PBI-05204, a supercritical CO2 extract of Nerium oleander, suppresses glioblastoma stem cells by inhibiting GRP78 and inducing programmed necroptotic cell death.

Successful treatment of glioblastoma multiforme (GBM), an aggressive form of primary brain neoplasm, mandates the need to develop new therapeutic strategies. In this study, we investigated the potential of PBI-05204 in targeting GBM stem cells (GSCs) and the underlying mechanisms. Treatment with PBI-05204 significantly reduced both the number and size of tumor spheres derived from patient-derived GSCs (GBM9, GSC28 and TS543), and suppressed the tumorigenesis of GBM9 xenografts. Moreover, PBI-05204 treatment led to a significant decrease in the expression of CD44 and NANOG, crucial markers of progenitor stem cells, in GBM9 and GSC28 GSCs. This treatment also down-regulated GRP78 expression in both GSC types. Knocking down GRP78 expression through GRP78 siRNA transfection in GBM9 and GSC28 GSCs also resulted in reduced spheroid size and CD44 expression. Combining PBI-05204 with GRP78 siRNA further decreased spheroid numbers compared to GRP78 siRNA treatment alone. PBI-05204 treatment led to increased expression of pRIP1K and pRIP3K, along with enhanced binding of RIPK1/RIPK3 in GBM9 and GSC28 cells, resembling the effects observed in GRP78-silenced GSCs, suggesting that PBI-05204 induced necroptosis in these cells. Furthermore, oleandrin, a principle active cardiac glycoside component of PBI-05204, showed the ability to inhibit the self-renewal capacity in GSCs. These findings highlight the potential of PBI-05204 as a promising candidate for the development of novel therapies that target GBM stem cells.

Evaluation of the Protective Effect of Compound Kushen Injection Against Radiation- induced Pneumonitis in Mice.

Background Radiation-induced lung injury (RILI) via inflammation is a common adverse effect of thoracic radiation that negatively impacts patient quality of life and survival. Compound kushen injection (CKI), a botanical drug treatment, was examined for its ability to reduce RILI, and inflammatory responses and improve survival in mice exposed total lung irradiation (TLI). CKI's specific mechanisms of action were also evaluated. Methods C3H mice underwent TLI and were treated with CKI (2, 4, or 8 mL/kg) intraperitoneally once a day for 8 weeks. The effects of CKI on survival were estimated by Kaplan-Meier survival analysis and compared by log-rank test. RILI damage was evaluated by histopathology and micro-computed tomography (CT). Inflammatory cytokines and cyclooxygenase metabolites were examined by IHC staining, western blot, and ELISA. Results Pre-irradiation treatment with 4 or 8 mL/kg CKI starting 2 weeks before TLI or concurrent treatment with 8 mL/kg CKI were associated with a significantly longer survival compared with TLI vehicle-treated group ( P < 0.05). Micro-CT images evaluations showed that concurrent treatment with 8 mL/kg CKI was associated with significantly lower incidence of RILI ( P < 0.05). Histological evaluations revealed that concurrent TLI treatment of CKI (4 and 8 mL/kg) significantly reduced lung inflammation (p < 0.05). Mechanistic investigation showed that at 72 hours after radiation, TLI plus vehicle mice had significantly elevated serum IL6, IL17A, and TGF-ß levels compared with non-irradiated, age-matched normal mice; in contrast, levels of these cytokines in mice that received TLI plus CKI treatment were lower than those in the TLI plus vehicle-treated mice ( P < 0.05) and similar to the nonirradiated mice. IHC staining showed that the CKI treatment led to a reduction of TGF-ß positive cells in the lung tissues of TLI mice (P < 0.01). The concurrent CKI with TLI treatment group had a significant reduction in COX-2 activity and COX-2 metabolites compared with the TLI vehicle-treated group ( P < 0.05). Conclusions These data suggest that CKI treatment was associated with reduced radiation-induced inflammation in lung tissues, reduced RILI, and improved survival. Further investigation of CKI in human clinical trials as a potential radioprotector against RILI to improve patients' quality of life and survival is warranted.

Targeting Glutamine Metabolism with a Novel Na+/K+-ATPase Inhibitor RX108 in Hepatocellular Carcinoma.

The poor prognosis and limited therapeutic options for human hepatocellular carcinoma (HCC), the most common form of liver cancer, highlight the urgent need to identify novel therapeutic modalities. Here, we describe the antitumor activity and underlying molecular mechanisms of a novel Na+/K+-ATPase inhibitor RX108 in human HCC cells and its xenograft model. RX108 dose-dependently inhibited HCC cell proliferation in vitro and tumor growth in a xenograft mouse model, and that the inhibition was associated with induction of apoptosis. Mechanistically, RX108 significantly downregulated alanine serine cysteine transporter 2 (ASCT2) protein expression and reduced glutamine and glutamate concentration in HCC cells and tumors. In addition, RX108 exposure led to a significant decrease in cell energy metabolism in Huh7 and Hep3B cells, including decreased levels of glutathione, NADH, NADPH, and mitochondrial respiration oxygen consumption rate. Furthermore, HCC cells exhibited evidence of glutamine addiction; the antiproliferative effect of RX108 was dependent on glutamine transport. Clinically, elevated ASCT2 mRNA expression in HCCs was associated with unfavorable survival. Taken together, these findings reveal a novel approach to target glutamine metabolism through inhibiting Na+/K+-ATPase and provide a rationale for using RX108 to treat HCC in patients whose tumors express ASCT2 at high levels. RX108 is currently under clinical development.

Heat-induced SIRT1-mediated H4K16ac deacetylation impairs resection and SMARCAD1 recruitment to double strand breaks.

Hyperthermia inhibits DNA double-strand break (DSB) repair that utilizes homologous recombination (HR) pathway by a poorly defined mechanism(s); however, the mechanisms for this inhibition remain unclear. Here we report that hyperthermia decreases H4K16 acetylation (H4K16ac), an epigenetic modification essential for genome stability and transcription. Heat-induced reduction in H4K16ac was detected in humans, Drosophila, and yeast, indicating that this is a highly conserved response. The examination of histone deacetylase recruitment to chromatin after heat-shock identified SIRT1 as the major deacetylase subsequently enriched at gene-rich regions. Heat-induced SIRT1 recruitment was antagonized by chromatin remodeler SMARCAD1 depletion and, like hyperthermia, the depletion of the SMARCAD1 or combination of the two impaired DNA end resection and increased replication stress. Altered repair protein recruitment was associated with heat-shock-induced γ-H2AX chromatin changes and DSB repair processing. These results support a novel mechanism whereby hyperthermia impacts chromatin organization owing to H4K16ac deacetylation, negatively affecting the HR-dependent DSB repair.

The Botanical Drug PBI-05204, a Supercritical CO2 Extract of Nerium Oleander, Is Synergistic With Radiotherapy in Models of Human Glioblastoma.

Glioblastoma multiforme (GBM) is the most common as well as one of the most malignant types of brain cancer. Despite progress in development of novel therapies for the treatment of GBM, it remains largely incurable with a poor prognosis and a very low life expectancy. Recent studies have shown that oleandrin, a unique cardiac glycoside from Nerium oleander, as well as a defined extract (PBI-05204) that contains this molecule, inhibit growth of human glioblastoma, and modulate glioblastoma patient-derived stem cell-renewal properties. Here we demonstrate that PBI-05204 treatment leads to an increase in vitro in the sensitivity of GBM cells to radiation in which the main mechanisms are the transition from autophagy to apoptosis, enhanced DNA damage and reduced DNA repair after radiotherapy (RT) administration. The combination of PBI-05204 with RT was associated with reduced tumor progression evidenced by both subcutaneous as well as orthotopic implanted GBM tumors. Collectively, these results reveal that PBI-05204 enhances antitumor activity of RT in preclinical/murine models of human GBM. Given the fact that PBI-05204 has already been examined in Phase I and II clinical trials for cancer patients, its efficacy when combined with standard-of-care radiotherapy regimens in GBM should be explored.

Fraction B From Catfish Epidermal Secretions Kills Pancreatic Cancer Cells, Inhibits CD44 Expression and Stemness, and Alters Cancer Cell Metabolism.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer related death in western countries. The successful treatment of PDAC remains limited. We investigated the effect of Fraction B, which is a fraction purified from catfish (Arius bilineatus, Val.) skin secretions containing proteins and lipids, on PDAC biology both in-vivo and in-vitro. We report here that Fraction B potently suppressed the proliferation of both human and mouse pancreatic cancer cells in vitro and significantly reduced the growth of their relevant xenograft (Panc02) and orthotopic tumors (human Panc-1 cells) (p < 0.05). The Reverse Phase Protein Array (RPPA) data obtained from the tumor tissues derived from orthotopic tumor bearing mice treated with Fraction B showed that Fraction B altered the cancer stem cells related pathways and regulated glucose and glutamine metabolism. The down-regulation of the cancer stem cell marker CD44 expression was further confirmed in Panc-1 cells. CBC and blood chemistry analyses showed no systemic toxicity in Fraction B treated Panc-1 tumor bearing mice compared to that of control group. Our data support that Fraction B is a potential candidate for PDAC treatment.

The Botanical Drug PBI-05204, a Supercritical CO2 Extract of Nerium Oleander, Inhibits Growth of Human Glioblastoma, Reduces Akt/mTOR Activities, and Modulates GSC Cell-Renewal Properties.

Glioblastoma multiform (GBM) is the most common primary glial tumor resulting in very low patient survival despite current extensive therapeutic efforts. Emerging evidence suggests that more effective treatments are required to overcome tumor heterogeneity, drug resistance and a complex tumor-supporting microenvironment. PBI-05204 is a specifically formulated botanical drug consisting of a modified supercritical C02 extract of Nerium oleander that has undergone both phase I and phase II clinical trials in the United States for treatment of patients with a variety of advanced cancers. The present study was designed to investigate the antitumor efficacy of this botanical drug against glioblastoma using both in vitro and in vivo cancer models as well as exploring efficacy against glioblastoma stem cells. All three human GBM cell lines, U87MG, U251, and T98G, were inhibited by PBI-05204 in a concentration dependent manner that was characterized by induction of apoptosis as evidenced by increased ANNEXIN V staining and caspase activities. The expression of proteins associated with both Akt and mTOR pathway was suppressed by PBI-05240 in all treated human GBM cell lines. PBI-05204 significantly suppressed U87 spheroid formation and the expression of important stem cell markers such as SOX2, CD44, and CXCR4. Oral administration of PBI-05204 resulted in a dose-dependent inhibition of U87MG, U251, and T98G xenograft growth. Additionally, PBI-05204-treated mice carrying U87-Luc cells as an orthotropic model exhibited significantly delayed onset of tumor proliferation and significantly increased overall survival. Immunohistochemical staining of xenograft derived tumor sections revealed dose-dependent declines in expression of Ki67 and CD31 positive stained cells but increased TUNEL staining. PBI-05204 represents a novel therapeutic botanical drug approach for treatment of glioblastoma as demonstrated by significant responses with in vivo tumor models. Both in vitro cell culture and immunohistochemical studies of tumor tissue suggest drug induction of tumor cell apoptosis and inhibition of PI3k/mTOR pathways as well as cancer stemness. Given the fact that PBI-05204 has already been examined in phase I and II clinical trials for cancer patients, its efficacy when combined with standard of care chemotherapy and radiotherapy should be explored in future clinical trials of this difficult to treat brain cancer.

Nonhomologous End Joining Is More Important Than Proton Linear Energy Transfer in Dictating Cell Death.

PURPOSE: This study seeks to identify biological factors that may yield a therapeutic advantage of proton therapy versus photon therapy. Specifically, we address the role of nonhomologous end-joining (NHEJ) and homologous recombination (HR) in the survival of cells in response to clinical photon and proton beams. METHODS AND MATERIALS: We irradiated HT1080, M059K (DNA-PKcs+/+), and HCC1937 human cancer cell lines and their isogenic counterparts HT1080-shDNA-PKcs, HT1080-shRAD51IND, M059J (DNA-PKcs-/-), and HCC1937-BRCA1 (BRCA1 complemented) to assess cell clonogenic survival and γ-H2AX radiation-induced foci. Cells were irradiated with either clinically relevant photons or 1 of 3 proton linear energy transfer (LET) values. RESULTS: Our results indicate that NHEJ deficiency is more important in dictating cell survival than proton LET. Cells with disrupted HR through BRCA1 mutation showed increased radiosensitivity only for high-LET protons whereas RAD51 depletion showed increased radiosensitivity for both photons and protons. DNA double strand breaks, assessed by γ-H2AX radiation-induced foci, showed greater numbers after 24 hours in cells exposed to higher LET protons. We also observed that NHEJ-deficient cells were unable to repair the vast majority of double strand breaks after 24 hours. CONCLUSIONS: BRCA1 mutation significantly sensitizes cells to protons, but not photons. Loss of NHEJ renders cells hypersensitive to radiation, whereas the relative importance of HR increases with LET across several cell lines. This may be attributable to the more clustered damage induced by higher LET protons, which are harder to repair through NHEJ. This highlights the importance of tumor biology in dictating treatment modality and suggests BRCA1 as a potential biomarker for proton therapy response. Our data also support the use of pharmacologic inhibitors of DNA repair to enhance the sensitivity to different radiation types, although this raises issues for normal tissue toxicity.

Pre-existing H4K16ac levels in euchromatin drive DNA repair by homologous recombination in S-phase.

The homologous recombination (HR) repair pathway maintains genetic integrity after DNA double-strand break (DSB) damage and is particularly crucial for maintaining fidelity of expressed genes. Histone H4 acetylation on lysine 16 (H4K16ac) is associated with transcription, but how pre-existing H4K16ac directly affects DSB repair is not known. To answer this question, we used CRISPR/Cas9 technology to introduce I-SceI sites, or repair pathway reporter cassettes, at defined locations within gene-rich (high H4K16ac/euchromatin) and gene-poor (low H4K16ac/heterochromatin) regions. The frequency of DSB repair by HR is higher in gene-rich regions. Interestingly, artificially targeting H4K16ac at specific locations using gRNA/dCas9-MOF increases HR frequency in euchromatin. Finally, inhibition/depletion of RNA polymerase II or Cockayne syndrome B protein leads to decreased recruitment of HR factors at DSBs. These results indicate that the pre-existing H4K16ac status at specific locations directly influences the repair of local DNA breaks, favoring HR in part through the transcription machinery.

SMARCAD1 Phosphorylation and Ubiquitination Are Required for Resection during DNA Double-Strand Break Repair.

The chromatin remodeling factor SMARCAD1, an SWI/SNF ATPase family member, has a role in 5' end resection at DNA double-strand breaks (DSBs) to produce single-strand DNA (ssDNA), a critical step for subsequent checkpoint and repair factor loading to remove DNA damage. However, the mechanistic details of SMARCAD1 coupling to the DNA damage response and repair pathways remains unknown. Here we report that SMARCAD1 is recruited to DNA DSBs through an ATM-dependent process. Depletion of SMARCAD1 reduces ionizing radiation (IR)-induced repairosome foci formation and DSB repair by homologous recombination (HR). IR induces SMARCAD1 phosphorylation at a conserved T906 by ATM kinase, a modification essential for SMARCAD1 recruitment to DSBs. Interestingly, T906 phosphorylation is also important for SMARCAD1 ubiquitination by RING1 at K905. Both these post-translational modifications are critical for regulating the role of SMARCAD1 in DNA end resection, HR-mediated repair, and cell survival after DNA damage.

MOF Suppresses Replication Stress and Contributes to Resolution of Stalled Replication Forks.

The human MOF (hMOF) protein belongs to the MYST family of histone acetyltransferases and plays a critical role in transcription and the DNA damage response. MOF is essential for cell proliferation; however, its role during replication and replicative stress is unknown. Here we demonstrate that cells depleted of MOF and under replicative stress induced by cisplatin, hydroxyurea, or camptothecin have reduced survival, a higher frequency of S-phase-specific chromosome damage, and increased R-loop formation. MOF depletion decreased replication fork speed and, when combined with replicative stress, also increased stalled replication forks as well as new origin firing. MOF interacted with PCNA, a key coordinator of replication and repair machinery at replication forks, and affected its ubiquitination and recruitment to the DNA damage site. Depletion of MOF, therefore, compromised the DNA damage repair response as evidenced by decreased Mre11, RPA70, Rad51, and PCNA focus formation, reduced DNA end resection, and decreased CHK1 phosphorylation in cells after exposure to hydroxyurea or cisplatin. These results support the argument that MOF plays an important role in suppressing replication stress induced by genotoxic agents at several stages during the DNA damage response.

Differentiation of Human Induced Pluripotent or Embryonic Stem Cells Decreases the DNA Damage Repair by Homologous Recombination.

The nitric oxide (NO)-cyclic GMP pathway contributes to human stem cell differentiation, but NO free radical production can also damage DNA, necessitating a robust DNA damage response (DDR) to ensure cell survival. How the DDR is affected by differentiation is unclear. Differentiation of stem cells, either inducible pluripotent or embryonic derived, increased residual DNA damage as determined by γ-H2AX and 53BP1 foci, with increased S-phase-specific chromosomal aberration after exposure to DNA-damaging agents, suggesting reduced homologous recombination (HR) repair as supported by the observation of decreased HR-related repair factor foci formation (RAD51 and BRCA1). Differentiated cells also had relatively increased fork stalling and R-loop formation after DNA replication stress. Treatment with NO donor (NOC-18), which causes stem cell differentiation has no effect on double-strand break (DSB) repair by non-homologous end-joining but reduced DSB repair by HR. Present studies suggest that DNA repair by HR is impaired in differentiated cells.

MCL-1 Depletion Impairs DNA Double-Strand Break Repair and Reinitiation of Stalled DNA Replication Forks.

Myeloid cell leukemia 1 (MCL-1) is a prosurvival BCL-2 protein family member highly expressed in hematopoietic stem cells (HSCs) and regulated by growth factor signals that manifest antiapoptotic activity. Here we report that depletion of MCL-1 but not its isoform MCL-1S increases genomic instability and cell sensitivity to ionizing radiation (IR)-induced death. MCL-1 association with genomic DNA increased postirradiation, and the protein colocalized with 53BP1 foci. Postirradiation, MCL-1-depleted cells exhibited decreased γ-H2AX foci, decreased phosphorylation of ATR, and higher levels of residual 53BP1 and RIF1 foci, suggesting that DNA double-strand break (DSB) repair by homologous recombination (HR) was compromised. Consistent with this model, MCL-1-depleted cells had a reduced frequency of IR-induced BRCA1, RPA, and Rad51 focus formation, decreased DNA end resection, and decreased HR repair in the DR-GFP DSB repair model. Similarly, after HU induction of stalled replication forks in MCL-1-depleted cells, there was a decreased ability to subsequently restart DNA synthesis, which is normally dependent upon HR-mediated resolution of collapsed forks. Therefore, the present data support a model whereby MCL-1 depletion increases 53BP1 and RIF1 colocalization at DSBs, which inhibits BRCA1 recruitment, and sensitizes cells to DSBs from IR or stalled replication forks that require HR for repair.

Cross-Sectional Study for the Detection of Mutations in the Beta-Globin Gene Among Patients with Hemoglobinopathies in the Bengali Population.

AIMS: Thalassemia is a common autosomal recessive blood disorder, which is most prevalent in South East Asian and Mediterranean populations. It is considered as a major health burden in the Indian population. The aims of the present study were to investigate the common, as well as uncommon, mutations responsible for thalassemia in the Bengali population. METHODS: The Bengali state was divided into four sampling zones. Mutation detection was done using Sanger sequencing of the HBB gene. RESULTS: A total of 14 different mutations were observed, including rare mutations IVS1-130(G>C), IVS1-129(A>C), -90(T>C), CD16(-C), -30(T>C), CD15(-T), and a novel mutation CD53(C>T). The frequencies of IVS1-5(G>C) and CD26(G>A) mutations were higher than other mutations. There were also some silent polymorphisms found in the studied group, CD3(T>C), CD10(C>A), IVSII-16(G>C), IVSII-74(T>G), -42(C>G). CONCLUSION: The present study is the first attempt to screen for ß-thalassemia-causing mutations by direct sequencing in different districts of West Bengal. The information obtained from the present study may be helpful for thalassemia management and prenatal mutation detection.

ß2-spectrin depletion impairs DNA damage repair.

ß2-Spectrin (ß2SP/SPTBN1, gene SPTBN1) is a key TGF-ß/SMAD3/4 adaptor and transcriptional cofactor that regulates TGF-ß signaling and can contribute to liver cancer development. Here we report that cells deficient in ß2-Spectrin (ß2SP) are moderately sensitive to ionizing radiation (IR) and extremely sensitive to agents that cause interstrand cross-links (ICLs) or replication stress. In response to treatment with IR or ICL agents (formaldehyde, cisplatin, camptothecin, mitomycin), ß2SP deficient cells displayed a higher frequency of cells with delayed γ-H2AX removal and a higher frequency of residual chromosome aberrations. Following hydroxyurea (HU)-induced replication stress, ß2SP-deficient cells displayed delayed disappearance of γ-H2AX foci along with defective repair factor recruitment (MRE11, CtIP, RAD51, RPA, and FANCD2) as well as defective restart of stalled replication forks. Repair factor recruitment is a prerequisite for initiation of DNA damage repair by the homologous recombination (HR) pathway, which was also defective in ß2SP deficient cells. We propose that ß2SP is required for maintaining genomic stability following replication fork stalling, whether induced by either ICL damage or replicative stress, by facilitating fork regression as well as DNA damage repair by homologous recombination.

Constitutive and ligand-induced EGFR signalling triggers distinct and mutually exclusive downstream signalling networks.

Epidermal growth factor receptor (EGFR) overexpression plays an important oncogenic role in cancer. Regular EGFR protein levels are increased in cancer cells and the receptor then becomes constitutively active. However, downstream signals generated by constitutively activated EGFR are unknown. Here we report that the overexpressed EGFR oscillates between two distinct and mutually exclusive modes of signalling. Constitutive or non-canonical EGFR signalling activates the transcription factor IRF3 leading to expression of IFI27, IFIT1 and TRAIL. Ligand-mediated activation of EGFR switches off IRF3-dependent transcription, activates canonical extracellular signal-regulated kinase (ERK) and Akt signals, and confers sensitivity to chemotherapy and virus-induced cell death. Mechanistically, the distinct downstream signals result from a switch of EGFR-associated proteins. EGFR constitutively complexes with IRF3 and TBK1 leading to TBK1 and IRF3 phosphorylation. Addition of epidermal growth factor dissociates TBK1, IRF3 and EGFR leading to a loss of IRF3 activity, Shc-EGFR association and ERK activation. Finally, we provide evidence for non-canonical EGFR signalling in glioblastoma.

MOF phosphorylation by ATM regulates 53BP1-mediated double-strand break repair pathway choice.

Cell-cycle phase is a critical determinant of the choice between DNA damage repair by nonhomologous end-joining (NHEJ) or homologous recombination (HR). Here, we report that double-strand breaks (DSBs) induce ATM-dependent MOF (a histone H4 acetyl-transferase) phosphorylation (p-T392-MOF) and that phosphorylated MOF colocalizes with γ-H2AX, ATM, and 53BP1 foci. Mutation of the phosphorylation site (MOF-T392A) impedes DNA repair in S and G2 phase but not G1 phase cells. Expression of MOF-T392A also blocks the reduction in DSB-associated 53BP1 seen in wild-type S/G2 phase cells, resulting in enhanced 53BP1 and reduced BRCA1 association. Decreased BRCA1 levels at DSB sites correlates with defective repairosome formation, reduced HR repair, and decreased cell survival following irradiation. These data support a model whereby ATM-mediated MOF-T392 phosphorylation modulates 53BP1 function to facilitate the subsequent recruitment of HR repair proteins, uncovering a regulatory role for MOF in DSB repair pathway choice during S/G2 phase.

Role of 53BP1 in the regulation of DNA double-strand break repair pathway choice.

The p53-binding protein 1 (53BP1) is a well-known DNA damage response (DDR) factor, which is recruited to nuclear structures at the site of DNA damage and forms readily visualized ionizing radiation (IR) induced foci. Depletion of 53BP1 results in cell cycle arrest in G2/M phase as well as genomic instability in human as well as mouse cells. Within the DNA damage response mechanism, 53BP1 is classified as an adaptor/mediator, required for processing of the DNA damage response signal and as a platform for recruitment of other repair factors. More recently, specific 53BP1 contributions to DSB repair pathway choice have been recognized and are being characterized. In this review, we have summarized recent advances in understanding the role of 53BP1 in regulating DNA DSBs repair pathway choice, variable diversity joining [V(D)J] recombination and class-switch recombination (CSR).

The role of NF-κB in the pathogenesis of glioma.

Activation of NF-κB affects multiple aspects of cancer biology including cell survival and resistance to treatment. Glioblastoma (GBM) is the most common primary malignant tumor of the brain in adults and is resistant to treatment. Recent studies have reported that NF-κB activation in GBM is widespread and have elucidated the underlying regulatory mechanisms. EGFR gene amplification and mutation are among the key genetic alterations in GBM, and aberrant EGFR signaling is a key activator of NF-κB in GBM. In this review we discuss the evidence for activation of NF-κB in GBM and the key signaling pathways involved. Substantial evidence suggests a role for NF-κB in the pathogenesis of GBM and its resistance to treatment, indicating that NF-κB pathways may be useful targets for treatment.

Opposing effect of EGFRWT on EGFRvIII-mediated NF-κB activation with RIP1 as a cell death switch.

RIP1 is a central mediator of cell death in response to cell stress but can also mediate cell survival by activating NF-κB. Here, we show that RIP1 acts as a switch in EGFR signaling. EGFRvIII is an oncogenic mutant that does not bind ligand and is coexpressed with EGFRWT in glioblastoma multiforme (GBM). EGFRvIII recruits ubiquitin ligases to RIP1, resulting in K63-linked ubiquitination of RIP1. RIP1 binds to TAK1 and NEMO, forming an EGFRvIII-RIP1 signalosome that activates NF-κB. RIP1 is essential for EGFRvIII-mediated oncogenicity and correlates with NF-κB activation in GBM. Surprisingly, activation of EGFRWT with EGF results in a negative regulation of EGFRvIII, with dissociation of the EGFRvIII-RIP1 signalosome, loss of RIP1 ubiquitination and NF-κB activation, and association of RIP1 with FADD and caspase-8. If EGFRWT is overexpressed with EGFRvIII, the addition of EGF leads to a RIP1 kinase-dependent cell death. The EGFRWT-EGFRvIII-RIP1 interplay may regulate oncogenicity and vulnerability to targeted treatment in GBM.