TRAF6 regulates melanoma invasion and metastasis through ubiquitination of Basigin

Abstract

TRAF6 plays a crucial role in the regulation of the innate and adaptive immune responses. Although studies have shown that TRAF6 has oncogenic activity, the role of TRAF6 in melanoma is unclear. Here, we report that TRAF6 is overexpressed in primary as well as metastatic melanoma tumors and melanoma cell lines. Knockdown of TRAF6 with shRNA significantly suppressed malignant phenotypes including cell proliferation, anchorage-independent cell growth and metastasis in vitro and in vivo. Notably, we demonstrated that Basigin (BSG)/CD147, a critical molecule for cancer cell invasion and metastasis, is a novel interacting partner of TRAF6. Furthermore, depletion of TRAF6 by shRNA reduced the recruitment of BSG to the plasma membrane and K63-linked ubiquitination, in turn, which impaired BSG-dependent MMP9 induction. Taken together, our findings indicate that TRAF6 is involved in regulating melanoma invasion and metastasis, suggesting that TRAF6 may be a potential target for therapy or chemo-prevention in melanoma.

Keywords: Basigin; TRAF6; invasion and metastasis; melanoma; ubiquitination.

Figure 1. TRAF6 is highly expressed in melanoma

A. A melanoma tissue array was stained using the TRAF6 antibody. IRIDI score was calculated and statistics analyzed as described in Materials and Methods. B. Representative images of immunohistochemical staining of TRAF6 in metastatic melanomas, primary melanomas and normal nevi were taken at 200× magnification. C. Immunoblot analysis was performed to examine TRAF6 expression in several normal and skin cancer cell lines by indicated antibodies.

Figure 2. Downregulation of TRAF6 expression in human melanoma cells attenuates proliferation in vitro and in vivo

A. Knockdown of TRAF6 was generated in SK-MEL-5 and SK-MEL-28 cell lines. TRAF6 protein expression was assessed by immunoblot (IB) analysis as indicated. B. shMock, shTRAF6#1 or #4 SK-MEL-5 and SK-MEL-28 cells were seeded (1 × 10³ per well/100 μL) into 96-well plates and proliferation was assessed as described in Materials and Methods. Data from multiple experiments are expressed as means ± SEM. Significant differences were evaluated using a two-way ANOVA and the asterisk (*) indicates a significant difference (p < 0.05). C. SK-MEL-5 (upper panel) or SK-MEL-28 (lower panel) cells expressing shMock, shTRAF6 #1 or #4 were seeded in 0.3% BME agar containing 10% FBS. The cultures were maintained in a 37°C, 5% CO₂ incubator for 10 days and then colonies were counted using a microscope and the Image J program. Data from multiple experiments were expressed as means ± SEM. Significant differences were evaluated using a one-way ANOVA and the respective significant differences are as indicated. D. SK-MEL-5 cells (5×10⁶/0.15mL) expressing shMock or sh-TRAF6 (#1 and #4) were injected into nude mice to establish subcutaneous xenografts. Once tumors were palpable, tumor volume was measured twice per week. Data represent means (n=5) ± SEM of each group. Bars, SD; *, P<0.05; **, P<0.01.

Figure 3. Knocking down TRAF6 inhibits melanoma invasiveness and metastasis in vitro and in vivo

A. Wound healing assay was performed to examine the migration capability in vitro as described in Materials and Methods. Images (at 40x magnification) were taken every 24 h up to 72 h. B. Cells that migrated across the membrane were stained with crystal violetand imaged at 100x magnification. Data represent the means (n=3) ± SEM of each group. C. Average number of lung micro-metastasis per mouse from each group was determined. Representative microscopic H&E images of lung section were shown. Positive immunohistochemical images indicative of melanoma (HMB-45 and S100) were shown.

Figure 4. TRAF6 interacts with BSG

A. TRAF6 binds to BSG. 293T cells were co-transfected with Flag-TRAF6 and BSG-Myc plasmids. Co-immunoprecipitation was performed with anti-Flag or anti-Myc, followed by immunoblotting with the indicated antibodies. B. TRAF6 binds to endogenous BSG. SK-MEL-5 and SK-MEL-28 cells lysates were immunoprecipitated using control IgG or anti-BSG antibody and the complex was detected by immunoblotting with anti-TRAF6. C. Schematic of the BSG/BSG mutant. D. The transmembrane domain mediates the interaction between BSG and TRAF6. Flag-TRAF6 and truncated BSG were co-transfected into 293T cells. At 36 h post-transfection, cell lysates were immunoprecipitated with anti-Flag antibody and then subjected to immunoblotting with anti-Myc or anti-Flag antibody.

Figure 5. TRAF6 regulates BSG ubiquitination and plasma membrane recruitment

A. BSG is recruited to the plasma membrane following FBS stimulation. SK-MEL-5 cells were starved for 16 h and treated with 30% FBS for the indicated time points. Membrane and cytosolic fractions were isolated as described in Materials and Methods and analyzed by immunoblotting with the indicated antibodies. B. SK-MEL-5 cells were serum starved, treated with 30% FBS for 5-30 min and fixed for immunofluorescence analysis. Nuclear DNA was stained with DAPI (blue). BSG subcellular translocation (red) was pointed by arrows. C. TRAF6 regulates the FBS-induced BSG plasma membrane recruitment. TRAF6-deficient SK-MEL-5 cells were starved for 16 h, and then treated with 30% FBS for indicated times. Membrane fraction extractions were examined by immunoblotting with indicated antibodies. D. TRAF6 is required for K63-mediated BSG polyubiquitination. 293T cells were co-transfected with Ub-K63-HA, along with TRAF6-WT-Flag or TRAF6-C70A-Flag and BSG-myc. At 36 h post-transfection, cell lysates were immunoprecipitated with anti-Myc. Ubiquitinated BSG was visualized by immunoblotting using anti-HA. E. FBS induces endogenous BSG ubiquitination. BSG-myc was transfected into SK-MEL-5 cells, at 24 h post-transfection, cells were starved for 16 h. After stimulation with 30% FBS, cell lysates were immunoprecipitated with anti-Myc. Endogenous ubiquitination of BSG was detected by P4D1 antibody.

Figure 6. Lysine residues at BSG cytoplasmic domain are responsible for BSG ubiquitination mediated by TRAF6

A. Cytoplasmic domain of BSG is ubiquitinated by TRAF6. 293T cells were co-transfected with Flag-TRAF6 and BSG-Myc or BSG-D231-269-Myc, along with Ub-K63-HA. At 36 h post-transfection, cell lysates were immunoprecipitated with anti-Myc. B. Schematic diagram of BSG mutant constructs, in which all of the lysine residues at the cytoplasmic domain were replaced with arginine (BSG-RRR). C. BSG-RRR-V5 mutants and Flag-TRAF6, along with Ub-K63-HA were co-transfected into 293T cells, detection was performed as described above.

Figure 7. TRAF6-BSG axis is required for MMP9 induction

A. and B. SK-MEL-5 cells expressing shMock and shTRAF6 (#1 and #4) were starved for 16 h. After stimulation with 30% FBS, cell lysates and mRNA were analyzed by immunoblotting (A), and qPCR (B). C. SK-MEL-5 cells were transfected with control, full length of BSG or BSG-RRR. At 36 h post-transfection, cells were starved for 16 h and stimulated with 30% FBS. Cell lysates were analyzed by immunoblotting with indicated antibodies. D. Transwell experiment was performed with SK-MEL-5 cells transfected with BSG-WT or BSG-RRR plasmids.