EGFR phosphorylation of DCBLD2 recruits TRAF6 and stimulates AKT-promoted tumorigenesis

Abstract

Aberrant activation of EGFR in human cancers promotes tumorigenesis through stimulation of AKT signaling. Here, we determined that the discoidina neuropilin-like membrane protein DCBLD2 is upregulated in clinical specimens of glioblastomas and head and neck cancers (HNCs) and is required for EGFR-stimulated tumorigenesis. In multiple cancer cell lines, EGFR activated phosphorylation of tyrosine 750 (Y750) of DCBLD2, which is located within a recently identified binding motif for TNF receptor-associated factor 6 (TRAF6). Consequently, phosphorylation of DCBLD2 Y750 recruited TRAF6, leading to increased TRAF6 E3 ubiquitin ligase activity and subsequent activation of AKT, thereby enhancing EGFR-driven tumorigenesis. Moreover, evaluation of patient samples of gliomas and HNCs revealed an association among EGFR activation, DCBLD2 phosphorylation, and poor prognoses. Together, our findings uncover a pathway in which DCBLD2 functions as a signal relay for oncogenic EGFR signaling to promote tumorigenesis and suggest DCBLD2 and TRAF6 as potential therapeutic targets for human cancers that are associated with EGFR activation.

Figure 8. Coexpression of p-EGFR^Y1172 and p-DCBLD2^Y750 correlates with poorer prognoses of gliomas or HNCs.

(A) A total of 132 clinical glioma specimens, including WHO grade II–IV gliomas, were analyzed by IHC. Representative images of serial sections of a GBM tissue using anti–p-EGFR^Y1172, anti–p-DCBLD2^Y750, anti-TRAF6, and anti–p-AKT^T308 antibodies are shown. Insets: IgG controls of the same area in serial sections of these specimens. (B) Kaplan-Meier analyses of patients with high p-Y-EGFR^Y1172/p-DCBLD2^Y750-expressing tumors (blue line) versus low p-EGFR^Y1172/p-DCBLD2^Y750-expressing tumors (red line) in IHC analyses in A. P values were calculated by using log-rank test. Black bars indicate censored data. (C) A total of 231 clinical HNC specimens on tumor TMAs were analyzed by IHC staining. Representative images of serial sections of a HNC tissue. Insets: IgG controls of the same area in serial sections of these samples. (D) Kaplan-Meier analyses of patients with high p-EGFR^Y1172/p-DCBLD^Y750-expressing HNCs (blue line) versus low p-EGFR^Y1172/p-DCBLD^Y750-expressing HNCs (red line) in IHC analyses. P values were calculated by using log-rank test. Black bars indicate censored data. (E) A working model of EGFR/DCBLD2/TRAF6/AKT signaling in tumorigenesis. Data in A and C are representative of 2 independent experiments. Scale bars: 50 μm. Arrows in A and C indicate positive staining.

Figure 7. Mutation of DCBLD2 from Y750 to D750 mimics EGFRvIII signaling on AKT activation and tumorigenesis.

(A) IP and/or IB assays of erlotinib treatments of U87/EGFRvIII/shD2 cells or (C) patient-derived GSC83/shD2 cells with exogenous expression of shRNA-resistant DCBLD2^WT or dominant activating DCBLD2^D750 mutant. (B) Erlotinib inhibited colony formation of U87/EGFRvIII/shD2/WT cells but had minimal effect on U87/EGFRvIII/shD2/D750 cells. (D) Erlotinib attenuated size of neurosphere and fold increase of cell numbers of GSC83/shD2/WT cells after 5 days of cell culture but had no appreciable effect on GSC83/shD2/D750 cells. Cells were seeded in 6 replicates. Scale bars: 50 μm. In B and D, error bars ± SD. *P < 0.05, compared with erlotinib-treated cells. In A and C, Flag-DCBLD2, EGFR and AKT were used as loading controls. Data are representative of 2 independent experiments.

Figure 6. EGFRvIII/DCBLD2/TRAF6/AKT signal pathway regulates glioma tumorigenesis.

(A) IB assays of knockdown of TRAF6 in U87/EGFRvIII cells with 2 different shRNAs, shT6#1 and shT6#3, and EGFRvIII-stimulated colony formation was inhibited by knockdown of TRAF6 with shT6. (B) Depletion of TRAF6 by shRNA inhibited EGFRvIII-promoted glioma tumorigenesis in the brains of animals. Scale bars: 1 mm. Images are representative of 5 mice per group of 2 independent experiments. (C) Treatment of U87/vIII cells with LY294002 inhibited EGFRvIII-stimulated AKT and colony formation, similar to knockdown of DCBLD2 (shD2) and TRAF6 (shT6) in U87/vIII cells. (D) Knockdown of AKT1 and AKT2 with siRNA (siAKT1&2) in U87/vIII cells blocked EGFRvIII-stimulated activity of AKT and colony formation, similar to knockdown of DCBLD2 (shD2) and TRAF6 (shT6) in U87/vIII cells. (E) Overexpression of constitutively active form of AKT (Myr.AKT) not only further stimulated colony formation of U87/vIII/shC cells, but also rescued the inhibited colony formation by knockdown of DCBLD2 (shD2) or by knockdown of TRAF6 (shT6). β-Actin was used as a loading control. Cells were seeded in 6 replicates. Error bars ± SD. *P < 0.05, compared with shC-treated cells (bars of shD2 and shT6 among pBABE) and compared with shC pBABE (bars of all myr.AKT). Data are representative of 2 independent experiments.

Figure 5. EGFR-stimulated p-DCBLD2^Y750 regulates TRAF6 E3 ligase activity.

IP-IB or IB analyses. (A) DCBLD2 promotes EGFR-stimulated TRAF6 E3 ligase activity. HA-TRAF6 and His-Ub were coexpressed with or without Flag-DCBLD2 and/or EGFRvIII in HEK293T cells. Proteins were pulled down with Ni-NTA beads. TRAF6 (Ub)n, polyubiquitinated TRAF6. (B) Mutation of P745Q, but not P641Q, in DCBLD2 inhibits EGFRvIII-stimulated TRAF6 E3 ligase activity. (C) Compared with DCBLD2^WT, expression of DCBLD2^F750 or DCBLD2^F621/F750, but not DCBLD2^F621, attenuates EGFRvIII-stimulated TRAF6 E3 ligase activity. (D) Reexpression of DCBLD2^WT or DCBLD2^F621, but not DCBLD2^F750 or DCBLD2^F621/F750, rescues EGFRvIII-stimulated TRAF6 E3 ligase activity in glioma U87/EGFRvIII/shD2 cells. HA-TRAF6, Flag-DCBLD2, EGFR, and β-actin were used as loading controls. Data are representative of 3 independent experiments with similar results.

Figure 4. EGFR-stimulated p-DCBLD2^Y750 regulates DCBLD2 association with TRAF6.

(A) EGFRvIII p-DCBLD2^Y750 and promotes the association of DCBLD2 with TRAF6. HA-TRAF6 was coexpressed with or without Flag-DCBLD2 and/or EGFRvIII in HEK293T cells. (B) EGF stimulates DCBLD2 and TRAF6 association in lung cancer 343T, HNC PCI-15B, melanoma A375, and glioma U87 cells. (C) Mutation of P745Q, but not P641Q, of DCBLD2 TIMs impairs EGFRvIII-induced association of DCBLD2 with TRAF6. (D) EGFR-stimulated p-DCBLD2^Y750 is critical for DCBLD2 binding to TRAF6. (E) EGFRvIII-activated p-Y750, but not p-Y621, of DCBLD2 is required for DCBLD2 binding to TRAF6. IP-IB or IB analyses. A specific anti–p-DCBLD2^Y750 antibody was used to detect EGFR-stimulated p-DCBLD2^Y750. Control, vector without DCBLD2. β-Actin was used as a loading control. Data are representative of 3 independent experiments.

Figure 3. EGFR phosphorylation of DCBLD2 at Y750, but not at Y621, is critical for EGFR-promoted tumorigenesis.

(A) EGFRvIII activates p-Y of DCBLD2 in glioma U87 and SNB19 cells. vIII or vIII-DK, U87 or SNB19 cells that express vIII or a mutant of vIII that lacks of cytoplasmic domain of vIII, respectively. p-Y of DCBLD2 was detected with a pan anti-tyrosine antibody, 4G10. p-Y of EGFR was detected with an anti–p-EGFRY1045 antibody. (B) EGF (50 ng/ml) stimulates p-Y of DCBLD2 in U87/EGFR cells. (C) Schematic of the DCBLD2^WT TIM P-X-E-X-X (Ar/Ac). CUB, C1r/C1s, Uegf, Bmp1 domain; LCCL, Limulus factor C, Coch-5b2, and Lgl1 domain; F5/8 type C, coagulation factor V/factor VIII homology (FV/VIII) domain. (D) DCBLD2^F621/F750 mutant abolishes, and DCBLD2^F621 or DCBLD2^F750 mutant attenuates, EGFRvIII-stimulated p-Y of DCBLD2 in U87/P or U87/EGFRvIII cells. (E) Reexpression of Flag-DCBLD2 shRNA-resistant DCBLD2^WT or DCBLD2^F621, but not DCBLD2^F750, DCBLD2^F621/F750 mutant or a vector control (C), rescues EGFRvIII-stimulated p-AKT^T308 and p-AKT^S473 in U87/EGFRvIII/shD2 cells. (F) Reexpression of Flag-DCBLD2 shRNA-resistant DCBLD2^WT and DCBLD2^F621, but not DCBLD2^F750, DCBLD2^F621/F750 mutant, or a vector control (C), rescues EGFRvIII-promoted U87 glioma growth in the brain of animals. Images represent results of 5 mice per group of 3 independent experiments. Scale bars: 1 mm. (G) Effect of reexpression of Flag-DCBLD2 shRNA-resistant DCBLD2^WT or indicated mutants on tumor size, cell proliferation, and cell apoptosis in U87/EGFRvIII/shD2 tumors. Error bars ± SD. **P < 0.01, compared with shControl + C tumors or shD2 tumors expressing DCBLD2^WT or DCBLD2^F621 mutant. In A, B, D–G, data are representative of 3 independent experiments. β-Actin or AKT was used as a loading control.

Figure 2. DCBLD2 is required for EGFR-driven tumorigenesis.

(A) IB analyses of DCBLD2 knockdown with 2 different shRNAs (shD2#1 and shD2#2) or a control shRNA in U87 and SNB19 cells. P, parental cells; vIII, U87 or SNB19 cells expressing EGFRvIII. β-Actin was used as a loading control. (B) Effects of knockdown of DCBLD2 by shD2 or shC on cell proliferation in vitro. (C) Effects of knockdown of DCBLD2 by shD2 or shC on cell apoptosis in vitro. (D) Effect of DCBLD2 knockdown by shD2 or shC on glioma cell colony formation in vitro. (E) shRNA knockdown of DCBLD2 inhibits EGFRvIII-promoted U87 glioma growth in the brain. Representative images of H&E, Ki-67, and TUNEL analyses of brain sections, with indicated U87 gliomas (arrows). Nuclei were stained with DAPI (blue). Ki-67 and TUNEL are in red. Scale bars: 1 mm (H&E staining); 50 μm (Ki-67 staining); 100 μm (TUNEL staining). Images represent results of 5 mice per group. (F) Quantification of tumor size, cell proliferation, and cell apoptosis. Data were from stained brain sections of 5 mice per group. (G) IB analyses of DCBLD2 knockdown by shD2 or shC in patient-derived GSCs (GSC83). (H) shRNA knockdown of DCBLD2 inhibits endogenous EGFRvIII-promoted tumorigenesis of gliomas established by patient-derived GSC83 cells in the brain. Quantification of tumor size is also shown. Scale bars: 1 mm. In B, C, D, F, and H, error bars ± SD. *P < 0.05, **P < 0.01, compared with parental or EGFRvIII cells or tumors treated with shC. Data and images are representative of 2 to 3 independent experiments.

Figure 1. DCBLD2 is required for EGF-stimulated cell proliferation and survival in cancer cell lines derived from glioma, lung cancer, HNC, and melanoma.

(A) DCBLD2 knockdown with a DCBLD2 shRNA (shD2) or a control shRNA (shC). EGF stimulation (50 ng/ml) of glioma SNB19 and U87 cells for 3 days. (B) Knockdown of DCBLD2 attenuates EGF-stimulated glioma cell proliferation. Glioma cells in 6 replicates were serum starved for 24 hours and then treated with or without EGF (50 ng/ml) for 3 days. (C) Knockdown of DCBLD2 inhibits glioma cell survival. (D) Knockdown of DCBLD2 by a shRNA (shD2) or a control shRNA (shC) in cell lines derived from lung cancer (343T), HNC (PCI-15B), and melanoma (A375). (E) Knockdown of DCBLD2 by shD2 inhibits EGF-stimulated 343T, PCI-15B, and A375 cell proliferation in vitro. (F) Effect of shRNA knockdown of DCBLD2 by shD2 on cell survival in vitro. (G) Effect of shRNA knockdown of DCBLD2 by shD2 on colony formation by 343T, PCI-15B, and A375 cells seeded on soft agar in triplicates. Scale bars: 1 mm. (H) Quantification of colony formation assays in G. DCBLD2 was knocked down by 2 separate shRNAs (shD2#1 and shD2#2; see Figure 2) in all experiments and only results of shD2#1 knockdown are shown. In B, C, E, F, and H, error bars ± SD. *P < 0.05, **P < 0.01, compared with parental with shC+EGF. Data and images are representative of 3 independent experiments. In A and D, β-actin and EGFR were used as loading controls.