Inhibition of Mesenchymal Phenotypes in Glioblastoma by Combined Targeting of NF-κB and STAT3 Pathways

Inhibition of Mesenchymal Phenotypes in Glioblastoma by Combined Targeting of NF-κB and STAT3 Pathways | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Inhibition of Mesenchymal Phenotypes in Glioblastoma by Combined Targeting of NF-κB and STAT3 Pathways Xiao Ren, Jiabo Li, Lei Chen, Xuya Wang, Jinhao Zhang, Yiming Zhang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5305574/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Glioblastoma, IDH-wildtype (GBM, WHO grade 4) is the most common and lethal type of brain cancer that is hard to cure. Among the 3 subtypes of GBM, the mesenchymal GBM is characterized by therapeutic resistance and poor outcomes. Here, we found that both STAT3 and NF-κB pathways are abnormally activated in mesenchymal GBM and the patients with higher expression of STAT3 and NF-κB had a poor prognosis in TCGA database. Using the STAT3 inhibitor, Stattic, to suppress the STAT3 signaling in GBM cells. However, it was observed that Stattic alone leads to compensatory activation of the NF-κB signaling. Therefore, we hypothesized that combined inhibition of STAT3 and NF-κB pathways may has a better anti-mesenchymal GBM effect than single signaling inhibition. ACT001, a novel NF-κB inhibitor, combined with Stattic has a synergistic anti-GBM effect, effectively inhibiting GBM proliferation, invasion, migration and promoting apoptosis. RNA-seq analysis showed that combined inhibition of the STAT3 and NF-κB pathways resulted in better suppression of downstream gene PLK4 expression compared to the inhibition of either pathway alone. Overexpression of PLK4 was found to enhance GBM cell proliferation, invasion and migration, while reducing apoptosis. Taken together, these findings suggest that combined targeting of NF-κB and STAT3 signaling pathways, by acting on PLK4, suppresses proliferation, invasion and migration, as well as promotes apoptosis in the mesenchymal subtype of GBM cells, offering a novel therapeutic strategy for mesenchymal GBM. glioblastoma mesenchymal phenotype NF-κB signaling STAT3 signaling ACT001 Stattic Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Glioblastoma, IDH-wildtype (GBM, WHO grade 4), is the most lethal and aggressive primary brain tumor in adults, making total resection nearly impossible[ 1 ] [ 2 ]. Despite standardized treatments for GBM, such as surgical resection, postoperative concurrent chemoradiotherapy and sequential chemotherapy, the mean survival time of GBM patients remains less than 15 months[ 3 ], with a five-year survival rate of only 7%[ 1 ]. Therefore, it is extremely urgent to further explore the survival characteristics and pathogenesis of GBM to address the disease suffering of patients. Signal transducer and activator of transcription protein 3 (STAT3) is a transcription factor phosphorylated by JAK kinases in response to cytokine activation, allowing it to dimerize and move into the nucleus to activate the transcription of cytokine-responsive genes[ 4 ] [ 5 ] [ 6 ]. As a member of the STAT family, STAT3 plays crucial roles in various cellular processes, including proliferation, survival, invasiveness[ 7 ] [ 8 ]. Our previous studies have highlighted that abnormal STAT3 activation is particularly prominent in tumor cells, especially in mesenchymal GBM, contributing to tumor growth, invasion, and immune-escape[ 9 ]. However, up to now, no drugs targeting the STAT3 signaling for GBM treatment have been approved in clinical trials. On the one hand, STAT3 is expressed in multiple systems complicates efforts to avoid side effects from broad inhibition[ 10 ]. On the other hand, GBMs exhibit abnormal activation of multiple signaling pathways, and bypass activation means inhibiting STAT3 alone cannot effectively halt tumor progression[ 11 ]. Therefore, it is necessary to further study the activation and feedback mechanism of STAT3 pathway in GBM and design a more reasonable treatment strategy. NF-κB pathway is another critical pathway mediating the transformation of GBM into mesenchymal subtype[ 12 ] [ 13 ] [ 14 ]. ACT001 (dimethylamino micheliolide, DMAMCL), a chemically improved product derived from parthenolide[ 15 ], which is a traditional inhibitor of NF-κB, has been certified as an orphan drug for recurrent GBMs by the Food and Drug Administration (FDA) in the United States and by the European Union. ACT001 is currently undergoing phase II clinical trials for recurrent GBM in China, Australia and the United States[ 16 ]. Our previous experiments have shown that ACT001 exerts a strong inhibitory effect on NF-κB signaling[ 17 ]. In this study, we found that Stattic increased the expression of NF-κB pathway transcription factor phosphorylated p65 while inhibiting the phosphorylation of STAT3 dimer into the nucleus. Therefore, we hypothesize that combined inhibition of NF-κB and STAT3 pathways can synergistically treat GBM and reverse the phenotypic transformation of mesenchymal GBM through PLK4. Combined Stattic and ACT001 reduce tumor growth in tumor-bearing mice and provide overall survival benefits. In short, compared with single drug treatment, combination therapy shows a better therapeutic prospect, paving the way for novel interventions of GBM. Materials and methods Materials Human GBM cell lines U251MG and SNB19 were obtained from Shanghai Institute of Biochemistry and Cell Biology. Stattic was purchased from Selleck Chemicals. ACT001 was provided by Accendatech Co., Ltd. (Tianjin, China). Cell culture and transfection of siRNAs Dulbecco’s modified Eagle’s medium (DMEM; Gibco USA) supplement with 10% fetal bovine serum (FBS; Gibco, USA) was used for cell culture. The cells were cultured in incubator containing 5% CO2 at 37℃. PLK4 siRNA and the negative control siRNA were purchased from GenPharma Pharmaceutical Technology Co., Ltd. The cells were seeded into 6-well plates (Corning, America) at a concentration of 1×10 5 cells per well and grown overnight before transfection. Cell counting kit-8 assay (CCK8) Cell viability with Stattic and ACT001 combined treatment was estimated using CCK8 (CK04, DOJINDO, Beijing, China), according to the manufacturer’s manual. Cells (2×10 3 cells per well) were seeded in 96-well plates and incubated for 24h, and then were treated with Stattic and ACT001 for 24, 48 or 72h. Next, the cells were incubated with CCK8 solution for 1h at room temperature, and absorbance was measured at 450 nm by using a microplate luminometer (BioTek, USA) Drug synergy assessment Cell viability was measured using the CCK8 assay. The cells were exposed to different concentrations of Stattic (0, 0.19, 0.38, 0.75, 1.5 and 3.0 µM) and ACT001 (0, 5, 10, 20 and 40 µM) and combinations of both drugs for 72 h. Synergy was assessed using the Bliss independence model as described in the literature[ 18 ]. Colony formation assay U251MG and SNB19 cell lines were seeded and cultured into 6-well plate at a density of 5×10 2 cells/well in DMEM supplement with 10% FBS for 24 hours. Then, the cells were treated with Stattic and ACT001, respectively. After the treatment, these disposed cells were then re‐suspended in DMEM containing 10% FBS and cultured in 5% CO 2 , 37℃ for 15 days to allow colony formation. The plate was washed with cold PBS. The colonies were fixed by 4% polyformaldehyde for 10 min at room temperature. Next, the colonies were dyed with 1% crystal violet for 5 minutes at room temperature. The colonies more than 100 cells were counted by microscope (Leica Microsystems, Germany). Each experiment was done thrice independently in this study. Colony formation rate = the number of each treatment/the number of control × 100%. Wound healing assays Cell migration was detected and evaluated by wounding healing (scratch) assays. Briefly, the cells were plated in 6-well plates (1.5×10 5 cells/plate) and incubated to generate confluent cultures. Scrapes were made with a 1000-µl sterile pipette tip, and the cells were washed with PBS and then treated with Stattic (2.0 µM), ACT001 (30 µM) or a combination of the two drugs for 48 h. For the ACT001 (10 µM)/siRNA PLK4 group, after the 24 h siRNA PLK4 transfection, scrapes were made, and the cells were treated with or without ACT001 for another 48 h. Finally, photos of cell migration were taken with a microscope. Transwell assays Migration assays were carried out using 24-well plates (Corning, America) and 8 µm transwell chambers (Corning, America). 10,000 cells in 300 µl of serum-free medium containing ACT001 (10 µM), Stattic (0.75 µM) or a combination of the two drugs were added to the top chamber. The lower chambers were filled with 500 µl of DMEM medium with 10% FBS. After being incubated in an incubator for 24 h, the filter membranes in the chambers were removed and washed with PBS three times to remove the medium. Then, the cells that crossed to the underside of the filter membrane were fixed with 4% polyformaldehyde for 10 min, stained with 0.1% crystal violet for 5 min, and counted under a microscope. Invasion assays were also carried out using 24-well plates (Corning, America) and 8 µm transwell chambers (Corning, America). Matrigel (Matrigel: medium, 1:4) was added to the refrigerator and incubated at 4°C for 1 h and then placed in an incubator at 37°C for 2 hours. After the Matrigel solidified, 30,000 cells in 300 µl of serum-free medium containing ACT001 (10 µM), Stattic (0.75 µM) or a combination of the two drugs were added to the top chamber. For the ACT001 (10 µM)/siRNA PLK4 group, after the 24 h siRNA PLK4 transfection, the cells were collected and suspended in serum-free medium (2.5×10 5 /ml) with or without ACT001. Then, 200 µl of cells were added to the top well. The lower chambers were filled with 500 µl of medium with 10% FBS. After being incubated in an incubator for 24 h, the filter membranes in the chambers were removed and washed with PBS three times to remove the medium and Matrigel. Then, the cells that crossed to the underside of the filter membrane were fixed with 4% polyformaldehyde for 30 min, stained with 0.1% crystal violet for 5min, and counted under a microscope. Western blotting analysis Western blotting was performed rely on relevant protocol[ 19 ]. Proteins were transferred onto PVDF membranes (ThermoFisher, USA), and incubated overnight in the 4 ◦ C fridge with the following primary antibodies: STAT3 (Cell Signaling, USA, 1:1000), phospho-STAT3 (Tyr705) (Cell Signaling, USA, 1:1000), NF-κB p65 (Cell Signaling, USA, 1:1000), phosphp-NF-κB p65 (Cell Signaling, USA, 1:1000), N-Cadherin (Cell Signaling, USA, 1:1000), Vimentin (Cell Signaling, USA, 1:1000), Bcl-2 (Cell Signaling, USA, 1:1000), Caspase3 (Cell Signaling, USA, 1:1000), PLK4 (Cell Signaling, USA, 1:1000), β-action (ZSGB-Bio, China, 1:3000). Secondary antibodies: HRP labeled goat anti-rabbit/mouse IgG (ZSGB-Bio, China, 1:3000). Chemiluminescent HRP substrate (Millipore, USA) and GBOX system (Syngene Company, UK) were used to detect protein expression. Implantation and oral administration of Stattic and ACT001 All animal experiments were approved by the Ethical Committee of the Tianjin Medical University General Hospital. In vivo experiments were performed using nude mice (6 weeks old). Intracranial tumors were established by stereotactically implanting 7×10 5 U251MG cells. Tumor burden was monitored by luciferase imaging every week starting on day 7 after implantation, and the nude mice were randomly divided into the following four groups (7 mice/group): (1) Control; (2) 200 mg/kg ACT001; (3) 5 mg/kg Stattic; and (4) 200 mg/kg ACT001 and 5 mg/kg Stattic. ACT001 was orally administered daily for 21 days. Stattic was dissolved in DMSO, diluted with Control (the concentration of DMSO was 1%), and orally administered 5 times every 7 days for 15 days. Overall survival of nude mice in all groups was monitored. Luciferin signal was detected with the in vivo imaging system (IVIS) every week. The brains of the mice were carefully extracted when mice died or on day 56. These brains were fixed in 4% polyformaldehyde and embedded in paraffin for IHC staining. Statistical analysis All experiments were performed at least three times and all quantitative data are presented as mean ± SD. Statistical analysis was performed using SPSS 20. An unpaired t-test was used to compare the means of two groups, and a two-tailed p value of < 0.05 was considered statistically significant. Results 1. Single-drug Stattic downregulates STAT3 signaling and compensatorily activates the NF-κB signaling The TCGA_Seq and TCGA_Microarray datasets were analyzed to assess the expression of STAT3 in patients with different subtypes of GBM. Both datasets indicated significantly higher STAT3 transcription levels in the mesenchymal subtype compared to the classical and proneural subtypes (Fig. 1 A-B). Kaplan-Meier (K-M) survival curves from these datasets revealed that glioma patients with high STAT3 expression exhibited significantly shorter survival times compared to those with low STAT3 expression (Fig. 1 C). These findings indicate that high STAT3 expression is associated with poor survival in glioma patients. Due to the poor efficacy of Stattic monotherapy in the treatment of GBM[ 20 ], we conducted RNA-seq analysis on Stattic-treated U251 MG cells. Compared with the control group, Stattic inhibited STAT3 dimer phosphorylation but increased NF-κB pathway transcription factor phosphorylated p65 (Fig. 1 D). Gene Set Enrichment Analysis (GSEA) and KEGG enrichment revealed significant NF-κB pathway enrichment in the Stattic treatment group (Fig. 1 E-F). To further verify the accuracy of the sequencing results, Western blotting with four Stattic concentrations (0, 0.75, 1.5, and 3.0 µM) demonstrated that STAT3 protein and phosphorylated STAT3 levels decreased with higher Stattic concentrations, while phospho-p65 levels increased and total p65 levels remained unchanged (Fig. 1 G-H). These results suggest that Stattic inhibits the expression and phosphorylation of STAT3 while inducing compensatory activation of the NF-κB pathway, indicating potential synergy in combined STAT3 and NF-κB inhibition for GBM treatment. 2. ACT001 combined with Stattic exhibits synergistic anti-mesenchymal GBM effects Furthermore, we hypothesize that ACT001 and Stattic may exhibit synergistic anti-tumor effects in mesenchymal GBM. Initially, we treated U251MG and SNB19 cells with ACT001 and Stattic separately. The IC50 values for ACT001 and Stattic in U251MG cells were 20.23 µmol/L and 1.11 µmol/L, respectively, while in SNB19 cells, they were 19.90 µmol/L and 0.74 µmol/L, respectively (Fig. 2 A). Next, we performed CCK8 assays to determine the concentrations of combined ACT001 and Stattic treatment and assessed the synergistic effect using the Bliss independence model. In U251MG cells, the strongest synergistic effect was observed at 10 µmol/L ACT001 and 0.75 µmol/L Stattic. In SNB19 cells, significant synergy was noted at 10 µmol/L ACT001 and 1.5 µmol/L Stattic (Fig. 2 B). Based on the IC50 values, we selected 10 µmol/L ACT001 and 1 µmol/L Stattic for further experiments. A CCK8 proliferation assay was conducted to evaluate the treatment of mesenchymal GBM with the dual drug combination. Measurements over 5 days indicated that the proliferation rate in the combination treatment group was the lowest compared to the control and single-drug groups (Fig. 2 C). A colony formation assay after 15 days of treatment revealed that the combined treatment group had the poorest ability to form clusters (Fig. 2 D-E). 3. Combination therapy inhibits migration, invasion, and EMT in mesenchymal GBM cells The effect of cell migration was assessed using a Wound healing assay. Compared to the control group, the combined treatment group exhibited the shortest cell crawling distance, followed by the single drug treatment group (Fig. 3 A-B). These results suggest that the migration ability of GBM cells in the dual drug treatment group was significantly weaker than in the single drug and control groups. Transwell experiment further confirmed that the combined treatment group had the fewest cells passing through the chamber, showing a stronger inhibitory effect on migration and invasion of mesenchymal GBM cells (Fig. 3 C-D). To further investigate the impact of combination therapy on the phenotype of GBM, Western blotting was used to analyze the protein levels of N-Cadherin, E-Cadherin, and Vimentin. The results demonstrated that the combined treatment group exhibited the lowest protein levels of N-Cadherin and Vimentin, while E-Cadherin levels were the highest (Fig. 3 E-F), suggesting that combination therapy may effectively inhibit cellular EMT. Additionally, the combined treatment group had the lowest protein levels of Bcl-2 and Caspase3 compared to other groups (Fig. 3 E-F), indicating that combination therapy may promotes cell apoptosis. 4. Combination of ACT001 and Stattic inhibits proliferation of GBM cells through targeting PLK4 To further explore the mechanism underlying the combination of ACT001 and Stattic, RNA-seq analysis was performed on the control group, single drug control groups, and combined treatment group. The results indicated that PLK4 was down-regulated in ACT001-treated group, Stattic-treated group, and particularly in the combined treatment group in both U251MG and SNB19 cells (Fig. 4 A). Western blotting revealed that PLK4 protein expression was significantly lower in the combined treatment group compared to control or single-drug groups (Fig. 3 E-F). These findings suggest that the NF-κB and STAT3 pathways jointly regulate PLK4 expression. Next, the effects of PLK4 knockdown and overexpression on GBM phenotypes were examined. In the PLK4 knockdown group, using sequences sh1 and sh2 for validation, CCK8 proliferation assays and colony formation assays were conducted. Results revealed that cell proliferation activity decreased in the PLK4 knockdown group and increased in the overexpression group compared to the PLK4 empty vector group, indicating that PLK4 expression significantly impacts GBM cell proliferation (Fig. 4 B-D). These findings suggest that the combination of ACT001 and Stattic shows a synergistic effect in inhibiting the growth of mesenchymal GBM cells through targeting PLK4. 5. Knock-down of PLK4 inhibit migration, invasion, EMT and promote apoptosis of GBM cells In order to explore the efforts of PLK4 on GBM migration and invasion. Wound healing assay showed that the crawling distance of cells in the PLK4 knockdown group was significantly shorter compared to the corresponding PLK4 empty vector group, while the overexpression group showed increased crawling distances (Fig. 5 A-B). In the Transwell assay, fewer cells passed through the chamber in the PLK4 knockdown group, and more in the overexpression group (Fig. 5 C-D), indicating that PLK4 levels significantly affect GBM migration and invasion capabilities. Western blotting validated that PLK4 knockdown group significantly reduced the expression of N-Cadherin, Vimentin, Caspase3, and Bcl-2 protein levels, while increased the expression of E-Cadherin level, compared to the untreated and corresponding PLK4 empty vector groups. Conversely, overexpression of PLK4 group had the opposite effects (Fig. 5 E-F). Thus, PLK4 knockdown may inhibit EMT process and promote apoptosis in GBM cells. In conclusion, these findings indicate that knockdown of PLK4 inhibits proliferation, migration, invasion, and EMT process, while promote apoptosis in GBM cells. 6. Combination of ACT001 and Stattic enhances anti-tumor effect. From our in vitro experiments, we found that the combination of ACT001 and Stattic inhibits the proliferation, invasion, migration, and EMT of GBM cells by reducing PLK4 expression, showing superior efficacy compared to single-drug treatments. We then evaluated the combined treatment's effects on tumor-bearing mice (Fig. 6 A). After 28 days of treatment, the fluorescence intensity in the brain tumors of the combination treatment group did not significantly increase. In contrast, the single-drug groups showed a slightly increased, and the control group exhibited a significantly increased (Fig. 6 B-C). Survival analysis over 56 days revealed that all 7 mice in the control group died, 5 mice in the ACT001 treatment group, 6 mice in the Stattic treatment group, and 3 mice in the combined treatment group, indicating a longer survival period for the combination treatment group (Fig. 6 D). Immunohistochemical analysis of paraffin-embedded mouse brain tissue sections revealed the highest expression of p-P65 in the Stattic group, followed by the control group, and the lowest in the combined treatment group. For p-STAT3 and PLK4, the control group had the highest expression, followed by the single-drug groups, with the combined treatment group showing the lowest expression (Fig. 6 E), consistent with the in vitro results. Additionally, H&E staining of the mouse brain tissue showed no significant morphological differences in the main organs (including the heart, liver, spleen, lungs, and kidneys) among all groups (Fig. 6 F). Importantly, no physiological or behavioral differences were observed among all the groups in our experiments. These results suggest that the combination of ACT001 and Stattic offers a more effective treatment approach for GBM, enhancing survival and reducing tumor progression without significant adverse effects. Discussion The STAT3 pathway is a critical signaling pathway abnormally activated in mesenchymal glioblastoma[ 21 ] [ 22 ], as confirmed by both the TCGA and CGGA databases. However, inhibiting the STAT3 pathway alone is insufficient for effective GBM treatment and may even induce resistance, leading to GBM recurrence[ 23 ]. To investigate the causes of resistance following STAT3 pathway inhibition, we used the small molecule inhibitor Stattic[ 24 ] for RNA-seq analysis. The results showed that in U251MG cells treated with Stattic, the downregulation of phosphorylated STAT3 was accompanied by an upregulation of phosphorylated p65 in the NF-κB pathway. This suggests that the upregulation of the NF-κB pathway following STAT3 pathway inhibition is a major cause of glioblastoma recurrence, highlighting the need for a combined strategy to inhibit both the STAT3 and NF-κB pathways. Although dual and multi-drug combination therapies are widely used in treating various diseases[ 25 ], such as hypertension[ 26 ] and viral infections[ 27 ], their application of dual-drug therapy in tumors, particularly GBM, remains relatively rare. Challenges include the inability of conventional drugs to cross the blood-brain barrier[ 28 ], poor specificity for disease targets, high doses leading to resistance[ 29 ], the temporal and spatial heterogeneity of tumors[ 30 ], difficulty in establishing animal models[ 31 ], and complex signaling pathways[ 32 ]. In our study, alongside Stattic, which targets the STAT3 pathway, we incorporated a novel drug, ACT001, specifically targeting the NF-κB pathway. ACT001 derived from the root bark of Magnolia delavayi Franch, was chemically modified to enhance water solubility and oral bioavailability to 75%. Like its precursor parthenolide[ 33 ], ACT001 significantly inhibits the NF-κB pathway[ 34 ]. The combination therapy of Stattic and ACT001 demonstrated a significant synergistic effect in the GBM U251MG cell line. In phenotypic experiments, combination therapy showed markedly better results compared to monotherapy and control groups. Thus, we believe that combining of Stattic and ACT001, targeting the STAT3 and NF-κB pathways respectively, holds promising potential for GBM treatment. This study provides valuable insight for tumor treatment research, demonstrating that dual-drug combination therapy, along with enhancing the efficacy of the novel drug ACT001, can achieve better therapeutic outcomes for GBM. PLK4, a crucial regulator of centriole replication during normal cell mitosis, can promote cell proliferation[ 35 ], activate invasion and migration[ 36 ], inhibit apoptosis[ 37 ], enhance tumor inflammatory infiltration, facilitate immune evasion, and promote angiogenesis in the tumor microenvironment[ 38 ], all closely associated with poor clinical prognosis[ 39 ]. Consequently, PLK4 has garnered widespread attention as a biomarker and therapeutic target for cancer[ 40 ]. Both RNA-seq data and our experimental validation indicate that the NF-κB and STAT3 pathways independently regulate the PLK4 gene. Simultaneous inhibition of these pathways produces a synergistic effect in suppressing PLK4 gene expression. Therefore, dual inhibition not only effectively blocks their downstream signaling but also further enhances therapeutic efficacy by inhibiting PLK4. Stattic, as a small molecule inhibitor of STAT3, has never been used clinically. likely due to its poor efficacy as monotherapy for GBM[ 41 ] [ 42 ], high recurrence rates, and intrinsic properties such as poor water solubility and limited blood-brain barrier penetration, ACT001, a novel inhibitor of the NF-κB pathway, also inhibits the STAT3[ 16 ] and PI3K-AKT pathways[ 15 ], limiting its specificity. Future research should aim to identify more effective STAT3 inhibitors with fewer side effects and better blood-brain barrier permeability. Additionally, exploring combination therapies with other treatments, such as immunotherapy[ 43 ], may enhance therapeutic outcomes. Further investigation into the specific mechanisms of PLK4 in GBM is needed to develop more precise targeted therapy strategies. In conclusion, this study successfully inhibited the mesenchymal phenotype of GBM cells by dual targeting of the NF-κB and STAT3 pathways and suppressing PLK4 (Fig. 6 G). This finding provides new insights and potential therapeutic strategies for GBM treatment. Although further research is necessary to validate its clinical value, this approach holds promise for offering new hope to GBM patients. Declarations Competing Interest Statement: The authors declare no competing interests. Fundings This work was supported by the National Natural Science Foundation of China (No.82373151); Beijing Natural Science Foundation (No.7232228); Beijing-Tianjin-Hebei Special Project (19JCZDJC64200(Z)); the Institute for Intelligent Healthcare, Tsinghua University (No. 2022ZLB007) and Tianjin Science and Technology Project (No.21JCYBJC00800). Author Contribution Xiao Ren: Data analysis ann Writing.Jiabo Li and Lei Chen: Data Curation and Formal analysis.Xuya Wang: Visualization.Jinhao Zhang: Methodology.Yiming Zhang: Resources.Jikang Fan and Debo Yun: Validation.Chen Zhang: Conceptualization.Shengping Yu: Supervision. Acknowledgement We thank CGGA and TCGA. We were also grateful to the online tool suppliers, including R and R studio. References Ostrom QT, Cioffi G, Waite K et al (2021) CBTRUS Statistical Report: Primary Brain and Other Central Nervous System Tumors Diagnosed in the United States in 2014–2018. Neurooncology 23:iii1–iii105. https://doi.org/10.1093/neuonc/noab200 Bikfalvi A, da Costa CA, Avril T et al (2023) Challenges in glioblastoma research: focus on the tumor microenvironment. Trends Cancer 9:9–27. https://doi.org/10.1016/j.trecan.2022.09.005 Alexander BM, Cloughesy TF (2017) Adult Glioblastoma. JCO 35:2402–2409. https://doi.org/10.1200/JCO.2017.73.0119 Zou S, Tong Q, Liu B et al (2020) Targeting STAT3 in Cancer Immunotherapy. Mol Cancer 19:145. https://doi.org/10.1186/s12943-020-01258-7 Bromberg JF, Wrzeszczynska MH, Devgan G et al (1999) Stat3 as an Oncogene. Cell 98:295–303. https://doi.org/10.1016/S0092-8674(00)81959-5 Schindler C, Darnell JE (1995) TRANSCRIPTIONAL RESPONSES TO POLYPEPTIDE LIGANDS: The JAK-STAT Pathway. Annu Rev Biochem 64:621–652 Johnson DE, O’Keefe RA, Grandis JR (2018) Targeting the IL-6/JAK/STAT3 signalling axis in cancer. Nat Reviews Clin Oncol 15:234–248. https://doi.org/10.1038/nrclinonc.2018.8 Frank DA (2007) STAT3 as a central mediator of neoplastic cellular transformation. Cancer Lett 251:199–210. https://doi.org/10.1016/j.canlet.2006.10.017 Yi L, Guo G, Li J et al (2020) IKBKE, a prognostic factor preferentially expressed in mesenchymal glioblastoma, modulates tumoral immunosuppression through the STAT3/PD-L1 pathway. Clin Translational Med 10:e130. https://doi.org/10.1002/ctm2.130 Tolomeo M, Cascio A (2021) The Multifaced Role of STAT3 in Cancer and Its Implication for Anticancer Therapy. Int J Mol Sci 22. https://doi.org/10.3390/ijms22020603 Han D, Yu T, Dong N et al (2019) Napabucasin, a novel STAT3 inhibitor suppresses proliferation, invasion and stemness of glioblastoma cells. J Experimental Clin Cancer Res 38:289. https://doi.org/10.1186/s13046-019-1289-6 Tang G, Luo L, Zhang J et al (2021) lncRNA LINC01057 promotes mesenchymal differentiation by activating NF-κB signaling in glioblastoma. Cancer Lett 498:152–164. https://doi.org/10.1016/j.canlet.2020.10.047 Vadla R, Miki S, Taylor B et al (2023) Glioblastoma Mesenchymal Transition and Invasion are Dependent on a NF-κB/BRD2 Chromatin Complex. https://doi.org/10.1101/2023.07.03.546613 . bioRxiv 2023.07.03.546613 Yamini B (2018) NF-κB, Mesenchymal Differentiation and Glioblastoma. https://doi.org/10.3390/cells7090125 . Cells 7: Hou Y, Sun B, Liu W et al (2021) Targeting of glioma stem-like cells with a parthenolide derivative ACT001 through inhibition of AEBP1/PI3K/AKT signaling. Theranostics 11:555–566. https://doi.org/10.7150/thno.49250 Tong L, Li J, Li Q et al (2020) ACT001 reduces the expression of PD-L1 by inhibiting the phosphorylation of STAT3 in glioblastoma. Theranostics 10:5943–5956. https://doi.org/10.7150/thno.41498 Li Q, Sun Y, Liu B et al (2020) ACT001 modulates the NF-κB/MnSOD/ROS axis by targeting IKKβ to inhibit glioblastoma cell growth. J Mol Med 98:263–277. https://doi.org/10.1007/s00109-019-01839-0 Zhao W, Sachsenmeier K, Zhang L et al (2014) A New Bliss Independence Model to Analyze Drug Combination Data. SLAS Discovery 19:817–821. https://doi.org/10.1177/1087057114521867 Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences 76:4350–4354. https://doi.org/10.1073/pnas.76.9.4350 Wang H, Tao Z, Feng M et al (2020) Dual PLK1 and STAT3 inhibition promotes glioblastoma cells apoptosis through MYC. Biochem Biophys Res Commun 533:368–375. https://doi.org/10.1016/j.bbrc.2020.09.008 Kim Y, Varn FS, Park S-H et al (2021) Perspective of mesenchymal transformation in glioblastoma. Acta Neuropathol Commun 9:50. https://doi.org/10.1186/s40478-021-01151-4 Fu W, Hou X, Dong L, Hou W (2023) Roles of STAT3 in the pathogenesis and treatment of glioblastoma. Front Cell Dev Biology 11 White J, White MPJ, Wickremesekera A et al (2024) The tumour microenvironment, treatment resistance and recurrence in glioblastoma. J Translational Med 22:540. https://doi.org/10.1186/s12967-024-05301-9 Leidgens V, Proske J, Rauer L et al (2016) Stattic and metformin inhibit brain tumor initiating cells by reducing STAT3-phosphorylation, vol 8. Oncotarget. No 5 Doostmohammadi A, Jooya H, Ghorbanian K et al (2024) Potentials and future perspectives of multi-target drugs in cancer treatment: the next generation anti-cancer agents. Cell Communication Signal 22:228. https://doi.org/10.1186/s12964-024-01607-9 Lu Y, Van Zandt M, Liu Y et al (2022) Analysis of Dual Combination Therapies Used in Treatment of Hypertension in a Multinational Cohort. JAMA Netw Open 5:e223877–e223877. https://doi.org/10.1001/jamanetworkopen.2022.3877 Koizumi Y, Iwami S (2014) Mathematical modeling of multi-drugs therapy: a challenge for determining the optimal combinations of antiviral drugs. Theoretical Biology Med Modelling 11:41. https://doi.org/10.1186/1742-4682-11-41 Pardridge WM (2005) The blood-brain barrier: Bottleneck in brain drug development. NeuroRX 2:3–14. https://doi.org/10.1602/neurorx.2.1.3 Sharom FJ (2011) The P-glycoprotein multidrug transporter. Essays Biochem 50:161–178. https://doi.org/10.1042/bse0500161 Marusyk A, Polyak K (2010) Tumor heterogeneity: Causes and consequences. Biochimica et Biophysica Acta (BBA) -. Reviews Cancer 1805:105–117. https://doi.org/10.1016/j.bbcan.2009.11.002 Hidalgo M, Amant F, Biankin AV et al (2014) Patient-Derived Xenograft Models: An Emerging Platform for Translational Cancer Research. Cancer Discov 4:998–1013. https://doi.org/10.1158/2159-8290.CD-14-0001 Hanahan D, Weinberg RA (2011) Hallmarks of Cancer: The Next Generation. Cell 144:646–674. https://doi.org/10.1016/j.cell.2011.02.013 Zhang Y, Feng W, Peng X et al (2022) Parthenolide alleviates peritoneal fibrosis by inhibiting inflammation via the NF-κB/ TGF-β/Smad signaling axis. Lab Invest 102:1346–1354. https://doi.org/10.1038/s41374-022-00834-3 Liu Y, Wang L, Liu J et al (2020) Anticancer Effects of ACT001 via NF-κB Suppression in Murine Triple-Negative Breast Cancer Cell Line 4T1. Cancer Manage Res 12:5131–5139. https://doi.org/10.2147/CMAR.S244748 Sillibourne JE, Bornens M (2010) Polo-like kinase 4: the odd one out of the family. Cell Div 5:25. https://doi.org/10.1186/1747-1028-5-25 Godinho SA, Pellman D (2014) Causes and consequences of centrosome abnormalities in cancer. Philosophical Trans Royal Soc B: Biol Sci 369:20130467. https://doi.org/10.1098/rstb.2013.0467 Fonseca I, Horta C, Ribeiro AS et al (2023) Polo-like kinase 4 (Plk4) potentiates anoikis-resistance of p53KO mammary epithelial cells by inducing a hybrid EMT phenotype. Cell Death Dis 14:133. https://doi.org/10.1038/s41419-023-05618-1 Marteil G, Guerrero A, Vieira AF et al (2018) Over-elongation of centrioles in cancer promotes centriole amplification and chromosome missegregation. Nat Commun 9:1258. https://doi.org/10.1038/s41467-018-03641-x Kawakami M, Mustachio LM, Zheng L et al (2018) Polo-like kinase 4 inhibition produces polyploidy and apoptotic death of lung cancers. Proceedings of the National Academy of Sciences 115:1913–1918. https://doi.org/10.1073/pnas.1719760115 Zhao Y, Wang X (2019) PLK4: a promising target for cancer therapy. J Cancer Res Clin Oncol 145:2413–2422. https://doi.org/10.1007/s00432-019-02994-0 Carro MS, Lim WK, Alvarez MJ et al (2010) The transcriptional network for mesenchymal transformation of brain tumours. Nature 463:318–325. https://doi.org/10.1038/nature08712 Siddiquee K, Zhang S, Guida WC et al (2007) Selective chemical probe inhibitor of Stat3, identified through structure-based virtual screening, induces antitumor activity. Proceedings of the National Academy of Sciences 104:7391–7396. https://doi.org/10.1073/pnas.0609757104 Mende AL, Schulte JD, Okada H, Clarke JL (2021) Current Advances in Immunotherapy for Glioblastoma. Curr Oncol Rep 23:21. https://doi.org/10.1007/s11912-020-01007-5 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5305574","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":369102952,"identity":"730f986f-5ddd-48c1-ab05-28dabab311a3","order_by":0,"name":"Xiao Ren","email":"","orcid":"","institution":"Sichuan Province Orthopedic Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiao","middleName":"","lastName":"Ren","suffix":""},{"id":369102953,"identity":"0f6f68d3-9677-447f-ad86-37d6fbf340eb","order_by":1,"name":"Jiabo Li","email":"","orcid":"","institution":"Northwestern University Feinberg School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jiabo","middleName":"","lastName":"Li","suffix":""},{"id":369102954,"identity":"db6db85f-8953-40c6-a7cf-de0182d9e3d5","order_by":2,"name":"Lei Chen","email":"","orcid":"","institution":"Tianjin Medical University General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Chen","suffix":""},{"id":369102956,"identity":"9f5bbb4a-4a29-4f88-974b-5754b7b40a8f","order_by":3,"name":"Xuya Wang","email":"","orcid":"","institution":"Tsinghua University Beijing Tsinghua Changgung Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xuya","middleName":"","lastName":"Wang","suffix":""},{"id":369102959,"identity":"90964150-493c-4566-a4f4-b31a827db60b","order_by":4,"name":"Jinhao Zhang","email":"","orcid":"","institution":"Capital Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jinhao","middleName":"","lastName":"Zhang","suffix":""},{"id":369102961,"identity":"f1949e20-a5ac-45a5-b4ee-a86c314539d1","order_by":5,"name":"Yiming Zhang","email":"","orcid":"","institution":"Tsinghua University Beijing Tsinghua Changgung Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yiming","middleName":"","lastName":"Zhang","suffix":""},{"id":369102962,"identity":"8f69e060-cdb5-4c95-98fc-255136d1a9b1","order_by":6,"name":"Jikang Fan","email":"","orcid":"","institution":"Huanhu Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jikang","middleName":"","lastName":"Fan","suffix":""},{"id":369102963,"identity":"dc2acde8-7f1e-4ea2-a1a1-839c87c1b334","order_by":7,"name":"Debo Yun","email":"","orcid":"","institution":"Nanchong Central Hospital","correspondingAuthor":false,"prefix":"","firstName":"Debo","middleName":"","lastName":"Yun","suffix":""},{"id":369102964,"identity":"e9b30bba-aff8-4b14-9cd2-422f811ed738","order_by":8,"name":"Chen Zhang","email":"","orcid":"","institution":"Tianjin Medical University General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Chen","middleName":"","lastName":"Zhang","suffix":""},{"id":369102965,"identity":"2dfde62c-95d1-4128-8e1e-95b96c7ef888","order_by":9,"name":"Shengping Yu","email":"","orcid":"","institution":"Tianjin Medical University General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Shengping","middleName":"","lastName":"Yu","suffix":""},{"id":369102966,"identity":"87b6a939-5f21-4e4e-8aad-f1ef423acdfd","order_by":10,"name":"Xuejun Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9ElEQVRIie3SsWrDMBCA4RMCeTmc1UKQ9BEUsmTIw5wxZDKhbxCXgCeFrjZ9iY4ZVTp4UXd1ax6g0OAloUMbQocuVTx20L8d3Ac3HEAs9j8joOPXeJQYAFadZzuEHAyfSeNYNZQAawTPH305kGhP+x5RsLZ9795OOxinnlh/GyLOFgrnyEdqld9tHcykJ66aEOkqUoiZkA/ldMNqOF9IgmOIPEPxiUKjfnUXsr5OumopG0GZ9nghpK8R6exyfjBWS7PK222dTVu336gQSX1ZeDra9X3yYj9O9WKSdsVTHyI3Fun3nMHPD/zdpEpscCEWi8Vi8A1tDVON9B8/0AAAAABJRU5ErkJggg==","orcid":"","institution":"Tsinghua University Beijing Tsinghua Changgung Hospital","correspondingAuthor":true,"prefix":"","firstName":"Xuejun","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2024-10-21 15:08:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5305574/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5305574/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":67443098,"identity":"7b9f357c-b8b1-4979-90c7-c4c0b6acf792","added_by":"auto","created_at":"2024-10-25 06:14:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":571471,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSingle-drug Stattic downregulates STAT3 signaling and compensatorily activates the NF-κB signaling.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Proportions of GBM Subtypes in the TCGA_Seq (inner ring) and TCGA_Microarray (outer ring) databases. \u003cstrong\u003eB.\u003c/strong\u003e Violin plots showing GSVA scores for GBM Subtypes in TCGA_Seq (left) and TCGA_Microarray (right) datasets. \u003cstrong\u003eC.\u003c/strong\u003e Kaplan-Meier survival curves for GBM patients with high versus low STAT3 expression in TCGA_Seq (left, p = 0.019) and TCGA_Microarray (right, p = 0.013) datasets. \u003cstrong\u003eD.\u003c/strong\u003e RNA-seq analysis of the Stattic-treated U251MG cell line revealed the relationship between STAT3 and P65. \u003cstrong\u003eE.\u003c/strong\u003e Enrichment of the NF-κB pathway in the Stattic-treated group within the Hallmark gene sets. \u003cstrong\u003eF.\u003c/strong\u003e KEGG enrichment bubble chart showing the rich ratio, gene number, and q-values for various pathways. \u003cstrong\u003eG-H.\u003c/strong\u003e Western blotting analysis to verify the expression levels of STAT3, pSTAT3, P65, and p-P65 proteins in U251MG cells under different concentrations of Stattic. Data are represented as the mean ± SEM (error bars) of three independent experiments. *P \u0026lt; 0.05, ****P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"20241021Figures1.png","url":"https://assets-eu.researchsquare.com/files/rs-5305574/v1/53df646b1bbde6b4c77a2d9e.png"},{"id":67444809,"identity":"16479efe-3e20-4dd5-9e56-e3143f7507aa","added_by":"auto","created_at":"2024-10-25 06:30:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":808210,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eACT001 combined with Stattic exhibits synergistic anti-mesenchymal GBM effects.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Dose-response curves showing the IC50 values of ACT001 and Stattic in U251MG and SNB19 cell lines. \u003cstrong\u003eB.\u003c/strong\u003e Heatmaps showing the interaction effects of ACT001 and Stattic on U251MG and SNB19 cell lines, where positive values indicate synergy, values near zero indicate additive effects, and negative values indicate antagonism. \u003cstrong\u003eC.\u003c/strong\u003eCCK8 proliferation assays of U251MG and SNB19 showing the effects of ACT001, Stattic, and their combination over five days. \u003cstrong\u003eD-E\u003c/strong\u003e. Colony formation assays of U251MG and SNB19 cells after 15 days, showing reduced colony numbers with ACT001, Stattic, and their combination compared to control. Data are represented as the mean ± SEM (error bars) of three independent experiments. ****P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"20241021Figures2.png","url":"https://assets-eu.researchsquare.com/files/rs-5305574/v1/54e99464ac62930da7cf285f.png"},{"id":67443101,"identity":"9f52062b-2227-4668-9f72-73df30ede97d","added_by":"auto","created_at":"2024-10-25 06:14:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1977843,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCombination therapy inhibits migration, invasion, and EMT in mesenchymal GBM cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA and C.\u003c/strong\u003e Wound healing assay comparing U251MG and SNB19 cell migration under Control, ACT001, Stattic, and Combo treatments at 0h, 24h, and 48h. Red lines mark the initial wound edges. \u003cstrong\u003eB and D.\u003c/strong\u003e Transwell assays showing the effects of Control, ACT001, Stattic, and Combo treatments on U251MG and SNB19 cell migration and invasion. Images are presented for both cell lines under each treatment condition. \u003cstrong\u003eE-F.\u003c/strong\u003e Western blotting analysis of STAT3, p-STAT3, P65, p-P65, PLK4, EMT markers (N-Cadherin, E-Cadherin, Vimentin), and apoptosis-related proteins (Caspase3, Bcl-2) in U251MG and SNB19 cells under different treatment conditions, with β-actin as a loading control. Data are represented as the mean ± SEM (error bars) of three independent experiments. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"20241021Figures3.png","url":"https://assets-eu.researchsquare.com/files/rs-5305574/v1/22952ed8724f735ffa7c2007.png"},{"id":67443637,"identity":"6385d2b0-6510-4511-b018-20c79b695064","added_by":"auto","created_at":"2024-10-25 06:22:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":980151,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCombination of ACT001 and Stattic inhibits proliferation of GBM cells through targeting PLK4.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e PLK4 gene expression across different treatments was measured in U251MG and SNB19 by RNA-seq. \u003cstrong\u003eB.\u003c/strong\u003e CCK8 cell proliferation curves of U251MG and SNB19 cells with PLK4 knockdown (sh1, sh2) and overexpression (OE), compared to empty vector controls, measured over five days. \u003cstrong\u003eC-D.\u003c/strong\u003e Colony formation assay showing the effects of PLK4 knockdown (sh1, sh2) and overexpression (OE) on U251MG and SNB19 cell growth, compared to empty vector controls. Data are represented as the mean ± SEM (error bars) of three independent experiments. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"20241021Figures4.png","url":"https://assets-eu.researchsquare.com/files/rs-5305574/v1/b8a23ecdfeeda4b9d60f4566.png"},{"id":67444810,"identity":"46bc82b5-2dc6-4571-b8e6-6ab1d033573d","added_by":"auto","created_at":"2024-10-25 06:30:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1997892,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKnock-down of PLK4 inhibit migration, invasion, EMT and promote apoptosis of GBM cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA-B.\u003c/strong\u003eWound healing assay showing the effects of PLK4 knockdown (sh1, sh2) and overexpression (OE) on the migration of U251MG and SNB19 cells over 48 hours, compared to empty vector controls. Red lines mark wound edges. \u003cstrong\u003eC-D.\u003c/strong\u003e Transwell migration and invasion assays showing the effects of PLK4 knockdown (sh1, sh2) and overexpression (OE) on U251MG and SNB19 cells, compared to empty vector controls. \u003cstrong\u003eE-F.\u003c/strong\u003e Western blotting analysis of EMT markers (N-Cadherin, E-Cadherin, Vimentin), apoptosis-related proteins (Caspase3, Bcl-2), and PLK4 expression in U251MG and SNB19 cells with PLK4 knockdown (sh1, sh2) and overexpression (OE), compared to empty vector controls, with β-actin as a loading control. Data are represented as the mean ± SEM (error bars) of three independent experiments. **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"20241021Figures5.png","url":"https://assets-eu.researchsquare.com/files/rs-5305574/v1/be3c5c109fbfe2d2f700bafe.png"},{"id":67443103,"identity":"ac723dc6-2783-4018-87b7-a02a187a1695","added_by":"auto","created_at":"2024-10-25 06:14:46","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2957174,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCombination of ACT001 and Stattic enhances anti-tumor effect.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Schematic representation of the experimental timeline for U251MG intracranial injection in mice, followed by daily gavage with ACT001 and Stattic, with bioluminescence imaging (IVIS) conducted at indicated time points. \u003cstrong\u003eB-C.\u003c/strong\u003e Bioluminescence images of mice bearing U251MG tumors at different time points post-treatment with control, ACT001, Stattic, or their combination. \u003cstrong\u003eD.\u003c/strong\u003e Kaplan-Meier survival analysis of mice after intracranial implantation of U251MG cells, comparing survival rates across different treatment groups. \u003cstrong\u003eE.\u003c/strong\u003e Immunohistochemical staining of p-P65, p-STAT3, and PLK4 in tumor sections from different treatment groups. \u003cstrong\u003eF.\u003c/strong\u003e H\u0026amp;E staining of heart, liver, spleen, lung, and kidney tissues from mice treated with control, ACT001, Stattic, or their combination. \u003cstrong\u003eG.\u003c/strong\u003eSchematic diagram showing ACT001 and Stattic dual-targeting NF-κB and STAT3 signaling pathways to inhibit mesenchymal GBM by suppressing the transcription of PLK4. Data are represented as the mean ± SEM (error bars) of three independent experiments. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ****P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"20241021Figures6.png","url":"https://assets-eu.researchsquare.com/files/rs-5305574/v1/d90d12805982da9d0a81db3d.png"},{"id":70829884,"identity":"2bf2c567-73a6-467b-9b95-e34f24db78de","added_by":"auto","created_at":"2024-12-07 13:01:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10382840,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5305574/v1/0c027c50-560e-4c79-b097-d96b1843ae47.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Inhibition of Mesenchymal Phenotypes in Glioblastoma by Combined Targeting of NF-κB and STAT3 Pathways","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGlioblastoma, IDH-wildtype (GBM, WHO grade 4), is the most lethal and aggressive primary brain tumor in adults, making total resection nearly impossible[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Despite standardized treatments for GBM, such as surgical resection, postoperative concurrent chemoradiotherapy and sequential chemotherapy, the mean survival time of GBM patients remains less than 15 months[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], with a five-year survival rate of only 7%[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Therefore, it is extremely urgent to further explore the survival characteristics and pathogenesis of GBM to address the disease suffering of patients.\u003c/p\u003e \u003cp\u003eSignal transducer and activator of transcription protein 3 (STAT3) is a transcription factor phosphorylated by JAK kinases in response to cytokine activation, allowing it to dimerize and move into the nucleus to activate the transcription of cytokine-responsive genes[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. As a member of the STAT family, STAT3 plays crucial roles in various cellular processes, including proliferation, survival, invasiveness[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Our previous studies have highlighted that abnormal STAT3 activation is particularly prominent in tumor cells, especially in mesenchymal GBM, contributing to tumor growth, invasion, and immune-escape[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, up to now, no drugs targeting the STAT3 signaling for GBM treatment have been approved in clinical trials. On the one hand, STAT3 is expressed in multiple systems complicates efforts to avoid side effects from broad inhibition[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. On the other hand, GBMs exhibit abnormal activation of multiple signaling pathways, and bypass activation means inhibiting STAT3 alone cannot effectively halt tumor progression[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Therefore, it is necessary to further study the activation and feedback mechanism of STAT3 pathway in GBM and design a more reasonable treatment strategy.\u003c/p\u003e \u003cp\u003eNF-κB pathway is another critical pathway mediating the transformation of GBM into mesenchymal subtype[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. ACT001 (dimethylamino micheliolide, DMAMCL), a chemically improved product derived from parthenolide[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], which is a traditional inhibitor of NF-κB, has been certified as an orphan drug for recurrent GBMs by the Food and Drug Administration (FDA) in the United States and by the European Union. ACT001 is currently undergoing phase II clinical trials for recurrent GBM in China, Australia and the United States[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Our previous experiments have shown that ACT001 exerts a strong inhibitory effect on NF-κB signaling[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we found that Stattic increased the expression of NF-κB pathway transcription factor phosphorylated p65 while inhibiting the phosphorylation of STAT3 dimer into the nucleus. Therefore, we hypothesize that combined inhibition of NF-κB and STAT3 pathways can synergistically treat GBM and reverse the phenotypic transformation of mesenchymal GBM through PLK4. Combined Stattic and ACT001 reduce tumor growth in tumor-bearing mice and provide overall survival benefits. In short, compared with single drug treatment, combination therapy shows a better therapeutic prospect, paving the way for novel interventions of GBM.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eHuman GBM cell lines U251MG and SNB19 were obtained from Shanghai Institute of Biochemistry and Cell Biology. Stattic was purchased from Selleck Chemicals. ACT001 was provided by Accendatech Co., Ltd. (Tianjin, China).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell culture and transfection of siRNAs\u003c/h3\u003e\n\u003cp\u003eDulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM; Gibco USA) supplement with 10% fetal bovine serum (FBS; Gibco, USA) was used for cell culture. The cells were cultured in incubator containing 5% CO2 at 37℃.\u003c/p\u003e \u003cp\u003ePLK4 siRNA and the negative control siRNA were purchased from GenPharma Pharmaceutical Technology Co., Ltd. The cells were seeded into 6-well plates (Corning, America) at a concentration of 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells per well and grown overnight before transfection.\u003c/p\u003e\n\u003ch3\u003eCell counting kit-8 assay (CCK8)\u003c/h3\u003e\n\u003cp\u003e Cell viability with Stattic and ACT001 combined treatment was estimated using CCK8 (CK04, DOJINDO, Beijing, China), according to the manufacturer\u0026rsquo;s manual. Cells (2\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells per well) were seeded in 96-well plates and incubated for 24h, and then were treated with Stattic and ACT001 for 24, 48 or 72h. Next, the cells were incubated with CCK8 solution for 1h at room temperature, and absorbance was measured at 450 nm by using a microplate luminometer (BioTek, USA)\u003c/p\u003e\n\u003ch3\u003eDrug synergy assessment\u003c/h3\u003e\n\u003cp\u003eCell viability was measured using the CCK8 assay. The cells were exposed to different concentrations of Stattic (0, 0.19, 0.38, 0.75, 1.5 and 3.0 \u0026micro;M) and ACT001 (0, 5, 10, 20 and 40 \u0026micro;M) and combinations of both drugs for 72 h. Synergy was assessed using the Bliss independence model as described in the literature[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eColony formation assay\u003c/h3\u003e\n\u003cp\u003eU251MG and SNB19 cell lines were seeded and cultured into 6-well plate at a density of 5\u0026times;10\u003csup\u003e2\u003c/sup\u003e cells/well in DMEM supplement with 10% FBS for 24 hours. Then, the cells were treated with Stattic and ACT001, respectively. After the treatment, these disposed cells were then re‐suspended in DMEM containing 10% FBS and cultured in 5% CO\u003csub\u003e2\u003c/sub\u003e, 37℃ for 15 days to allow colony formation. The plate was washed with cold PBS. The colonies were fixed by 4% polyformaldehyde for 10 min at room temperature. Next, the colonies were dyed with 1% crystal violet for 5 minutes at room temperature. The colonies more than 100 cells were counted by microscope (Leica Microsystems, Germany). Each experiment was done thrice independently in this study. Colony formation rate\u0026thinsp;=\u0026thinsp;the number of each treatment/the number of control \u0026times; 100%.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eWound healing assays\u003c/h2\u003e \u003cp\u003eCell migration was detected and evaluated by wounding healing (scratch) assays. Briefly, the cells were plated in 6-well plates (1.5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/plate) and incubated to generate confluent cultures. Scrapes were made with a 1000-\u0026micro;l sterile pipette tip, and the cells were washed with PBS and then treated with Stattic (2.0 \u0026micro;M), ACT001 (30 \u0026micro;M) or a combination of the two drugs for 48 h. For the ACT001 (10 \u0026micro;M)/siRNA PLK4 group, after the 24 h siRNA PLK4 transfection, scrapes were made, and the cells were treated with or without ACT001 for another 48 h. Finally, photos of cell migration were taken with a microscope.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTranswell assays\u003c/h3\u003e\n\u003cp\u003eMigration assays were carried out using 24-well plates (Corning, America) and 8 \u0026micro;m transwell chambers (Corning, America). 10,000 cells in 300 \u0026micro;l of serum-free medium containing ACT001 (10 \u0026micro;M), Stattic (0.75 \u0026micro;M) or a combination of the two drugs were added to the top chamber. The lower chambers were filled with 500 \u0026micro;l of DMEM medium with 10% FBS. After being incubated in an incubator for 24 h, the filter membranes in the chambers were removed and washed with PBS three times to remove the medium. Then, the cells that crossed to the underside of the filter membrane were fixed with 4% polyformaldehyde for 10 min, stained with 0.1% crystal violet for 5 min, and counted under a microscope. Invasion assays were also carried out using 24-well plates (Corning, America) and 8 \u0026micro;m transwell chambers (Corning, America). Matrigel (Matrigel: medium, 1:4) was added to the refrigerator and incubated at 4\u0026deg;C for 1 h and then placed in an incubator at 37\u0026deg;C for 2 hours. After the Matrigel solidified, 30,000 cells in 300 \u0026micro;l of serum-free medium containing ACT001 (10 \u0026micro;M), Stattic (0.75 \u0026micro;M) or a combination of the two drugs were added to the top chamber. For the ACT001 (10 \u0026micro;M)/siRNA PLK4 group, after the 24 h siRNA PLK4 transfection, the cells were collected and suspended in serum-free medium (2.5\u0026times;10\u003csup\u003e5\u003c/sup\u003e/ml) with or without ACT001. Then, 200 \u0026micro;l of cells were added to the top well. The lower chambers were filled with 500 \u0026micro;l of medium with 10% FBS. After being incubated in an incubator for 24 h, the filter membranes in the chambers were removed and washed with PBS three times to remove the medium and Matrigel. Then, the cells that crossed to the underside of the filter membrane were fixed with 4% polyformaldehyde for 30 min, stained with 0.1% crystal violet for 5min, and counted under a microscope.\u003c/p\u003e\n\u003ch3\u003eWestern blotting analysis\u003c/h3\u003e\n \u003cp\u003eWestern blotting was performed rely on relevant protocol[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Proteins were transferred onto PVDF membranes (ThermoFisher, USA), and incubated overnight in the 4\u003csup\u003e◦\u003c/sup\u003eC fridge with the following primary antibodies: STAT3 (Cell Signaling, USA, 1:1000), phospho-STAT3 (Tyr705) (Cell Signaling, USA, 1:1000), NF-κB p65 (Cell Signaling, USA, 1:1000), phosphp-NF-κB p65 (Cell Signaling, USA, 1:1000), N-Cadherin (Cell Signaling, USA, 1:1000), Vimentin (Cell Signaling, USA, 1:1000), Bcl-2 (Cell Signaling, USA, 1:1000), Caspase3 (Cell Signaling, USA, 1:1000), PLK4 (Cell Signaling, USA, 1:1000), β-action (ZSGB-Bio, China, 1:3000). Secondary antibodies: HRP labeled goat anti-rabbit/mouse IgG (ZSGB-Bio, China, 1:3000). Chemiluminescent HRP substrate (Millipore, USA) and GBOX system (Syngene Company, UK) were used to detect protein expression.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImplantation and oral administration of Stattic and ACT001\u003c/h2\u003e \u003cp\u003e All animal experiments were approved by the Ethical Committee of the Tianjin Medical University General Hospital. \u003cem\u003eIn vivo\u003c/em\u003e experiments were performed using nude mice (6 weeks old). Intracranial tumors were established by stereotactically implanting 7\u0026times;10\u003csup\u003e5\u003c/sup\u003e U251MG cells. Tumor burden was monitored by luciferase imaging every week starting on day 7 after implantation, and the nude mice were randomly divided into the following four groups (7 mice/group): (1) Control; (2) 200 mg/kg ACT001; (3) 5 mg/kg Stattic; and (4) 200 mg/kg ACT001 and 5 mg/kg Stattic. ACT001 was orally administered daily for 21 days. Stattic was dissolved in DMSO, diluted with Control (the concentration of DMSO was 1%), and orally administered 5 times every 7 days for 15 days. Overall survival of nude mice in all groups was monitored. Luciferin signal was detected with the \u003cem\u003ein vivo\u003c/em\u003e imaging system (IVIS) every week. The brains of the mice were carefully extracted when mice died or on day 56. These brains were fixed in 4% polyformaldehyde and embedded in paraffin for IHC staining.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll experiments were performed at least three times and all quantitative data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Statistical analysis was performed using SPSS 20. An unpaired t-test was used to compare the means of two groups, and a two-tailed p value of \u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e1. Single-drug Stattic downregulates STAT3 signaling and compensatorily activates the NF-κB signaling\u003c/h2\u003e \u003cp\u003eThe TCGA_Seq and TCGA_Microarray datasets were analyzed to assess the expression of STAT3 in patients with different subtypes of GBM. Both datasets indicated significantly higher STAT3 transcription levels in the mesenchymal subtype compared to the classical and proneural subtypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-B). Kaplan-Meier (K-M) survival curves from these datasets revealed that glioma patients with high STAT3 expression exhibited significantly shorter survival times compared to those with low STAT3 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). These findings indicate that high STAT3 expression is associated with poor survival in glioma patients.\u003c/p\u003e \u003cp\u003eDue to the poor efficacy of Stattic monotherapy in the treatment of GBM[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], we conducted RNA-seq analysis on Stattic-treated U251 MG cells. Compared with the control group, Stattic inhibited STAT3 dimer phosphorylation but increased NF-κB pathway transcription factor phosphorylated p65 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Gene Set Enrichment Analysis (GSEA) and KEGG enrichment revealed significant NF-κB pathway enrichment in the Stattic treatment group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-F). To further verify the accuracy of the sequencing results, Western blotting with four Stattic concentrations (0, 0.75, 1.5, and 3.0 \u0026micro;M) demonstrated that STAT3 protein and phosphorylated STAT3 levels decreased with higher Stattic concentrations, while phospho-p65 levels increased and total p65 levels remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG-H). These results suggest that Stattic inhibits the expression and phosphorylation of STAT3 while inducing compensatory activation of the NF-κB pathway, indicating potential synergy in combined STAT3 and NF-κB inhibition for GBM treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2. ACT001 combined with Stattic exhibits synergistic anti-mesenchymal GBM effects\u003c/h2\u003e \u003cp\u003eFurthermore, we hypothesize that ACT001 and Stattic may exhibit synergistic anti-tumor effects in mesenchymal GBM. Initially, we treated U251MG and SNB19 cells with ACT001 and Stattic separately. The IC50 values for ACT001 and Stattic in U251MG cells were 20.23 \u0026micro;mol/L and 1.11 \u0026micro;mol/L, respectively, while in SNB19 cells, they were 19.90 \u0026micro;mol/L and 0.74 \u0026micro;mol/L, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Next, we performed CCK8 assays to determine the concentrations of combined ACT001 and Stattic treatment and assessed the synergistic effect using the Bliss independence model. In U251MG cells, the strongest synergistic effect was observed at 10 \u0026micro;mol/L ACT001 and 0.75 \u0026micro;mol/L Stattic. In SNB19 cells, significant synergy was noted at 10 \u0026micro;mol/L ACT001 and 1.5 \u0026micro;mol/L Stattic (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Based on the IC50 values, we selected 10 \u0026micro;mol/L ACT001 and 1 \u0026micro;mol/L Stattic for further experiments.\u003c/p\u003e \u003cp\u003eA CCK8 proliferation assay was conducted to evaluate the treatment of mesenchymal GBM with the dual drug combination. Measurements over 5 days indicated that the proliferation rate in the combination treatment group was the lowest compared to the control and single-drug groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). A colony formation assay after 15 days of treatment revealed that the combined treatment group had the poorest ability to form clusters (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-E).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3. Combination therapy inhibits migration, invasion, and EMT in mesenchymal GBM cells\u003c/h2\u003e \u003cp\u003eThe effect of cell migration was assessed using a Wound healing assay. Compared to the control group, the combined treatment group exhibited the shortest cell crawling distance, followed by the single drug treatment group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-B). These results suggest that the migration ability of GBM cells in the dual drug treatment group was significantly weaker than in the single drug and control groups. Transwell experiment further confirmed that the combined treatment group had the fewest cells passing through the chamber, showing a stronger inhibitory effect on migration and invasion of mesenchymal GBM cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-D).\u003c/p\u003e \u003cp\u003eTo further investigate the impact of combination therapy on the phenotype of GBM, Western blotting was used to analyze the protein levels of N-Cadherin, E-Cadherin, and Vimentin. The results demonstrated that the combined treatment group exhibited the lowest protein levels of N-Cadherin and Vimentin, while E-Cadherin levels were the highest (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-F), suggesting that combination therapy may effectively inhibit cellular EMT. Additionally, the combined treatment group had the lowest protein levels of Bcl-2 and Caspase3 compared to other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-F), indicating that combination therapy may promotes cell apoptosis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e4. Combination of ACT001 and Stattic inhibits proliferation of GBM cells through targeting PLK4\u003c/h2\u003e \u003cp\u003eTo further explore the mechanism underlying the combination of ACT001 and Stattic, RNA-seq analysis was performed on the control group, single drug control groups, and combined treatment group. The results indicated that PLK4 was down-regulated in ACT001-treated group, Stattic-treated group, and particularly in the combined treatment group in both U251MG and SNB19 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Western blotting revealed that PLK4 protein expression was significantly lower in the combined treatment group compared to control or single-drug groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-F). These findings suggest that the NF-κB and STAT3 pathways jointly regulate PLK4 expression.\u003c/p\u003e \u003cp\u003eNext, the effects of PLK4 knockdown and overexpression on GBM phenotypes were examined. In the PLK4 knockdown group, using sequences sh1 and sh2 for validation, CCK8 proliferation assays and colony formation assays were conducted. Results revealed that cell proliferation activity decreased in the PLK4 knockdown group and increased in the overexpression group compared to the PLK4 empty vector group, indicating that PLK4 expression significantly impacts GBM cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-D). These findings suggest that the combination of ACT001 and Stattic shows a synergistic effect in inhibiting the growth of mesenchymal GBM cells through targeting PLK4.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e5. Knock-down of PLK4 inhibit migration, invasion, EMT and promote apoptosis of GBM cells\u003c/h2\u003e \u003cp\u003eIn order to explore the efforts of PLK4 on GBM migration and invasion. Wound healing assay showed that the crawling distance of cells in the PLK4 knockdown group was significantly shorter compared to the corresponding PLK4 empty vector group, while the overexpression group showed increased crawling distances (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B). In the Transwell assay, fewer cells passed through the chamber in the PLK4 knockdown group, and more in the overexpression group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-D), indicating that PLK4 levels significantly affect GBM migration and invasion capabilities. Western blotting validated that PLK4 knockdown group significantly reduced the expression of N-Cadherin, Vimentin, Caspase3, and Bcl-2 protein levels, while increased the expression of E-Cadherin level, compared to the untreated and corresponding PLK4 empty vector groups. Conversely, overexpression of PLK4 group had the opposite effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE-F). Thus, PLK4 knockdown may inhibit EMT process and promote apoptosis in GBM cells. In conclusion, these findings indicate that knockdown of PLK4 inhibits proliferation, migration, invasion, and EMT process, while promote apoptosis in GBM cells.\u003c/p\u003e\u003cp\u003e 6. \u003cb\u003eCombination of ACT001 and Stattic enhances anti-tumor effect.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFrom our \u003cem\u003ein vitro\u003c/em\u003e experiments, we found that the combination of ACT001 and Stattic inhibits the proliferation, invasion, migration, and EMT of GBM cells by reducing PLK4 expression, showing superior efficacy compared to single-drug treatments. We then evaluated the combined treatment's effects on tumor-bearing mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eAfter 28 days of treatment, the fluorescence intensity in the brain tumors of the combination treatment group did not significantly increase. In contrast, the single-drug groups showed a slightly increased, and the control group exhibited a significantly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-C). Survival analysis over 56 days revealed that all 7 mice in the control group died, 5 mice in the ACT001 treatment group, 6 mice in the Stattic treatment group, and 3 mice in the combined treatment group, indicating a longer survival period for the combination treatment group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Immunohistochemical analysis of paraffin-embedded mouse brain tissue sections revealed the highest expression of p-P65 in the Stattic group, followed by the control group, and the lowest in the combined treatment group. For p-STAT3 and PLK4, the control group had the highest expression, followed by the single-drug groups, with the combined treatment group showing the lowest expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE), consistent with the \u003cem\u003ein vitro\u003c/em\u003e results. Additionally, H\u0026amp;E staining of the mouse brain tissue showed no significant morphological differences in the main organs (including the heart, liver, spleen, lungs, and kidneys) among all groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). Importantly, no physiological or behavioral differences were observed among all the groups in our experiments. These results suggest that the combination of ACT001 and Stattic offers a more effective treatment approach for GBM, enhancing survival and reducing tumor progression without significant adverse effects.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe STAT3 pathway is a critical signaling pathway abnormally activated in mesenchymal glioblastoma[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], as confirmed by both the TCGA and CGGA databases. However, inhibiting the STAT3 pathway alone is insufficient for effective GBM treatment and may even induce resistance, leading to GBM recurrence[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. To investigate the causes of resistance following STAT3 pathway inhibition, we used the small molecule inhibitor Stattic[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] for RNA-seq analysis. The results showed that in U251MG cells treated with Stattic, the downregulation of phosphorylated STAT3 was accompanied by an upregulation of phosphorylated p65 in the NF-κB pathway. This suggests that the upregulation of the NF-κB pathway following STAT3 pathway inhibition is a major cause of glioblastoma recurrence, highlighting the need for a combined strategy to inhibit both the STAT3 and NF-κB pathways.\u003c/p\u003e \u003cp\u003eAlthough dual and multi-drug combination therapies are widely used in treating various diseases[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], such as hypertension[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and viral infections[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], their application of dual-drug therapy in tumors, particularly GBM, remains relatively rare. Challenges include the inability of conventional drugs to cross the blood-brain barrier[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], poor specificity for disease targets, high doses leading to resistance[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], the temporal and spatial heterogeneity of tumors[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], difficulty in establishing animal models[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], and complex signaling pathways[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In our study, alongside Stattic, which targets the STAT3 pathway, we incorporated a novel drug, ACT001, specifically targeting the NF-κB pathway. ACT001 derived from the root bark of Magnolia delavayi Franch, was chemically modified to enhance water solubility and oral bioavailability to 75%. Like its precursor parthenolide[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], ACT001 significantly inhibits the NF-κB pathway[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The combination therapy of Stattic and ACT001 demonstrated a significant synergistic effect in the GBM U251MG cell line. In phenotypic experiments, combination therapy showed markedly better results compared to monotherapy and control groups. Thus, we believe that combining of Stattic and ACT001, targeting the STAT3 and NF-κB pathways respectively, holds promising potential for GBM treatment. This study provides valuable insight for tumor treatment research, demonstrating that dual-drug combination therapy, along with enhancing the efficacy of the novel drug ACT001, can achieve better therapeutic outcomes for GBM.\u003c/p\u003e \u003cp\u003ePLK4, a crucial regulator of centriole replication during normal cell mitosis, can promote cell proliferation[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], activate invasion and migration[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], inhibit apoptosis[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], enhance tumor inflammatory infiltration, facilitate immune evasion, and promote angiogenesis in the tumor microenvironment[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], all closely associated with poor clinical prognosis[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Consequently, PLK4 has garnered widespread attention as a biomarker and therapeutic target for cancer[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Both RNA-seq data and our experimental validation indicate that the NF-κB and STAT3 pathways independently regulate the PLK4 gene. Simultaneous inhibition of these pathways produces a synergistic effect in suppressing PLK4 gene expression. Therefore, dual inhibition not only effectively blocks their downstream signaling but also further enhances therapeutic efficacy by inhibiting PLK4.\u003c/p\u003e \u003cp\u003eStattic, as a small molecule inhibitor of STAT3, has never been used clinically. likely due to its poor efficacy as monotherapy for GBM[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], high recurrence rates, and intrinsic properties such as poor water solubility and limited blood-brain barrier penetration, ACT001, a novel inhibitor of the NF-κB pathway, also inhibits the STAT3[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and PI3K-AKT pathways[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], limiting its specificity. Future research should aim to identify more effective STAT3 inhibitors with fewer side effects and better blood-brain barrier permeability. Additionally, exploring combination therapies with other treatments, such as immunotherapy[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], may enhance therapeutic outcomes. Further investigation into the specific mechanisms of PLK4 in GBM is needed to develop more precise targeted therapy strategies.\u003c/p\u003e \u003cp\u003eIn conclusion, this study successfully inhibited the mesenchymal phenotype of GBM cells by dual targeting of the NF-κB and STAT3 pathways and suppressing PLK4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). This finding provides new insights and potential therapeutic strategies for GBM treatment. Although further research is necessary to validate its clinical value, this approach holds promise for offering new hope to GBM patients.\u003c/p\u003e"},{"header":"Declarations","content":" \u003ch2\u003eCompeting Interest Statement:\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003ch2\u003eFundings\u003c/h2\u003e \u003cp\u003eThis work was supported by the National Natural Science Foundation of China (No.82373151); Beijing Natural Science Foundation (No.7232228); Beijing-Tianjin-Hebei Special Project (19JCZDJC64200(Z)); the Institute for Intelligent Healthcare, Tsinghua University (No. 2022ZLB007) and Tianjin Science and Technology Project (No.21JCYBJC00800).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eXiao Ren: Data analysis ann Writing.Jiabo Li and Lei Chen: Data Curation and Formal analysis.Xuya Wang: Visualization.Jinhao Zhang: Methodology.Yiming Zhang: Resources.Jikang Fan and Debo Yun: Validation.Chen Zhang: Conceptualization.Shengping Yu: Supervision.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank CGGA and TCGA. We were also grateful to the online tool suppliers, including R and R studio.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eOstrom QT, Cioffi G, Waite K et al (2021) CBTRUS Statistical Report: Primary Brain and Other Central Nervous System Tumors Diagnosed in the United States in 2014\u0026ndash;2018. Neurooncology 23:iii1\u0026ndash;iii105. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/neuonc/noab200\u003c/span\u003e\u003cspan address=\"10.1093/neuonc/noab200\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBikfalvi A, da Costa CA, Avril T et al (2023) Challenges in glioblastoma research: focus on the tumor microenvironment. Trends Cancer 9:9\u0026ndash;27. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.trecan.2022.09.005\u003c/span\u003e\u003cspan address=\"10.1016/j.trecan.2022.09.005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlexander BM, Cloughesy TF (2017) Adult Glioblastoma. JCO 35:2402\u0026ndash;2409. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1200/JCO.2017.73.0119\u003c/span\u003e\u003cspan address=\"10.1200/JCO.2017.73.0119\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZou S, Tong Q, Liu B et al (2020) Targeting STAT3 in Cancer Immunotherapy. Mol Cancer 19:145. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s12943-020-01258-7\u003c/span\u003e\u003cspan address=\"10.1186/s12943-020-01258-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBromberg JF, Wrzeszczynska MH, Devgan G et al (1999) Stat3 as an Oncogene. Cell 98:295\u0026ndash;303. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0092-8674(00)81959-5\u003c/span\u003e\u003cspan address=\"10.1016/S0092-8674(00)81959-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchindler C, Darnell JE (1995) TRANSCRIPTIONAL RESPONSES TO POLYPEPTIDE LIGANDS: The JAK-STAT Pathway. Annu Rev Biochem 64:621\u0026ndash;652\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJohnson DE, O\u0026rsquo;Keefe RA, Grandis JR (2018) Targeting the IL-6/JAK/STAT3 signalling axis in cancer. Nat Reviews Clin Oncol 15:234\u0026ndash;248. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nrclinonc.2018.8\u003c/span\u003e\u003cspan address=\"10.1038/nrclinonc.2018.8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFrank DA (2007) STAT3 as a central mediator of neoplastic cellular transformation. Cancer Lett 251:199\u0026ndash;210. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.canlet.2006.10.017\u003c/span\u003e\u003cspan address=\"10.1016/j.canlet.2006.10.017\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYi L, Guo G, Li J et al (2020) IKBKE, a prognostic factor preferentially expressed in mesenchymal glioblastoma, modulates tumoral immunosuppression through the STAT3/PD-L1 pathway. Clin Translational Med 10:e130. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/ctm2.130\u003c/span\u003e\u003cspan address=\"10.1002/ctm2.130\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTolomeo M, Cascio A (2021) The Multifaced Role of STAT3 in Cancer and Its Implication for Anticancer Therapy. Int J Mol Sci 22. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ijms22020603\u003c/span\u003e\u003cspan address=\"10.3390/ijms22020603\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHan D, Yu T, Dong N et al (2019) Napabucasin, a novel STAT3 inhibitor suppresses proliferation, invasion and stemness of glioblastoma cells. J Experimental Clin Cancer Res 38:289. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s13046-019-1289-6\u003c/span\u003e\u003cspan address=\"10.1186/s13046-019-1289-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTang G, Luo L, Zhang J et al (2021) lncRNA LINC01057 promotes mesenchymal differentiation by activating NF-κB signaling in glioblastoma. Cancer Lett 498:152\u0026ndash;164. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.canlet.2020.10.047\u003c/span\u003e\u003cspan address=\"10.1016/j.canlet.2020.10.047\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVadla R, Miki S, Taylor B et al (2023) Glioblastoma Mesenchymal Transition and Invasion are Dependent on a NF-κB/BRD2 Chromatin Complex. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1101/2023.07.03.546613\u003c/span\u003e\u003cspan address=\"10.1101/2023.07.03.546613\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. bioRxiv 2023.07.03.546613\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYamini B (2018) NF-κB, Mesenchymal Differentiation and Glioblastoma. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/cells7090125\u003c/span\u003e\u003cspan address=\"10.3390/cells7090125\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Cells 7:\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHou Y, Sun B, Liu W et al (2021) Targeting of glioma stem-like cells with a parthenolide derivative ACT001 through inhibition of AEBP1/PI3K/AKT signaling. Theranostics 11:555\u0026ndash;566. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.7150/thno.49250\u003c/span\u003e\u003cspan address=\"10.7150/thno.49250\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTong L, Li J, Li Q et al (2020) ACT001 reduces the expression of PD-L1 by inhibiting the phosphorylation of STAT3 in glioblastoma. Theranostics 10:5943\u0026ndash;5956. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.7150/thno.41498\u003c/span\u003e\u003cspan address=\"10.7150/thno.41498\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Q, Sun Y, Liu B et al (2020) ACT001 modulates the NF-κB/MnSOD/ROS axis by targeting IKKβ to inhibit glioblastoma cell growth. J Mol Med 98:263\u0026ndash;277. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00109-019-01839-0\u003c/span\u003e\u003cspan address=\"10.1007/s00109-019-01839-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao W, Sachsenmeier K, Zhang L et al (2014) A New Bliss Independence Model to Analyze Drug Combination Data. SLAS Discovery 19:817\u0026ndash;821. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1177/1087057114521867\u003c/span\u003e\u003cspan address=\"10.1177/1087057114521867\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTowbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences 76:4350\u0026ndash;4354. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1073/pnas.76.9.4350\u003c/span\u003e\u003cspan address=\"10.1073/pnas.76.9.4350\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang H, Tao Z, Feng M et al (2020) Dual PLK1 and STAT3 inhibition promotes glioblastoma cells apoptosis through MYC. Biochem Biophys Res Commun 533:368\u0026ndash;375. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bbrc.2020.09.008\u003c/span\u003e\u003cspan address=\"10.1016/j.bbrc.2020.09.008\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim Y, Varn FS, Park S-H et al (2021) Perspective of mesenchymal transformation in glioblastoma. Acta Neuropathol Commun 9:50. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s40478-021-01151-4\u003c/span\u003e\u003cspan address=\"10.1186/s40478-021-01151-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFu W, Hou X, Dong L, Hou W (2023) Roles of STAT3 in the pathogenesis and treatment of glioblastoma. Front Cell Dev Biology 11\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWhite J, White MPJ, Wickremesekera A et al (2024) The tumour microenvironment, treatment resistance and recurrence in glioblastoma. J Translational Med 22:540. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s12967-024-05301-9\u003c/span\u003e\u003cspan address=\"10.1186/s12967-024-05301-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeidgens V, Proske J, Rauer L et al (2016) Stattic and metformin inhibit brain tumor initiating cells by reducing STAT3-phosphorylation, vol 8. Oncotarget. No 5\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDoostmohammadi A, Jooya H, Ghorbanian K et al (2024) Potentials and future perspectives of multi-target drugs in cancer treatment: the next generation anti-cancer agents. Cell Communication Signal 22:228. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s12964-024-01607-9\u003c/span\u003e\u003cspan address=\"10.1186/s12964-024-01607-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu Y, Van Zandt M, Liu Y et al (2022) Analysis of Dual Combination Therapies Used in Treatment of Hypertension in a Multinational Cohort. JAMA Netw Open 5:e223877\u0026ndash;e223877. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1001/jamanetworkopen.2022.3877\u003c/span\u003e\u003cspan address=\"10.1001/jamanetworkopen.2022.3877\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoizumi Y, Iwami S (2014) Mathematical modeling of multi-drugs therapy: a challenge for determining the optimal combinations of antiviral drugs. Theoretical Biology Med Modelling 11:41. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/1742-4682-11-41\u003c/span\u003e\u003cspan address=\"10.1186/1742-4682-11-41\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePardridge WM (2005) The blood-brain barrier: Bottleneck in brain drug development. NeuroRX 2:3\u0026ndash;14. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1602/neurorx.2.1.3\u003c/span\u003e\u003cspan address=\"10.1602/neurorx.2.1.3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSharom FJ (2011) The P-glycoprotein multidrug transporter. Essays Biochem 50:161\u0026ndash;178. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1042/bse0500161\u003c/span\u003e\u003cspan address=\"10.1042/bse0500161\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarusyk A, Polyak K (2010) Tumor heterogeneity: Causes and consequences. Biochimica et Biophysica Acta (BBA) -. Reviews Cancer 1805:105\u0026ndash;117. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bbcan.2009.11.002\u003c/span\u003e\u003cspan address=\"10.1016/j.bbcan.2009.11.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHidalgo M, Amant F, Biankin AV et al (2014) Patient-Derived Xenograft Models: An Emerging Platform for Translational Cancer Research. Cancer Discov 4:998\u0026ndash;1013. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1158/2159-8290.CD-14-0001\u003c/span\u003e\u003cspan address=\"10.1158/2159-8290.CD-14-0001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHanahan D, Weinberg RA (2011) Hallmarks of Cancer: The Next Generation. Cell 144:646\u0026ndash;674. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cell.2011.02.013\u003c/span\u003e\u003cspan address=\"10.1016/j.cell.2011.02.013\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Y, Feng W, Peng X et al (2022) Parthenolide alleviates peritoneal fibrosis by inhibiting inflammation via the NF-κB/ TGF-β/Smad signaling axis. Lab Invest 102:1346\u0026ndash;1354. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41374-022-00834-3\u003c/span\u003e\u003cspan address=\"10.1038/s41374-022-00834-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu Y, Wang L, Liu J et al (2020) Anticancer Effects of ACT001 via NF-κB Suppression in Murine Triple-Negative Breast Cancer Cell Line 4T1. Cancer Manage Res 12:5131\u0026ndash;5139. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2147/CMAR.S244748\u003c/span\u003e\u003cspan address=\"10.2147/CMAR.S244748\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSillibourne JE, Bornens M (2010) Polo-like kinase 4: the odd one out of the family. Cell Div 5:25. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/1747-1028-5-25\u003c/span\u003e\u003cspan address=\"10.1186/1747-1028-5-25\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGodinho SA, Pellman D (2014) Causes and consequences of centrosome abnormalities in cancer. Philosophical Trans Royal Soc B: Biol Sci 369:20130467. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1098/rstb.2013.0467\u003c/span\u003e\u003cspan address=\"10.1098/rstb.2013.0467\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFonseca I, Horta C, Ribeiro AS et al (2023) Polo-like kinase 4 (Plk4) potentiates anoikis-resistance of p53KO mammary epithelial cells by inducing a hybrid EMT phenotype. Cell Death Dis 14:133. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41419-023-05618-1\u003c/span\u003e\u003cspan address=\"10.1038/s41419-023-05618-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarteil G, Guerrero A, Vieira AF et al (2018) Over-elongation of centrioles in cancer promotes centriole amplification and chromosome missegregation. Nat Commun 9:1258. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41467-018-03641-x\u003c/span\u003e\u003cspan address=\"10.1038/s41467-018-03641-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKawakami M, Mustachio LM, Zheng L et al (2018) Polo-like kinase 4 inhibition produces polyploidy and apoptotic death of lung cancers. Proceedings of the National Academy of Sciences 115:1913\u0026ndash;1918. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1073/pnas.1719760115\u003c/span\u003e\u003cspan address=\"10.1073/pnas.1719760115\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao Y, Wang X (2019) PLK4: a promising target for cancer therapy. J Cancer Res Clin Oncol 145:2413\u0026ndash;2422. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00432-019-02994-0\u003c/span\u003e\u003cspan address=\"10.1007/s00432-019-02994-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarro MS, Lim WK, Alvarez MJ et al (2010) The transcriptional network for mesenchymal transformation of brain tumours. Nature 463:318\u0026ndash;325. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nature08712\u003c/span\u003e\u003cspan address=\"10.1038/nature08712\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSiddiquee K, Zhang S, Guida WC et al (2007) Selective chemical probe inhibitor of Stat3, identified through structure-based virtual screening, induces antitumor activity. Proceedings of the National Academy of Sciences 104:7391\u0026ndash;7396. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1073/pnas.0609757104\u003c/span\u003e\u003cspan address=\"10.1073/pnas.0609757104\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMende AL, Schulte JD, Okada H, Clarke JL (2021) Current Advances in Immunotherapy for Glioblastoma. Curr Oncol Rep 23:21. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11912-020-01007-5\u003c/span\u003e\u003cspan address=\"10.1007/s11912-020-01007-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"glioblastoma, mesenchymal phenotype, NF-κB signaling, STAT3 signaling, ACT001, Stattic","lastPublishedDoi":"10.21203/rs.3.rs-5305574/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5305574/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGlioblastoma, IDH-wildtype (GBM, WHO grade 4) is the most common and lethal type of brain cancer that is hard to cure. Among the 3 subtypes of GBM, the mesenchymal GBM is characterized by therapeutic resistance and poor outcomes. Here, we found that both STAT3 and NF-κB pathways are abnormally activated in mesenchymal GBM and the patients with higher expression of STAT3 and NF-κB had a poor prognosis in TCGA database. Using the STAT3 inhibitor, Stattic, to suppress the STAT3 signaling in GBM cells. However, it was observed that Stattic alone leads to compensatory activation of the NF-κB signaling. Therefore, we hypothesized that combined inhibition of STAT3 and NF-κB pathways may has a better anti-mesenchymal GBM effect than single signaling inhibition. ACT001, a novel NF-κB inhibitor, combined with Stattic has a synergistic anti-GBM effect, effectively inhibiting GBM proliferation, invasion, migration and promoting apoptosis. RNA-seq analysis showed that combined inhibition of the STAT3 and NF-κB pathways resulted in better suppression of downstream gene PLK4 expression compared to the inhibition of either pathway alone. Overexpression of PLK4 was found to enhance GBM cell proliferation, invasion and migration, while reducing apoptosis. Taken together, these findings suggest that combined targeting of NF-κB and STAT3 signaling pathways, by acting on PLK4, suppresses proliferation, invasion and migration, as well as promotes apoptosis in the mesenchymal subtype of GBM cells, offering a novel therapeutic strategy for mesenchymal GBM.\u003c/p\u003e","manuscriptTitle":"Inhibition of Mesenchymal Phenotypes in Glioblastoma by Combined Targeting of NF-κB and STAT3 Pathways","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-25 06:14:41","doi":"10.21203/rs.3.rs-5305574/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d571bdec-108c-4743-bd44-813d140caaee","owner":[],"postedDate":"October 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-12-07T12:53:44+00:00","versionOfRecord":[],"versionCreatedAt":"2024-10-25 06:14:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5305574","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5305574","identity":"rs-5305574","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}