Correction: Molecular Heterogeneity in a Patient-Derived Glioblastoma Xenoline is Regulated by Different Cancer Stem Cell Populations.

[This corrects the article DOI: 10.1371/journal.pone.0125838.].

Molecular heterogeneity in a patient-derived glioblastoma xenoline is regulated by different cancer stem cell populations.

Malignant glioblastoma (GBM) is a highly aggressive brain tumor with a dismal prognosis and limited therapeutic options. Genomic profiling of GBM samples has identified four molecular subtypes (Proneural, Neural, Classical and Mesenchymal), which may arise from different glioblastoma stem-like cell (GSC) populations. We previously showed that adherent cultures of GSCs grown on laminin-coated plates (Ad-GSCs) and spheroid cultures of GSCs (Sp-GSCs) had high expression of stem cell markers (CD133, Sox2 and Nestin), but low expression of differentiation markers (ßIII-tubulin and glial fibrillary acid protein). In the present study, we characterized GBM tumors produced by subcutaneous and intracranial injection of Ad-GSCs and Sp-GSCs isolated from a patient-derived xenoline. Although they formed tumors with identical histological features, gene expression analysis revealed that xenografts of Sp-GSCs had a Classical molecular subtype similar to that of bulk tumor cells. In contrast xenografts of Ad-GSCs expressed a Mesenchymal gene signature. Adherent GSC-derived xenografts had high STAT3 and ANGPTL4 expression, and enrichment for stem cell markers, transcriptional networks and pro-angiogenic markers characteristic of the Mesenchymal subtype. Examination of clinical samples from GBM patients showed that STAT3 expression was directly correlated with ANGPTL4 expression, and that increased expression of these genes correlated with poor patient survival and performance. A pharmacological STAT3 inhibitor abrogated STAT3 binding to the ANGPTL4 promoter and exhibited anticancer activity in vivo. Therefore, Ad-GSCs and Sp-GSCs produced histologically identical tumors with different gene expression patterns, and a STAT3/ANGPTL4 pathway is identified in glioblastoma that may serve as a target for therapeutic intervention.

Constitutive activation of signal transducer and activator of transcription 3 (STAT3) and nuclear factor κB signaling in glioblastoma cancer stem cells regulates the Notch pathway.

Malignant gliomas are locally aggressive, highly vascular tumors that have a dismal prognosis, and present therapies provide little improvement in the disease course and outcome. Many types of malignancies, including glioblastoma, originate from a population of cancer stem cells (CSCs) that are able to initiate and maintain tumors. Although CSCs only represent a small fraction of cells within a tumor, their high tumor-initiating capacity and therapeutic resistance drives tumorigenesis. Therefore, it is imperative to identify pathways associated with CSCs to devise strategies to selectively target them. In this study, we describe a novel relationship between glioblastoma CSCs and the Notch pathway, which involves the constitutive activation of STAT3 and NF-κB signaling. Glioma CSCs were isolated and maintained in vitro using an adherent culture system, and the biological properties were compared with the traditional cultures of CSCs grown as multicellular spheres under nonadherent culture conditions. Interestingly, both adherent and spheroid glioma CSCs show constitutive activation of the STAT3/NF-κB signaling pathway and up-regulation of STAT3- and NF-κB-dependent genes. Gene expression profiling also identified components of the Notch pathway as being deregulated in glioma CSCs, and the deregulated expression of these genes was sensitive to treatment with STAT3 and NF-κB inhibitors. This finding is particularly important because Notch signaling appears to play a key role in CSCs in a variety of cancers and controls cell fate determination, survival, proliferation, and the maintenance of stem cells. The constitutive activation of STAT3 and NF-κB signaling pathways that leads to the regulation of Notch pathway genes in glioma CSCs identifies novel therapeutic targets for the treatment of glioma.

Curcumin potentiates rhabdomyosarcoma radiosensitivity by suppressing NF-κB activity.

Ionizing radiation (IR) is an essential component of therapy for alveolar rhabdomyosarcoma. Nuclear factor-kappaB (NF-κΒ) transcription factors are upregulated by IR and have been implicated in radioresistance. We evaluated the ability of curcumin, a putative NF-κΒ inhibitor, and cells expressing genetic NF- κΒ inhibitors (IκBα and p100 super-repressor constructs) to function as a radiosensitizer. Ionizing radiation induced NF-κΒ activity in the ARMS cells in vitro in a dose- and time-dependent manner, and upregulated expression of NF-κΒ target proteins. Pretreatment of the cells with curcumin inhibited radiation-induced NF-κΒ activity and target protein expression. In vivo, the combination of curcumin and IR had synergistic antitumor activity against Rh30 and Rh41 ARMS xenografts. The greatest effect occurred when tumor-bearing mice were treated with curcumin prior to IR. Immunohistochemistry revealed that combination therapy significantly decreased tumor cell proliferation and endothelial cell count, and increased tumor cell apoptosis. Stable expression of the super-repressor, SR-IκBα, that blocks the classical NF-κB pathway, increased sensitivity to IR, while expression of SR-p100, that blocks the alternative pathway, did not. Our results demonstrate that curcumin can potentiate the antitumor activity of IR in ARMS xenografts by suppressing a classical NF-κΒ activation pathway induced by ionizing radiation. These data support testing of curcumin as a radiosensitizer for the clinical treatment of alveolar rhabdomyosarcoma. IMPACT OF WORK: The NF-κΒ protein complex has been linked to radioresistance in several cancers. In this study, we have demonstrated that inhibiting radiation-induced NF-κΒ activity by either pharmacologic (curcumin) or genetic (SR-IκBα) means significantly enhanced the efficacy of radiation therapy in the treatment of alveolar rhabdomyosarcoma cells and xenografts. These data suggest that preventing the radiation-induced activation of the NF-κΒ pathway is a promising way to improve the antitumor efficacy of ionizing radiation and warrants clinical trials.

Inhibition of type I interferon-mediated antiviral action in human glioma cells by the IKK inhibitors BMS-345541 and TPCA-1.

The nuclear factor-kappa B (NFκB) signal transduction pathway plays an important role in immunity, inflammation, cell growth, and survival. Since dysregulation of this pathway results in high, constitutive NFκB activation in various cancers and immune disorders, the development of specific drugs to target this pathway has become a focus for treating these diseases. NFκB regulates various aspects of the cellular response to interferon (IFN). However, the role of the upstream regulator of the NFκB signaling pathway, the inhibitor of κB kinase (IKK) complex, on IFN function has not been examined. In the present study, we examined the effects of 2 IKK inhibitors, N-(1,8-Dimethylimidazo[1,2-a]quinoxalin-4-yl)-1,2-ethanediamine hydrochloride (BMS-345541) and 2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide (TPCA-1), on IFN action in several human glioma cell lines. IKK inhibitors inhibit glioma cell proliferation, as well as TNF-induced RelA (p65) nuclear translocation and NFκB-dependent IL8 gene expression. Importantly, BMS-345541 and TPCA-1 differentially inhibit IFN-induced gene expression, completely suppressing MX1 and GBP1 gene expression, while having only a minor effect on ISG15 expression. Furthermore, these IKK inhibitors displayed marked differences in blocking IFN-induced antiviral action against cytopathic effects and replication of vesicular stomatitis virus (VSV) and encephalomyocarditis virus (EMCV). Our results show that the IKK complex plays an important function in IFN-induced gene expression and antiviral activity. Since VSV and EMCV are oncolytic viruses used in cancer therapy, our results indicate the potential synergy in combining IKK inhibitors with oncolytic viruses.