CD9 inhibition reveals a functional connection of extracellular vesicle secretion with mitophagy in melanoma cells.

Tetraspanins are often used as Extracellular Vesicle (EV) detection markers because of their abundance on these secreted vesicles. However, data on their function on EV biogenesis are controversial and compensatory mechanisms often occur upon gene deletion. To overcome this handicap, we have compared the effects of tetraspanin CD9 gene deletion with those elicited by cytopermeable peptides with blocking properties against tetraspanin CD9. Both CD9 peptide or gene deletion reduced the number of early endosomes. CD9 peptide induced an increase in lysosome numbers, while CD9 deletion augmented the number of MVB and EV secretion, probably because of compensatory CD63 expression upregulation. In vivo, CD9 peptide delayed primary tumour cell growth and reduced metastasis size. These effects on cell proliferation were shown to be concomitant with an impairment in mitochondrial quality control. CD9 KO cells were able to compensate the mitochondrial malfunction by increasing total mitochondrial mass reducing mitophagy. Our data thus provide the first evidence for a functional connection of tetraspanin CD9 with mitophagy in melanoma cells.

CD9 knockdown suppresses cell proliferation, adhesion, migration and invasion, while promoting apoptosis and the efficacy of chemotherapeutic drugs and imatinib in Ph+ ALL SUP­B15 cells.

Philadelphia chromosome­positive acute lymphoblastic leukemia (Ph+ ALL) is regarded as a prognostically unfavorable subgroup, as this ALL subgroup has an increased risk of relapse/refractory disease. CD9, which belongs to the tetraspanin membrane proteins, is implicated in several pathological processes, including tumor progression. However, the role of CD9 in the pathogenesis of Ph+ ALL and the potential benefit of applying CD9­targeted RNA interference strategies for treatment of Ph+ ALL require further investigation. The aim of the present study was to determine the effects of CD9 on leukemic cell progression and the efficacy of therapeutic agents in Ph+ ALL cells, in addition to assessing the in vitro anti­leukemia activity of CD9­targeted RNA interference in Ph+ ALL cells. In the present study, a lentiviral short hairpin RNA (shRNA) expression vector targeting CD9 gene in Ph+ ALL SUP­B15 cells was constructed. The present results demonstrated that treatment of SUP­B15 cells with lentiviral­mediated shRNA against CD9 decreased CD9 mRNA and protein expression compared with the shControl cells transduced with a blank vector. In addition, CD9 knockdown could suppress cell proliferation, adhesion, migration and invasion, and promote apoptosis and the efficacy of chemotherapeutic drugs (such as vincristine, daunorubicin, cyclophosphamide and dexamethasone) and the tyrosine kinase inhibitor imatinib in SUP­B15 cells. Furthermore, CD9 knockdown suppressed cell proliferation and promoted apoptosis in SUP­B15 cells via a p53­dependent pathway. These findings suggested that gene silencing of CD9 using a shRNA­expressing lentivirus vector may provide a promising treatment for Ph+ ALL.

CD9 blockade suppresses disease progression of high-risk pediatric B-cell precursor acute lymphoblastic leukemia and enhances chemosensitivity.

CD9 has been implicated in cancer progression but its prognostic relevance and therapeutic potential in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) are largely unknown. In a cohort of pediatric BCP-ALL patients, we found that CD9+ cases had a significantly lower 5-year relapse-free survival rate than CD9- cases. Multivariate analysis demonstrated that CD9 positivity independently predicted inferior survival outcomes, and could be applied with established prognostic features, including prednisone response and cytogenetic status, to refine patient stratification. Administration of CD9 antibody substantially suppressed disease progression in NOD/SCID mice xenografted with CD9+ cell lines and primary leukemic blasts from patients with high-risk and refractory BCP-ALL, without compromising hematopoietic stem cell engraftment. Combination of anti-CD9 with conventional chemotherapy further reduced leukemic burden and prolonged animal survival. Mechanistically, CD9 blockade inhibited leukemic cell proliferation, induced G0/G1 cell cycle arrest, activated p38, and enhanced chemotherapeutic agent-induced apoptosis. Further, CD9 physically interacted with integrin very late antigen-4, regulated affinity to vascular cell adhesion molecule-1, and was involved in leukemia-stroma interaction. Collectively, our study established CD9 as a new prognostic marker, validated the preclinical efficacy of CD9 antibody, and laid the foundation for clinical development of CD9-targeted therapy for high-risk and refractory pediatric BCP-ALL.

CD9 regulates keratinocyte differentiation and motility by recruiting E-cadherin to the plasma membrane and activating the PI3K/Akt pathway.

During keratinocyte stratification and wound healing, keratinocytes undergo a switch between differentiation and motility. However, limited knowledge exists on the mechanisms of the switch. We have previously demonstrated that the expression of CD9 was changed in different wound stages and involved in the regulation of keratinocyte migration. In this study, we showed that CD9 expression was increased in both human and mouse keratinocytes undergoing differentiation. CD9 overexpression in keratinocytes stimulated terminal differentiation and reduced cell motility. CD9 silencing inhibited calcium-induced keratinocyte differentiation and increased cell motility. Furthermore, CD9 overexpression recruited E-cadherin to the plasma membrane and subsequently activated PI3K/Akt signaling, while CD9 knockdown inhibited the recruitment of E-cadherin to the plasma membrane and PI3K/Akt activation. Importantly, silencing E-cadherin expression or inhibiting PI3K/Akt signaling reversed CD9 overexpression-induced differentiation and -reduced motility. These results demonstrate that CD9 acts as an important node that regulates keratinocyte differentiation and motility. The recruitment of E-cadherin to the plasma membrane and activation of the PI3K/Akt signaling pathway mediated by CD9 play an important role in these processes.

Inhibition of CD9 expression reduces the metastatic capacity of human hepatocellular carcinoma cell line MHCC97-H.

Metastasis is a characteristic of malignant tumors and may be a fatal clinical factor for many patients with cancer. Hepatocellular carcinoma (HCC) cells are highly metastatic; the mechanism of metastasis is complicated and may be influenced by a number of factors. Membrane proteins may block receptors or inhibit important enzymes, thus inhibiting tumor progression, and may be potential therapeutic targets for tumor prognosis and treatment. The present study aimed to use proteomics to analyze the dynamic changes of membrane proteins in HCC cells, to improve our understanding of membrane protein functions and to clarify the important components of the mechanisms of HCC metastasis. The present study used the highly metastatic MHCC97-H and the lowly metastatic MHCC97-L HCC cell lines, and the isobaric tags for relative and absolute quantitation (iTRAQ) approach was used for high-throughput screening of metastasis-related membrane proteins. A total of 22 membrane proteins were identified as differentially expressed between the MHCC97-H and MHCC97-L cell lines; these results were verified by reverse transcription-quantitative polymerase chain reaction and western blotting. A number of the identified proteins were revealed to be related to tumor metastasis, including the tetraspan in transmembrane protein CD9. CD9 was demonstrated to be highly expressed in MHCC97-H cells compared with MHCC97-L cells. The functional role of CD9 was characterized by inhibiting its expression using a small interfering RNAs, which demonstrated that reduced CD9 expression inhibited cell migration and metastasis, as determined by wound-healing and invasion assays. Results from the present study demonstrated that CD9 was highly expressed in the highly metastatic HCC cells and promoted HCC cell migration. This protein may be a novel target for regulating the invasive phenotype in HCC.

Tetraspanin blockage reduces exosome-mediated HIV-1 entry.

HIV-1 is one of the most studied retroviruses. The role of exosomes in HIV-1 entry and pathogenesis are beginning to be appreciated. Exosomes can incorporate host proteins that are also contained in viruses (e.g., tetraspanins).

Tetraspanin CD9 stabilizes gp130 by preventing its ubiquitin-dependent lysosomal degradation to promote STAT3 activation in glioma stem cells.

Glioblastoma (GBM) is the most malignant and lethal brain tumor harboring glioma stem cells (GSCs) that promote tumor propagation and therapeutic resistance. GSCs preferentially express several critical cell surface molecules that regulate the pro-survival signaling for maintaining the stem cell-like phenotype. Tetraspanin CD9 has recently been reported as a GSC biomarker that is relevant to the GSC maintenance. However, the underlying molecular mechanisms of CD9 in maintaining GSC property remain elusive. Herein, we report that CD9 stabilizes the IL-6 receptor glycoprotein 130 (gp130) by preventing its ubiquitin-dependent lysosomal degradation to facilitate the STAT3 activation in GSCs. CD9 is preferentially expressed in GSCs of human GBM tumors. Mass spectrometry analysis identified gp130 as an interacting protein of CD9 in GSCs, which was confirmed by immunoprecipitation and immunofluorescent analyses. Disrupting CD9 or gp130 by shRNA significantly inhibited the self-renewal and promoted the differentiation of GSCs. Moreover, CD9 disruption markedly reduced gp130 protein levels and STAT3 activating phosphorylation in GSCs. CD9 stabilized gp130 by preventing its ubiquitin-dependent lysosomal degradation to promote the BMX-STAT3 signaling in GSCs. Importantly, targeting CD9 potently inhibited GSC tumor growth in vivo, while ectopic expression of the constitutively activated STAT3 (STAT3-C) restored the tumor growth impaired by CD9 disruption. Collectively, we uncovered a critical regulatory mechanism mediated by tetraspanin CD9 to maintain the stem cell-like property and tumorigenic potential of GSCs.

Novel CD9-targeted therapies in gastric cancer.

There are 33 human tetraspanin proteins, emerging as key players in malignancy, the immune system, fertilization, cellular signaling, adhesion, morphology, motility, proliferation, and tumor invasion. CD9, a member of the tetraspanin family, associates with and influences a variety of cell-surface molecules. Through these interactions, CD9 modifies multiple cellular events, including adhesion, migration, proliferation, and survival. CD9 is therefore considered to play a role in several stages during cancer development. Reduced CD9 expression is generally related to venous vessel invasion and metastasis as well as poor prognosis. We found that treatment of mice bearing human gastric cancer cells with anti-CD9 antibody successfully inhibited tumor progression via antiproliferative, proapoptotic, and antiangiogenic effects, strongly indicating that CD9 is a possible therapeutic target in patients with gastric cancer. Here, we describe the possibility of CD9 manipulation as a novel therapeutic strategy in gastric cancer, which still shows poor prognosis.

[Tetraspanins: a new target for antiangiogenic therapy?]. / Les tétraspanines : une nouvelle cible pour la thérapie anti-angiogénique ?

Inhibition of the vascular endothelial growth factor A or inhibition of its receptors are currently used for the treatment of cancer. However, the results are still modest, especially because of the multitude and redundancy of angiogenic factors. It can be hypothesized that therapies targeting directly endothelial cells themselves could be more effective. The tetraspanins are transmembrane molecules, which are devoid of intrinsic enzymatic activity but can associate with each other and with other molecules such as integrins or proteins of the immunoglobulin superfamily to form a network. The tetraspanins are present on the surface of endothelial cells and in vitro, inhibition of these molecules by antibodies or small interfering RNA suggests that tetraspanins play a role in angiogenesis. These preliminary data have been confirmed by the study of cancer xenografts in tetraspanin-deficient mice, which have a significant decrease in tumor size and tumor angiogenesis. In vivo, it has been shown that intravenous administration of a monoclonal antibody (ALB6) directed against CD9 decreases the tumor growth and angiogenesis and that intravitreal injection of a small interfering RNA decreasing CD9 significantly inhibits choroidal neovascularization induced by laser. Finally, anti-angiogenic effects and potent anti-tumor activity are observed by the intraperitoneal administration of GS-168AT2, a peptide derived from CD9-Partner 1, a molecule belonging to the immunoglobulin superfamily, which interacts strongly with the CD9 and CD81. These data suggest that the pharmacological modulation of the tetraspanin web could play a new promising anti-angiogenic strategy.

CD9 negatively regulates CD26 expression and inhibits CD26-mediated enhancement of invasive potential of malignant mesothelioma cells.

CD26/dipeptidyl peptidase IV is a cell surface glycoprotein which consists of multiple functional domains beside its ectopeptidase site. A growing body of evidence indicates that elevated expression of CD26 correlates with disease aggressiveness and invasive potential of selected malignancies. To further explore the molecular mechanisms involved in this clinical behavior, our current work focused on the interaction between CD26 and CD9, which were recently identified as novel markers for cancer stem cells in malignant mesothelioma. We found that CD26 and CD9 co-modulated and co-precipitated with each other in the malignant mesothelioma cell lines ACC-MESO1 and MSTO-211H. SiRNA study revealed that depletion of CD26 led to increased CD9 expression, while depletion of CD9 resulted in increased CD26 expression. Consistent with these findings was the fact that gene transfer of CD26 into CD26-negative MSTO-211H cells reduced CD9 expression. Cell invasion assay showed that overexpression of CD26 or gene depletion of CD9 led to enhanced invasiveness, while CD26 gene depletion resulted in reduced invasive potential. Furthermore, our work suggested that this enhanced invasiveness may be partly mediated by α5ß1 integrin, since co-precipitation studies demonstrated an association between CD26 and α5ß1 integrin. Finally, gene depletion of CD9 resulted in elevated protein levels and tyrosine phosphorylation of FAK and Cas-L, which are downstream of ß1 integrin, while depletion of CD26 led to a reduction in the levels of these molecules. Collectively, our findings suggest that CD26 potentiates tumor cell invasion through its interaction with α5ß1 integrin, and CD9 negatively regulates tumor cell invasion by reducing the level of CD26-α5ß1 integrin complex through an inverse correlation between CD9 and CD26 expression. Our results also suggest that CD26 and CD9 serve as potential biomarkers as well as promising molecular targets for novel therapeutic approaches in malignant mesothelioma and other malignancies.

Cigarette smoke impairs phagocytosis of apoptotic neutrophils by alveolar macrophages via inhibition of the histone deacetylase/Rac/CD9 pathways.

Efferocytosis, which is the homeostatic phagocytosis of apoptotic cells, prevents the release of toxic intracellular contents and subsequent tissue damage. Impairment of efferocytosis was reported in alveolar macrophages (AMs) of patients with chronic obstructive pulmonary disease (COPD), a common disease caused by smoking. In COPD, histone deacetylase (HDAC) activity is reduced in AMs. We investigated whether the reduction of HDAC activity is associated with the impairment of efferocytosis. Murine AMs were collected by bronchoalveolar lavage and their ability to efferocytose apoptotic human polymorphonuclear leukocytes was assessed. Pre-treatment of AMs with cigarette smoke extract (CSE) or trichostatin A (TSA), an HDAC inhibitor, suppressed efferocytosis and CSE reduced HDAC activity. TSA inhibited the activity of Rac, a key mediator of efferocytosis. These TSA-induced impairments were restored by treatment of AMs with aminophylline, a potent activator of HDAC. To further elucidate the underlying mechanism, we explored a role of CD9 in TSA-induced impairment of efferocytosis. CD9 is a transmembrane protein of the tetraspanin family that facilitates the uptake of several pathogens and other material. TSA profoundly down-regulated the expression of CD9 on AMs. The expression of CD9 was partly down-regulated by the Rac inhibitor. Pretreatment with an anti-CD9 mAb or CD9 small interfering RNA inhibited efferocytosis, which was attributable to the reduced binding of AMs to apoptotic cells. These results suggest that smoking impairs efferocytosis via inhibition of HDAC/Rac/CD9 pathways. Aminophylline/theophylline is effective in restoring the impairment of efferocytosis and might have benefit for the treatment of patients with COPD.

ALCAM/CD166 adhesive function is regulated by the tetraspanin CD9.

ALCAM/CD166 is a member of the immunoglobulin superfamily of cell adhesion molecules (Ig-CAMs) which mediates intercellular adhesion through either homophilic (ALCAM-ALCAM) or heterophilic (ALCAM-CD6) interactions. ALCAM-mediated adhesion is crucial in different physiological and pathological phenomena, with particular relevance in leukocyte extravasation, stabilization of the immunological synapse, T cell activation and proliferation and tumor growth and metastasis. Although the functional implications of ALCAM in these processes is well established, the mechanisms regulating its adhesive capacity remain obscure. Using confocal microscopy colocalization, and biochemical and functional analyses, we found that ALCAM directly associates with the tetraspanin CD9 on the leukocyte surface in protein complexes that also include the metalloproteinase ADAM17/TACE. The functional relevance of these interactions is evidenced by the CD9-induced upregulation of both homophilic and heterophilic ALCAM interactions, as reflected by increased ALCAM-mediated cell adhesion and T cell migration, activation and proliferation. The enhancement of ALCAM function induced by CD9 is mediated by a dual mechanism involving (1) augmented clustering of ALCAM molecules, and (2) upregulation of ALCAM surface expression due to inhibition of ADAM17 sheddase activity.

CD9 expression as a favorable prognostic marker for patients with malignant mesothelioma.

CD9 is involved in cell growth, adhesion and motility and its expression is reported to be of prognostic significance in various types of human malignancies. We found increased cell migration in the mesothelioma cell lines MSTO-211H and TUM1 following in vitro shRNA-mediated knockdown of CD9 expression. We investigated CD9 expression in 112 malignant pleural mesotheliomas. CD9 expression was observed in 62 of 71 epithelioid, 13 of 20 biphasic and only 1 of 21 sarcomatoid mesotheliomas. Among the epithelioid mesotheliomas (EMs), CD9 expression was observed in all of the 33 cases with a differentiated type (EM-D) and in 29 of the 38 cases with a less-differentiated type (EM-LD). Patients with CD9 expression showed higher 1- and 2-year survival rates (63 and 25%) compared to the patients without CD9 expression (39 and 11%). Univariate analysis revealed that patients with CD9 expression demonstrated a more favorable survival (P=0.0025) along with other clinicopathological factors, including age younger than 60 years, IMIG stage I-II, epithelioid histology, EM-D and patients who underwent extrapleural pneumonectomy or received chemotherapy. Multivariate analysis identified CD9 expression as an independent prognostic factor with a hazard ratio (HR) of 1.99 in the analysis of all mesotheliomas (P=0.0261) and an HR of 2.60 in the analysis of EMs (P=0.0376). CD9 expression is an independent favorable prognostic marker of malignant mesothelioma.

Impaired cell adhesion, apoptosis, and signaling in WASP gene-disrupted Nalm-6 pre-B cells and recovery of cell adhesion using a transducible form of WASp.

Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency disease affecting cell morphology and signal transduction in hematopoietic cells. The function of Wiskott-Aldrich syndrome protein (WASp) and its partners in protein interaction have been studied intensively in mice; however, detailed biochemical characterization of its signal transduction and assessment of its functional consequence in human WASp-deficient lymphocytes remain difficult. In this study, we generated Nalm-6 cells in which the WAS protein gene (WASP) was disrupted by homologous recombination-based gene targeting and a cell-permeable form of recombinant WASp for functional study. The WASP⁻/⁻ cells showed impaired adhesive capacity and polarization to plate-bound anti-CD47 mAb, anti-CD9 mAb, or to fibronectin. The defective morphological changes were accompanied by impaired intracellular signaling. In addition, the WASp-deficient cells displayed augmented apoptosis induced by CD24 cross-linking. A recombinant fusion protein composed of Hph-1 cell-permeable peptide and WASp prepared in Escherichia coli. Hph-1-WASp was efficiently transduced and expressed in WASP⁻/⁻ Nalm-6 cells in a dose-dependent manner. The wild-type WASp, but not the mutant restored adhesion capacity, spreading morphology, and cytoskeletal reorganization. Additionally, the recombinant protein was successfully transduced into normal lymphocytes. These findings suggest that gene-disrupted model cell lines and cell-permeable recombinant proteins may serve as important tools for the detailed analysis of intracellular molecules involved in PID.