Acetate Supplementation Induces Growth Arrest of NG2/PDGFRα-Positive Oligodendroglioma-Derived Tumor-Initiating Cells

<div><p>Cancer is associated with globally hypoacetylated chromatin and considerable attention has recently been focused on epigenetic therapies. N-acetyl-L-aspartate (NAA), the primary storage form of acetate in the brain, and aspartoacylase (ASPA), the enzyme responsible for NAA catalysis to generate acetate and ultimately acetyl-Coenzyme A for histone acetylation, are reduced in oligodendroglioma. The short chain triglyceride glyceryl triacetate (GTA), which increases histone acetylation and inhibits histone deacetylase expression, has been safely used for acetate supplementation in Canavan disease, a leukodystrophy due to ASPA mutation. We demonstrate that GTA induces cytostatic G₀ growth arrest of oligodendroglioma-derived cells <i>in vitro</i>, without affecting normal cells. Sodium acetate, at doses comparable to that generated by complete GTA catalysis, but not glycerol also promoted growth arrest, whereas long chain triglycerides promoted cell growth. To begin to elucidate its mechanism of action, the effects of GTA on ASPA and acetyl-CoA synthetase protein levels and differentiation of established human oligodendroglioma cells (HOG and Hs683) and primary tumor-derived oligodendroglioma cells that exhibit some features of cancer stem cells (grade II OG33 and grade III OG35) relative to an oligodendrocyte progenitor line (Oli-Neu) were examined. The nuclear localization of ASPA and acetyl-CoA synthetase-1 in untreated cells was regulated during the cell cycle. GTA-mediated growth arrest was not associated with apoptosis or differentiation, but increased expression of acetylated proteins. Thus, GTA-mediated acetate supplementation may provide a safe, novel epigenetic therapy to reduce the growth of oligodendroglioma cells without affecting normal neural stem or oligodendrocyte progenitor cell proliferation or differentiation. </p> </div

OG33 and OG35 cells are recalcitrant to differentiation.

<p>OG33 (A) and OG35 (B-D) cells were induced to differentiate over 6 days <i>in </i><i>vitro</i> via activation of adenylyl cyclase (1 mM dibutyryl cAMP or 10 μM forskolin) or inhibition of MEK1/2 (1 μM PD035901), ErbB2 (1 μM PD174285), PI3K (5 μM and 10 μM LY294002), or mTOR (10 nM and 20 nM rapamycin [RAP]) signaling. DMSO (0.2% DMSO) was used as a control for PD174285, PD035901 and LY294002 treatments. Despite inhibition of PI3K-Akt-mTOR and ERK signaling pathways, both cells failed to differentiate into cells with oligodendroglial morphology (A, B) or increase immunoreactivity for GPAF or CNPase (not shown). MEK and ErbB2 inhibition actually increased OG35 proliferation in DM. While inhibition of PI3K-Akt-mTOR signaling induced morphological alterations in OG35 cells (B), this was not associated with increased CNPase (C) or ASPA (D) protein levels. In fact, ASPA expression decreased in the presence of 10 μM LY294002 and 20 nM rapamycin. Differentiation of Oli-Neu cells with cAMP for 4 days or inhibition of ErbB2 signaling for 2 days served as controls for oligodendrocyte cell morphology. DM - differentiation medium, SCM - stem cell medium. n = 3 independent cultures. *p < 0.05. Scale bar = 100 μm.</p

OG33 and OG35 cells exhibit a mesenchymal profile.

<p>A) PCR of OG33 and OG35 cells (grade II and grade III, respectively, primary oligodendroglioma-derived cells that exhibit self-renewal and tumorigenicity) maintained as floating spheres in stem cell medium for 4 days. A grade III oligodendroglioma tumor (Oligo tumor) and proneural (PN) GBM GSCs served as controls. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. OG cells expressed the OPC marker PDGFRα and Notch1, but lacked the neural stem cell markers CD133, SOX2, and Olig2. In contrast, they abundantly expressed the mesenchymal markers CD44, BCL2A1, Wilms Tumor 1 (WT1). B) Principal component analysis (PCA) of SNP raw intensity data from GeneChip® Human Mapping 250K Nsp Arrays. OG cells share similar gene amplifications/deletions to the established HOG oligodendroglioma cell line. Furthermore, the OG cells were more similar to mesenchymal GBM GSCs (GBM12, GBM9, and GBM34) than proneural GSCs (GBM44, GBM8, and GBM2). The Hs683 cell line, which was derived from a GBM tumor but shares features of oligodendroglioma tumors, failed to cluster with either tumor type. C) Immunocytochemistry after 3 days growth in stem cell medium (adherent on PLL) or differentiation medium revealed that the OG cells express a transition mesenchymal profile. In stem cell medium, cells expressed OPC markers (NG2 and PDGFRα), although less abundantly than Oli-Neu OPCs. In contrast, they abundantly expressed the mesenchymal markers CD44 and glutathione S-transferase π (GSTπ). In differentiation medium, OG33 and OG35 cells expressed lower levels of CNPase than Oli-Neu cells, and, unlike Oli-Neu cells, the OG cells failed to express myelin basic protein (MBP), a well-accepted marker of mature oligodendrocytes. Oli-Neu and PN GBM GSCs served as controls. Scale bar = 100 µm. </p

GTA increases protein acetylation of OG cells <i>in</i><i>vitro</i>.

<p>OG33 (A) and OG35 (B) cells were treated with 0.25% GTA in the absence of an HDACi, the cells were harvested at the indicated time points, and analyzed by western blot analysis (25 μg protein, whole cell Triton Lysis Buffer lysate) with an antibody specific to acetylated lysine residues. Both cells displayed a time-dependent increase in acetylation of several proteins. C) Cells were treated with 0.25% GTA in the absence or presence of the HDACi SAHA (1 μM) for 24 hours and analyzed by western blot analysis (25 μg protein, whole cell RIPA Buffer lysate). There was a significant increase in the number of acetylated proteins in SAHA treated cells. Even in the absence of SAHA, GTA enhanced acetylation of several proteins, including those within the histone molecular weight range (H1-21.3kDa, H2a-14.3kDa, H2b-13.8kDa, H3-15.0kDa, and H4-11.3kDa).</p

GTA inhibits OG33 and OG35 cell differentiation.

<p>Western blots (25 μg whole cell lysate) and densitometric analysis of ASPA (E), AceCS1 (F) and CNPase (G) protein levels normalized to actin. A) GTA treatment induced the presence of a novel immunoreactive Aspa species in OG33 cells (densitometry only shows the putative 36 kDa ASPA protein) and reduced ASPA protein levels in OG35 cells. B) GTA increased AceCS1 protein levels in OG33 cells only in SCM. C) GTA blunted the temporal increase in CNPase protein levels. *p < 0.05, **p ≤ 0.01, #p ≤ 0.001, ##p < 0.0001 unless otherwise indicated symbols represent significance relative to untreated cells at the same time point. n = 3 independent cultures.</p

GTA inhibits OG33 and OG35 cell growth.

<p>A, B) Cell cycle profile of PI-labeled cells as floating spheres in stem cell medium (SCM) or in differentiation medium (DM) after 24 hours of treatment with 0.25% glycerol, 0.25% triglycerides (canola oil), 12 mM sodium acetate (NaAc), 36 mM sodium acetate (equivalent acetate to 0.25% GTA), or 0.25% GTA. In SCM (A), only 0.25% GTA reduced the proportion of dividing OG cells via G₀ growth arrest. In DM (B), GTA had no effect on OG35 cells, but increased the percent of OG33 cells in S phase, without alterations in G₀/G₁ or G₂/M. In contrast, 36 mM sodium acetate induced G₀ growth arrest in both OG cells. C, D) Cells were treated for up to 5 days as described above with medium replenished every 48 hours. Growth dynamics were assessed by unbiased trypan blue exclusion based cytometry after 1, 3, and 5 days of treatment. In SCM (C), triglycerides promoted cell growth, glycerol had no effect on growth, and sodium acetate displayed a dose-dependent reduction in growth. GTA was more effective than 36 mM sodium acetate at growth reduction of OG33 cells. In DM (D), neither triglycerides nor glycerol affected growth, while sodium acetate displayed a dose-dependent growth reduction. GTA-mediated growth reduction was greater than sodium acetate in both OG cells. E) Photograph depicting representative medium coloration after 5 days in culture. F) The percent viable cells after 5 days of treatment was determined by unbiased trypan blue exclusion based cytometry. Only 12 mM sodium acetate and triglycerides in DM significantly reduced cell viability. *p < 0.05, **p ≤ 0.01, #p ≤ 0.001, ##p < 0.0001. n = 3 - 6.</p