Signaling In Glioblastoma Cells Research Articles

The Expression of Serglycin Is Required for Active Transforming Growth Factor β Receptor I Tumorigenic Signaling in Glioblastoma Cells and Paracrine Activation of Stromal Fibroblasts via CXCR-2.

Serglycin (SRGN) is a pro-tumorigenic proteoglycan expressed and secreted by various aggressive tumors including glioblastoma (GBM). In our study, we investigated the interplay and biological outcomes of SRGN with TGFβRI, CXCR-2 and inflammatory mediators in GBM cells and fibroblasts. SRGN overexpression is associated with poor survival in GBM patients. High SRGN levels also exhibit a positive correlation with increased levels of various inflammatory mediators including members of TGFβ signaling pathway, cytokines and receptors including CXCR-2 and proteolytic enzymes in GBM patients. SRGN-suppressed GBM cells show decreased expressions of TGFβRI associated with lower responsiveness to the manipulation of TGFβ/TGFβRI pathway and the regulation of pro-tumorigenic properties. Active TGFβRI signaling in control GBM cells promotes their proliferation, invasion, proteolytic and inflammatory potential. Fibroblasts cultured with culture media derived by control SRGN-expressing GBM cells exhibit increased proliferation, migration and overexpression of cytokines and proteolytic enzymes including CXCL-1, IL-8, IL-6, IL-1β, CCL-20, CCL-2, and MMP-9. Culture media derived by SRGN-suppressed GBM cells fail to induce the above properties to fibroblasts. Importantly, the activation of fibroblasts by GBM cells not only relies on the expression of SRGN in GBM cells but also on active CXCR-2 signaling both in GBM cells and fibroblasts.

Fibronectin Type III Domain Containing 3B as a Potential Prognostic and Therapeutic Biomarker for Glioblastoma.

Glioblastoma (GBM) is a representative malignant brain tumor characterized by a dismal prognosis, with survival rates of less than 2 years and high recurrence rates. Despite surgical resection and several alternative treatments, GBM remains a refractory disease due to its aggressive invasiveness and resistance to anticancer therapy. In this report, we explore the role of fibronectin type III domain containing 3B (FNDC3B) and its potential as a prognostic and therapeutic biomarker in GBM. GBM exhibited a significantly higher cancer-to-normal ratio compared to other organs, and patients with high FNDC3B expression had a poor prognosis (p < 0.01). In vitro studies revealed that silencing FNDC3B significantly reduced the expression of Survivin, an apoptosis inhibitor, and also reduced cell migration, invasion, extracellular matrix adhesion ability, and stem cell properties in GBM cells. Furthermore, we identified that FNDC3B regulates PTEN/PI3K/Akt signaling in GBM cells using MetaCore integrated pathway bioinformatics analysis and a proteome profiler phospho-kinase array with sequential western blot analysis. Collectively, our findings suggest FNDC3B as a potential biomarker for predicting GBM patient survival and for the development of treatment strategies for GBM.

STAC1 is required for glioblastoma cell invasion and survival

Glioblastoma Multiforme (GBM) is a highly malignant brain tumor with poor prognosis. Understanding the molecular mechanisms driving GBM tumorigenesis is crucial for developing effective therapeutic strategies. This study investigates the role of STAC1, a gene belonging to the SH3 and cysteine-rich domain family, in glioblastoma cell invasion and survival. Computational analyses of patient samples reveal that STAC1 expression is elevated in GBM tissues, and higher STAC1 expression is associated with lower overall survival rates. Consistently, we find that overexpression of STAC1 in glioblastoma cells enhances invasion, while knockdown of STAC1 reduces invasion and the expression of genes associated with epithelial-to-mesenchymal transition (EMT). STAC1 depletion also induces apoptosis in glioblastoma cells. Furthermore, we show that STAC1 regulates AKT and calcium channel signaling in glioblastoma cells. Collectively, our study provides valuable insights into the pathogenic roles of STAC1 in GBM and highlights its potential as a promising target for the treatment of high-grade glioblastoma.

TRIM22 promotes the proliferation of glioblastoma cells by activating MAPK signaling and accelerating the degradation of Raf-1

The tripartite motif (TRIM) 22 and mitogen-activated protein kinase (MAPK) signaling pathways play critical roles in the growth of glioblastoma (GBM). However, the molecular mechanism underlying the relationship between TRIM22 and MAPK signaling remains unclear. Here, we found that TRIM22 binds to exon 2 of the sphingosine kinase 2 (SPHK2) gene. An ERK1/2-driven luciferase reporter construct identified TRIM22 as a potential activator of MAPK signaling. Knockout and overexpression of TRIM22 regulate the inhibition and activation of MAPK signaling through the RING-finger domain. TRIM22 binds to Raf-1, a negative regulator of MAPK signaling, and accelerates its degradation by inducing K48-linked ubiquitination, which is related to the CC and SPRY domains of TRIM22 and the C1D domain of Raf-1. In vitro and in vivo, an SPHK2 inhibitor (K145), an ERK1/2 inhibitor (selumetinib), and the nonphosphorylated mutant Raf-1S338A inhibited GBM growth. In addition, deletion of the RING domain and the nuclear localization sequence of TRIM22 significantly inhibited TRIM22-induced proliferation of GBM cells in vivo and in vitro. In conclusion, our study showed that TRIM22 regulates SPHK2 transcription and activates MAPK signaling through posttranslational modification of two critical regulators of MAPK signaling in GBM cells.

Paeoniflorin Inhibits EMT and Angiogenesis in Human Glioblastoma via K63-Linked C-Met Polyubiquitination-Dependent Autophagic Degradation.

Epithelial-to-mesenchymal transition (EMT) and angiogenesis have emerged as two pivotal events in cancer progression. Paeoniflorin has been widely studied in experimental models and clinical trials for cancer treatment because of its anti-cancer property. However, the underlying mechanisms of paeoniflorin in EMT and angiogenesis in glioblastoma was not fully elucidated. The present study aimed to investigate whether paeoniflorin inhibits EMT and angiogenesis, which involving c-Met suppression, while exploring the potential ways of c-Met degradation. In our study, we found that paeoniflorin inhibited EMT via downregulating c-Met signaling in glioblastoma cells. Furthermore, overexpressing c-Met in glioblastoma cells abolished the effects of paeoniflorin on EMT. Moreover, paeoniflorin showed anti-angiogenic effects by suppressing cell proliferation, migration, invasion and tube formation through downregulating c-Met in human umbilical vein endothelial cells (HUVECs). And c-Met overexpression in HUVECs offset the effects of paeoniflorin on angiogenesis. Additionally, paeoniflorin induced autophagy activation involving mTOR/P70S6K/S6 signaling and promoted c-Met autophagic degradation, a process dependent on K63-linked c-Met polyubiquitination. Finally, paeoniflorin suppressed mesenchymal makers (snail, vimentin, N-cadherin) and inhibited angiogenesis via the identical mechanism in an orthotopic xenograft mouse model. The in vitro and in vivo experiments showed that paeoniflorin treatment inhibited EMT, angiogenesis and activated autophagy. What’s more, for the first time, we identified c-Met may be a potential target of paeoniflorin and demonstrated paeoniflorin downregulated c-Met via K63-linked c-Met polyubiquitination-dependent autophagic degradation. Collectively, these findings indicated that paeoniflorin inhibits EMT and angiogenesis via K63-linked c-Met polyubiquitination-dependent autophagic degradation in human glioblastoma.

β2-Microglobulin Maintains Glioblastoma Stem Cells and Induces M2-like Polarization of Tumor-Associated Macrophages

β2-microglobulin signaling in glioblastoma cells activates a PI3K/AKT/MYC/TGFβ1 axis that maintains stem cells and induces M2-like macrophage polarization, highlighting potential therapeutic strategies for targeting tumor cells and the immunosuppressive microenvironment in glioblastoma.

ACTL6A deficiency induces apoptosis through impairing DNA replication and inhibiting the ATR-Chk1 signaling in glioblastoma cells

Actin-like 6A (ACTL6A) is a core subunit of the SWI/SNF chromatin remodeling complex and is highly expressed in several types of human cancers including glioblastoma. Recent studies verified that ACTL6A regulates the proliferation, differentiation, and migration of cancer cells. In this study, we identified ACTL6A as an important regulator of DNA replication. ACTL6A knockdown could impair the DNA replication initiation in glioblastoma cells. The regulation of DNA replication by ACTL6A was mediated through regulating the expression of the CDC45-MCM-GINS (CMG) complex genes. Further investigation revealed that ACTL6A transcriptionally regulates MCM5 expression. Furthermore, ACTL6A knockdown induced DNA damage and diminished the activity of the ATR-Chk1 pathway, which ultimately led glioblastoma cells to apoptosis and death. Taken together, our findings highlight the critical role of ACTL6A in DNA replication and ATR-Chk1 pathway, and reveal a potential target for therapeutic intervention in glioblastoma.

The transcript ENST00000444125 of lncRNA LINC01503 promotes cancer stem cell properties of glioblastoma cells via reducing FBXW1 mediated GLI2 degradation

LINC010503 is a novel oncogenic lncRNA in multiple cancers. In this study, we further explored the expression of LINC010503 transcripts and their regulations on the glioblastoma (GBM) stem cell (GSC) properties. LINC01503 transcription patterns in GBM and normal brain tissues were compared using RNA-seq data from Genotype-Tissue Expression (GTEx) and The Cancer Genome Atlas (TCGA)-GBM. GBM cell lines (U251 and U87) were used as in vitro cell models for cellular and molecular studies. The results showed that ENST00000444125 was the dominant transcript of LINC01503 in both normal and tumor tissues. Its expression was significantly elevated in the tumor group and associated with poor survival outcomes. LINC01503 had both cytoplasmic and nuclear distribution. It positively modulated the expression of multiple GSC markers, including CD133, SOX2, NESTIN, ALDH1A1, and MSI1, and tumorsphere formation in U251 and U87 cells. RNA pull-down and RIP-qPCR assay confirmed an interaction between ENST00000444125 and GLI2. ENST00000444125 positively regulated the half-life of the GLI2 protein in GBM cells. ENST00000444125 overexpression reduced GLI2 ubiquitination and partially attenuated FBXW1 overexpression induced GLI2 ubiquitination. ENST00000444125 overexpression could activate Wnt/β-catenin signaling in GBM cells. However, these activating effects were remarkedly hampered when GLI2 was knocked down. In conclusion, this study revealed that LINC01503 might have isoform-specific dysregulation in GBM. Among the two major transcripts expressed in GBM cells, ENST00000444125 might be the major functional transcript. Its upregulation might enhance the GSC properties of GBM cells via reducing FBXW1-mediated proteasomal degradation of GLI2.

Novel Insights into the Antagonistic Effects of Losartan against Angiotensin II/AGTR1 Signaling in Glioblastoma Cells.

Simple SummaryPatients with high-grade glioma (HGG) such as glioblastoma (GBM) who undergo surgical resection with adjuvant therapy have a mean overall survival of 14.6 months and 100% of recurrence. Thus, these disappointing outcomes in terms of glioblastoma life expectancy require seeking novel pharmacological tools, including drug repurposing. In the present study, we identify a novel molecular mechanism through which Losartan antagonizes Angiotensin II (Ang II)/Angiotensin II type I receptor (AGTR1) signaling, overexpressed in GBM cells. For instance, we demonstrate how Losartan drastically inhibits the stimulatory effects of Ang II on aromatase activity and consequently reduces local estrogen production, sustaining cancer progression. Thus, it is reasonable to repurpose Losartan as an adjuvant pharmacological tool to be implemented prospectively in the novel therapeutic strategies adopted in GBM patients.New avenues for glioblastoma therapy are required due to the limited mortality benefit of the current treatments. The renin-angiotensin system (RAS) exhibits local actions and works as a paracrine system in different tissues and tumors, including glioma. The glioblastoma cell lines U-87 MG and T98G overexpresses Angiotensin II (Ang II)/Angiotensin II type I receptor (AGTR1) signaling, which enhances in vitro and in vivo local estrogen production through a direct up-regulation of the aromatase gene promoters p I.f and p I.4. In addition, Ang II/AGTR1 signaling transactivates estrogen receptor-α in a ligand-independent manner through mitogen-activated protein kinase (MAPK) activation. The higher aromatase mRNA expression in patients with glioblastoma was associated with the worst survival prognostic, according to The Cancer Genome Atlas (TCGA). An intrinsic immunosuppressive glioblastoma tumor milieu has been previously documented. We demonstrate how Ang II treatment in glioblastoma cells increases programmed death-ligand 1 (PD-L1) expression reversed by combined exposure to Losartan (LOS) in vitro and in vivo. Our findings highlight how LOS, in addition, antagonizes the previously documented neoangiogenetic, profibrotic, and immunosuppressive effects of Ang II and drastically inhibits its stimulatory effects on local estrogen production, sustaining glioblastoma cell growth. Thus, Losartan may represent an adjuvant pharmacological tool to be repurposed prospectively for glioblastoma treatment.

Pericytes augment glioblastoma cell resistance to temozolomide through CCL5-CCR5 paracrine signaling

Glioblastoma (GBM) is a prevalent and highly lethal form of glioma, with rapid tumor progression and frequent recurrence. Excessive outgrowth of pericytes in GBM governs the ecology of the perivascular niche, but their function in mediating chemoresistance has not been fully explored. Herein, we uncovered that pericytes potentiate DNA damage repair (DDR) in GBM cells residing in the perivascular niche, which induces temozolomide (TMZ) chemoresistance. We found that increased pericyte proportion correlates with accelerated tumor recurrence and worse prognosis. Genetic depletion of pericytes in GBM xenografts enhances TMZ-induced cytotoxicity and prolongs survival of tumor-bearing mice. Mechanistically, C-C motif chemokine ligand 5 (CCL5) secreted by pericytes activates C-C motif chemokine receptor 5 (CCR5) on GBM cells to enable DNA-dependent protein kinase catalytic subunit (DNA-PKcs)-mediated DDR upon TMZ treatment. Disrupting CCL5-CCR5 paracrine signaling through the brain-penetrable CCR5 antagonist maraviroc (MVC) potently inhibits pericyte-promoted DDR and effectively improves the chemotherapeutic efficacy of TMZ. GBM patient-derived xenografts with high CCL5 expression benefit from combined treatment with TMZ and MVC. Our study reveals the role of pericytes as an extrinsic stimulator potentiating DDR signaling in GBM cells and suggests that targeting CCL5-CCR5 signaling could be an effective therapeutic strategy to improve chemotherapeutic efficacy against GBM.

COXIBs and 2,5-dimethylcelecoxib counteract the hyperactivated Wnt/\u03b2-catenin pathway and COX-2/PGE2/EP4 signaling in glioblastoma cells

BackgroundGlioblastoma (GBM) is the deadliest and the most common primary brain tumor in adults. The invasiveness and proliferation of GBM cells can be decreased through the inhibition of Wnt/β-catenin pathway. In this regard, celecoxib is a promising agent, but other COXIBs and 2,5-dimethylcelecoxib (2,5-DMC) await elucidation. Thus, the aim of this study was to analyze the impact of celecoxib, 2,5-DMC, etori-, rofe-, and valdecoxib on GBM cell viability and the activity of Wnt/β-catenin pathway. In addition, the combination of the compounds with temozolomide (TMZ) was also evaluated. Cell cycle distribution and apoptosis, MGMT methylation level, COX-2 and PGE2 EP4 protein levels were also determined in order to better understand the molecular mechanisms exerted by these compounds and to find out which of them can serve best in GBM therapy.MethodsCelecoxib, 2,5-DMC, etori-, rofe- and valdecoxib were evaluated using three commercially available and two patient-derived GBM cell lines. Cell viability was analyzed using MTT assay, whereas alterations in MGMT methylation level were determined using MS-HRM method. The impact of COXIBs, in the presence and absence of TMZ, on Wnt pathway was measured on the basis of the expression of β-catenin target genes. Cell cycle distribution and apoptosis analysis were performed using flow cytometry. COX-2 and PGE2 EP4 receptor expression were evaluated using Western blot analysis.ResultsWnt/β-catenin pathway was attenuated by COXIBs and 2,5-DMC irrespective of the COX-2 expression profile of the treated cells, their MGMT methylation status, or radio/chemoresistance. Celecoxib and 2,5-DMC were the most cytotoxic. Cell cycle distribution was altered, and apoptosis was induced after the treatment with celecoxib, 2,5-DMC, etori- and valdecoxib in T98G cell line. COXIBs and 2,5-DMC did not influence MGMT methylation status, but inhibited COX-2/PGE2/EP4 pathway.ConclusionsNot only celecoxib, but also 2,5-DMC, etori-, rofe- and valdecoxib should be further investigated as potential good anti-GBM therapeutics.

SKA3 promotes glioblastoma proliferation and invasion by enhancing the activation of Wnt/β-catenin signaling via modulation of the Akt/GSK-3β axis

Spindle and kinetochore-related complex subunit 3 (SKA3) is a key modulator of the progression of multiple tumor types. However, the involvement of SKA3 in glioblastoma (GBM) has not been well studied. The current study aimed to explore the role of SKA3 expression and the potential function of the protein in GBM. Our data showed that SKA3 expression was significantly up-regulated in GBM. Functional assays demonstrated that the knockdown of SKA3 impeded the proliferation, colony formation and invasion of GBM cells, while SKA3 overexpression produced the opposite effects. Further investigation revealed that SKA3 overexpression enhanced the activation of Wnt/β-catenin signaling, which was associated with the enhanced phosphorylation of Akt and glycogen synthase kinase-3β (GSK-3β). Notably, the inhibition of Akt markedly abrogated the SKA3 overexpression-induced promotion of Wnt/β-catenin signaling in GBM cells. Further, the inhibition of Wnt/β-catenin signaling markedly abrogated the SKA3 overexpression-induced promotion of tumor growth. In addition, the knockdown of SKA3 significantly retarded tumor formation and GBM progression in vivo. In summary, these data demonstrate that SKA3 exerts promotes tumor growth in GBM by enhancing the activation of Wnt/β-catenin signaling via modulation of the Akt/GSK-3β axis. This work highlights the pivotal role of SKA3/Akt/GSK-3β/Wnt/β-catenin signaling in the progression of GBM and suggests that SKA3 is an attractive therapeutic target with potential to be used to treat GBM.

PI3Kγ inhibition suppresses microglia/TAM accumulation in glioblastoma microenvironment to promote exceptional temozolomide response

Precision medicine in oncology leverages clinical observations of exceptional response. Toward an understanding of the molecular features that define this response, we applied an integrated, multiplatform analysis of RNA profiles derived from clinically annotated glioblastoma samples. This analysis suggested that specimens from exceptional responders are characterized by decreased accumulation of microglia/macrophages in the glioblastoma microenvironment. Glioblastoma-associated microglia/macrophages secreted interleukin 11 (IL11) to activate STAT3-MYC signaling in glioblastoma cells. This signaling induced stem cell states that confer enhanced tumorigenicity and resistance to the standard-of-care chemotherapy, temozolomide (TMZ). Targeting a myeloid cell restricted an isoform of phosphoinositide-3-kinase, phosphoinositide-3-kinase gamma isoform (PI3Kγ), by pharmacologic inhibition or genetic inactivation disrupted this signaling axis by reducing microglia/macrophage-associated IL11 secretion in the tumor microenvironment. Mirroring the clinical outcomes of exceptional responders, PI3Kγ inhibition synergistically enhanced the anti-neoplastic effects of TMZ in orthotopic murine glioblastoma models. Moreover, inhibition or genetic inactivation of PI3Kγ in murine glioblastoma models recapitulated expression profiles observed in clinical specimens isolated from exceptional responders. Our results suggest key contributions from tumor-associated microglia/macrophages in exceptional responses and highlight the translational potential for PI3Kγ inhibition as a glioblastoma therapy.

Abstract A15: The selective role of PIK3CB/p110β in temozolomide resistance in glioblastoma

Abstract Background: The divergent roles of phosphatidylinositide 3-kinase (PI3K) family genes in cancer pathogenesis and progression have been highly studied. PI3K genes are divided into three classes (I, II, and III). Class IA PI3K includes three highly homologous catalytic subunits p110α, β, or δ, encoded by the genes PIK3CA, B, and D, respectively. Studies in glioblastoma (GBM), the most lethal and most common brain cancer, have shown PI3K to promote resistance to the front-line chemotherapeutic drug, temozolomide (TMZ) (1). We recently investigated these isoforms in GBM and found that levels of PIK3CB/p110β, but not other PI3K isoforms, present prognostic significance for GBM recurrence and that this isoform selectively activates GBM cell survival pathways (2). Our previous work also shows that a gap-junction protein, Connexin 43 (Cx43), promotes TMZ resistance in GBM by activating PI3K signaling (3). Given that PIK3CB/p110β is the most important isoform in GBM PI3K signaling, it is therefore critical to investigate whether PI3K isoforms have divergent assignments in Cx43-mediated TMZ resistance. Understanding the role of PI3K isoforms in drug resistance will foster the rational design of more effective treatments for GBM. Hypothesis: Based upon the results described above, we hypothesize that PIK3CB/p110β is selectively important for Cx43-mediated TMZ resistance in GBM. Approaches: The expression and activity of PI3K genes and Cx43 are manipulated by overexpression of active PI3K mutants, short hairpin RNA (shRNA)-mediated knockdown, a peptide compound called αCT1, or specific chemical inhibitors. Activity of signaling is assessed by immunoblotting. Cx43 channel activity is monitored by ATP release. Protein-protein interactions are measured using fluorescence microscopy and immunoprecipitation. Cell viability is determined using the MTS viability assay. Results: We find that (1) shRNA knockdown of Cx43 inactivates PI3K signaling in GBM cells, while constitutive activation of PI3K diminishes TMZ sensitization by the Cx43 peptide inhibitor, αCT1; (2) GBM patients with high levels of Cx43 also express more p110β, active AKT, and display TMZ resistance; (3) Cx43 activates PI3K independent of Cx43-formed channels; (4) p110β, but not p110α or p110δ, interacts with Cx43; and (5) selective inhibition of p110β circumvents TMZ resistance together with αCT1. Conclusion/Impact: We conclude that PIK3CB/p110β is selectively important for Cx43-mediated temozolomide resistance, presenting the possibility of selectively targeting PIK3CB/p110β as a new and effective approach to overcoming TMZ resistance in GBM.

A novel molecular link between HOXA9 and WNT6 in glioblastoma identifies a subgroup of patients with particular poor prognosis.

Despite much effort to improve treatments, patients with malignant glioma still present a very poor prognosis that has not changed significantly in the last decades. In this context, it is crucial to better understand glioma pathogenesis to identify new molecular prognostic subgroups and therapeutic targets. WNT6 was recently identified as a new oncogenic molecule in glioblastoma (GBM), with prognostic value in patients, but the mechanisms underlying WNT6 aberrant expression in glioma are still unknown. WNT6 was overexpressed in a subset of gliomas independently of IDH mutations, 1p/19q codeletion status, and WNT6 gene copy number. Interestingly, WNT6 expression is associated with the DNA methylation levels of particular CpG regions at both the WNT6 promoter and the gene body in glioma patient samples. HOXA9, a transcription factor previously associated with poorer clinical outcome in GBM, was identified as a novel transcriptional regulator of WNT6, activating the WNT/β‐catenin pathway in vitro and in vivo. In various cohorts of glioma patients, mRNA levels of WNT6 and HOXA9 were significantly correlated, extending our in vitro and in vivo findings into the clinical setting. Interestingly, this novel molecular link between WNT6 and HOXA9 was not limited to glioma, as they were co‐expressed also in patients with other tumor types. Clinically, WNT6 was a prognostic biomarker of shorter survival in GBM, independently of HOXA9 expression. Concomitant high expression of both WNT6 and HOXA9 identified a subgroup of patients with particularly dismal survival. These findings describe novel WNT6 regulatory mechanisms in GBM, establishing particular DNA methylation patterns and HOXA9 as critical regulators of WNT6 expression in glioma. This HOXA9‐WNT6 molecular link supports WNT signaling in GBM cells and is a powerful prognostic biomarker, highlighting the clinical relevance of this axis in patients. Novel therapies targeting WNT6‐HOXA9 signaling may thus be useful for this deadly disease.

Crosstalk between GBM cells and mesenchymal stemlike cells promotes the invasiveness of GBM through the C5a/p38/ZEB1 axis.

Mesenchymal stemlike cells (MSLCs) have been detected in many types of cancer including brain tumors and have received attention as stromal cells in the tumor microenvironment. However, the cellular mechanisms underlying their participation in cancer progression remain largely unexplored. The aim of this study was to determine whether MSLCs have a tumorigenic role in brain tumors. To figure out molecular and cellular mechanisms in glioma invasion, we have cultured glioma with MSLCs in a co-culture system. Here, we show that MSLCs in human glioblastoma (GBM) secrete complement component C5a, which is known for its role as a complement factor. MSLC-secreted C5a increases expression of zinc finger E-box-binding homeobox 1 (ZEB1) via activation of p38 mitogen-activated protein kinase (MAPK) in GBM cells, thereby enhancing the invasion of GBM cells into parenchymal brain tissue. Our results reveal a mechanism by which MSLCs undergo crosstalk with GBM cells through the C5a/p38 MAPK/ZEB1 signaling loop and act as a booster in GBM progression. 1. MSLCs activate p38 MAPK-ZEB1 signaling in GBM cells through C5a in a paracrine manner, thereby boosting the invasiveness of GBM cells in the tumor microenvironment.2. Neutralizing of C5a could be a potential therapeutic target for GBM by inhibition of mesenchymal phenotype.

Transcriptional modulation of calcium-permeable AMPA receptor subunits in glioblastoma by MEK-ERK1/2 inhibitors and their role in invasion.

Glioblastoma is the most common primary brain tumor. Glioblastoma cells secrete a significant amount of glutamate, which serve as a potential growth factor in glioma pathobiology through their specific receptor subtypes including α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR). Glioblastoma express AMPAR subunits; however, its regulation and activation with downstream intracellular signaling are not well-understood. Phosphorylated-extracellular signaling-regulated kinase (ERK)1/2 is known to regulate the ionotropic glutamate receptors in cortical neurons. The mitogen-activated protein kinase cascade is frequently activated in several tumors, including glioma. Nonetheless, the association of ERK signaling with AMPAR subunits in glioblastoma is undetermined. Here, we demonstrated potential role of AMPAR in invasion, and the modulation of AMPAR subunits at transcript level by ERK signaling in glioblastoma cells. Inhibition of ERK signaling specifically downregulated the expression of calcium-permeable AMPAR subunits, GluA1 and GluA4, and upregulated calcium-impermeable AMPAR subunit GluA2 implying differential regulation of the expression of calcium-permeable AMPAR subunits of glioblastoma. Concomitantly, it significantly decreased the invasion of U87MG cells. Taken together, these findings suggest that the AMPAR enhances invasion of glioblastoma, and ERK signaling modulates the differential expression of calcium-permeable AMPAR phenotype that might play a crucial role in the invasive propensity of glioblastoma cells.

MYST1/KAT8 contributes to tumor progression by activating EGFR signaling in glioblastoma cells.

With short survival time, glioblastoma (GBM) is the most malignant tumor in the central nervous system. Recently, epigenetic enzymes play essential roles in the regulation of tumorigenesis and cancer development of GBM. However, little is known about MYST1/KAT8/MOF, a histone acetylation enzyme, in GBM. The present study shows that MYST1 promotes GBM progression through activating epidermal growth factor receptor (EGFR) signaling. MYST1 expression was increased in GBM and was negatively correlated with prognosis in patients with glioma and GBM. Knockdown of MYST1 reduced cell proliferation and BrdU incorporation in LN229, U87, and A172 GBM cells. Besides, MYST1 downregulation also induced cell cycle arrest at G2M phase, as well as the reduced expression of CDK1, Cyclin A, Cyclin B1, and increased expression of p21CIP1/Waf1. Meanwhile, Self‐renewal capability in vitro and tumorigenecity in vivo were also impaired after MYST1 knockdown. Importantly, MYST1 expression was lowly expressed in mesenchymal subtype of GBM and was positively correlated with EGFR expression in a cohort from The Cancer Genome Atlas. Western blot subsequently confirmed that phosphorylation and activation of p‐Try1068 of EGFR, p‐Ser473 of AKT and p‐Thr202/Tyr204 of Erk1/2 were also decreased by MYST1 knockdown. Consistent with the results above, overexpression of MYST1 promoted GBM growth and activated EGFR signaling in vitro and in vivo. In addition, erlotinib, a US Food and Drug Administration approved cancer drug which targets EGFR, was able to rescue MYST1‐promoted cell proliferation and EGFR signaling pathway. Furthermore, the transcription of EGF, an EFGR ligand, was shown to be positively regulated by MYST1 possibly via H4K16 acetylation. Our findings elucidate MYST1 as a tumor promoter in GBM and an EGFR activator, and may be a potential drug target for GBM treatment.

ERK1/2 signaling regulates the immune microenvironment and macrophage recruitment in glioblastoma.

The tumor microenvironment is an important determinant of glioblastoma (GBM) progression and response to treatment. How oncogenic signaling in GBM cells modulates the composition of the tumor microenvironment and its activation is unclear. We aimed to explore the potential local immunoregulatory function of ERK1/2 signaling in GBM. Using proteomic and transcriptomic data (RNA seq) available for GBM tumors from The Cancer Genome Atlas (TCGA), we show that GBM with high levels of phosphorylated ERK1/2 have increased infiltration of tumor-associated macrophages (TAM) with a non-inflammatory M2 polarization. Using three human GBM cell lines in culture, we confirmed the existence of ERK1/2-dependent regulation of the production of the macrophage chemoattractant CCL2/MCP1. In contrast with this positive regulation of TAM recruitment, we found no evidence of a direct effect of ERK1/2 signaling on two other important aspects of TAM regulation by GBM cells: (1) the expression of the immune checkpoint ligands PD-L1 and PD-L2, expressed at high mRNA levels in GBM compared with other solid tumors; (2) the production of the tumor metabolite lactate recently reported to dampen tumor immunity by interacting with the receptor GPR65 present on the surface of TAM. Taken together, our observations suggest that ERK1/2 signaling regulates the recruitment of TAM in the GBM microenvironment. These findings highlight some potentially important particularities of the immune microenvironment in GBM and could provide an explanation for the recent observation that GBM with activated ERK1/2 signaling may respond better to anti-PD1 therapeutics.

Afatinib and Temozolomide combination inhibits tumorigenesis by targeting EGFRvIII-cMet signaling in glioblastoma cells

BackgroundGlioblastoma (GBM) is an aggressive brain tumor with universal recurrence and poor prognosis. The recurrence is largely driven by chemoradiation resistant cancer stem cells (CSCs). Epidermal growth factor receptor (EGFR) and its mutant EGFRvIII are amplified in ~ 60% and ~ 30% of GBM patients, respectively; however, therapies targeting EGFR have failed to improve disease outcome. EGFRvIII-mediated cross-activation of tyrosine kinase receptor, cMET, regulates GBM CSC maintenance and promote tumor recurrence. Here, we evaluated the efficacy of pan-EGFR inhibitor afatinib and Temozolomide (TMZ) combination on GBM in vitro and in vivo.MethodsWe analyzed the effect of afatinib and temozolomide (TMZ) combination on GBM cells U87MG and U251 engineered to express wild type (WT) EGFR, EGFRvIII or EGFRvIII dead kinase, CSCs isolated from U87 and U87EGFRvIII in vitro. The therapeutic utility of the drug combination was investigated on tumor growth and progression using intracranially injected U87EGFRvIII GBM xenografts.ResultsAfatinib and TMZ combination synergistically inhibited the proliferation, clonogenic survival, motility, invasion and induced senescence of GBM cells compared to monotherapy. Mechanistically, afatinib decreased U87EGFRvIII GBM cell proliferation and motility/invasion by inhibiting EGFRvIII/AKT, EGFRvIII/JAK2/STAT3, and focal adhesion kinase (FAK) signaling pathways respectively. Interestingly, afatinib specifically inhibited EGFRvIII-cMET crosstalk in CSCs, resulting in decreased expression of Nanog and Oct3/4, and in combination with TMZ significantly decreased their self-renewal property in vitro. More interestingly, afatinib and TMZ combination significantly decreased the xenograft growth and progression compared to single drug alone.ConclusionOur study demonstrated significant inhibition of GBM tumorigenicity, CSC maintenance in vitro, and delayed tumor growth and progression in vivo by combination of afatinib and TMZ. Our results warrant evaluation of this drug combination in EGFR and EGFRvIII amplified GBM patients.