ABSTRACT
The Wnt signaling pathways play pivotal roles in carcinogenesis. Modulation of the cell-surface abundance of Wnt receptors is emerging as an important mechanism for regulating sensitivity to Wnt ligands. Endocytosis and degradation of the Wnt receptors Frizzled (Fzd) and lipoprotein-related protein 6 (LRP6) are regulated by the E3 ubiquitin ligases zinc and ring finger 3 (ZNRF3) and ring finger protein 43 (RNF43), which are disrupted in cancer. In a genome-wide small interfering RNA screen, we identified the deubiquitylase ubiquitin-specific protease 6 (USP6) as a potent activator of Wnt signaling. USP6 enhances Wnt signaling by deubiquitylating Fzds, thereby increasing their cell-surface abundance. Chromosomal translocations in nodular fasciitis result in USP6 overexpression, leading to transcriptional activation of the Wnt/ß-catenin pathway. Inhibition of Wnt signaling using Dickkopf-1 (DKK1) or a Porcupine (PORCN) inhibitor significantly decreased the growth of USP6-driven xenograft tumors, indicating that Wnt signaling is a key target of USP6 during tumorigenesis. Our study defines an additional route to ectopic Wnt pathway activation in human disease, and identifies a potential approach to modulate Wnt signaling for therapeutic benefit.
Subject(s)
DNA-Binding Proteins/metabolism , Frizzled Receptors/metabolism , Neoplasms, Experimental/metabolism , Oncogene Proteins/metabolism , Proto-Oncogene Proteins/metabolism , Ubiquitin Thiolesterase/metabolism , Ubiquitination , Wnt Signaling Pathway , Animals , DNA-Binding Proteins/genetics , Frizzled Receptors/genetics , HEK293 Cells , HeLa Cells , Humans , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , Low Density Lipoprotein Receptor-Related Protein-6/genetics , Low Density Lipoprotein Receptor-Related Protein-6/metabolism , Mice , Neoplasms, Experimental/genetics , Oncogene Proteins/genetics , Proto-Oncogene Proteins/genetics , Ubiquitin Thiolesterase/genetics , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolismABSTRACT
Ewing sarcoma is a rare and deadly pediatric bone cancer for which survival rates and treatment options have stagnated for decades. Ewing sarcoma has not benefited from immunotherapy due to poor understanding of how its immune landscape is regulated. We recently reported that ubiquitin-specific protease 6 (USP6) functions as a tumor suppressor in Ewing sarcoma, and identified it as the first cell-intrinsic factor to modulate the Ewing sarcoma immune tumor microenvironment (TME). USP6 induces intratumoral infiltration and activation of multiple innate immune lineages in xenografted nude mice. Here we report that natural killer (NK) cells are essential for its tumor-inhibitory functions, as NK cell depletion reverses USP6-mediated suppression of Ewing sarcoma xenograft growth. USP6 expression in Ewing sarcoma cells directly stimulates NK cell activation and degranulation in vitro, and functions by increasing surface levels of multiple NK cell-activating ligands. USP6 also induces surface upregulation of the receptor for the apoptosis-inducing ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), providing an additional route for enhanced sensitivity to NK cell killing. Furthermore, USP6-expressing Ewing sarcoma and NK cells participate in a paracrine immunostimulatory feedforward loop, wherein IFNγ secreted by activated NK cells feeds back on USP6/Ewing sarcoma cells to induce synergistic expression of chemokines CXCL9 and CXCL10. Remarkably, expression of USP6 in subcutaneous Ewing sarcoma xenografts induces systemic activation and maturation of NK cells, and induces an abscopal response in which growth of distal tumors is inhibited, coincident with increased infiltration and activation of NK cells. This work reveals how USP6 reprograms the Ewing sarcoma TME to enhance antitumor immunity, and may be exploited for future therapeutic benefit. Significance: This study provides novel insights into the immunomodulatory functions of USP6, the only cancer cell-intrinsic factor demonstrated to regulate the immune TME in Ewing sarcoma. We demonstrate that USP6-mediated suppression of Ewing sarcoma tumorigenesis is dependent on NK cells. USP6 directly activates NK cell cytolytic function, inducing both intratumoral and systemic activation of NK cells in an Ewing sarcoma xenograft model.
Subject(s)
Bone Neoplasms , Neuroectodermal Tumors, Primitive, Peripheral , Sarcoma, Ewing , Humans , Animals , Mice , Intrinsic Factor , Ligands , Mice, Nude , Apoptosis Inducing Factor , Ubiquitin-Specific Proteases , Tumor Microenvironment , Ubiquitin ThiolesteraseABSTRACT
Ewing sarcoma is the second most common pediatric bone cancer, with a 5-year survival rate for metastatic disease of only 20%. Recent work indicates that survival is strongly correlated with high levels of tumor-infiltrating lymphocytes (TIL), whose abundance is associated with IFN-inducible chemokines CXCL10 and CCL5. However, the tumor-intrinsic factors that drive chemokine production and TIL recruitment have not been fully elucidated. We previously showed that ubiquitin-specific protease 6 (USP6) directly deubiquitinates and stabilizes Jak1, thereby inducing an IFN signature in Ewing sarcoma cells. Here, we show that this gene set comprises chemokines associated with immunostimulatory, antitumorigenic functions, including CXCL10 and CCL5. USP6 synergistically enhanced chemokine production in response to exogenous IFN by inducing surface upregulation of IFNAR1 and IFNGR1. USP6-expressing Ewing sarcoma cells stimulated migration of primary human monocytes and T lymphocytes and triggered activation of natural killer (NK) cells in vitro. USP6 inhibited Ewing sarcoma xenograft growth in nude but not NSG mice and was accompanied by increased intratumoral chemokine production and infiltration and activation of NK cells, dendritic cells, and macrophages, consistent with a requirement for innate immune cells in mediating the antitumorigenic effects of USP6. High USP6 expression in patients with Ewing sarcoma was associated with chemokine production, immune infiltration, and improved survival. This work reveals a previously unrecognized tumor-suppressive function for USP6, which engenders an immunostimulatory microenvironment through pleiotropic effects on multiple immune lineages. This further raises the possibility that USP6 activity may be harnessed to create a "hot" tumor microenvironment in immunotherapy. SIGNIFICANCE: This study reveals a novel tumor-suppressive function for USP6 by inducing an immunostimulatory microenvironment, suggesting that USP6 activity may be exploited to enhance immunotherapy regimens.
Subject(s)
Bone Neoplasms/genetics , Lymphocytes, Tumor-Infiltrating , Sarcoma, Ewing/genetics , Tumor Suppressor Proteins/physiology , Ubiquitin Thiolesterase/physiology , Animals , Bone Neoplasms/immunology , Bone Neoplasms/metabolism , Bone Neoplasms/mortality , Cell Movement/drug effects , Chemokine CCL5/biosynthesis , Chemokine CXCL10/biosynthesis , Dendritic Cells/drug effects , Humans , Immunotherapy , Interferons/pharmacology , Janus Kinase 1/metabolism , Killer Cells, Natural/drug effects , Macrophages/drug effects , Mice , Mice, Nude , Neoplasm Transplantation , Receptor, Interferon alpha-beta/metabolism , Receptors, Interferon/metabolism , Sarcoma, Ewing/immunology , Sarcoma, Ewing/metabolism , Sarcoma, Ewing/mortality , Tumor Microenvironment/immunology , Ubiquitin Thiolesterase/immunology , Ubiquitin Thiolesterase/metabolism , Up-Regulation/drug effects , Xenograft Model Antitumor Assays , Interferon gamma ReceptorABSTRACT
Ewing sarcoma is the second most common sarcoma of the bone, afflicting predominantly the pediatric population. Although patients with localized disease exhibit favorable survival rates, patients with metastatic disease suffer a dismal 5-year rate of approximately 25%. Thus, there is a great need to develop treatments to combat the disseminated disease. Ubiquitin-specific protease 6 (USP6/TRE17) has been implicated as the key etiologic factor in several benign mesenchymal tumors, including nodular fasciitis and aneurysmal bone cyst (ABC). However, the role of USP6 in the biology of malignant entities remains unexplored. Previously, it was observed that USP6 is sufficient to drive formation of tumors mimicking ABC and nodular fasciitis, and that it functions through JAK1/STAT3 signaling. However, in the context of Ewing sarcoma, USP6 does not enhance the transformation, but rather triggers an IFN response signature, both in cultured Ewing sarcoma cells in vitro and in clinical specimens in vivo. Not only does USP6 independently induce activation of the IFN signaling mediators, JAK1 and STAT1, but it also renders Ewing sarcoma cells exquisitely responsive to exogenous IFNs, potentiating activation of STAT1 and STAT3. Furthermore, IFNß (a type I IFN) induces apoptosis specifically in USP6-positive but not USP6-negative Ewing sarcoma cells. Finally, apoptosis is mediated through the proapoptotic ligand TRAIL, which is synergistically induced by type I IFN and USP6. IMPLICATIONS: These findings provide the first insights into USP6 functions in a clinically relevant malignant entity, and raise the possibility of using IFN for targeting USP6-positive Ewing sarcoma.
Subject(s)
Bone Neoplasms/metabolism , Interferons/pharmacology , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Sarcoma, Ewing/metabolism , TNF-Related Apoptosis-Inducing Ligand/metabolism , Ubiquitin Thiolesterase/genetics , Ubiquitin Thiolesterase/metabolism , Bone Neoplasms/drug therapy , Bone Neoplasms/genetics , Cell Line, Tumor , Cell Survival/drug effects , Child , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/drug effects , Humans , Sarcoma, Ewing/drug therapy , Sarcoma, Ewing/genetics , Sequence Analysis, RNA , Signal Transduction/drug effects , TNF-Related Apoptosis-Inducing Ligand/genetics , Up-Regulation/drug effectsABSTRACT
Bone and soft tissue tumors (BSTT) are relatively poorly understood, hampering the development of effective therapies. Here we report a role for the ubiquitin-specific protease 6 (USP6)/TRE17 oncogene, which is overexpressed upon chromosome translocation in various human tumors, including aneurysmal bone cyst (ABC), and the related benign lesion nodular fasciitis. Ectopic expression of USP6 is known to drive formation of tumors, which recapitulate key features of ABC and nodular fasciitis; however, the identity of USP6's relevant substrates has been obscure. Here we report that the Jak1-STAT3 signaling pathway serves as an essential effector of USP6 in BSTT formation. We found that USP6 directly deubiquitinated Jak1, leading to its stabilization and activation of STAT3. The tumorigenic potential of USP6 was attenuated significantly by CRISPR-mediated deletion of Jak1 or STAT3, or by administration of a Jak family inhibitor. Analysis of primary clinical samples of nodular fasciitis confirmed the activation of a Jak1-STAT3 gene signature in vivo Together, our studies highlight Jak1 as the first identified substrate for USP6, and they offer a mechanistic rationale for the clinical investigation of Jak and STAT3 inhibitors as therapeutics for the treatment of bone and soft tissue tumors along with other neoplasms driven by USP6 overexpression. Cancer Res; 76(18); 5337-47. ©2016 AACR.