The International Journal of Biochemistry & Cell Biology 43 (2011) 10301044

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The International Journal of Biochemistry
& Cell Biology
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Mechanisms involved in lung cancer development in COPD

Gaetano Caramori
a,c,
, Paolo Casolari
a
, Giorgio Narciso Cavallesco
b
, Sarah Giuffr
a
,
Ian Adcock
c
, Alberto Papi
a
a
Centro di Ricerca su Asma e BPCO, Universit di Ferrara, Ferrara, Italy
b
Modulo di Chirurgia Toracica, Universit di Ferrara, Ferrara, Italy
c
Airways Disease Section, National Heart and Lung Institute, Imperial College London, London, UK
a r t i c l e i n f o
Article history:
Available online 14 October 2010
Keywords:
COPD
Airway inammation
Lung cancer
p21
Squamous cell carcinoma
a b s t r a c t
Lung cancer and chronic obstructive pulmonary disease (COPD) are leading causes of morbidity and
mortality worldwide. They share a common environmental risk factor in cigarette smoke exposure and
a genetic predisposition represented by the incidence of these diseases in only a fraction of smokers.
COPD is also a major independent risk factor for lung carcinoma, among long-term smokers. Smokers
with COPD also have a higher risk of developing a specic histological subtype of non-small cell lung
cancer termed squamous cell carcinoma. For these reasons the focus of this review is on the potential
pathogenic molecular links between tobacco smoking-related COPD and squamous cell carcinoma. We
believe that we needtopromote more studies onthe molecular andcellular pathobiologyof smokers with
premalignant bronchial lesions of the squamous cell lung carcinoma compared with a control group of
smokers with and without COPDto unravel the complex molecular interactions between COPDand early
squamous cell lung carcinoma. These studies shouldalso look at younger healthy smokers incombination
withriskmodels of lungcancer andCOPD. Overall these studies mayallowthe discoveryof newmolecular
targets of the early carcinogenesis process that in the foreseeable future may render the early diagnosis
and treatment, and may be even the prevention, of invasive squamous cell lung carcinoma a reality.
2010 Elsevier Ltd. All rights reserved.
1. Introduction
Lung cancer and chronic obstructive pulmonary disease (COPD)
are leading causes of morbidity and mortality worldwide. They
share a commonenvironmental riskfactor incigarette smoke expo-
sure and a genetic predisposition represented by the incidence
of these diseases in only a fraction of smokers (Punturieri et al.,
2009). BothCOPDandlungcancer incidencearegrowinginwomen.
Although some of this trend can be attributed to changing smoking
habits, there is emerging evidence that women may be biologically
more susceptible to the harmful effects of cigarette smoke than are
men. A recent National Heart, Lung, and Blood Institute (NHLBI)
and National Cancer Institute (NCI) workshop has suggested to fos-
ter the research on the pathogenetic links between COPD and lung
cancer (Punturieri et al., 2009).

Supported by Associazione per la Ricerca e la Cura dellAsma (ARCA, Padova,

Italy), Fondo per Ricerca Scientica di Interesse Locale 20072009 of the University
of Ferrara (ex60%), Chiesi Farmaceutici (Italy), GlaxoSmithKline (UK), Novartis (UK)
and Pzer (UK).

Corresponding author at: Centro di Ricerca su Asma e BPCO, Universit di Fer-

rara, Via Savonarola 9, 44121 Ferrara, Italy. Tel.: +39 0532 236674;
fax: +39 0532 210297.
E-mail addresses: gaetano.caramori@unife.it, crm@unife.it (G. Caramori).
Worldwide lung cancer is the leading cause of deathfromcancer
both in men and women, and tobacco smoking is associated with
more of 90% of cases of lung cancer (Jemal et al., 2009; Parkin et
al., 2005; Stellman et al., 2001). Lung cancer accounts for 12% of all
cancers diagnosed worldwide, making it the most common malig-
nancy, other than non melanoma skin cancer (Abidoye et al., 2007).
In 2002, it has been estimated that 1.35 million people throughout
the world were diagnosed with lung cancer, and 1.18 million died
of lung cancer per year, more than for any other type of cancer. In
more developedcountries, incidence andmortality rates are gener-
ally declining among males and are starting to plateau for females,
reecting previous trends in smoking prevalence. In contrast, there
are some populations in emerging (such as China) and less devel-
oped countries where increasing lung cancer rates are predicted to
continue, due to endemic use of tobacco (Youlden et al., 2008).
Fifteenpercent of lifetime smokers developlung cancer, but 10%
of lung cancers occur in never-smokers (where represents the sev-
enth most common cause of cancer worldwide). The majority of
lung cancers among non-smokers occur in women (Egleston et al.,
2009). Interestingly, overall 10% of men and 20% of women, with
newly diagnosed non-small cell lung cancer (NSCLC), are never
smokers, and an overwhelming proportion of these have adeno-
carcinoma (Scagliotti et al., 2009). This proportion rises in Asiatic
women (Scagliotti et al., 2009).
1357-2725/$ see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biocel.2010.08.022
G. Caramori et al. / The International Journal of Biochemistry & Cell Biology 43 (2011) 10301044 1031
Cigarette smoking is also the most important risk factor for
chronic obstructive pulmonary disease (COPD) (GOLD, 2009). COPD
is a preventable and treatable disease with some signicant extra-
pulmonary effects that may contribute to the severity in individual
patients. Its pulmonary component is characterised by airow
limitation that is not fully reversible. The airow limitation is usu-
ally progressive and associated with an abnormal inammatory
response of the lung to noxious particles or gases (GOLD, 2009).
COPD prevalence, morbidity and mortality vary across countries
but in general are directly related to the prevalence of tobacco
smoking. However, air pollution resulting from the burning of
biomass fuels has also been identied as a COPDrisk factor in many
countries (GOLD, 2009).
COPD is currently a leading cause of morbidity and mortality
worldwide. The prevalence and burden of COPD are projected to
increase in the coming decades due to continued exposure to COPD
risk factors and the changing age structure of the world popula-
tion (GOLD, 2009; Lopez et al., 2006). Female smokers appear to be
more susceptible than male smokers to developing COPD(Sin et al.,
2007). Smoking-related lung diseases such as COPD and lung can-
cer are growing epidemics inwomenworldwide (Ben-ZakenCohen
et al., 2007). An animal model that displays all of the morphologic
and functional aspects of COPD has yet to be established, although
cigarette smoke-induced lung injury has been suggested to be the
closest. In this regard, it is interesting to note that exposure of mice
to cigarette smoke for up to 22 weeks resulted in emphysematous-
like changes that occurred earlier in females than in males (Card
and Zeldin, 2009).
Interestingly, COPD which is characterised by a chronic inam-
mation of the lower airway, is also a major independent risk factor
for lung carcinoma, among long-term smokers. In fact, the pres-
ence of COPD increases the risk of lung cancer up to 4.5-fold
(Islam and Schottenfeld, 1994; Mannino et al., 2003; Punturieri
et al., 2009; Purdue et al., 2007; Skillrud et al., 1986; Tockman
et al., 1987) and even more so in 1-antrypsin deciency car-
riers (Yang et al., 2008). Even a relatively modest reduction in
forced expiratory volume in one second (FEV
1
), a marker of air-
owobstruction, is a signicant predictor of lung cancer, especially
among women (Wasswa-Kintu et al., 2005). Interestingly lung can-
cer risk increases signicantly for white women with a history of
COPD, but not inAfricanAmericanwomen(Schwartzet al., 2009). In
addition, repeatedcigarette smoke exposure inmice promotes lung
tumour development following induction of airway inammation
(Takahashi et al., 2010).
Lung cancer is also a leading cause of morbidity and mortality in
patients with COPD (Anthonisen et al., 2005). This evidence clearly
emerges from a large longitudinal study of asymptomatic smokers
withmild-to-moderateCOPD, whereafter afollow-upof 14.5years,
33% of these subjects died of lung cancer (Anthonisen et al., 2005).
Furthermore 5070% of patients diagnosed with lung cancer have
spirometric evidence of COPD (Yao and Rahman, 2009). Smokers
with COPDalso have a higher risk of developing a specic histolog-
ical subtype of NSCLC termed squamous cell carcinoma (Malhotra
et al., 2006; Papi et al., 2004; Purdue et al., 2007). NSCLC accounts
for 85% of all lung cancer cases in the United States (Molina et al.,
2008) and importantly, squamous cell carcinoma still represents
the most common histological subtype of lung cancer in European
men (Janssen-Heijnen and Coebergh, 2001).
When feasible, surgical resection remains the single most con-
sistent and successful option for the treatment of NSCLC. However,
close to 70% of patients with lung cancer present with locally
advanced or metastatic disease at the time of diagnosis (Molina
et al., 2008). Despite signicant advances in diagnostic techniques
and understanding of the molecular biology, even the most recent
therapeutic innovations for NSCLC have yielded little improve-
ment in prognosis with overall 5-year survival rates still averaging
around 15%in most countries: being <14%among males and <18%
among females (Carney, 2002; Parkin et al., 2005; Youlden et al.,
2008).
Lung cancer and COPD therefore share a common risk
factortobacco smokingthrough which they may also share
similar pathogenic mechanisms. However, urbanandhouse air pol-
lution is also recognized as a strong risk factor for both COPD and
lung cancer (Zhang and Smith, 2007) and self-reported physician-
diagnosedpulmonary emphysema, whichis oftenpresent insevere
COPD, represents another risk factor for lung cancer, even in life-
long non-smokers (Turner et al., 2007). Whether women are more
likely to develop both COPD and lung cancer remains an open
question, but developing a better understanding of how sex and
hormonal differences inuence disease development and progres-
sion can hopefully lead to improved interventions and outcomes
for our patients (Mannino, 2007).
Since (a) more than 90% of all cases of COPDand lung cancer are
tobaccosmoking-relatedand(b) onlyaproportionof lifelongsmok-
ers will develop COPD and/or squamous cell carcinoma we focus
our review on the potential pathogenic molecular links between
tobacco smoking-related COPD and squamous cell carcinoma.
2. Field carcinogenesis in the bronchial/bronchiolar
epithelium and preneoplastic lesions of the squamous cell
lung carcinoma in the COPD patients
Astepwise progression of smoking-associated pathological pre-
malignant changes (from basal cell hyperplasia to dysplasia and
nally to in situ squamous cell carcinoma) was described in the
normal bronchial epitheliummore than three decades ago and this
occurs before the development of an invasive squamous cell lung
carcinoma (Auerbach et al., 1979). In one study, the entire tracheo-
bronchial tree of a subject with widespread dysplastic changes
in the bronchial epithelium but no overt lung carcinoma was
examined. The deoxyribonucleic acid (DNA) extracted from the
microdissected bronchial epithelial cells was analysed for point
mutations in the protein TP53, a tumour suppressor gene (see
below), showing the widespread presence of a single somatic TP53
point mutation in the bronchi suggesting that a single progenitor
bronchial epithelial clone may have expanded to populate broad
areas of the bronchial mucosa. This concept is known as eld car-
cinogenesis (Franklin et al., 1997).
Although there is a eld effect phenomenon for preneoplastic
lung lesions, recent data suggest that there are at least two dis-
tinct lung airway compartments (central and peripheral) involved
in lung cancer pathogenesis. Lower airways inammation may also
play an important role in lung cancer development and could be
an important component of the eld effect phenomenon (Wistuba,
2007).
It is conceivable that such a stepwise progression of the process
of carcinogenesis couldbefacilitatedinsmokers whodevelopCOPD
because of their impaired clearance of carcinogenic substances
resulting fromchronic airowobstruction. Indeed increased depo-
sition of particulate matter in the major bronchi has recently been
described in a computer model of the lungs of patients with COPD
(Segal et al., 2002).
However, on the basis of the recent novel molecular ndings
in this eld, we believe that the explanation of the link between
COPD and squamous cell lung carcinoma is probably more com-
plex than a simple mechanistic one. In fact there are also many
possible molecular links between tobacco-smoking related COPD
and squamous cell lung carcinoma. Unfortunately, the precise tim-
ing of these alterations during the sequential phases of squamous
cell lung carcinoma development inCOPDpatients is still unknown.
1032 G. Caramori et al. / The International Journal of Biochemistry & Cell Biology 43 (2011) 10301044
Fig. 1. In mouse models of lung carcinogenesis there is a niche of stem cells localized at the bronchioloalveolar junction which may be involved in the development of both
adenocarcinoma and squamous cell lung carcinomas. These proliferating stem cells in the human small airways are Ki67+, a marker of cell proliferation, as indicated by the
closed arrow in the inset (immunoperoxidase staining with brown color, personal observation of the authors, 400 magnication).
3. Pulmonary stem cells as a target of the carcinogenic
process in human squamous cell lung carcinoma
Despite current data supporting a direct relationship between
proximal airway basal progenitors and cells associated with
carcinogenesis in murine models, the precise stem cell tar-
get of this process in the development of human squa-
mous cell lung carcinoma remains unknown (Giangreco et al.,
2007).
In murine models a pulmonary stem cell population is found
at the bronchioalveolar duct junction. These cells, termed bron-
chioalveolar stem cells (BASCs), are resistant to bronchiolar and
alveolar damage and proliferate during epithelial cell renewal in
vivo and their transformed counterparts give rise to adenocarci-
noma (Dovey et al., 2008; Kim, 2007; Kim et al., 2005). BASCs
can be functionally dened in vivo on the basis of their resistance
to chemical (naphthalene) injury, their infrequent proliferation
relative to other progenitor cell types, and their co-expression
of the bronchiolar and alveolar secretory cell markers Clara
cell secretory protein and pro-surfactant protein C, respectively.
Their immunophenotype is CD45(neg), CD31(neg), CD34(neg), Sca-
l(low), autouorescence (low) (Teisanu et al., 2009). Interestingly,
theproteinkinaseCiota(PKCiota) is anoncogenerequiredfor main-
tenance of the transformed phenotype of NSCLC cells (Regala et al.,
2009). A mouse model in which oncogenic Kras (G12D) is activated
by Cre-mediated recombination in the lung with or without simul-
taneous genetic loss of the mouse PKCiota gene, Prkci, has recently
been established. Genetic loss of Prkci dramatically inhibits Kras-
initiated hyperplasia and subsequent lung tumour formation in
vivo. This effect correlates with a defect in the ability of Prkci-
decient bronchioalveolar stem cells to undergo Kras-mediated
expansion and morphologic transformation in vitro and in vivo
(Regala et al., 2009), suggesting that both squamous cell lung carci-
noma and lung adenocarcinoma may originate fromthe same stem
cells (Fig. 1). This is an area which clearly requires more human
translational research.
4. Genetic and epigenetic abnormalities in the human
squamous cell lung carcinoma and in the
bronchial/bronchiolar epithelium from COPD patients
Carcinogenesis is a complex process characterised by the
accumulation of multiple independent genetic alterations, often
involving overexpressionof oncogenes andloss of tumour suppres-
sor genes. These genetic alterations disrupt the normal regulation
of cell signalling pathways, essential for the control of cell growth,
differentiation and apoptosis (Ocak et al., 2009).
Molecular genetic studies of squamous cell lung carcinoma
have revealed that clinically evident squamous cell lung cancers
have multiple genetic and epigenetic abnormalities, including DNA
sequencealterations, copynumber changes, andaberrant promoter
hypermethylation (Sato et al., 2007). Together, these abnormal-
ities result in the activation of oncogenes and inactivation of
tumour-suppressor genes (Sato et al., 2007). In many cases these
abnormalities can also be found in premalignant lesions and in his-
tologically normal lung bronchial epithelial cells as well (Sato et al.,
2007).
In the multistage development of lung squamous cell carci-
noma there is an early, multiple, and sequentially occurring loss
of heterozygosity [LOH (i.e., deletion of one copy of allelic DNA
sequences)] and microsatellite alterations in widely dispersed,
apparently clonally independent foci (of approximately 90.000
cells) in normal and hyperplastic bronchial epithelium (Park et al.,
1999; Wistuba et al., 1999, 2000). LOH on chromosomes 3p and
9p is more frequent than LOH on chromosomes 5q, 17p (17p13;
TP53 gene), and 13q (13q14; retinoblastoma gene) (Wistuba et al.,
1997). Importantly, in comparison to small cell lung cancer (SCLC),
patients with squamous cell carcinoma have less allelic loss and
microsatellite alterations in their preneoplastic bronchial epithe-
lium (Wistuba et al., 1997). These changes may persist for many
years after smoking cessation (Franklin et al., 1997; Wistuba et al.,
1997; Yashima et al., 1997). More research relating to both DNA
and histone modications is required in this area.
G. Caramori et al. / The International Journal of Biochemistry & Cell Biology 43 (2011) 10301044 1033
Table 1
Genes potentially involved in lung carcinogenesis and COPD pathogenesis
a
.
- CHRNA3
- CHRNA5
- CHRNB4
- TP53
- p21
WAP/CIP1
- RB1
a
It is still unknown if there is an increased risk for the squamous cell carcinoma
histological subtype.
5. Genes and inherited susceptibility to human squamous
cell lung carcinoma and COPD
Both lung cancer and COPD full the criteria for a complex
genetic disease in which environmental factors (mainly tobacco
smoking) interact with multiple polymorphic genes to inuence
susceptibility to the diseases. Only a fraction of smokers (around
15%) will develop lung cancer and/or COPDin their lifetime (GOLD,
2009; Spitz et al., 2003) which suggests a different individual sus-
ceptibility to the risk of lung cancer and/or COPDor time of disease
onset.
There is evidence of familial aggregation [relative risk
(RR) =1.71)] of lung cancer among relatives of late-onset lung can-
cer cases, and even after adjustment for smoking behaviour of
probands and their relatives, the risk of lung cancer (RR=1.33) is
still statistically signicant. There is no evidence of familial aggre-
gation of lung cancer among young onset (55 years of age) lung
cancer cases or among relatives of never-smokers (Etzel et al.,
2003). In a more recent study, smokers with a family history of
early-onset lung cancer in a rst-degree relative had a higher risk
of developing lung cancer with increasing age than smokers with-
out a family history. An increase in risk occurs after age 60 years
in these individuals, with a mean of 17.1% of white case relatives
and 25.1% of black case relatives diagnosed with lung cancer by age
70 years. Relatives of black cases were at a statistically signicant
increasedriskof lungcancer comparedwithrelatives of white cases
(odds ratio, 2.07) (Cot et al., 2005).
Familial clustering of COPD has been known for many years,
particularly in subjects with a severe genetic deciency of 1 antit-
rypsin, who usually also develop severe pulmonary emphysema
(GOLD, 2009). However, less of 1% of COPD patients have this de-
ciency and more recent studies have also demonstrated familial
aggregationinsomesubjects withCOPD, evenintheabsenceof pul-
monary emphysema (Molno and Coyle, 2008; Patel et al., 2008).
Lung cancer is mainly attributable to tobacco use, and few
large families with multiple cases of lung cancer are suitable for
linkage analysis (Foulkes, 2008). Nevertheless, one locus on chro-
mosome 6q23-25 has been suggested by a traditional linkage study
(Bailey-Wilson et al., 2004) although no specic gene has yet been
identied (Foulkes, 2008). The same region is also involved in the
genetic susceptibility to COPD (Schwartz and Ruckdeschel, 2006).
Recent studies have discovered several genes (Table 1 and text
below) that are associated with substantial increases in the risk of
both lung cancer and COPD. In persons carrying mutations in these
genes, tobacco smoking may be particularly dangerous.
6. Genetic abnormalities in the carcinogen-metabolizing
enzymes and the pathogenesis of human squamous cell
lung carcinoma and COPD
Human lung is a major target organ for all inhaled carcino-
gens contained in the tobacco smoking. Many of these compounds
require enzymatic activation to exert their deleterious effects on
pulmonary cells (Zhang et al., 2006). Although metabolism of for-
eign substances is usually benecial in eliminating potential toxins
from the body, in some instances the metabolic process can trans-
form harmless substances into toxic chemicals through a process
called metabolic bioactivation (Ben-Zaken Cohen et al., 2007).
Individual differences in in situ activation and inactivation of xeno-
biotics may contribute to the risk of developing both COPD and
squamous cell lung carcinoma associated with these compounds
(Gresner et al., 2007; Rotunno et al., 2009).
The major xenobiotic metabolizing enzymes, including both
phase I [cytochromes P (CYPs), microsomal epoxide hydrolases
(EPHX), avin monooxygenases (such as heme oxygenase-1;
HMOX1) and myeloperoxidase (MPO)] and phase II enzymes
[conjugation enzymes, including several transferases (such as glu-
tathione S-transferases (GSTs) and arylamine N-acetyltransferases
(CoASAc; NAT, EC 2.3.1.5)] are expressed in human lung tissues
(Zhang et al., 2006). Xenobiotics undergo metabolic activation by
phase I enzymes whereas phase II enzymes transform compounds
activated by phase I enzymes into inactivated hydrophilic com-
pounds that are eventually excreted (Arif et al., 2008).
The combination of several genetic polymorphisms in these
enzymes that activate or detoxify the tobacco smoke carcinogens,
such as CYPs, EPHX, HMOX1 and GSTs, might modulate the risk
of chronic smokers of developing both COPD and squamous cell
lung carcinoma (Caramori and Adcock, 2006). There are at least 57
different CYP genes and 24 pseudogenes, which can be further sub-
divided into 17 different families. The main CYP responsible for the
rst phase reaction is CYP2A6, which belongs to the CYP2 subfam-
ily. The human CYP2 family is a heterogeneous group of enzymes.
It contains the subfamilies CYP2A, CYP2B, CYP2C, CYP2D, CYP2E,
CYP2F, and CYP2J. CYP2B6, CYP2D6, CYP2E1, CYP2F1, and CYP2J2
are the only functional members in their respective subfamilies
(Ben-Zaken Cohen et al., 2007).
One important xenobiotic substrate for CYP enzymes in
cigarettesmokeis polycyclic aromatic hydrocarbon(PAH), whichin
its native formis relatively harmless in small doses but upon bioac-
tivation by CYP enzymes, can become very toxic towards the lungs.
Animal studies have raised the possibility that there may be impor-
tant sex-relateddifferences inthe metabolism(mediatedlargely by
CYP enzymes) of some constituents of cigarette smoke, leading to
increased production of carcinogenic and airway-toxic molecules
in females (Ben-Zaken Cohen et al., 2007). In fact, estrogen and
related compounds may up-regulate the expression of cytochrome
P450 enzymes in lungs (Ben-Zaken Cohen et al., 2007).
Thehomozygous *2Aalleleof CYP1A1is anindependent riskfac-
tor for very severe COPD (Cheng et al., 2009). In contrast, CYP1A1
m1homozygous genotype is a risk factor for lung cancer (Dialyna et
al., 2003). However, the CYP1B1*3 and CYP1B1*4 genotypes do not
associate with the risk of lung cancer and/or COPD (Kaur-Knudsen
et al., 2009). CYP2E1 gene polymorphism are associated with an
increased risk of lung cancer (Wu et al., 1998) and COPD patients
have an higher frequency of the 1053T and 1293C alleles in the
promoter of the CYP2E1 gene which are associated with higher
transcription, increased protein levels and increased enzymatic
activity (Arif et al., 2008).
Two haplotypes in the EPHX1 gene are signicantly associ-
ated with lung cancer risk in the overall population (Rotunno et
al., 2009). In a recent meta-analysis of the literature (Hu et al.,
2008a), the EPHX1 139 heterozygote was found to protect against
the development of COPD in Asian populations, but not in Cau-
casians. The other gene types of EPHX1 113 and EPHX1 139 are not
associated with an increased risk of COPD. The slow activity phe-
notype of EPHX1 is also associated with an increased risk of COPD.
In contrast, the fast activity phenotype of EPHX1 is a protective fac-
tor for developing COPD in Asian, but not Caucasian, populations.
Finally, the very slow activity phenotype of EPHX1 is a risk factor
for developing COPD in the Caucasian population, but not in the
Asian population (Hu et al., 2008a).
1034 G. Caramori et al. / The International Journal of Biochemistry & Cell Biology 43 (2011) 10301044
Polymorphisms of HOX-1 gene promoter can be grouped into
three classes: small (S) [25 (GT)(n) repeats], M (26-31 repeats),
and L (32 repeats). The class L promoter may be associated with
the development of lung adenocarcinoma in Japanese male smok-
ers (Kikuchi et al., 2005). The allele frequencies of class L and the
genotypic frequencies of the group with L are also signicantly
higher in Chinese COPD patients than in controls (Fu et al., 2007).
MPOis a lysosomal phase I enzyme expressed predominantly in
neutrophil granulocytes, monocytes and macrophages. It converts
a benzo(a)pyrene into highly reactive and carcinogenic metabo-
lite benzo(a)pyrene 7,8-diol-9,10 epoxide (BPDE). It also activates
tobacco smoke-derived carcinogens such as PAHs, aromatic and
heterocyclic amines and induces the formation of endogenous
carcinogenic free radicals (Gresner et al., 2007). The majority of
literature available nowadays claim a protective effect of the MPO
variant allele (i.e. A/A and A/Ggenotypes) against lung cancer, with
the risk reduction of approximately 50% (Gresner et al., 2007).
Smoking status is believed to modulate the link between MPO
polymorphism and lung cancer. The possession of at least one
variant A allele was shown to be associated with a 46%, 58% and
70% reduction in risk of squamous cell carcinoma among non-
smokers, light- and heavy-smokers, respectively (Gresner et al.,
2007). Nitrosativestress, mainlymediatedbyMPOandneutrophilic
inammation, may also contribute to the pathogenesis of severe
COPD (Ricciardolo et al., 2005).
Polymorphisms of the GSTs genes are associated with the risk of
lung cancer and interestingly are more frequent in SCLC and in the
squamous cell carcinoma compared with the lung adenocarcinoma
(Gresner et al., 2007).
The Val allele of GSTP1 may have a protective effect for devel-
opment of COPD, whereas smokers with the GSTP1 Ile allele have
an increased risk for the development of COPD (Calikoglu et al.,
2006). GSTP1 Ile/Val and Val/Val genotypes are associated with a
decreased risk of COPD when compared to the Ile/Ile genotype.
Among the combinations of the genotypes, the combination of
GSTM1, GSTT1 null, and GSTP1 Val/Val is associated with the max-
imal increased risk (12-fold) of COPD (Calikoglu et al., 2006).
The 481C/T, 590G/A, 803A/Gand 857G/Aare the functional vari-
ations of exon 2 of the NAT2 gene that lead to the amino acid
changes like Ile114Thr, Gly268Arg, Arg197Gln and Lys268Arg (Arif
et al., 2008). Homozygous alleles at all 4 locus without any varia-
tions are known as the fast acetylators and are represented as the
NAT24 allele, whereas, variations categorized as slow acetylators
are represented as NAT25, NAT26 and NAT27 alleles depending
upon the variants. There is a higher frequency of slow acety-
lator alleles (may detoxify fewer xenobiotics) in COPD patients
compared to controls (Arif et al., 2008). Evidence for a putative
association of NAT1 polymorphism and lung cancer, has also been
reported (Agndez, 2008).
Recent molecular studies investigating gene expression in the
bronchial epithelial cells of a group of control smokers with normal
lung function compared with smokers with COPD using microar-
rays (Pierrou et al., 2007) are very useful and are providing novel
clues on the possible molecular links between COPD and squa-
mous cell carcinoma of the lung. Among the others, the protein
aldo-keto reductase family 1 member B10 (AKR1B10) seems of
particular interest since it is overexpressed in the bronchial epithe-
lium of COPD patients and in the neoplastic cells of squamous
cell carcinoma of the lung (Fukumoto et al., 2005; Pierrou et al.,
2007) suggesting its involvement in the multistep carcinogene-
sis of squamous cell carcinoma of the lung. Aldo-keto reductases
are NADPH-dependent oxidoreductases that catalyze the reduc-
tion of a variety of carbonyl compounds. AKR1B10 is a member of
this superfamily and reduces aromatic and aliphatic aldehyde sub-
strates and has putative physiologic roles in steroid metabolism
or detoxication of reactive aldehydes (Fukumoto et al., 2005).
Data suggests that AKR1B10 translocates to the nucleus during the
cell cycle and is involved in cell cycle regulation by an unknown
mechanism. Another possibility is that AKR1B10promotes carcino-
genesis of squamous cell lung carcinoma through its enzymatic
activity that counteracts the conversion of -carotene to retinoic
acid (Fukumoto et al., 2005).
The transcription factor nuclear factor E2-related factor 2 (Nrf2)
increases the expression of several protective phase-2 detoxifying
enzymes [such as such as GSTs and NAD(P)H:quinone oxidore-
ductase (NQO1)] and antioxidant enzymes such as peroxiredoxins
(Shibata et al., 2008). Kelch-like ECH-associated protein 1 (KEAP1)
negatively regulates Nrf2 activity by targeting it to proteasomal
degradation (Singh et al., 2006) and under normal conditions, low
cellular concentrations of Nrf2 are maintained by proteasomal
degradation through a Keap1-dependent mechanismin which two
amino-terminal motifs, DLG and ETGE, promote efcient ubiquiti-
nation and rapid turnover (Shibata et al., 2008).
Common somatic mutations in the coding region of the Nrf2
gene, altering amino acids in the DLG or ETGE motifs, resulting in
increased cellular accumulation of Nrf2 are associated with poor
prognosis insquamous cell lungcarcinoma. Thesemutant Nrf2cells
have constitutive induction of cytoprotective enzymes and drug
efux pumps, which are insensitive to Keap1-mediated inactiva-
tion (Shibata et al., 2008). It is interesting to note that mutations
resulting with the loss of Keap1 function (also causing greater
nuclear accumulation of Nrf2) are more common in lung adeno-
carcinomas (Ohta et al., 2008; Padmanabhan et al., 2006; Singh
et al., 2006). Interestingly, disruption of the Nrf2 gene in mice
enhances oxidative damage and inammation in lungs after expo-
sure to cigarette smoke (Rangasamy et al., 2004) and a signicant
decrease of the Nrf2 protein level as been described in the COPD
lungs (Goven et al., 2008; Malhotra et al., 2008; Suzuki et al., 2008)
suggesting that different changes in the Nrf2 pathway are involved
in the development of COPD and lung cancer.
7. Genetic abnormalities in the genes with an impact on
smoking behaviour and the carcinogenic process of the
human squamous cell lung carcinoma and COPD
susceptibility
Recent studies have found two single-nucleotide polymor-
phisms (SNPs) signicantly associated with lung cancer risk, both
of them located in a region of the long arm of the chromosome 15
(15q25.1) which contains a cluster of 6 genes (a) nicotinic acetyl-
choline receptor alpha subunits 3 (CHRNA3) and 5 (CHRNA5), and
the 4 nicotinic acetylcholine receptor (nAChR) subunit (CHRNB4),
(b) proteasome alpha 4 subunit isoform 1 (PMSA4) (c) the IREB2
iron-sensing response element, and d) LOC123688, a gene of
unknown function (Amos et al., 2008; Falvella et al., 2009; Hung
et al., 2008; Thorgeirsson et al., 2008). This region is hypothesized
toaccount for 14%of theattributableriskof lungcancer cases (Hung
et al., 2008). A non-synonymous variant (rs16969968 in exon 5) of
CHRNA5 that induces an amino acid substitution (D398N) in the
protein is among the markers with the strongest disease associ-
ations. This SNP substitutes aspartic acid (D) for asparagine (N)
at amino acid position 398 (D398N) in CHRNA5 protein, which is
located in the highly conserved site in the central part of the sec-
ond intracellular loop. The function of the second intracellular loop
and the possible biological consequences of the D398N alteration
remain to be elucidated, but its conservation across species, sug-
gests that it could have functional importance (Hung et al., 2008).
CHRNA3 contained a synonymous variant in exon 5 (rs1051730)
that is also strongly associated with disease. In another study the
risk of lung cancer was more than 5-fold higher among those sub-
jects who had both a family history of lung cancer and two copies
G. Caramori et al. / The International Journal of Biochemistry & Cell Biology 43 (2011) 10301044 1035
of high-risk alleles rs8034191 (odds ratio [OR] =7.20) or rs1051730
(OR=5.67), both of which were located in the 15q24-25.1 locus,
than among control subjects (Liu et al., 2008).
These SNPs inthe 15q24-25.1 locus are extremely rare inAsians,
and the three risk SNPs reported in Caucasians are not associ-
ated with lung cancer risk in Chinese (Wu et al., 2009). However,
other SNPs (rs2036534C>T, rs667282C>T, rs12910984G>A, and
rs6495309T>C) are associated with signicantly increased lung
cancer risk and smoking behaviour. The rs6495309T>C change in
the CHRNA3 promoter is a functional variant because it affects the
Oct-1 binding ability, resulting in increased CHRNA3 expression
(Wu et al., 2009).
It is a matter of contention as to whether the effect of these
genes on the risk of lung cancer is independent of any effect
they may have on smoking behaviour leading to increased expo-
sure to tobacco carcinogens. Indeed, studies have subsequently
demonstrated that the non-synonymous CHRNA5 SNP rs16969968
is also the most signicant SNP associated with nicotine depen-
dence in both AfricanAmericans and in EuropeanAmericans.
Other SNPs that affect CHRNA5 mRNA levels are associated
with nicotine dependence in AfricanAmericans but not in
EuropeanAmericans. The CHRNA3 SNP rs578776, which has a low
correlation with rs16969968, is associated with nicotine depen-
dence in EuropeanAmericans but not in AfricanAmericans. Less
common SNPs (frequency 5%) are also associated with nicotine
dependence (Saccone et al., 2009).
In contrast, one of the 15q SNP (rs16969968) has a strong
association with the risk of lung cancer that is independent of
the association with smoking quantity (Lips et al., 2009) whereas
another study has concluded that the CHRNA5 SNP variants are
implicated both in smoking behaviour and more directly in lung
cancer risk (Spitz et al., 2008). This is an area of active research.
Normal human bronchial epithelium and squamous cell lung
cancer cell lines are able to produce acetylcholine (Racke and
Matthiesen, 2004; Song et al., 2003a,b) that in vitro has been shown
to promote the proliferation of neoplastic cells acting on nicotinic
acetylcholine receptors (nAChRs) (Song et al., 2003a,b) and the
proliferation of lung broblasts and myobroblasts acting on mus-
carinic acetylcholinereceptors (mAChRs) (Pieper et al., 2007). There
are multiple subtypes of nAChRs and mAChRs. Most small-cell lung
carcinoma (SCLC) and NSCLC express 7 nAChR, and heteromeric
3, 4, and5 containing nAChR(Song andSpindel, 2008b; Song et
al., 2008a; West et al., 2003) whereas SCLCcells express all vemus-
carinic receptor subtypes, squamous cell lung carcinomas express
only M
2
R, M
3
R, and M
4
R (Song and Spindel, 2008b; Song et al.,
2003a,b, 2008a).
In squamous cell lung carcinomas up-regulation of 5 and 3
nAChR subunit mRNAs, increased levels of ACh and choline acetyl-
transferase mRNA and decreased cholinesterase activity have been
reported (Song and Spindel, 2008b; Song et al., 2008a). Notably,
acetylcholinesterase activityinsquamous cell carcinoma is 80%less
than in its adjacent non-cancerous tissue (Martnez-Moreno et al.,
2006). Levels of nAChR expressed in lung cancers are changed by
smoking (Lam et al., 2007). Lynx1, an allosteric inhibitor of nAChR
activity, is also decreased in squamous cell lung carcinomas (Song
and Spindel, 2008b; Song et al., 2008a). In vitro stimulation of a
squamous cell lung carcinoma cell line with nicotine increases ACh
secretion, expression and activity of nAChR and stimulates prolif-
eration of these cells (Song and Spindel, 2008b; Song et al., 2008a).
In vitro, nicotine, acting on nicotinic receptors, may stim-
ulate mitogen-activated protein kinase (MAPK) (p44/42) and
Akt-dependent proliferation and NF-B-dependent survival of
squamous cell lung cancer cell lines conferring a survival advantage
to these cells (Carlisle et al., 2007; Tsurutani et al., 2005).
Nicotine also increases the expression of the peroxisome
proliferatoractivated receptor (PPAR) / protein acting through
the 7 nAChR and mediated by activation of the PI3K/Akt/mTOR
pathway and changes in Sp1 promoter binding (Sun et al., 2009).
Activation of PPAR / induces NSCLC cell growth (Han et al.,
2008). In addition nicotine can induce lung broblasts to produce
bronectin, a major component of the extracellular matrix (Roman
et al., 2004). Interestingly, in the same in vitro model an M3 mAChR
selectiveantagonist (darifenacin), prevents bothnicotinic andmus-
carinic receptor-inducedMAPKactivationandgrowthof squamous
cell lung cancer cell lines in vitro and in vivo (Song and Spindel,
2008b; Song et al., 2007; Song et al., 2008a) (Fig. 2).
Hypoxia-inducible factor (HIF)-1 is overexpressed in NSCLC,
andis closely associatedwithanadvancedtumour grade, increased
angiogenesis, and resistance to chemotherapy and radiotherapy. In
vitro, nicotine increases HIF-1 and vascular endothelial growth
factor (VEGF) expression in NSCLC (squamous cell carcinoma and
adenocarcinoma) cell lines. Functionally, nicotine potently stim-
ulated in vitro tumour angiogenesis by promoting tumour cell
migration and invasion (Zhang et al., 2007).
There are no studies on the potential effect of smoking or its
components (such as nicotine) on the release of acetylcholine from
the non-neuronal cholinergic system in the airways. Theoretically,
an increased release of acetylcholine in the lung of smokers, via an
autocrine or paracrine loop, may cause both small airway brosis
[a characteristic of the COPDlung (GOLD, 2009)], thus contributing
to the development of COPD, and enhance cholinergic signalling on
bronchial/bronchiolar epitheliumthus contributing to the carcino-
genesis.
Thus chronic nicotine inhalation and/or endogenous acetyl-
choline released locally in the lung might both play a role in both
COPDand squamous cell lung carcinoma development in long term
smokers.
8. Genes involved in DNA damage/repair mechanisms and
the carcinogenic process of the human squamous cell lung
carcinoma and COPD susceptibility
Tobaccosmoking-relatedCOPDis alsoassociatedwithincreased
oxidative stress inthe lower airways whichcancause deoxyribonu-
cleic acid (DNA) damage and carcinogenesis (Psarras et al., 2005).
Damage to DNA induces several cellular responses that enable cell
either to eliminate or cope with the damage or to activate a pro-
grammed cell death process, presumably to eliminate cells with
potentially catastrophic mutations (Sancar et al., 2004). DNArepair
mechanisms include direct repair, base excision repair, nucleotide
excision repair, double-strand break repair, and cross-link repair
(Sancar et al., 2004). A lack of DNA repair may be another common
mechanism linking both COPD and squamous cell lung carcinoma
(Caramori and Papi, 2007). For example one possibility is that her-
itable genetic polymorphisms inuence the efciency of both DNA
repair damage in the bronchial epithelium and connective tissue
damage repair. It is also likely that genetic factors modulating the
quality of DNA repair explain the absence of both COPD and squa-
mous cell lung cancer in most smokers (Brody and Spira, 2006).
Several polymorphisms in DNA repair genes have already been
reported to be associated with squamous cell lung carcinoma risk
(Popanda et al., 2004). Interestingly, women have a 1015% lower
capacity for repairing tobacco carcinogen-induced DNA damage
than men (Wei et al., 2000). DNA repair mechanisms also play
an important role in the epigenetic regulation of cell function by
mediating DNA demethylation (Gehring et al., 2009) and may, in
turn, be regulated by the epigenetic changes seen in lung cancer as
discussed below. Clearly due to the complexity and the interrela-
tionships of the many different pathways involved in the process
of DNA repair in the lung this area deserves to be fully explored in
large ad hoc studies.
1036 G. Caramori et al. / The International Journal of Biochemistry & Cell Biology 43 (2011) 10301044
Fig. 2. Scheme summarizing the potential role of acetylcholine released by the squamous cell lung cancer cells to promote their proliferation acting on muscarinic (mAChRs)
and nicotinic (nAChRs) acetylcholine receptors.
9. Genes involved in cell cycle regulation and apoptosis and
the carcinogenic process of the human squamous cell lung
carcinoma and COPD susceptibility
Although cigarette smoke may be directly mutagenic (Anderson
and Bozinovski, 2003), polymorphisms in the genes controlling
acquired somatic mutations may also contribute, at least to some
extent, to the observed differences in susceptibility to COPD and
squamous cell lung carcinoma. Alterations in cell cycle regulation
and apoptosis leading to malignant transformationcould be caused
by common genetic variants in tumour suppressor genes, such as
the protein TP53 (Tuder et al., 2008).
DNA damage triggers a danger response coordinated by the
ataxia telengectasia mutated gene (ATM) and TP53 proteins. This
physiological andtemporaryresponse, as seeninresponsetooxida-
tive stress, aims to lessen the cellular damage as cells go into a
waiting period for DNA repair, but can become permanent when
cells have dangerously shortened telomeres (the DNA-protective
TTAGGrepeats at the endof chromosomes). This ultimately leads to
cell senescence (replicative senescence), which is apoptosis resis-
tant and metabolically active but unable to proliferate beyond the
G1 stage of the cell cycle (Tuder et al., 2008).
Li-Fraumeni syndrome, a hereditary cancer syndrome, is char-
acterisedbythe earlyonset of various cancers andmultiple primary
tumours. Indeed, the name SBLA has been proposed for this con-
dition because it describes familial development of sarcoma (S),
breast and brain tumour (B), leukaemia, lung, and laryngeal can-
cer (L), and adrenal cortical carcinoma (A) (Hwang et al., 2003).
Studies of Li-Fraumeni syndrome indicate that 5070% of families
carry a germline TP53 mutation. In the families with germline TP53
mutations lung cancer accounts for only 4% of the tumours and it is
diagnosed at a relatively later age in TP53 mutation carriers com-
paredwithother cancers, includingbreast cancer andsarcoma. This
suggests that environmental factors might contribute to lung can-
cer risk in mutation carriers. In fact, cigarette smoking signicantly
increases lung cancer risk in carriers of a germline TP53 mutation
(Hwang et al., 2003). However, some TP53 SNPs have been associ-
atedwithanincreasedriskof developingCOPD(Arif et al., 2008; Lee
et al., 2006). Insmokers withCOPDthe presence of serumanti-TP53
antibodies may be surrogate markers for both TP53 alterations and
preclinical lung cancer (Trivers et al., 1996).
A critical downstream mediator of the protein TP53 is the
tumour suppressor gene p21
WAP/CIP1
, which is transcriptionally
activated by TP53 to induce cell cycle arrest and DNA repair. Cell
cycle progression to the S phase is regulated by complexes of
cyclins and cyclin-dependent kinases (CDKs). p21
WAP/CIP1
acting as
a central downstream target of TP53 activation inhibits the cyclin
E-CDK2 and cyclin D1-CDK4 complexes, and therefore can induce
G1 arrest and block entry into the S phase of the cell cycle. In
the cytoplasm, p21
WAP/CIP1
remains in quaternary complexes with
cyclin and Cdks. p21
WAP/CIP1
also interacts with proliferating cell
nuclear antigen (PCNA), an accessory protein of DNA polymerase,
and thereby inhibits DNAsynthesis (Tuder et al., 2008). p21
WAP/CIP1
is highlyresponsivetooxidativestress. Translocationof p21
WAP/CIP1
into the nucleus may occur through either a TP53-dependent or -
independent pathway and cytoplasmic p21
WAP/CIP1
itself acts as an
inhibitor of apoptosis (Tomita et al., 2002).
One p21
WAP/CIP1
SNPresults inanaminoacidchange incodon31
fromserine to arginine and is a moderate-risk allele for squamous-
cell carcinoma (Popanda et al., 2007). This polymorphismis located
in a highly conserved region of p21
WAP/CIP1
and is expected to affect
its molecular function. In a case control study involving 206 Tai-
wanese subjects with COPD and 210 healthy smokers as control
subjects Lee and colleagues (Lee et al., 2006) reported that the dis-
tribution frequencies of genotypes of p21
WAP/CIP1
codon 31 were
signicantly different between the COPD and the control groups.
Furthermore, a higher risk for COPD was seen in persons with
p21
WAP/CIP1
R/R and R/S genotypes against S/S genotype [odd ratios
(OR) =2.07] (Lee et al., 2006).
p21
WAP/CIP1
protein expression is increased in alveolar
macrophages [a key cell in the pathogenesis of COPD (Barnes,
2004)] and bronchial epithelial cells of patients with COPD(Tomita
et al., 2002) and, in vitro, hydrogen peroxide, an oxidative stress,
induces cytoplasmic expression of p21
WAP/CIP1
in airway epithe-
lial cells and fails to induce their apoptosis (Tomita et al., 2002).
These data suggest that an increased expression of p21
WAP/CIP1
in
the lower airways caused by chronic exposure to tobacco smoking
may represent another potential molecular link between COPDand
G. Caramori et al. / The International Journal of Biochemistry & Cell Biology 43 (2011) 10301044 1037
squamous cell lung carcinoma. In animal models targeted disrup-
tion of the p21
WAP/CIP1
gene attenuates cigarette-smoke-mediated
lung inammatory responses (Yao et al., 2008) and p21
WAP/CIP1
knock-out mice develop spontaneous tumours including lung can-
cer (Martn-Caballero et al., 2001).
Retinoblastoma (RB) is a rare childhood tumour that arises
in the retina of one (unilateral) or both (bilateral) eyes and can
occur with (i.e., familial retinoblastoma) or without (i.e., spo-
radic retinoblastoma) a family history of the disease (Fletcher et
al., 2004). Inactivating mutations in the RB1 gene are critical for
the development of these tumours. Bilateral retinoblastoma arises
when a germline mutation in one copy of RB1 is followed by the
somatic loss of, or a mutation in, the remaining wild-type copy.
Although most patients with unilateral sporadic disease acquire
somatic mutations in both copies of RB1, some patients carry a
new germline mutation in one copy and acquire a somatic muta-
tion in the other copy (Fletcher et al., 2004). Compared with the
general population, carriers of germline mutations in RB1 who sur-
vive retinoblastoma (i.e., hereditary retinoblastoma survivors) are
at increased risk of early-onset second cancers, particularly sar-
comas, brain tumours, and melanoma, but also of late-onset lung
cancer (Fletcher et al., 2004; Marees et al., 2008; Sanders et al.,
1989; Yu et al., 2009).
The protein encoded by RB1, p105 Rb, functions in multiple cel-
lular processes relevant in the carcinogenesis including cell-cycle
checkpoint control. Duringthecell cycle, p105Rbis phosphorylated
by the CDK4 and cyclin D-CDK6 complexes, whose activities are
themselves regulated by a family of polypeptide inhibitors, which
includes p16
INK4a
. Interestingly, there are no studies investigating
p105 Rb expression in the COPDlung but the peripheral lung of the
COPD patients contains higher percentages of pro-inammatory
senescent type 2 alveolar cells that co-express p16
INK4a
and phos-
phorylated NF-B (Tsuji et al., 2006, 2009). Also p16
INK4a
promoter
methylation (see below) in the bronchial epithelium is very fre-
quent among NSCLC patients and cancer-free controls and persists
after smoking cessation (Belinsky et al., 2002; Russo et al., 2005;
Soria et al., 2002). This area mandates more detailed studies.
10. Epigenetic alterations and the carcinogenic process of
the human squamous cell lung carcinoma and COPD
susceptibility
Epigenomics is thelarge-scalestudyof epigenetic modications,
i.e. heritable changes in gene expression without DNA sequence
alterations (Ocak et al., 2009). These epigenetic changes include
DNA methylation and post-translational modications of histones
(histone acetylation, methylation, ubiquitination, sumoylation and
phosphorylation), which together control chromatin structure
and remodelling and that ultimately control the transcriptional
outcome of the cell (Guil and Esteller, 2009). The main epige-
netic alterations associated with lung cancer are DNA promoter
hypermethylation, DNA global hypomethylation, posttranslational
modicationof histones andmicroRNAsilencing by DNAhyperme-
thylation (Dubey and Powell, 2009).
DNAmethylation has critical roles in the control of gene activity
and the architecture of the nucleus of the cell because it is usually
associated with gene silencing (Esteller, 2008). In humans, DNA
methylation occurs on cytosines that precede guanines; these are
called dinucleotide CpGs. CpG sites are not randomly distributed
in the genome; instead, there are CpG-rich regions known as CpG
islands, which span the 5

end of the regulatory region of many

genes (Esteller, 2008). These islands are not usually methylated
in normal cells. The methylation of particular subgroups of pro-
moter CpG islands can, however, be detected in normal tissues
(Esteller, 2008). The maintenance of these methyl CpG marks is
due to the action of a number of DNA methyltransferases (DNMTs)
which add the universal methyl donor S-adenosyl-l-methionine
(SAM) tocytosine. DNMT1is consideredtobe a maintenance DNMT
whereas DNMT2 has little or no DNMT activity in vitro (Adcock et
al., 2006a,b).
In a recent study performed on lung squamous cell carcino-
mas and matched normal lung tissue, normal tissues fromdifferent
individuals showed very similar DNA methylation patterns over-
all. Whereas each lung squamous cell carcinoma contained several
hundred hypermethylated CpG islands. Eleven CpG islands were
methylated in 80100% of the lung squamous cell carcinomas. In
addition, extensive DNA hypomethylation in the lung squamous
cell carcinomas occurs specically at repetitive sequences, includ-
ingshort andlonginterspersednuclear elements andLongterminal
repeat (LTR) elements, segmental duplications, and subtelomeric
regions, but single-copy sequences rarely become demethylated
(Rauch et al., 2008). In one study aberrant methylation of the
p16
INK4a
(p16 inactivation is the most effective mechanism of
blocking the cyclin DRb pathway) tumour suppressor gene
and/or O
6
-methylguanine-DNA methyltransferase (MGMT) pro-
moters can be detected in DNA from sputum in 100% of patients
with squamous cell lung carcinoma up to 3 years before clinical
diagnosis (Palmisano et al., 2000), and has thus been proposed
as a biomarker for early detection of squamous cell lung cancer
(Belinsky et al., 1998; Nuovo et al., 1999).
Interestingly MGMT hypermethylation is more common in
squamous cell carcinomas in males, and smokers than in adeno-
carcinomas in females, and non-smokers. 17-estradiol decreases
DNMT1 and HDAC1 protein expressions and their binding activity
on the MGMT promoter in vitro in lung cancer cell lines and this
may partially contribute to the MGMT hypermethylation gender
difference seen in lung cancer (Lai et al., 2009).
The DNA hypermethylation status of the p16
INK4a
, CDH13,
RASSF1A, and APC genes is also associated with increased risk
for recurrence following surgical resection of early NSCLC and
with detection of methylated DNA in histologically negative lymph
nodes (Brock et al., 2008).
MicroRNAs (miRNAs) are small non-coding RNAs that regulate
protein expression either through actions on mRNAs synthesis or
translation and function as key controllers in a myriad of cellu-
lar processes, including proliferation, differentiationandapoptosis.
miRNAs can function as tumour suppressors and oncogenes (Hu
et al., 2008b) and mutation, misexpression, and altered miRNA
processing are all implicated in carcinogenesis and/or tumour pro-
gression. Aberrant expression or function of miRNAs may result
fromSNPs affecting their sequence, expression, or binding to target
sites (Hu et al., 2008b) or through epigenetic alterations, resulting
in aberrant patterns of expression (Guil and Esteller, 2009).
Patients with early NSCLC who are homozygous CC at the SNP
rs11614913 in hsa-mir-196a2 have a signicantly decreased sur-
vival (Hu et al., 2008b) and an increased susceptibility to lung
cancer in Chinese populations (Tian et al., 2009). This SNP is asso-
ciated with an increase in mature hsa-mir-196a expression but
not with changes in levels of the precursor, suggesting enhanced
processing of the pre-miRNA to its mature form and can affect
binding of mature hsa-mir-196a2-3p to its target mRNA (Hu et al.,
2008b).
11. Lower airways inammation in COPD and the
carcinogenic process of the human squamous cell lung
carcinoma
COPD is recognized as a chronic inammation of the lower air-
ways characterised by the accumulation of macrophages, CD4
+
and
CD8
+
T cells, dendritic cells, B cells and neutrophil granulocytes,
1038 G. Caramori et al. / The International Journal of Biochemistry & Cell Biology 43 (2011) 10301044
particularlyinthe smaller airways, andthe severityof COPDis asso-
ciated with the degree of inltration by these inammatory cells
(GOLD, 2009). A causal relation between inammation and cancer
has already been proposed by Galen and later by Rudolf Virchow,
who noticed the inltration of leukocytes in malignant tissues and
suggested that cancers arise at regions of chronic inammation.
The longer the inammation persists, the higher the risk of asso-
ciated carcinogenesis [reviewed by Balkwill and Mantovani (2001)
and Shacter and Weitzmann (2002)].
However, the mechanism by which chronic lower airway
inammationis linkedto lung carcinogenesis is still not completely
understood (Lee et al., 2008; Peebles et al., 2007).
Inammatory mediators in the microenvironment around the
stem cells of the bronchial/bronchiolar epithelium can pro-
mote neoplasia by inducing proneoplastic mutations, proliferation,
resistance to apoptosis, angiogenesis, invasion, metastasis, and
secretion of immunosuppressive factors. In fact, inammatory cells
suchas macrophages andTlymphocytes cancommunicatewiththe
neoplastic cells through a reciprocal and self-perpetuating inter-
action resulting in increased growth and resistance to immune
destructionbyloss of tumour immunogenicityand/or reducedanti-
tumour immune response in the local milieu (Reiman et al., 2007).
All these changes confer a survival advantage to the neoplastic
cell. An animal model of COPD-like airway inammation promotes
lung carcinogenesis (adenocarcinoma) in a background of a G12D-
activated K-ras allele in airway secretory cells (Moghaddam et al.,
2009b).
We will reviewbelowsome of the inammatory mediators and
intracellular signalling pathways potentially relevant in the patho-
genetic link between COPD and lung cancer.
12. Inammatory mediators in COPD and lung cancer
12.1. Adhesion molecules in COPD and lung cancer
Integrin-induced cell adhesion is regulated by galectin-3, a
galactose-binding protein which is also involved in cell cycle
regulation, apoptosis and angiogenesis. Galectin-3 expression is
increased in small airway epithelial cells of patients with COPD
suggesting a role of galectin-3 in the pathogenesis of COPD (Pilette
et al., 2007). Nuclear expression of galectin-3 is also a signif-
icant prognostic predictor for the recurrence of squamous cell
carcinoma. Furthermore, the susceptibility to tobacco carcinogen
[4-(methylnitrosamino)-1-(3-pyridle)-1-butanone]-induced lung
tumourigenesis is decreased in galectin-3 decient mice sug-
gesting an important role of galectin-3 in the progression
of lung cancer (Abdel-Aziz et al., 2008; Yao and Rahman,
2009).
12.2. Oxidants in COPD and lung cancer
Oxidative stress increases cytoplasmic expression of
p21
WAP/CIP1
and promotes transition from the G1 to the G2/M
phase of the cell cycle resulting in an imbalance of apopto-
sis/proliferation towards hyperproliferation in lung epithelial cells
(Tomita et al., 2002). This may enhance the epithelial transition
from normal to hyperplastic and nally to carcinomatous status in
smokers and patients with COPD. Oxidative stress may also induce
somatic mutations in DNA (Anderson and Bozinovski, 2003) and
may also affect DNA methylation by the formation of 8-hydroxy-2

-deoxyguanosine (8-OHdG) residues since the presence of these

residues affects DNMT1 DNA binding to the TGF-1 promoter
(Turk et al., 1995; Valinluck et al., 2004).
12.3. Chemokines, their receptors and heterogeneous nuclear
ribonucleoproteins in COPD and lung cancer
Activation of the CXCR4/CXCL12 (SDF-1) axis may have a role in
the pathogenesis of lung cancer. In fact, neutralization of CXCL12
by anti-CXCL12 or anti-CXCR4 monoclonal antibody in preclinical
in vivo studies results in a signicant decrease of NSCLC metastases
(Otsuka and Bebb, 2008). Activation of the CXCR4/CXCL12 (SDF-
1) axis also induces nuclear export of the heterogeneous nuclear
ribonucleoprotein (hnRNP) A2/B1 (Tauler and Mulshine, 2009) and
hnRNP A2/B1 may have a role in cell migration (Pan et al., 2008).
HnRNPs comprise a family of multifunctional proteins that regulate
mRNA processing, transport and subcellular localisation, telom-
ere stability, cell senescence and cell cycle regulation (Tauler and
Mulshine, 2009). Most hnRNPs may mediate cancer progression
and only one, hnRNP E4, has been proposed so far as a tumour
suppressor gene. Interestingly, hnRNPs regulate mRNA stability
by binding to 3

-UTR elements of a variety of mRNAs (Tauler and

Mulshine, 2009).
HnRNP A2/B1 may have a role in cell migration (Pan et al.,
2008) and overexpression of hnRNP A2/B1 (a splicing variant of
hnRNP A2 mRNA) in plasma and bronchial epithelium (obtained
with brushings and/or bronchial biopsies) has a high sensitivity
for the presence of NSCLC, particularly squamous cell carcinoma,
and is also present in high-risk smokers years before they develop
lung cancer (Katsimpoula et al., 2009; Sueoka et al., 2005). Fur-
thermore, squamous cell carcinoma patients with hnRNP A2/B1
overexpression have a better prognosis compared with that of the
patients without hnRNP A2/B1 expression, whereas in adenocar-
cinoma patients, there is no such a difference between them (Wu
et al., 2003). The role of the CXCR4/CXCL12 axis and of the hnRNP
A2/B1 in the pathogenesis of COPD and in the associated lung car-
cinogenesis should be explored.
12.4. Prostaglandins in COPD and lung cancer
The enzyme cyclooxygenase-2 (COX-2) and its product
prostaglandin E
2
(PGE
2
) may modulate the inammatory response
as well as carcinogenesis through effects on cell proliferation,
apoptosis and angiogenesis and may be involved in the patho-
genesis of both COPD and lung cancer. Prostaglandin D
2
(PGD
2
)
is a biologically active lipid derived from arachidonic acid via
the action of COX-1 and COX-2. PGD
2
binds primarily to two
receptors termed DP1 and DP2 [also known as chemoattractant
receptor-homologous molecule expressed on Th2 cells (CRTh2)].
The sputumlevels of PGE2, COX-2 and matrix metalloproteinase-2
(MMP-2; a protease associated with both inammation and
airway remodelling in COPD) are increased in smokers with COPD
compared to non-smoking control subjects and the sputum levels
of PGE
2
and MMP-2 are inversely correlated with FEV
1
% predicted
in COPD patients (Chen et al., 2008). COX-2 expression is also
increased in the peripheral lung of COPD patients compared to
control subjects (Xaubet et al., 2004).
Many, but not all (Hayes et al., 2006), epidemiological stud-
ies suggest that there is a decreased incidence of lung cancer,
particularly of adenocarcinoma, in subjects who regularly use
aspirin or other nonsteroidal anti-inammatory drugs (NSAIDs)
(Schreinemachers andEverson, 1994; Slatore et al., 2009; VanDyke
et al., 2008) and a reviewof the literature suggests that daily intake
of NSAIDs, primarily aspirin, produced risk reductions of 36% for
lung cancer (Harris et al., 2005). NSAID effects became apparent
after ve or more years of use and were stronger with longer dura-
tion (Harris et al., 2005). A few studies suggest that ibuprofen has
stronger anticancer effects than aspirin against lung cancer (Harris
et al., 2005). Carriers of the C allele of a common polymorphism
in the 3

-UTR of COX-2, associated with reduced production of

G. Caramori et al. / The International Journal of Biochemistry & Cell Biology 43 (2011) 10301044 1039
COX-2, have a signicantly increased risk of lung cancer (Campa
et al., 2004). The COX2.8473 SNP is located downstream of the
stop codon, in the 3

-UTR region. Binding of proteins to the 3

-
UTR can control mRNA stability and degradation. This region is
characterised by multiple repeats of AU-rich elements, which are
alsofoundinseveral other genes encoding inammatory mediators
suchas cytokines andalsoproto-oncogenes, whosemRNAs arevery
unstable. It is possible that the T/C substitution at COX2.8473 stabi-
lizes the COX2 mRNA, thus resulting in higher expression (Campa
et al., 2004).
Both lung adenocarcinomas and squamous cell carcinoma show
cytoplasmic staining for COX-2 in neoplastic cells but the level of
staining is stronger in adenocarcinomas (Wolff et al., 1998). COX-
2 expression has also been shown in the bronchial preneoplastic
lesions (Hida et al., 1998).
12.5. Intracellular signalling pathways in COPD and lung cancer
The transcription factor, nuclear factor B (NF-B), could well
be an important player in airways inammation and it is activated
in the bronchial epithelium and in inammatory cells both in the
lower airways of COPD patients and in the premalignant lesions of
the bronchial epitheliumand neoplastic cells of squamous cell lung
carcinoma (Caramori et al., 2004; Di Stefano et al., 2002; Tang et al.,
2006; Tichelaar et al., 2005).
NF-B activation and subsequent transactivation of
inammation-related genes may play a central role in both
COPD and squamous cell lung carcinoma (Karin, 2009). Indeed,
in addition to its tumour-promoting role, which depends on
stimulation of cell proliferation and inhibition of cell death, NF-B
may also participate in tumour initiation (Greten and Karin,
2004). For instance, NF-B-activated macrophages in the bronchial
tissues of COPD patients can release oxidants in the proximity of
the basal bronchial epithelial cells to cause their DNA damage.
Recent evidence suggests that prolonged exposure to cigarette
smoke induces lung cancer in animals following induction of an
NF-B-dependent inammatory response in the lower airways
(Takahashi et al., 2010).
Sirtuin 1 (SIRT1), a member of the silent information regulator 2
in mammals, has recently been found to be reduced in the periph-
eral lung of COPD patients (Nakamaru et al., 2009; Rajendrasozhan
et al., 2008). SIRT1 is known to be able to deacetylate in vitro
substrates such as NF-B and deacetylation attenuates NF-B tran-
scriptional activation (Spange et al., 2009).
Peroxisome proliferator-activated receptors (PPARs) are ligand-
activated transcription factors that belong to the nuclear hormone
receptor superfamily. PPAR regulates several metabolic path-
ways by binding to sequence-specic PPAR response elements
in the promoter region of the target genes (Belvisi and Mitchell,
2009). PPAR regulates cell growth by inducing differentiation
and apoptosis. These effects are mediated through inhibition of
transcription factors, including NF-B. PPAR ligands inhibit the
release of pro-inammatory cytokines from airway epithelial cells
and play an important role in regulating their differentiation. In
an animal model of COPD-like airway inammation the PPAR
agonist, rosiglitazone, inhibits lipopolysaccharide (LPS)-induced
neutrophilia and reduces chemoattractants and survival factors
(Belvisi and Mitchell, 2009). Decreased expression of PPAR has
also been observed in lung cancer.
13. High-throughput genome-wide technologies for
measuring gene expression for an early diagnosis of lung
cancer in COPD patients
Given the heterogeneous nature of lung cancer, monitoring of
only one or a fewgenes is probably of limited value. Apan-genomic
Table 2
New potential compounds for the treatment of both lung cancer and COPD.
Wide spectrum anti-inammatory compounds:
- Inhaled glucocorticoids
- COX-2 selective inhibitors
- Novel anti-inammatory compounds (curcumin, resveratrol, statins).
Selective antagonists of inammatory mediators:
- Muscarinic M3 receptor antagonists
- CXCR4/CXCL12 axis blockers
Transcription factor modulators:
- NF-B blockers
- PPAR agonists
analysis using high-throughput gene expression analysis seems
more efcient for identifying specic events in lung carcinogen-
esis (Beane et al., 2009). Since 2000, several studies have proposed
a molecular classication of human lung carcinomas on the basis
of gene expression panels and have described numerous putative
biological markers of lung cancer (Lacroix et al., 2008). Using a
gene-microarray on histologically normal large-airway epithelial
cells obtainedat bronchoscopyfromsmokers withsuspicionof lung
cancer, one study has identied an 80-gene biomarker that distin-
guishes smokers with and without lung cancer with an accuracy of
83%(80%sensitive, 84%specic), andapproximately90%sensitivity
for stage 1 lung cancer (Spira et al., 2007). In a similar vein, stud-
ies are now being conducted to look at whole genomes from lung
cancer cells using deep sequencing technologies (Pleasance et al.,
2010a,b). Furthermore, global analysis of epigenetic marks includ-
ing histone and DNA methylation (Goto et al., 2009) and miRNA
proles (Landi et al., 2010) are beginning to demonstrate distinct
proles between cancer cell types. The potential for personalized
medicine based on precise delineation of an individuals global
genomic and epigenomic maps is expensive but may be viable in
the future. At this time, improving the biological signicance of
microarray data is still an important clinical challenge (Lacroix et
al., 2008). Similar studies in COPDare required to distinguish these
proles from that of COPD.
14. New potential pharmacological therapies for both lung
cancer and COPD
At present there are many compounds in development or under
study (Table 2) that either prevent lung cancer in animal models or
demonstrate efcacy in human epidemiological and small clinical
studies. However, few of these have been proven effective in large
controlled clinical trials. Recently, the oral epidermal growth fac-
tor receptor (EGFR) tyrosine-kinase inhibitor erlotinib was shown
to be well tolerated and signicantly prolongs progression-free
survival in a phase 3 placebo controlled study of 889 subjects
with unresectable NSCLC (SATURN; BO18192) following rst-line
platinum-doublet chemotherapy (Cappuzzo et al., 2010).
In animal models of lung cancer inhaled glucocorticoids have a
protective effect and reverse DNA hypomethylation and modulate
mRNA expression of oncogenes (Alyaqoub et al., 2006; Balansky
et al., 2006, 2010; DAgostini et al., 2009; Pereira et al., 2007;
Wattenberg et al., 2000) and epidemiological studies suggest that
regular use of inhaled glucocorticoids, with and without long-
acting inhaled
2
agonists (LABA), may reduce the risk of lung
cancer among former smokers with diagnosed COPD (Khan and
Agarwal, 2007; Kiri et al., 2009; Miller et al., 2007; Parimon et al.,
2007). However, a 6-month treatment with high doses of inhaled
glucocorticoids does not cause any regression of bronchial dyspla-
sia or secondary markers of carcinogenesis in smokers (Lam et al.,
2004; van den Berg et al., 2007) and a long-term clinical trial in
moderate to severe COPD patients treated for 3 years with inhaled
glucocorticoids has not demonstrated a decreased risk of lung can-
1040 G. Caramori et al. / The International Journal of Biochemistry & Cell Biology 43 (2011) 10301044
cer (Calverley et al., 2007). There is an ongoing chemoprevention
trial measuring the effect of inhaled uticasone propionate in high-
risksmokers (however patients withFEV
1
less of onelitrehavebeen
excluded from this study) (van den Berg et al., 2008).
In a series of case control studies, daily use of a selective COX-
2 inhibitor, either celecoxib or rofecoxib, signicantly reduces the
risk for lung cancer, however the use of these drugs increased the
relative risk of cardiovascular disease (Harris, 2009).
In an animal model of COPD-like airway inammation, cur-
cumin suppresses bronchoalveolar lavage (BAL) inammation and
protects against lung carcinogenesis (Moghaddamet al., 2009a). In
vitro curcumin has also direct anti-tumoural effects reducing cell
viability, colony formation and inducing apoptosis (Moghaddam
et al., 2009a). However, in another animal model curcumin pro-
motes lung cancer, probably by enhancing oxidant formation and it
has been proposed that current smokers and ex-smokers should be
excluded from chemopreventive trials of curcumin (Dance-Barnes
et al., 2009).
Moderate wine ingestion has benecial effects on the risk of
developing COPD in long term smokers and in the rate of progres-
sion of airow obstruction in COPD patients and on the risk of
developing lung cancer (Doll et al., 2005; Kamholz, 2006; Tabak
et al., 2001). Resveratrol [3,5,4

-trihydroxystilbene], a polyphe-
nolic compound found in red wine, has both anti-oxidant and
anti-inammatory effects and this may be responsible for some
of these benets of moderate wine intake described in epidemio-
logical studies (Kamholz, 2006). In vitro in bronchial epithelial cell
lines resveratrol inhibits the constitutive andinducedexpressionof
cytochrome P450 1A1 and 1B1 genes with a reduced overall level
of benzo{a}pyrene metabolism. In vitro, in lung cancer cell lines,
resveratrol has also direct anti-cancer effects through cell cycle
arrest, and inhibition of transcription factors such as NF-B, and
potentiates the apoptotic effects of cytokines (Kamholz, 2006).
Normal bronchial epithelium and squamous cell lung carcino-
mas synthesize ACh. AChsecretedby the neoplastic cells stimulates
tumours growth by binding to nicotinic and muscarinic receptors
expressed on lung cancers. Thus antagonists to nicotinic and mus-
carinic receptors can inhibit lung cancer growth. The muscarinic
receptor (mAChR) subtype utilized for cell proliferation is the M3
subtype and consistent with this M3 mAChR antagonists inhibit
growth of squamous cell carcinomas (Song and Spindel, 2008b;
Song et al., 2008a). However, a long-term clinical trial in moderate
to severe COPDpatients treated for 3 years with inhaled tiotropium
(a strong M3blocker) has not demonstrateda decreasedrisk of lung
cancer (Tashkin et al., 2008). Due to the safety of these drugs and
their wide use in clinical practice for the treatment of COPD, we
need more long-termcontrolled clinical trials specically designed
to address this issue.
Many different signal transduction pathways, originating from
a wide variety of cellular stresses and stimuli, converge on a single
target, the NFB/IB complex and its activating kinase (inhibitor of
B kinase, IKK). IKK2 inhibitors are in development as novel ther-
apies for the treatment of COPD (Adcock et al., 2006a) and lung
cancer (Lin et al., 2010). Interestingly, in vitro, inhibition of NF-B
using bortezomib or the compound BAY-11-7085 sensitizes squa-
mous cell lung carcinoma cell lines to death induced by histone
deacetylases inhibitors (Denlinger et al., 2004; Rundall et al., 2004).
In the animal lungs, NF-B inhibition suppresses airway inam-
mation (Newton et al., 2007) and urethane-induced lung cancer
(Stathopoulos et al., 2007). Interestingly, TP53 may also suppress
NF-Bactivityinanimal models of airwayinammation(Komarova
et al., 2005).
Thereis accumulatingevidencethat statins haveimportant anti-
inammatory effects in the lung. Although randomised control
trials are yet to be reported, non-randomised studies have consis-
tentlyshownbenet inCOPDpatients takingstatins comparedwith
those not on the drug. These benets include reductions in both
cardiovascular and respiratory morbidity/mortality. Other poten-
tial benets include a reduced decline in FEV
1
and reduced risk of
lung cancer (van Gestel et al., 2009; Young et al., 2009).
A long-term clinical trial in moderate to severe COPD patients
treated for 3 years with the anti-oxidant N-acetylcysteine (NAC;
600mg daily) did not demonstrated a decreased risk of lung cancer
(Decramer et al., 2005). Similarly in the large EUROSCAN trial, nei-
ther vitamin A nor N-acetylcysteine (600mg daily) could prevent
tumour recurrence or the occurrence of second primary tumours in
patients with lung cancer during the 2-year follow-up period (van
Zandwijk et al., 2000). However, the doses used in these studies
may not have produced a local anti-oxidant effect.
15. Conclusions
Many new compounds that target the molecular pathology of
advanced squamous cell lung carcinoma are now undergoing clin-
ical trials, however, we believe that we need to promote more
studies onthe molecular and cellular pathobiology of smokers with
premalignant bronchial lesions of the squamous cell lung carci-
noma compared with a control group of smokers with and without
COPD to unravel the complex molecular interactions between
COPD and early squamous cell lung carcinoma. These studies
should also look at younger healthy smokers in combination with
risk models of lung cancer and COPD. Overall these studies may
allow the discovery of new molecular targets of the early carcino-
genesis process that in the foreseeable future may render the early
diagnosis and treatment, and may be even the prevention, of inva-
sive squamous cell lung carcinoma a reality.
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