Foci of Tumor Heterogeneity in Diffuse Low-Grade Gliomas

IDH1-mutated gliomas are slow-growing brain tumors which progress into high-grade gliomas. The early molecular events causing this progression are ill-defined. Previous studies revealed that 20% of these tumors already have transformation foci. These foci offer opportunities to better understand malignant progression. The investigators used immunohistochemistry and high throughput RNA profiling to characterize foci cells. These have higher p-STAT3 staining revealing activation of JAK/STAT signaling. They downregulate genes involved in Hippo/Yap pathway (AMOT, CCDC80, LIX1), Wnt signaling (CPE, DAAM2, GPR37, SFRP2), EGFR signaling (EPS15, MLC1), cytoskeleton and cell-cell communication (EZR, GJA1) while increasing SKA3, a kinetochore-associated protein. In addition, foci cells show reduced levels of the lipid metabolic ethanolamine-phosphate phospho-lyase (ETNPPL/AGXT2L1). This enzyme is involved in the catabolism of phosphoethanolamine involved in membrane synthesis. The investigators detected ETNPPL protein in glioma cells as well as in astrocytes in the human brain. Its nuclear localization suggests additional roles for this enzyme. ETNPPL expression is inversely correlated to glioma grade and the investigators found no ETNPPL protein in glioblastomas.

Overexpression of ETNPPL reduces the growth of glioma stem cells indicating that this enzyme opposes gliomagenesis. Collectively, these results suggest that a combined alteration in membrane lipid metabolism and STAT3 pathway promotes IDH1-mutated glioma malignant progression.

Tumors with foci of at least four millimeters in diameter, assessed by hematoxylin & eosin stainings, were selected. Four drills (two in foci and two in the other part of the tumor ) were performed in the FFPE tumor blocks using a two millimetres punch from a Tissue Micro Array apparatus in RNAse-free conditions. After the punches, the adequate selection of tumor areas was checked by hematoxylin & eosin stainings of sections. Total RNA was extracted using the Qiagen RNeasy FFPE kit, quantified with Nanodrop 1000 (Thermo Fisher) and the RNA integrity number (RIN) was determined using a Bioanalyzer 2100. The RIN was on average 2.5 but were still suitable for labelling and hybridization on DNA chips according to the Affymetrix technical department. After amplification and labelling with an Affymetrix WT Pico Kit, cDNA were hybridized on Human Gene 2.1 ST chips. Data were normalized with the Affymetrix Expression Console software (GC-RMA algorithm) and the RNA profiles were generated using the Affymetrix Transcriptome Analysis Console (3.1.0.5) software. Differentially expressed genes were selected on the basis of a linear fold change ≥ 1.1 and p-value ≤ 0.05 (n=8 tumors). The data that support the findings of our study are openly available at the functional genomics data Gene Expression Omnibus.