Background: Medulloblastoma (MB) represents the most prevalent malignant paediatric brain tumour, characterised by the sonic Hedgehog molecular subtype (SHH-MB), which is driven by aberrant activation of the SHH signalling cascade. Suppressor of fused (SUFU), a core member of SHH signal transduction, functions as a tumour suppressor by inhibiting the activity of transcription factors glioma-associated oncogene homologue (GLI)-triggered SHH signalling pathway. While ubiquitin-mediated proteasomal degradation of SUFU has been shown to modulate SHH signalling, the regulatory factors involved in SUFU deubiquitination and their specific roles in MB pathogenesis remain largely undefined.
Methods: Mass spectrometry and co-immunoprecipitation were employed to identify the interaction between USP9X and SUFU. Clinical correlation analyses were conducted using MB tissue microarrays and publicly available datasets. Ubiquitination assays, functional cell-based experiments, and orthotopic xenograft models were performed to evaluate the biological role of USP9X in SHH-MB. Chaperone-mediated autophagy (CMA) inhibitors were utilised to investigate their regulatory effects on USP9X expression and SHH-MB progression.
Results: Mass spectrometry identified the deubiquitinase ubiquitin-specific protease 9X (USP9X) as a previously unrecognised SUFU-binding partner. Notably, USP9X and SUFU exhibit a positive correlation in a MB tissue array, with both exhibiting low expression levels that are associated with adverse prognostic outcomes. Loss of USP9X in SHH-MB enhances cell proliferation in vitro as well as orthotopic MB xenograft tumourgenicity in vivo. Mechanically, USP9X deubiquitinates and stabilises SUFU, thereby negatively regulating SHH signal transduction. Interestingly, SHH signalling promotes SUFU ubiquitination through CMA-dependent degradation of USP9X protein levels, facilitating pathway activation. Combined inhibition of CMA and SHH pathway had a synergically therapeutic effect on SHH-MB.
Conclusions: These results establish CMA and USP9X as pivotal regulators of SHH medulloblastoma MB progression, emphasising their potential as novel therapeutic targets for medulloblastoma, and combinatorial inhibition of CMA and Smoothened (SMO) may provide a strategy to overcome intrinsic or acquired resistance to SMO monotherapy in SHH-MB.
Key points:
| [1] |
Northcott PA, Robinson GW, Kratz CP, et al. Medulloblastoma. Nat Rev Dis Primers. 2019; 5(1): 11.
|
| [2] |
Hovestadt V, Ayrault O, Swartling FJ, Robinson GW, Pfister SM, Northcott PA. Medulloblastomics revisited: biological and clinical insights from thousands of patients. Nat Rev Cancer. 2020; 20(1): 42-56.
|
| [3] |
Northcott PA, Buchhalter I, Morrissy AS, et al. The whole-genome landscape of medulloblastoma subtypes. Nature. 2017; 547(7663): 311-317.
|
| [4] |
Abdel-Rasol M, El-Sayed W. Advancing medulloblastoma treatment: molecular mechanisms, drug repurposing, and precision therapies. Mol Diagn Ther. 2026; 30(1): 75-90.
|
| [5] |
Teglund S, Toftgard R. Hedgehog beyond medulloblastoma and basal cell carcinoma. Biochim Biophys Acta. 2010; 1805(2): 181-208.
|
| [6] |
Liu X, Ding C, Tan W, Zhang A. Medulloblastoma: molecular understanding, treatment evolution, and new developments. Pharmacol Ther. 2020; 210:107516.
|
| [7] |
Rohatgi R, Milenkovic L, Scott M. Patched1 regulates Hedgehog signaling at the primary cilium. Science. 2007; 317(5836): 372-376.
|
| [8] |
Corbit KC, Aanstad P, Singla V, Norman AR, Stainier DYR, Reiter JF. Vertebrate Smoothened functions at the primary cilium. Nature. 2005; 437(7061): 1018-1021.
|
| [9] |
Ingham P, Nakano Y, Seger C. Mechanisms and functions of Hedgehog signalling across the metazoa. Nat Rev Genetics. 2011; 12(6): 393-406.
|
| [10] |
Jiang J, Hui C. Hedgehog signaling in development and cancer. Dev Cell. 2008; 15(6): 801-812.
|
| [11] |
Kogerman P, Grimm T, Kogerman L, et al. Mammalian suppressor-of-fused modulates nuclear-cytoplasmic shuttling of Gli-1. Nat Cell Biol. 1999; 1(5): 312-319.
|
| [12] |
Chen M, Wilson CW, Li Y, et al. Cilium-independent regulation of Gli protein function by Sufu in Hedgehog signaling is evolutionarily conserved. Genes Dev. 2009; 23(16): 1910-1928.
|
| [13] |
Zhang Z, Shen L, Law K, et al. Suppressor of fused chaperones Gli proteins to generate transcriptional responses to sonic Hedgehog signaling. Mol Cell Biol. 2017; 37(3): e00421-16.
|
| [14] |
Huang D, Wang Y, Tang J, Luo S. Molecular mechanisms of suppressor of fused in regulating the hedgehog signalling pathway. Oncol Lett. 2018; 15(5): 6077-6086.
|
| [15] |
Cooper AF, Yu KP, Brueckner M, et al. Cardiac and CNS defects in a mouse with targeted disruption of suppressor of fused. Development. 2005; 132(19): 4407-4417.
|
| [16] |
Svärd J, Henricson KH, Persson-Lek M, et al. Genetic elimination of Suppressor of fused reveals an essential repressor function in the mammalian Hedgehog signaling pathway. Dev Cell. 2006; 10(2): 187-197.
|
| [17] |
Lee Y, Kawagoe R, Sasai K, et al. Loss of suppressor-of-fused function promotes tumorigenesis. Oncogene. 2007; 26(44): 6442-6447.
|
| [18] |
Guerrini-Rousseau L, Masliah-Planchon J, Waszak SM, et al. Cancer risk and tumour spectrum in 172 patients with a germline SUFU pathogenic variation: a collaborative study of the SIOPE Host Genome Working Group. J Med Genet. 2022; 59(11): 1123-1132.
|
| [19] |
Taylor MD, Liu L, Raffel C, et al. Mutations in SUFU predispose to medulloblastoma. Nat Genet. 2002; 31(3): 306-310.
|
| [20] |
Han B, Sun Z, Yu T, et al. SPOP‒PTEN‒SUFU axis promotes progression of clear cell renal cell carcinoma via activating SHH and WNT pathway. Cell Death Discov. 2021; 7(1): 120.
|
| [21] |
Brugières L, Remenieras A, Pierron G, et al. High frequency of germline SUFU mutations in children with desmoplastic/nodular medulloblastoma younger than 3 years of age. J Clin Oncol. 2012; 30(17): 2087-2093.
|
| [22] |
Popovic D, Vucic D, Dikic I. Ubiquitination in disease pathogenesis and treatment. Nat Med. 2014; 20(11): 1242-1253.
|
| [23] |
Yue S, Chen Y, Cheng S. Hedgehog signaling promotes the degradation of tumor suppressor Sufu through the ubiquitin-proteasome pathway. Oncogene. 2009; 28(4): 492-499.
|
| [24] |
Raducu M, Fung E, Serres S, et al. SCF (Fbxl17) ubiquitylation of Sufu regulates Hedgehog signaling and medulloblastoma development. EMBO J. 2016; 35(13): 1400-1416.
|
| [25] |
Infante P, Faedda R, Bernardi F, et al. Itch/beta-arrestin2-dependent non-proteolytic ubiquitylation of SuFu controls Hedgehog signalling and medulloblastoma tumorigenesis. Nat Commun. 2018; 9(1): 976.
|
| [26] |
Wang W, Qiu J, Qu P, et al. Regulator of cullins-1 (ROC1) negatively regulates the Gli2 regulator SUFU to activate the hedgehog pathway in bladder cancer. Cancer Cell Int. 2021; 21(1): 75.
|
| [27] |
Yan Z, Cheng M, Hu G, et al. Positive feedback of SuFu negating protein 1 on Hedgehog signaling promotes colorectal tumor growth. Cell Death Dis. 2021; 12(2): 199.
|
| [28] |
Murtaza M, Jolly LA, Gecz J, Wood SA. La FAM fatale: USP9X in development and disease. Cell Mol Life Sci. 2015; 72(11): 2075-2089.
|
| [29] |
Chen Z, Wang H, Wang S, et al. USP9X deubiquitinates ALDH1A3 and maintains mesenchymal identity in glioblastoma stem cells. J Clin Invest. 2019; 129(5): 2043-2055.
|
| [30] |
Biber P, Carminati A, Mohager W, et al. USP9X is a mechanosensitive deubiquitinase that controls tumor cell invasiveness and drug response through YAP stabilization. Cell Rep. 2025; 44(10):116372.
|
| [31] |
Paudel P, Zhang Q, Leung C, et al. Crystal structure and activity-based labeling reveal the mechanisms for linkage-specific substrate recognition by deubiquitinase USP9X. Proc Natl Acad Sci U S A. 2019; 116(15): 7288-7297.
|
| [32] |
Huang J, Wang J. Selective protein degradation through chaperone‑mediated autophagy: implications for cellular homeostasis and disease (review). Mol Med Rep. 2025; 31(1): 13.
|
| [33] |
Kaushik S, Cuervo A. The coming of age of chaperone-mediated autophagy. Nat Rev Mol Cell Biol. 2018; 19(6): 365-381.
|
| [34] |
Bufalieri F, Severini LL, Caimano M, Infante P, Di Marcotullio L. DUBs activating the Hedgehog signaling pathway: a promising therapeutic target in cancer. Cancers (Basel). 2020; 12(6): 1518.
|
| [35] |
Yang H, Chen X, Li K, et al. Repurposing old drugs as new inhibitors of the ubiquitin‒proteasome pathway for cancer treatment. Semin Cancer Biol. 2021; 68: 105-122.
|
| [36] |
Zhang Q, Jiang J. Regulation of Hedgehog signal transduction by ubiquitination and deubiquitination. Int J Mol Sci. 2021; 22(24):13338.
|
| [37] |
Lu H, Lyu Y, Tran L, et al. HIF-1 recruits NANOG as a coactivator for TERT gene transcription in hypoxic breast cancer stem cells. Cell Rep. 2021; 36(13):109757.
|
| [38] |
Khan OM, Carvalho J, Spencer-Dene B, et al. The deubiquitinase USP9X regulates FBW7 stability and suppresses colorectal cancer. J Clin Invest. 2018; 128(4): 1326-1337.
|
| [39] |
Toloczko A, Guo F, Yuen H, et al. Deubiquitinating enzyme USP9X suppresses tumor growth via LATS kinase and core components of the Hippo pathway. Cancer Res. 2017; 77(18): 4921-4933.
|
| [40] |
Zhu C, Ji X, Zhang H, et al. Deubiquitylase USP9X suppresses tumorigenesis by stabilizing large tumor suppressor kinase 2 (LATS2) in the Hippo pathway. J Biol Chem. 2018; 293(4): 1178-1191.
|
| [41] |
Kaushik S, Cuervo A. Chaperone-mediated autophagy: a unique way to enter the lysosome world. Trends Cell Biol. 2012; 22(8): 407-417.
|
| [42] |
Kon M, Kiffin R, Koga H, et al. Chaperone-mediated autophagy is required for tumor growth. Sci Transl Med. 2011; 3(109):109ra117.
|
| [43] |
Li B, Ming H, Qin S, et al. HSPA8 activates Wnt/beta-catenin signaling to facilitate BRAF V600E colorectal cancer progression by CMA-mediated CAV1 degradation. Adv Sci (Weinh). 2024; 11(3):e2306535.
|
| [44] |
Anguiano J, Garner TP, Mahalingam M, Das BC, Gavathiotis E, Cuervo AM. Chemical modulation of chaperone-mediated autophagy by retinoic acid derivatives. Nat Chem Biol. 2013; 9(6): 374-382.
|
| [45] |
Rudin CM, Hann CL, Laterra J, et al. Treatment of medulloblastoma with hedgehog pathway inhibitor GDC-0449. N Engl J Med. 2009; 361(12): 1173-1178.
|
| [46] |
Yauch RL, Dijkgraaf GJP, Alicke B, et al. Smoothened mutation confers resistance to a Hedgehog pathway inhibitor in medulloblastoma. Science. 2009; 326(5952): 572-574.
|
| [47] |
Lo-Coco F, Avvisati G, Vignetti M, et al. Retinoic acid and arsenic trioxide for acute promyelocytic leukemia. N Engl J Med. 2013; 369(2): 111-121.
|
| [48] |
Henning NJ, Boike L, Spradlin JN, et al. Deubiquitinase-targeting chimeras for targeted protein stabilization. Nat Chem Biol. 2022; 18(4): 412-421.
|
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2026 The Author(s). Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.
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