Glioblastoma Multiforme (GBM) is the most aggressive form of primary brain tumour, with a median survival of 12–14 months after diagnosis. Although GBM has been extensively characterised on the molecular level during the past decades, many targeted therapies have been proved to be ineffective due to high heterogeneity of GBM. Thus, novel therapies targeting the altered metabolism which is exhibited by all cancer cells have gained more attentions. Our strategy is the therapeutic ketogenic diet (KD), a high fat, low carbohydrate and adequate protein diet, which has been recognized as a treatment for refractory paediatric epilepsy. Recent studies have shown that KD reduces tumour growth and potentiates the effects of radiotherapy in some glioma animal models. However, the underlying mechanism is still unclear. To unravel the mechanism of action, we carried out small-RNA sequencing and chromatin modifying enzyme analysis using brain tumour samples from GBM mice model, fed with either KD or standard diet (SD). Our results highlighted an overall upregulation of tumour suppressor miRNAs and key chromatin modifying enzymes that are typically downregulated in GBM in mice fed with KD compared with those fed with SD. We also observed a corresponding downregulation in target genes of these miRNAs and chromatin modifying enzymes. These genes have been reported as key regulators in tumour growth, tumour invasion and chemo/radio-resistance in previous publications. Therefore, our study indicates that targeting cancer metabolism using the KD alters the epigenetic landscape of GBM and induces changes in key genes that potentiate the effects of chemo/radiotherapy.
Diffuse intrinsic pontine glioma (DIPG) is a devastating pediatric brainstem tumor with no efficacious treatment. The most prevalent mutation occurs on the amino terminal tail at Lysine 27 (K27M) resulting in global hypomethylation of H3K27. The H3.1-K27M mutation is highly co-expressed with an activating mutation (G328V) in Activin-A receptor Type 1 (ACVR1), a bone morphogenetic protein (BMP) receptor. We used the Sleeping Beauty Transposase (SB) system to deliver plasmids encoding NRASV12 and a short hair pin for TP53 (shp53) in combination with ACVR1-G328V or H3.1-K27M, or both, into the lateral ventricle of neonatal mice, to generate endogenous tumors with DIPG mutations. Tumor development was monitored by measuring luciferase activity in vivo. Moribund stage animals were perfused and processed for histology. Additionally, tumor neurospheres (NS) were generated from moribund stage tumors. H3.1 K27M (MS=99 dpi) did not have a statistically significant effect on survival compared to the control group NRAS/ shp53 (MS=80), however, tumors induced with NRAS/shp53/ACVR1 G328V (MS=119 dpi) had enhanced survival compared to control group NRAS/ shp53 (p=0.0029). Tumors harboring NRAS/shp53/ACVR1 G328V/H3.1 K27M (MS=154) exhibit a synergistic increase in survival compared to the NRAS/shp53/H3.1 group (p<0.0001) and the NRAS/shp53/ACVR1G328V group (p=0.0137). In vitro and in vivo, tumors or tumor NS harboring ACVR1-G328V exhibit elevated phopho-Smad1/5, transducer of the BMP pathway, while those harboring H3.1-K27M exhibit decreased H3K27me3 and increased H3K27 acetylation levels compared to controls. In conclusion, the SB transposase system can be used to develop a mouse model that accurately represents the molecular biology of DIPGs with ACVR1-G328V and H3.1-K27M mutations. ACVR1-G328V and H3.1-K27M may induce a protective effect that prolongs survival through upregulation of the BMP-Smad1/5 pathway and epigenetic deregulation. In the future we aim to elucidate the mechanisms by which ACVR1 and H3.1-K27M mutations contribute to tumor initiation and progression to develop targeted therapies for DIPG.
Aims Glioblastoma Multiforme (GBM) is the most aggressive form of primary brain tumour, with a median survival of 12-14 months after diagnosis. Although GBM has been extensively characterised on the molecular level during the past decades, many targeted therapies have been proved ineffective due, in part, to high heterogeneity of GBM. Thus, novel therapies targeting the altered metabolism which is exhibited by all cancer cells have gained much attention. The therapeutic ketogenic diet (KD) is a high fat, low carbohydrate and adequate protein diet. It has been recognized as a treatment for refractory paediatric epilepsy for decades. Recent studies have shown that a KD reduced tumour growth and potentiated the effects of therapy in some glioma animal models. However, the underlying mechanism(s) is still unclear. Thus, the aim of this study was to understand the mechanism of action behind the KD’s effects in inhibiting tumour growth and potentiating chemotherapy and radiotherapy. Method To unravel the mechanism of action, we analyzed the expression of genes encoding chromatin modifying enzymes in brain tumour samples from mice fed either a KD or standard diet (SD), using the Mouse Epigenetic Chromatin Modification Enzyme PCR Array (Qiagen, Germany). The expression of genes of interest selected from the array were validated by qRT-PCR. Human GBM cell lines and primary cells from GBMs were used to validate the results of the GBM mouse model. Beta-hydroxybutyrate, the main physiological ketone body found in the circulation of patients during KD, was used in in vitro experiments to mimic the in vivo physiological effect of a KD. The effect of protein arginine methyltransferase 8 (PRMT8) overexpression in GBM cells was studied using a lentiviral system. Cell proliferation was measured by Sulforhodamine B assay (Sigma, USA). Spheroid growth and invasion was measured in GBM spheroids cultured in Matrigel matrix (Corning, USA). Results Our results highlighted changes in the expression of a number of key chromatin modifying enzymes in mice fed a KD compared to those fed a SD. PRMT8, a gene highly downregulated in GBM, was upregulated in tumors from mice fed a KD, with corresponding downregulation of its target genes, dihydrofolate reductase (DHFR) and C-X-C chemokine receptor type 4 (CXCR4). Our results also showed that overexpression of PRMT8 in GBM cells reduced cell proliferation and invasiveness. Conclusion PRMT8, DHFR and CXCR4 have been shown to play key roles in tumour growth, invasion, migration and chemo/radio-resistance. Moreover, therapeutic strategies to downregulate these genes have been investigated in the form of methotrexate for DHFR inhibition and small molecule inhibitors of CXCR4. Thus, our results suggest that one mechanism through which the KD exerts its therapeutic effects may be through altering the expression of chromatin modifying enzymes. This provides additional support for the use of a KD as an adjuvant in combination with existing therapeutic approaches.
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