Biomarkers of Response to PI3K Inhibitors Figure 3. Relationship between de novo phosphorylation status of multiple signaling pathways and tumor cell line sensitivity to GDC-0941. A, relative activities of multiple signaling pathways in tumor cell lines with differential sensitivities to GDC-0941. The heat map indicates the relative phosphorylation levels 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) as assessed by quantitative phosphorylation analysis using a sandwich ELISA with epitope-specific antibodies. Cell lines are ordered (left to right) in increasing order of resistance, with sensitivity of each cell line indicated below cell line label as sensitive ‘‘S’’ or resistant ‘‘R’’. Color bar indicates relative levels (z-score) of phosphorylation. B–E, correlation between tumor cell line sensitivity to GDC-0941 and phosphorylation status of Akt1 (S473), Akt1 (T308), 4E-BP1 (T37/T46), and cleaved PARP. Data in scatter plots (left panels) represent z- score of phosphorylation status of (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 z-score and GI50 as estimated by Spearman’s rank correlation (rs) is indicated. Data in box plots (right panels) represents mean 6 SEM relative level of phosphorylation for indicated protein, for all sensitive cell lines (n = 8) versus all resistant cell lines (n = 8);*, P,0.05;***, P,0.001. doi:10.1371/journal.pone.0046518.g003 In the NVP-BEZ235-resistant MDA-MB-231 breast tumor cell line, loss of OGT expression resulted in increased sensitivity to NVP-BEZ235 (Fig. S5A). In contrast, loss of OGT expression had no effect in the sensitivity of the highly NVP-BEZ235-resistant OVCAR-4 ovarian tumor cell line to NVP-BEZ235 (Fig. S5B). Increased cellular levels of O-GlcNAc results in resistance to GDC-0941 and increased phosphorylation of ERBB3 As a corollary, we next tested the overall role of the O-GlcNAc modification in mediating tumor cell line response to inhibition of PI3K by GDC-0941, by using PUGNAc, a pharmacological inhibitor of OGA, the glycosidase that removes the O-GlcNAc moiety, thus increasing the overall cellular levels of O-GlcNAc [25]. Treatment of the highly GDC-0941-sensitive T-47D tumor cell line with PUGNAc resulted in significantly increased resistance to GDC-0941 as compared to control cells treated with either GDC-0941 or PUGNAc alone (Fig. S6A). Immunoblotting of cell lysates from T-47D cells treated with GDC-0941 and/or PUGNAc, show that PUGNAc treatment results in increased total cellular levels of O-GlcNAc (Fig. S6B) and increased phosphor- ylation of the receptor tyrosine kinase ERBB3 (Fig. S6D), relative to control treated cells. Finally, increased levels of O-GlcNAc, upon treatment with PUGNAc with GDC-0941, results in the decreased phosphorylation of MEK1/2 (Fig. S6E). Loss of DDN expression sensitizes breast tumor cell lines to GDC-0941 and alters multiple signaling pathways Also amongst the top differentially expressed genes we identified between GDC-0941-sensitive and GDC-0941–resistant tumor cell lines was dendrin (DDN) (Table 1; Fig. 2A and 2C). While its function in transformed human tumor cells is not known, two studies in untransformed human and mouse kidney podocytes have put forth that DDN encodes a proline-rich protein that may play a role in TGF-b1-induced apoptosis [29], possibly through its PLOS ONE | www.plosone.org 7 September 2012 | Volume 7 | Issue 9 | e46518