SMURF2 phosphorylation at Thr249 modifies the stemness and tumorigenicity of glioma stem cells by regulating TGF-β receptor stability

Glioma stem cells (GSCs) contribute to the pathogenesis of glioblastoma, the most malignant form of glioma. The implication and underlying mechanisms of SMAD specific E3 ubiquitin protein ligase 2 (SMURF2) on the GSC phenotypes remain unknown. We previously demonstrated that SMURF2 phosphorylation at Thr249 (SMURF2Thr249) activates its E3 ubiquitin ligase activity. Here, we demonstrate that SMURF2Thr249 phosphorylation plays an essential role in maintaining GSC stemness and tumorigenicity. SMURF2 silencing augmented the self-renewal potential and tumorgenicity of patient-derived GSCs. The SMURF2Thr249 phosphorylation level was low in human glioblastoma pathology specimens. Introduction of the SMURF2T249A mutant resulted in increased stemness and tumorgenicity of GSCs, recapitulating the SMURF2 silencing. Moreover, the inactivation of SMURF2Thr249 phosphorylation increases TGF-β receptor (TGFBR) protein stability. Indeed, TGFBR1 knockdown markedly counteracted the GSC phenotypes by SMURF2T249A mutant. These findings highlight the importance of SMURF2Thr249 phosphorylation in maintaining GSC phenotypes, thereby demonstrating a potential target for GSC-directed therapy.


50
SMAD specific E3 ubiquitin protein ligase 2 (SMURF2) is the E3 ubiquitin ligase 51 responsible for specifying the substrates for ubiquitination and degradation by proteasomes (1,52 2). Accumulating evidence indicates SMURF2 regulates a wide array of physiological 53 processes, including cell proliferation, invasion, self-renewal, and migration, through its 54 regulation of a variety of signaling pathways (3)(4)(5). The E3 ubiquitin ligase activity of SMURF2 55 is regulated at the post-transcriptional level through SUMOylation, methylation, and 56 phosphorylation (6-8), as well as at the transcriptional level (9). We recently demonstrated that 57 the phosphorylation of SMURF2 at Thr 249 (SMURF2 Thr249 ) by extracellular signal-regulated 58 kinase 5 (ERK5) plays an essential role in maintaining the stemness of mesenchymal stem cells 59 (MSCs), which contributes to skeletogenesis (10). Mechanistically, SMURF2 Thr249 60 phosphorylation activates its E3 ubiquitin ligase activity, which modifies the stability of SMAD 61 proteins, which in turn transcriptionally activate the expression of SOX9, the principal 62 transcription factor of skeletogenesis in MSCs. 63 Gliomas, which represent approximately 80% of all primary malignant brain tumors 64 in humans, can be categorized into four grades according to the World Health Organization 65 (WHO) classification criteria: grade I, grade II, grade III, and grade IV (glioblastoma, GBM) 66 (11,12). GBM, the most malignant form of glioma, is one of the most aggressive and deadly 67 types of cancer. Patients with GBM have a very poor prognosis, with a five-year survival rate 68 of only 5. 1% (13, 14). Glioma stem cells (GSCs), also known as glioma-initiating cells, are a 69 subpopulation of tumor cells that exhibit stem cell-like capacities such as self-renewal and 70 tumor-initiating capacities (15)(16)(17). Recent studies have determined that GSCs contribute to 71 high rates of therapeutic resistance and rapid recurrence (18,19), cancer invasion, immune 72 evasion, tumor angiogenesis, and the recruitment of tumor-associated macrophages, which 73 indicates that targeting GSCs is an efficacious strategy for improving GBM treatment (20)(21)(22). Transforming growth factor-β (TGF-β) signaling, which is tightly regulated through 75 protein ubiquitination (23,24), has been shown to play a crucial role in maintaining the 76 stemness and tumorigenicity of GSCs through several pathways including the SMAD-SOX4-77 SOX2 axis and the SMAD-LIF-JAK-STAT pathway (25,26). SMAD7 acts as a scaffold protein 78 to recruit SMURF2 to the TGF-β receptor (TGFBR) complex to facilitate its ubiquitination (27). 79 This leads to the proteasome-mediated degradation of TGFBRs and the attenuation of TGF-β 80 signaling. Ubiquitin-specific peptidase 15 (USP15), a deubiquitinating enzyme, binds to the 81 SMAD7-SMURF2 complex and deubiquitinates and stabilizes TGFBR1, resulting in enhanced 82 TGF-β signaling (28,29). The balance between USP15 and SMURF2 activities determines the 83 activity of TGF-β signaling and subsequent oncogenesis in GBM. Indeed, a deficiency in 84 USP15 decreases the oncogenic capacity of GSCs due to the repression of TGF-β signaling 85 (28); conversely, USP15 amplification confers poor prognosis in individuals with GBM (30). 86 However, although SMURF2 should be assumed to play an opposite role from that of USP15, 87 no reports have yet directly addressed the implication and underlying mechanisms of SMURF2 88 on the GSC phenotypes and subsequent glioma pathogenesis both in vivo and in vitro. 89 In this study, we reveal that SMURF2 silencing by shRNA resulted in an augmentation 90 of the self-renewal potential and tumorigenicity of GSCs. The SMURF2 Thr249 phosphorylation 91 level was downregulated in GBM patients, regardless of the lack of marked changes in its 92 mRNA and protein levels. Additionally, the SMURF2 Thr249 phosphorylation level was lower in 93 GSCs than that in differentiated glioma cells. The inactivation of SMURF2 Thr249 94 phosphorylation by a non-phosphorylatable mutant (SMURF2 T249A mutant) increased the self-95 renewal potential and tumorigenicity of GSCs, thus mimicking the GSC phenotype in SMURF2 96 silencing. Mechanistically, SMURF2 Thr249 phosphorylation activates its E3 ubiquitin ligase 97 activity, which decreases the protein stability of TGFBR1 via proteasome-mediated degradation.

98
Finally, TGFBR1 silencing rescues the increased self-renewal potential and tumorigenicity of 99 6 GSCs by inactivating SMURF2 Thr249 phosphorylation. Collectively, these findings highlight the 100 importance of SMURF2 Thr249 phosphorylation in maintaining the stemness and tumorigenicity 101 of GSCs; these findings also indicate that SMURF2 Thr249 phosphorylation could be an important 102 posttranslational modification in treatment strategies aimed at disrupting GSCs.

105
Targeting SMURF2 promotes the self-renewal potential and tumorigenicity of GSCs 106 We first elucidated the functional significance of SMURF2 in maintaining GSCs in 107 vitro by targeting SMURF2 expression using lentiviral shRNA (shSMURF2) in TGS-01 and 108 TGS-04 GSCs, which are human GBM patient-derived GSCs. Disruption of SMURF2 with 109 shRNA significantly increased GSC tumorsphere formation in both TGS-01 and TGS-04 GSCs 110 (Fig. 1A). Additionally, an in vitro limiting dilution assay demonstrated that the self-renewal 111 potential of GSCs was significantly increased by SMURF2 silencing in both TGS-01 and TGS-112 04 GSCs (Fig. 1B). Furthermore, SMURF2 knockdown resulted in the significant upregulation 113 of the stem cell transcription factors SOX2 and SOX4 in TGS-01 GSCs, along with a marked 114 reduction in the SMURF2 protein level (Fig. 1C). Conversely, disrupting SMURF2 did not 115 significantly alter cell apoptosis in TGS-01 GSCs (Fig. 1D). 116 We next examined whether SMURF2 silencing could affect the tumorigenic potential  We next assessed whether our findings were relevant to clinical data in glioma patients 130 using publicly available datasets and our clinical samples. No marked alterations of SMURF2 131 mRNA levels were found among grades II, III, and IV cancer or among classical, mesenchymal, 132 and proneural tumors, according to the Cancer Genome Atlas (TCGA) ( Fig. 2A). Moreover, in 133 accordance with the lack of marked alterations of the SMURF2 mRNA levels in glioma 134 specimens in the TCGA database, we confirmed that the SMURF2 protein level was 135 comparable between control nonneoplastic brain tissue (NB), diffuse astrocytoma (grade II),    (Fig. 2F). Therefore, these results indicated that the SMURF2 Thr249 156 phosphorylation level was significantly lower in GSCs than that in differentiated glioma cells.

157
Our experimental findings aligned with publicly available clinical data suggest that 158 SMURF2 Thr249 phosphorylation rather than SMURF2 levels (protein and mRNA) might be 159 associated with tumor grade and glioma stemness in humans. Thus, SMURF2 Thr249 160 phosphorylation may serve as a prognostic marker of GBM.  Additionally, an immunoblotting analysis revealed that the protein levels of SOX2 and SOX4 172 were significantly upregulated by SMURF2 T249A but significantly downregulated by 173 SMURF2 WT (Fig. 3C). Conversely, cell apoptosis was not markedly altered by either 174 SMURF2 T249A or SMURF2 WT in TGS-01 GSCs (Fig. 3D). 175 We next examined the impact of SMURF2 Thr249 phosphorylation on the tumorigenic  The self-renewal potential and tumorigenicity of GSCs were activated by inactivating 194 SMURF2 Thr249 phosphorylation, thus recapitulating GSC phenotypes by SMURF2 silencing.

195
The phosphorylation of SMURF2 Thr249 activates its ubiquitin E3 ligase ability to accelerate the 196 proteasomal degradation of SMAD proteins (SMAD1, SMAD2, and SMAD3) in MSCs to 197 control the stemness; furthermore, the TGF-β/SMAD and BMP/SMAD axes play a crucial role 198 in regulating the stemness and tumorigenicity of GSCs through the SMAD pathway (25,33,199 34). We therefore investigated whether SMURF2 Thr249 phosphorylation could regulate the TGF-   Imaging System (Bio-Rad). Quantification was performed by densitometry using ImageJ.   Table), annealed, and inserted into the mCherry vector, and the shRNA vector for TGFBR1 was

459
Values are expressed as the mean ± S.E. and statistical significance was determined using the 460 one-way ANOVA using the Bonferroni post hoc test.