17 research outputs found
List of top-scoring genes that are up-regulated in GDC-0941-resistant or GDC-0941-sensitive tumor cell lines.
<p>List of top-scoring genes that are up-regulated in GDC-0941-resistant or GDC-0941-sensitive tumor cell lines.</p
<i>In vitro</i> sensitivity of the NCI-60 tumor cell line collection to GDC-0941.
<p>A, GI<sub>50</sub> (in ĀµM) of GDC-0941 for 60 cell lines are ordered from lowest to highest, with PIK3CA mutation status indicated with orange box underneath name of cell line. B, z-scores of cell line sensitivity are ordered from lowest to highest. z-scores of 0.8 and ā0.8 are indicated by dashed orange line. C, GI<sub>50</sub> (in ĀµM) of GDC-0941 for 16 sensitive and resistant cell lines of epithelia origin are ordered from lowest to highest. D, z-scores of cell line sensitivity for 16 sensitive and resistant cell lines of epithelia origin are ordered from lowest to highest. z-scores of 0.8 and ā0.8 are indicated by dashed orange line.</p
Loss of OGT expression increases sensitivity of the MDA-MB-231 and OVCAR-4 tumor cell lines to GDC-0941 and alters the phosphorylation state of Chk2.
<p>A, MDA-MB-231 cells transfected with OGT-targeting siRNA or non-silencing siRNA, in the presence or absence of 1 ĀµM GDC-0941. Cell viability was assayed at 0, 24, 48, and 72 hours post-transfection and treatment with GDC-0941. B, Immunoblot analysis of whole cell lysates from MDA-MB-231 cells using anti-OGT or anti-O-GlcNAc antibodies. Cells were treated with 1 ĀµM GDC-0941 or DMSO control for 24 hours, as indicated. C, Immunoblot analysis of whole cell lysates from MDA-MB-231 cells with indicated antibodies. Cell treatments are as indicated. D, Quantitative phosphorylation analysis of Chk2 (T68) in MDA-MB-231 cells transfected with OGT-targeting siRNA or non-silencing siRNA, in the presence or absence of 1 ĀµM GDC-0941 for 24 hours. Cells were treated with 1 ĀµM GDC-0941 or DMSO control for 24 hours, as indicated. E, OVCAR-4 cells transfected with OGT-targeting siRNA or non-silencing siRNA, in the presence or absence of 4 ĀµM GDC-0941. Cell viability was assayed at 0, 24, 48, and 72 hours post-transfection and treatment with GDC-0941. F, Immunoblot analysis of whole cell lysates from OVCAR-4 cells using anti-OGT and anti-OGlcNAc antibodies. Cells were treated with 4 ĀµM GDC-0941 or DMSO control for 24 hours, as indicated. G, Immunoblot analysis of whole cell lysates from OVCAR-4 cells with indicated antibodies. Cell treatments are as indicated. H, Quantitative phosphorylation analysis of Chk2 (T68) in OVCAR-4 cells transfected with OGT-targeting siRNA or non-silencing siRNA, in the presence or absence of 4 ĀµM GDC-0941 for 24 hours. Cells were treated with 4 ĀµM GDC-0941 or DMSO control for 24 hours, as indicated. All above data represent mean Ā± SEM (<i>n</i>ā=ā3);*, <i>P</i><0.05;**, <i>P</i><0.01;***, P<0.001.</p
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.
<p>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 Ī²-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 ĀµM 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 ĀµM GDC-0941. Plotted is expression of DDN mRNA relative to expression of Ī²-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 ĀµM GDC-0941 for 24 hours. Cells were treated with 1 ĀµM GDC-0941 or DMSO control for 24 hours, as indicated. Data in panels B and E represents mean Ā± SD (nā=ā3). Data in panels C and F represent mean Ā± SEM (<i>n</i>ā=ā3);*, <i>P</i><0.05;**, <i>P</i><0.01;***, P<0.001.</p
Gene expression signature predictive of <i>in vitro</i> tumor cell line response to GDC-0941.
<p>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 (nā=ā8) and resistant (nā=ā8) 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 āSā or resistant āRā. Cell lines are shown on the horizontal axis and genes are shown on the vertical axis. Color bar indicates relative levels (log<sub>2</sub>) of gene expression, following median centering. BāC, 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 log<sub>2</sub> mRNA expression of DDN or OGT (y-axis) versus GI<sub>50</sub> (ĀµM) of GDC-041 (x-axis) for each of 16 cell lines (nā=ā3 for each cell line tested). Correlation between log<sub>2</sub> mRNA expression and GI<sub>50</sub> as estimated by Spearmanās rank correlation (r<sub>s</sub>) is indicated. Data in box plots (right panels) represent mean Ā± SD log<sub>2</sub> level of mRNA expression of DDN or OGT in all sensitive cell lines (nā=ā8) versus all resistant cell lines (nā=ā8);*, <i>P</i><0.05;**, P<0.01.</p
Integration of RNAseq, ChIPseq and TCGA data.
<p><sup>a</sup>Amplified/highly-expressed <i>vs</i>. non-amplified/highly-expressed (FDR<10%)</p><p>Integration of RNAseq, ChIPseq and TCGA data.</p
Integrative Genomics Implicates EGFR as a Downstream Mediator in <i>NKX2-1</i> Amplified Non-Small Cell Lung Cancer
<div><p><i>NKX2-1</i>, encoding a homeobox transcription factor, is amplified in approximately 15% of non-small cell lung cancers (NSCLC), where it is thought to drive cancer cell proliferation and survival. However, its mechanism of action remains largely unknown. To identify relevant downstream transcriptional targets, here we carried out a combined NKX2-1 transcriptome (NKX2-1 knockdown followed by RNAseq) and cistrome (NKX2-1 binding sites by ChIPseq) analysis in four <i>NKX2-1</i>-amplified human NSCLC cell lines. While NKX2-1 regulated genes differed among the four cell lines assayed, cell proliferation emerged as a common theme. Moreover, in 3 of the 4 cell lines, epidermal growth factor receptor (EGFR) was among the top NKX2-1 upregulated targets, which we confirmed at the protein level by western blot. Interestingly, EGFR knockdown led to upregulation of NKX2-1, suggesting a negative feedback loop. Consistent with this finding, combined knockdown of NKX2-1 and EGFR in NCI-H1819 lung cancer cells reduced cell proliferation (as well as MAP-kinase and PI3-kinase signaling) more than knockdown of either alone. Likewise, NKX2-1 knockdown enhanced the growth-inhibitory effect of the EGFR-inhibitor erlotinib. Taken together, our findings implicate EGFR as a downstream effector of NKX2-1 in <i>NKX2-1</i> amplified NSCLC, with possible clinical implications, and provide a rich dataset for investigating additional mediators of NKX2-1 driven oncogenesis.</p></div
NKX2-1 regulates EGFR levels, with negative feedback.
<p>(A) NKX2-1 knockdown leads to reduced EGFR protein levels quantified by western blot (% residual indicated). Levels normalized to Ī±-tubulin loading control. (B) EGFR knockdown by siRNA reduces cell proliferation comparable to NKX2-1 knockdown (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142061#pone.0142061.g001" target="_blank">Fig 1A</a>). **, <i>P</i>-value < 0.01 (two tailed Studentās t-test). (C) EGFR knockdown leads to elevated NKX2-1 protein levels (% increase indicated; levels normalized to Ī±-tubulin loading control), suggesting negative feedback regulation.</p
Combined NKX2-1 and EGFR knockdown reduces cell proliferation and MAPK/PI3K signaling.
<p>(A) Combined knockdown of NKX2-1 and EGFR reduces H1819 cell proliferation more than either alone. **, <i>P</i>-value < 0.01; ***, <i>P</i>-value < 0.001 (two tailed Studentās t-test). (B) Combined knockdown of NKX2-1 and EGFR in H1819 cells diminishes MAPK signaling (p-MAPK) and PI3K signaling (p-AKT) more than either alone. Percent residual indicated; levels normalized to Ī±-tubulin loading control. Note, in the particular western shown, EGFR knockdown does not appear to increase NKX2-1 levels appreciably, although the increase has been reproducibly observed in multiple other experiments (e.g. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142061#pone.0142061.g004" target="_blank">Fig 4C</a>, and Figure B, Panel H in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142061#pone.0142061.s002" target="_blank">S2 File</a>). (C) NKX2-1 knockdown collaborates with EGFR inhibitor erlotinib to inhibit H1819 cell growth ***, <i>P</i>-value ā¤ 0.001 (two tailed Studentās t-test).</p
Defining the NKX2-1 cistrome by ChIPseq.
<p>(A) Validating the NKX2-1 antibody for ChIP: ChIP-PCR identifies the known NKX2-1 binding site in the SFTPB promoter. Note the enrichment of SFTPB compared to an irrelevant gene (WNT5A). (B) Overlap among the four NSCLC cell lines of NKX2-1 binding site-associated genes (within 100Kb). (C) <i>De novo</i> motif analysis re-discovers the known NKX2-1 consensus binding motif, and identifies enrichment of other transcription factor binding motifs nearby NKX2-1 binding sites. (D) NKX2-1 binding peaks identified at the EGFR locus in H1819 cells. The two called peaks are identified by blue triangles, and supporting reads are shown in the close-up inset. Binding peaks at EGFR in other cell lines are shown in Figure D, Panel B in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142061#pone.0142061.s002" target="_blank">S2 File</a>.</p