Fig 1: CXADR serves as the SFRP2 receptor in retinoblastoma(A) CXADR is highly expressed in retinoblastoma cell lines and tumors. CXADR RNA expression was analyzed by qRT-PCR and normalized to the levels in human ARPE-19 retinal pigment epithelial cells (n = 3).(B) SFRP2 co-immunoprecipitates with endogenous CXADR in 293T cells. The indicated FLAG-tagged proteins were transfected into 293T cells, and their interaction with endogenous CXADR was assessed by anti-FLAG immunoprecipitation followed by anti-CXADR immunoblotting. C-terminally FLAG-tagged SFRP2 specifically co-immunoprecipitated with endogenous CXADR.(C) CXADR serves as the SFRP2 receptor in retinoblastoma. C-terminally FLAG-tagged SFRP2 secreted from transfected 293T cells was incubated with Y79 cells that were transfected with CXADR siRNA or control siRNA as indicated. Cells were washed, and the whole-cell lysate was prepared to assess the binding of SFRP2 to the cells. SFRP2-FLAG bound to control siRNA-transfected cells, and the binding was abolished by CXADR silencing. The silencing of CXADR was verified by immunoblotting.(D) Microscale thermophoresis analysis of SFRP2-CXADR interaction. The interaction between recombinant SFRP2 (6838-FR-025, R&D Systems) and recombinant CXADR (3336-CX-05, R&D Systems) was analyzed by microscale thermophoresis, which determined the KD of interaction to be 11.9 nM.(E) CXADR silencing rescues growth inhibition following SFRP2 silencing. RB383 and Y79 cells were transfected with SFRP2 siRNA, CXADR siRNA, and/or control siRNA as indicated. Cell proliferation was assessed by IncuCyte.(F) CXADR silencing abrogates CREB phosphorylation upon SFRP2 silencing. RB383 and Y79 cells were transfected with SFRP2 siRNA, CXADR siRNA, and/or control siRNA as indicated. The levels of SFRP2, CXADR, phosphorylated CREB, and tubulin were assessed by immunoblotting 48 h after transfection. A representative result from three independent experiments is shown.(G) CXADR silencing abrogates NO production upon SFRP2 silencing. RB383 and Y79 cells were transfected with SFRP2 siRNA, CXADR siRNA, and/or control siRNA as indicated. The NO production in cell lysate was assessed 12 h and 24 h after siRNA transfection (n = 3).(H) CXADR overexpression inhibits retinoblastoma growth. Y79 cells were infected with lentiviruses expressing CXADR or empty vector, and cell proliferation was assessed by IncuCyte. CXADR expression was verified by anti-CXADR immunoblotting (inset).(I) CXADR overexpression results in NO production, which can be suppressed by recombinant SFRP2. NO production is enhanced in CXADR-overexpressing Y79 cells, which was rapidly suppressed by addition of recombinant SFRP2 (250 ng/mL) to the culture medium (n = 3).(J) Growth inhibition by CXADR overexpression can be rescued by recombinant SFRP2. Y79 cells expressing CXADR or empty vector were treated with recombinant SFRP2 (250 ng/mL) or left untreated, and cell proliferation was assessed by IncuCyte.(K) Model for the regulation of NO production and proliferation by SFRP2 and CXADR. SFRP2 is abundantly secreted from retinoblastoma, binds CXADR, and prevents NO production (1). SFRP2 silencing triggers NO production through CXADR, leading to growth arrest (2). When both SFRP2 and CXADR are silenced, NO production is not induced, and retinoblastoma can proliferate (3). CXADR silencing does not induce NO production and allows retinoblastoma proliferation (4).