GSK343, an Inhibitor of Enhancer of Zeste Homolog 2, Reduces Glioblastoma Progression through Inflammatory Process Modulation: Focus on Canonical and Non-Canonical NF-κB/IκBα Pathways

Glioblastoma (GB) is a tumor of the central nervous system characterized by high proliferation and invasiveness. The standard treatment for GB includes radiotherapy and chemotherapy; however, new therapies are needed. Particular attention was given to the role of histone methyltransferase enhancer of zeste-homolog-2 (EZH2) in GB. Recently, several EZH2-inhibitors have been developed, particularly GSK343 is well-known to regulate apoptosis and autophagy processes; however, its abilities to modulate canonical/non-canonical NF-κB/IκBα pathways or an immune response in GB have not yet been investigated. Therefore, this study investigated for the first time the effect of GSK343 on canonical/non-canonical NF-κB/IκBα pathways and the immune response, by an in vitro, in vivo and ex vivo model of GB. In vitro results demonstrated that GSK343 treatments 1, 10 and 25 μM significantly reduced GB cell viability, showing the modulation of canonical/non-canonical NF-κB/IκBα pathway activation. In vivo GSK343 reduced subcutaneous tumor mass, regulating canonical/non-canonical NF-κB/IκBα pathway activation and the levels of reactive oxygen species (ROS), malondialdehyde (MDA), and superoxide dismutase (SOD). Ex vivo results confirmed the anti-proliferative effect of GSK343 and also demonstrated its ability to regulate immune response through CXCL9, CXCL10 and CXCL11 expression in GB. Thus, GSK343 could represent a therapeutic strategy to counteract GB progression, thanks to its ability to modulate canonical/non-canonical NF-κB/IκBα pathways and immune response.


Introduction
Glioblastoma (GB) is the most common and aggressive cancer of the central nervous system (CNS) with a global incidence of 10 cases per 100,000 people per year [1]. The vast majority of GB develops rapidly de novo in elderly patients, without clinical or histologic evidence of a less malignant precursor lesion [2]; whereas secondary GB progresses from low-grade diffuse astrocytoma or anaplastic astrocytoma [2]. Its histopathological features include cellular polymorphism, nuclear atypia, mitotic activity, vascular thrombosis, microvascular proliferation, and necrosis [3]. Current standard therapy for GB includes surgical resection, followed by radiotherapy and chemotherapy with temozolomide (TMZ), an oral alkylating agent [4]. Although the clinical treatment options are multiple, the survival rate for patients with GB remains very low and additional therapies are needed [1]. Studies have demonstrated that more than 140 gene mutations are involved in GB [5,6]; molecular structure with GSK126, this could be a future objective to evaluate in the context of brain tumors [30]. However, although some of the biological properties of GSK343 in GB have been investigated over the past decade [25,32], many molecular interactions and cross-talks remain unknown such as the regulation of canonical and non-canonical NF-κB/IκBα pathways or immune response. Therefore, considering these assumptions, we aimed to evaluate for the first time the effect of GSK343 on canonical and non-canonical NF-κB/IκBα pathways and immune response by using in vitro, in vivo and ex vivo models of GB, so as to provide a new perspective on this complex clinical scenario.  Figure 1A,B). However, the treatment with GSK343 at 50 µM exerted an elevated toxicity in all three GB cell lines both at 24 h and 48 h, reducing viability by more than 40% and 20%, respectively, compared to the control groups. Furthermore, we decided to evaluate the cytotoxicity of GSK343 also in normal human astrocytes (NHA) cells as a control, showing that the treatment with GSK343 at the higher concentration of 50 µM decreased NHA viability both at 24 h and 48 h (73% and 69%, respectively) (Supplementary Figure S1A,B). Therefore, based on these results, we decided to continue analyzing GSK343 only at the concentrations of 1, 10 and 25 µM to reduce the level of toxicity. The IC 50  GSK343 powerfully inhibits EZH2 activity, suppressing the progression of various cancer types including ovarian cancer [27], osteosarcoma [28] neuroblastoma and glioma [26], promoting programmed cell death and autophagy processes [29]. Pharmacokinetic studies revealed that some EZH2 inhibitors such as GSK126 and MC3629 cross the BBB [30,31]; however, not much is known about the ability of GSK343 to traverse BBB, nevertheless, considering the similar molecular structure with GSK126, this could be a future objective to evaluate in the context of brain tumors [30]. However, although some of the biological properties of GSK343 in GB have been investigated over the past decade [25,32], many molecular interactions and cross-talks remain unknown such as the regulation of canonical and non-canonical NF-κB/IκBα pathways or immune response. Therefore, considering these assumptions, we aimed to evaluate for the first time the effect of GSK343 on canonical and non-canonical NF-κB/IκBα pathways and immune response by using in vitro, in vivo and ex vivo models of GB, so as to provide a new perspective on this complex clinical scenario.

In Vitro Studies
2.1.1. Effect of GSK343 Treatment on GB Cell Viability GB cell viability was assessed following 24 h and 48 h of treatment with GSK343 at increasing concentrations (1, 10, 25 and 50 μM). GSK343 treatment significantly reduced viability in all GB cell lines in the same way and in a concentration-dependent manner both at 24 h and 48 h compared to the control groups ( Figure 1A,B). However, the treatment with GSK343 at 50 μM exerted an elevated toxicity in all three GB cell lines both at 24 h and 48 h, reducing viability by more than 40% and 20%, respectively, compared to the control groups. Furthermore, we decided to evaluate the cytotoxicity of GSK343 also in normal human astrocytes (NHA) cells as a control, showing that the treatment with GSK343 at the higher concentration of 50 μM decreased NHA viability both at 24 h and 48 h (73% and 69%, respectively) (Supplementary Figure S1A,B). Therefore, based on these results, we decided to continue analyzing GSK343 only at the concentrations of 1, 10 and Since GSK343 showed similar effects on cell viability in all GB cell cultures, we decided to continue to analyze the effect of GSK343 only on the U87 cell line, because it represented one of the most frequently used cell lines in the field of GB [33]. Since GSK343 showed similar effects on cell viability in all GB cell cultures, we decided to continue to analyze the effect of GSK343 only on the U87 cell line, because it represented one of the most frequently used cell lines in the field of GB [33].

Effect of GSK343 Treatment on Canonical and Non-Canonical NF-κB/IκBα Pathways in U87 Cell Lysates
The effect of GSK343 on the NF-κB/IκBα pathway was evaluated in U87 cell lysates. Our results demonstrated that the treatment with GSK343 for 24 h at the concentrations of 10 and 25 µM was able to significantly reduce NF-κB expression and also at the lower concentration of 1 µM, restored IκB-α expression and decreased IKKβ in a concentration-dependent manner compared to the control group (Figure 2A-C). Additionally, we detected the effect of GSK343 on NF-κB-inducing kinase (NIK) level, a kinase that regulates the activation of the non-canonical NF-κB pathway, by ELISA kit, demonstrating that GSK343 treatment for 24 h was able to reduce its level ( Figure 2D). Moreover, we decided to investigate the effect of GSK343 on pro-inflammatory cytokines expression such as IL-1β and TNF-α by Western blot analysis, showing that GSK343 treatment for 24 h at the concentrations of 1, 10 and 25 µM significantly decreased their expression compared to control group ( Figure 2E,F). The effect of GSK343 on the NF-κB/IκBα pathway was evaluated in U87 cell lysates. Our results demonstrated that the treatment with GSK343 for 24 h at the concentrations of 10 and 25 μM was able to significantly reduce NF-κB expression and also at the lower concentration of 1 μM, restored IκB-α expression and decreased IKKβ in a concentrationdependent manner compared to the control group (Figure 2A-C). Additionally, we detected the effect of GSK343 on NF-κB-inducing kinase (NIK) level, a kinase that regulates the activation of the non-canonical NF-κB pathway, by ELISA kit, demonstrating that GSK343 treatment for 24 h was able to reduce its level ( Figure 2D). Moreover, we decided to investigate the effect of GSK343 on pro-inflammatory cytokines expression such as IL-1β and TNF-α by Western blot analysis, showing that GSK343 treatment for 24 h at the concentrations of 1, 10 and 25 μM significantly decreased their expression compared to control group ( Figure 2E,F).

Figure 2.
Effect of GSK343 treatment on NF-κB/IκBα pathway in U87 cell lysates. Our data demonstrated that the treatment with GSK343 at the concentrations of 1, 10 and 25 μM significantly reduced NF-κB (A), IKKβ expression (C) and restored IκBα expression (B). Moreover, GSK343 treatment at the concentrations of 1, 10 and 25 μM significantly reduced NIK level (D), and pro-inflammatory cytokines IL-1β (E) and TNFα expression (F The effect of GSK343 on the apoptotic pathway was investigated in U87 cell lysates. After 24 h of treatment, our data demonstrated that GSK343 at the concentrations of 1, 10 and 25 μM was able to increase the expression of pro-apoptotic protein as Bax, while p53 expression was notably increased only at the concentrations of 10 and 25 μM ( Figure  3A,B); instead, anti-apoptotic Bcl2 protein expression was significantly reduced at 10 and 25 μM concentrations, as shown in Figure 3C. The effect of GSK343 on the apoptotic pathway was investigated in U87 cell lysates. After 24 h of treatment, our data demonstrated that GSK343 at the concentrations of 1, 10 and 25 µM was able to increase the expression of pro-apoptotic protein as Bax, while p53 expression was notably increased only at the concentrations of 10 and 25 µM ( Figure 3A,B); instead, anti-apoptotic Bcl2 protein expression was significantly reduced at 10 and 25 µM concentrations, as shown in Figure 3C.

GSK343 Treatment Modulated Epithelial-Mesenchymal Transition and Metalloproteinases Expression in U87 Cell Lysates
Considering the involvement of EZH2 in epithelial-mesenchymal transition (EMT) [34], we decided to investigate the effect of GSK343 treatment on E-cadherin and N-cadherin protein levels by Western blot analysis. Our data demonstrated that the treatment with GSK343 for 24 h at the concentrations of 1, 10 and 25 µM was able to increase E-cadherin expression and reduce N-cadherin expression ( Figure 4A,B).
Additionally, we investigated the effect of GSK343 treatment on matrix metalloproteinases (MMPs), particularly MMP2 and MMP9 expression, showing that the treatment with GSK343 for 24 h at the concentrations of 1, 10 and 25 µM was able to notably reduce their expression compared to the control group ( Figure 4C,D). To confirm the effect of GSK343 on EMT and

GSK343 Treatment Modulated Epithelial-Mesenchymal Transition and Metalloproteinases Expression in U87 Cell Lysates
Considering the involvement of EZH2 in epithelial-mesenchymal transition (EMT) [34], we decided to investigate the effect of GSK343 treatment on E-cadherin and N-cadherin protein levels by Western blot analysis. Our data demonstrated that the treatment with GSK343 for 24 h at the concentrations of 1, 10 and 25 μM was able to increase Ecadherin expression and reduce N-cadherin expression ( Figure 4A,B).
Additionally, we investigated the effect of GSK343 treatment on matrix metalloproteinases (MMPs), particularly MMP2 and MMP9 expression, showing that the treatment with GSK343 for 24 h at the concentrations of 1, 10 and 25 μM was able to notably reduce their expression compared to the control group ( Figure 4C,D). To confirm the effect of GSK343 on EMT and MMPs, we performed the cell migration assay, showing that the treatment with GSK343 significantly decreased U87 cell migration ( Figure 4E).

In Vivo Studies
The histological evaluation demonstrated that the control group was characterized by nuclear pleomorphism and high cellular density ( Figure 5A,A1,A2). However, the treatment with GSK343, at doses of 5 mg/kg and 10 mg/kg, was able to significantly reduce

Effect of GSK343 Treatment on Tumor Growth
The histological evaluation demonstrated that the control group was characterized by nuclear pleomorphism and high cellular density ( Figure 5A,A1,A2). However, the treatment with GSK343, at doses of 5 mg/kg and 10 mg/kg, was able to significantly reduce it compared to the control group ( Figure 5B1,B2,C1,C2). To better appreciate the histological evaluation, we showed the images in other 3 different areas, as demonstrated in the Supplementary Figure S2. Moreover, the treatment with GSK343 at doses of 5 mg/kg and 10 mg/kg significantly reduced the subcutaneous tumor mass (see macroscopic Figure 5A3,B3,C3), tumor weight as well as the tumor burden compared to the control group ( Figure 5D,E). During the experiment, no important changes in animals' weight were observed ( Figure 5F). Based on the in vitro results, we evaluated the effect of GSK343 treatment on canonical and non-canonical NF-κB/IκBα pathways also in the U87-xenograft model. Our results demonstrated that the control group was characterized by an increase in nuclear NF-κB expression and a decrease in cytosolic IκBα expression ( Figure 6A,B). Moreover, the

Effect of GSK343 Treatment on Canonical and Non-Canonical NF-κB/IκBα Pathways in U87-Xenograft Model
Based on the in vitro results, we evaluated the effect of GSK343 treatment on canonical and non-canonical NF-κB/IκBα pathways also in the U87-xenograft model. Our results demonstrated that the control group was characterized by an increase in nuclear NF-κB expression and a decrease in cytosolic IκBα expression ( Figure 6A,B). Moreover, the GB group demonstrated a significant increase in IKKβ expression ( Figure 6C); however, the treatment with GSK343 at both doses was able to reduce NF-κB translocation into the nucleus and restore IκB-α cytosolic expression compared to the control group ( Figure 6A,B), also reducing IKKβ expression ( Figure 6C). Additionally, to prove that GSK343 was able to modulate non-canonical NF-κB/IκBα pathway activation, we also investigated NIK level by ELISA kit, demonstrating that the treatment with GSK343 at doses of 5 mg/kg and 10 mg/kg significantly decreased its level ( Figure 6D). Our data on the NF-κB/IκBα pathway were also confirmed by evaluating phosphorylated proteins p-NF-κB and p-IκBα by Western blot analysis ( Figure 6E,F). Moreover, we decided to evaluate the effect of GSK343 treatment also on pro-inflammatory cytokines levels as IL-1β and TNF-α by ELISA kit, demonstrating that the control group was characterized by high levels of IL-1β and TNF-α; however, the treatment with GSK343 at doses of 5 mg/kg and 10 mg/kg significantly reduced their levels ( Figure 6G,H). α by Western blot analysis ( Figure 6E,F). Moreover, we decided to evaluate the effect of GSK343 treatment also on pro-inflammatory cytokines levels as IL-1β and TNF-α by ELISA kit, demonstrating that the control group was characterized by high levels of IL-1β and TNF-α; however, the treatment with GSK343 at doses of 5 mg/kg and 10 mg/kg significantly reduced their levels ( Figure 6G,H).

Effect of GSK343 Treatment on the Apoptotic Process in U87-Xenograft Model
Apoptosis plays a key role in GB progression [35]. Therefore, to confirm the in vitro results, we decided to investigate the effect of GSK343 on apoptosis also in the U87-xenograft model by evaluating Bax, BID, caspase-9 and Bcl2 expression by Western blot analysis. Our results showed that the treatment with GSK343 at doses of 5 mg/kg and 10 mg/kg significantly increased pro-apoptotic Bax, BID and caspase-9 protein expression ( Figure  7A-C), while Bcl2 expression was significantly reduced compared to control group (Figure 7D).

Effect of GSK343 Treatment on the Apoptotic Process in U87-Xenograft Model
Apoptosis plays a key role in GB progression [35]. Therefore, to confirm the in vitro results, we decided to investigate the effect of GSK343 on apoptosis also in the U87-xenograft model by evaluating Bax, BID, caspase-9 and Bcl2 expression by Western blot analysis. Our results showed that the treatment with GSK343 at doses of 5 mg/kg and 10 mg/kg significantly increased pro-apoptotic Bax, BID and caspase-9 protein expression ( Figure 7A-C), while Bcl2 expression was significantly reduced compared to control group ( Figure 7D).

Effect of GSK343 Treatment on Epithelial-Mesenchymal Transition and Metalloproteases Expression in U87-Xenograft Model
Considering the in vitro results, we investigated the effect of GSK343 on the EMT process also in the U87-xenograft model by evaluating E-cadherin and N-cadherin expression. Immunohistochemical localization demonstrated that the control group was characterized by a decrease in E-cadherin expression and consequently an increase in N-cadherin expression (Figures 8A,F). However, the treatment with GSK343 at both doses was able to significantly increase E-cadherin and reduce N-cadherin expression ( Figure  8B,C,G,H, respectively). The data for E-cadherin and N-cadherin were also confirmed by Western blot analysis, as shown in Figure 8E,J, respectively. Furthermore, we decided to evaluate metalloproteases (MMPs) levels also in the U87-xenograft model by Western blot analysis. Our data demonstrated that the treatment with GSK343 at doses of 5 mg/kg and 10 mg/kg significantly reduced MMP2 and MMP9 expression compared to the control group ( Figure 8K,L), confirming the previous results.

Effect of GSK343 Treatment on Epithelial-Mesenchymal Transition and Metalloproteases Expression in U87-Xenograft Model
Considering the in vitro results, we investigated the effect of GSK343 on the EMT process also in the U87-xenograft model by evaluating E-cadherin and N-cadherin expression. Immunohistochemical localization demonstrated that the control group was characterized by a decrease in E-cadherin expression and consequently an increase in N-cadherin expression ( Figure 8A,F). However, the treatment with GSK343 at both doses was able to significantly increase E-cadherin and reduce N-cadherin expression (Figure 8B,C,G,H, respectively). The data for E-cadherin and N-cadherin were also confirmed by Western blot analysis, as shown in Figure 8E,J, respectively. Furthermore, we decided to evaluate metalloproteases (MMPs) levels also in the U87-xenograft model by Western blot analysis. Our data demonstrated that the treatment with GSK343 at doses of 5 mg/kg and 10 mg/kg significantly reduced MMP2 and MMP9 expression compared to the control group ( Figure 8K,L), confirming the previous results.

Effect of GSK343 on Oxidative Stress in U87-Xenograft Model
Oxidative stress (OS) has been considered as one of many contributors in developing risk of cancer, including GB [36]; therefore, we decided to investigate the properties of GSK343 to modulate OS by evaluating the levels of reactive oxygen species (ROS) and malondialdehyde (MDA), a well-known marker of lipid peroxidation. Our data demonstrated that GSK343 at doses of 5 and 10 mg/kg was able to decrease ROS and MDA levels compared to the control group ( Figure 9A,B). Moreover, we investigated the level of superoxide dismutase (SOD) enzyme, an endogenous antioxidant enzyme which protects cells against toxic levels of free radicals, showing that GSK343 at both doses increased its level, counteracting OS ( Figure 9C). risk of cancer, including GB [36]; therefore, we decided to investigate the properties of GSK343 to modulate OS by evaluating the levels of reactive oxygen species (ROS) and malondialdehyde (MDA), a well-known marker of lipid peroxidation. Our data demonstrated that GSK343 at doses of 5 and 10 mg/kg was able to decrease ROS and MDA levels compared to the control group ( Figure 9A,B). Moreover, we investigated the level of superoxide dismutase (SOD) enzyme, an endogenous antioxidant enzyme which protects cells against toxic levels of free radicals, showing that GSK343 at both doses increased its level, counteracting OS ( Figure 9C).

Effect of GSK343 Treatment on Primary GB Cell Viability
GSK343 cytotoxicity was evaluated in primary GB cell culture obtained from patients, demonstrating that GSK343 treatment for 24 h at the concentrations of 1, 10 and 25 μM was able to significantly reduce primary GB cells viability compared to the control group, confirming its antiproliferative effect ( Figure 10).  2.2.6. Effect of GSK343 Treatment on Primary GB Cell Viability GSK343 cytotoxicity was evaluated in primary GB cell culture obtained from patients, demonstrating that GSK343 treatment for 24 h at the concentrations of 1, 10 and 25 µM was able to significantly reduce primary GB cells viability compared to the control group, confirming its antiproliferative effect ( Figure 10).

Effect of GSK343 on Oxidative Stress in U87-Xenograft Model
Oxidative stress (OS) has been considered as one of many contributors in developing risk of cancer, including GB [36]; therefore, we decided to investigate the properties of GSK343 to modulate OS by evaluating the levels of reactive oxygen species (ROS) and malondialdehyde (MDA), a well-known marker of lipid peroxidation. Our data demonstrated that GSK343 at doses of 5 and 10 mg/kg was able to decrease ROS and MDA levels compared to the control group ( Figure 9A,B). Moreover, we investigated the level of superoxide dismutase (SOD) enzyme, an endogenous antioxidant enzyme which protects cells against toxic levels of free radicals, showing that GSK343 at both doses increased its level, counteracting OS ( Figure 9C). 2.2.6. Effect of GSK343 Treatment on Primary GB Cell Viability GSK343 cytotoxicity was evaluated in primary GB cell culture obtained from patients, demonstrating that GSK343 treatment for 24 h at the concentrations of 1, 10 and 25 μM was able to significantly reduce primary GB cells viability compared to the control group, confirming its antiproliferative effect ( Figure 10).

Effect of GSK343 Treatment on Immune Response
Considering the role of EZH2 in immune response, we decided to evaluate the levels of chemokines CXCL9, CXCL10 and CXCL11, key mediators for the trafficking of antitumor immune cells, on primary GB cell culture by ELISA assay. Our data demonstrated that GSK343 treatment at the concentrations of 10 and 25 µM was able to increase CXCL9, CXCL10 and CXCL11 levels compared to the control group ( Figure 11A-C Considering the role of EZH2 in immune response, we decided to evaluate the levels of chemokines CXCL9, CXCL10 and CXCL11, key mediators for the trafficking of antitumor immune cells, on primary GB cell culture by ELISA assay. Our data demonstrated that GSK343 treatment at the concentrations of 10 and 25 μM was able to increase CXCL9, CXCL10 and CXCL11 levels compared to the control group ( Figure 11A-C).

Discussion
GB is the most common and aggressive malignant tumor of the central nervous system (CNS) [3]. In the last decade, many studies have focused on the role of EZH2 in GB [37,38], suggesting that its inhibition could represent a valid and alternative strategy to contrast cancer progression through apoptosis and autophagy modulation [32]. However, the relationship between EZH2 expression and canonical/non-canonical NF-κB pathway or immune response is not yet fully investigated in GB. Therefore, in this paper, we decided to evaluate for the first time the beneficial effect of GSK343, a highly potent and

Discussion
GB is the most common and aggressive malignant tumor of the central nervous system (CNS) [3]. In the last decade, many studies have focused on the role of EZH2 in GB [37,38], suggesting that its inhibition could represent a valid and alternative strategy to contrast cancer progression through apoptosis and autophagy modulation [32]. However, the relationship between EZH2 expression and canonical/non-canonical NF-κB pathway or immune response is not yet fully investigated in GB. Therefore, in this paper, we decided to evaluate for the first time the beneficial effect of GSK343, a highly potent and selective EZH2 inhibitor, in GB through canonical and non-canonical NF-κB pathway and immune response modulation, by an in vitro, in vivo and ex vivo model of GB. Firstly, we evaluated the cytotoxic effect of GSK343 at different concentrations in an in vitro model of GB using U87, U138 and A172 cell lines. Clearly, our data demonstrated that GSK343 treatment significantly reduced U87, U138 and A172 cell viability in a concentration-dependent manner both at 24 h and 48 h in the same way in all three cell cultures, highlighting its cytotoxic effect.
Various studies revealed that EZH2 is able to modulate many cellular processes, including inflammation and cell adhesion by targeting genes such as IL-1β and CDH13 [37]. Particularly, scientific evidence has focused on the crosstalk between EZH2 and NF-κB in cancer [39]. NF-κB is the major regulator of various cell processes, such as inflammation, proliferation, and apoptosis [14]. It has been demonstrated that aberrant activation of NF-κB could be associated with EZH2 overexpression or dysregulation in cancer [40]. Therefore, in this study we decided to investigate for the first time the effect of GSK343 on canonical and non-canonical NF-κB/IκBα pathways in GB. The activation of NF-κB involves two major signaling pathways, the canonical and non-canonical (or alternative) pathways, both being important for regulating immune and inflammatory responses despite their differences in signaling mechanism [14]. In the canonical NF-κB pathway, the activation of the IκB kinase (IKK/IKBK) complex (IKK) (composed of IKKα, IKKβ, and NF-κB essential modulator (NEMO)/IKKγ subunits) leads to IκBα phosphorylation by IKKβ [14]. In vitro, GSK343 treatment demonstrated the ability to reduce NF-κB and IKKβ expression, and consequently increase IκB-α expression in a concentration-dependent manner. Once activated, NF-κB induces the expression of various pro-inflammatory cytokines such as IL-1β and TNF-α which consequently promote the inflammatory process in GB [41]. In this context, our data revealed that the treatment with GSK343 at higher concentrations significantly reduced their levels compared to the control group, counteracting inflammation. On the other hand, we detected the capacity of GSK343 in GB to modulate the non-canonical NF-κB signaling pathway which is regulated by NF-κB-inducing kinase (NIK), demonstrating for the first time that in vitro GSK343 was able to significantly reduce NIK level and consequently the non-canonical NF-κB pathway activation. Additionally, in an in vivo study, we confirmed the ability of GSK343 to modulate canonical and non-canonical NF-κB/IκBα pathway activation, also evaluating the expression of phosphorylated proteins p-NF-κB and p-IκB-α. Despite the biological pathways underlying GB malignancy are still unclear, scientific evidence revealed the interconnection between the NF-κB/IκBα pathway and proto-oncogene non-receptor tyrosine kinase SRC [42,43]. SRC, a non-receptor tyrosine kinase protein that in humans is encoded by the SRC gene, drives GB invasion and progression through various processes modulation, such as epithelial-to-mesenchymal transition, angiogenesis, phosphorylation of transcription factors NF-κB and OS, which consequently promote tumor growth [42,43]. NF-κB plays a key role in cancer-related oxidative stress (OS), including GB [44]. Brain tumorigenesis has been associated with OS that is reflected by an imbalance between free radicals' production and antioxidant mechanism which in turn induces damage to protein, lipid and deoxyribonucleic acid (DNA), promoting genomic instability [44]. Reactive oxygen species (ROS), mainly superoxide anion radical (O 2 −), hydroxyl radical (·OH), and hydrogen peroxide (H 2 O 2 ) are involved in carcinogenesis, particularly in the tumor initiation and promotion phases [45]. Despite OS having a controversial role in GB [46,47], studies indicate that antioxidant therapy may be a means of treating tumors [44,47]. In this context, scientific evidence demonstrated that EZH2 overexpression has been linked with an increase of OS [47][48][49]. Indeed, we found an increase of ROS and MDA levels in GB, probably correlated to NF-κB/IκBα and EZH2 over-expression, and a decrease of antioxidant enzyme SOD level. However, GSK343 treatment was able to decrease ROS and MDA level and increase SOD enzyme level, reducing OS. Moreover, it has been demonstrated that EZH2 overexpression could suppress apoptosis in a variety of cancers, promoting cancer cell survival [32]. In this context, studies revealed that the downregulation of EZH2 expression in cancer is associated with an increase of apoptosis and a cell cycle arrest in the G0/G phases [34]. Therefore, considering the key role of apoptosis in GB pathogenesis [18], we decided to evaluate the effect of GSK343 on the apoptotic pathway in GB cells. In this context, our results demonstrated that GSK343 treatment significantly increased the expression of pro-apoptotic proteins such as Bax and p53, whereas Bcl2 expression was significantly reduced. Moreover, data on programmed cell death obtained in the in vitro model were confirmed by the in vivo xenograft model, through a significant increase in pro-apoptotic Bax, BID and caspase-9 protein expression, and a decrease in anti-apoptotic Bcl2 expression following GSK343 treatment. Studies demonstrated that overexpression of EZH2 in cancer is associated with epithelial-tomesenchymal transition (EMT) [24,37]. EMT has been shown to be crucial in tumorigenesis enhancing metastasis, chemoresistance and tumor stemness [50]. It is characterized by the loss of epithelial characteristics and the gain of mesenchymal attributes in epithelial cells; this process is regulated by a complex network of signaling pathways and transcription factors [50]. Due to its emerging role as a pivotal driver of tumorigenesis, targeting EMT is of great therapeutic interest in counteracting metastasis and chemoresistance in cancer patients [50,51]. Our results demonstrated that GSK343 treatment in a concentrationdependent manner was able to reduce N-cadherin and increase E-cadherin expression. The in vitro results on EMT were also confirmed through the in vivo xenograft model, in which we proved that GSK343 treatment, in a dose-dependent manner, significantly restored E-cadherin expression and reduced N-cadherin expression. Scientific evidence revealed that several proteases as MMP2 and MMP9 are involved in many steps of cancer, including primary tumor growth, angiogenesis, invasion of the basement membrane and stroma, and metastatic process [52,53]. The expression of MMP2 and MMP9 in GB found that the treatment with GSK343 was able to significantly reduce their expression both in vitro and in vivo, counteracting cancer cell migration. Although GSK343 demonstrated numerous beneficial effects, including the ability to reduce subcutaneous tumor mass in vivo, we decided to perform an ex vivo model on primary GB cells obtained from patients to confirm the effects of GSK343. According to previous results, GSK343 exerted a cytotoxic effect also on primary GB cells, confirming its antiproliferative effect. Despite the modulatory effects of GSK343 on EMT, apoptosis and autophagy are well-known examples [26,32], not enough was identified about the ability of GSK343 to act on the immune system in GB environment. The host immune response to cancer cells is a potent mechanism for tumor suppression. In this regard, previous studies exploring the mechanisms of EZH2-mediated oncogenesis have largely focused on the cell-intrinsic mechanisms by which EZH2 regulates the expression of genes that are necessary for cancer cell proliferation and survival, promoting tumor development and progression [22]. It has been demonstrated that EZH2 promotes tumor development through a cell-extrinsic mechanism involving inhibition of the antitumor activity of NK cells, the major components of the innate immune response, which are generally recruited to tumor sites with chemokines, such as CXCL9, CXCL10 and CXCL11, to explicate their immune function [22,54]. In this context, for the first time, we found that GSK343 treatment was able to modulate the innate immune response, increasing CXCL9, CXCL10 and CXCL11 levels in GB, as a result of EZH2 inhibition, enhancing NK cell migration to tumor sites and consequently NK cell-mediated tumor growth inhibition. Therefore, the obtained results demonstrated the beneficial effect of GSK343, proposing that it could be an alternative therapeutic strategy to counteract GB progression thanks to its ability to modulate canonical and non-canonical NF-κB/IκBα pathways and immune response in GB.

Experimental Groups
Control groups: U87, U138 and A172 cell lines were treated with culture medium. Vehicle groups: U87, U138 and A172 cell lines were treated with 0.001% of DMSO dissolved in culture medium. GSK343 1 µM group: U87, U138 and A172 cell lines were treated with GSK343 1µM dissolved in culture medium with 0.001% of DMSO. GSK343 10 µM group: U87, U138 and A172 cell lines were treated with GSK343 10 µM dissolved in culture medium with 0.001% of DMSO. GSK343 25 µM group: U87, U138 and A172 cell lines were treated with GSK343 25 µM dissolved in culture medium with 0.001% of DMSO. GSK343 50 µM group: U87, U138 and A172 cell lines were treated with GSK343 50 µM dissolved in culture medium with 0.001% of DMSO.
Furthermore, we evaluated the cytotoxicity of GSK343 at the concentrations of 1, 10, 25 and 50 µM in NHA cells as a control for 24 h and 48 h (Supplementary Figure S1).

MTT Assay
Cell viability was measured using a mitochondria-dependent dye for live cells (tetrazolium dye; MTT), as previously described by Campolo et al. [57]. Cells were pre-treated with increasing concentrations of GSK343 (1 µM, 10 µM, 25 µM and 50 µM) dissolved in a culture medium with 0.001% of DMSO for 24 h and 48h to determine high concentrations with high toxicity on cell viability. After 24h and 48h, cells were incubated at 37 • C with MTT (0.2 mg/mL) for 1 h. The medium was removed by aspiration and the cells were lysed with DMSO (100 µL). The extent of reduction of MTT to formazan was quantified by measurement of optical density at 550 nm (OD550) with a microplate reader. The half maximal inhibitory concentration (IC 50 ) of GSK343 from the percentage viability of U87 cells was calculated using Graph Pad prism 7.04 software by the interpolation of the values in dose-response curves.

Western Blot Analysis
Western blot analysis in U87 cell lysates was performed as previously described [58].

Cell Migration Assay
Cell migration in U87 cells was evaluated using Cell Migration Assay Kit (Abcam, ab235693) according to the manufacturer's instructions.

Statistical Analysis
All values are expressed as mean ± standard error of the mean (SEM) of "n" observations. Each analysis was performed three times with three samples replicates for each one. The results were analyzed by one-way analysis of variance (ANOVA) followed by a Bonferroni post hoc test for multiple comparisons. A p-value of less than 0.05 was considered significant.

Experimental Design
Xenograft tumor model was performed, as previously described [59]. Mice were inoculated subcutaneously with 3 × 10 6 human U87 cells in 0.2 mL of Phosphate-Buffered Saline (PBS) and 0.1 mL Matrigel (BD Bioscience, Bedford, MA). Once tumors were palpable (100 mm 3 ), the animals were randomly divided into groups to receive vehicle or GSK343 treatment. GSK343 was administered at doses of 5 mg/kg and 10 mg/kg once daily via intraperitoneal (ip) injection every day for 21 days, as described by Bownes et al. [29]. The animals were humanely euthanized after 21 days of treatment and the subcutaneous tumors were excised and processed for several analysis such as histological evaluation and Western blot analysis. Tumor volumes were measured non-invasively by using an electronic caliper. The tumor burden was calculated using the following formula: 0.5 × length × width. The tumor volume was calculated using an empirical formula, V = 1/2 × ((the shortest diameter)2 × (the longest diameter)). The experiments were performed three times to verify the data.

Experimental Groups
Animals were randomly divided into 3 groups, as described below: All values are expressed as mean ± standard error of the mean (SEM) of "n" observations. Each analysis was performed three times with three samples replicates for each one. The results were analyzed by one-way analysis of variance (ANOVA) followed by a Bonferroni post hoc test for multiple comparisons. A p-value of less than 0.05 was considered significant.

Patient-Derived Glioblastoma Cell Culture
Primary tumor GB cells from patients were acquired according to protocol approved by the Regional Ethical Board at the University of Messina. All subjects gave their informed consent for inclusion before they participated in the study. The study was performed in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of AOU "G. Martino," Hospital of Messina (No. 47/19 of 05/02/2019). Tumor samples were processed aseptically, and primary cell cultures were initiated using DMEM (Catalog No. D5030; Sigma-Aldrich) with 15% heat-inactivated fetal calf serum (FCS) (Catalog No. 12103C; Sigma-Aldrich), 2 mM GlutaMAX-I (Catalog No. 35050061; Ther-moFisher Scientific), 1% insulin-transferrin-selenium-X supplement (Catalog No. 41400045; ThermoFisher Scientific), and 1% penicillin-streptomycin mixture (Catalog No. 15640055; Invitrogen, Carlsbad, CA, USA). Cells were used within 7 days of plating or established as primary cell lines, as previously described [63].

1.
Control group: primary GB cells obtained from patients were treated with culture medium.

2.
Vehicle group: primary GB cells obtained from patients were treated with 0.001% of DMSO dissolved in culture medium. 3. GSK343 1 µM: GB cells from patients were treated with GSK343 at concentration of 1 µM dissolved in culture medium with 0.001% of DMSO. 4. GSK343 10 µM: GB cells from patients were treated with GSK343 at concentration of 10 µM dissolved in culture medium with 0.001% of DMSO. 5. GSK343 25 µM: GB cells from patients were treated with GSK343 at concentration of 25 µM dissolved in culture medium with 0.001% of DMSO.

Cell Viability Assay
Primary GB cells culture obtained from patients were treated with GSK343 at the concentrations of 1, 10 and 25 µM dissolved in culture medium with 0.001% of DMSO for 24 h. After 24 h, primary GB cells were incubated at 37 • C with MTT (0.2 mg/mL; M5655; Sigma-Aldrich) for 1 h. The medium was removed by aspiration, and the cells were lysed with 100 µL of DMSO (sc-358801; Santa Cruz Biotechnology). The extent of reduction of MTT to formazan was quantified by measurement of optical density at 550 nm (OD550) with a microplate reader as described [63]

Materials
GSK343 and other chemical reagents are purchased by Sigma-Aldrich (Milan, Italy). All stock solutions were prepared in non-pyrogenic saline (0.9% NaCl; Baxter, Liverpool, UK).

Statistical Analysis
All values are expressed as mean ± standard error of the mean (SEM) of "n" observations. Each analysis was performed three times with three samples replicates for each one. The results were analyzed by one-way analysis of variance (ANOVA) followed by a Bonferroni post hoc test for multiple comparisons. A p-value of less than 0.05 was considered significant.

Conclusions
In conclusion, our data demonstrated, for the first time, that GSK343 treatment was able to modulate canonical and non-canonical NF-κB/IκBα pathway activation in GB. Moreover, in the field of cancer research, our preliminary data obtained about the capacity of GSK343 to modulate OS and immune response in GB appears very interesting. Therefore, based on the obtained results, GSK343, thanks to its numerous abilities, could represent a possible therapeutic strategy to contrast or reduce GB growth, which has a extremely high mortality due to its resistance to currently used therapies. However, considering the limitations of preclinical models, further studies are needed for a fuller understanding of the mechanism of action of GSK343 in GB pathology.