblasts could evolve multiple machineries to evade immune patrol and hinder immunotherapies. Here, we present a new mechanism involving the tetraspanin protein CD9 in orchestrating differentiation arrest and immune escape in pediatric AML. We first examined CD9 expression and its prognostic impact in patient cohorts of childhood leukemia. The expression of CD9 on blasts of AML patients (12.2%, n=82) was significantly lower than those of ALL patients (90.4%, n=219, P&amp;lt;0.001) or stem cells from normal bone marrow donors (48.4%, n=22, P=0.014). Among AML cases, the blasts of 32 patients (39%) were CD9+. The expression pattern of CD9 was highly associated with cytogenetic/genetic anomalies and FAB subtypes, for instance enrichment of CD9 in M4, M5 and M7 AML. The 5-year relapse-free survival rate of CD9- patients was significantly lower than CD9+ patients (34.1% vs. 61.2%, P=0.018), with its prognostic significance exhibited distinctively in M4/M5 AML. To elucidate the mechanism governing CD9 silencing, DNA and histone methylation status of CD9 were evaluated by bisulfite and ChIP sequencing for the classical histone marks H3K9/27me3, which indicated methylation may not contribute to CD9 repression. However, a marked decrease of H3K9/27Ac occupancy at the CD9 locus was observed in AML than in ALL cells (4.8-14.2-fold, P&amp;lt;0.05), and strongly correlated with CD9 repression ( r=0.585-0.719, P&amp;lt;0.01). Exposure of CD9- AML cell lines (n=8) or samples (n=9) to the histone deacetylase inhibitor panobinostat significantly elevated CD9 mRNA and protein expression (3.1-32.2-fold, P&amp;lt;0.05), restored activating histone acetylation marks (4.1-41.6-fold, P&amp;lt;0.05), and potently suppressed myeloblast proliferation ex vivo (median IC50: 21.4 nM). An epigenetic compound library screen confirmed histone hypoacetylation as the key mechanism driving CD9 silencing. Enforced CD9 expression in MV4-11 cells significantly suppressed proliferation ( P&amp;lt;0.01) and colony formation ( P=0.002). NOD/SCID mice transplanted with CD9+ cells exhibited a drastically reduced leukemic load in hematopoietic organs by 70.7-91.8% ( P&amp;lt;0.05), a significantly prolonged survival duration ( P&amp;lt;0.001), and a marked regression of extramedullary myeloid sarcoma when compared with animals receiving CD9- cells. Global transcriptome profiling of pediatric AML (n=31) revealed decreased stemness (NES: -1.7, P=0.01) and increased monocyte (NES: 1.8, P=0.034) gene signatures in CD9+ samples. Concordantly, we observed a profound up-regulation of CD9 (9.4-51.1-fold, P&amp;lt;0.01) preceding the appearance of lineage markers (CD14, CD36, CD86, CD115, CD300e, iCD68 and iTNFα) in PMA-mediated monocyte/macrophage differentiation but not ATRA-mediated neutrophil differentiation of myeloblasts. Importantly, the gain of CD9 in THP-1 cells and primary myeloblasts (n=6, P&amp;lt;0.05) consistently augmented the expression of specific monocytic markers, corroborating its cell-intrinsic function in AML differentiation. Single-cell transcriptomic analyses of bone marrow cells from MV4-11-transplanted mice detected a significant enrichment of differentially regulated genes functioning in antigen processing and presentation. Confirmatory overexpression of CD9 promoted basal and IFNγ-induced MHC-I/II expression ( P&amp;lt;0.01) through the JAK2/STAT5 axis. Inter-patient comparisons (n=31) revealed a higher MHC-I expression in CD9+ AML ( P&amp;lt;0.001). Interestingly, CD9 physically bound to MHC-I/II and formed an immune complex at the cell membrane as revealed by IP-MS, co-IP and confocal microscopy. In NSG mice, co-transplantation of human PBMCs mounted an effective immunity against CD9+ but not CD9- AML (MV4-11 and MOLM-13, P&amp;lt;0.05), concomitant with a robust bone marrow infiltration of cytotoxic T cells. Taken together, our data provided molecular, cellular and clinical evidence showing the plausible function of CD9 as a key driver intertwining monocytic differentiation and immune recognition in pediatric AML, and inspired a new combinatorial epigenetic/immunotherapy for this rare but aggressive malignancy.

CD9 negatively regulates collective electrotaxis of the epidermal monolayer by controlling and coordinating the polarization of leader cells.

Endogenous electric fields (EFs) play an essential role in guiding the coordinated collective migration of epidermal cells to the wound centre during wound healing. Although polarization of leadercells is essential for collective migration, the signal mechanisms responsible for the EF-induced polarization of leader cells under electrotactic collective migration remain unclear. This study aims to determine how the leader cells are polarized and coordinated during EF-guided collective migration of epidermal cell sheets. Collective migration of the human epidermal monolayer (human immortalized keratinocytes HaCaT) under EFs was observed via time-lapse microscopy. The involvement of tetraspanin-29 (CD9) in EF-induced fibrous actin (F-actin) polarization of leader cells as well as electrotactic migration of the epidermal monolayer was evaluated by genetic manipulation. Blocking, rescue and co-culture experiments were conducted to explore the downstream signalling of CD9. EFs guided the coordinated collective migration of the epithelial monolayer to the anode, with dynamic formation of pseudopodia in leader cells at the front edge of the monolayer along the direction of migration. F-actin polarization, as expected, played an essential role in pseudopod formation in leader cells under EFs. By confocal microscopy, we found that CD9 was colocalized with F-actin on the cell surface and was particularly downregulated in leader cells by EFs. Interestingly, genetic overexpression of CD9 abolished EF-induced F-actin polarization in leader cells as well as collective migration in the epidermal monolayer. Mechanistically, CD9 determined the polarization of F-actin in leader cells by downregulating a disintegrin and metalloprotease 17/heparin-binding epidermal growth factor-like growth factor/epidermal growth factor receptor (ADAM17/HB-EGF/EGFR) signalling. The abolished polarization of leader cells due to CD9 overexpression could be restored in a co-culture monolayer where normal cells and CD9-overexpressing cells were mixed; however, this restoration was eliminated again by the addition of the HB-EGF-neutralizing antibody. CD9 functions as a key regulator in the EF-guided collective migration of the epidermal monolayer by controlling and coordinating the polarization of leader cells through ADAM17/HB-EGF/EGFR signalling.

A role for tetraspanin proteins in regulating fusion induced by Burkholderia thailandensis

Burkholderia pseudomallei is the causative agent of melioidosis, a disease with high morbidity that is endemic in South East Asia and northern Australia. An unusual feature of the bacterium is its ability to induce multinucleated giant cell formation (MNGC), which appears to be related to bacterial pathogenicity. The mechanism of MNGC formation is not fully understood, but host cell factors as well as known bacterial virulence determinants are likely to contribute. Since members of the tetraspanin family of membrane proteins are involved in various types of cell:cell fusion, their role in MNGC formation induced by Burkholderia thailandensis, a mildly pathogenic species closely related to B. pseudomallei, was investigated. The effect of antibodies to tetraspanins CD9, CD81, and CD63 in MNGC formation induced by B. thailandensis in infected mouse J774.2 and RAW macrophage cell lines was assessed along with that of recombinant proteins corresponding to the large extracellular domain (EC2) of the tetraspanins. B. thailandensis-induced fusion was also examined in macrophages derived from CD9 null and corresponding WT mice, and in J774.2 macrophages over-expressing CD9. Antibodies to CD9 and CD81 promoted MNGC formation induced by B. thailandensis, whereas EC2 proteins of CD9, CD81, and CD63 inhibited MNGC formation. Enhanced MNGC formation was observed in CD9 null macrophages, whereas a decrease in MNGC formation was associated with overexpression of CD9. Overall our findings show that tetraspanins are involved in MNGC formation induced by B. thailandensis and by implication, B. pseudomallei, with CD9 and CD81 acting as negative regulators of this process.

KLF4-mediated upregulation of CD9 and CD81 suppresses hepatocellular carcinoma development via JNK signaling

Tetraspanins CD9 and CD81 frequently serve as the surface markers of exosomes, which are involved in intercellular communication during tumor progression. KLF4 is a well-known tumor suppressor in various cancers. This study aims to investigate the relationship between KLF4 and CD9/CD81 in hepatocellular carcinoma (HCC). The results showed that CD9 and CD81 were transcriptionally activated by KLF4 in HCC cell lines. Decreased expressions of CD9 and CD81 were found in most HCC tumor tissues and predicted advanced stages. Furthermore, KLF4 expression was positively associated with CD9 and CD81 expression in HCC specimens. Functionally, overexpression of CD9 and CD81 inhibited HCC cell proliferation in vitro and in vivo and silencing CD9 and CD81 displayed opposite phenotypes. Mechanistically, we found that JNK signaling pathway may be involved in the growth suppression mediated by CD9 and CD81. In addition, increased expression of KLF4, CD9 or CD81 had no obvious impact on exosome secretion from HCC cells. Collectively, we identified CD9 and CD81 as new transcriptional targets of KLF4 and the dysregulated KLF4-CD9/CD81-JNK signaling contributes to HCC development. Our findings will provide new promising targets against this disease.

Integrative transcriptomic and proteomic analysis reveals CD9/ITGA4/PI3K-Akt axis mediates trabecular meshwork cell apoptosis in human glaucoma.

Glaucoma has been the leading cause of irreversible blindness worldwide. High intraocular pressure (IOP) is a high‐risk factor of glaucoma, repression of which has been the important treatment of glaucoma in clinic. Trabecular meshwork is crucial for maintaining IOP in aqueous humour out‐flow system. It is urgent to reveal the molecular mechanism of trabecular meshwork in glaucoma. Previous studies found that some pathways were related to glaucoma, such as extracellular matrix (ECM)‐receptor interaction, phosphatidylinositol 3‐kinase (PI3K)‐protein kinase B (Akt) and apoptosis. To identify novel molecules in glaucoma, we performed high‐throughput transcriptome and proteome analysis to immortal human trabecular meshwork cells (iHTM) and glaucomatous human trabecular meshwork cells (GTM3), respectively. Twenty‐six up‐regulated genes/proteins and 59 down‐regulated genes/proteins were identified as the high‐risk factors based on differential analysis, including some known factors of glaucoma. Furthermore, a glaucoma‐related protein‐protein interaction (PPI) network was constructed for investigating the function roles of risk factors. Some genes were identified as potential regulator in the pathogenesis of glaucoma based on the topology analysis and module analysis to the network. Importantly, we identified and demonstrated that CD9 played key roles in glaucoma by biological experiment. CD9 is down‐regulated in glaucoma, overexpression of CD9 can active integrin α4 (ITGA4), PI3K and Akt, which lead to the decreased apoptosis and attenuate glaucoma. All these results provide a novel molecular therapy of glaucoma.

PF222 EPIGENETIC SILENCING OF CD9 CONFERS POOR SURVIVAL IN PEDIATRIC ACUTE MYELOID LEUKEMIA AND AGGRAVATES DISEASE PROGRESSION

Background:Acute myeloid leukemia (AML) accounts for ∼5% of pediatric malignancies with inferior survival rates of 50–70% despite optimal application of risk‐directed, chemotherapy‐based treatment protocols. It is of prime importance to identify novel molecular targets that are amenable to therapeutic interventions, which relies critically on the dissection of key regulatory pathways governing disease progression and/or pathogenesis. CD9, a tetraspanin family protein, has been implicated in cancer progression but its expression, prognostic impact and functions in pediatric AML remain largely unknown.Aims:(1) To characterize the expression and epigenetic regulation of CD9 in a cohort of pediatric AML patients and its association with long‐term survival outcomes; and (2) to elucidate the multi‐faceted functions of CD9 in pediatric AML, focusing on the crucial cellular properties affecting leukemia progression.Methods:Bone marrow (BM) samples were collected from pediatric AML patients consecutively enrolled in the NOPHO‐AML 2004 and NOPHO‐DBH AML 2012 studies. Immunophenotypes of isolated leukemic blasts were characterized with CD9, CD33, CD34 and CD45 antibodies by flow cytometry. Patients were stratified into CD9+ and CD9‐ subgroups for comparison of overall survival (OS) and relapse‐free survival (RFS). Epigenetic control of CD9 in AML cell lines was investigated by bisulfite sequencing of CD9 promoter, and confirmed by decitabine and panobinostat treatment. The impact of CD9 on leukemic cell proliferation, cell division and chemosensitivity was measured by Trypan Blue exclusion, competition, colony formation, SNARF‐1 and Annexin V/7‐AAD assays, subsequent to transduction of a CD9‐expressing lentiviral vector. The influence of CD9 on leukemia progression was evaluated in the NOD/SCID mouse xenograft model.Results:The median CD9 expression on leukemic blasts of AML patients was significantly lower than that on CD34+hematopoietic stem cells of normal BM donors (11.6% vs.46.9%, P &lt; 0.001). Higher abundance of methylated CpG sites in CD9 promoter was detected in AML compared with B‐ALL cell lines (7.3% vs. 1.7%). Treatment of CD9‐ AML cells with the DNA methyltransferase inhibitor decitabine or histone deacetylase inhibitor panobinostat increased CD9 expression by 4.9–64.1‐fold (P &lt; 0.05). Of 58 cases of pediatric AML, blasts of 19 patients (32.8%) were CD9+ (≥20% CD9 expressing cells). Kaplan‐Meier analyses revealed that the 5‐year survival rates of CD9‐ patients were significantly lower than those of CD9+ patients (OS: 48.1% vs.88.2%, P = 0.004; RFS: 35.7% vs.66.7%, P = 0.009). Overexpression of CD9 in the AML cell line MV4–11 reduced proliferation, cell division and clonogenic potential, and enhanced cytarabine‐induced apoptosis (P &lt; 0.05). NOD/SCID mice xenografted with GFP‐tagged, CD9‐overexpressing MV4–11 cells exhibited significantly reduced leukemic load in bone marrow, spleen, blood and liver by ≥71.4% (P &lt; 0.01), and prolonged survival by 1.4‐fold (P = 0.002) when compared with animals transplanted with control, GFP‐only MV4–11 cells.Summary/Conclusion:CD9 was epigenetically silenced in pediatric AML and associated with dismal survival outcomes. Reexpression of CD9 substantially suppressed AML progression and enhanced sensitivity to conventional chemotherapy. Our study collectively established CD9 as a novel prognostic factor and tumor suppressor in pediatric AML, which could potentially be applied for refinement of clinical risk group stratification and derivation of new targeted therapies.

PF167 THE TETRASPANIN CD9 REGULATES GLUCOCORTICOID SENSITIVITY IN PEDIATRIC B‐PRECURSOR ACUTE LYMPHOBLASTIC LEUKEMIA

Background:De novo or acquired resistance to glucocorticoids, the backbone regimens for remission‐induction in pediatric B‐precursor acute lymphoblastic leukemia (B‐ALL), poses a significant therapeutic hurdle and is a strong predictor of inferior survival. With the mechanisms being insufficiently understood, strategies for restoring glucocorticoid sensitivity have been limited. We previously reported that CD9 negativity was associated with poorer prednisone response in a cohort of pediatric B‐ALL patients (Leung et al, EHA, 2017), but the functional linkage remains undefined.Aims:(1) To establish the glucocorticoid sensitivity pattern of B‐ALL cell lines and its relationship with CD9 expression; (2) to investigate the definitive role of CD9 in regulation of glucocorticoid response using gain‐ and loss‐of‐function approaches; and (3) to determine if CD9 could affect the expression of glucocorticoid receptor.Methods:An in vitro drug sensitivity profiling, comprising 7 standard chemotherapeutic agents (6‐mercaptopurine, cytarabine, daunorubicin, vincristine, methotrexate, prednisolone and dexamethasone), was performed on 4 CD9+ (697, BV‐173, RS4;11, SUP‐B15) and 3 CD9− (REH, SEM, KOPN‐8) B‐ALL cell lines. Responses to prednisolone and dexamethasone were re‐assessed after lentiviral transduction of CD9 overexpression or knockout vectors into B‐ALL cell lines. The expression of glucocorticoid receptor NR3C1 in B‐ALL cell lines was measured by qPCR and Western blot.Results:Unsupervised clustering analysis of drug profiling data revealed a unique pattern of glucocorticoid response, which was manifested by the preferential resistance of CD9− cells to prednisolone and dexamethasone, but not to 6‐mercaptopurine, cytarabine, daunorubicin, vincristine and methotrexate. Overexpression of CD9 in the CD9− SEM cell line significantly enhanced its sensitivity to prednisolone (IC50: 6,231 vs. 55,346 nM; P = 0.017) and dexamethasone (IC50: 351.5 vs. 990.6 nM; P = 0.005), when compared to cells transduced with control vector. In contrast, CD9 knockout in the CD9+ 697 cell line significantly decreased its sensitivity to prednisolone (IC50: 321.1 vs. 242.7 nM; P = 0.018) and dexamethasone (IC50: 16.2 vs. 13.8 nM; P = 0.032). Such difference was not observed for other chemotherapeutic agents, indicating the specific influence of CD9 on glucocorticoid response. The glucocorticoid receptor NR3C1 was ubiquitously expressed in CD9+ and CD9− cell lines, and was not significantly altered upon CD9 overexpression/knockout.Summary/Conclusion:Our results collectively indicate that CD9 negativity was definitively linked to glucocorticoid resistance, which could be partially reversed by CD9 reactivation through a NR3C1‐independent mechanism. Comprehensive understanding of the interaction between CD9 and glucocorticoid susceptibility could lead to improved therapeutic strategies for resistant pediatric B‐ALL. The findings could also be exploited for other related malignancies (e.g. AML), of which glucocorticoids remain largely inefficacious.

CD9 regulates keratinocyte migration by negatively modulating the sheddase activity of ADAM17.

CD9 is a trans-membrane protein, and has recently been implicated in different physiological and cellular processes, such as cell migration and adhesion. According to previous study, down-regulation of CD9 contributes to keratinocyte migration, critical for wound re-epithelialization. Nevertheless, it is widely believed that tetraspanin CD9 does not have ligands or function as the cell surface receptor, rather it is thought to associate with other transmembrane molecules, thereby mediate keratinocyte migration. Little is known about how CD9 associates with transmembrane molecules in migratory keratinocytes. Here, using confocal microscopy, we observed that tetraspanin CD9 and ADAM17 co-localized on the surface of keratinocytes in the course of wound repair in vivo and in vitro. Co-immunoprecipitation experiments demonstrated a direct association between CD9 and ADAM17 in HaCaT cells and C57-MKs. Functional studies revealed that down-regulation or over-expression of CD9 exerted negative regulatory effects on ADAM17 sheddase activity. This activity is involved in CD9-regulated cell motility and migration. Further studies found that ADAM17 inhibitor-TAPI-2 or siADAM17 significantly abolished the enhanced effect of keratinocyte migration induced by CD9 down-regulation. Meanwhile, the sheddase activity of ADAM17 was inhibited by TAPI-2, which decreased this release of AREG and HB-EGF in CD9-silenced HaCat cells and C57-MKs. Importantly, neutralizing antibody against HB-EGF significant weakened keratinocyte migration and motility in CD9-silenced keratinocytes, and the inhibition of CD9-regulated keratinocyte migration by siADAM17 was rescued by addition of recombinant HB-EGF, activating EGFR/ERK pathway. Collectively, our results suggest that ADAM17 sheddase activity is activated by down-regulation of CD9, thereby mediating shedding of HB-EGF and activation of EGFR/ERK signaling, which crucially affects the keratinocyte migration and wound healing.

Characterization of CD9-CXCL12-CXCR4 Expression in Biological Subtypes of Childhood ACUTE Lymphoblastic Leukemia: Preliminary Findings and Future Perspectives

Background. Although the prognosis of acute lymphoblastic leukemia (ALL) has improved considerably in recent years, some cases still exhibit therapy-resistance and late relapse. CD9-CXCL12-CXCR4 pathway has been implicated in hematopoietic and leukemic stem cell homing, promoting cell adhesion and survival in bone marrow stromal niches and mediating cell dissemination to secondary lymphoid organs. We characterized the CD9-CXCL12-CXCR4 expression in specific biological subgroups of childhood ALL, comparing, in some cases, diagnosis vs relapse, in order to investigate the role of these genes, in the process of leukemia relapse and to preliminarily evaluate their impact as prognostic markers, even for those subtypes with well-established good outcome.Materials and Methods. We analyzed bone marrow (BM) samples from 68 children with ALL, 3 cases with chronic myeloid leukemia, as control for t(9;22) positive leukemia, and 4 healthy donors (HDs). Patients were enrolled and treated at our Center from 2000 to 2010. We reverse-transcribed 500 ng of patients’ diagnostic RNA and performed a Real-time PCR using the SYBRTM Green PCR Master Mix (Applied Biosystems®), calculating the median fold-changes (MFCs) among different reactions and comparing with HDs. GUS gene mRNA was used as an internal positive control, showing no significant variation in our experiments. Each PCR experiment was carried out in duplicate. Sequences of oligonucleotides used for experiments, together with RQ-PCR protocol were previously published (Gandemer V. et al Leukemia Research 2010)Results. We analyzed for CD9, CXCL12 and CXCR4 expression 39 children with t(12;21) positive ALL, 12 cases with t(1;19) positive ALL, 8 cases with t(9;22) positive ALL, and 9 cases resulted negative for chromosomal translocation screening. Among the t(12;21) positive ALL we found that CD9 was overexpressed in 13 out of 39 patients (33%), CXCL12 in 14 out of 39 (35%) and CXCR4 in 9 out of 39 (23%), respectively. We noted that in 2 cases who presented a late relapse, one (CT13) showed a normal expression of the analyzed genes, conversely the other case (CT24), who suffered from a isolated extramedullary relapse, showed a high overexpression of CD9. In the t(1;19) subgroup we found only one case with overexpression of both CD9 and CXCR4 genes, respectively. Moreover we noted that comparing diagnosis vs relapse in one case, we observed a dramatic increased expression of CXCL12 (CT70 diagnosis 3,33 FCs vs relapse 38 FCs). In the subgroup of children with t(9;22) positive ALL, we observed an extremely high expression of the CD9-CXCL12-CXCR4 genes. In particular, we found an overexpression of CD9 in 4 out of 8 (50%) cases with this subtype of ALL; interestingly, all these case showed a BM relapse. One of these (CT56) showed an overexpression of all the three pathway’s components. As negative counterpart, we also analyzed three cases with a t(9;22) positive CML, finding a normal expression of all genes. In order to determine this pathway’s expression in children presenting an ALL without known chromosomal aberration, we analyzed other 9 cases. In only one (CT44) we found an overexpression of both CD9 and CXCL12. This patient suffered from an early relapse (<24 months from diagnosis).Conclusions. Our preliminary findings demonstrated that the CD9-CXCL12-CXCR4 pathway is mainly altered in children with t(12;21) and t(9;22) positive ALL. In the latter subgroup we found a linear correlation between overexpression and impending relapse, confirming that this pathway can be potentially used as prognostic marker. Among t(1;19) positive ALL, we found few cases with single gene aberration. More importantly, we found an overexpression of one or two pathway's components in diagnostic samples of those cases, who presented a subsequent relapse. These data need to be confirmed in a larger population and will pave the way to identify new prognostic marker and/or therapeutic target, since CXCR4 has already antagonist drugs in clinical use, showing very recent promising results (Randhawa S et al. British Journal of Hematology 2016). DisclosuresNo relevant conflicts of interest to declare.

The Tetraspanin CD9 Regulates Engraftment and Mobilization of Human CD34+ Hematopoietic Stem/Progenitor Cells and Modulates VLA-4 Activity

Migration, homing and engraftment of hematopoietic stem/progenitor cells depend critically on the SDF-1/CXCR4 axis. We previously identified the tetraspanin CD9 as a downstream signal of this axis, and it regulates short-term homing of cord blood (CB) CD34+ cells (Leung et al, Blood, 2011). However, its roles in stem cell engraftment, mobilization and the underlying mechanisms have not been described. Here, we provided evidence that CD9 blockade profoundly reduced long-term bone marrow (BM; 70.9% inhibition; P = .0089) and splenic engraftment (87.8% inhibition; P = .0179) of CB CD34+ cells (n = 6) in the NOD/SCID mouse xenotransplantation model, without biasing specific lineage commitment. Interestingly, significant increase in the CD34+CD9+ subsets were observed in the BM (9.6-fold; P < .0001) and spleens (9.8-fold; P = .0014) of engrafted animals (n = 3-4), indicating that CD9 expression on CD34+ cells is up-regulated during engraftment in the SDF-1-rich hematopoietic niches. Analysis of paired BM and peripheral blood (PB) samples from healthy donors revealed higher CD9 expressions in BM-resident CD34+ cells (46.0% CD9+ cells in BM vs 26.5% in PB; n = 13, P = .0035). Consistently, CD34+ cells in granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood (MPB) expressed lower levels of CD9 (32.3% CD9+ cells; n = 25), when compared with those in BM (47.7% CD9+ cells; n = 16, P = .0030). In vitro exposure of MPB CD34+ cells to SDF-1 significantly enhanced CD9 expression (1.5-fold increase; n = 4, P = .0060). Treatment of NOD/SCID chimeric mice with G-CSF decreased the CD34+CD9+ subsets in the BM from 79.2% to 62.4% (n = 8, P = .0179). These data indicate that CD9 expression is down-regulated during egress or mobilization of CD34+ cells. To investigate the possible mechanisms, we performed a VCAM-1 (counter receptor of the VLA-4 integrin) binding assay on BM CD34+ cells. Our results demonstrated that CD34+CD9+ cells preferentially bound to soluble VCAM-1 (17.2%-51.4% VCAM-1-bound cells in CD9+ cells vs 12.8%-25.9% in CD9- cells; n = 10, P ≤ .0003), suggesting that CD9+ cells possess higher VLA-4 activity. Concomitant with decreased CD9 expression, MPB CD34+ cells exhibited lower VCAM-1 binding ability (2.8%-4.0% VCAM-1-bound cells; n = 3), when compared to BM CD34+ cells (15.5%-37.7%; n = 10, P < .0130). In vivo treatment of NOD/SCID chimeric mice with G-CSF reduced VCAM-1 binding of CD34+ cells in the BM by 49.0% (n = 5, P = .0010). Importantly, overexpression of CD9 in CB CD34+ cells promoted VCAM-1 binding by 39.5% (n = 3, P = .0391), thus providing evidence that CD9 regulates VLA-4 activity. Preliminary results also indicated that enforcing CD9 expression in CB CD34+ cells could enhance their homing and engraftment in the NOD/SCID mouse model. Our findings collectively established that CD9 expression and associated integrin VLA-4 activity are dynamically regulated in the BM microenvironment, which may represent important events in governing stem cell engraftment and mobilization. Strategies to modify CD9 expression could be developed to enhance engraftment or mobilization of CD34+ cells. DisclosuresNo relevant conflicts of interest to declare.

Downregulation of CD9 promotes pancreatic cancer growth and metastasis through upregulation of epidermal growth factor on the cell surface.

The expression of CD9 has been shown to be inversely associated with pancreatic cancer metastasis but the underlying mechanism remains incompletely understood. Using the two closely associated pancreatic cancer cell lines, PaTu-8898s and PaTu-8898t, which are metastatic and non-metastatic, respectively, we showed that the PaTu-8988s cells expressed a lower level of CD9 but had higher proliferation and migration rates than the PaTu-8898t cells. An inverse correlation between CD9 expression and the cell surface level of epidermal growth factor receptor (EGFR) was observed in both cell lines. In the PaTu-8898s cells, overexpression of CD9 decreased the cell surface expression of EGFR, associated with increased expression of dynamin-2, whereas in the PaTu-8898t cells, knockdown of CD9 with RNA interference (RNAi) increased the cell surface expression of EGFR, associated with decreased expression of dynamin-2. However, the total EGFR level did not change by manipulation of CD9 expression, suggesting that CD9 plays a role in EGFR endocytosis. Furthermore, in the PaTu-8898ts cells, CD9 overexpression decreased the cell proliferation and migration, which were reversed by EGFR overexpression, whereas in the PaTu-8898t cells, CD9 knockdown enhanced the cell proliferation and migration which were blocked by EGFR RNAi both in vitro and in vivo. Thus, in pancreatic cancer cells, downregulation of CD9 may play a role in cancer growth and metastasis through, at least in part, enhancing cell surface expression of EGFR.

Human Endometrial CD98 Is Essential for Blastocyst Adhesion

BackgroundUnderstanding the molecular basis of embryonic implantation is of great clinical and biological relevance. Little is currently known about the adhesion receptors that determine endometrial receptivity for embryonic implantation in humans.Methods and Principal FindingsUsing two human endometrial cell lines characterized by low and high receptivity, we identified the membrane receptor CD98 as a novel molecule selectively and significantly associated with the receptive phenotype. In human endometrial samples, CD98 was the only molecule studied whose expression was restricted to the implantation window in human endometrial tissue. CD98 expression was restricted to the apical surface and included in tetraspanin-enriched microdomains of primary endometrial epithelial cells, as demonstrated by the biochemical association between CD98 and tetraspanin CD9. CD98 expression was induced in vitro by treatment of primary endometrial epithelial cells with human chorionic gonadotropin, 17-β-estradiol, LIF or EGF. Endometrial overexpression of CD98 or tetraspanin CD9 greatly enhanced mouse blastocyst adhesion, while their siRNA-mediated depletion reduced the blastocyst adhesion rate.ConclusionsThese results indicate that CD98, a component of tetraspanin-enriched microdomains, appears to be an important determinant of human endometrial receptivity during the implantation window.

Expression and function of CD9 in melanoma cells

CD9, a member of the tetraspanin family, functions as an organizer in "tetraspanin webs," through interacting with other cell adhesion molecules. It plays a role in differentiation, fertilization, and cell migration. We investigated the expression and function of CD9 in melanoma. CD9 protein expression in B16 mouse melanoma and six human melanoma cell lines was decreased compared to normal melanocytes. B16F1 clones stably overexpressing CD9 had reduced ability to form colonies in soft agar; however, paradoxically these overexpressing clones had increased ability to invade Matrigel. Similarly, transient overexpression of CD9 in the human metastatic melanoma cell line WM9 dramatically decreased anchorage-independent growth, while transient overexpression of CD9 in the radial growth phase cell line SbCl2 resulted in the gain of Matrigel invasion activity. DNA sequencing of CD9 cDNA from all six human melanoma cell lines did not show deletions, insertions, or mutations. Treatment of all six human melanoma cell lines with the histone deacetylase inhibitor trichostatin A increased CD9 levels. The DNA methylation inhibitor 5-aza-cytidine also increased CD9 protein levels with greater increases seen in cell lines derived from more malignant melanomas.

Tetraspanins regulate cell-to-cell transmission of HIV-1

BackgroundThe presence of the tetraspanins CD9, CD63, CD81 and CD82 at HIV-1 budding sites, at the virological synapse (VS), and their enrichment in HIV-1 virions has been well-documented, but it remained unclear if these proteins play a role in the late phase of the viral replication cycle. Here we used overexpression and knockdown approaches to address this question.ResultsNeither ablation of CD9, CD63 and/or CD81, nor overexpression of these tetraspanins was found to affect the efficiency of virus release. However, confirming recently reported data, tetraspanin overexpression in virus-producing cells resulted in the release of virions with substantially reduced infectivity. We also investigated the roles of these tetraspanins in cell-to-cell transmission of HIV-1. Overexpression of CD9 and CD63 led to reduced cell-to-cell transmission of this virus. Interestingly, in knockdown experiments we found that ablation of CD63, CD9 and/or CD81 had no effect on cell-free infectivity. However, knockdown of CD81, but not CD9 and CD63, enhanced productive particle transmission to target cells, suggesting additional roles for tetraspanins in the transmission process. Finally, tetraspanins were found to be downregulated in HIV-1-infected T lymphocytes, suggesting that HIV-1 modulates the levels of these proteins in order to maximize the efficiency of its transmission within the host.ConclusionAltogether, these results establish an active role of tetraspanins in HIV-1 producer cells.

Colocalisation of CD9 and mortalin in CD9-induced mitotic catastrophe in human prostate cancer cells

CD9, a member of the tetraspanin family of proteins, is involved in a variety of cellular interactions with many other proteins and molecules. Although CD9 has been implicated in cell fusion, migration and cancer progression, the detailed function of this protein is not completely understood and likely depends on interactions with different protein partners, which are not yet all known. Using co-immunoprecipitation and mass-spectrometric protein sequencing, we have identified in prostate cancer cells, a novel CD9 partner, the 75-kDa protein HSPA9B, also known as mortalin. We further show that introduction and overexpression of wild-type CD9 into human PC-3 prostate cancer cells induces mitotic catastrophe. We also demonstrate, by immunocolocalisation studies, the interaction of CD9 and mortalin in PC-3 cells undergoing mitotic catastrophe. Our results not only identified mortalin as a new CD9 partner, but also clarify the mechanisms by which CD9 may control prostate cancer progression.

CD9 promotes adeno-associated virus type 2 infection of mammary carcinoma cells with low cell surface expression of heparan sulphate proteoglycans

Recombinant adeno-associated virus (AAV) is a promising non-pathogenic vector in the emerging field of gene therapy. For AAV serotype 2 (AAV-2) infection, experimental evidence points to an involvement of heparan sulphate proteoglycans (HSPG), but also to the existence of additional receptors. We investigated a potential role of the tetraspanin CD9 in AAV-2 infection of breast cancer cells mainly because it binds to the heparin-binding EGF-like growth factor, suggesting that it may also interact with a heparin-binding virus. Among breast cancer cell lines, expression of HSPG or potential AAV-2 (co)-receptors was not found to correlate with transduction efficiency. In complete accordance with the role of CD9, blocking with anti-CD9 antibodies resulted in drastically decreased AAV-2 transduction efficiencies in cell lines with low expression of HSPG. Furthermore, specific inhibition of CD9 expression with siRNA resulted in fewer transgene-positive cells, whereas overexpression of CD9 in the breast cancer cell line T47D as well as in BT8Ca and BT12Ca rat glioma cells (with low background expression of HSPG and CD9) increased the number of AAV-transduced cells. The minimal epitope recognized by antibody 72F6, which most efficiently blocked AAV-mediated transgene expression, was deduced from the specific binding to peptides immobilized on colour-encoded microspheres consisting of the amino acid sequence PKKDV located in the large extracellular loop of CD9. Our results clearly point to an involvement of CD9 in the attachment, uptake or processing of AAV-2 by target cells expressing low amounts of HSPG, which may help to define cell populations accessible in AAV-based therapeutic applications.