Biomarkers of Response to PI3K Inhibitors Figure 5. Loss of DDN expression increases sensitivity of the MCF-7 and MDA-MB-231 tumor cell lines to GDC-0941 and alters the phosphorylation state of p38 MAPK. A, MCF-7 cells transfected with DDN-targeting siRNA or non-silencing siRNA, in the presence or absence of 300 nM GDC-0941. Cell viability was assayed at 0, 24, 48, and 72 hours post-transfection and treatment with GDC-0941. B, Validation of decreased levels of DDN mRNA following transfection of MCF-7 cells with DDN-targeting siRNA or non-silencing siRNA, in the presence or absence of 300 nM GDC-0941. Plotted is expression of DDN mRNA relative to expression of b-actin mRNA, as determined by qRT-PCR. C, Quantitative phosphorylation analysis of p38 MAPK (T180/Y182) in MCF-7 cells transfected with DDN-targeting siRNA or non-silencing siRNA, in the presence or absence of 300 nM GDC-0941 for 24 hours. Cells were treated with 300 nM GDC-0941 or DMSO control for 24 hours, as indicated. D, MDA-MB-231 cells transfected with DDN-targeting siRNA or non-silencing siRNA, in the presence or absence of 1 mM GDC-0941. Cell viability was assayed at 0, 24, 48, and 72 hours post- transfection and treatment with GDC-0941. E, Validation of decreased levels of DDN mRNA following transfection of MDA-MB-231 cells with DDN- targeting siRNA or non-silencing siRNA, in the presence or absence of 1 mM GDC-0941. Plotted is expression of DDN mRNA relative to expression of b- actin mRNA, as determined by qRT-PCR. F, Quantitative phosphorylation analysis of p38 MAPK (T180/Y182) in MDA-MB-231 cells transfected with DDN-targeting siRNA or non-silencing siRNA, in the presence or absence of 1 mM GDC-0941 for 24 hours. Cells were treated with 1 mM GDC-0941 or DMSO control for 24 hours, as indicated. Data in panels B and E represents mean 6 SD (n = 3). Data in panels C and F represent mean 6 SEM (n = 3); *, P,0.05;**, P,0.01;***, P,0.001. doi:10.1371/journal.pone.0046518.g005 regulation of cyclins, up-regulation of CDK-inhibitors, and/or modulation of p53. p38 MAPK may also prevent cell proliferation through the positive regulation of apoptosis via transcriptional and post-transcriptional mechanisms [36]. Additional work has dem- onstrated that pharmacological inhibition of mTORC1 alone can lead to the activation of the MAPK pathway in a PI3K-dependent feedback loop in human cancer [37]. Our work suggests that, in the context of PI3K inhibition, the activation of p38 MAPK may have a putative role in the chemosensitization of tumor cells. It is of note that only one gene, VAV3, a guanine nucleotide exchange factor which has been shown to be an effector for multiple receptor tyrosine kinases [38,39], overlapped between the present work and the work by O’Brien et al. [6], as a candidate biomarker of de novo sensitivity of breast tumor cell lines to GDC- 0941. A detailed analysis of VAV3 as a modulator of response to GDC-0941 and other PI3K inhibitors in specific tumor subtypes is the subject of a separate study (manuscript in preparation). One potentially important cause of the low overlap between the genes identified here and in the study by O’Brien et al. is that in the latter case the investigators confined their search to breast tumor cell lines, whereas we did not introduce this restriction. The biomarkers identified in our work and other recent studies using panels of diverse tumor cell lines may have increased likelihood to overcome heterogeneity in clinical specimens [40,41]. This work serves as a starting point for further in vitro and in vivo characterization of these candidate biomarkers as predictors of de novo sensitivity or resistance to PI3K inhibitors, as pharmacody- namics markers, as well providing additional insights into the PLOS ONE | www.plosone.org 10 September 2012 | Volume 7 | Issue 9 | e46518