(reverse), 59-FAM-AGGAGTATGACGAGTCCGGCCCC-39- BHQ (probe)). Phosphoryation, apoptosis assays, and western blotting Phosphorylation and/or cleavage status of Erk1/2 (T202/ Y204), Mek1/2 (S217/S221), p38 MAPK (T180/Y182), Stat3 (Tyr705), Erbb2 (Y1221/Y1222), Erbb3 (panY), Akt1 (S473), Akt1 (T308), mTOR (S2448), 4E-BP1 (T37/T46), p70 S6 Kinase (T389), S6 (S235/S236), IRS1 (S307), IRS2 (panY), p53 (S15), Bad (S112), cleaved caspase-3, cleaved PARP, and Chk2 (T68) were determined using the PathScan Sandwich ELISA kits (Cell Signaling Technology) as per manufacturer’s instructions. Raw signal intensity (OD450) was normalized to total Akt protein levels. For visualization as a heatmap, data were background-subtracted, median normalized, and converted into a heatmap using Java TreeView. Assays were performed in triplicate. For Western analysis, cells were washed with PBS, and lysate was collected using the M-PER extraction reagent (Thermo Scientific) containing Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific). 20 mg of protein per sample were separated by electrophoresis on 4–12% Bis-Tris NuPAGE Novex acrylamide gels (Invitrogen) and were then transferred to nitrocellulose membranes using the iBlot system (Invitrogen). Antibodies used were OGT, O-GlcNAc, b-actin, GSK-3b (S9), p27Kip1, FoxM1, FoxO1 (T24)/FoxO3a (T32), and total Cyclin D1. Anti-OGT and anti-O-GlcNAc antibodies were obtained from Sigma. All other antibodies were obtained from Cell Signaling. Quantitative analysis of pixel density for O-GlcNAc staining was determined using AlphaVIEW SA software (Cell Biosciences). Results Determination of in vitro sensitivity to GDC-0941 in the NCI-60 tumor cell line collection As shown in Figure 1, the NCI-60 tumor cell lines [9] exhibited over a 2-log range in GDC-0941 GI50 values, ranging from 0.011 mM to 3.80 mM (mean = 0.66 mM; Fig. 1A; Table S1), with all tumor types displaying a wide range of sensitivities to GDC- 0941 (Figs. S1A and S1F). As others previously reported based on analysis of breast cancer cell lines [6], we found an association between PI3KCA mutational status amongst cells in the NCI-60 tumor cell line collection and sensitivity to GDC-0941 (P = 0.044; Figs. S1B and S1C), whereas there is no association between loss of PTEN protein and response to GDC-0941 (P = 0.31; Figs. S1B and S1C). Furthermore, we found no association between mutational status of additional effectors in the PI3K-signaling pathway (AKT1, PDK1, MTOR) amongst cells in the NCI-60 tumor cell line collection and sensitivity to GDC-0941 (Figs. S1D and S1E). Following standardization of all GI50 values, we deemed fourteen cell lines with standardized GI50 values $0.8 SD from the mean GI50 as resistant and eleven cell lines with standardized GI50 values #20.8 SD from the mean GI50 as sensitive (Figure 1B). Identification of genes correlating with response to GDC- 0941 amongst the NCI-60 tumor cell line collection We next sought to identify those genes whose expression may be a priori predictive of in vitro sensitivity or resistance to GDC-0941. Profiles of basal mRNA gene expression for the NCI-60 tumor cell line collection (in the absence of GDC-0941) were generated using publicly available data [11] at the CellMiner database(http:// discover.nci.nih.gov/cellminer). Following preliminary efforts to identify genes differentially expressed between all GDC-0941- sensitive and –resistant tumor cell lines (n = 25; Fig. 1B) in the Biomarkers of Response to PI3K Inhibitors NCI-60 collection (not shown), we chose to focus only on those GDC-0941-sensitive and-resistant tumor cell lines that were epithelial in origin (breast, colon, NSCLC, renal, ovarian, prostate, and melanoma), and omit from further analyses tumor cell lines derived from leukemias and tumors of CNS-origin (Figs. 1C and 1D). This segregation of the cell lines used allowed us to profile a subset of the NCI-60 tumor cell line collection (T-47D, IGROV1, BT-549, MCF7, HS 578T, UACC-257, HOP-92, SK-OV-3, M14, NCI-H226, SK-MEL-5, HCC-2998, MDA-MB-231, NCI/ ADR-RES, NCI-H23, and OVCAR-4) that still represents a heterogeneous population of tumor types while avoiding the significant variability in gene expression resulting from inclusion of mesenchymal or other tumors (not shown). Using the Comparative Marker Selection Module [21] of GenePattern 3.3, we identified 221 genes differentially expressed between GDC-0941-sensitive and GDC-0941-resistant tumor cell lines with a P value,0.01 (Table S2). Unsupervised hierarchical clustering of the top 50 differentially expressed genes (rank order based on P value) shows strong discrimination between sensitive and resistant tumor cell lines the 16 analyzed NCI-60 tumor cell lines (T-47D, IGROV1, BT-549, MCF7, HS 578T, UACC-257, HOP-92, SK-OV-3, M14, NCI-H226, SK-MEL-5, HCC-2998, MDA-MB-231, NCI/ADR-RES, NCI-H23, and OVCAR-4) into GDC-0941-sensitive or GDC-0941-resistant subgroups (Fig. 2A). Table 1 lists the top differentially expressed genes in GDC-0941- sensitive and GDC-0941-resistant tumor cell lines, along with associated metrics used in the analysis. In order to assess more subtle contributions of coherent cellular pathways in distinguishing GDC-0941-sensitive and GDC-0941- resistant tumor cell lines, we next performed Gene Set Enrichment Analysis [22]. Using an FDR cut-off of ,25%, we identified several KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways specifically enriched in GDC-0941-resistant tumor cell lines, including the proteasome and cysteine and methionine metabolic pathways (Fig. S2). Correlation between activation of key signaling nodes and response to GDC-0941 amongst the NCI-60 tumor cell lines We next examined the baseline phosphorylation levels of multiple downstream effectors of the PI3K pathway, including Akt1 (S473), Akt1 (T308), mTOR (S2448), 4E-BP1 (T37/T46), p70 S6 Kinase (T389), and S6 (S235/S236). In addition, we assayed the activation status of MAP kinases, Erk1/2 (T202/ Y204), Mek1/2 (S217/S221), and p38 MAPK (T180/Y182); transcriptional activators, Stat3 (Tyr705); growth factor and nutrient signaling, Erbb2 (Y1221/Y1222), Erbb3 (panY), IRS1 (S307), IRS2 (panY); and apoptosis signaling molecules, p53 (S15), Bad (S112), cleaved caspase-3, and cleaved PARP (Fig. 3A). It is notable that levels of phosphorylated Akt1, either at S473 (Fig. 3B; rs = 20.50, P = 0.02) or T308 (Fig. 3C; rs = 20.64, P = 0.004), significantly correlated with sensitivity to GDC-0941. We also observed significant correlation between the level of phosphorylated 4E-BP1, at T37/T46, and sensitivity to GDC- 0941 (Fig. 3D; rs = 20.73, P,0.001). These results suggest that increased baseline activation of several downstream effectors in the PI3K pathway (phosphorylated Akt1 (at S473 and/or T308), and phosphorylated 4E-BP1 at T37/T46) may serve as predictive markers for de novo sensitivity to GDC-0941. Finally, we observed a significant correlation between baseline levels of cleaved PARP and sensitivity to GDC-0941 (Fig. 3E; rs = 20.65, P = 0.003). PLOS ONE | www.plosone.org 4 September 2012 | Volume 7 | Issue 9 | e46518