Abstract
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.
Highlights
Glioblastoma (GBM), the most lethal form of primary intrinsic brain tumor in both pediatric and adult populations, has the highest mortality rate among all malignant nervous system neoplasms, with a median survival of only 12–15 months
To further compare the proliferation capacity of parental and TMZresistant GBM cells, the cells were treated with 100 μM TMZ for 3 or 5 days
After full-length and truncated fused in sarcoma (FUS) proteins were expressed in HEK293T cells, IP analysis determined that all of FUS fragments containing the RRM or Znf_RanBP2 domains immunoprecipitated murine double minute 2 (MDM2) (Figure 7F). These results demonstrate that ADAMTS9-AS2 might attenuate the interaction between FUS and MDM2, inhibiting MDM2mediated FUS K48-ubiquitination and degradation
Summary
Glioblastoma (GBM), the most lethal form of primary intrinsic brain tumor in both pediatric and adult populations, has the highest mortality rate among all malignant nervous system neoplasms, with a median survival of only 12–15 months. In 2005, the US Food and Drug Administration (FDA) approved combination temozolomide (TMZ) and radiotherapy treatment in adults with newly diagnosed glioblastoma, as well as the use of TMZ alone as a maintenance treatment (HombachKlonisch et al, 2018). Over time, the majority of patients with GBM gradually develop resistance to TMZ during treatment, leading to GBM recurrence and treatment failure (Kaur et al, 2018; Teng et al, 2018). Epigenetic variations have been shown to play major roles in mediating the resistance to targeted therapies and conventional cytotoxic agents. LncRNAs have been shown to play essential roles in cancer therapeutic response. The detailed mechanism of lncRNAs in temozolomide (TMZ) resistance in glioblastoma (GBM) remain to be elucidated
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