IHC and FISH analysis of PI3KCA in NSCLCs.

<p>A, left: SCC negative for PIK3CA expression; right: SCC positive for PIK3CA expression. B, left: ADC negative for PIK3CA expression; right: ADC positive for PI3KCA expression. Magnification 10× and 40×, respectively. C. Dual-color fluorescence in situ hybridization analysis of PIK3CA gene copy number. FISH analysis of PIK3CA (red signals) and chromosome region 3p14.1 (green signals). Left, NSCLC sample with diploid cells; right, NSCLC sample with multiple clustered spots of red signals of PIK3CA with 2 chromosome region 3p14.1 signals (gene amplification). Original magnification 100×.</p

IHC and FISH analysis of AKT2 in NSCLCs.

<p>A, left: SCC negative for AKT2 expression; right: SCC positive for AKT2 expression. B, left: ADC negative for AKT2 expression; right: ADC positive for AKT2 expression. Magnification 10× and 40×, respectively. C. Dual-colour fluorescence in situ hybridization analysis of AKT2 gene copy number. FISH analysis of AKT2 (red signals) and chromosome region 19p13.1 (green signals). Left, NSCLC sample with diploid cells; right, NSCLC sample with multiple clustered spots of red signals of AKT2 with 2 chromosome region 19p13.1 signals (gene amplification). Original magnification 100×.</p

The top 11 canonical signalling pathways influenced by constitutive PI3K signaling.

<p>Active PIK3CA (E545K)-expressing lentivirus was transduced in non-transformed lung epithelial cells (BEAS-2B). Transduced cells were selected in blasticydin, checked for expression of the exogenous PIK3CA-E545K, were analysed for their transcriptomes as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030427#s2" target="_blank">Materials and methods</a>. <b>A.</b> Immunoblot for PIK3CA expression in transfected BEAS-2B cells. <b>B.</b> Heat map showing fold change patterns of DEGs induced by constitutive PI3K signalling. The heat map was generated in Matlab (Mathworks), and compares fold change patterns of DEGs in BEAS-2B-PI3K-E545K cells compared to parental BEAS-2B (p<0.01). Red: up-regulated genes; green: down-regulated genes. Fold changes of all down-regulated DEGs and all but one up-regulated DEG are #8 (central color spectrum bar). <b>C.</b> The top 11 functional categories determined by IPA, that were significantly up-regulated or down-regulated in BEAS-2B-PI3K-E545K cells compared to parental BEAS-2B are shown. The 2126 DEGs in BEAS-2B-PI3K-E545K were mapped to the IPA-defined network. The significance p-values that determine the probability that the association between the genes in the dataset and the canonical pathway is by chance alone were calculated by Fisher's exact test, and are expressed as −log (p-value). <b>D.</b> Bio-functions identified by IPA in the 2126 DEGs from BEAS-2B-PI3K-E545K compared with BEAS-2B. <b>E.</b> Sub-Categories and Functions identified through IPA showing the genes associated to lung cancer in the 2126 DEGs from BEAS-2B-PI3K-E545K compared with BEAS-2B.</p

Correlation between AKT activation and clinico-pathological features of NSCLC patients.

a<p>AKT activation was evaluated with phospho-specific antibodies (pS473) and scored as negative (<10% of the tumour cells with weak, focal immunopositivity or absence of staining) and high (>10% of tumour cells with strong or diffuse immunopositivity).</p>§<p>G1–G2 vs G3–G4.</p><p>*Stage II vs Stage III.</p

IHC and FISH analysis of AKT1 in NSCLCs.

<p>A, left: SCC negative for AKT1 expression; right: SCC positive for AKT1 expression. B, left: ADC negative for AKT1 expression; right: ADC positive for AKT1 expression. Magnification 10× and 40×, respectively. C. Dual-colour fluorescence in situ hybridization analysis of AKT1 gene copy number. FISH analysis of AKT1 (red signals) and centromere of chromosome 14 (green signals). Left, NSCLC sample with diploid cells; right, NSCLC sample with multiple clustered spots of red signals of AKT1 with 2 centromere signals (gene amplification). Original magnification 100×.</p

Activation of PI3KCA regulates expression of HMGA1, c-Fos, c-MYC in primary NSCLC.

<p>Quantitative real-time RT-PCR analysis of c-Fos, HMGA1, c-Myc gene expression in primary NSCLCs.</p

Mutation analysis of PIK3CA and KRAS genes in NSCLCs.

<p><b>A.</b> Mutation detection in the exons 9 and 20 of PIK3CA from NSCLC. The negative derivative of the fluorescence (−dF/dT) versus temperature graph shows peaks with different Tm. The wild type sample showed a single Tm at 66°C. The heterozygous mutant sample showed an additional peak at 57°C. <b>B.</b> Point mutation in the PI3KCA gene involving a GAG→AAG transition in codon 545 of exon 9 inducing the substitution of a glutammic acid with a lysine (E545K). <b>C.</b> Point mutations in the KRAS gene involving a GGT→GCT, GGT→TGT, GGT→GTT; GGC→TGC transition in codon 12 of exon 2 inducing the substitution of a glycine by an alanine, a cysteine and a valine (G12A G12C G12V) transition in codon 13 of exon 2 inducing the substitution of a glycine by a cysteine (G13C). <b>D.</b> pAKT staining of sample ADC-30.</p

pS473 AKT immunostaining (IHC) in NSCLCs.

<p>A, left: SCC negative for pAKT phosphorylation; right: SCC positive for pS473 phosphorylation. B, left: ADC negative for pAKT phosphorylation; right: ADC positive for pS473 phosphorylation. Magnification 10× and 40×, respectively.</p

Correlation between AKT activation and expression of the different members of the PI3K pathway in NSCLCs.

a<p>AKT activation was evaluated with as pS473 positivity and scored as negative (<10% of positive tumour cells) and high (>10% of positive tumour cells).</p>b<p>PIK3CA was graded as positive (>25% of tumour cells showed strong or diffuse immunopositivity) as moderate (>10% of tumour cells showed moderate immunopositivity) or negative (0–10% of the tumour cells showed weak, focal immunopositivity or absence of staining).</p>c<p>PTEN expression was classified as (+) when staining was detected in >50% of the cells, (+/−) when staining was detected in 25–50% of cells and (−) when staining was detected in 0–25% of cells. For statistical analysis PTEN expression was considered lost when samples were classified as (−).</p>d<p>AKT1 was graded as positive (>25% of positive tumour cells) as moderate (>10% of of positive tumour cells) or negative (0–10% of positive tumour cells).</p>e<p>AKT2 was graded as positive (>25% of positive tumour cells) as moderate (>10% of positive tumour cells) or negative (0–10% of positive tumour cells).</p>§<p>Statistically significant.</p

Activation of PI3KCA regulates expression of HMGA1, c-Fos, c-MYC in NSCLC cell lines.

<p><b>A.</b> Quantitative RT-PCR analysis of HMGA1, c-Fos, c-MYC gene expression in control BEAS-2B cells and in the corresponding cells transduced with active PIK3CA. <b>B.</b> Quantitative real-time RT-PCR analysis of c-Fos, HMGA1, c-Myc gene expression in different NSCLC cell lines.</p