The GSK3β/β-catenin/MnSOD axis promotes resistance to GD-induced cell death.

<p>(A) Wild type HME cells were cotransfected with equal amounts of the indicated expression vectors. 24 hours after transfection, cells were glucose-starved for 48 hours and the percentage of dead cells was quantified by FACS analysis of propidium iodide positive cells. Graphs report the average derived from three independent experiments ± SD (<i>t-</i>test, **p<0.01). The inset shows an anti-HA immunoblot of total protein extracts from cells transfected with the HA-GSK3β or HA-GSK3β(K85R) vectors and used for the GD experiments. β-actin was used as loading control. (B) Wild type HME cells stably transfected with an empty vector (pcDNA#1 and pcDNA#2) or with an expression vector for MnSOD (MnSOD#1 and MnSOD#2) were exposed to GD for 60 hours and the percentage of dead cells were quantified by FACS analysis of propidium iodide positive cells. At least three independent empty vector- or MnSOD-transfected clones were analyzed and gave similar results. The inset represents an immunoblot analysis showing the expression of MnSOD in total protein extracts from two representative clones used for the experiments. β-actin was used as loading control. Graphs report the average derived from three independent experiments ± SD (<i>t-</i>test, **p<0.01 or ***p<0.001, empty vector Vs MnSOD).</p

Cells expressing <i>^delE746-A750EGFR</i> or <i>^E545KPIK3CA</i> cancer alleles have functional AMP/LKB1/AMPK sensor machinery.

<p>(A) Regulation of LKB1 kinase in HME cells after GD. Equal amounts of total protein lysates of wild type or isogenic cells were analyzed by immunoblot with antibodies against LKB1 and pLKB1(Ser428); β-actin was used as loading control. The graph indicates the densitometry analysis of the data derived from at least three independent experiments (<i>t-</i>test, *p<0.05, Not treated Vs GD-treated cells. <i>ns</i>: not significant). (B) AICAR treatment increases pAMPKα(T172) in wild type and in oncogene-carrying cells and does not influence LKB1 expression. The indicated isogenic cell lines were treated for 2 hours with 1 mM AICAR and total protein lysates were analyzed by immunoblot with the indicated antibodies.</p

ATP content and GSH/GSSG ratio in wild type or oncogene-expressing HME cells after glucose deprivation.

<p>(A) Total ATP content after GD in wild type HME cells and isogenic clones carrying <i>^delE746-A750EGFR</i>, <i>^E545KPIK3CA</i> or <i>^G13DKRAS</i> cancer alleles. At the indicated time, cells were harvested and the total ATP content was analyzed; ATP amount was normalized for the number of nuclei. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037526#s2" target="_blank">Results</a> (mean ± SD, n = 3) are expressed as percentages of the ATP amount respect to T = 0. WTp: wild type HME parental cells; PI3K_cnt and EGFRdel_cnt are HME control cells carrying the wild type alleles obtained through somatic homologous recombination (see text for details). (B) Reduced and oxidized glutathione ratio (GSH/GSSG) in wild type, control and oncogenes-carrying cells after GD. Cells were glucose starved for 10 hours, and then the GSH/GSSG ratio was measured. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037526#s2" target="_blank">Results</a> report the average of eight independent experiments ± SD (<i>t-</i>test, **p<0.01, Not treated Vs GD-treated cells. <i>ns</i>: not significant).</p

GD-induced oxidative stress control AMPKα activation.

<p>(A) Wild type HME or isogenic cells carrying the indicated cancer alleles were glucose deprived for 10 hours and equal amounts of protein lysates were assayed by immunoblot with the indicated antibodies. The graph reports the densitometry analysis of the pAMPKα/AMPKα_total. (B) Time course analysis of AMPKα phosphorylation after GD. Wild type HME and isogenic cells carrying <i>^delE746-A750EGFR</i> or <i>^E545KPIK3CA</i> or <i>^G13DKRAS</i> alleles were glucose starved for the indicated time. Total proteins were analyzed by immunoblot as indicated. The graph reports the densitometry analysis of the pAMPKα/AMPKα_total. <i>Wild type</i>: black square; <i>^E545KPIK3CA</i>: white square; <i>^delE746-A750EGFR</i>: gray square; <i>^G13DKRAS</i>: dark gray square. Data are representative of three independent experiments that gave similar results. (C and D) Treatment with antioxidants attenuates GD-induced pAMPKα(T172). Wild type HME cells were pre-treated with N-acetyl-L-Cysteine (C) or mimetic SOD (D) 1 hour before glucose starvation. Proteins extracts were analyzed by immunoblot with anti-pAMPKα(T172) and anti-AMPKα antibodies. The graph reports the densitometry analysis of the pAMPKα/AMPKα_total; average from at least three independent experiments ± SD are reported as relative fold to not-treated cells. (E) Catalase treatment reduces GD-induced pAMPKα. Wild type HME cells were treated with catalase at the indicated concentrations and harvested after 3 hours of GD. Equal amount of protein lysates were analyzed by immunoblot. (F) G6PDH inhibitor induces AMPKα(T172) phosphorylation through ROS generation. Wild type HME cells were treated as indicated with 6-AN in glucose- and serum-containing media (left panels) or were pre-treated with NAC before 6-AN treatment (right panels) and total protein extracts were analyzed by immunoblot with anti-pAMPKα(T172) and anti-AMPKα antibodies. Histograms in E and F report the densitometry analysis of the pAMPKα/AMPKα_total. Average from at least three independent experiments ± SD were plotted.</p

HME cells carrying the <i>^delE746-A750EGFR</i> or the <i>^E545KPIK3CA</i> allele are resistant to GD-induced cell death.

<p>Wild type or isogenic clones carrying <i>^delE746-A750EGFR</i> or <i>^E545KPIK3CA</i> or <i>^G13DKRAS</i> alleles were glucose-starved (GD) for the indicated hours and the percentage of dead cells were quantified by FACS analysis of propidium iodide positive cells. Graphs report the average of three independent experiments ± SD (<i>t-</i>test, **p<0.05 or ***p<0.001, Not treated Vs GD-treated cells). The diagrams are representative of a FACS analysis performed 24 hours after GD.</p

Upregulation of FOXO4 and β-catenin by EGFR and PIK3CA oncogenes contributes to <i>MnSOD</i> expression in response to GD.

<p>(A) GD controls β-catenin phosphorylation and stability. Wild type HME or oncogene carrying cells were glucose deprived for 4 hours in the presence or not of lithium chloride and equal amounts of proteins were assayed by immunoblot. The anti-pβ-catenin antibody recognizes the phosphorylated Ser33,37 and Thr41 residues of β-catenin. The graph reports the densitometry analysis of the pβ-catenin/β-catenin (gray bars) and the β-catenin/β-actin (black bars). (B) Time course analysis of β-catenin phosphorylation after GD. HME clones were GD for the indicated time. Total proteins were analyzed by immunoblot as indicated. The graph reports the densitometry analysis of pβ-catenin/β-catenin. (C) Regulation of GSK3β phosphorylation in response to GD. The indicated HME clones were glucose starved for 6 hours and total protein extracts were analyzed by immunoblot as indicated. pGSK3β antibody recognizes specifically the phospho-Ser9 residue of GSK3β. The graph reports the densitometry analysis of the pGSK3β/β-catenin signals. (D) Regulation of nuclear β-catenin and FOXO4 accumulation by oncogenes in response to GD. Wild type HME or isogenic cells carrying <i>^delE746-A750EGFR</i> or <i>^E545KPIK3CA</i> cancer alleles were glucose starved for 5 hours and equal amounts of nuclear protein extracts were assayed by immunoblot as indicated. MCM7 antibody recognizes the nuclear minichromosome maintenance protein 7 and is here used as loading control. Graphs report the densitometry analysis of indicated protein/MCM7 signals ratio. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037526#s2" target="_blank">Results</a> report the average of three independent experiments ± SD (<i>t-</i>test, **p<0.01; *p<0.05). (E) Intracellular localization of active β-catenin (ABC) after GD exposure. Wild type HME or isogenic cells carrying the <i>^delE746-A750EGFR</i> or the <i>^E545KPIK3CA</i> cancer alleles were glucose-starved for 5 hours. Cell were fixed and stained with a specific anti active, not phosphorylated β-catenin antibody (Red) and DAPI (Blue) for the nuclear staining and analyzed by fluorescence microscopy. Graphs report the percentage of β-catenin positive nuclei (average ± SD of 10 different fields containing at least 40 cells/field. <i>t-</i>test, ***p<0.001, **p<0.01, *p<0.05). The exposure time was kept constant through the images analysis. (F) β-catenin contributes to <i>MnSOD</i> promoter activation under GD. (Left graph) Wild type HME cells were transfected with vectors expressing the indicated proteins together with a wild type MnSOD promoter luciferase-reporter (−3340+1<i>MnSOD-luc)</i>, or with a mutant derivative which contains mutated FOXO binding sites (−3340+1mut<i>MnSOD-luc</i>) and analyzed after 8 hours of GD. Data represent means ± SD derived from four independent experiments. (Right graph) Wild type HME cells or isogenic clones expressing the delE746-A750EGFR or the E545KPIK3CA allele were transfected with vectors expressing the indicated proteins in presence of wild type MnSOD promoter luciferase-reporter and analyzed after 8 hours of GD. Data represent means ± SD. The efficiency of transfection was normalized by the cotransfection of CMV-Renilla luciferase reporter.</p

GD induces the transcription of antioxidant enzymes in cells carrying the <i>^delE746-A750EGFR</i> or the <i>^E545KPIK3CA</i> alleles.

<p>(A) Upregulation of <i>MnSOD</i> expression by oncogenes. Total RNA was harvested from wild type HME or the indicated isogenic clones. The expression of <i>MnSOD</i> normalized to β-actin mRNA was analyzed by quantitative real time PCR. Data (means ± SD) indicate fold relative to wild type cells. (B) Wild type or the indicated isogenic HME cells were glucose starved and RNA was harvested at the indicated time points. The relative expression of the <i>MnSOD</i> or <i>Catalase</i> genes, normalized to β-actin mRNA, was analyzed by quantitative real time PCR. Data show means ± SD.</p

NAC and Catalase confer resistance to GD-induced cell death.

<p>(A) Antioxidant but not pyruvate treatment inhibits GD-induced cell death. Wild type HME cells were glucose starved for 36 hours with or without 5 mM N-acetyl-L-Cysteine or sodium pyruvate at the indicated concentrations. The percentage of dead cells was quantified by FACS as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037526#pone-0037526-g001" target="_blank">Fig 1</a>. The data are derived on the average from at least three independent experiments ± SD (<i>t-</i>test, **p<0.05 or **p<0.001). (B) Purified human catalase treatment induces cellular resistance to GD-induced cell death. Wild type HME cells were glucose starved for 36 hours with or without 12 hours of pre-treatment with purified human catalase (600 U/ml) or heat inactivated catalase (HI). The percentage of dead cells was quantified as in (A).</p

Total glycogen content in oncogene-expressing HME cells after glucose deprivation.

<p>Total glycogen content after GD in wild type HME cells and isogenic clones carrying <i>^delE746-A750EGFR, ^E545KPIK3CA</i> or <i>^G13DKRAS</i> cancer alleles. At the indicated time, cells were harvested and total glycogen content was analyzed. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037526#s2" target="_blank">Results</a> (average of three independent experiments ± SD) are expressed as glycogen content normalized for protein mass. WT: wild type HME cells; PIK3CA_cnt and EGFR_cnt are HME control cells carrying the wild type alleles (see text for details).</p

Supplementary Figure Legends from TGFα and Amphiregulin Paracrine Network Promotes Resistance to EGFR Blockade in Colorectal Cancer Cells

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