function as a transcription factor that promotes the cytosolic expression of cathepsin L [30]. To assess the functionality of DDN in mediating sensitivity to inhibition of PI3K by GDC-0941, and to further probe the observed linear relationship between basal DDN gene expression (Fig. 2C) and de novo sensitivity to GDC- 0941, we used siRNA pools targeting DDN to knockdown the expression of DDN in the GDC-0941-resistant MDA-MB-231 breast tumor cell line, and in a second breast tumor cell line, the GDC-0941-sensitive MCF-7 breast tumor cell line. Following knockdown of DDN and treatment with GDC-0941, we then assayed for cell proliferation and phosphorylation status of multiple signaling pathways, including downstream effectors of PI3K (Fig. 5). Knockdown of DDN in both cell lines (Fig. 5B and 5E; Fig. S4C and S4D) resulted in statistically significant increased sensitivity to GDC-0941 as compared to control cells only treated with either GDC-0941 or DDN siRNA (Fig. 5A and 5D; Fig. S4C and S4D). We then analyzed the effects of loss of DDN expression by examining the phosphorylation status of additional signaling nodes, including key regulators of PI3K, apoptotic and MAPK pathways. Amongst all effectors analyzed (not shown), increased phosphorylation of the cell cycle checkpoint regulator p38 MAPK at T180/Y182 was observed in both the MCF-7 and MDA-MD- 231 cell lines upon treatment with GDC-0941 and DDN siRNA (Fig. 5C and 5F). Discussion The PI3K (phosphoinositide 3-kinase) signaling cascade is one of the most frequently de-regulated pathways in human cancer [1], resulting in aberrant cell proliferation and migration. Conse- quently, there are widespread efforts to develop selective inhibitors against the PI3K pathway as a chemotherapeutic strategy in cancer, and a number of these compounds are undergoing clinical development [2], including GDC-0941. Here we describe the identification and characterization of several putative genomic and phosphoproteomic biomarkers of response to the PI3K inhibitor, GDC-0941. To this end, we used the NCI-60 human tumor cell line collection to identify candidate predictive biomarkers of sensitivity or resistance to GDC-0941, providing a starting point for the rational selection of patients for whom treatment with PI3K inhibitors will provide the greatest clinical benefit. We observed a wide range of sensitivities (0.011 mM to 3.80 mM) to GDC-0941 across the NCI-60 human tumor cell line collection. In accordance with previous results in a large panel of breast tumor cell lines [6], while PI3K mutational status was also predictive of sensitivity to GDC-00941 in the more diverse NCI-60 panel, PI3K mutational status was not 100% specific for prediction of sensitivity, suggesting a more complex relationship between PI3K activation and de novo sensitivity to GDC-0941. As such, one caveat is that there may be heretofore uncharacterized mutations in additional genes that also drive de novo sensitivity to GDC-0941. Only three of the eight GDC-0941-sensitive tumor cell lines studied in-depth in this work had activating mutations in PI3KCA. Nevertheless, our results demonstrate that increased levels of phosphorylation of key downstream effectors of the PI3K pathway significantly correlate with increased sensitivity to GDC- 0941 (Fig. 3) and that alternative modes of PI3K pathway activation (e.g. PI3K-independent Akt1 phosphorylation) may lead to sensitivity to GDC-0941. Also, the increased PARP cleavage we also observed in GDC-0941 sensitive cell lines (Fig. 3) may reflect predisposition of sensitive tumor cell lines to trigger the pro- apoptotic machinery upon chemotherapeutic treatment. To this end, multivariate models incorporating PI3K mutational status and PI3K-Akt pathway activation status (or other proteomic Biomarkers of Response to PI3K Inhibitors markers) may be more predictive of sensitivity to GDC-0941 than PI3K mutational status alone, consistent with a recent study on the selective mTOR inhibitors, rapamycin and everolimus [31]. These insights may facilitate the identification those patients lacking activating mutations in PI3KCA who are still de novo sensitive to GDC-0941 or other inhibitors of the PI3K and/or mTOR pathways. OGT regulates nutrient sensing and multiple signaling path- ways though its reversible posttranslational modification of proteins by the addition of the O-GlcNAc moiety and is now being appreciated as a possible regulatory switch, influencing protein-protein interactions, glucose metabolism, proteasome function, the transcriptional machinery, and mechanisms of cellular invasion and proliferation in breast cancer in cancer biology [24–27,32]. OGT may function as an oncogene, as it required for the survival of several breast tumor cell lines, and may regulate levels of p27Kip1 and FoxM1 [26]. The work presented presents multiple lines of evidence to support the hypothesis that OGT and O-GlcNAc glycosylation may modulate tumor cell line response to PI3K inhibitors. First, we show that levels of OGT mRNA are significantly associated with sensitivity to GDC-0941 and that loss of OGT expression and the concomitant decreases in cellular levels of O-GlcNAc result in increased sensitivity to GDC- 0941 and NVP-BEZ235 (Fig. 4; Fig. S5A). Loss of OGT expression, upon treatment with GDC-0941, results in significant decreases in levels of phosphorylated p-GSK-3b, p-FoxO1/ FoxO3a and/or Cyclin D1, all of which are key downstream effectors of receptor tyrosine kinase and PI3K signaling. It is interesting to note that Akt is not modified by O-GlcNAc in the MDA-MB-231 or OVCAR-4 tumor cell lines and that the loss of OGT expression does not alter levels of phosphorylated Akt (at S473) in both the OVCAR-4 and MDA-MB-231 tumor cell lines (not shown), the latter of which is consistent with previous work [26]. Second, we provide data showing that increasing cellular levels of O-GlcNAc results in increased resistance to GDC-0941 (Fig. S6). Both lines of evidence suggests that O-GlcNAc glycosylation may represent an attractive target for the modulation of patient response to PI3K inhibitors and other molecules targeting pathways with effectors similarly modified by the addition of the O-GlcNAc moiety. Common to the cell lines in which loss of OGT expression results in sensitization to PI3K inhibitors was increased phosphor- ylation of Chk2. This cell cycle regulatory protein has overlapping substrate specificity with the cell cycle checkpoint Chk1 but its role in regulating cell cycle progression is less well understood, and is likely to be subjective based on tissue and/or tumor type [33]. Generally, Chk2 is activated by phosphorylation at T68 and acts primarily at G2/M and S phase, through its inhibition of CDC25A, leading to cell cycle arrest. Chk2 is also thought to have a modulatory role in stimulating apoptosis in response to DNA damage resulting from chemotherapeutic agents and in the regulation of IR-induced p53-dependent apoptosis [33]. It is not known if Chk2 itself is a target for O-GlcNAc glycosylation; however, recent work has shown that Polo-like kinase 1, a known regulator of Chk2 activity [34], is targeted for O-GlcNAc glycosylation by OGT [35]. Upon loss of DDN expression in both the de novo sensitive MCF- 7 and de novo resistant MDA-MB-231 cell lines (Fig. 5), we observed significantly increased levels of activated p38 MAPK. p38 MAPK is known to be activated by environmental stress and has a complex, and often context-dependent role in regulating cell signaling. Context-dependence aside, p38 MAPK generally seems to antagonize cell proliferation by negatively regulating cell cycle progression at G1/S and G2/M transitions though the down- PLOS ONE | www.plosone.org 9 September 2012 | Volume 7 | Issue 9 | e46518