Altered expression of polycomb group genes in glioblastoma multiforme

Abstract

The Polycomb group (PcG) proteins play a critical role in histone mediated epigenetics which has been implicated in the malignant evolution of glioblastoma multiforme (GBM). By systematically interrogating The Cancer Genome Atlas (TCGA), we discovered widespread aberrant expression of the PcG members in GBM samples compared to normal brain. The most striking differences were upregulation of EZH2, PHF19, CBX8 and PHC2 and downregulation of CBX7, CBX6, EZH1 and RYBP. Interestingly, changes in EZH2, PHF19, CBX7, CBX6 and EZH1 occurred progressively as astrocytoma grade increased. We validated the aberrant expression of CBX6, CBX7, CBX8 and EZH2 in GBM cell lines by Western blotting and qRT-PCR, and further the aberrant expression of CBX6 in GBM tissue samples by immunohistochemical staining. To determine if there was functional significance to the diminished CBX6 levels in GBM, CBX6 was overexpressed in GBM cells resulting in decreased proliferative capacity. In conclusion, aberrant expression of PcG proteins in GBMs may play a role in the development or maintenance of the malignancy.

Figure 1. Interrogation of The Cancer Genome Atlas (TCGA) database of mRNA expression of PcG genes in glioblastoma multiforme (GBM).

PcG gene expression statuses in glioblastoma patients were allocated into 3 different categories, overexpression (Tumor/Normal Ratio≥2, red); downregulation (Tumor/Normal Ratio≤0.5, green), and no change (0.5≤Tumor/Normal Ratio≤2, yellow). The stacked bar graph depicts the percentage of each category, calculated out of total 424 patients.

Figure 2. Expression of PcG genes in different glioblastoma subtypes and astrocytoma grades.

(A) PcG gene expression in TCGA glioblastoma multiforme (GBM) subtypes. Using the TCGA data for GBM, pair-wise comparisons were conducted between all 4 of the molecular subtypes (Mesenchymal, Classical, Neural and Proneural, color coded). ** P<0.01. (B) Continuum of abnormal expression in PcG genes in different histological grades of astrocytomas. The Y-axis measures the background adjusted Robust Multi-array Average (RMA) which indicates the normalized expression level. Box-and-whisker plots show the distribution of mRNA expression in normal brain and different grades of astrocytomas (color coded). For detailed statistical analyses, please see Table S3 & S4.

Figure 3. CBX6 expression is downregulated in human glioblastoma multiforme (GBM) cell Lines.

(A) qRT-PCR analysis of the mRNA levels of multiple PcG genes in primary human astrocytes, U251MGand T98G human GBM cell lines. Expression was normalized to the GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) gene and the average of each PcG expression in primary human astrocytes was arbitrarily set to one. Data represent the average of three independent experiments. Error bars represent standard deviation. ** (P<0.01). (B) Western blot analysis of the expression of multiple PcG proteins in primary human astrocytes, T98G and U251MG GBM cell lines. GAPDH was used as a loading control.

Figure 4. Aberrant expression of CBX6 in human glioblastoma multiforme (GBM).

Human GBM tissue array was stained by immunohistochemistry using anti-CBX6 antibody. (A) Representative pictures of normal cerebrum (1, 2) and GBMs (3, 4) stained with CBX6 antibodies. (B) CBX6 levels were evaluated, H scores were calculated as in Materials and Methods and graphed. *** (P<0.001).

Figure 5. Overexpressing CBX6 gene inhibits the growth of U251MG cells.

(A) Western blot analysis of the expression of CBX6 in U251MG-Luc/TR-CBX6^TO cell lines. 0.5ug/ml of Doxycycline was added to induce CBX6 expression. GAPDH was used as a loading control. Top labels indicate assigned number of each cell line. (B) Colony formation assay, U251MG-Luc/TR-CBX6^TO cells were treated with 0.5 µg/ml doxycycline to induce CBX6 expression. The colonies were stained with 0.05% crystal violet (CV). Shown is a representative of two independent experiments performed in triplicate with line 17. (C) Methanol was added to solubilize the crystal violet dye. Absorbance at 540 nm was read using DTX 880 plate reader (B.D.) Error bars represent standard deviation. * P<0.05. (D) U251MG-Luc-CBX6^TO cells (line 6 and 17) and U251MG-Luc-Vector^TO (vector control) cells were treated with doxycycline and cell proliferation was assessed using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega). Shown is a representative of three independent experiments performed in quadruplicate. ** P<0.01.