Figure 2. Gene expression signature predictive of in vitro tumor cell line response to GDC-0941. A, gene expression signature identifies tumor cell lines that are sensitive (left) or resistant (right) to GDC-0941. Unsupervised hierarchical clustering was performed for 50 genes most differentially expressed between 16 GDC-0941 sensitive Biomarkers of Response to PI3K Inhibitors (n = 8) and resistant (n = 8) tumor cell lines (as determined by the Comparative Marker Selection suite in GenePattern with a P value,0.01). Sensitivity of each cell line to GDC-0941 is indicated below cell line label as sensitive ‘‘S’’ or resistant ‘‘R’’. Cell lines are shown on the horizontal axis and genes are shown on the vertical axis. Color bar indicates relative levels (log2) of gene expression, following median centering. B–C, correlation between tumor cell line sensitivity to GDC- 0941 and mRNA expression levels of DDN and OGT. Data in scatter plots (left panels) represent mean log2 mRNA expression of DDN or OGT (y- axis) versus GI50 (mM) of GDC-041 (x-axis) for each of 16 cell lines (n = 3 for each cell line tested). Correlation between log2 mRNA expression and GI50 as estimated by Spearman’s rank correlation (rs) is indicated. Data in box plots (right panels) represent mean 6 SD log2 level of mRNA expression of DDN or OGT in all sensitive cell lines (n = 8) versus all resistant cell lines (n = 8);*, P,0.05;**, P,0.01. doi:10.1371/journal.pone.0046518.g002 Loss of OGT expression and concomitant decreased O- GlcNAc levels sensitizes tumor cell lines to phosphoinositide 3-kinase inhibitors and alters multiple signaling pathways Amongst the most differentially expressed genes we identified between GDC-0941-sensitive and GDC-0941–resistant tumor cell lines is O-linked b-N-acetylglucosamine transferase (OGT) (Table 1; Fig. 2). This encodes is a glycosyltransferase that, in response to cellular glucose levels and receptor tyrosine kinase activation, reversibly modifies diverse cytosolic and nuclear proteins through the addition of O-linked b-N-acetylglucosamine (O-GlcNAc), with the O-GlcNAc modification functioning as a regulatory switch in a manner analogous to phosphorylation [24– 27]. To assess the functionality of OGT in mediating sensitivity to inhibition of PI3K by GDC-0941, we used siRNA pools targeting OGT to knockdown the expression of OGT in the GDC-0941- resistant MDA-MB-231 and OVCAR-4 tumor cell lines, and then assayed for cell proliferation and phosphorylation status of multiple signaling pathways, including downstream effectors of PI3K (Fig. 4). Knockdown of OGT in the MDA-MB-231 cell line resulted in dramatically increased sensitivity to GDC-0941 as compared to control cells only treated with either GDC-0941 or OGT siRNA (Fig. 4A and Fig. S4A). Furthermore, knockdown of OGT in the highly GDC-0941-resistant OVCAR-4 cell line resulted in a modest, yet statically significant, increase in sensitivity to GDC-0941 as compared to control cells only treated with either GDC-0941 or OGT siRNA (Fig. 4E and Fig. S4B). We next probed for alterations in cellular signaling pathways that may be driving the increased sensitivity observed upon loss of OGT expression in the MDA-MB-231 and OVCAR-4 tumor cell lines. Immunoblotting of cell lysates from both MDA-MB-231 and OVCAR-4 cell lines treated with GDC-0941 and OGT siRNA, show that upon the loss of OGT expression, there is concomitant decrease in total cellular levels of O-GlcNAc (Fig. 4B and 4F, arrowheads; Fig. S3). In the MDA-MB-231 cell line treated with OGT-siRNAs and 1 mM GDC-0941, we observed significant decreases in phosphorylation of the Akt1 substrate GSK-3b and FoxO1/FoxO3a and increased levels of total p27Kip1, relative to GDC-0941- or OGT siRNA-alone treated cells (Fig. 4C). In contrast, while we observed similar decreased levels of phosphor- ylated FoxO1/FoxO3a in OVCAR-4 cells treated with OGT- siRNAs and 4 mM GDC-0941, levels of phosphorylated GSK-3b and total p27Kip1were unchanged (Fig. 4G). However, we did observe decreased levels of total Cyclin D1 in OVCAR-4 tumor cells treated with OGT siRNA and 4 mM GDC-0941, relative to GDC-0941- or OGT siRNA-alone treated cells (Fig. 4G). We further analyzed the effects of loss of OGT expression by examining the phosphorylation status of additional signaling nodes, including key regulators of PI3K, apoptotic and MAPK PLOS ONE | www.plosone.org 5 September 2012 | Volume 7 | Issue 9 | e46518