Signaling networks associated with AKT activation in non-small cell lung cancer (NSCLC): new insights on the role of phosphatydil-inositol-3 kinase.

Abstract

Aberrant activation of PI3K/AKT signalling represents one of the most common molecular alterations in lung cancer, though the relative contribution of the single components of the cascade to the NSCLC development is still poorly defined. In this manuscript we have investigated the relationship between expression and genetic alterations of the components of the PI3K/AKT pathway [KRAS, the catalytic subunit of PI3K (p110α), PTEN, AKT1 and AKT2] and the activation of AKT in 107 surgically resected NSCLCs and have analyzed the existing relationships with clinico-pathologic features. Expression analysis was performed by immunohistochemistry on Tissue Micro Arrays (TMA); mutation analysis was performed by DNA sequencing; copy number variation was determined by FISH. We report that activation of PI3K/AKT pathway in Italian NSCLC patients is associated with high grade (G3-G4 compared with G1-G2; n = 83; p<0.05) and more advanced disease (TNM stage III vs. stages I and II; n = 26; p<0.05). In addition, we found that PTEN loss (41/104, 39%) and the overexpression of p110α (27/92, 29%) represent the most frequent aberration observed in NSCLCs. Less frequent molecular lesions comprised the overexpression of AKT2 (18/83, 22%) or AKT1 (17/96, 18%), and KRAS mutation (7/63, 11%). Our results indicate that, among all genes, only p110α overexpression was significantly associated to AKT activation in NSCLCs (p = 0.02). Manipulation of p110α expression in lung cancer cells carrying an active PI3K allele (NCI-H460) efficiently reduced proliferation of NSCLC cells in vitro and tumour growth in vivo. Finally, RNA profiling of lung epithelial cells (BEAS-2B) expressing a mutant allele of PIK3 (E545K) identified a network of transcription factors such as MYC, FOS and HMGA1, not previously recognised to be associated with aberrant PI3K signalling in lung cancer.

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Signaling Networks Associated with AKT Activation in
Non-Small Cell Lung Cancer (NSCLC): New Insights on the
Role of Phosphatydil-Inositol-3 kinase
Marianna Scrima1, Carmela De Marco1,2, Fernanda Fabiani2, Renato Franco3, Giuseppe Pirozzi3, Gaetano
Rocco3, Maria Ravo4, Alessandro Weisz4, Pietro Zoppoli1,5, Michele Ceccarelli1,5, Gerardo Botti3,
Donatella Malanga1,2, Giuseppe Viglietto1,2*
1Biogem scarl, Institute for Genetic Research ‘‘Gaetano Salvatore’’, Ariano Irpino (Avellino), Italy, 2Department of Experimental and Clinical Medicine, University Magna
Graecia Catanzaro, Italy, 3Fondazione ‘‘G Pascale’’, National Cancer Institute, Naples, Italy, 4Molecular Medicine Laboratory, Faculty of Medicine and Surgery, University of
Salerno, Baronissi, Italy, 5Department of Biological and Environmental Studies, University of Sannio, Benevento, Italy
Abstract
Aberrant activation of PI3K/AKT signalling represents one of the most common molecular alterations in lung cancer, though
the relative contribution of the single components of the cascade to the NSCLC development is still poorly defined. In this
manuscript we have investigated the relationship between expression and genetic alterations of the components of the
PI3K/AKT pathway [KRAS, the catalytic subunit of PI3K (p110a), PTEN, AKT1 and AKT2] and the activation of AKT in 107
surgically resected NSCLCs and have analyzed the existing relationships with clinico-pathologic features. Expression analysis
was performed by immunohistochemistry on Tissue Micro Arrays (TMA); mutation analysis was performed by DNA
sequencing; copy number variation was determined by FISH. We report that activation of PI3K/AKT pathway in Italian
NSCLC patients is associated with high grade (G3–G4 compared with G1–G2; n=83; p,0.05) and more advanced disease
(TNM stage III vs. stages I and II; n=26; p,0.05). In addition, we found that PTEN loss (41/104, 39%) and the overexpression
of p110a (27/92, 29%) represent the most frequent aberration observed in NSCLCs. Less frequent molecular lesions
comprised the overexpression of AKT2 (18/83, 22%) or AKT1 (17/96, 18%), and KRAS mutation (7/63, 11%). Our results
indicate that, among all genes, only p110a overexpression was significantly associated to AKT activation in NSCLCs
(p=0.02). Manipulation of p110a expression in lung cancer cells carrying an active PI3K allele (NCI-H460) efficiently reduced
proliferation of NSCLC cells in vitro and tumour growth in vivo. Finally, RNA profiling of lung epithelial cells (BEAS-2B)
expressing a mutant allele of PIK3 (E545K) identified a network of transcription factors such as MYC, FOS and HMGA1, not
previously recognised to be associated with aberrant PI3K signalling in lung cancer.
Citation: Scrima M, De Marco C, Fabiani F, Franco R, Pirozzi G, et al. (2012) Signaling Networks Associated with AKT Activation in Non-Small Cell Lung Cancer
(NSCLC): New Insights on the Role of Phosphatydil-Inositol-3 kinase. PLoS ONE 7(2): e30427. doi:10.1371/journal.pone.0030427
Editor: Alfredo Fusco, Consiglio Nazionale delle Ricerche (CNR), Italy
Received July 25, 2011; Accepted December 16, 2011; Published February 17, 2012
Copyright: ? 2012 Scrima et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from the Associazione Italiana Ricerca sul Cancro (AIRC) to GV and AW and by European Union (I.P: CRESCENDO,
contract n.er LSHM-CT-2005-018652), Ministero dell’Universita ` e della Ricerca (PRIN, Grant n.er 2008CJ4SYW_004) to A.W. The funders had no role in study design,
data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: viglietto@unicz.it
Introduction
Lung cancer is the leading cause of cancer deaths worldwide
[1,2]. Epithelial lung cancer is classified into two main groups:
small-cell lung cancer (SCLC) (about 15% of all lung cancers) and
non–small-cell lung cancer (NSCLC) (about 85% of all lung
cancers) [3]. NSCLC comprises squamous-cell carcinoma (SCC),
adenocarcinoma (ADC), and large-cell lung cancer (LCC) [3].
Despite advances in early detection and standard treatment,
NSCLC is often diagnosed at an advanced stage and patients often
have poor prognosis, with five-year survival rate less than 15%
[4,5]. For this reason a better understanding of the molecular
origins of the disease will contribute to improve therapeutic
treatment of lung cancer patients.
Recent studies have shown that the phosphatidylinositol 3-
kinase (PI3K) signalling cascade is frequently overactivated in
human cancer [6–8] playing a critical role both in the initiation
and progression of NSCLC [9,10]. The PI3K pathway regulates
cellular functions such as proliferation, survival, motility and
angiogenesis that are critical to the growth and/or maintenance of
tumours [11,12]. The end-point of the PI3K pathway is AKT, a
serine/threonine protein kinase that mediates most signals
funnelled through the PI3K pathway. AKT is activated by
recruitment to cell membrane via binding of its PH domain to 39-
phosphorylated phosphatidylinositols generated by PI3K and
subsequent phosphorylation at T308 and S473 [12,13]. Con-
versely, the lipid phosphatase PTEN attenuates AKT activation by
dephosphorylating the 39 position of phosphatidylinositols [14].
Aberrant AKT activation contributes to lung carcinogenesis
[9,10]. Hyperactivation of AKT is detected in most NSCLC cell
lines [15–17], and in 30–75% NSCLCs [18–22] and promotes
resistance to chemotherapy and radiation therapy [16]. AKT
activation in cancer is currently evaluated using phospho-specific
antibodies against S473 in immunohistochemical analyses of
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tumour specimens. Although phosphorylation of AKT at S473 has
been correlated with poor clinical outcomes in many tumour
types, results in lung cancer are apparently inconsistent [7–10]
having been associated with either poor or good prognosis [20–
22]. AKT can be activated through several mechanisms, which
result from distinct and often mutually exclusive events that
include activating mutations (KRAS, PIK3CA or AKT1),
increased expression (PIK3CA, AKT1, AKT2) or loss of PTEN
[10]. However, the relative contribution of the single components
within the PI3K pathway to AKT activation in NSCLCs is still
unclear. In this manuscript we have investigated the relationship
between the genetic alterations present in these genes and the
activation of AKT in NSCLC.
Materials and Methods
Ethics Statement
Patient accrual was conducted according to internal Review
Board of the INT Fondazione Pascale (Naples, Italy) (CEI 556/10
of 12/3/2010). The study was approved by the internal Review
Board of the AOU Mater Domini/University Magna Graecia
(Catanzaro, Italy) in the meeting of 16/3/2011. Written informed
consent was obtained from all participants to the study. All animal
work was conducted according to the relevant Italian guidelines
and was approved by the Internal Committee for Animal Study
(CESA) of the Institute for Genetic Research ‘‘Gaetano Salvatore
on April 7th2008 (CESA 10-08).
Patients
Archive material from 107 patients diagnosed of NSCLC [3]
was obtained from INT Fondazione Pascale (Naples, Italy).
Median age was 64 year old (range 28–82). Among patients with
clinical data available, women were 18 and males 83. Stage was
known for 81 patients: 67 patients had stage I–II disease and 14
had stage III–IV disease. Grade was known for 83 patients: 35
cases were G1–G2 and 48 were G3–G4. See Table S1, Table S2
and Table S3 for more detailed clinical characteristics of all
patients.
TMA slides were deparaffinized, heated in a pressure cooker
with 1 mM EDTA, pH 8.0 for 10 min, and incubated with pepsin
at 37uC for 30 min. The slides were then dehydrated in increasing
ethanol concentrations, and then air-dried. The probes were
denatured at 96uC for 5 min, and hybridization solution was
applied on each slide and incubated at 75uC for 1 min. After
overnight incubation at 37uC in a humid chamber, slides were
washed with 0.46 SSC and 0.3% NP40 for 2 min at 75uC, air-
dried in darkness, counterstained with DAPI, and a coverslip was
applied.
Tissue Microarray (TMA) and Immunohistochemistry
TMAs were constructed in collaboration with the Unit of
Immunostaining at the Centro Nacional de Investigaciones
Oncologicas (Madrid, Spain) according to established methods
[23] using a Tissue Arrayer (Beecher Instruments, Gene Micro-
Array Technologies, Silver Spring, MD). Immunostaining was
performed using the avidin-biotin-peroxidase method (LSAB kit;
DAKO, Glostrup, Denmark) as described previously [24].
Antibodies used for immunostaining were selected according to
previously published work [25–29]. Anti-pS473 (#9277), anti-
AKT1 (#2938), anti-AKT2 (#4057), anti-PIK3CA (#4249), anti-
PTEN (#9559) were all from Cell Signaling Technology (Danvers,
MA, USA).
The anti-Akt1 and anti-Akt2 have been shown to be isoform-
specific antibodies in previous work [25]. In addition, by using
NCI-H460 cells interfered for Akt1 or Akt2, respectively, we
confirmed that the anti-Akt1 antibody recognizes only the Akt1
isoform and the anti-Akt2 antibody recognizes only the Akt2
isoform (Figure S1A).
The immunohistochemical score of pAKT and PTEN used in
this work was selected on the basis of the widely established criteria
existing in the literature [28,30,31]: pAKT was scored as positive
when .10% of tumour cells were positive with strong or diffuse
immunopositivity. PTEN expression was classified as (+) when
staining was detected in .50% of the cells, (+/2) when staining
was detected in 25–50% of cells and (2) when staining was
detected in 0–25% of cells. For statistical analysis PTEN
expression was considered lost when samples were classified as (2).
Also for the immunostaining scores of AKT1, AKT2 and
PIK3CA, we selected criteria described in previous reports
[27,28,32]. Tumor specimens were divided into four groups
according to the percentage of positive cells: (2) comprised
completely negative samples; (+) comprised samples with up to
10% of positive cells; (++) comprised samples with 11–50% of
positive cells; and (+++) comprised samples with .50% of positive
cells, respectively. For statistical reasons, tumours were classified
into a low expression group comprising (2) and (+) and a high
expression group that comprises (++) and (+++).
For each one immunohistochemical round a negative control
has been included, by replacing the primary antibody with solvent
at the same volume of that with the primary antibody resuspended
in it. All controls gave satisfactory results. Stained TMA sections
were evaluated by two expert pathologists (RF, GB) using uniform
criteria. Discrepancies were resolved through simultaneous
inspection and discussion of the results. Discrepancies between
two cores from the same case were resolved through a joint
analysis of the two cores.
Fluorescence In Situ Hybridization (FISH)
FISH analysis was performed on TMAs. BAC clones were
designed according to the Ensembl database (www.ensembl.org).
BAC clones covering the AKT1 gene were RP11-982M15, RP11-
477I4 and RP11-556J09. Control BAC probes covering chromo-
some region 14q11 was RP11-324B11. BAC clones covering the
AKT2 gene were RP11-36B02, RP11-688J23, RP11-725P04.
Control BAC probes covering chromosome region 19p13.1 were
RP11-737I1, RP11-520G3. BAC clones covering the PIK3CA
gene were RP11-360P21 and RP11-245C23. Control BAC probes
covering chromosome region 3p14.1 were RP11-175F9 and
RP11-15B21. All BAC clones were labelled with dUTP-Sprectrum
Orange (Vysis Inc., DownersGrove, IL; USA). All Control probes
were labelled with dUTP-Sprectrum Green (Vysis Inc., Down-
ersGrove, IL; USA).
Two different investigators that had no previous knowledge of
the genetic, clinical and IHC results evaluated FISH analysis. All
FISH were scored in an average of 130 (60–210) nuclei.
For evaluation of copy number of the genes encoding AKT1,
AKT2 and PIK3CA, a gene-to-control ratio of 1.0 was classified
as disomy; ratios between 1.0 and 2.0 were considered gene low-
level gains; ratios .2.0 were considered as high polysomy and/or
gene amplification [33,34].
Accordingly, tumours were divided into different classes:
disomy, trisomy (3 copies of chromosomes in .40% of cells),
low polysomy ($3 copies of chromosomes in .40% of cells), high
polysomy ($4 copies of chromosomes in $40% of cells), and gene
amplification (presence of gene clusters with a ratio of gene-to-
chromosome of $2 per cell in $40% of cells or presence of small
or nonenumerable clusters of the gene signal). This allowed the
classification of patients into two groups: FISH-negative (disomy
Molecular Alterations in NSCLC
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and gains) and FISH-positive (high polysomy and/or gene
amplification).
PCR, RT-PCR and mutation analysis
Total RNA and genomic DNA were prepared as described
[35,36]. Q-RT-PCR and Q-PCR were performed using the Power
SYBR Green PCR Master Mix in an ABI Prism 7300
thermocycler (Applied Biosystems, Foster City, CA, USA). cDNAs
were synthesized from 1 mg of total RNA using QuantiTect
Reverse Trascription (Qiagen, The Netherlands, Venlo). Normal-
ization was performed to GAPDH mRNA content. The relative
amounts of mRNA or DNA were calculated by the comparative
cycle threshold (CT) method by Livak and Schmittgen [37].
Mutation analysis for PIK3CA using LightCycler was performed
with DNA Master/Hybridization probes kit (Roche Molecular
Biochemicals, Mannheim, Germany). Direct sequencing was
performed using the BigDye v3.03 cycle sequencing kit (Applied
Biosystems) in a capillary automatic sequencer (ABI PRISM 3100
Genetic Analyzer; Applied Biosystems). Protocols and primers for
Q-PCR, Q-RT-PCR and sequencing KRAS (exons 2 and 3) and
PIK3CA (exons 9 and 20) are reported in Appendix S1.
Antibodies and Western Blot
Western blot analysis was carried out by standard methods [38].
Whole cell extracts were prepared by homogenizing cells in NP-40
lysis buffer (10 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1% NP-
40) containing protease inhibitors. Lysates were cleared by
centrifugation and proteins were separated by SDS-PAGE.
Antibodies used were from Cell Signaling Technology: anti-
AKT1 (#2938); anti-S473 (#9277), anti-PIK3CA (#4249).
Cell lines
NCI-H460 was purchased from ATCC-LGC Promochem
(South West London, UK) and maintained in RPMI1640
(Gibco-Invitrogen, Carlsbad, CA, USA), supplemented with
10% of fetal bovine serum and 100 U/ml penicillin-streptomycin
(Invitrogen, Carlsbad, CA, USA). BEAS-2B cells were purchased
from Cambrex (Milan, Italy) and grown as suggested by the
manufacturer [39].
Virus generation and Infection
To generate p110a encoding lentivirus, the cDNA encoding
human p110a (Addgene, Cambridge, MA, USA) was cloned in
pENTR1A vector (Invitrogen) and recombined in pLenti6.2/C-
LumioTM/V5-DEST Vector by making use of the Gateway
Technology (Invitrogen). pLenti vector was used to generate
lentiviral particles in HEK293T packaging cells as described [40].
Transduced BEAS-2B cells underwent three rounds of infection
and were selected in medium containing 5 mg/ml blasticidin
(Invitrogen).TheHuman PIK3CA
(NM_005163) and AKT2 (NM_001626) MISSION shRNA set
(Sigma-Aldrich, St.Luis, MO) and the Mission non-target control
transduction viruses (SHC002V) were used to generate lentiviral
particles in HEK293T packaging cells [40]. After transfection,
supernatants were collected at 8-hour intervals, filtered and used
for three rounds of transduction of NCI-H460 cells in the presence
of 8 mg/ml of polybrene (Sigma).
(NM_006218),AKT1
In Vitro Proliferation Assay
Cells proliferation was assayed by MTT [3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide; Sigma] reduction. Cells
were plated in 96-well flat-bottomed microtiter plates (200 ml cell
suspensions, 26103/well for NCI-H460) and incubated with MTT
substrate (5 mg/ml) for 4 h. Every 24 hours, the culture medium
was removed and anhydrous 2-propanol was added. The optical
density was measured at 570 nm.
Tumourigenic assays
Cells (16106) were suspended in 100 ml 10% FBS and 100 ml
Matrigel (BD Biosciences, NJ, USA) and subcutaneously injected
into the right flank of 6-week-old athymic nude mice (Charles
River, West Germany) in triplicates. Every 7 days tumour size was
measured with a caliper.
RNA profiling analysis
RNA concentration was determined with a Nanodrop (Nano-
Drop, Wilmington, Delaware, USA) spectrophotometer and its
quality was assessed with an Agilent 2100 Bioanalyzer (Agilent
Technologies, Milano, Italy). For each sample, 500 ng of total
RNA were synthesized to biotinylated cRNA using the Illumina
RNA Amplification Kit (Ambion, Inc., Austin, TX). Synthesis was
carried out according to the manufacturers’ instructions. cRNA
concentration and the quality were assessed out as described
above. From each sample, technical replicates were produced and
750 ng cRNA were hybridized for 18 hrs to Human HT-
12_V3_0_R1 Expression BeadChips (Illumina Inc., San Diego,
CA, USA) according to the protocol provided by the manufac-
turer. Hybridized chips were washed and stained with streptavi-
din-conjugated Cy3 (GE Healthcare Milano, Italy). BeadChips
were dried and scanned with an Illumina BeadArray Reader
(Illumina Inc.).
Microarrays data analysis: RNA profiling, genes’
characterization, enriched pathways and bibliographic
networks discovery
Expression files were normalized and analyzed using Gene-
Spring 10.1 (Agilent Technologies, Santa Clara, CA). Differen-
tially expressed (DEGs) genes between BEAS-2B and BEAS-PI3K-
CA cells were selected on the basis of the fold change (the ratio
between the expression levels in the two conditions) and the
statistical significance. We filtered the lists using fold change 1.5
and T-test (p-value (0.01) as threshold. The DEGs list (composed
by 2126 probesets) was used to evaluate the functional behavior in
terms of Biological Processes and Molecular Function, Develop-
ment Function and Disease and Disorder terms. The degree of
enrichment was statistically evaluated to determine whether an
observed level of annotation for a group of genes is significant. In
particular, for each term, a q-value was computed by the
Hypergeometric test (p#0.05) and corrected using False Discovery
Rate (FDR) [41]. The terms with a q-value exceeding the
significance threshold were then selected as representative.
Pathway and network analysis were performed using Ingenuity
Pathway Analysis (IPA, Ingenuity Systems).
The dataset was mined for significant pathways with the IPA
library of canonical pathways, and networks were generated by
using IPA as graphical representation of the molecular relation-
ships between genes and gene products. The significance of the
association between the list of DEGs and the Canonical Pathway
was measured using a Fisher’s exact test to calculate a p-value
(p#0.05). Fisher’s exact test results were also corrected for multiple
testing using FDR.
In networks, genes or gene products are represented as nodes,
and the biological relationship between two nodes is represented as
an edge (line). All edges are supported by at least one reference
from the literature, from a textbook, or from canonical
information stored in the IPA Knowledge Base. Human, mouse,
Molecular Alterations in NSCLC
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and rat orthologs of a gene are stored as separate objects, but are
represented as a single node in the network. The network
building’s algorithm determines a statistical score for each
network. This is done by comparing the number of focus genes
that contribute to a given network relative to the total number of
occurrences of those genes in all networks or pathways stored in
the IPA Knowledge Base. The intensity of genes (node) colour in
the networks indicates the degree of downregulation (green) or
upregulation (red) of gene expression. Nodes are displayed using
various shapes that represent the functional class of gene products.
Results
AKT activation in NSCLCs
As a read-out of PI3K/AKT signalling in NSCLC we
determined the phosphorylation status of residue S473 of AKT1
(pAKT). pAKT was evaluated on TMAs containing duplicated
core biopsies of 107 NSCLCs. As controls 45 matched normal
samples were used. Patients’ clinico-pathological characteristics
are described in Materials and Methods and summarized in
Tables S1, S2 and S3. The results obtained from pAKT staining in
NSCLC are summarized in Table 1. pAKT staining was barely
detectable in the epithelial cells from normal alveolar epithelium
and from upper airways (39 out of 45 samples) (See Figure S1). In
contrast, AKT activation was observed in 60 out of 97 of NSCLC
analysed (Table 1). Positive pAKT staining was significantly higher
in the carcinoma samples than either normal alveolar or bronchial
epithelium (P,0.001; Chi square test). pAKT staining was
observed in 23/37 SCCs and 30/44 ADCs (Figure 1A and B,
respectively). We found a significant association between pAKT
staining and the grade or the stage of the disease (Table 2): pAKT
staining was significantly more represented in patients with grades
G3–G4 compared with patients with grades G1–G2 (p,0.05) and
in patients with TNM stage III compared with patients with stage
II disease (p,0.05). See Tables S4 and S5 for distribution of
patients into SCCs and ADCs. These results demonstrate that, in
agreement with work in other populations, in Italian NSCLC
patients AKT activation occurs in tumour tissue and correlates
with a more advanced stage of disease [20–22]. See also Table S9
and S10 for a detailed, patient-by-patient, list of pAKT positivity.
Mechanisms of AKT activation in NSCLCs:
immunohistochemistry
To investigate the molecular mechanisms leading to AKT
activation in Italian patients affected by NSCLC we performed a
comprehensive analysis of the expression and/or the genetic status
of AKT1 and AKT2 and their closest regulators (KRAS, PIK3CA
and PTEN). Of the 107 cases present on the TMAs 96 could be
properly analysed for AKT1, 83 for AKT2, 104 for PTEN and 92
for PIK3CA.
See Materials and Methods for the evaluation criteria used for
AKT1. Briefly, samples defined (2) were completely negative for
AKT1; samples defined (+) contained up to 10% of positive cells;
samples defined (++) comprised 11–50% of positive cells; samples
Table 1. AKT activation in NSCLCs.
AKT activation (pS473)a
HISTOLOGYSAMPLE NUMBERLOWHIGH
NORMAL45 396
ADC 441430
SCC37 14 23
ASQ743
LCC633
CAR321
Total9737 60
aAKT 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).
ADC (adenocarcinoma), SCC (squamous cell carcinoma), ASQ (adenosquamous
carcinoma), LCC (large cell carcinoma) CAR (carcinoid tumour).
doi:10.1371/journal.pone.0030427.t001
Figure 1. pS473 AKT immunostaining (IHC) in NSCLCs. 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 106 and 406,
respectively.
doi:10.1371/journal.pone.0030427.g001
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defined (+++) comprised .50% of positive cells, respectively.
Figure S2 shows representative stainings of (+), (++) or (+++)
AKT1 expression in SCCs and ADCs. Tumours were classified
into a low expression group comprising (2) and (+) and a high
expression group that comprises (++) and (+++). Analysis of TMAs
258.1 and 258.2 showed that AKT1 was over-expressed in 17/96
NSCLC cases (,19%) (Figure 2), with SCCs and ADCs showing
similar results: 7/37 AKT1 positive tumors were SCCs (19%) and
7/44 AKT1 positive tumours were ADCs (16%). See Figure 2A
and B, respectively. Nine out of 15 (60%) NSCLCs overexpressing
AKT1 showed AKT activation (Table 3).
We then analysed the expression of AKT2 in NSCLCs. See
Materials and Methods for the evaluation of AKT2 staining.
Samples defined (2) were completely negative for AKT2; samples
defined (+) were with up to 10% of positive cells; samples defined
(++) comprised 11–50% of positive cells; and samples defined
(+++) comprised .50% of positive cells, respectively. Figure S3
shows representative stainings of (+), (++) or (+++) AKT2
expression in SCCs and ADCs. Tumours were classified into a
low expression group comprising (2) and (+) and a high expression
group that comprises (++) and (+++). AKT2 was overexpressed in
18/83 NSCLCs (,22%) (Figure 3). At difference with AKT1,
AKT2 overexpression was observed more frequently in SCCs (10/
31 SCCs, 32%; 4/33 ADCs, 12%). See Figure 3A and B,
respectively. In addition, most AKT2 positive tumours (12/17,
71%) showed AKT activation (Table 3).
Patients accrued for this study had already been characterised
for PTEN expression [38]: complete loss occurred in 41 of 104
(39%) NSCLCs and partial down-regulation was observed in 41
additional cases. PTEN loss was more frequently observed in
SCCs (22/40, 55%) than in ADCs (14/51, 27%) (See Figure S5).
However, when correlated with AKT activation, the loss or the
reduction of the levels of PTEN protein was not associated with
AKT activation (n=95; p=0.832) (Table 3).
Finally, we analysed the expression of the catalytic subunit of
PI3K, p110a. Evaluation criteria are reported in Materials and
Methods. Samples defined (2) were completely negative for
p110a; samples defined (+) contained up to 10% of p110a positive
cells; samples defined (++) comprised 11–50% of p110a positive
cells; and samples defined (+++) comprised .50% of p110a
positive cells. Figure S4 shows representative stainings of (+), (++)
or (+++) AKT1 expression in SCCs and ADCs. Tumours were
classified into a low expression group comprising (2) and (+) and a
high expression group that comprises (++) and (+++). We observed
p110a overexpression in ,29% of NSCLCs (27/92): 12 out of 34
were SCCs (35%) and 12 out of 43 were ADCs (28%) (Figure 4A
and 4B). At difference with other genes within the pathway that
have been analysed, we found that NSCLCs with overexpressed
p110a presented significantly activated AKT (18 out of 26;
p=0.02) (Table 3).
Notably, from the integrated analysis of the TMAs we found
that AKT activation was more frequently observed in tumours
showing aberrant expression of more than a single gene within the
PI3K pathway (PTEN loss, or overexpression of AKT1, AKT2,
p110a respectively). In fact, AKT activation was detected in 15–
64% of tumours showing aberration in a single gene, 44–89% of
tumours with aberrant expression of two genes, 67–100% of
Table 2. Correlation between AKT activation and clinico-
pathological features of NSCLC patients.
Akt activation (pS473)a
Low (n) High (n)P value
Gender
Male27 49
Female99
Grade
G1–G21916 0.0351
G3–G4 1533
TNM stage
Stages I2035 0.049*
Stage II66
Stage III2 12
aAKT 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).
1G1–G2 vs G3–G4.
*Stage II vs Stage III.
doi:10.1371/journal.pone.0030427.t002
Figure 2. IHC and FISH analysis of AKT1 in NSCLCs. 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 106 and 406, 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 1006.
doi:10.1371/journal.pone.0030427.g002
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tumours with aberrant expression of three genes and 100% of
tumours with aberrant expression of all four genes. Conversely,
aberrant expression of the members of PI3K pathway was less
common in tumours showing no activation of AKT signalling (see
Table 4).
Mechanisms of protein overexpression: FISH analysis
FISH analysis in NSCLCs was performed for AKT1, AKT2
and PIK3CA to determine the molecular mechanisms of the
overexpression of the corresponding proteins. See Materials and
Methods for classification of tumours by FISH. We found 20/82
NSCLC (24%) with copy number gain of the AKT1 gene at
chromosome 14, of which 16 were high polysomy (.4 copies) and
4 focal amplification (SCC-11, SCC-12, SCC-14 and SCC-21)
(Figure 2C). Expectedly, several AKT1 FISH-positive NSCLCs
(12 out of 20 cases, 60%) showed moderate or high AKT1
expression. See Tables S9 and S10 for a detailed list of genetic
alterations detected in single SCC and ADC patients. In the case
of AKT2, we observed 24/73 NSCLCs (31%) with copy number
gain of the gene at chromosome 19, of which 23 patients had high
polysomy and 1 patient had focal amplification (SCC-11). See
Figure 3C for a representative example. However, the significance
of AKT2 amplification in lung cancer remains unclear, since 13/
24 (54%) cases of AKT2 FISH-positive tumours did not show
increased expression of the corresponding protein.
FISH analysis with chromosome 3q26.32 probes revealed the
presence of an increase in the PIK3CA gene copy number in 19
cases (,26%), all of which presented high polysomy, with 7 cases
showing also focal amplification (ADC-5, SCC-4, SCC-14, SCC-
16, SCC-19, SCC-30, SCC-34) (Figure 4C). The majority of
NSCLCs with increased copy number of PIK3CA (13 out of 19
cases, 68%) showed moderate or high expression of p110a.
However, not all FISH-positive NSCLCs resulted in the
activation of AKT signalling. As shown in Tables S6, S7 and
S8, 11/18, 10/19 and 14/23 cases that were FISH-positive for
PIK3CA, AKT1 and AKT2 resulted positive for pAKT,
respectively.
Mechanisms of AKT activation: mutation analysis of
PIK3CA and KRAS
Patients accrued for this study had already been analysed for
AKT1 mutations [24]. Patient SCC-29 presented a somatic
mutation in the gene encoding AKT1 resulting in a glutamic acid
to lysine substitution at amino acid 17 (E17K) [24]. The tumour
from this patient showed increased AKT expression and activity.
Similarly, missense mutations in PIK3CA have been rarely
Table 3. Correlation between AKT activation and expression
of the different members of the PI3K pathway in NSCLCs.
pAKT
negativea
pAKT
positivea
Total
numberP value
PIK3CAb
negative12517 0.021
moderate 13 2437
high8 18 26
PTENc
positive9 1221
reduced1622 38
negative1323 36
AKT1d
negative2123 45
moderate827 35
high6915
AKT2e
negative 201939
moderate5 1722
high5 1217
aAKT activation was evaluated with as pS473 positivity and scored as negative
(,10% of positive tumour cells) and high (.10% of positive tumour cells).
bPIK3CA 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).
cPTEN expression was classified as (+) when staining was detected in .50% of
the cells, (+/2) when staining was detected in 25–50% of cells and (2) when
staining was detected in 0–25% of cells. For statistical analysis PTEN expression
was considered lost when samples were classified as (2).
dAKT1 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).
eAKT2 was graded as positive (.25% of positive tumour cells) as moderate
(.10% of positive tumour cells) or negative (0–10% of positive tumour cells).
1Statistically significant.
doi:10.1371/journal.pone.0030427.t003
Figure 3. IHC and FISH analysis of AKT2 in NSCLCs. 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 106 and 406, 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 1006.
doi:10.1371/journal.pone.0030427.g003
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reported [42–45]. We found a GAG1633RAAG substitution that
leads to the amino acid change E545K in one SCC (SCC-6)
(Figure 5A and B). Conversely, 7 NSCLCs showed mutations in
KRAS: G12A (GGTRGCT) (n=1); G12C (GGTRTGT) (n=4);
G12V(GGTRGTT)(n=1);
(Figure 5C). KRAS mutations were detected mainly in ADCs (6
of 32; 19%) as described [46,47]. Five out of 7 of cases with
mutationsin KRASshowed
(Figure 5D).
G13C(GGCRTGC)(n=1)
significantAKT activation
Activated PI3K contributes to cell proliferation and
tumourigenicity of NSCLC cells
Given the importance of PI3K signalling in NSCLCs, we
investigated the role of constitutive PIK3CA activation on the
tumourigenic potential of human lung epithelial cells. To this aim,
we made use of a mutant cell line (NCI-H460) that harbours a
heterozygous activating mutation (E545K) in PIK3CA. Cells were
transduced with a lentivirus expressing shRNA for PIK3CA
(Figure 6A). Silencing of p110a expression was assessed by
immunoblot (Figure 6B). Here we show that suppression of p110a
expression in NCI-H460 cells markedly reduced in vitro anchorage-
dependent and in vivo tumour growth of cells subcutaneously
injected into immunodeficient mice (n=6/group) (Figure 6C and
D, respectively), indicating that PI3K activation plays a significant
role in the malignant behaviour of NSCLC cells.
Molecular profiling of PI3K activation in lung epithelial
cells
To further characterize the role played by PIK3CA in
development of NSCLC, we performed RNA profiling analysis
of human lung epithelial cells expressing an active PI3KCA
mutant (E545K) to identify cellular targets of constitutive PI3K
signalling. Expression of exogenous PI3KCA allele was deter-
mined by immunoblot (Fig. 7A). Expression values obtained were
filtered for fold change greater than 1.5 and subjected to t-test (p-
value cut-off of 0.01) with Benjamini-Hochberg (B–H) FDR
correction [41], obtaining a total of 2126 differentially expressed
probe sets, of which 1005 were down-regulated and 1121 were up-
regulated. The complete microarray data for all probe sets with
the respective normalised values will be available at ArrayExpress
and are provided in additional files (Table S11).
We used Ingenuity Pathway Analysis (IngenuityHSystems,
http://www.ingenuity.com, IPA) to investigate the biological
relevance of the PI3K-dependent expression changes by catego-
rizing our dataset into biological pathways and/or functions and
diseases (Figure 7B, 7C, 7D, 7E; Figure S6; Table S11). The
function ‘‘Cancer’’ was most frequent and associated with 466
genes, followed by ‘‘Cell Death’’ (392 genes), ‘‘Cellular Growth
and Proliferation’’ (357 genes), ‘‘Cellular Movement’’ (196 genes),
‘‘Cell Cycle’’ (161 genes), ‘‘Cell-to-cell Signalling and Interaction’’
(112 genes), and ‘‘Cellular Morphology’’ (97 genes), respectively.
We found that active PI3K regulates expression of most cell cycle
molecules such as CCND1, CCND2, Cdk6 and Cdk inhibitors as
well as of several apoptosis-related genes such as BAG3, IGFBP7,
IGFBP3, TRADD and TRIB1. As to ‘‘Cell movement’’ function,
Figure 4. IHC and FISH analysis of PI3KCA in NSCLCs. 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 106 and 406, respec-
tively. 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 1006.
doi:10.1371/journal.pone.0030427.g004
Table 4. Alteration in the expression of PTEN, PI3K, AKT1 and
AKT2 in pAKT positive NSCLCs.
AlterationpAKT positivepAKT negative
AKT1b
9/59 (15%)6/35 (17%)
AKT2c
12/48 (25%) 5/30 (17%)
PI3Kd
18/47 (38%)8/33 (24%)
PTENe
23/36 (64%) 9/38 (24%)
AKT1, PTEN 4/7 (57%)3/7 (43%)
AKT2, PTEN 8/9 (89%)1/9 (11%)
PI3K, PTEN4/9 (44%)5/9 (56%)
AKT1, AKT2 3/4 (75%) 1/4 (25%)
PI3K, AKT13/6 (50%) 3/6 (50%)
PI3K, AKT25/8 (62%)3/8 (37%)
AKT1, AKT2, PTEN 3/3 (100%)0
AKT1, PI3K, PTEN2/3 (67%) 1/3 (33%)
AKT2, PI3K, PTEN 3/3 (100%)0
AKT1, AKT2, PI3K2/3 (67%) 1/3 (33%)
AKT1, AKT2, PI3K, PTEN 2/2 (100%)0
bModerate and high AKT1 expression as defined in Table 3.
cModerate and high AKT2 expression as defined in Table 3.
dModerate and high PIK3CA expression as defined in Table 3.
ePTEN loss as defined in Table 3.
doi:10.1371/journal.pone.0030427.t004
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Figure 5. Mutation analysis of PIK3CA and KRAS genes in NSCLCs. A. Mutation detection in the exons 9 and 20 of PIK3CA from NSCLC. The
negative derivative of the fluorescence (2dF/dT) versus temperature graph shows peaks with different Tm. The wild type sample showed a single Tm
at 66uC. The heterozygous mutant sample showed an additional peak at 57uC. B. Point mutation in the PI3KCA gene involving a GAGRAAG
transition in codon 545 of exon 9 inducing the substitution of a glutammic acid with a lysine (E545K). C. Point mutations in the KRAS gene involving a
GGTRGCT, GGTRTGT, GGTRGTT; GGCRTGC 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). D. pAKT staining of sample
ADC-30.
doi:10.1371/journal.pone.0030427.g005
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IPA analysis of PI3K-regulated Functions identified growth factors
(TGFA), cytokines (IL1A, IL1B, IL6 and IL8) and chemokines
(CXCL2) that are involved in stromal-to-epithelial signalling,
invasion, angiogenesis and metastasis. It is of note that IPA
analysis of DEGs in PIK3CA-transduced BEAS-2B cells retrieved
several functions linked to ADCs and SCCs (Figure 7D, 7E),
indicating that the adoptive expression of active PIK3CA elicit a
transcriptional response that is associated to lung cancer.
Activated PI3KCA regulates the expression of the
oncogenic transcription factors HMGA1, FOS and MYC
IPA analysis of DEGs demonstrated that PI3K activation
induced the up-regulation of several oncogenic transcription
factors (i.e. MYC, JUN, JUN-B, FOS, HMGA1, HES1), with
each transcription factor being the node of networks involving 30–
40 down-regulation or up-regulation events (Figures S7A, S7B,
S7C, S7D, S7E, S7F, S7G, S7H).
Figure 6. Interference with PIK3CA decreases growth and tumorigenesis of human NSCLC cells carrying activated p110a. A.
Immunoblot analysis of phosphorylated S473 (pAKT) and total AKT in NSCLC cells. B. Immunoblot analysis of PI3KCA expression in parental (NCI-
H460), scrambled-transduced (SCR) or PI3KCA-specific shRNA-transduced lentiviruses (shPIK3CA). C. NCI-H460 cells transduced with the control
lentivirus (SCR) or with lentivirus carrying shRNA to PIK3CA (shPIK3CA) were seeded in flat-bottom 96-well plates and the relative number of viable
cells was measured by MTT assay. Absorbance was read at 570 nm and the data are mean of triplicates. D. SCR- and shPIK3CA transduced NCI-H460
cells were subcutaneously injected into the flank of athymic nude mice and the growth of xenotransplated tumour was measured as described in
Material and Methods.
doi:10.1371/journal.pone.0030427.g006
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We confirmed the results obtained from the array analysis by
quantitative RT-PCR on a selected panel of genes (HMGA1,
FOS, MYC) (Figure 8A). Subsequently, we performed Q-RT-
PCR analysis of the mRNA expression of HMGA1, FOS and
MYC genes in ADC- or SCC-derived cell lines (n=8) and
correlated them with the AKT activation (S473 phosphorylation)
status as a read-out of PI3K activity. The cell lines used were as
follows: ADC-derived cell lines, A549, NCI-H522, NCI-H23,
NCI-H460, NCI-H596; SCC-derived cell lines, NCI-H226,
CALU1,BEN1.Q-RT-PCR
NSCLC-derived cell lines with activated AKT (A549, NCI-
H460, NCI-H596, NCI-H226, CALU1) presented, on average,
increased expression of HMGA1, FOS and MYC compared with
cells with low AKT activation (NCI-H522, NCI-H23, BEN1). For
HMGA1 the average values were: 0.6 in the case of AKT negative
cells and 2 in the case of AKT positive cells; for FOS the average
values were: 0.9 in the case of AKT negative cells and 18 in the
case of AKT positive cells; for MYC the average values were: 2.5
in the case of AKT negative cells and 3.9 in the case of AKT
positive cells (Figure 8B).
Subsequently, we extended the analysis performed in cancer cell
lines to primary NSCLC. To this aim, expression of HMGA1,
FOS and MYC was determined in a representative panel of
NSCLC (n=14; 4 ADC, 8 SCC and 2 ADS) and correlated with
the status of AKT activation (Figure 9). Primary NSCLC with
activated AKT presented (n=10), on average, increased expres-
sion of HMGA1, FOS and MYC compared with tumors that
showed low AKT activation (n=4). For HMGA1 the average
values were: 5.4 in the case of AKT negative cells and 13.5 in the
case of AKT positive cells; for FOS the average values were: 14.1
in the case of AKT negative cells and 30.5 in the case of AKT
positive cells; for MYC the average values were: 3.5 in the case of
AKT negative cells and 6.5 in the case of AKT positive cells
(Figure 8B). However, it is to be noted that in both cell lines and
tumours, data showed a trend that was not statistically significant
given the low number of samples analysed and the huge
heterogeneity of expression shown by HMGA1, FOS and MYC
in tumors.
analysis demonstratedthat
Discussion
We report a detailed analysis of the contribution of the different
members of PI3K/AKT pathway to AKT deregulation in lung
cancer. The most interesting findings of this study were that in
Italian NSCLC patients activation of AKT was associated with
advanced stage and higher grade and that, in these tumours, the
major determinant of AKT activation was the over-expression of
the catalytic subunit of phosphatidylinositol 3-kinase, p110a.
Experimental evidence obtained by manipulation of PI3K
signalling in NSCLC cells also indicated that p110a is required
for in vitro and in vivo growth and disclosed a network of PI3K-
regulated transcription factors that may be responsible for the
oncogenic effects exerted by aberrant PI3K signalling in cancer
[48].
To our knowledge this is the first comprehensive analysis aimed
at determining the role of AKT signalling performed on a cohort
of Italian NSCLC patients. So far, little information concerning
AKT activation in Italian NSCLC patients was available. In the
cohort of NSCLC patients studied here, AKT pathway is activated
in 62% of cases, with significant S473 phosphorylation detected
more frequently in patients with advanced disease (TNM stage III
vs. stage II; n=26; p,0.05) and higher grade (G3–G4 compared
with G1–G2; n=83; p,0.05). Several NSCLCs analysed in this
study over-expressed PIK3CA, implying that the deregulated
expression of wild type p110a might represent an oncogenic event
during cancer development in the lung. Conversely, we found
PIK3CA mutation in only one SCC patient, confirming that,
although frequent in breast, gastric and hepatocellular cancers,
PIK3CA mutations are rare in NSCLCs [49]. Other molecular
lesions detected in NSCLC patients comprise PTEN loss (39%)
and AKT1 or AKT2 over-expression (18% and 22%, respective-
ly). It is of note that although PTEN loss in NSCLCs is more
common than overexpression of p110a, our results indicate that
the latter is the unique alteration that is significantly associated to
AKT activation (p=0.02).
Interestingly, simultaneous aberrant expression of two or more
members within the PI3K pathway was relatively infrequent in
unselected NSCLCs but was significantly more frequent in
NSCLCs with activated AKT (see Table 4 for details). This
observation suggests that p110a over-expression alone is not
sufficient to activate AKT signalling and hence requires other
alterations to be fully oncogenic in NSCLCs. Moreover, at
difference with the significant AKT activation shown by NSCLCs
with mutant KRAS or AKT1, the tumour that harboured mutant
PIK3CA was negative for pAKT, suggesting that the type or the
position of the alteration within the pathway may influence
mechanisms and effects of pathway deregulation [45,49–51].
Accordingly, KRAS mutations were mutually exclusive with other
genetic alterations (except for ADC-23 who presented simulta-
neous presence of KRAS mutation and polysomy of AKT1 and
AKT2) whereas copy number variations of PIK3CA, AKT1 and
AKT2 were not [52]. These findings are reminiscent of breast or
endometrial cancer, in which PIK3CA mutations are frequently
detected in settings of low PTEN expression or mutations [53,54],
and suggest that genetic alterations of the PI3K/AKT pathway in
NSCLCs are not functionally redundant.
In addition, this manuscript provides novel experimental
evidence to the observation that SCCs and ADCs develop by
different genetic alterations: i) mutations in PIK3CA and AKT1
(3% altogether) were detected only in SCCs [this manuscript; 24]
whereas KRAS mutations were observed in ADCs (19%); ii) SCC
patients (85%) presented at least one genetic alteration in
PI3KCA, AKT1, AKT2 or PTEN more frequently than ADC
Figure 7. The top 11 canonical signalling pathways influenced by constitutive PI3K signaling. 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 Materials and methods. A. Immunoblot for PIK3CA expression in
transfected BEAS-2B cells. 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). C. 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 2log (p-value). D. Bio-functions identified by IPA in the 2126 DEGs from BEAS-2B-PI3K-E545K
compared with BEAS-2B. E. 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.
doi:10.1371/journal.pone.0030427.g007
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patients (50%); iii) PIK3CA copy gains occurred more frequently
in SCCs (25%) than in ADCs (9%) as described previously [15,49];
iv) coexistence of at least two alterations in the members of the
PI3K pathway occurred more frequently in SCC patients (45%)
than in ADC patients (8%).
FISH results indicated that gene amplification of the PIK3CA
gene at 3p21 is responsible for ,20% of cases with enhanced
p110a expression, in agreement with previous reports indicating
that gains of part or of the entire long arm of chromosome 3,
where the PIK3CA gene maps, are recurrent in NSCLCs
[15,55,56]. Yet, since several NSCLCs overexpress p110a in the
absence of gene amplification other mechanisms must be involved
in the dysregulation of PIK3CA expression in NSCLCs.
The functional effects of mutant or amplified PIK3CA in lung
cancer are unclear [49]. Our data indicated that in NSCLC cells,
PIK3 signalling is required in vitro and in vivo since suppression of
p110a expression inhibits the growth of xenografted cells carrying
an activated PIK3CA allele. However, it is likely that PI3K might
act in concert with other oncogenic hits to promote malignant
transformation of lung epithelial cells since several NSCLCs
present aberrant expression of AKT1, AKT2 or loss of PTEN in
addition to PIK3CA overexpression (7%, 10% and 21%,
respectively) and PIK3CA mutations are not mutually exclusive
with EGFR and KRAS mutations in lung cancer [49–51,54].
Finally, RNA profiling experiments led to the identification of
.2000 differentially regulated transcripts that likely contributes to
the oncogenic effects of aberrant PI3K signalling in lung epithelial
cells. Categorization of differentially expressed genes into
biological pathways and/or functions identified gene expression
changes induced by the constitutive activation of PI3K-dependent
Figure 8. Activation of PI3KCA regulates expression of HMGA1, c-Fos, c-MYC in NSCLC cell lines. A. 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. Quantitative real-
time RT-PCR analysis of c-Fos, HMGA1, c-Myc gene expression in different NSCLC cell lines.
doi:10.1371/journal.pone.0030427.g008
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signalling in lung epithelial cells. Interestingly, analysis of DEGs
retrieved several functions linked to lung cancer of both ADC and
SCC histotypes, suggesting that the activation of PI3K signalling
induces a transcriptional programme that is characteristic of lung
cancer cells. In this sense, it is worth noting that IPA analysis
identified a network of transcription factors that are linked to PI3K
activation - such as MYC, JUN, JUN-B, FOS, HMGA1 and
HES1- that are the central nodes of multiple molecular networks
up-regulated by constitutive PI3K signalling. These findings
suggest that part of the oncogenic activity exerted by PI3K in
lung epithelial cells is dependent on the ability of PI3K to
reprogram transcription. The existence of a correlation between
PI3K signalling and the expression of oncogenic transcription
factors is confirmed by the finding that cell lines and primary
tumours with high AKT activation present, on average,
consistently higher expression of MYC, FOS and HMGA1 than
cell lines and tumours with low AKT activation.
It is of note that in agreement with its role in promoting cells
cycle progression, active PI3K up-regulates the expression of
several cell cycle promoting molecules – CCND1, CCND2, Cdk6
– as well as down-regulates Cdk inhibitors. To this regard, it is
worth noting that PI3K-dependent regulation of CCND2
expression may occur indirectly through MYC [57].
In conclusion, the results reported in this manuscript indicate
that PI3KCA over-expression occur at a much higher frequency in
lung cancers than do activating mutations, apparently representing
the major determinant of AKT activation in NSCLC. PI3KCA
overexpression in NSCLCs occurs, at least in part, through gene
copy gains, which occur more often in SCCs than in ADCs.
Finally, it is of particular interest the identification of a network of
transcription factors that are upregulated by constitutive PI3K
signalling and may represent critical mediators of the oncogenic
effects exerted by aberrant PI3K.
Supporting Information
Figure S1
normal lung. A. Immunoblot for anti-AKT1 and anti-AKT2
antibodies in NCI-H460 cells interfered for AKT1 and AKT2,
respectively. B. Top left: normal lung negative for pAKT pS473
phosphorylation; top right: normal lung negative for AKT1.
Bottom left: normal lung negative for AKT2; bottom right: normal
lung negative for PIK3CA. Magnification 106.
(TIF)
IHC for pAKT, AKT1, AKT2 and PI3KCA in
Figure
NSCLCs. A. AKT1 expression in SCCs: from left to right,
negative, (+), (++), (+++). SCC positive for AKT1 expression. B.
AKT1 expression in ADCs: from left to right, negative, (+), (++),
(+++). Magnification 106and 406, respectively.
(TIFF)
S2
Immunostaining analysis ofAKT1 in
Figure
NSCLCs. A. AKT2 expression in SCCs: from left to right,
negative, (+), (++), (+++). SCC positive for AKT1 expression. B.
AKT2 expression in ADCs: from left to right, negative, (+), (++),
(+++). Magnification 106and 406, respectively.
(TIFF)
S3
Immunostaininganalysis ofAKT2 in
Figure
NSCLCs. A. PIK3CA expression in SCCs: from left to right,
negative, (+), (++), (+++). SCC positive for AKT1 expression. B.
PIK3CA expression in ADCs: from left to right, negative, (+), (++),
(+++). Magnification 106and 406, respectively.
(TIFF)
S4
Immunostaining analysis of PIK3CA in
Figure S5
expression in normal lung tissue. B. Left, SCC negative for
PTEN expression; right: ADC positive for PTEN expression.
Magnification 106and 406, respectively. C. Q-reverse transcrip-
tase PCR analysis of PTEN mRNA expression in normal lung
tissues and NSCLC. D. Q-PCR analysis of PTEN gene number in
normal lung tissues and NSCLC. DNA from peripheral blood
leukocytes (PBL) was used as control. PTEN copy number in PBL
was set arbitrarily as 2. The average value of the PTEN gene in
normal tissues was similar to the PBL value (2).
(TIFF)
PTEN expression in NSCLCs. A. PTEN
Figure 9. Activation of PI3KCA regulates expression of HMGA1,
c-Fos, c-MYC in primary NSCLC. Quantitative real-time RT-PCR
analysis of c-Fos, HMGA1, c-Myc gene expression in primary NSCLCs.
doi:10.1371/journal.pone.0030427.g009
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Figure S6
DEGs from BEAS-2B-PI3K-E545K compared with BEAS-2B.
(TIFF)
Top Bio-functions identified by IPA in the 2122
Figure S7
graphical representation of genes having known biolog-
ical relationships. Green icons indicate down-regulated genes
and red icons indicates up-regulated genes.
(PDF)
Network analysis was performed to provide a
Table S1
tients.
(DOCX)
Clinico-pathological features of NSCLC pa-
Table S2
(DOCX)
Clinico-pathological features of SCC patients.
Table S3
(DOCX)
Clinico-pathological features of ADC patients.
Table S4
AKT positivity) and clinico-pathological features of SCC
patients.
(DOCX)
Correlation between AKT activation (pS473
Table S5
AKT positivity) and clinico-pathological features of ADC
patients.
(DOCX)
Correlation between AKT activation (pS473
Table S6
presence of genetic alterations in PI3K, AKT1 and AKT2
in NSCLCs.
(DOCX)
Correlation between AKT activation and the
Table S7
presence of genetic alterations of PI3K, AKT1 and AKT2
in SCCs.
(DOCX)
Correlation between AKT activation and the
Table S8
presence of genetic alterations in PI3K, AKT1 and AKT2
in ADCs.
(DOCX)
Correlation between AKT activation and the
Table S9
PI3K/AKT pathway in SCC patients. Copy number gains in
AKT1, AKT2, PI3KCa genes were determined by FISH: high
polysomy (HP) and gene amplification (A). Mutation analysis
Summary of the genetic alterations in the
identified activating mutations of PI3KCA (E545K), KRAS
(G12C, G12V, G12A, G13C) and AKT1(E17K). PTEN expres-
sion was classified as (+) when staining was detected in .50% of
the cells, (+/2) when staining was detected in 25–50% of cells and
(2) when staining was detected in 0–25% of cells. AKT activation
was evaluated with phospho-specific antibodies (pS473), scored as
negative (,10% of the tumour cells with weak, focal immunopo-
sitivity or absence of staining) and high (.10% of tumour cells
with strong or diffuse immunopositivity).
(DOCX)
Table S10
PI3K/AKT pathway in ADC patients. Copy number gains in
AKT1, AKT2, PI3KCa genes were determined by FISH: high
polysomy (HP) and gene amplification (A). Mutation analysis
identified activating mutation of PI3KCA (E545K), KRAS (G12C,
G12V, G12A, G13C) and AKT1(E17K). PTEN expression was
classified as (+) when staining was detected in .50% of the cells,
(+/2) when staining was detected in 25–50% of cells and (2)
when staining was detected in 0–25% of cells. AKT activation was
evaluated with phospho-specific antibodies (pS473), scored as
negative (,10% of the tumour cells with weak, focal immunopo-
sitivity or absence of staining) and high (.10% of tumour cells
with strong or diffuse immunopositivity.
(DOCX)
Summary of the genetic alterations in the
Table S11
BEAS-2B vs BEAS-PI3KCA-E545K. Expression microarray
(HT-12_V3_0_R1) data were prefiltered to remove genes
changing less than 1.5 fold, and a t-test was run to determine
significant (p,0.01) changers. A multiple testing correction using
the algorithm of Benjamini and Hochberg was used to reduce the
false discovery rate. See file attached.
(XLS)
Genes significantly increased or decreased in
Appendix S1
PCR (PTEN, c-Fos, HMGA-1, c-Myc, Jun-B) and sequencing
KRAS (exons 2 and 3) and PIK3CA (exons 9 and 20).
(DOC)
Protocols and primers for Q-PCR (PTEN), Q-RT-
Author Contributions
Conceived and designed the experiments: GV MS. Performed the
experiments: MS FF CDM MR DM. Analyzed the data: MS RF GV
MR AW PZ MC. Contributed reagents/materials/analysis tools: GP GR
RF GB. Wrote the paper: GV MS AW.
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