Volume 12, Issue 4, Pages 889-901 (October 2003) Oncogenic Ras and Akt Signaling Contribute to Glioblastoma Formation by Differential Recruitment of Existing mRNAs to Polysomes  Vinagolu K. Rajasekhar, Agnes Viale, Nicholas D. Socci, Martin Wiedmann, Xiaoyi Hu, Eric C. Holland  Molecular Cell  Volume 12, Issue 4, Pages 889-901 (October 2003) DOI: 10.1016/S1097-2765(03)00395-2

Figure 1 Correlation of Combined Ras and Akt Signaling with the Phosphorylation of Proteins Regulating Translation Initiation (A) Mouse glial progenitor cells. Whole cell protein extracts of nestin expressing glial progenitor cells infected with activated Ras (Ntva-R), or Akt (Ntva-A), or both Ras and Akt (Ntva-RA) or LacZ (Ntva-L as control [C]) were resolved on SDS PAGE and immunoblotted using antibodies against downstream signaling components representing Ras pathway (ERK and eIF4E) as well as Akt pathway (Akt, p70S6K, S6RP, and 4E-BP1). Reactivity to anti-hemaglutinin (HA) antibody shows the HA-epitope tagged Akt-specific infection. (B) Human normal brain and GBM tumor tissues. Total cell extracts of normal brain (lanes 1and 2) and GBM (lanes 3–10) tissues were separated by electrophoresis and immunoblotted using antibodies against signaling proteins representing Ras pathway (ERK, p38, JNK, eIF4E, and cMyc) as well as Akt pathway (Akt, S6RP, 4E-BP1, and eNOS). Phospho-specific antibody was denoted with a prefix “p” and anti-GAPDH antibody reactivity represents loading control in all of the protein blots. Molecular Cell 2003 12, 889-901DOI: (10.1016/S1097-2765(03)00395-2)

Figure 2 Blockade of Ras and Akt Signaling on Phosphorylation of Translation Initiation Components in U373- and U87-Human Glioma Cell Lines and in Mouse Glial Progenitor Cells (A) Inhibitor specificity. Two hours postadministration of 20 μM U0126 (U), 10 μM LY294002 (L), or 10 nM rapamycin (R), or solvent mock control (O), the whole cell extracts were prepared from Ntva-RA cells (nestin-expressing glial progenitors infected with activated Ras and Akt) and immunoblot analysis was performed using antibodies against ERK, eIF4E, Akt, p70S6 kinase, S6RP, 4E-BP1, eIF4G, and GSK3 (α/β). (B–D) Combinatorial inhibitory effects in Ntva-RA cells and in human GBM cells, U373 (C) and U87 (D). CCl-779 (C) (1 ng/ml), an additional functional analog of rapamycin, was also included. Molecular Cell 2003 12, 889-901DOI: (10.1016/S1097-2765(03)00395-2)

Figure 3 Scatter Plots of Gene Probes as Affected by Inhibition of Ras or Akt Signaling Total and polysomal RNAs were hybridized to Affymetrix Murine Genome U74 (RV-U74Av2) oligonucleotide 12,488-gene microarray chip. Relative gene probe intensities (X-axis) of total RNA (A and B), and polysomal mRNA (C and D) of Ntva-RA cells were plotted against corresponding values (Y-axis) obtained upon treatment with inhibitors of signaling downstream of Ras (Ras Blockade denoted with downward arrow: [A] and [C]) and Akt (Akt Blockade denoted with downward arrow: [B] and [D]) as indicated in Figure 2. The scatter plots were also generated comparing the same gene probe fluorescence intensities of polysomal mRNA (X-axis) and total RNA (Y-axis) to depict the normalized polysomal mRNA pattern (NPR) in Ntva-RA cells (E) and as a function of Akt blockade (F) or in two independent cultures of uninfected cells, Ntva-C1(G) and Ntva-C2 (H). Raw intensity of each spot without background subtraction or normalization is shown. Expressed genes (X-axis) in this group were further plotted against their net fold change values (Y-axis) of normalized polysomal mRNA (CNPR) either in Ntva-RA background as a function of Akt blockade (I) or in between Ntva-C1 and Ntva-C2 (J) samples. Molecular Cell 2003 12, 889-901DOI: (10.1016/S1097-2765(03)00395-2)

Figure 4 Cluster Analysis of Normalized Polysomal mRNA Profiles as Affected by Ras and Akt Signaling Total RNA and polysomal mRNA from Ntva (uninfected control:C), Ntva-L (mock infected control:L), Ntva-R, Ntva-A, and Ntva-RA cells were measured with Murine Genome U74 (RV-U74Av2) oligonucleotide microarray chip. Sets of U0126 (Ras Blockade), or LY294002 and rapamycin (Akt Blockade) administered Ntva-RA cells were also included in these experiments. (A) Seven hundred five genes from the normalized polysomal mRNAs (NPR) were identified to have significant fold changes (>3 and p < 0.0025) in at least 4 of the 11 comparisons and were subjected to unsupervised clustering as described in the text. The CNPR values of all these genes in these comparisons were shown in the unsupervised cluster image on the left with the color scale given at the bottom. The green bars to the left indicate the 42 clusters and the purple bars to the far left show grouping of these clusters into “patterns” of similar character. (B) Box-whisker plots showing the median expression profile for a number of selected clusters. The box shows the 25%–75% quantile range with the horizontal line indicating the median, the vertical bars are 1.5 times the interquantile width and the circles show any data points more extreme than those. The lines connecting the mean values are for pattern recognition only. The “soft filter” genes were generated by “patterns” i, iii, and v. (C) Pie chart represents the full set of 705 genes selected via the noise filter were classified with functional annotation. (D) Table illustrating 78 of 221 named mRNAs in the union set found to be associated with glioma and or cancer biology. The genes known to be misexpressed in human cancers and GBMs are highlighted. The full clustering results including the named genes in the screening filter, the hard filter, soft filter, and the union set, in addition to other clustering analysis and information are available at http://www.mskcc.org/GCL/GBMRNA. Molecular Cell 2003 12, 889-901DOI: (10.1016/S1097-2765(03)00395-2)

Figure 5 Effect of Blockade of Ras and Akt Signaling on Synthesis of Oncogenic Proteins SDS-PAGE analysis and autoradiography of immunoprecipitations from whole cell extracts of metabolically labeled Ntva-RA cells using antibodies against Id4 (A), Miz1 (B), ATM (C), Cyclin D2 (D), VEGF (E), Notch1 (F), Pak3(G), p27Kip1(H), IGFBP4 (I), IGFBP5 (J), β-Actin (K) and transferrin receptor (L) were preformed either without (Control), or with therapeutic Akt blockade (downward arrow) as in Figure 2. The histograms on the top of these protein autoradiographs represent corresponding microarray-based virtual northern blots representing total RNA (gray bar) or normalized polysomal mRNA (orange bar). Molecular Cell 2003 12, 889-901DOI: (10.1016/S1097-2765(03)00395-2)