USP14-mediated stabilization of ACTN1 maintains mesenchymal characteristics in glioblastoma

Qiu, Wenjin; Shi, Xueping; Wei, Ruting; Song, Jiaquan; Ling, Yuanguo; Hu, Yunjia; Song, Shibin; Chen, Yimin; Yang, Hua; Luo, Peng; Chu, Liangzhao

doi:10.1038/s42003-025-09083-8

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Published: 25 November 2025

USP14-mediated stabilization of ACTN1 maintains mesenchymal characteristics in glioblastoma

Wenjin Qiu ORCID: orcid.org/0000-0002-6290-3681^1,2^na1,
Xueping Shi¹^na1,
Ruting Wei¹^na1,
Jiaquan Song¹,
Yuanguo Ling¹,
Yunjia Hu³,
Shibin Song¹,
Yimin Chen¹,
Hua Yang¹,
Peng Luo ORCID: orcid.org/0000-0001-5113-6176²^na2 &
…
Liangzhao Chu ORCID: orcid.org/0000-0002-1861-2473¹^na2

Communications Biology volume 8, Article number: 1680 (2025) Cite this article

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Abstract

Glioblastoma (GBM) is a highly aggressive primary brain tumor with a dismal prognosis, particularly in its mesenchymal (MES) subtype, which correlates strongly with poor survival. Despite this, the mechanisms preserving MES identity remain poorly understood. Here, we show that alpha-actinin 1 (ACTN1) is upregulated in MES GBM and drives proneural-to-mesenchymal transition (PMT). Using patient samples and multiple GBM cell lines, we find that ACTN1 overexpression promotes proliferation, invasion, and tumorigenesis, while its silencing diminishes these malignant traits and shifts gene expression away from MES markers. Mechanistically, we identify ubiquitin-specific peptidase 14 (USP14) as a pivotal deubiquitinase (DUB) that stabilizes ACTN1 by removing its ubiquitin chains. Pharmacological inhibition of USP14 with IU1 reduces ACTN1 protein levels, impairs MES-associated phenotypes, and suppresses tumor progression in vitro and in intracranial xenograft models. Clinically, elevated USP14 and ACTN1 expression correlates with poorer survival in GBM patients, highlighting the USP14–ACTN1 axis as a key driver of PMT and a promising therapeutic target for this devastating disease.

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Introduction

Glioblastoma (GBM) is the most aggressive primary brain tumor in adults, with a median survival of only 15 to 20 months¹. Standard treatment consists of maximal surgical resection, followed by high-dose radiation therapy and concurrent or adjuvant temozolomide chemotherapy. Despite these intensive interventions, GBM prognosis remains poor, with a 5-year survival rate under 5%^2,3. Gene expression profiling has categorized GBM into three subtypes: classical, mesenchymal (MES), and proneural (PN)^4,5,6. Of these, the PN subtype is linked with relatively better outcomes, while MES represents the most malignant form, closely associated with worse survival rates⁷. Similar to the epithelial-mesenchymal transition (EMT) observed in other cancers, GBM’s PN subtype can transition to the MES subtype^8,9. This proneural-to-mesenchymal transition (PMT) is a pivotal factor driving glioblastoma invasion, progression, and poor prognosis¹⁰. However, the specific mechanisms underlying PMT in GBM are largely unexplored and warrant further investigation.

Alpha-actinins (ACTNs) are cytoskeletal proteins involved in various non-muscle functions, including cytokinesis, cell adhesion, and migration, in addition to their established role in sarcomere structure¹¹. Four isoforms of α-actinin exist: ACTN1, ACTN2, ACTN3, and ACTN4¹². Among these, ACTN1 plays a critical role in controlling cellular processes, such as cell division, adhesion, and migration¹³. Elevated ACTN1 levels are associated with poor prognosis in cancers such as oral squamous cell carcinoma and acute myeloid leukemia^14,15. Recent studies reveal ACTN1’s involvement in the progression of cancers like head and neck squamous cell carcinoma¹³, breast cancer¹⁶, hepatocellular carcinoma¹⁷, and gastric cancer¹⁸, as well as its prognostic significance in malignant gliomas¹⁹. However, the exact mechanisms by which ACTN1 drives these malignant behaviors, particularly in supporting the mesenchymal identity of GBM, are not fully understood. ACTN1 is known to be regulated by post-translational modifications (PTMs), including the ubiquitin-proteasome pathway mediated by E3-ubiquitin ligase Cullin3²⁰. The stability of ACTN1 in GBM cells and its impact on PMT, however, require further investigation.

Deubiquitinases (DUBs) are essential enzymes that reverse ubiquitination, a process critical for regulating the ubiquitin signaling pathway involved in cancer initiation and progression²¹. Human DUBs are divided into six families: ubiquitin-specific proteases (USPs), ubiquitin carboxyterminal hydrolases (UCHs), ovarian tumor proteases (OTUs), Machado-Joseph disease proteases (MJDs), JAB1/MPN/Mov34 metalloenzymes (JAMMs), and the MINDY family^22,23. Among these, ubiquitin-specific peptidase 14 (USP14), a notable member of the USP family, plays a significant role in modulating several oncogenic processes, such as cell invasion, proliferation, drug resistance, immune evasion, apoptosis, and autophagy^24,25,26,27. Recent studies highlight USP14’s ability to deubiquitinate NAP1L1, which promotes glioma cell proliferation²⁸. In glioblastoma stem cells, USP14 enhances stem-like properties, tumorigenicity, and resistance to radiotherapy by stabilizing MST4-phosphorylated ALKBH5²⁹. However, whether USP14 regulates ACTN1, a potential driver of the mesenchymal identity in glioblastoma, remains unknown. Exploring USP14’s role in ACTN1 regulation could provide valuable insights into tumor progression and therapeutic approaches.

In this study, we observed that ACTN1 is upregulated in GBM, correlating positively with MES subtype markers and inversely with PN markers, indicating its role in the MES phenotype. Elevated ACTN1 levels suggest that it may promote malignant GBM progression. We also identified USP14 as a critical post-translational regulator of ACTN1 stability in GBM cells. Mechanistically, USP14 binds to and stabilizes ACTN1, thereby supporting GBM’s mesenchymal identity. Furthermore, we demonstrated that IU1, a small-molecule inhibitor targeting USP14’s catalytic activity^24,30, promotes ACTN1 degradation and suppresses the mesenchymal phenotype of GBM in both in vitro and in vivo models. Our findings suggest that IU1 holds promise as a lead compound for developing anti-cancer drugs targeting glioblastoma with high ACTN1 expression. Collectively, this study positions the USP14/ACTN1 axis as a potential therapeutic target to counter mesenchymal identity and malignant progression in GBM.

Results

High ACTN1 expression in the Mesenchymal (MES) subtype GBM reflects MES characteristics

To investigate ACTN1’s function in glioblastoma (GBM), we analyzed its protein levels across 10 normal brain tissues (NBTs) and 64 GBM patient samples, including three subtypes: 25 mesenchymal (MES), 20 classical (CL), and 19 proneural (PN) samples, by IB analysis. Results indicated that ACTN1 expression was significantly reduced in NBTs compared to GBM subtypes, with distinct variations observed among GBM subtypes. The MES subtype displayed the highest levels of ACTN1, while PN had the lowest (Fig. 1A; Supplementary Fig. 1A). This pattern suggests a specific upregulation of ACTN1 in the MES subtype, possibly contributing to its aggressive features.

**Fig. 1: ACTN1 Association with MES Characteristics and Its Role in Invasion, Migration, Proliferation, and Tumor Formation in GBM.**

To deepen our understanding of ACTN1 expression, we assessed it in normal human astrocytes (NHAs) alongside multiple GBM cell lines. Here, U251, T98G, and primary GBM1 cells represented PN subtypes, while U87MG, LN229, and primary GBM2 cells were classified as MES subtypes³¹. Notably, ACTN1 expression was significantly higher in GBM cells compared to NHAs, with particularly high levels in MES subtype cells (U87MG, LN229, and GBM2) (Fig. 1B). This observation suggests a link between ACTN1 expression and MES subtype-specific markers, potentially indicating a role in promoting aggressive traits in MES GBM cells. To explore the association between ACTN1 and subtype-specific markers, we compared ACTN1 levels in MES (LN229, GBM2) and PN (U251, GBM1) cells. MES cells (LN229, GBM2) exhibited higher ACTN1 and MES markers (YKL-40, MET, COL5A1), whereas PN cells (U251, GBM1) displayed reduced ACTN1 levels and increased PN marker expression (Olig2, PDGFRα) (Fig. 1C). These results suggest ACTN1 may have a functional role in mesenchymal features in GBM.

ACTN1 enhances invasion, migration, proliferation, and tumorigenesis in GBM cells

Given that the proneural-to-mesenchymal transition (PMT) contributes to the invasiveness and growth of glioblastoma, we explored ACTN1’s role in this process. Depleting ACTN1 by two non-overlapping lentiviral shRNAs in LN229 and GBM2 cells led to decreased expression of MES markers (YKL-40, MET, COL5A1) while increasing PN markers (Olig2, PDGFRα) (Fig. 1D). In contrast, ACTN1 overexpression in U251 and GBM1 cells increased MES markers while reducing PN markers (Supplementary Fig. 1B). Transwell assays further demonstrated that ACTN1 silencing reduced invasion and migration in LN229 and GBM2 cells (Fig. 1E, F), whereas its overexpression enhanced these capabilities in U251 and GBM1 cells (Supplementary Fig. 1C, D). These findings indicate that ACTN1 positively influences GBM cell invasiveness and migration, aligning with MES subtype characteristics. Additionally, ACTN1 knockdown decreased proliferation in LN229 and GBM2 cells, while its overexpression significantly boosted proliferation in U251 and GBM1 cells (Fig. 1G; Supplementary Fig. 1E), suggesting a role in promoting tumorigenic features. To examine tumorigenic potential in vivo, equal numbers of luciferase-labeled LN229 and GBM2 cells, transduced with either shCtrl or shACTN1, were injected into mouse brains. Xenografts derived from ACTN1-depleted cells showed notably slower tumor growth (Fig. 1H, I), and mice with these xenografts had significantly improved survival rates compared to controls (Fig. 1J). These results support that ACTN1 contributes to tumor growth and aggressiveness in GBM, specifically in the MES subtype.

Identification of USP14 as a potential DUB of ACTN1

To investigate whether ACTN1 stability is regulated by the ubiquitin-proteasome system (UPS), we performed a cycloheximide (CHX) chase assay in U251 and GBM1 cells. ACTN1 protein levels declined rapidly post-CHX treatment, becoming nearly undetectable within 8 hours. However, treatment with the UPS inhibitor MG132 effectively prevented this degradation, indicating that ACTN1 stability is regulated by proteasomal pathways (Supplementary Fig. 2A). To identify deubiquitinases (DUBs) that may interact with ACTN1, we utilized the BioGRID database, identifying USP2, USP14, BAP1, and ZRANB1 as potential candidates. Of these, only USP14 was observed to interact with ACTN1 in HEK293T cells, suggesting it as the primary DUB associated with ACTN1 (Fig. 2A). Analysis of USP14 expression across NBTs and GBM subtypes showed its highest expression in MES GBM (Fig. 2B; Supplementary Fig. 2B), further indicating its relevance in aggressive GBM subtypes. Moreover, correlation analysis of clinical specimens encompassing NBTs and GBM subtypes revealed a significant positive correlation between ACTN1 (Fig. 1A) and USP14 (Fig. 2B) protein levels (Supplementary Fig. 2C).

**Fig. 2: USP14 as a Potential DUB of ACTN1 and Its Role in Maintaining ACTN1 Stability.**

Given USP14’s known deubiquitinating activity, we hypothesized it might stabilize ACTN1 by removing ubiquitin chains. In HEK293T cells, overexpression of wild-type USP14 (USP14-WT), but not the inactive mutant C114A, led to a dose-dependent increase in ACTN1 levels (Fig. 2C). Further experiments revealed that silencing USP14 in LN229 and GBM2 cells significantly decreased ACTN1 protein levels without affecting mRNA levels, implying that USP14 stabilizes ACTN1 post-translationally (Fig. 2D, E). Treatment with MG132 restored ACTN1 levels, reinforcing that USP14 stabilizes ACTN1 by preventing proteasomal degradation (Fig. 2E). Furthermore, compared to control LN229 and GBM2 cells, USP14-deficient cells exhibited significantly reduced levels of ACTN1, which could be effectively restored by USP14-WT overexpression, but not USP14-C114A (Fig. 2F). CHX chase assays revealed that USP14 depletion accelerated ACTN1 degradation in MES GBM (Fig. 2G, H; Supplementary Fig. 2D, E), whereas ectopic expression of USP14-WT, but not USP14-C114A, markedly increased ACTN1 stability in both HEK293T and PN GBM cells (Fig. 2I; Supplementary Fig. 2F, G). These findings establish USP14 as a stabilizing factor for ACTN1 in GBM.

USP14 interacts with ACTN1

To confirm a direct interaction between USP14 and ACTN1, double immunofluorescence staining was performed, showing co-localization in the nucleus through confocal microscopy (Fig. 3A). Co-immunoprecipitation (Co-IP) assays demonstrated that His-tagged ACTN1 could be detected in Flag-USP14 WT or Flag-USP14 C114A precipitates in HEK293T cells, suggesting the interaction does not require USP14’s deubiquitinase (DUB) activity (Fig. 3B). Additionally, we examined endogenous USP14 and ACTN1 in LN229 and GBM2 cell lines, which showed a consistent association (Fig. 3C). Next, GST pull-down assay demonstrated that GST-ACTN1 can be directly bound to USP14-WT or USP14-C114A, but not to GST alone (Fig. 3D), indicating that DUB activity of USP14 is not required for its interaction with ACTN1. To verify the binding domains, we generated truncation mutants of Flag-USP14 and His-ACTN1 to narrow down their interaction sites (Fig. 3E, G). Results showed that the M4 region of USP14 (amino acids 352–428) and the M1 segment of ACTN1 (amino acids 31–245) were both necessary and sufficient for this interaction (Fig. 3F, H), confirming a robust direct association.

**Fig. 3: Interaction Between USP14 and ACTN1.**

USP14 deubiquitinates ACTN1 and specifically cleaves Lys27-, Lys48-, and Lys63-linked polyubiquitin chains

Considering USP14’s function as a deubiquitinase (DUB), we next examined its effect on ACTN1 ubiquitination. Loss of USP14 resulted in a significant increase in ACTN1 ubiquitination in LN229 and GBM2 cells (Fig. 4A). To further investigate, we co-expressed His-ACTN1 and HA-ubiquitin with either USP14-WT or USP14-C114A in U251 and GBM1 cells. Wild-type USP14 reduced ACTN1 ubiquitination, whereas the C114A mutant had no effect (Fig. 4B). In addition, we assessed endogenous ACTN1 ubiquitination in GBM cells: USP14 knockdown increased endogenous ACTN1 ubiquitination (Supplementary Fig. 3A), while overexpression of USP14-WT markedly decreased endogenous ACTN1 ubiquitination, with no change upon overexpression of USP14-C114A (Supplementary Fig. 3B). Previous studies suggest that ACTN1 can be ubiquitylated and targeted for degradation by the E3 ligase Cullin3. To determine if USP14 counteracts this activity, we overexpressed USP14, which largely reversed the ubiquitination induced by Cullin3 (Fig. 4C).

**Fig. 4: USP14 Deubiquitinates ACTN1, Targeting Lys27-, Lys48-, and Lys63-Linked Polyubiquitin Chains.**

In vitro ubiquitination assays confirmed that only wild-type USP14, and not the C114A variant, effectively removed ubiquitin from ACTN1 (Fig. 4D). We further assessed USP14’s ability to cleave different ubiquitin linkages—seven distinct types formed via specific lysine residues³² (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, Lys63). USP14 selectively cleaved Lys27-, Lys48-, and Lys63-linked polyubiquitin chains on ACTN1 (Fig. 4E), suggesting that USP14 targets these specific linkages in ACTN1 ubiquitination.

To further verify, we used ubiquitin mutants with single and combined lysine mutations (K27R, K48R, K63R, and a triple-mutant 3KR, which lacks all three Lys residues). While each single mutation (27R, 48R, 63R) reduced deubiquitination by USP14, the 3KR mutant completely abolished it (Fig. 4F, G). In an in vitro deubiquitination assay, USP14-WT efficiently removed the Lys27-, Lys48-, and Lys63-linked chains on ACTN1 (Fig. 4H, I; Supplementary Fig. 4A). Expressing of lysine-resistant ubiquitin variants (Lys27R, Lys48R, Lys63R) in USP14-depleted LN229 and GBM2 cells partially mitigated the USP14 loss–induced downregulation of ACTN1 (Supplementary Fig. 4C–H), whereas the triple lysine-resistant ubiquitin mutant (Lys27/48/63R) completely reversed the decrease in ACTN1 protein upon USP14 knockdown (Fig. 4J; Supplementary Fig. 4B). These findings indicate that USP14 targets the Lys27-, Lys48-, and Lys63-linked ubiquitin chains on ACTN1, and that these three linkages are critical for USP14-mediated stabilization of ACTN1. Collectively, our findings demonstrate that USP14 is essential for ACTN1 deubiquitination, selectively cleaving key ubiquitin linkages to stabilize the protein.

ACTN1 Mediates the Impact of USP14 on PMT in GBM

To further understand USP14’s role in glioblastoma, we examined its effects on mesenchymal (MES) subtype cells. MES cells (LN229, GBM2) showed higher USP14 expression compared to proneural (PN) cells (U251, GBM1) (Supplementary Fig. 5A). To explore if ACTN1 could counteract USP14 loss in MES cells, we performed knockdown experiments. Reduced USP14 expression led to decreased MES markers (YKL-40, MET, COL5A1) and an increase in PN markers (Olig2, PDGFRα), changes that were reversed by ACTN1 overexpression (Fig. 5A). Conversely, USP14 upregulation increased MES markers and reduced PN markers, which was counteracted by ACTN1 knockdown (Fig. 5B).

**Fig. 5: ACTN1-Dependent Role of USP14 in PMT in GBM.**

Our findings led us to hypothesize that ACTN1 mediates USP14’s effects on PMT. Transwell and CCK8 assays demonstrated that USP14 overexpression enhanced invasiveness, migration, and proliferation in U251 and GBM1 cells, effects that were nullified by ACTN1 knockdown (Fig. 5C; Supplementary Fig. 5B-D). Conversely, USP14 inhibition reduced the invasiveness, migration, and proliferation of LN229 and GBM2 cells, which was restored by ACTN1 overexpression (Fig. 5D–F). To verify this effect in vivo, we used an orthotopic xenograft model. Bioluminescence imaging revealed that tumors from USP14-depleted LN229 and GBM2 cells showed significantly slower growth compared to controls, but ACTN1 overexpression reversed this effect (Fig. 5G, H; Supplementary Fig. 5E). Kaplan-Meier survival analysis showed that mice injected with USP14-depleted cells had prolonged survival compared to controls; however, survival decreased significantly when ACTN1 was overexpressed in these cells (Fig. 5I). These results underscore ACTN1 as a mediator of USP14’s role in promoting PMT in GBM cells.

Pharmacological Inhibition of USP14 by IU1 Enhances ACTN1 Ubiquitination and Suppresses PMT

To further validate our findings on USP14, we used IU1, a small-molecule inhibitor of USP14’s catalytic activity, to assess its impact on ACTN1 stabilization. First, we confirmed IU1’s effectiveness in inhibiting USP14’s deubiquitinating activity. As shown in Fig. 6A, IU1 treatment markedly reduced USP14’s ability to remove ubiquitin from ACTN1. Accordingly, ACTN1 protein levels decreased in LN229 and GBM2 cells following IU1 treatment, with partial restoration by co-treatment with MG132 (Fig. 6B). Additionally, IU1 and CHX co-treatment significantly shortened ACTN1’s half-life (Fig. 6C), indicating that IU1, similar to USP14 depletion, promotes ACTN1 ubiquitination and proteasomal degradation.

**Fig. 6: IU1 Inhibition of USP14 Promotes ACTN1 Ubiquitination and Blocks PMT.**

IU1 treatment also impaired the invasive, migratory, and proliferative abilities of LN229 and GBM2 cells, effects that were counteracted by ACTN1 overexpression (Fig. 6D–F). Furthermore, IU1 treatment reduced ACTN1 levels and MES markers while increasing PN markers, with ACTN1 overexpression reversing these effects (Fig. 6G). In an in vivo model, mice with intracranial tumors derived from luciferase-labeled LN229 and GBM2 cells treated with 40 mg/kg IU1 exhibited reduced tumor growth compared to vehicle-treated controls; ACTN1 overexpression reversed this effect (Fig. 6H; Supplementary Fig. 6A, B). Kaplan-Meier survival curves demonstrated that IU1 treatment significantly extended survival compared to vehicle-treated mice, with survival benefits negated by ACTN1 overexpression (Fig. 6I). We also assessed whether combining IU1 treatment with ACTN1 knockdown confers additional benefit in vivo. In intracranial models using LN229 and GBM2 cells, IU1 treatment produced similar intracranial tumor progression dynamics and survival regardless of ACTN1 knockdown status (Supplementary Fig. 6C–F), likely because inhibition of USP14 activity by IU1 maintained ACTN1 at persistently low levels in tumor cells. Taken together, these results indicate that IU1-induced ACTN1 ubiquitination disrupts PMT and thereby limits GBM progression.

USP14 correlates positively with ACTN1 levels and links to poor prognosis in GBM patients

To assess the clinical significance of USP14 in relation to ACTN1, we evaluated USP14 and ACTN1 expression in PN and MES subtype GBM tissues. MES samples showed significantly higher USP14 and ACTN1 levels compared to primary PN tissues (Fig. 7A). Using IHC staining across 19 PN and 25 MES subtype GBM specimens, we confirmed that MES tumors predominantly expressed high levels of both USP14 and ACTN1 (Fig. 7B, C), with a strong positive correlation in expression levels (Fig. 7D–F). Kaplan-Meier survival analysis of 64 GBM samples showed that patients with elevated USP14 expression had significantly shorter overall and progression-free survival compared to those with low USP14 levels (Fig. 7G). Likewise, Kaplan-Meier analysis stratified by ACTN1 expression demonstrated that high ACTN1 expression corresponded with reduced overall and progression-free survival, indicating poorer outcomes for patients with elevated ACTN1 levels (Fig. 7H). Together, these findings underscore the importance of USP14 as a key deubiquitinating enzyme stabilizing ACTN1, which promotes PMT and negatively impacts prognosis in GBM through the USP14/ACTN1 axis.

**Fig. 7: Correlation of USP14 with ACTN1 and Poor Prognosis in MES GBM Patients.**

Discussion

Intratumor heterogeneity, characterized by diverse genotypes and cell subtypes within a single tumor, is influenced by intrinsic cellular factors, inflammatory cytokines, and therapeutic interventions such as radiotherapy, chemotherapy, and anti-angiogenesis therapy^33,34. This heterogeneity remains a significant barrier in developing effective treatment plans for glioblastoma (GBM). PMT, a process in GBM that mimics the epithelial-mesenchymal transition (EMT) seen in other cancers, endows GBM cells with enhanced aggressive traits linked to increased invasion, proliferation, recurrence, therapy resistance, and poor prognosis^10,35. Thus, deciphering the molecular mechanisms that drive PMT is crucial for identifying therapeutic targets and designing new GBM treatment strategies.

Previous studies have shown that ACTN1 expression in grade II/III gliomas correlates negatively with patient survival, indicating that targeting ACTN1 could potentially improve outcomes in these patients¹⁹. However, ACTN1’s specific expression patterns and functions in GBM remain unclear. Our results reveal that ACTN1 plays a pivotal role in driving the transition from PN to MES differentiation, which is associated with aggressive malignancy in GBM. We observed that ACTN1 expression is notably higher in MES cells (LN229, GBM2) than in PN cells (U251, GBM1), with MES cells expressing MES markers and PN cells expressing PN markers. Notably, ectopic expression of ACTN1 in PN cells induced MES phenotypes, while ACTN1 knockdown in MES cells led to a reduction in MES markers, an increase in PN markers, and significant inhibition of tumorigenic properties in both in vitro and in vivo models, underscoring ACTN1’s role in driving PMT. Collectively, our data implicate ACTN1 as a key contributor to the mesenchymal phenotype in GBM, although the downstream circuitry remains to be fully defined. As an actin-filament crosslinker, ACTN1 likely promotes mesenchymal traits by modulating cell morphology, motility, and focal-adhesion–associated signaling. Consistent with this, ACTN1 depletion reduced MES markers and increased PN markers, indicating that ACTN1 helps sustain a MES gene-expression program—likely as a downstream consequence of these signaling changes.

ACTN1 stability is known to be regulated by post-translational modifications (PTMs) like ubiquitination, with the E3-ubiquitin ligase Cullin3 mediating this process in conditions like nemaline myopathy²⁰. However, how ACTN1 stability is maintained in GBM remains unclear. The counter-process of ubiquitination, or deubiquitination, has yet to be fully explored for ACTN1 regulation. Through BioGRID database analysis³⁶, we identified USP14 as a novel deubiquitinase that stabilizes ACTN1. USP14 has emerged as a broadly oncogenic deubiquitinase across multiple tumor contexts. In gastric cancer, USP14 stabilizes the m6A reader IMP2, boosting CXCL2 secretion to drive resistance to anti–PD-1 therapy³⁷. In hepatocellular carcinoma, disrupting the AKT–USP14–TUBA1A axis promotes TUBA1A degradation and suppresses liver tumorigenesis in vivo³⁸. In triple-negative breast cancer, lovastatin disrupts the USP14–Survivin complex, increasing Survivin polyubiquitination; silencing or inhibiting USP14 with IU1 synergizes with lovastatin to curb tumor growth³⁹. In head and neck squamous cell carcinoma, USP14 bound to both RELA and IκBα and reduced IκBα K48-ubiquitination, leading to the degradation of IκBα, thereby activating NF-κB signaling⁴⁰. These studies reinforce a convergent theme: USP14 stabilizes pro-tumor substrates (e.g., IMP2, TUBA1A, Survivin) and pathways (e.g., NF-κB). By analogy, our GBM data identify ACTN1 as a USP14 substrate, suggesting that therapeutically targeting USP14 can destabilize ACTN1 and blunt mesenchymal progression in GBM.

Our study revealed that USP14 interacts with ACTN1 in GBM, regulating its protein levels without affecting mRNA levels, suggesting a post-translational mechanism. USP14 knockdown significantly reduced ACTN1 expression, downregulated MES markers, and increased PN markers, while USP14 overexpression elevated ACTN1 and MES marker levels. Depleting USP14 also substantially reduced GBM tumorigenicity and improved survival in mice models, effects that were reversed with ectopic ACTN1 expression. This strongly supports that USP14-mediated ACTN1 stabilization is crucial for maintaining MES traits and facilitating GBM progression.

DUBs like USP14 counteract E3-ubiquitin ligases by removing ubiquitin modifications from proteins⁴¹. Our findings showed that USP14’s role as a DUB requires its catalytic activity since a catalytically inactive mutant, USP14-C114A, failed to stabilize ACTN1. To confirm this mechanism, we explored whether USP14 deubiquitinates ACTN1. USP14 knockdown increased ACTN1 ubiquitination, while USP14-WT overexpression (but not USP14-C114A) reduced it. Among the seven polyubiquitin linkages, Lys48 and Lys63 are primarily associated with proteasomal degradation^42,43, and our data showed that USP14 directly cleaves Lys27-, Lys48-, and Lys63-linked chains from ACTN1. Utilizing IU1, a small-molecule inhibitor of USP14’s catalytic activity, we observed significant anti-tumor activity in GBM preclinical models, suggesting potential for USP14-targeted therapeutic development. Clinical data revealed a positive correlation between USP14 and ACTN1 levels in GBM patients, where elevated USP14 was linked to decreased patient survival, highlighting the USP14/ACTN1 axis as a potential prognostic marker in GBM. Our study identifies ACTN1 as a bona fide USP14 substrate that enforces mesenchymal programs in GBM and shows that pharmacologic DUB inhibition with IU1 provides proof of concept for the druggability of this axis. Complementing these findings, recent work in patient-derived GSCs demonstrates that USP14 also drives stemness and radioresistance by stabilizing ALKBH5, underscoring the breadth of USP14’s tumor-promoting roles in glioblastoma²⁹. Together, these observations nominate USP14—and its substrate circuitry—as a therapeutically actionable node in GBM, supporting the development of selective, brain-penetrant USP14 inhibitors and biomarker-guided strategies to target USP14-dependent tumors.

In summary, our findings reveal USP14 as a key enzyme that stabilizes ACTN1 through deubiquitination, thereby playing an essential role in the proneural-to-mesenchymal transition of glioblastoma. By maintaining mesenchymal characteristics, the USP14/ACTN1 axis promotes tumor growth and progression. This work not only enhances our understanding of PMT at the molecular level but also highlights USP14 inhibition as a promising therapeutic approach to potentially improve outcomes for patients with GBM.

Methods

Clinical samples and database

We obtained 10 normal brain tissues (NBTs) and 64 glioblastoma samples from the Department of Neurosurgery at the Affiliated Hospital of Guizhou Medical University. NBTs were derived from patients undergoing craniotomy decompression for traumatic brain injury. Glioblastoma samples were collected from patients at the time of surgery before chemo- and radiotherapy, and two neuropathologists identified the histological specimens independently according to the WHO criteria. Of the glioblastoma samples, 25 showed high expression of MES GBM markers (YKL-40, MET, COL5A1) and low expression of PN GBM markers (Olig2, PDGFRα)³¹. Clinicopathological details are provided in Supplementary Table 1. All ethical regulations relevant to human research participants were followed. All procedures for tumor collection and analysis were approved by the Guizhou Medical University Institutional Research Medical Ethics Committee, with informed consent obtained from all participants.

Cell culture

U87MG, U251, LN229, and T98G cells were purchased from the American Type Culture Collection (ATCC). Human embryonic kidney HEK293T cells were obtained from Bena Culture Collection Technology (China). The primary GBM cell lines were derived from GBM surgical specimens and maintained in primary serum-free cultures grown on laminin-coated plates. All cells were cultured in FBS (10%)- and antibiotics-contained Dulbecco’s modified Eagle’s medium (DMEM) at 37 °C. Normal human astrocytes (NHAs) were originally from Lonza and maintained per the manufacturer’s instructions. All cell lines were authenticated by STR profiles and tested for mycoplasma contamination every 2 months.

RNA extraction and qRT-PCR analysis

Following the manufacturer’s protocol, total RNA was extracted with TRIzol reagent (Invitrogen), and first-strand cDNA synthesis was conducted using PrimeScript RT Master Mix (TaKaRa). Real-time quantitative PCR was then carried out with SYBR Green (Applied Biosystems), adhering to the provided guidelines. The relative expression levels of USP14 and ACTN1 mRNAs were normalized to GAPDH and calculated using the 2^-ΔΔCt method. The following primers were used for qRT-PCR: USP14, 5’-TGGCTTCAGCGCAGTATATTAC-3’ (forward) and 5’-CCTTGTTCACCTTTCTCGGCA-3’ (reverse); ACTN1, 5’-TGAGGAGTGGTTGCTGAATGAG-3’ (forward) and 5’-AACTTCTCTGCCAGGTGGTCC-3’ (reverse).

Antibodies and reagents

The study utilized the following antibodies: USP14 (Proteintech, #14517-1-AP; WB 1:1000, IF 1:100), ACTN1 (Santa Cruz, #sc-17829; WB 1:500, IF 1:100), FLAG-tag (Abcam, #ab205606; WB 1:1000), His-tag (Cell Signaling Technology, #12698; WB 1:1000), HA-tag (Abcam, #ab9110; WB 1:1000), YKL-40 (Novus Biologicals, #AF2599; WB 1:1000), Olig2 (Santa Cruz, #sc-293163; WB 1:500), PDGFRα (Cell Signaling Technology, #3174; WB 1:1000), MET (Santa Cruz, #sc-8057; WB 1:200), COL5A1 (Cell Signaling Technology, #86903; WB 1:1000), and β-actin (Abcam, #ab8227; WB 1:5000). The USP14 inhibitor IU1 (#662210) and the protein synthesis inhibitor cycloheximide (CHX, #C7698) were obtained from Sigma-Aldrich, while MG132 (#S2619), a proteasome inhibitor, was sourced from Selleckchem.

Transfection

USP14, ACTN1, and control shRNAs were procured from Sigma–Aldrich, with targeting sequences detailed in Supplementary Table 2. Flag-tagged wild-type USP14 (USP14-WT), its catalytically inactive mutant (USP14-C114A), and His-tagged ACTN1 overexpression plasmids were custom-synthesized by Jikai Gene Biotechnology (Shanghai, China) and validated via DNA sequencing. Plasmids encoding various HA-tagged ubiquitin forms, including Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, and Lys63 (pRK5-HA series), were obtained from Addgene. Cells were seeded in 6-well plates and incubated for 24 hours to reach 70–80% confluency before transfection. Transfections utilized Lipofectamine 3000 (Invitrogen, cat. L3000150) in accordance with the manufacturer’s protocol, and stable cell lines were subsequently selected using puromycin.

Transwell invasion and migration assays

In the invasion assay, 2 × 10⁴ cells were seeded into the upper chambers of 24-well Transwell inserts with 8 µm pores (Corning, NY, USA) in 200 µL of serum-free medium. The lower chambers contained 500 µL of DMEM with 10% FBS to stimulate invasion. After 24 hours of incubation, cells were fixed with 4% paraformaldehyde for 15 minutes and stained with crystal violet. Six randomly selected fields were captured by microscopy for analysis. In the migration assay, filters were used without Matrigel pre-coating.

CCK-8 assay

The CCK-8 assay (Beyotime) was utilized to assess glioblastoma cell proliferation in line with the manufacturer’s protocol. Transfected cells were seeded into 96-well plates at a concentration of 5000 cells per well, and 10 µL of CCK-8 reagent was added at 24, 48, 72, and 96 hours. Cell viability was determined by measuring absorbance at 450 nm. Each experiment included three biological replicates, with measurements repeated in triplicate to ensure accuracy.

Immunoblotting (IB)

Immunoblot (IB) analysis was performed to analyze target protein expression. Cellular proteins were extracted using RIPA buffer with protease inhibitors (Roche), separated by SDS-PAGE, and transferred to PVDF membranes. The membranes were incubated overnight with primary antibodies, followed by HRP-conjugated secondary antibodies after washing. Signals were detected using a SuperSignal West Femto Maximum Sensitivity Substrate Trial Kit (Thermo Fisher Scientific).

Immunofluorescence (IF)

Cells were fixed in 4% paraformaldehyde for 20 minutes, permeabilized with 0.25% Triton X-100, and blocked with 1% BSA for 1 hour. Cells were then incubated overnight with primary antibodies, followed by washing with PBS-T and a 1-hour incubation with secondary antibodies. Nuclei were stained with DAPI, and images were captured using Zeiss LSM 5 confocal microscopy. Colocalization of USP14 and ACTN1 was quantified using the Manders overlap coefficient.

Immunohistochemistry (IHC)

Immunohistochemistry (IHC) was conducted following established protocols. Paraffin-embedded 4-µm tissue sections were baked at 60°C for 1 hour, deparaffinized in xylene, and rehydrated through graded ethanol solutions (100%, 95%, and 85%). Antigen retrieval was performed in pH 6.0 citric buffer for 20 minutes, followed by treatment with 3% hydrogen peroxide in PBS and blocking with goat serum. Sections were sequentially incubated with primary and HRP-conjugated secondary antibodies, developed with DAB chromogen (Vector Laboratories), and counterstained with hematoxylin. Staining intensity for USP14 and ACTN1 was scored from 0 to 3 under ×40 magnification. For analysis of protein expression and survival in the clinical GBM cohort, cases were dichotomized by the cohort median of the per-sample IHC intensity score: High = above the median; Low = at or below the median.

Protein half-life assay

For the ACTN1 half-life assay, GBM cells were treated with the protein synthesis inhibitor cycloheximide (CHX, Sigma-Aldrich, 100 μM) for specified time intervals before sample collection.

Co-immunoprecipitation

Cells transfected with the specified constructs were lysed in NETN buffer containing protease inhibitors. Cell lysates were pre-cleared with protein A/G agarose (Santa Cruz) at 4 °C for 1 hour, then incubated with the target antibodies overnight at 4 °C for immunoprecipitation. The resulting immunocomplexes were bound to protein A/G agarose for an additional 2 hours at 4 °C, washed four times with NETN buffer, and boiled to elute proteins. The samples were then analyzed by SDS-PAGE and IB.

GST pulldown assay

To test direct binding, GST pull-downs were performed exclusively with purified proteins. Full-length human ACTN1 was cloned into pGEX-4T-1, expressed in E. coli BL21 (DE3) at 16 °C with 0.4 mM isopropyl β-d-1-thiogalactopyranoside (IPTG), and purified on glutathione Sepharose 4B followed by gel filtration (Superdex 200) in binding buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM DTT, 5% glycerol, 0.05% NP-40). Purification followed manufacturer protocols for GST-tagged proteins.

Human USP14 (WT or catalytic inactive mutant C114A) bearing an N-terminal Flag tag was expressed in E. coli BL21 (DE3) under the same induction conditions and purified on ANTI-FLAG® M2 affinity gel with competitive elution using 3×Flag peptide, then polished by gel filtration in the same buffer.

For pull-downs, 5 µg GST-ACTN1 (or GST control) was immobilized on 20 µL glutathione beads pre-blocked with 1% BSA (30 min, 4 °C), washed 3× with binding buffer, and incubated with 1–2 µg purified Flag-USP14 (WT or C114A) in 200 µL binding buffer for 1–2 h at 4 °C with rotation. Beads were washed 4–5 times (including two high-salt washes at 300 mM NaCl) and boiled in 2× SDS sample buffer. Bound USP14 was detected by SDS–PAGE/IB with anti-Flag; equal bait loading was verified by Coomassie staining.

In vivo and in vitro ACTN1 ubiquitylation assay

For the in vivo ACTN1 ubiquitylation assay, cells were transfected with the indicated plasmids, then treated with 20 μM MG132 for 8 hours. Cells were collected and lysed in NETN buffer plus 0.1% SDS, 20 μM MG132, and protease inhibitors. Lysates were incubated with anti-ACTN1 antibody for 3 hours and protein A/G agarose beads for a further 8 hours at 4 °C. Reactions were subjected to IB analysis.

For the in vitro ACTN1 ubiquitylation assay, HEK293T cells were transfected with His-ACTN1 and HA-ubiquitin, HA-ubiquitin-Lys27, HA-ubiquitin-Lys48, or HA-ubiquitin-Lys63, and were treated with 20 μM MG132 for 8 hours. Ubiquitylated ACTN1 was purified from the cell extracts with an anti-His affinity column and then incubated with the recombinant GST-USP14 WT or GST-USP14 C114A protein in a deubiquitylation buffer for 2 hours at 37 °C. Reactions were subjected to IB analysis.

In vivo xenografts

For intracranial xenograft studies, male BALB/c-A nude mice aged 5 to 6 weeks were randomly assigned to experimental groups (n = 10 per group). Using a stereotactic apparatus, 5 × 10⁵ luciferase-tagged LN229 or GBM2 cells—modified as required—were injected into the right caudate putamen. Tumor initiation and growth were monitored via bioluminescence imaging (IVIS 200 Spectrum Imaging System) on days 0, 7, 14, and 28 post-implantation. For survival analysis, mice were observed up to 70 days or until severe symptoms (e.g., weight loss, impaired movement) manifested.

To assess the effects of the USP14 inhibitor IU1, ALZET micro-osmotic pumps (DURECT Corp.) were surgically implanted in tumor-bearing mice, administering IU1 (40 mg/kg) or a vehicle at a constant rate of 0.5 μL/h over seven days. Tumor progression was continuously monitored with in vivo bioluminescent imaging, with the study endpoint set at 70 days or upon the development of significant clinical signs.

Sample coding was applied to maintain blinding for the experimenters during analysis. We have complied with all relevant ethical regulations for animal use. All animal procedures adhered to national guidelines and received approval from the Guizhou Medical University Institutional Committee for Animal Research.

Statistics and reproducibility

Data are reported as mean ± s.e.m. Sample sizes and replicates were determined via G*Power based on effect size and Type I/II error levels. Normality and variance were assessed using the Shapiro–Wilk and F tests. For data with normal distribution, Student’s t-test (two groups) or one/two-way ANOVA (> two groups) was applied; otherwise, the Mann-Whitney U test was used. Outliers (>3 standard deviations from the mean) were excluded. Pearson correlation analyzed the relationship between USP14 and ACTN1 expression, and Kaplan-Meier analysis was used for survival in GBM patients and mouse models. GraphPad Prism 9 performed all analyses, with p < 0.05 as the significance threshold.

Ethics approval and consent to participate

All animal procedures adhered to national guidelines and received approval from the Guizhou Medical University Institutional Committee for Animal Research (no. 2302336). All procedures for tumor collection and analysis were approved by the Guizhou Medical University Institutional Research Medical Ethics Committee, with informed consent obtained from all participants.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

Numerical source data for all figures are provided as Supplementary Data 1 (Excel); all other data are available from the corresponding author upon reasonable request.

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Acknowledgements

This work was funded by the following grants: Project of Science and Technology Foundation of Guizhou Health Commission (Grant No. gzwkj [2022-090] and Grant No. gzwkj [2024-146]); Guizhou Provincial Science and Technology Projects (Grant No. QianKeHe Foundation-ZK [2024] General-244); Doctoral Research Launch Foundation, the Affiliated Hospital of Guizhou Medical University (Grant No. gyfybsky-2024-16). We extend our sincere appreciation to Dr. Xiaomin Cai from Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine for graciously supporting us.

Author information

These authors contributed equally: Wenjin Qiu, Xueping Shi, Ruting Wei.
These authors jointly supervised this work: Peng Luo, Liangzhao Chu.

Authors and Affiliations

Department of Neurosurgery, The Affiliated Hospital of Guizhou Medical University, Guiyang, China
Wenjin Qiu, Xueping Shi, Ruting Wei, Jiaquan Song, Yuanguo Ling, Shibin Song, Yimin Chen, Hua Yang & Liangzhao Chu
The Key Laboratory of Environmental Pollution Monitoring and Disease Control of Ministry of Education, School of Public Health, Guizhou Medical University, Guiyang, China
Wenjin Qiu & Peng Luo
School of Clinical Medicine, Guizhou Medical University, Guiyang, China
Yunjia Hu

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Contributions

W.J.Q., P.L., and L.Z.C. conceptualized and designed the study. W.J.Q. X.P.S. and R.T.W. carried out the majority of the experiments. Y.J.H., S.B.S., and Y.M.C. gathered the clinical samples. J.Q.S., Y.G.L., and L.Z.C. conducted data analysis and interpretation. W.J.Q. and X.P.S. authored the manuscript, with H.Y., P.L., and L.Z.C. providing critical review and revisions. All authors read and approved the final manuscript.

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Correspondence to Peng Luo or Liangzhao Chu.

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Qiu, W., Shi, X., Wei, R. et al. USP14-mediated stabilization of ACTN1 maintains mesenchymal characteristics in glioblastoma. Commun Biol 8, 1680 (2025). https://doi.org/10.1038/s42003-025-09083-8

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Received: 29 March 2025
Accepted: 17 October 2025
Published: 25 November 2025
Version of record: 25 November 2025
DOI: https://doi.org/10.1038/s42003-025-09083-8