A soluble form of B cell maturation antigen, a receptor for the tumor necrosis factor family member APRIL, inhibits tumor cell growth

P Rennert, P Schneider, T G Cachero, J Thompson, L Trabach, S Hertig, N Holler, F Qian, C Mullen, K Strauch, J L Browning, C Ambrose, J Tschopp, P Rennert, P Schneider, T G Cachero, J Thompson, L Trabach, S Hertig, N Holler, F Qian, C Mullen, K Strauch, J L Browning, C Ambrose, J Tschopp

Abstract

A proliferation-inducing ligand (APRIL) is a ligand of the tumor necrosis factor (TNF) family that stimulates tumor cell growth in vitro and in vivo. Expression of APRIL is highly upregulated in many tumors including colon and prostate carcinomas. Here we identify B cell maturation antigen (BCMA) and transmembrane activator and calcium modulator and cyclophilin ligand (CAML) interactor (TACI), two predicted members of the TNF receptor family, as receptors for APRIL. APRIL binds BCMA with higher affinity than TACI. A soluble form of BCMA, which inhibits the proliferative activity of APRIL in vitro, decreases tumor cell proliferation in nude mice. Growth of HT29 colon carcinoma cells is blocked when mice are treated once per week with the soluble receptor. These results suggest an important role for APRIL in tumorigenesis and point towards a novel anticancer strategy.

Figures

Figure 1
Figure 1
APRIL is a ligand for the TNF receptors BCMA and TACI. (A) Various receptor–Fc fusion proteins of the TNF receptor family (0.5 μg) were mixed with Flag-tagged soluble hAPRIL (0.2 μg), immunoprecipitated with protein-A, and detected by Western blotting with antibodies to human Ig (top panel). Coimmunoprecipitating APRIL was detected with anti-Flag antibodies (bottom panel). (B) Binding of Flag-APRIL and Flag-BAFF to plastic-coated BCMA–Fc, TACI–Fc, or TRAILR2–Fc (control) was assessed by ELISA. Flag–ligands were added at the indicated concentrations, and their binding was revealed using anti-Flag M2 antibody. (C) Binding of BCMA–Fc to 293 cells (ctrl) and to 293 cells stably transfected with full-length hBAFF (BAFF) or with a fusion protein in which the TNF homology domain of BAFF had been replaced by that of APRIL. The same cells were also stained with anti-hBAFF mAb (Buffy-1) to determine the absolute amount of surface expressed ligands (right panel). The epitope recognized by Buffy-1, which is close to the membrane, is retained in the APRIL fusion protein, as shown in the scheme. (D) Mixtures of Flag-tagged BAFF (10 μg) and Myc-tagged APRIL (10, 1, 0.1, or 0.01 μg) were immunoprecipitated with substoechiometric amounts of BCMA–Fc (0.2 μg) and analyzed by Western blotting. Ligands were detected simultaneously with anti-Flag and anti-Myc antibodies (top panel), and BCMA–Fc with antibodies to hIg (bottom panel).
Figure 1
Figure 1
APRIL is a ligand for the TNF receptors BCMA and TACI. (A) Various receptor–Fc fusion proteins of the TNF receptor family (0.5 μg) were mixed with Flag-tagged soluble hAPRIL (0.2 μg), immunoprecipitated with protein-A, and detected by Western blotting with antibodies to human Ig (top panel). Coimmunoprecipitating APRIL was detected with anti-Flag antibodies (bottom panel). (B) Binding of Flag-APRIL and Flag-BAFF to plastic-coated BCMA–Fc, TACI–Fc, or TRAILR2–Fc (control) was assessed by ELISA. Flag–ligands were added at the indicated concentrations, and their binding was revealed using anti-Flag M2 antibody. (C) Binding of BCMA–Fc to 293 cells (ctrl) and to 293 cells stably transfected with full-length hBAFF (BAFF) or with a fusion protein in which the TNF homology domain of BAFF had been replaced by that of APRIL. The same cells were also stained with anti-hBAFF mAb (Buffy-1) to determine the absolute amount of surface expressed ligands (right panel). The epitope recognized by Buffy-1, which is close to the membrane, is retained in the APRIL fusion protein, as shown in the scheme. (D) Mixtures of Flag-tagged BAFF (10 μg) and Myc-tagged APRIL (10, 1, 0.1, or 0.01 μg) were immunoprecipitated with substoechiometric amounts of BCMA–Fc (0.2 μg) and analyzed by Western blotting. Ligands were detected simultaneously with anti-Flag and anti-Myc antibodies (top panel), and BCMA–Fc with antibodies to hIg (bottom panel).
Figure 2
Figure 2
In vitro proliferation induced by APRIL expression is blocked by soluble hBCMA–Fc fusion protein. (A) Western blot analysis of APRIL expression in the presence or absence of doxycycline (dox), a tetracycline analogue that induces expression. Two independently derived lines are shown. The major APRIL bands at 17 and 22 kD are indicated. (B) [3H]thymidine incorporation in the presence or absence of doxycycline with or without 20 μg/ml of hBCMA–Fc added. Values shown are 3H incorporation (inc.) expressed as percent of control. Two different experiments are shown. P < 0.02; n = 3 wells per data point.
Figure 2
Figure 2
In vitro proliferation induced by APRIL expression is blocked by soluble hBCMA–Fc fusion protein. (A) Western blot analysis of APRIL expression in the presence or absence of doxycycline (dox), a tetracycline analogue that induces expression. Two independently derived lines are shown. The major APRIL bands at 17 and 22 kD are indicated. (B) [3H]thymidine incorporation in the presence or absence of doxycycline with or without 20 μg/ml of hBCMA–Fc added. Values shown are 3H incorporation (inc.) expressed as percent of control. Two different experiments are shown. P < 0.02; n = 3 wells per data point.
Figure 3
Figure 3
Expression of APRIL receptors. Northern blot showing expression of TACI and BCMA mRNA in various human (A) and murine (B) cell lines. Probing with β-actin gave comparable signals for all cell lines (data not shown). B cell lines: J558, BW5147, A20, U266, RPMI8226, Ramos, Namalwa, NC-37, IM-9, SKW6.4, Raji, Daudi, BJAB; monocytic line: U937; T cell line: Jurkat; adenocarcinoma cell lines: HT29, SW480; lung carcinoma cell line: A549; melanoma cell line: ME260; fibroblast-derived cell line: NIH-3T3. (C) Flow cytometric analysis of NIH-3T3, HT-29, A459, and Raji cells stained with Flag–BAFF (BAFF), Flag–APRIL (APRIL), Flag–APRIL depleted with BCMA–Fc (+BCMA) or, as control, Flag–APRIL depleted with TRAILR2–Fc (+TRAILR2). Bound ligands were detected with anti-Flag mAb.
Figure 3
Figure 3
Expression of APRIL receptors. Northern blot showing expression of TACI and BCMA mRNA in various human (A) and murine (B) cell lines. Probing with β-actin gave comparable signals for all cell lines (data not shown). B cell lines: J558, BW5147, A20, U266, RPMI8226, Ramos, Namalwa, NC-37, IM-9, SKW6.4, Raji, Daudi, BJAB; monocytic line: U937; T cell line: Jurkat; adenocarcinoma cell lines: HT29, SW480; lung carcinoma cell line: A549; melanoma cell line: ME260; fibroblast-derived cell line: NIH-3T3. (C) Flow cytometric analysis of NIH-3T3, HT-29, A459, and Raji cells stained with Flag–BAFF (BAFF), Flag–APRIL (APRIL), Flag–APRIL depleted with BCMA–Fc (+BCMA) or, as control, Flag–APRIL depleted with TRAILR2–Fc (+TRAILR2). Bound ligands were detected with anti-Flag mAb.
Figure 4
Figure 4
Growth of human adenocarcinoma and lung carcinoma cell lines in nude (Nu/Nu) mice. (A) Growth rate of HT29 tumors implanted subcutaneously. Mice were left untreated or were treated with purified BCMA–Fc (200 μg) or control hIgG (200 μg) on day of inoculation and weekly thereafter. The scattergram showing tumor volume in pooled control mice and BCMA–Fc-treated mice 6 wk after HT29 tumor implantation is shown in the right panel. (B) Scattergrams showing tumor volume in pooled control mice and BCMA–Fc-treated mice 7 wk (left panel) and 10 wk (right panel) after subcutaneous implantation of A549 lung carcinoma cells. *P = 0.06; **P = 0.01; ***P < 0.005.

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