O⁶-Methylguanine-DNA-methyltransferase (MGMT) and nuclear factor κB (NF-κB) are two key effectors associated with the development of resistance to alkylating agent–based chemotherapy. This prompted us to hypothesize that NF-κB might be involved in MGMT regulation. Consistent with this hypothesis, we have discovered two putative NF-κB binding sites within the MGMT promoter region and showed a specific and direct interaction of NF-κB at each of these sites. Forced expression of the NF-κB subunit p65 in HEK293 cells induced an increase in MGMT expression whereas addition of the NF-κB super repressor ΔNIκB completely abrogated the induction. We also found a significant correlation between the extent of NF-κB activation and MGMT expression in the glioma cell lines and the human glial tumors tested and showed that it was independent of MGMT promoter methylation. Our results are of potential clinical significance because we show that cell lines with ectopic p65 or high constitutive NF-κB activity are less sensitive to nitrosourea treatment and that suppression of MGMT activity with O⁶-benzylguanine completely abolishes the chemoresistance acquired by NF-κB. The findings of our study strongly suggest that NF-κB plays a major role in MGMT regulation and that MGMT is most probably the major player in NF-κB–mediated chemoresistance to alkylating agents. [Cancer Res 2007;67(18):8952–9]

Gliomas express many genes that play a role in neural precursor cells (NPCs), but no direct comparison between glioma and stem cell (SC) gene expression profiles has been performed. To investigate the similarities and differences between gliomas and SCs, we compared the microRNA (miRNA) expression signatures of glial tumors, embryonic SCs (ESCs), NPCs, and normal adult brains from both human and mouse tissues. We demonstrated that both human gliomas (regardless of their grade) and methylcholanthrene-induced mouse glioma shared an miRNA expression profile that is reminiscent of NPCs. About half of the miRNAs expressed in the shared profile clustered in seven genomic regions susceptible to genetic/epigenetic alterations in various cancers. These clusters comprised the miR17 family, mir183-182, and the SC-specific clusters mir367-302 and mir371-373, which are upregulated in gliomas, ESCs, and NPCs. The bipartite cluster of 7 + 46 miRNAs on chromosome 14q32.31, which might represent the largest tumor suppressor miRNA cluster, was downregulated in the shared expression profile. This study provides the first evidence for association between these clusters and gliomas. Despite the broad similarity in the miRNA expression profiles, 15 miRNAs showed disparate expression between SC and gliomas. Ten miRNAs belong to the 2 SC-specific clusters and the remaining (mir135b, mir141, mir205, mir200C, and mir301a) have been previously shown to associate with malignancies. Our finding showed that all gliomas displayed NPC-like miRNA signatures, which may have implications for studies of glioma origins. Furthermore, careful study of the 15 miRNAs that differ in expression between SCs and gliomas, particularly those 5 that are not SC-specific, may enhance our understanding of gliomagenesis.

Androgen receptor: a potential therapeutic target for glioblastoma Androgen receptor: a potential therapeutic target for glioblastoma

Neuromyelitis optica (NMO) is a chronic autoimmune disease of the central nervous system (CNS). The main immunological feature of the disease is the presence of autoantibodies to Aquaporin 4 (AQP4+), identified in about 82 % of cases. Currently, there are no reliable biomarkers for monitoring treatment response in patients with NMO. In an effort to identify biomarkers, we analyzed microRNAs (miRNAs) in the blood of rituximab-treated NMO patients before and after therapy. Total RNA extracted from whole blood of nine rituximab-responsive NMO patients before and 6 months following treatment was subjected to small RNAseq analysis. The study included an additional group of seven untreated AQP4+ seropositive NMO patients and 15 healthy controls (HCs). Fourteen miRNAs were up regulated and 32 were downregulated significantly in the blood of NMO patients following effective therapy with rituximab (all p < 0.05). Furthermore, we show that expression of 17 miRNAs was significantly higher and of 25 miRNAs was significantly lower in untreated NMO patients compared with HCs (all p < 0.05). Following rituximab treatment, the expression levels of 10 of the 17 miRNAs that show increased expression in NMO reverted to the levels seen in HCs. Six of these “normalized” miRNAs are known as brain-specific/enriched miRNAs. Specific miRNA signatures in whole blood of patients with NMO might serve as biomarkers for therapy response. Furthermore, monitoring the levels of brain-specific/enriched miRNAs in the blood might reflect the degree of disease activity in the CNS of inflammatory demyelinating disorders.

Androgen receptor (AR) is a ligand-mediated transcription factor that belongs to the superfamily of steroid receptors. AR is overexpressed in most glioblastomas and is a potential therapeutic target. In prostate and breast cancers, AR activation can be achieved also by a ligand-independent signaling through receptor tyrosine kinases such as epidermal growth factor receptor (EGFR). Considering its major role in glioblastoma, we explored whether EGFR is involved in AR signaling in this tumor. Analysis of mRNA expression in 28 glioblastoma samples with quantitative real-time reverse-transcription polymerase chain reaction revealed a positive and significant correlation between AR and EGFR mRNA expression levels (R = 0.47, p = 0.0092), which was validated by The Cancer Genome Atlas dataset (n = 671) analysis (R = 0.3, p = 0.00006). Using Western blotting and immunofluorescence staining, we showed that the transduced overexpression of EGFR or its variant EGFRvIII in the U87MG cells induced AR protein overexpression and nuclear translocation and Protein kinase B (AKT) S473 and AR S210/213 phosphorylation. The EGFR kinase inhibitor afatinib and the AKT inhibitor MK2206 reduced AR nuclear translocation. Afatinib diminished AKT phosphorylation at 30 min and 6 h in the EGFR- and EGFRvIII-overexpressing cells, respectively, and decreased AR phosphorylation in EGFR-overexpressing cells at 4 h. Afatinib or MK2206 combination therapy with the AR antagonist enzalutamide in the EGFR and EGFRvIII-overexpressing cells had synergistic efficacy. Our findings suggest that EGFR signaling is involved in AR activation in glioblastoma and buttresses the concept of combining an EGFR signaling inhibitor with AR antagonists as a potential glioblastoma treatment.