TRE17/USP6 oncogene translocated in aneurysmal bone cyst induces matrix metalloproteinase production via activation of NF-kappaB

Abstract

Aneurysmal bone cyst (ABC) is an aggressive, pediatric bone tumor characterized by extensive destruction of the surrounding bone. Although first described over 60 years ago, its molecular etiology remains poorly understood. Recent work revealed that ABCs harbor translocation of TRE17/USP6, leading to its transcriptional upregulation. TRE17 encodes a ubiquitin-specific protease (USP), and a TBC domain that mediates binding to the Arf6 GTPase. However, the mechanisms by which TRE17 overexpression contributes to tumor pathogenesis, and the role of its USP and TBC domains, are unknown. ABCs are characterized by osteolysis, inflammatory recruitment and extensive vascularization, the processes in which matrix proteases have a prominent role. This led us to explore whether TRE17 regulates the production of matrix metalloproteinases (MMPs). In this study we show that TRE17 is sufficient to induce expression of MMP-9 and MMP-10, in a manner requiring its USP activity, but not its ability to bind Arf6. TRE17 induces transcription of MMP-9 through activation of nuclear factor-kappaB (NF-kappaB), mediated in part by the GTPase RhoA and its effector kinase, ROCK. Furthermore, xenograft studies show that TRE17 induces formation of tumors that reproduce multiple features of ABC, including a high degree of vascularization, with an essential role for the USP domain. In sum, these studies reveal that TRE17 is sufficient to initiate tumorigenesis, identify MMPs as novel TRE17 effectors that likely contribute to ABC pathogenesis and define the underlying signaling mechanism of their induction.

Figure 1. TRE17 induces secretion of MMP-9 and MMP-10 in a USP-dependent manner

(A) hFOB1.19 human fetal osteoblasts were transfected with the indicated HA-tagged TRE17 constructs, then serum-starved. MMP-9 and MMP-10 in the CM were concentrated as described in Experimental Procedures, and detected by immunoblotting. Whole cell lysates were blotted for TRE17 (arrowheads indicate migration of TRE17(long) and TRE17(short) isoforms; asterisk denotes non-specific (ns) band recognized by antibody), and actin as a loading control. (B) Domain structure of TRE17 alleles. TBC, TBC domain; C and H, cysteine and histidine subdomains of the USP domain. TRE17(long)/USP- and TRE17/A6- harbor point mutations in the indicated domains, as detailed in Experimental Procedures. (C) MC3T3 osteoblasts stably expressing HA-tagged TRE17(long) or TRE17(A6-) in a doxycycline (dox)-inducible manner were treated with dox (2 μg/ml) for 24 hrs. Cell extracts were immunoprecipitated with anti-HA, then blotted with anti-Arf6 (top panel) or anti-TRE17 (bottom panel). I.P., anti-HA immunoprecipitate; WCL, whole cell lysate. (D) Stable MC3T3 cell lines expressing the indicated TRE17 alleles were grown with or without dox for 24 hrs, then starved for 24 hrs in the continued absence or presence of dox. MMP-9 was purified from the CM then subjected to blotting, or zymography to detect gelatinolytic activity. (E) HeLa cells were transfected as indicated, and accumulation of MMP-9 and MMP-10 in the CM was monitored as in (A).

Figure 2. TRE17 induces MMP expression on a transcriptional level

(A) hFOB1.19 or (B) HeLa cells were transfected, then serum-starved for 24-48 hours. MC3T3-derived cell lines (C) were treated with or without dox for 24 hrs, then starved for an additional 24 hrs. For all three cell systems RNA was isolated, and RT-qPCR was performed to monitor MMP-9 and MMP-10 mRNA, using two independent primer pairs for each (denoted MMP9-1 and -2, and MMP10-1 and -2), normalizing against GAPDH. For hFOB1.19 and HeLa cells, data represent the mean ± SD of three experiments. Samples with statistically significant differences from vector control cells are indicated (*, p<0.05; **, p<0.01). D) hFOB1.19 or HeLa cells were co-transfected with the indicated TRE17 constructs and a luciferase reporter driven a fragment of the human MMP-9 promoter encoding base pairs −674 to +3 relative to the transcription start site. Luciferase assays were performed; data represent the mean ± SD of 4 experiments. Samples with statistically significant differences from vector control cells are indicated (*, p<0.05; **, p<0.01).

Figure 3. NFκB is required for induction of MMP-9 by TRE17

A) hFOB1.19 or HeLa cells were transfected with the indicated TRE17 constructs and either a luciferase reporter driven by the WT MMP-9 promoter, or a mutant in which the NFκB response element was ablated. Luciferase assays were performed; data represent the mean ± SD of 4 experiments (*, p<0.05; **, p<0.01). (B) Left, hFOB1.19 were transfected as indicated, then starved for 24 hrs. Middle, control or TRE17(long)/MC3T3 cells were treated with dox, then starved in the presence of dox. Bay11-7082 was added at the indicated micromolar concentration during starvation. Right, control or TRE17(long)/MC3T3 cells were treated with Bay11-7082 (B, 10 μM) or U0126 (U, 50 μM) where indicated. For all three, MMP-9 in the CM was detected; whole cell lysates were blotted with TRE17 and actin antibodies. (C) MMP-9 was assayed as in (B), except the IKK inhibitors wedelolactone (W, 50 μM), SC514 (SC, 100 μM) or IKK Inhibitor XI (XI, 20 μM) were used where indicated.

Figure 4. TRE17(long) activates NFκB in a USP-dependent manner

(A) hFOB1.19 or Hela cells were co-transfected with the indicated TRE17 construct and a luciferase reporter driven by tandem NFκB response elements. Cells were starved and luciferase assays were performed. Data represent the mean ± SD of four independent experiments (*, p<0.05; **, p<0.01). (B) EMSAs were performed in transiently transfected HeLa (top), or MC3T3 lines expressing vector, TRE17(long), or TRE17(long)/USP-(bottom). As a positive control, vector control cells were treated with TNFα (10 ng/ml, 30 minutes) where indicated. (C) hFOB1.19 cells were transfected with the indicated TRE17 construct, starved for 24 hr, then stained with anti-HA, anti-p65, and DAPI. Arrows highlight transfected cells. Scale bar, 20 μm.

Figure 5. RhoA and ROCK partially mediate activation of NFκB by TRE17

(A) RhoA activity was measured in transiently transfected hFOB1.19 (left), HeLa (middle), and MC3T3 cell lines (right). Cell extracts were subjected to pulldowns using a GST fusion of the RhoA·GTP-binding domain (RBD) of Rhotekin, followed by blotting with anti-RhoA. Active RhoA, RBD pulldowns; total RhoA, whole cell lysates. (B) hFOB1.19 or HeLa were transfected with NFκB-luciferase and TRE17(long), in the absence or presence of dominant negative RhoA (RhoAN19) or Y27632, and subjected to luciferase assays. (C) Control or TRE17(long)/MC3T3 cells were treated with dox for 24 hrs, then starved with dox and the indicated concentration of Y27632 (μM) for 24 hrs. MMP-9 in the CM was detected by immunoblotting and zymography. Whole cell extracts were blotted to confirm TRE17 and actin levels.

Figure 6. TRE17(long) induces formation of tumors that recapitulate multiple features of ABC in a USP-dependent manner

(A) In vivo bioluminescence imaging of mice injected with MC3T3 cell lines expressing TRE17(long), TRE17(long)/USP-, or luciferase alone. (B) Left, macroscopic images of tumor and injection site of mice injected with vector or TRE17(long)-expressing MC3T3. Right, H&E stain of TRE17(long) tumor section. Arrowheads highlight vascular elements. (C) Immunohistochemistry was performed on serial sections of TRE17(long)-induced tumor using antibodies against TRE17 and MMP-9. Nuclear staining for TRE17 is non-specific; only central cells exhibit specific cytoplasmic staining of TRE17.