Glioblastoma (GBM), the most severe and common brain tumor in adults, is characterized by multiple somatic mutations and aberrant activation of inflammatory responses. Immune cell infiltration and subsequent inflammation cause tumor growth and resistance to therapy. Somatic loss-of-function mutations in the gene encoding tumor suppressor protein p53 (TP53) are frequently observed in various cancers. However, numerous studies suggest that TP53 regulates malignant phenotypes by gain-of-function (GOF) mutations. Here we demonstrate that a TP53 GOF mutation promotes inflammation in GBM. Ectopic expression of a TP53 GOF mutant induced transcriptomic changes, which resulted in enrichment of gene signatures related to inflammation and chemotaxis. Bioinformatics analyses revealed that a gene signature, upregulated by the TP53 GOF mutation, is associated with progression and shorter overall survival in GBM. We also observed significant correlations between the TP53 GOF mutation signature and inflammation in the clinical database of GBM and other cancers. The TP53 GOF mutant showed upregulated C-C motif chemokine ligand 2 (CCL2) and tumor necrosis factor alpha (TNFA) expression via nuclear factor kappa B (NFκB) signaling, consequently increasing microglia and monocyte-derived immune cell infiltration. Additionally, TP53 GOF mutation and CCL2 and TNFA expression correlated positively with tumor-associated immunity in patients with GBM. Taken together, our findings suggest that the TP53 GOF mutation plays a crucial role in inflammatory responses, thereby deteriorating prognostic outcomes in patients with GBM.

BackgroundGenetic engineering technology such as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system provides a powerful tool for developing disease models and determining gene functions. Recent interests in canine cancer models have highlighted the necessity of developing genetic engineering tools for dogs. In this study, we attempted to generate optimized CRISPR/Cas9 system to target canine tumor protein 53 (TP53), one of the most crucial tumor suppressor genes, to establish TP53 knockout canine cells for canine cancer research.ResultsWe constructed CRISPR/Cas9 vectors using each of three TP53 gene-targeting guide RNAs (gRNAs) with minimal off-target potential. After transfection, we obtained several clones of TP53 knockout cells containing “indel” mutations in the targeted locus which had infinite cellular life span, resistance to genotoxicity, and unstable genomic status in contrast to normal cells. Of the established TP53 knockout cells, TP53KO#30 cells targeted by TP53 gRNA #30 showed non-cancerous phenotypes without oncogenic activation both in vitro and in vivo. More importantly, no off-target alteration was detected in TP53KO#30 cells. We also tested the developmental capacity of TP53 knockout cells after application of the somatic cell nuclear transfer technique.ConclusionsOur results indicated that TP53 in canine cells was effectively and specifically targeted by our CRISPR/Cas9 system. Thus, we suggest our CRISPR/Cas9-derived canine TP53 knockout cells as a useful platform to reveal novel oncogenic functions and effects of developing anti-cancer therapeutics.Electronic supplementary materialThe online version of this article (10.1186/s12896-018-0491-5) contains supplementary material, which is available to authorized users.

Recent advances in somatic cell nuclear transfer (SCNT) in canines facilitate the production of canine transgenic models. Owing to the importance of stable and strong promoter activity in transgenic animals, we tested human elongation factor 1α (hEF1α) and cytomegalovirus (CMV) promoter sequences in SCNT transgenic dogs. After transfection, transgenic donor fibroblasts with the hEF1α-enhanced green fluorescence protein (EGFP) transgene were successfully isolated using fluorescence-activated cell sorting (FACS). We obtained four puppies, after SCNT, and identified three puppies as being transgenic using PCR analysis. Unexpectedly, EGFP regulated by hEF1α promoter was not observed at the organismal and cellular levels in these transgenic dogs. EGFP expression was rescued by the inhibition of DNA methyltransferases, implying that the hEF1α promoter is silenced by DNA methylation. Next, donor cells with CMV-EGFP transgene were successfully established and SCNT was performed. Three puppies of six born puppies were confirmed to be transgenic. Unlike hEF1α-regulated EGFP, CMV-regulated EGFP was strongly detectable at both the organismal and cellular levels in all transgenic dogs, even after 19 months. In conclusion, our study suggests that the CMV promoter is more suitable, than the hEF1α promoter, for stable transgene expression in SCNT-derived transgenic canine model.

Glioblastoma (GBM) is the most aggressive and malignant brain tumor, resulting in a poor prognosis. The current therapy for GBM consists in concurrent radiation and chemotherapy following removal of the tumor. Although the therapy prolongs patient survival, recurrence often occurs. The major cause of tumor recurrence is thought to be GBM stem cells (GSCs), which aid the development of chemo-radiotherapy resistance, and can self-renew and aberrantly differentiate. Therefore, GSCs should be targeted to eradicate the tumor and prevent recurrence. Transcriptomic analysis has categorized GBM into proneural (PN), mesenchymal and classical subtypes, and the outcome of recurrence and prognosis markedly depends on subtype. To identify specific GSC markers, the present study analyzed public microarray and RNA-seq data and identified dihydropyrimidinase-related protein 5 (DRP5) as a candidate GSC marker. DRP5 is known to mediate semaphorin 3A signaling and is involved in the regulation of neurite outgrowth and axon guidance during neuronal development. In the present study, DRP5 was specifically upregulated in the PN-subtype GSCs and served crucial roles in maintaining GSC properties, including tumor sphere formation, stem cell marker expression and xenograft tumor growth. Furthermore, bioinformatics analysis revealed that DRP5 expression was positively correlated with signatures of stemness, including Notch, Hedgehog and Wnt/β-catenin expression, which are also known to be positively correlated with PN-subtype gene signatures. Conversely, DRP5 expression was negatively correlated with NF-κB and signal transducer and activator of transcription 3 stemness signatures, which are negatively correlated with PN-subtype gene signatures. Taken together, these findings suggested that DRP5 was specifically expressed in PN-subtype GSCs and may be used as a functional marker of PN-subtype GSCs.

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