Molecular Analysis of a Multistep Lung Cancer Model Induced by
Chronic Inflammation Reveals Epigenetic Regulation of p16 and
Activation of the DNA Damage Response Pathway1,2
David Blanco*,y, Silvestre Vicent*,y, Mario F. Fraga z, Ignacio Fernandez-Garcia*,y, Javier Freire*,y, Amaia Lujambio z,
Manel Esteller z, Carlos Ortiz-de-Solorzano*, Ruben Pio*,§, Fernando Lecanda*,y and Luis M. Montuenga*,y
*Division of Oncology, Center for Applied Medical Research (CIMA), Pamplona, Spain; yDepartment of Histology
and Pathology, School of Medicine, University of Navarra, Pamplona, Spain; zCancer Epigenetics Laboratory,
Molecular Pathology Program, Spanish National Cancer Center (CNIO), Madrid, Spain; §Department of
Biochemistry, School of Medicine, University of Navarra, Pamplona, Spain
Abstract
The molecular hallmarks of inflammation-mediated
lung carcinogenesis have not been fully clarified, mainly
due to the scarcity of appropriate animal models. We
have used a silica-induced multistep lung carcinogen-
esis model driven by chronic inflammation to study the
evolution of molecular markers and genetic alterations.
We analyzed markers of DNA damage response (DDR),
proliferative stress, and telomeric stress: ;-H2AX, p16,
p53, and TERT. Lung cancer–related epigenetic and
genetic alterations, including promoter hypermethyla-
tion status of p16(CDKN2A), APC, CDH13, Rassf1, and
Nore1A, as well as mutations of Tp53, epidermal growth
factor receptor, K-ras, N-ras, and c-H-ras, have been
also studied. Our results showed DDR pathway activa-
tion in preneoplastic lesions, in association with induc-
ible nitric oxide synthase and p53 induction. p16 was
also induced in early tumorigenic progression and was
inactivated in bronchiolar dysplasias and tumors. Re-
markably, lack of mutations of Ras and epidermal
growth factor receptor, and a very low frequency of
Tp53 mutations suggest that they are not required for
tumorigenesis in this model. In contrast, epigenetic
alterations in p16(CDKN2A), CDH13, and APC, but not in
Rassf1 and Nore1A, were clearly observed. These data
suggest the existence of a specific molecular signature
of inflammation-driven lung carcinogenesis that shares
some, but not all, of the molecular landmarks of chem-
ically induced lung cancer.
Neoplasia (2007) 9, 840–852
Keywords: lung cancer, inflammation, animal model, preneoplastic lesions,
DNA damage response.
Introduction
Lung cancer is a complex disease that develops through the
progressive accumulation of both genetic and epigenetic
alterations. A number of molecular changes potentially
leading to lung cancer have already been described [1].
These changes may be induced by several genotoxic carcino-
gens [2] to which a smoker’s airways are continuously exposed.
Aside from direct carcinogens, inhaled smoke contains other
constituents that can stimulate a chronic inflammatory re-
sponse in the lung [3]. Strong evidence supports that chronic
inflammation promotes tumorigenesis [4]. Inflammatory cellular
response can induce cell proliferation and tissue repair, cre-
ating a fertile ‘‘soil’’ enriched with cytokines and growth or
angiogenic factors. Inflammatory foci are also a continuous
source for reactive oxygen species (ROS) and reactive ni-
trogen species, which may induce DNA damage, including
DNA strand breaks and adducts, mismatches, and mutations
[5,6]. These events may eventually lead to transformation and
tumorigenesis [4].
There are strong epidemiological evidences linking pulmo-
nary chronic inflammation to a higher risk of lung cancer [7]:
Chronic obstructive airway disease has been shown in many
series of patients to be an independent predictor of lung cancer
risk, and various studies have reported increased cancer risk
among adults with asthma, tuberculosis, or postinflammatory
pulmonary interstitial fibrosis, such as in patients with silicosis
and asbestosis [7]. Chronic inflammation may promote some
of the molecular changes observed in airway epithelial cells
that lead to cancer. Molecules involved in both inflammation
and tumorigenesis include proteins of the COX-2 and NF-nB
Abbreviations: AC, adenocarcinoma; DDR, DNA damage response; EGFR, epidermal growth
factor receptor; g-H2AX, Ser139-phosphorylated histone 2AX; iNOS, inducible nitric oxide synthase;
mC, 5-methylcytosine; NSCLC, non–small cell lung cancer; ROS, reactive oxygen species; SCC,
squamous cell carcinoma; TERT, catalytic subunit of telomerase reverse transcriptase
Address all correspondence to: Luis M. Montuenga, PhD, Division of Oncology, Department of
Histology and Pathology, Center of Applied Medical Research, University of Navarra, CIMA Building,
Pio XII, 55, Pamplona 31008, Spain. E-mail: lmontuenga@unav.es
1This article refers to supplementary material, which is designated by ‘‘W’’ (i.e., Table W1,
Figure W1) and is available online at www.bcdecker.com.
2This work was funded through the ‘‘UTE project CIMA’’ and the Spanish Ministry of Health
(ISCIII: RTICC, FIS-PI 04/2128). S.V. is a postdoctoral fellow of the ‘‘Jose y Ana Royo’’
Foundation. C.O. was supported by the Spanish Ministry of Science and Education (MCYT
TEC2005-04732), the EU Marie Curie Program (MIRG-CT-2005-028342), and a Ramo´n y
Cajal Fellowship. F.L. was supported by ‘‘Fundacio´n Mutua Madrilen˜a,’’ ISCIII-RTICC RD06/
0020, PI042282, and the Spanish Ministry of Industry, Tourism, and Commerce (FIT-090100-
2005-46).
Received 27 June 2007; Revised 10 August 2007; Accepted 14 August 2007.
Copyright D 2007 Neoplasia Press, Inc. All rights reserved 1522-8002/07/$25.00
DOI 10.1593/neo.07517
Neoplasia . Vol. 9, No. 10, October 2007, pp. 840–852 840
www.neoplasia.com
RESEARCH ARTICLE
signaling pathways, chemokines and cytokines (TNF-a, IL-1,
IL-6, and IL-8), growth and angiogenic factors (PDGF, TGF-b,
and EGF), proteolytic enzymes, and cytotoxic agents such
as ROS and nitric oxide (NO) [4,8,9]. The role of inflamma-
tion in lung carcinogenesis has not yet been clarified mainly
due to the scarcity of appropriate chronic inflammation
models of lung cancer. Several animal models have been
developed in the last decades to better understand lung car-
cinogenesis, but most of these do not provide unambiguous
information on the specific role of inflammation. Malkinson
[10] has studied a mouse model in which chronic inflamma-
tion, induced by butylated hydroxytoluene, promotes lung
carcinogenesis previously initiated by a chemical carcinogen
(methylcholanthrene). Very few animal models of cancer in
which chronic inflammation drives carcinogenesis with no
other carcinogenic insult have been molecularly character-
ized [11–13]. In the silica-induced rat lung carcinogenesis
model, a single intratracheal instillation of crystalline silica
dust suspended in saline leads to silicotic chronic inflamma-
tion and progressive proliferative epithelial lesions. Silica
leads to the formation of silicotic granulomas, which consist
of aggregates of activated macrophages that phagocytose
dust mineral, induce collagen deposition, and recruit lym-
phoid cells. The rat silicotic granulomas remain stable or
increase in size over time and, thus, are essentially irrevers-
ible. They produce several molecular mediators (interleu-
kins, TNF-a, TGF-b, NO, and so on), which promote further
inflammatory stress and fibrogenic responses within the lung
and induce progressive proliferative epithelial reactivity in
neighboring areas [14]. Therefore, this is a very useful model
for studying this type of pure chronic inflammation–driven
carcinogenesis. The severity of epithelial lesions grows over
time from alveolar and bronchiolar epithelial hyperplasias to
advanced preneoplastic lesions, and finally to adenocarci-
nomas (AC) and squamous cell carcinomas (SCC) [14,15].
According to the literature and our own experiments, lung
tumors are already induced by the 11th month in around 40%
of the rats. In later stages, after month 17, an increase in
incidence (90%) is clearly observed [14]. Most of the tumors
found in this model are AC (84%), compared to mixed car-
cinomas (8%) or SCC (8%). At the histopathological level,
this model recapitulates a number of features of the multistep
carcinogenic process observed in human peripheral lung
cancer and provides access to a wealth of preneoplastic
lesions amenable for molecular analysis. In the last decade,
several groups have studied the silicotic rat model, focusing
mainly on the short-term activation of classic inflammation-
related molecular hallmarks, including the activation of the
NF-nB and PKC signaling pathways, as well as the produc-
tion of growth factors IL-1, IL-6, TNF-a, ROS, NO, and so on
[16–19]. However, a detailed analysis of molecular altera-
tions in the progression of the preneoplastic and neoplastic
epithelial lesions in this model is still lacking.
The aim of our study was to improve our understanding of
the molecular pathways involved in inflammation-mediated
carcinogenesis and to follow molecular changes along the
process from normal epithelial cells to cancer in the silica-
induced lung cancer model. We focused our study on the
evolution of a variety of molecular markers that are already
associated with human lung cancer. Recently, several
reports have emphasized the existence of tumorigenesis
barriers that slow or inhibit the progression of preneoplastic
lesions to neoplasia [20,21]. One such barrier involves DNA
replication stress, which leads to activation of the DNA
damage checkpoint and, thereby, to apoptosis or cell cycle
arrest mediated by either p53 or p16 [22,23]. We thus have
analyzed preneoplastic and neoplastic lung lesions for a
number of well-characterized markers of DNA damage re-
sponse (DDR), proliferative stress, and telomeric stress such
as Ser139-phosphorylated histone 2AX (g-H2AX), p53, p16,
and the catalytic subunit of telomerase reverse transcriptase
(TERT). In order to dissect the molecular profile, we also
studied lung cancer–related epigenetic and genetic alter-
ations, including promoter hypermethylation status of p16
(CDKN2A), APC, CDH13, Rassf1, and Nore1A, and muta-
tions of Tp53, epidermal growth factor receptor (EGFR),
K-ras, N-ras, and c-H-ras. In brief, our study shows the rel-
evance of a set of molecular and epigenetic alterations, rather
than of ‘‘classic’’ mutations, in the multistep progression of
an inflammation-mediated lung cancer model.
Materials and Methods
Carcinogenesis Protocol
Fisher F344/NCr female rats fromHarlanUKLimited (Oxon,
UK) were used. Rats were housed in specific pathogen-free
conditions with access to food and water ad libitum. Proce-
dures were carried out in strict compliance European Union
and University of Navarra (Institutional Animal Care and
Use Committee) relevant guidelines for the use of laboratory
animals. The crystalline silica sample was 99% pure a-quartz
(Min-U-Sil 5; US Silica Co., Berkeley Springs, WV), with a
particle size of < 5 mm [14]. The silica sample was suspended
in sterile neutral-buffered saline and briefly sonicated to
provide full dispersion. Eight-week-old rats were anesthe-
tized with a mixture of oxygen and isoflurane, and placed on
their backs on a metal board slanted at a 60j angle, with the
mouth kept open. In this position, at the end of an expiration,
rats received a single intratracheal instillation of 16 mg of
quartz in 0.3 ml of saline through a cannula connected to a
syringe [14]. Epithelial reactions to silica started with foci of
hyperplastic epithelial cells adjacent to silicotic granulomas,
including hyperplasias of type II pneumocytes from month 1
and hyperplasias of bronchiolar epithelial cells from month 4.
Preneoplastic adenomatoid lesions (AL) and dysplastic
bronchiolar lesions were observed from month 8. AL in large
clusters of completely lined alveolar spaces that had pro-
gressed to become more conspicuous and include areas
of adenomatoid proliferation separated by thick fibrous
septa were formed by alveolar epithelial hyperplastic reac-
tion. The shape of hyperplastic adenomatoid cells was usu-
ally less cuboidal and more flattened. AC and SCC were
observed from month 11. AC were fibrotic or nonfibrotic, with
an acinar, papillary, or alveolar pattern showing varying de-
grees of differentiation.
Molecular Analysis of Inflammation-Driven Lung Cancer Blanco et al. 841
Neoplasia . Vol. 9, No. 10, 2007
Samples
Seventy rats were instilled with 16 mg of quartz, and the
lungs were obtained on months 3 to 4 (n = 10), month 6 (n =
5), month 8 (n = 5), month 10 (n = 5), month 12 (n = 5),
months 16 to 17 (n = 20), and month 21 (n = 20) after
instillation. Several lesions were studied throughout these
time periods: hyperplasias of type II pneumocytes, hyper-
plasias of bronchiolar epithelial cells, preneoplastic AL and
dysplastic bronchiolar epithelium, and AC (fibrotic and non-
fibrotic) and SCC. Hyperplastic and advanced preneoplastic
lesions were also observed in late stages. Tissues from
control rats (n = 8), instilled with 0.3 ml of saline without
quartz, were collected on month 6 (n = 4) and month 12 (n =
4). Rats were killed by exsanguination under anesthesia with
Ketolar (Parke Davis, Madrid, Spain) and Rompun (Bayer
AG, Leverkusen, Germany). The trachea was exposed by
dissection and ligated during maximal inspiration. The larynx,
trachea, bronchi, lungs, lymph nodes, and heart were re-
moved en bloc and fixed by immersion in 4% formaldehyde
in phosphate buffer for 24 hours. Lung lobes were sectioned
along their main bronchial axis, embedded in paraffin, and
sectioned at 4 mm thickness. Paraffin-embedded lungs from
treated and control rats were used.
A subset of the tumors was isolated from the fresh unfixed
lungs and split into two halves. One was rapidly snap frozen
in liquid nitrogen and kept at 
80jC for DNA extraction. The
other was processed for histologic analysis. Normal lungs
from control rats were processed in the same manner.
Methylation Analysis
Quantification of global DNA methylation. Genomic DNA
was extracted by conventional methods from frozen control
lungs (n = 6); frozen treated lungs obtained on months 3 to 4
(n = 5), months 6 to 8 (n = 5), and months 10 to 12 (n = 5);
and frozen isolated tumors obtained onmonths 16 to 21 (n = 9;
five AC and four SCC). 5-Methylcytosine (mC) DNA content
in all the samples was determined by high-performance cap-
illary electrophoresis, as previously described [24]. Briefly,
genomic DNA (3–5 mg) was obtained from the different
tissues, and DNA hydrolysis was carried out with 1.25 ml
(200 U/ml) of nuclease P1 for 16 hours at 37jC. Subse-
quently, alkaline phosphatase was added, and mixtures were
incubated for 2 hours at 37jC. Hydrolyzed samples were
injected under pressure (0.3 psi) for 3 seconds into an un-
coated fused-silica capillary in a CE system (P/AC MDQ;
Beckman Coulter, Palo Alto, CA). Quantification of the relative
methylation of each DNA sample was determined as: %mC =
(mC peak area  100)/(C peak area + mC peak area). All
samples were analyzed in duplicate, and three analytical
measurements were made per replicate.
Bisulfite genomic analysis of the p16(CDKN2A), APC,
CDH13, Rassf1, and Nore1A CpG islands. DNA samples
from control lungs (n = 6); treated lungs obtained on months
3 to 4 (n = 5), months 6 to 8 (n = 5), and months 10 to 12 (n =
5); and tumors obtained on months 16 to 21 (n = 9; five AC
and four SCC) were treated with sodium bisulfite, as previ-
ously described [25]. Primers (Table W1) spanning the CpG
island of the rat p16(CDKN2A), APC, H-cadherin (CDH13),
Rassf1, and Nore1A promoters were used for bisulfite ge-
nomic sequencing. At least six individual clones were se-
quenced for each sample. A single CpG was considered to
be methylated when more than half of the clones retained an
unmodified cytosine at that position.
Mutational Analysis
DNA extraction after laser capture microdissection. To ob-
tain near-pure cancer cell populations for mutation analy-
ses, we performed laser capture microdissection (CTRMIC/
ASLMD, Leica, Germany). Two consecutive 5-mm sections
were cut from formalin-fixed paraffin-embedded tissue
blocks. One section was stained with hematoxylin and eosin
(H&E), which was used for histologic evaluation and control;
another section was stained with methyl green for microdis-
section (Figure W1). Tumor tissue and adjacent normal lung
tissue for control were obtained by microdissection. The
microdissected cells were collected in a 0.5-ml microcentri-
fuge tube containing 40 ml of DNA lysis buffer (10 mM Tris–
HCl pH 8.0, 1 mM EDTA, 1% Tween-20, and 20 mg/ml
proteinase K). Tubes were then incubated for 14 hours at
55jC. Proteinase was inactivated by incubation at 95jC for
10 minutes.
Polymerase chain reaction exon amplification. Eight micro-
liters of genomic DNA was used for very-high-fidelity polymer-
ase chain reaction (PCR) amplification with AccuPrime Pfx
DNA Polymerase System (Invitrogen, Carlsbad, CA). PCR
was carried out in a total volume of 30 ml containing 0.2 mM
of each primer (Table W2), 1 U of AccuPrime Pfx DNA
Polymerase, and 1 AccuPrime Pfx Reaction Mix (50 mM
Tris–HCl pH 8.0, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA,
1mMMgSO4, and 3mMdNTPs). PCRswere performed on a
PTC-100 thermocycler (MJResearch, Watertown, MA) using
the following protocol: 95jC for 3 minutes; 37 cycles of 95jC
for 30 seconds, primers’ specific annealing temperature for
35 seconds, and 68jC for 35 seconds; and 7 minutes at
68jC for extension. PCR products, which were confirmed to
have a single target band in a 2% agarose gel, were purified
using the Qiagen MinElute PCR Purification Kit (Qiagen,
Valencia, CA).
Sequencing and analysis. The purified products were then
subjected to direct sequencing by an ABI377 sequencer
(Perkin-Elmer Applied Biosystems, Foster City, CA). Exons
were sequenced upstream and downstream, and compared
to a control sequence. They were carefully analyzed first with
ChromasPro 1.33 (Tewantin, Australia) and, later, manually. If
a mutation was detected, additional microdissection and
sequencing of the tumor and the surrounding normal tissue
were carried out to verify the mutation.
Immunostaining
Immunostaining was performed with antibodies to p16
(F-12, 1:50; Santa Cruz Biotechnology, Santa Cruz, CA),
842 Molecular Analysis of Inflammation-Driven Lung Cancer Blanco et al.
Neoplasia . Vol. 9, No. 10, 2007
p53 (FL-393, 1:100; Santa Cruz Biotechnology; or Pab 240,
1:100; Abcam, Cambridge, UK), EGFR (2232, 1:50; Cell
Signaling, Danvers, MA), TERT (Ab-2, 1:500; Calbiochem,
San Diego, CA), g-H2AX (2577, 1:200; Cell Signaling), and
inducible nitric oxide synthase (iNOS) (Sc-650, 1:100; Santa
Cruz Biotechnology). An indirect avidin–biotin–peroxidase
method (Dako, Barcelona, Spain) was used for EGFR, TERT,
g-H2AX, and iNOS analysis. In the case of p53 and p16
immunostaining, we used the EnVision (K4001; Dako) signal
enhancement system. Immunohistochemical technique was
carried out as follows. Slides were deparaffinized and incu-
bated for 10 minutes with 3% H2O2 in water to quench
endogenous peroxidase activity. Heat-mediated antigen re-
trieval was used for antibodies to EGFR and g-H2AX (15 min-
utes at 375 W in 1 mM EDTA, pH 8.0) and p53 (15 minutes at
750 W and 15 minutes at 375 W in 10 mM sodium citrate, pH
6.0). Tissues were incubated with 5% normal rabbit serum in
Tris-buffered saline (TBS) (500 mM NaCl and 50 mM Tris–
HCl pH 7.4) for 30 minutes at room temperature. After blot-
ting excess serum, sections were incubated at 4jC over-
night, with the primary antibody diluted in TBS for p53, p16,
telomerase, and iNOS, and diluted in mixed sera for EGFR
and g-H2AX.
Tissues were washed in TBS and incubated with the
appropriate secondary antibody. For the indirect avidin–
biotin–peroxidase method, biotinylated rabbit–anti-mouse
Ig antiserum was added at a 1:200 dilution for 30 minutes at
room temperature; after the slides had been washed, they
were incubated for 30 minutes at room temperature with the
avidin–biotin complex at a 1:100 dilution. For the EnVision
signal enhancement system, the secondary monoclonal
complex was applied for 30 minutes at room temperature.
After the slides had been washed in TBS, development of
peroxidase with diaminobenzidine and H2O2 was performed.
The slides were counterstained with hematoxylin, dehy-
drated, and mounted. As negative controls, the primary
antibody was replaced with mouse IgG1 or IgG2a specific
for Aspergillus niger glucose oxidase (Dako).
Figure 1. Oxidative stress and DDR in silica-induced lesions measured by immunohistochemistry in serial consecutive sections. The figures in this plate show the
activation of the iNOS/p53/c-H2AX pathway in the multistep progression from normal tissue to tumors. Low levels of iNOS and p53 proteins were found in
morphologically normal bronchial epithelial cells, whereas c-H2AX was completely absent (A). In normal bronchioli, iNOS expression was also found in smooth
muscle cells (arrow; A). A clear increase in the coexpression of iNOS/p53/c-H2AX was observed from hyperplastic (B and C) to dysplastic (D) bronchiolar cells. In
tumors, colocalization was still present in some areas, although c-H2AX levels were reduced compared to hyperplastic and advanced preneoplastic tissues (E and
F). Counterstaining by Harris hematoxylin. Original magnifications, 420 (C, E, and F); 630 (A, B, and D).
Molecular Analysis of Inflammation-Driven Lung Cancer Blanco et al. 843
Neoplasia . Vol. 9, No. 10, 2007
Staining criteria
The immunostaining level of the following histologic
structures was analyzed in each section: normal bronchioles
(NB), normal type II alveolar cells (NA), hyperplastic bron-
chioles (HB), hyperplastic type II alveolar cells (HA), dys-
plastic bronchioles (DB), AL, and tumors AC and SCC. The
total numbers of normal and pathological structures ana-
lyzed by each antibody are indicated in the Results section.
p53, p16, or TERT-immunostained area in a given tissue
was expressed as the percentage of positive nuclei and cal-
culated as: (positive nuclei/total nuclei)  100. This quanti-
tative method has been previously published [26]. For p53
and p16 quantification, although some cytoplasmic staining
was found at some stages, only nuclear immunostaining
was considered. For p53, we considered overexpression
when > 50% of the nuclei were immunostained. The result
was scored as negative immunostaining if < 10% of the nuclei
were positive. In the case of p16, loss of expression was
recorded when < 25% of positive nuclei were found. For
telomerase, we considered overexpression when > 75% of
the nuclei were positive. The mean percentage of nuclear
protein expression and its 95% confidence interval were
calculated for p53, p16, and TERT in all the histologic struc-
tures studied. In the evaluation of EGFR and iNOS immu-
nostaining, the intensity was classified into four categories:
(
) negative, (+) weak, (++) moderate, and (+++) strong.
Immunostaining was estimated independently by two experi-
enced researchers.
Statistical analysis
Statistical analysis was performed with the SPSS 13.0
software (SPSS, Inc., Chicago, IL). Chi-square analysis was
applied to study the differences of p53, p16, or TERT ex-
pression between the normal, preneoplastic, and tumoral
tissues. Fisher’s exact test was applied to study differences
Figure 2. p53, p16, and TERT nuclear expression by immunohistochemistry in the multistep progress to lung cancer. (A) Normal lung epithelial cells showed
low levels of nuclear protein expression for p53, p16, and TERT. (B and C) Hyperplastic bronchiolar (B) and alveolar (C) cells showed a significant increase for
p53, p16, and TERT nuclear immunostaining. (D) DB at 10 to 12 months showed a higher percentage of positive nuclear cells for p53 and TERT. However, p16 was
significantly decreased in these advanced preneoplastic lesions. (E and F) p53 and TERT overexpression was commonly observed in AC (E) and SCC (F),
whereas p16 overexpression was observed in SCC (F) and in a subset of more fibrotic AC. Loss of p16 protein expression (< 25% positive nuclei) was detected
in 44% of AC (p16; E). Counterstaining by Harris hematoxylin. Original magnifications, 420 (p53-TERT; B) (p16; D, E, and F); 560 (p16; B); 700 (A and C)
(p53-TERT; D).
844 Molecular Analysis of Inflammation-Driven Lung Cancer Blanco et al.
Neoplasia . Vol. 9, No. 10, 2007
in DNA global hypomethylation analysis. Differences were
considered significant when P < .05.
Results
DDR and Oxidative Stress from Early Hyperplastic
Tissues to Tumors
In serial consecutive tissue sections of the silica-induced
lung cancer model, we analyzed the expression of p53,
iNOS, and g-H2AX, a marker of activated DDR. We studied
the expression of these three markers through five stages
(n = 8 for each stage), including normal, preneoplastic, and
neoplastic tissues. In the normal bronchiolar epithelial cells
of treated animals, p53 and iNOS expressions were found
at low levels, whereas there was no signal for g-H2AX
(Figure 1A). iNOS expression was also observed in alveolar
and parenchymal macrophages present in the early phases
of the silicotic response, as well as in epithelioid macro-
phages at the core of granulomas. iNOS expression in
granulomas was maintained up to the latest stages studied
(21 months) (Figure W2). p53, iNOS, and g-H2AX were
clearly induced in the same tissue elements of early preneo-
plastic lesions such as hyperplastic bronchioloalveolar cells
and persisted in advanced preneoplastic stages such as
dysplastic bronchiolar cells (Figure 1, B–D). In tumors, co-
localization was still present in some areas, although g-H2AX
levels were reduced compared to hyperplastic and advanced
preneoplastic tissues (Figure 1, E and F ). These data sug-
gest that (nitro)oxidative stress coexists with p53 accumula-
tion in tumorigenic progression, and both are associated with
DNA damage measured by the DDR marker g-H2AX.
p53 Stabilization and Loss of p16 Expression in Multistage
Progression to Silica-Induced Lung Tumors
We analyzed the evolution of the p53 and p16 protein
expressions along the multistep progression from the normal
epithelium to non–small cell lung cancer (NSCLC) tumors
(50 AC and 9 SCC). p53 nuclear immunostaining was
negative in normal alveoli (mean percentage of positive
nuclei: 5 ± 0.1%; n = 35) and bronchiolar epithelial tissues
(12 ± 3%; n = 32) (Figures 2A and 3). A significant increase
for p53 nuclear expression was observed in hyperplastic
bronchiolar (56 ± 4%; n = 40) and alveolar (51 ± 6%; n = 29)
epithelial cells (Figures 2, B and C, and 3; Table 1; P < .001),
and this overexpression was maintained in epithelial hyper-
plasias over time, at least up to month 12. In dysplastic
bronchiolar lesions (87 ± 7%; n = 12), p53 was significantly
higher when compared to hyperplastic bronchiolar epithe-
lium onmonths 10 to 12 (Figures 2D and 3; Table 1; P < .001).
The percentage of cells with nuclear p53 was significantly
lower in tumors (54 ± 8%; n = 59) compared to advanced
preneoplastic lesions such as bronchiolar dysplasia (P <
.001) and adenomatoid lesions (P = .008) (Figure 3; Table 1).
In tumors, p53 overexpression was observed in 26 of 50
(52%) AC and in 6 of 9 (67%) SCC (Figure 2, E and F ). No
statistical differences were observed between the two histo-
logic tumor types.
p16 protein expression was negative in normal alveolar
(2 ± 0.6%; n = 36) and bronchiolar (5.6 ± 1.8%; n = 35)
epithelial tissues (Figures 2A and 3). We observed a signif-
icant increase in p16 nuclear overexpression in hyperplastic
bronchiolar (61 ± 4%; n = 40) and alveolar (34 ± 10%; n = 29)
epithelial cells compared to normal tissues (Figures 2, B and
C, and 3; Table 1; P < .001). This overexpression was
maintained in bronchiolar and type II hyperplasias from
months 6 to 12. Interestingly, p16 was significantly de-
creased in dysplastic bronchiolar epithelium (13 ± 4%; n =
12) compared to neighboring bronchiolar hyperplasias (Fig-
ures 2D and 3; Table 1; P < .001). In tumors, the percentage
of cells with nuclear p16 was significantly lower (42 ± 8.4%;
n = 59) than in bronchiolar hyperplasias (Figure 3; Table 1;
P < .001). Loss of p16 protein expression (< 25% of positive
nuclei) was detected in 22 of 50 (44%) AC and in 2 of 9 (22%)
SCC (Figure 2E ). Nonfibrotic AC showed a significantly
lower score for p16 staining (30 ± 8% of positive nuclei)
compared to fibrotic AC (58 ± 9%; P = .016) and SCC (76 ±
24%; P = .027).
All these data confirm that p53 nuclear levels increase in
epithelial cells during the multistep progression from normal
to hyperplastic and advanced preneoplastic tissues, and
remain high in most tumors. Conversely, p16 is overex-
pressed in hyperplastic tissues, but its expression is de-
creased or lost in advanced bronchiolar dysplasias and in
40% of the tumors.
TERT Protein Overexpression in Multistep Progress to
Lung Tumors
Nuclear TERT protein overexpression was observed from
months 3 to 12 in hyperplastic bronchiolar (86 ± 2%; n = 19)
Figure 3. Differential expression for p53, p16, and TERT between different stages of cancer progression. Graphs show the mean percentage of nuclear protein
expression and its 95% confidence interval for p53, p16, and TERT immunostaining in all the histologic structures studied. TUMOR, AC and SCC.
Molecular Analysis of Inflammation-Driven Lung Cancer Blanco et al. 845
Neoplasia . Vol. 9, No. 10, 2007
and alveolar (87 ± 3%; n = 20) tissues compared to normal
bronchiolar (50 ± 6%; n = 48) and alveolar (7 ± 1%; n = 38)
epithelial cells (Figures 2, A–C, and 3; Table 1; P < .001). A
high percentage of immunostained nuclei was also observed
from month 10 in advanced preneoplastic adenomatoid
lesions (81 ± 5%; n = 9) and dysplastic bronchiolar cells
(91 ± 2%; n = 12) compared to normal tissues (Figures 2D
and 3). At the late stage, lung tumors also showed a
significant overexpression of TERT (77 ± 2%; n = 59) com-
pared to control tissues (Figures 2, E and F, and 3; Table 1;
P < .001). The percentage of nuclei stained for TERT at 16
to 21 months was significantly lower in tumors than in hy-
perplastic bronchioloalveolar and dysplastic bronchiolar
lesions (Table 1; P < .001), showing a decrease in TERT nu-
clear detection from preneoplastic tissues to cancer. No sta-
tistical differences were observed between AC and SCC.
We conclude that TERT is highly expressed in reactive
epithelial tissues during multistep carcinogenesis, with the
highest percentage of expression in hyperplastic and dysplas-
tic tissues, and a slight decrease of its expression in tumors.
Mutational Profile of Lung Tumors
Tp53. We analyzed the mutational status of the Tp53 gene,
the most commonly mutated gene in human cancer. We
sequenced exons 5 to 9—those more frequently mutated in
human lung cancer and also found mutated in rodent tumors
[27,28]. To avoid contamination with nontumor DNA, we
microdissected tumors from paraffin sections. We selected
a subset of 32 tumors showing either high or low p53 protein
nuclear accumulation, as determined by immunohistochem-
istry. Five mutations were identified in 3 of 32 (9%) tumors
(one AC and two SCC). Two mutations were detected in
exon 5 of one AC sample: a missense mutation (K162R) and
a nonsense mutation in codon 164. In two SCC, three
mutations were located in exon 6, involving codons 254
and 255 (both sense mutations), and codon 246 (a missense
mutation leading to an arginine-to-serine substitution) (Fig-
ure 4, A and B ). Therefore, Tp53 mutations are rare events
in tumors induced by silica-mediated chronic inflammation.
Ras proto-oncogene family. We then aimed to determine
how relevant is the status of the Ras family gene in the
development of NSCLC in the rat silica model. First, we
analyzed by sequencing the mutational status of exons 1
and 2 of K-ras (codons 6–37 and 44–97), N-ras (codons
1–23 and 48–74), and c-H-ras (codons 1–82) in 23 laser-
microdissected tumors (15 AC and 8 SCC). We did not
identify any mutation in the tumors (Figure 4A). These data
strongly suggest the lack of genetic alterations in these
Ras family genes in this chronic inflammation–driven lung
cancer model.
EGFR. EGFR mutations and gene amplification, along with
protein overexpression, are relatively frequent events in
human lung cancer and have been the basis for new molec-
ular targeted therapies [29]. We analyzed the EGFR muta-
tional status and protein pattern expression in 32 NSCLC
tumors (25 AC and 7 SCC). First, immunohistochemical
analyses using a validated antibody specific to EGFR
revealed that 92% of AC and 71% of SCC were positive for
this receptor, with both membrane and cytoplasmic local-
izations (Figure W3). Strong or moderate staining for EGFR
was found in 14 of 25 (55%) AC (Figure W3, A and B) and
in 1 of 7 (14%) SCC, whereas only 2 of 25 (8%) AC (Fig-
ure W3C) and 2 of 7 (29%) SCC were completely negative.
Next, we analyzed the DNA mutational status of EGFR
through exons 18 to 21 in 32 microdissected tumors previ-
ously analyzed for protein expression. We did not find any
gene mutation in any of the codons analyzed (Figure 4A).
These data suggest that EGFR overexpression, but not mu-
tation, may be a relevant biologic event in the progression of
silica-induced tumors.
Epigenetic Alterations in Multistep Progression to Cancer
Global DNA methylation status. The epigenetic regulation
of gene expression has been shown to be very frequent in
lung carcinogenesis. As we have previously reported [15],
silica-induced tumors show clear global genomic hypometh-
ylation, with an average loss of 25% in mC DNA content. In
this study, we extend this analysis to explore whether global
hypomethylation was found in previous stages of the lung
carcinogenesis process, when only preneoplastic lesions,
but not tumors, are present. The global DNA methylation
status in the samples obtained on months 3 to 4 (n = 5),
months 6 to 8 (n = 5), and months 10 to 12 (n = 5), and in
tumors obtained on months 16 to 21 (n = 9) showed that
Table 1. Statistical Analysis of the Differences in p53, p16, and TERT Expression between Different Stages of Cancer Progression.
Comparisons p53 p16 TERT
Normal versus hyperplasia NA versus HA v2 = 74.00, P < .001 v2 = 53.79, P < .001 v2 = 77.00, P < .001
NB versus HB v2 = 89.44, P < .001 v2 = 71.38, P < .001 v2 = 91.38, P < .001
Hyperplasia versus advanced preneoplasia HA versus AL v2 = 4.63, P = 2.01 v2 = 2.10, P = .718 v2 = 4.56, P = .041
HB versus DB v2 = 39.74, P < .001 v2 = 46.93, P < .001 v2 = 1.89, P = .169
Nonneoplastic versus neoplastic lesions NA versus TUMOR v2 = 78.00, P < .001 v2 = 57.48, P < .001 v2 = 95.00, P < .001
HB versus TUMOR v2 = 54.40, P < .001 v2 = 40.12, P < .001 v2 = 48.64, P < .001
HA versus TUMOR v2 = 11.39, P = .010 v2 = 6.32, P = .177 v2 = 12.68, P < .001
HB versus TUMOR v2 = 11.34, P = .010 v2 = 28.39, P < .001 v2 = 18.22, P < .001
AL versus TUMOR v2 = 11.94, P = .008 v2 = 2.43, P = .657 v2 = 0.67, P = .412
DB versus TUMOR v2 = 17.31, P < .001 v2 = 18.16, P < .001 v2 = 19.48, P < .001
TUMOR, AC and SCC.
846 Molecular Analysis of Inflammation-Driven Lung Cancer Blanco et al.
Neoplasia . Vol. 9, No. 10, 2007
hypomethylation only occurs in tumors, but not in lung
extracts containing preneoplastic tissues (Figure 5).
CpG island hypermethylation in p16(CDKN2A), APC, and
CDH13 gene promoters. We determined the role of pro-
moter hypermethylation in the transcriptional repression of
p16(CDKN2A), APC, and H-cadherin (CDH13) genes,
whose promoters are frequently hypermethylated in human
lung cancer [30,31]. We analyzed preneoplasia-containing
tissues obtained on months 3 to 4 (n = 5), months 6 to 8 (n =
5), and months 10 to 12 (n = 5), and tumors obtained on
months 16 to 21 (five AC and four SCC).
All samples analyzed in the time course from months 3
to 12 were unmethylated. However, we observed a strong
CpG island hypermethylation in seven of nine tumors in
the promoter region of CDH13 (78%); in six of nine tumors
in p16(CDKN2A) (67%); and in five of nine tumors in APC
(56%) (Figure 6). We found concurrent promoter hyper-
methylation of all three genes analyzed in four of nine tumors.
Furthermore, p16 promoter hypermethylation showed a
strong inverse association, with the p16 protein expression
measured by immunohistochemistry. Promoter hypermeth-
ylation was associated with low or negative p16 protein
expression, whereas unmethylated cases showed p16 over-
expression (Figures 6 and W4). These data suggest that
epigenetic alterations are relevant biologic events in tumors
generated in the context of the inflammatory insult.
CpG island hypermethylation in Rassf1 and Nore1A gene
promoters. We next tested whether the Ras pathway could
be activated by promoter hypermethylation of Ras effectors,
as has been demonstrated for Rassf1 [32] or Nore1A [33]
in human lung cancer. For this approach, we studied the
methylation status of the promoters of the rat homologues
for Ras effectors Rassf1 and Nore1A in the silica-induced
tumors. We did not observe epigenetic alterations in the
promoters of these two Ras effectors (Figure W5). These
data, together with the mutational Ras status, strongly sug-
gest the lack of genetic and epigenetic alterations in the
Ras family members in this inflammation-mediated lung can-
cer model.
Discussion
In this report, we have characterized some of the key
molecular alterations leading to lung cancer in the context
of chronic inflammation (Figure 7). Using a rat silica model
of inflammation-induced lung cancer [15], our results showed
an activation of the DDR pathway in preneoplastic lesions,
in association with proliferative and telomeric stress. Sur-
prisingly, we found a very low frequency of mutations in the
Tp53 gene and a total absence of mutations in the main Ras
signaling family genes and EGFR. In contrast, epigenetic
events in tumor suppressors, including p16, APC, and CDH13,
were found with high frequencies in this lung cancer model.
Our data show that p16 loss is significantly associated
with inflammation-induced lung carcinogenesis. In the silica-
induced lung cancer model, downregulation of p16 in late
preneoplastic lesions could be the landmark for the escape
of some epithelial cells from the tight growth-regulatory
mechanisms found in preneoplasia to loosely regulated
tumorigenic pathways. In fact, in vitro studies of lung cells
have already suggested that the loss of p16 expression,
rather than the alteration of the ARF/p53 pathway, is the key
event in bypassing growth arrest and in acquiring a trans-
formed phenotype with genetic instability and tumoral poten-
tial [34]. Accordingly, a significant decrease in p16 protein
was also observed in 40% of silica-induced tumors, and p16
Figure 5. DNA global hypomethylation analysis in controls (n = 6);
preneoplasia-containing lungs on months 3 to 4 (n = 5), months 6 to 8 (n =
5), and months and 10 to 12 (n = 5) after treatment; and in tumors (n = 9). A
clear global genomic hypomethylation with an average loss of 25% in mC
DNA content only occurs in tumors. Results are expressed as mean ± S.D.
Fisher’s exact test was applied, and differences were considered significant
when P < .05.
Figure 4. Mutational analysis for Ras (n = 23), Tp53, and EGFR (n = 32) in
tumor DNA extracts. (A) No mutations were found in K-ras, N-ras, c-H-ras, or
EGFR in any of the silica-induced tumors analyzed after microdissection. For
Tp53, five mutations were found in three tumors of 32 cases analyzed (9%).
(B) Electropherogram of the DNA sequence of the tumor (mt) versus the cor-
responding adjacent normal tissue (wt) showing the mutations found in exon
5 in AC and exon 6 in SCC.
Molecular Analysis of Inflammation-Driven Lung Cancer Blanco et al. 847
Neoplasia . Vol. 9, No. 10, 2007
protein downregulation correlated with gene promoter hyper-
methylation. Some, but not all, lung cancer experimental mod-
els show p16 promoter hypermethylation [35,36]. Belinsky
et al. [11] have shown that tumors produced by instillation of
carbon black particles in rats arise, in part, through the
epigenetic inactivation of p16, suggesting that exposure to
some particular carcinogens may be associated with specific
gene inactivation through methylation [37]. More importantly,
loss of p16 expression was also found in human lung carci-
nogenesis, starting at the moderate dysplasia stage [38].
Moreover, p16 loss in preneoplastic lesions occurred exclu-
sively in patients who also showed loss of p16 expression in
their related invasive carcinoma [38]. The inactivation of p16
has been reported in approximately 50% of human NSCLC,
and this inactivation is frequently associated with p16 pro-
moter hypermethylation [39].
Other classic oncogenic events present in human NSCLC
were found at low frequency or were absent in this model.
The incidence of Tp53 mutations observed in silica-induced
tumors was significantly lower (9%) than the incidence
reported for NSCLC in human smokers (50%) and non-
smokers (28%) [40]. Studies conducted by several groups
have consistently reported a very low frequency or a com-
plete lack of mutations at the Tp53 gene locus in rodent lung
tumors [41,42]. The low prevalence of Tp53 mutations in
murine versus human lung tumors is striking because the
gene is highly conserved and the tumors’ morphologic
features are very similar. In contrast to Tp53, Ras mutations
have been frequently found in chemically induced murine
lung tumors and have been strongly correlated with promu-
tagenic adducts generated from the metabolism of chemical
carcinogens present in tobacco [43]. In the silica model,
mutations in K-ras, N-ras, or H-Ras, including codons 12,
13, and 61 (which are known to transform ras into an
oncogenic protein in both human and rodent cells), were
completely absent. Ras mutations are also infrequent in
other rat lung tumor models induced by particulate carcino-
gens (diesel exhaust and carbon black) [41]. The lack of
mutations in the silica model supports the current view that
the existence of Ras mutations is highly dependent on the
type of carcinogen. This view is further supported by the lack
of mutations in codon 12 of K-ras in lung cancer found in
workers with silicosis [44]. Moreover, promoter hypermeth-
ylation of Ras effectors Nore1A and Rassf1 present in
Figure 6. Bisulfite genomic sequencing of the p16(CDKN2A), H-cadherin (CDH13), and APC gene promoters in normal tissues (n = 6); preneoplasia-containing
tissues on months 3 to 4 (n = 5), months 6 to 8 (n = 5), and months 10 to 12 (n = 5); and nine representative lung tumors. Vertical bars show the distribution of CpG
islands at p16, H-cadherin, and APC. The vertical arrow indicates the transcriptional start point. Black dots indicate methylated CpG islands; white dots indicate
unmethylated CpG islands. The position of the bisulfite sequencing primers is represented with white horizontal arrows. We observed H-cadherin promoter
hypermethylation in seven of nine silica-induced tumors (T1, T2, T3, T4, T5, T6, and T9), and APC promoter hypermethylation in five of nine tumors (T2, T3, T4, T5,
and T9). Hypermethylation in the p16 promoter was also an important event in six of nine tumors studied (T1, T2, T3, T5, T8, and T9).
848 Molecular Analysis of Inflammation-Driven Lung Cancer Blanco et al.
Neoplasia . Vol. 9, No. 10, 2007
humans [32,33] was not found in the silica-induced tumors.
These data strongly support the view that inflammation-
driven carcinogenesis is not mediated by genetic alterations
in the Ras pathway. Silica-mediated chronic inflammation
may also activate the EGFR pathway. EGFR is expressed in
a range from 40% to 70% of human NSCLC [45]. We have
observed EGFR expression in 87% of silica-induced tumors,
which could reflect the autocrine or paracrine stimulation of
tumor growth by several EGFR ligands. NSCLC patients who
show clinical responses to EGFR tyrosine kinase inhibitors
frequently harbor somatic mutations in the EGFR gene.
These EGFR mutations are more commonly found in fe-
males, nonsmokers, and ACwith bronchioloalveolar features
[29]. Moreover, EGFR and Ras mutations seem to be mu-
tually exclusive in NSCLC [46]. We thus looked for EGFR
mutations in DNA frommicrodissected inflammation-induced
tumors. Although a great proportion of the tumors arising in
the rat silica model are AC with bronchoalveolar-like fea-
tures, we did not find EGFR mutations in any of the cases
studied, suggesting that mutations in Ras or EGFR path-
ways are not involved in this inflammation-driven lung carci-
nogenesis model.
Tobacco and nontobacco-related human lung cancers
harbor different combinations of mutations and gene pro-
moter methylation events [47]. One of the most interesting
findings of this study was the wealth of epigenetic events.
Previously, chronic inflammation had been associated with
high levels of methylation in ulcerative colitis and chronic
gastritis [48,49]. However, a direct causal relationship of
promoter hypermethylation has not yet been unequivocally
shown in lung tumors. In our study, aberrant methylation
events have been detected in the p16(CDKN2A), APC, and
CDH13 promoters in the silica-induced tumors but were not
present in lung tissues containing preneoplastic lesions. This
is the first description of APC gene promoter hypermethyla-
tion in a lung cancer animal model. Epigenetic alteration of
CDH13 has recently been described in mouse lung tumor
models [50]. In human NSCLC, aberrant promoter hyper-
methylation for these tumor-suppressor genes has been
reported by several independent studies [30,31]. An associ-
ation between CDH13 methylation and tumor progression in
human NSCLC has also been suggested [51]. Previously, we
have shown a strong epigenetic alteration of E-cadherin and
b-catenin accumulation in the context of the epithelial–
mesenchymal transition in this model [15]. One could spec-
ulate that the loss of function of APC and CDH13 through
gene promoter hypermethylation may disrupt cells’ ability to
regulate adhesion; in turn, APC loss may also activate the
Wnt signaling pathway through stabilization of the b-catenin
protein [52]. Deregulation of b-catenin causes cells to remain
in a less differentiated and more proliferative state [53].
These findings suggest that simultaneous epigenetic inacti-
vation of several putative tumor-suppressor genes (p16,
APC, E-cadherin, and CDH13) may have a strong tumori-
genic effect. Nevertheless, mutations of other potential
oncogenes not analyzed in this study or loss of other putative
tumor-suppressor genes cannot be ruled out.
The current view about the acquisition of a tumorigenic
phenotype and progression through carcinogenesis de-
scribes a strong association between DNA damage events,
genomic instability, and selective genomic pressure. Recent
studies in several human tumor types have shown that the
DNA damage checkpoint was activated in epithelial early
preneoplastic lesions either by DNA replication stress or by
oncogenic stress [20,21,54]. According to these studies,
DDR acts as a tumorigenesis barrier inducing cell cycle
Figure 7. Summary of the molecular events found in the silica-induced lung carcinogenesis model. DDR, p16, TERT, and p53 overexpressions are observed from
early preneoplastic lesions. p16 inactivation occurs in dysplastic bronchiolar lesions and tumors. No mutations were observed in tumors, with the only exception of
Tp53 mutations with a very low frequency. Epigenetic alterations were found in tumors in the tumor-suppressor genes p16(CDKN2A), APC, and CDH13.
Molecular Analysis of Inflammation-Driven Lung Cancer Blanco et al. 849
Neoplasia . Vol. 9, No. 10, 2007
arrest, senescence, or apoptosis, thus protecting from pro-
gression to malignancy. In the present study, we found an
association between the presence of local inflammatory me-
diators and the activation of the DDR pathway. It is well-
known that silica exposure rapidly induces inflammatory
mediators in the lung, which results in oxidative stress and
DNA alterations, including single-strand breaks, mutagenic
base modification by 8-oxoguanine, and various other DNA
oxidation products [55,56]. Oxidative stress elicited by crys-
talline silica was associated with a remarkable increase in
the expression of the inflammatory mediator iNOS in the
rat airways, in agreement with previous studies [17,57,58].
The reactions to silica particles in the lungs stimulate the
activation of a number of molecular mediators, including
those that are primarily involved in inflammatory and fibro-
genic responses and their immunological aspects, such as
interleukins and TNF-a. Silica particles, either by direct sur-
face effects or during phagocytosis, generate ROS, which
seem to trigger critical signaling events for both NF-nB and
AP-1 activation [16,59]. The pathogenic model proposed for
asbestos-induced mesothelioma is similar to this proposal
for silica-induced tumors. In both cases, mineral dust/fiber
causes accumulation of cytokine-releasing (especially TNF-a)
and NO-releasing active macrophages [16,60,61]. A recent
report has also suggested that NF-nB–dependent inflam-
mation, combined with asbestos-induced genetic instability,
may be a potential cause of mesothelial carcinogenesis
[61]. The current hypothesis is that, in iNOS-expressing
epithelial cells, chronic exposure to nitrogen and oxygen
radicals may induce (nitro)oxidative stress, leading to DNA
damage and activation of a p53 response [22,62]. In the lung
epithelial lesions found in the rat silica model, iNOS was
commonly coexpressed with g-H2AX and was associated
with p53 accumulation. Moreover, this coexpression was
more frequently observed in preneoplastic tissues than in
tumors, suggesting the relevance of DDR in epithelial cells
during chronic inflammation. DNA damage and other cellu-
lar stresses can induce the expression of p53 and p16, en-
forcing cell cycle arrest [22,63]. We observed a clear induction
of p53 and p16 expression occurring in parallel with chronic
inflammatory stress and the replicative activation of hyper-
plastic lesions. Activated p53 inhibits cell proliferation and
protects against genomic instability by controlling cell cycle
checkpoints, DNA repair, apoptosis, and senescence [64].
Overexpression of p53 and p16 in hyperplastic lesions may
depend on the activation of the DDR and may mutually re-
inforce the barrier toward tumorigenesis. In contrast, in most
advanced preneoplastic lesions, p53 expression remains
high, whereas a very significant decrease of p16 occurs. This
further emphasizes the potential role of p16 loss as a key
molecular event.
Besides oxidative stress, the activation of DDR and cell
cycle arrest could also be mediated by telomere shortening
due to replicative stress [23]. TERTactivity contributes to the
elongation of shortening telomeres in preneoplastic and
tumor cells escaping from telomere crisis mediated by crit-
ically short telomeres [65]. Previous studies in breast and
lung tumors have also shown an increase in telomerase
expression in preneoplastic lesions [65,66]. Our data are in
accordance with previous studies on rodents where telo-
merase is detectable in normal tissues and significantly
increased in preneoplastic and neoplastic cells [67,68].
We observed a clear induction of TERT expression in the
silica-induced multistep progression that was more evident
in preneoplastic tissues than in tumoral tissues. The over-
expression of telomerase, together with inactivation of the
p16–Rb pathway, could bypass DDR-mediated cell cycle
arrest and extend the lifespan of tissues. Furthermore, telo-
merase overexpression may affect the proliferation of epi-
thelial cells not only by stabilizing telomeres but also by
affecting the expression of growth-promoting genes. Smith
et al. [69] reported that activation of telomerase in p16-
inactivated tumoral cells altered the expression of a set of
genes that have a profound effect on cell proliferation, in-
cluding EGFR, which is indeed overexpressed in this model.
In summary, we have explored the specific molecular pro-
file of chronic inflammation–mediated neoplastic transfor-
mation of the lung. Our data suggest that induction of iNOS
expression in the inflammatory context is associated with
activation of the DDR and p53 accumulation in preneoplastic
epithelial cells. p16 is also induced in the early steps of tu-
morigenic progression, followed by a clear loss of expression
in late dysplastic bronchiolar lesions. Among the recently
reported barriers to preneoplasia-to-neoplasia transition,
p16 seems to be the one bypassed in this inflammation-
driven model, very likely as a consequence of a strong epi-
genetic regulation of p16 gene expression. TERTand EGFR
are also overexpressed in this model. Lack of mutations of
Ras signaling components and EGFR, and a very low fre-
quency for Tp53 suggest that they are not major players in
inflammation-driven carcinogenesis, whereas epigenetic al-
terations in tumor-suppressor genes such as p16(CDKN2A),
CDH13, and APC are more relevant. Our results suggest
that chronic inflammation– induced tumors have a specific
molecular signature that shares some, but not all, of the al-
terations found in chemically induced lung cancer.
Acknowledgements
The authors want to express their special gratitude to Umberto
Saffiotti (National Cancer Institute, National Institutes of Health)
for his invaluable help in establishing the model at the start
of this project, and for his constant advice, encouragement,
and suggestions. We thank Paz Zamora, Elena Remirez, and
Gabriel de Biurrun for technical assistance.
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Table W1. Primers Used for Methylation Analysis.
Gene Sequence Product Size (bp) Annealing Temperature (jC)
p16(CDKN2A) F: 5V-GGTAATAGTGTTTTTAGAGGTG-3V 259 60
R: 5V-CTACCCTAACTAATCTATCTAC-3V
APC F: 5V-TAGGGGTTTGAAGGTGTATAGG-3V 305 58
R: 5V-CTCCTATAACAAACCTAATCATCAC-3V
CDH13 F: 5V-TTTATTTGGGAAGTTGGTTGGTTG-3V 442 62
R: 5V-TATCCTTCTCAAAATAAACACACAC-3V
Rassf1 F: 5V-AGGTTGAGATGTTTTTGAGATG-3V 326 59
R: 5V-TCCTCCTAACTACAATAACCACTAC-3V
Nore1A F: 5V-AGGGTTGGAGATAGAGGTAGAAG-3V 246 60
R: 5V-ACAACAACTCCAAAACCTAACC-3V
Primers used for bisulfite genomic sequencing of the CpG islands of the rat p16(CDKN2A), APC, H-cadherin (CDH13), Rassf1, and Nore1A gene promoters.
Figure W1. Microdissection process in SCC mutated in Tp53 (codon 246). One section was stained with H&E, which was used for histologic evaluation and control;
another section was stained with methyl green (MG) for microdissection (MD).
Table W2. Primers Used for Mutational Analysis.
Gene Exon Sequence Product Size (bp) Annealing Temperature (jC)
Tp53 5 F: 5V-TCCGCTGACCTTTGATTCTT-3V 268 58
R: 5V-AGACCCTGGACAACCAGTTC-3V
6 F: 5V-CTCCCCGGCCTCTGACTTATT-3V 274 58
R: 5V-CCTGGCACACAGCTTCCTAC-3V
7 F: 5V-CTTGTGCTGTGCCTCCTCTT-3V 198 58
R: 5V-GCCTCCACCTTCTTTGTCCT-3V
8 F: 5V-CAAAGTCACCCCTTGCTCTC-3V 210 58
R: 5V-CATGCGCTCTGACGATAATG-3V
9 F: 5V-TTTGTCCAGCACTTCTGTCCT-3V 250 58
R: 5V-CGATGGACATCTGGTGGAGT-3V
K-ras 1 F: 5V-AGGCCTGCTGAAATGACTG-3V 177 59
R: 5V-AGGATGACTGCCACCCTTTA-3V
2 F: 5V-TCTCAGGACTCCTACAGGAAAC-3V 267 59
R: 5V-GCAGGCCTAACAACTAGCAAA-3V
N-ras 1 F: 5V-GGTCTGCGGAGTTTGAGATT-3V 125 57
R: 5V-CATCCACAAAGTGGTTCTGG-3V
2 F: 5V-CCGAAAACAAGTGGTGATTG-3V 125 57
R: 5V-ACACACAGAGGAACCCTTCG-3V
c-H-ras 1 F: 5V-GTTTGGCAACCCCTGTAGAA-3V 193 59
R: 5V-TGGGACTCTAACCCATGACC-3V
2 F: 5V-AGGGTAGGCGGATTCTCTGT-3V 217 59
R: 5V-AGGACTTGGTGTTGTTGATGG-3V
EGFR 18 F: 5V-GCCCACTCTTGCACTGAATAA-3V 251 58
R: 5V-TCCCAGAAGCCTAGTCCAGA-3V
19 F: 5V-TAATGCAGAGCCCTTGAGGAT-3V 249 58
R: 5V-GGAAACCGTGGTTAGCAAGAC-3V
20 F: 5V-CCCATCAGCCAAGAAACAAT-3V 303 58
R: 5V-TCCTGCTTCTGAAACCTGCT-3V
21 F: 5V-CTGGATGGTTCACTCCCTCA-3V 245 58
R: 5V-TCTGGGCTGTCAGGAAAATG-3V
Primers used for mutational analysis by genomic sequencing of the rat p53 (Tp53), K-ras, N-ras, c-H-ras, and EGFR genes.
Figure W2. Immunohistochemical expression of iNOS in macrophages of silica-treated lungs. iNOS was observed in alveolar and parenchymal macrophages of
silicotic rats at early stages (A). iNOS expression at the core of granulomas was observed in all the stages of the model (B: month 4; C: month 12; D: month 21).
Counterstaining by Harris hematoxylin. Original magnifications, 140 (D); 280 (B and C); and 420 (A).
Figure W3. EGFR expression by immunohistochemistry in silica-induced tumors. Strong or moderate staining of EGFR was found in 55% of AC (A and B) and in
14% of SCC. Only 8% of AC (C) and 29% of SCC were negative. EGFR was localized in the cell membrane and cytoplasm of tumoral cells (arrow; inset, A).
Counterstaining by Harris hematoxylin. Original magnifications, 140 (B); 420 (A and C); 920 (inset, A).
Figure W4. Direct bisulfite sequencing analyses of the promoter region of p16 revealed a strong correlation between the status of p16 promoter hypermethylation
and protein expression in tumors measured by immunohistochemistry. Promoter hypermethylation– positive tumors showed a good association with the loss of
nuclear p16 protein expression (tumor 3). This association was also observed in cases without p16 promoter hypermethylation that showed high p16 nuclear
protein expression (tumor 7).
Figure W5. Bisulfite genomic sequencing of the Rassf1 and Nore1A promoters in nine representative silica-induced lung tumors and six normal tissues. Vertical
bars represent the distribution of the CpG islands at the Rassf1 and Nore1A CpG islands. The vertical arrow indicates the transcriptional start point. Black dots
indicate methylated CpG islands; white dots indicate unmethylated CpG islands. The position of the bisulfite sequencing primers used is represented with white
horizontal arrows. We did not observe significant Ras effectors promoter hypermethylation in any of the tumors analyzed.