Epitope Specificity Determines Pathogenicity and Detectability of Anti-Platelet-Derived Growth Factor Receptor α Autoantibodies in Systemic Sclerosis.

OBJECTIVE: To identify the epitopes recognized by autoantibodies targeting platelet-derived growth factor receptor α (PDGFRα) in systemic sclerosis (SSc) and develop novel assays for detection of serum anti-PDGFRα autoantibodies. METHODS: Epstein-Barr virus-immortalized B cells from 1 patient with SSc (designated PAM) were screened for expression of IgG binding to PDGFRα and induction of reactive oxygen species in fibroblasts. The variable regions of anti-PDGFRα IgG were cloned into an IgG expression vector to generate distinct recombinant human monoclonal autoantibodies (mAb), which were characterized by binding and functional assays. The epitopes of anti-PDGFRα recombinant human mAb were defined by molecular docking, surface plasmon resonance binding assays, screening of a conformational peptide library spanning the PDGFRα extracellular domains, and expression analyses of alanine-scanned PDGFRα mutants. Direct or competitive enzyme-linked immunosorbent assays were established to detect all serum anti-PDGFRα autoantibodies or, selectively, the agonistic ones. RESULTS: Three types of anti-PDGFRα recombinant human mAb, with the same VH but distinct VL chains, were generated. Nonagonistic VH PAM-Vκ 13B8 recognized 1 linear epitope, whereas agonistic VH PAM-Vλ 16F4 and VH PAM-Vκ 16F4 recognized 2 distinct conformational epitopes. Serum anti-PDGFRα antibodies were detected in 66 of 70 patients with SSc, 63 of 130 healthy controls, 11 of 26 patients with primary Raynaud's phenomenon (RP), and 13 of 29 patients with systemic lupus erythematosus (SLE). Serum VH PAM-Vκ 16F4-like antibodies were found in 24 of 34 patients with SSc, but not in healthy controls, patients with primary RP, or patients with SLE. Peptides composing the VH PAM-Vκ 16F4 epitope inhibited PDGFRα signaling triggered by serum IgG from SSc patients. CONCLUSION: Agonistic anti-PDGFRα autoantibodies are enriched in SSc sera and recognize specific conformational epitopes that can be used to discriminate agonistic from nonagonistic antibodies and block PDGFRα signaling in patients with SSc.

Overexpression of CD157 contributes to epithelial ovarian cancer progression by promoting mesenchymal differentiation.

Epithelial ovarian carcinoma (EOC) is an aggressive tumor often diagnosed at an advanced stage, when there is little or no prospect of cure. Despite advances in surgical and chemotherapeutic strategies, only marginal improvements in patient outcome have been obtained. Hence, unraveling the biological mechanisms underpinning EOC progression is critical for improving patients' survival. Recently, we reported that CD157 (an ectoenzyme regulating leukocyte diapedesis) is expressed in EOC and that high expression of the molecule is negatively correlated with the disease outcome in patients. Here, we demonstrate that forced overexpression of CD157 in OVCAR-3, TOV-21G, A2780 and OV-90 ovarian cancer cell lines promotes morphological and phenotypic changes characterized by disruption of intercellular junctions, downregulation of epithelial markers and upregulation of mesenchymal ones. These changes in cell shape and phenotype bring to reduced sensitivity to anoikis, increased anchorage-independent growth, cell motility and mesothelial invasion. Conversely, knockdown of CD157 in OV-90 and OC314 cells reverts the mesenchymal phenotype and reduces the cells' migratory potential. Transcriptome profiling analysis highlighted 378 significantly differentially expressed genes, representing the signature of CD157-overexpressing OVCAR-3 and OV-90 cells. The modulation of selected genes translates into alteration of protein expression that give cells a highly malignant phenotype. The overall picture deduced from the analysis of the modulated transcripts is that high expression of CD157 strengthens a number of biological processes favoring tumor progression (including development and cell motility), and weakens several biological processes hindering tumor progression (such as apoptosis, cell death and response to stress). Together, these findings implicate CD157 in the progression of EOC to metastatic disease and suggest that CD157 may represent a valuable therapeutic target.

The CD157-integrin partnership controls transendothelial migration and adhesion of human monocytes.

CD157, a member of the CD38 gene family, is an NAD-metabolizing ectoenzyme and a signaling molecule whose role in polarization, migration, and diapedesis of human granulocytes has been documented; however, the molecular events underpinning this role remain to be elucidated. This study focused on the role exerted by CD157 in monocyte migration across the endothelial lining and adhesion to extracellular matrix proteins. The results demonstrated that anti-CD157 antibodies block monocyte transmigration and adhesion to fibronectin and fibrinogen but that CD157 cross-linking is sufficient to overcome the block, suggesting an active signaling role for the molecule. Consistent with this is the observation that CD157 is prevalently located within the detergent-resistant membrane microdomains to which, upon clustering, it promotes the recruitment of ß(1) and ß(2) integrin, which, in turn, leads to the formation of a multimolecular complex favoring signal transduction. This functional cross-talk with integrins allows CD157 to act as a receptor despite its intrinsic structural inability to do so on its own. Intracellular signals mediated by CD157 rely on the integrin/Src/FAK (focal adhesion kinase) pathway, resulting in increased activity of the MAPK/ERK1/2 and the PI3K/Akt downstream signaling pathways, which are crucial in the control of monocyte transendothelial migration. Collectively, these findings indicate that CD157 acts as a molecular organizer of signaling-competent membrane microdomains and that it forms part of a larger molecular machine ruled by integrins. The CD157-integrin partnership provides optimal adhesion and transmigration of human monocytes.

Functional role and prognostic significance of CD157 in ovarian carcinoma.

BACKGROUND: CD157, an ADP-ribosyl cyclase-related cell surface molecule, regulates leukocyte diapedesis during inflammation. Because CD157 is expressed in mesothelial cells and diapedesis resembles tumor cell migration, we investigated the role of CD157 in ovarian carcinoma. METHODS: We assayed surgically obtained ovarian cancer and mesothelial cells and both native and engineered ovarian cancer cell lines for CD157 expression using flow cytometry and reverse transcription-polymerase chain reaction (RT-PCR), and for adhesion to extracellular matrices, migration, and invasion using cell-based assays. We investigated invasion of human peritoneal mesothelial cells by serous ovarian cancer cells with a three-dimensional coculture model. Experiments were performed with or without CD157-blocking antibodies. CD157 expression in tissue sections from ovarian cancer patients (n = 88) was examined by immunohistochemistry, quantified by histological score (H score), and categorized as at or above or below the median value of 60, and compared with clinical parameters. Statistical tests were two-sided. RESULTS: CD157 was expressed by ovarian cancer cells and mesothelium, and it potentiated the adhesion, migration, and invasion of serous ovarian cancer cells through different extracellular matrices. CD157-transfected ovarian cancer cells migrated twice as much as CD157-negative control cells (P = .001). Blockage of CD157 inhibited mesothelial invasion by serous ovarian cancer cells in a three-dimensional model. CD157 was expressed in 82 (93%) of the 88 epithelial ovarian cancer tissue specimens. In serous ovarian cancer, patients with CD157 H scores of 60 or greater had statistically significantly shorter disease-free survival and overall survival than patients with lower CD157 H scores (CD157 H score > or =60 vs <60: median disease-free survival = 18 months, 95% confidence interval [CI] = 5.92 to 30.07 vs unreached, P = .005; CD157 H score > or =60 vs <60: median overall survival = 45 months, 95% CI = 21.21 to 68.79 vs unreached, P = .024). Multivariable Cox regression showed that CD157 is an independent prognostic factor for recurrence (hazard ratio of disease recurrence = 3.01, 95% CI = 1.35 to 6.70, P = .007) and survival (hazard ratio of survival = 3.44, 95% CI = 1.27 to 9.31, P = .015). CONCLUSIONS: CD157 plays a pivotal role in the control of ovarian cancer cell migration and peritoneal invasion, and it may be clinically useful as a prognostic tool and therapeutic target.

Ectoenzymes and innate immunity: the role of human CD157 in leukocyte trafficking.

CD157 is a glycosylphosphatidylinositol-anchored molecule encoded by a member of the CD38/ADP-ribosyl cyclase gene family, involved in the metabolism of NAD. Expressed mainly by cells of the myeloid lineage and by vascular endothelial cells, CD157 has a dual nature behaving both as an ectoenzyme and as a receptor. Although it lacks a cytoplasmic domain, and cannot transduce signals on its own, the molecule compensates for this structural limit by interacting with conventional receptors. Recent experimental evidence suggests that CD157 orchestrates critical functions of human neutrophils. Indeed, CD157-mediated signals promote cell polarization, regulate chemotaxis induced through the high affinity fMLP receptor and control transendothelial migration.