Table_3_Novel Function of lncRNA ADAMTS9-AS2 in Promoting Temozolomide Resistance in Glioblastoma via Upregulating the FUS/MDM2 Ubiquitination Axis.DOCX

BackgroundLncRNAs have been shown to play essential roles in cancer therapeutic response. However, the detailed mechanism of lncRNAs in temozolomide (TMZ) resistance in glioblastoma (GBM) remain to be elucidated.
MethodsTo elucidate the mechanism maintaining TMZ resistance, we constructed two TMZ-resistant GBM cell lines (T98G-R/U118-R). LncRNAs from four public datasets were reanalyzed, and the candidate lncRNA ADAMTS9-AS2 was evaluated in TMZ-treated GBM patients and in vitro cell lines.
ResultsReanalysis of lncRNA expression profiles identified ADAMTS9-AS2 as significantly overexpressed in TMZ-resistant GBM cells and as positively associated with the IC50 of TMZ in GBM cells. Overexpression of ADAMTS9-AS2 was also significantly associated with poor TMZ response and shorter progression-free survival (PFS) in TMZ-treated GBM patients. Knockdown of ADAMTS9-AS2 inhibited proliferation and attenuated the IC50 of TMZ, as well as mitigating invasion and migration in TMZ-resistant GBM cells. Subsequent investigations indicated that reduced expression of ADAMTS9-AS2 significantly suppressed expression of the FUS protein, which was predicted as a direct substrate of ADAMTS9-AS2. Expression trends of FUS were directly correlated with those of ADAMTS9-AS2, as shown by increasing concentrations and prolonged treatment with TMZ. RNA pull-down and RIP assays indicated that both endogenous and exogenous ADAMTS9-AS2 directly binds to the RRM and Znf_RanBP2 domains of FUS, consequently increasing FUS protein expression. Knockdown of ADAMTS9-AS2 reduced the half-life of FUS and decreased FUS protein stability via K48 ubiquitin degradation. Moreover, the E3 ubiquitin-protein ligase MDM2 interacts with and down regulates FUS, while the RRM and Znf_RanBP2 domains of FUS facilitate its binding with MDM2. ADAMTS9-AS2 decreased the interaction between MDM2 and FUS, which mediates FUS K48 ubiquitination. Additionally, knockdown of the ADAMTS9-AS2/FUS signaling axis significantly alleviated progression and metastasis in TMZ-resistant cells.
ConclusionADAMTS9-AS2 possessed a novel function that promotes TMZ resistance via upregulating the FUS/MDM2 axis in GBM cells. The RRM or Znf_RanBP2 domains of FUS facilitate the combination of ADAMTS9-AS2 and FUS, competitively inhibiting MDM2-dependent FUS K48 ubiquitination and resulting in enhanced FUS stability and TMZ resistance. Our results suggest that the ADAMTS9-AS2/FUS/MDM2 axis may represent a suitable prognostic biomarker and a potential target in TMZ-resistant GBM therapy.

背景：已有研究表明，长链非编码RNA（long non-coding RNA, lncRNA）在癌症治疗应答中发挥关键作用。然而，长链非编码RNA在胶质母细胞瘤（glioblastoma, GBM）对替莫唑胺（temozolomide, TMZ）耐药中的具体分子机制仍有待阐明。
方法：为阐明维持替莫唑胺耐药的分子机制，我们构建了两株替莫唑胺耐药的胶质母细胞瘤细胞系（T98G-R/U118-R）。重新分析了四个公共数据集的长链非编码RNA表达谱，并在替莫唑胺处理的胶质母细胞瘤患者及体外细胞系中对候选长链非编码RNA ADAMTS9-AS2进行了验证。
结果：重新分析长链非编码RNA表达谱发现，ADAMTS9-AS2在替莫唑胺耐药胶质母细胞瘤细胞中显著高表达，且与胶质母细胞瘤细胞的替莫唑胺半最大效应浓度（half maximal inhibitory concentration, IC50）呈正相关。ADAMTS9-AS2高表达还与替莫唑胺治疗的胶质母细胞瘤患者不良治疗应答及更短的无进展生存期（progression-free survival, PFS）显著相关。敲低ADAMTS9-AS2可抑制替莫唑胺耐药胶质母细胞瘤细胞的增殖，降低其替莫唑胺IC50，同时减弱细胞侵袭与迁移能力。后续研究表明，ADAMTS9-AS2表达下调可显著抑制FUS蛋白的表达，而FUS被预测为ADAMTS9-AS2的直接作用底物。随着替莫唑胺处理浓度升高、作用时间延长，FUS的表达趋势与ADAMTS9-AS2的表达趋势直接相关。RNA下拉实验（RNA pull-down）及RNA免疫沉淀实验（RNA immunoprecipitation, RIP）证实，内源性与外源性ADAMTS9-AS2均可直接结合FUS的RRM结构域与Znf_RanBP2结构域，进而上调FUS蛋白的表达。敲低ADAMTS9-AS2可缩短FUS的半衰期，并通过K48泛素化降解途径降低FUS的蛋白稳定性。此外，E3泛素蛋白连接酶（E3 ubiquitin-protein ligase）MDM2可与FUS相互作用并下调其表达，而FUS的RRM与Znf_RanBP2结构域可促进其与MDM2的结合。ADAMTS9-AS2可减弱MDM2与FUS之间的相互作用，从而抑制FUS的K48泛素化过程。进一步研究发现，敲低ADAMTS9-AS2/FUS信号轴可显著缓解替莫唑胺耐药细胞的增殖与转移能力。
结论：本研究揭示ADAMTS9-AS2具有全新功能：通过上调胶质母细胞瘤细胞中的FUS/MDM2信号轴，促进替莫唑胺耐药。FUS的RRM或Znf_RanBP2结构域可促进ADAMTS9-AS2与FUS的结合，竞争性抑制MDM2依赖的FUS K48泛素化，进而增强FUS的蛋白稳定性并诱导替莫唑胺耐药。研究结果表明，ADAMTS9-AS2/FUS/MDM2信号轴有望成为替莫唑胺耐药胶质母细胞瘤的潜在预后生物标志物与治疗靶点。