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REVIEW
HTTP://DX.DOI.ORG/10.5504/BBEQ.2013.0062
MEDICAL BIOTECHNOLOGY
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MOLECULAR AND GENETIC ASPECTS OF LUNG CANCER
Mohammad Mohawsh AL Zeyadi1,2
1
Kufa University, Faculty of Science, Najaf, Iraq
2
Sofia University “ Kliment Ohridski”, Faculty of Biology, Sofia, Bulgaria
Correspondence to: Mohammad Mohawsh AL Zeyadi
E-mail: [email protected]
ABSTRACT
Lung cancer is a serious health problem. It is one of the leading causes of death worldwide. There are over one million deaths
worldwide due to lung cancer. Molecular-cytogenetic studies could give reliable information about genetic alterations which
could be related to lung cancer pathogenesis. Such alterations could serve as the basis for better prognosis and treatment
strategies. Lung carcinogenesis is known to be a multistage process involving alterations in multiple genes and diverse pathways.
These genes include proto-oncogenes, tumor suppressor genes (TSG), and DNA repair genes. We present a review of the genetic
aspect of these alterations. Knowledge of the genetic aspects will aid the understanding of the essence of lung carcinogenesis in
the search for appropriate markers for early diagnosis and adequate treatment.
Biotechnol. & Biotechnol. Eq. 2013, 27(5), 4051-4060
Keywords: lung cancer, cytogenetic aberrations, oncogenes,
tumor suppressor genes
Introduction
Lung cancer is one of the leading causes of death from cancer
in economically developed countries and in developing ones
(2, 39). Today, lung cancer is classified into two major types:
small-cell lung cancer (SCLC) and non-small cell lung cancer
(NSCLC). NSCLC is more common and makes up about 85 %
of all lung cancers. NSCLC is the leading cause of cancer deaths
around the world (15, 76). Although the majority of the NSCLC
cases are caused or induced by cigarette smoking, 10 % of these
patients are non-smokers. Almost all patients suffering from
SCLC are smokers. SCLC metastasizes more quickly, which is
why it is more dangerous than NSCLC. Lung carcinogenesis,
like the development of other cancers, is a multistage process
involving alterations in multiple genes and diverse pathways
(22). These genes include proto-oncogenes, which are positive
growth regulators; tumor suppressor genes (TSG), which are
negative growth regulators; and genes involved in apoptotic
control and DNA repair genes (16). Inherited polymorphisms
in a variety of genes, most notably the carcinogen metabolism
genes, affect the susceptibility of an individual to develop lung
cancer, with accumulation of environmental exposures. Genes
involved in DNA repair, cell growth, signal transduction and
cell cycle control may all be damaged at different stages of
lung tumor progression (22). More generalized changes consist
of chromosomal rearrangements, microsatellite instability,
deregulated expression of telomerase and angiogenesis (16).
Mutational activation of oncogenes and inactivation of tumor
suppressor genes, and subsequent increased genetic instability
are major genetic events in lung carcinogenesis. By the time
lung cancer is clinically diagnosed, as many as 10 to 20 genetic
alterations may have accumulated (22).
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Cytogenetic Aberrations
Cytogenetic and molecular studies have provided evidence
that multiple genetic lesions occur during the pathogenesis of
lung cancer. Chromosome instability occurs early in cancer
development and is associated with poor prognosis (104).
In fact, chromosomal abnormalities, activation of dominant
oncogenes, and inactivation of tumor suppressor genes have
been described. The chromosomal aberrations present in
NSCLC and SCLC cells, include aneuploidy, chromosomal
translocations, and amplifications (16).
Aneuploidy. Numeric aberrations in chromosomes,
referred to as aneuploidy, are commonly observed in human
cancer (71). Whether aneuploidy is a cause or consequence
of lung cancer has long been debated (75). As reviewed by
Cherneva and Dimova (16), at the cytogenetic level, aneuploidy
distinguishes between cancer cells and pre-malignant lesions
from normal lung epithelium. Pre-invasive tissues (primary
tumor) tend to be diploid with only a few aberrations (gain
in chromosome 7, loss in 3p and 9p). A single aneuploidy has
been detected in squamous dysplasia and in high-risk smokers,
whereas invasive and metastatic lung carcinoma are generally
aneuploid, the aneuploidy involving several chromosomes.
Therefore, accumulation of chromosomal imbalances during
carcinogenesis has been considered as a cause of progression
from normal through pre-invasive to invasive tumors. Many
new technologies, such as spectral karyotyping (SKY),
fluorescent in situ hybridization (FISH) and comparative
genomic hybridization (CGH), provide useful information
about the chromosomal abnormalities in lung cancer. For
example, FISH assessment of aneuploidies in lung cancer
has revealed that the most frequent gains (82 %) are seen in
chromosome 7.
Yen et al. (103) found chromosomal aberrations by CGH
analysis of primary tumors of malignant cells of 31 lung
adenocarcinoma cases: more frequently loss in 19q and 22q,
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and gain in 11p. Interestingly, occurrences of 3p loss and 11p
gain appear to be higher in smokers than in non-smokers.
Pooled CGH of these results shows frequent amplifications on
chromosome arms 1p, 1q, 2q, 3q, 4q, 5p, 5q, 6p, 6q, 7p, 7q,
8q, 12q, 13q, 14q, 17q, Xp, and Xq. Frequent deletions are
found on 1p, 3p, 4q, 8p, 9p, 9q, 10q, 13q, 16p, 16q, 17p, 18q,
19p, 19q, 20p, and 22q (103). The deletion of 3p and increased
copy number of 11p and Xp appear more often in male than
in female patients (103). The results from CGH studies show
common regions of amplification on 1q, 3q 5p, 8q, 11q 12p 17q,
20q in NSCLC (16). Some studies indicate that there is not a
unique chromosome abnormality present in all cases of SCLC,
although loss of chromosome 13 and structural abnormalities of
chromosome 3 were frequently found. A feature of these SCLC
cell lines established from metastatic deposits is the complexity
of the karyotypes due to numerical and structural chromosomal
abnormalities (56). Amplification of 3q is most prevalent in
squamous cell carcinoma, being present in 75 % to 94 % of
the tumors. A common amplicon at 3q26-q28 that codes the
catalytic subunit of phosphatidylinositol 3-kinase (PIK3CA)
has been identified by array CGH. p63, which regulates cell
growth and differentiation, is also a commonly amplified gene
in squamous cell carcinoma. An increased copy number for
p63 has been detected by FISH and immunohistochemistry on
tissue microarray of a large collection of NCSLC (16).
Berrieman et al. (8), by means of M-FISH, found
translocations between chromosomes 5 and 14, 5 and 11
and, 1 and 6, in NSCLC. Chromosomes 4, 5, 8, 11, 12 and
19 were most frequently involved in interchromosomal
translocations (8). Cherneva and Dimova (16) highlighted
that a specific translocation in NSCLC is t(15;19), which
results in overexpression of Notch3; with a high frequency
of translocation t(15;19)(q11;p13). Studies on some types of
cancer show that chromosomal aberrations can be induced as a
result of certain environmental and occupational exposures as
well as therapy with cytotoxic drugs (29).
Genetic Aberrations
Molecular abnormalities in lung cancer
Only about 1 % of the cancers arise in individuals with a
hereditary cancer syndrome resulting from the inheritance
of a germline mutation (18); e.g. Knudson (43) showed
that retinoblastoma was caused by a germ-line mutation in
approximately 40 % of U.S. cases. A recent meta-analysis
suggests that individuals with a family history of lung cancer
are at an increased risk of developing the disease (53). The
vast majority of cancer-associated mutations are somatic.
Such lesions are now known to occur in several different
types of genes (18): i. oncogenes; ii. tumor suppressor genes;
iii. cell cycle control genes; iv. mutator (DNA repair) genes;
v. apoptosis regulatory genes; and vi. telomerase-associated
genes. Approximately 50 tumor suppressor genes and over
100 oncogenes had already been described by the year 2000.
Because tumor suppressor genes, telomeres, and oncogenes
are intimately involved in the regulation of cell growth and
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division, cancer can be considered a disease of deregulation
of the cell cycle. Oncogenes result from gain-of-function
mutations in their normal cellular counterpart proto-oncogenes
and act in a dominant fashion (66).
Oncogenes
Protooncogenes are wild-type universally present genes that
stimulate carcinogenesis when mutated. The mutant forms are
designated as oncogenes (105). Oncogenes encode proteins
whose normal function is to promote cellular proliferation
(18). They act as dominant genes, in which a mutation in
only one copy of the gene leads to activation usually through
overexpression of a protein product. A variety of mutational
events can transform a protooncogene into an oncogene,
including point mutations, translocations, amplifications, and
deletions (105). Oncogene proteins play different cellular
roles: signal transduction (e.g. Ras), transcriptional regulation
(e.g. Myc), cell surface receptors (e.g. epidermal growth factor
receptor) or components of the cyclin/cdk system that control
the cell cycle (e.g. cyclin D1) (18).
RAS oncogene activation. The ras gene family (K-RAS,
H-RAS and N-RAS) encodes similar 21 kD membrane-bound
proteins and is activated by point mutations at codons 12, 13 or
61 in approximately 20 % to 30 % lung adenocarcinomas and
15 % to 20 % of all NSCLCs, but very rarely in SCLCs (70).
Activating mutations in the K-ras proto-oncogene are found in
about 90 % of ras mutations in lung adenocarcinomas. Point
mutations at codon 12 are the most frequent, with 85 % of
K-RAS mutations affecting this codon; followed by mutations
at codons 13 and 61 (94). Once activated, K-Ras stimulates
multiple downstream effectors (27). K-Ras plays role as a
key signal transducer, and may also promote cell survival
and suppress apoptosis. K-RAS mutations are common in
lung adenocarcinoma and have been associated with cigarette
smoking, asbestos exposure, and female gender (1).
MYC family genes. The MYC proto-oncogene family
genes (C-MYC, N-MYC and L-MYC) (46), or P-MYC, R-MYC
and B-MYC (17), consist of three exons containing 6 kb to
7 kb. The C-MYC gene has been mapped to the long arm of
chromosome 8, 8q24. N-MYC promotes cell proliferation
(62); N- and L-MYC genes have been identified at 2p2324 and 1p32 , respectively (7). MYC encode cycle-specific
nuclear phosphoproteins which function as basic helix-loophelix leucinezipper (bHLHZ) transcription factors and are
involved in the regulation of expression of other cell growth,
differentiation and apoptosis-related genes. In contrast to the
RAS oncogenes, point mutations of MYC family genes are
rare in human lung cancers. Activation of the MYC family
genes has been observed mainly by gene amplification or
transcriptional dysregulation, with the C-MYC gene being
most frequently activated in lung cancer, particularly in SCLCs
(46). Amplification and overexpression of MYC family genes
appear to be more prevalent in tumor cell lines than in tumor
tissues, and in SCLCs than in NSCLCs (63). Furthermore,
while low levels of MYC expression can induce proliferation,
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overexpression of MYC can stimulate the p53 pathway and
apoptosis (57). Results of numerous studies of MYC gene
amplification show that 18 % of SCLC tumors, 8 % of NSCLC
tumors, and 31 % of SCLC cell lines have gene amplification
of one member of the MYC family (63). Whereas N-MYC
alterations are commonly present in SCLC (16) and 20 % of
NSCLC cell lines bear a gene amplification of one member of
the MYC family (C-MYC) (46, 63), N-MYC amplification is not
found in NSCLC (16).
EGFR and ERBB2. EGFR is a well studied oncogene and
its product belongs to a family of closely related growth factor/
receptor tyrosine kinases that includes EGFR (ErbB1), HER2/
neu (ErbB2), HER3 (ErbB3), and HER4 (ErbB4) (65, 101).
Together with Ras, EGFR receptor tyrosine kinases are key
components of signaling cascades. Important examples are the
heregulin (neuregulin) receptor (ErbB2/HER2/neu) encoded
by the ERBB2 gene and EGFR encoded by the EGFR gene
(32). These receptors have a common structure that consists
of an N-terminal ligand-binding extracellular domain, a single
transmembrane helix, and an intracelular tyrosine kinase
domain with a C-terminal tail (16). Upon ligand binding,
these receptors homodimerize or heterodimerize resulting in
autophosphorylation, activation, and subsequent activation
of intracellular signaling cascades such as the RAS/RAF/
MEK/ERK, PI3K/Akt, and Jak/Stat signaling pathways.
EGFR can contribute to oncogenesis via at least three major
mechanisms: overexpression of EGFR ligands, amplification
of EGFR, and mutational activation of EGFR (106). EGFR
mutations occur in highly selected subpopulations of lung
cancer patients: adenocarcinoma histology (78). EGFR
regulates epithelial proliferation with EGF, TGFa as specific
ligands. ErbB2 forms heterodimers with other members of
the ErbB family and stimulates various signaling pathways
including phosphatidylinositol 3-kinase/Akt, JAK-STAT (82)
and Ras/Raf/MAPK (7). The ERBB2 (HER/neu) oncogene has
been mapped to chromosome 17q21.1 (86). In normal lung
epithelium, expression of EGFR is found in the basal layer where
proliferation occurs. In response to tobacco smoke, progressive
atypical changes are noted in the bronchial epithelium,
followed by squamous metaplasia with mild through moderate
and severe dysplasia and carcinoma in situ (16). Activation
of ERBB2 (86) and EGFR (18) occurs by gene amplification
and/or overexpression and can be detected in several types of
human tumors (86), especially in breast and ovarian cancers.
In lung cancers, the ERBB2 gene is highly expressed in over
a third of NSCLCs, especially adenocarcinomas (95). On the
other hand, ERBB2 overexpression is also correlated to the
responsiveness of lung tumors to chemotherapy, especially
in NSCLCs (90). The TGFA and AREG genes, encoding,
respectively, the EGFR ligands TGF-α and amphiregulin,
are also overexpressed in NSCLC (48). Overexpression of
the ERBB2 gene is associated with a poor prognosis (95).
A different type of mutant EGFR is provided by EGFRvIII,
a variant found in a number of different cancers, including
NSCLC, and caused by an in-frame deletion of exons 2–7
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of the EGFR gene, corresponding to the extracellular ligandbinding domain. There is a significant association between
EGFR mutations, especially exon 19 deletion, and response to
TKIs (72), and EGFR mutation testing is being evaluated as
a biomarker of response to EGFR tyrosine kinase inhibitors
(65). It is thought that such frequent somatic rearrangements
are brought about by homologous recombination between an
Alu repeat sequence in intron 7 of the EGFR gene and Alu-like
sequences in intron 1 (28). EGFRvIII constitutively activates
PI3K signaling, and promotes the constitutive phosphorylation
of extracellular-regulated kinases (ERKs) (28). Recent analyses
have reported EGFR overexpression in 62 % of NSCLC cases,
and its expression is correlated with a poor prognosis (59).
EGFR is commonly expressed in adenocarcinoma (40 % to
60 %), in virtually all cases of squamous cell carcinoma, in
approiximately 65 % of large cell carcinoma cases, but is not
expressed in small cell carcinoma (16), making EGFR and
its family members prime candidates for the development
of targeted therapeutics. EGFR mutation accounts for
approximately 40 % to 45 % of all EGFR-TK–activating
mutations in NSCLC (72). In lung cancer patients, mutations in
the TK domain of the EGFR gene are more common in neversmokers than in smokers (51 % vs. 10 %), in adenocarcinomas
versus other types of lung cancer (40 % vs. 3 %), in patients of
East Asian ancestry than in other ethnicities (30 % versus 8 %),
and in females versus males (42 % versus 14 %). The mutation
status seems not to be associated with age at diagnosis, clinical
stage, the presence of certain histologic features, or overall
survival, and such mutations are not found in normal tissues
or tissues from other cancer types (2). Mutations are found in
the KRAS gene—an EGFR signaling pathway gene—in 8 % of
lung cancers but not in those that have an EGFR gene mutation
(23). This shows that the pathogenesis of adenocarcinoma
is different (16). EGFR gene amplification is found in only
9 % of lung cancers. A high gene copy number is associated
with a poorer prognosis, while EGFR overexpression is not
consistently connected with a poor outcome (35).
MET gene. The MET proto-oncogene has come to
prominence by virtue of the germline and somatic mutations
that characterize hereditary papillary renal cell carcinoma (18).
The MET gene is located in 7q31 (25) and encodes a receptor
TK. It is the cell surface receptor for hepatocyte growth factor/
scatter factor (HGF), a known mitogen for both epithelial
cells and many types of tumor cells. MET transduces, via
PI3K, HGF-mediated signals that are involved in regulating
differentiation, cellular motility, angiogenesis and tissue
invasion. Gain-of-function MET mutations are known to be
associated with increased levels of tyrosine phosphorylation
and enhanced kinase activity as compared with the wild-type
(18). Engelman et al. (25) described a gefitinib-sensitive lung
cancer cell line that developed resistance to gefitinib as a result
of focal amplification of the MET proto-oncogene. Inhibition
of MET signaling in these cells restored their sensitivity
to gefitinib. MET amplification was detected in 4 (22 %) of
18 lung cancer specimens that had developed resistance
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to gefitinib or erlotinib (25). Ma et al. (50) showed that the
level of MET expression did not correlate with the presence
of MET mutations (50, 52). Engelman et al. (25) found that
amplification of MET caused gefitinib resistance by driving
ERBB3-dependent activation of phosphoinositide 3-kinase,
a pathway thought to be specific to the EGFR/ERBB family
receptors. Thus, they proposed that MET amplification may
promote drug resistance in other ERBB-driven cancers as well
(25).
CDKN1A polymorphism and susceptibility to lung cancer
The CDKN1A gene is located on chromosome 6 (6p21.2)
and encodes a potent cyclin-dependent kinase inhibitor. The
encoded protein binds to and inhibits the activity of cyclinCDK2 or -CDK4 complexes, and thus functions as a regulator
of cell cycle progression at G1. The expression of this gene is
tightly controlled by the tumor suppressor protein p53, through
which this protein mediates the p53-dependent cell cycle G1
phase arrest in response to a variety of stress stimuli (58).
p53 polymorphisms and haplotypes have been found to be
associated with different types of cancer. Sjalander et al. (79)
found an increased frequency of the p21 arg31 allele in lung
cancer patients, especially in comparison with patients with
chronic obstructive pulmonary disease (COPD). Thus, allelic
variants of both p53 and its effector protein p21 may have
an influence on lung cancer. The results of this and previous
studies indicate that allelic variants of both p53 and its effector
protein p21 may have an influence on lung cancer (79).
Tumor suppressor genes
Tumor suppressor genes (TSGs) in the wild-type state have
two active copies (alleles). These genes are involved in the
regulation of a diverse array of different cellular functions
including cell cycle checkpoint control, detection and repair
of DNA damage, protein ubiquitination and degradation,
mitogenic signaling, cell specification, differentiation and
migration, and tumor angiogenesis (77). TSGs are thought to
functionally suppress carcinogenesis in their routine cellular
activity. Because both copies of the TSG must be mutated
for tumorigenesis, TSGs are designated as recessive genes.
This is in contrast to dominant protooncogenes, in which
only one mutation is needed. The most common assays for
identifying TSGs include LOH, CGH, array CGH, and FISH.
LOH studies have provided clues to the localization of the
many TSGs involved in lung carcinogenesis (105). In general,
alterations of chromosomes 3p, 9p21, 13q14, and 17p13 are
frequently observed, even in the early precursor lesions. Most
of these molecular alterations are thought to also correlate with
smoking-related damage and may not necessarily indicate an
increased risk for development of invasive carcinoma (97).
TSGs often encode proteins with a regulatory role in cell cycle
progression (e.g. Rb). Rb gene expression can be regulated by
the p53 protein (76). TSGs may also encode DNA-binding
transcription factors (e.g. p53) and inhibitors of cyclindependent kinases required for cell cycle progression (e.g.
p16). Knudson (43) found the “two hit hypothesis” provides
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the basis for a mechanistic understanding of tumor suppressor
gene mutagenesis.
TP53. The TP53 gene, located at 17p13.1, contains 11
exons. It is the most frequently mutated gene in a wide variety
of different cancers and is without doubt the best-studied
tumor suppressor gene in lung cancer pathogenesis. It encodes
a nuclear phosphoprotein, p53, which performs multiple
functions in the cell (18), cell cycle control, DNA repair, cell
differentiation, genomic instability, programmed cell death,
and cell senescence (75). Mutation of the TP53 gene is the
main event that leads to the inactivation of the p53 stressresponse pathway in lung cancers (64). TP53 mutations are
among the most frequent abnormalities occurring in 75 % to
100 % of SCLCs and ~50 % of NSCLCs (30). The TP53 gene
is inactivated either by allele loss or by more subtle lesions in
~40 % to 60 % of NSCLC tumors and ~80 % to 95 % of SCLC
tumors (80). Such lesions are generally considered to occur
at an early stage in lung cancer (32) and appear to represent
a poor prognostic marker for survival in NSCLC (80). It has
been found that mutations of TP53 are detected in dysplastic
and premalignant lesions in the lung and they are an early
event in lung cancer and occur before micro invasion (16, 40,
66). Specific point mutations in lung cancer are associated with
tobacco exposure and formation of DNA adducts (21). Most
mutations are missense mutations occurring in the mid region
of the gene (exon 5–8), leading to both gain of new function
and loss of wild type p53 function, due to its short half-life
(6). Among these mutations associated with lung cancer, G,T
transversions are common (~30 % of the total) (61), are mostly
G:C to T:A transversions. This type of mutation is known
to be induced by benzo(a)pyrene and aflatoxin B1 which
are associated with the etiology of lung cancer (61). Other
mutations occur in radon-induced cancer, showing carcinogen
specificity of mutations (16). The mutated p53 protein is
consistently associated with a poorer prognosis (21).
p16Ink4a/Cyclin
D1/CDK4/RB
pathyway.
The
retinoblastoma gene (RB) is located in human chromosomal
region 13q14 and encodes a nuclear phosphoprotein that was
initially identified as a tumor suppressor gene in childhood
retinoblastomas (24). The hypophosphorylated RB protein acts
as a growth suppressor by inactivating a number of proteins
including the transcription factor E2F1, which promote
transcription of genes required for DNA replication, thus
blocking the G1/S transition. RB mutations are found in 90 %
of SCLCs and 15 % to 30 % of NSCLCs (24). The p16INK4a
gene (P16) is inactivated in >70 % of cell lines derived from
all histologic types of human NSCLCs (5). Most mutations
result in truncated proteins (55). The p16INK4a gene is located
on chromosome 9q21 and encodes a cell cycle inhibitor that
prevents Rb protein hyperphosphorylation by inhibiting
Cyclin D1/CDK4 kinase activity. While RB inactivation is the
preferential mechanism in SCLCs, p16Ink4a abnormalities are
found frequently in NSCLC. Inactivation of p16INK4a is caused
by a variety of mechanisms including mutations, deletions or
promoter hypermethylation. The rate of mutation of p16INK4a is
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relatively low in NSCLC, while homozygous deletions have
been observed in 10 % to 40 % of the tumors (74). Inactivation
of p16 may result from homozygous or hemizygous deletion
(12, 67), inactivation of the remaining P16 allele by point
mutation (68), or gene silencing through methylation of CpG
islands surrounding the first exon of P16 (5). Methylation of
CpG sequences in the P16 gene provides a way of suppressing
expression of P16 in the absence of any mutation in the
DNA and has been referred to as epigenetic regulation (5).
Hypermethylation in the 5’-CpG island of the p16INK4a gene
has been suggested to cause the downregulation of p16INK4a
in lung cancer carrying no genetic mutations of p16INK4a (54).
Taken together, the inactivation rate of p16INK4a is 30 % to 70 %
in NSCLC (70). P16 may be silenced by DNA methylation in
the early stages of NSCLC, whereas homozygous deletions
and/or mutations may occur more frequently in later stages
of NSCLC development. The CyclinD1/Cyclin Dependent
Kinase 4 (CDK4) complex phosphorylates Rb and inactivates
the RB-binding activity of the transcription factors essential
for the G1/S entry (e.g. E2F1 protein). Overexpression of
Cyclin D1 is found in the majority of the NSCLC cell lines as
a result of abnormal gene amplification. In primary NSCLC
tumors, 12 % to 40 % of NSCLC tumors overexpress CyclinD1
through gene amplification or other mechanisms (69). The role
of CDK4 in lung cancer is unknown, but it has been reported
that CDK4 is expressed in about 90 % of NSCLCs and the
expression is associated with poor differentiation (49).
3P deletion and other chromosomal abnormalities.
Allelic loss at the short arm of chromosome 3 is one of the
most common and earliest events in the pathogenesis of lung
cancer (96). For example, loss of heterozygosity (LOH) of 3p
is present in 70 % to 100 % of NSCLC (44) and is observed
in more than 90 % of SCLCs (96). Interestingly, there is a
progressive increase in the frequency and size of 3p allele
loss with increasing histopathological changes. This suggests
that chromosome 3p may “harbor” multiple TSGs, which are
involved in the early stages of bronchial carcinogenesis (17,
102). Many candidate genes have been identified in these
regions: e.g. the von Hippel-Lindau gene (VHL) (70) located at
3p25, the FHIT/FRA3B gene located at 3p14.2, the RASSF1A
gene located at 3p21.3 (34), the retinoic acid receptor-ß
(RAR-ß) located at 3p24, and semaphorin SEMA3F and
ß-catenin gene, both located at 3p21.3 (11). The FHIT/FRA3B
(Fragile Histidine Triad) gene encodes a dinucleoside (Ap3A)
hydrolase (4). It has been mapped to chromosome 3p14.2 (97,
98) and encompasses approximately 1 Mb of genomic DNA
which includes the human common fragile site (FRA3B) (81).
FHIT is a candidate TSG on the basis of frequent LOH in
lung cancer (53) and homozygous deletions in NSCLC cell
lines (82). Small cell lung tumors (80 %) and non-small cell
lung tumors (40 %) show abnormalities in RNA transcripts of
FHIT, and 76 % of the studied tumors exhibit loss of FHIT
alleles (83). FHIT protein is absent in some precancerous
dysplastic lesions, suggesting that FHIT inactivation may
occur at an early phase of lung carcinogenesis (83, 98). More
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extensive genetic loss of FHIT is associated with increasing
severity of the histopathological grade (11, 98). There is also a
relation between tobacco use and loss of FHIT (98); however
it is not clear whether FHIT is a target or a marker of NSCLC
carcinogenesis (11).
RASSF1A (Ras ASSociation domain Family 1A). The
RASSF1A gene encodes a 38.8 kD protein containing a RAS
association (RalGDS/AF-6) domain (RA). RASSF1A is mapped
to 3p21.3. The expression of RASSF1A has been reported to be
epigenetically inactivated in 40 % to 63 % (49) of primary
NSCLCs and in 80 % to 100 % SCLC cell lines through
hypermethylation of the CpG island promoter sequence (19).
Conflicting data with regard to the clinical implications of the
loss of RASSF1A have been described. Tomizawa et al. (87)
reported that methylation of the RASSF1A promoter region
correlates with adverse survival, which could not be confirmed
by others (91).
Von Hippel–Lindau gene, (VHL) gene, located at
3p25. The gene is named after the familial cancer syndrome.
However, since the gene is rarely altered in NSCLC, it seems
not an important target in NSCLC carcinogenesis (70).
The retinoic acid receptor beta (RAR-beta). The RARB
gene mapped to chromosome 3, located at 3p24, encodes one
of the primary receptors for retinoic acid (92), which inhibits
the transcription of AP-1 and thus inhibits proliferation and
induces apoptosis. When silenced, the gene does not code a
receptor and retinoic acid cannot exert its effects (16). This
makes it an important signaling molecule in lung growth,
differentiation and carcinogenesis (92). In vitro experiments
revealed that the receptor shows growth-suppression activity
in a lung cancer cell line. RARB is inactivated in 72 % of
SCLC (16), and expression is downregulated in about 40 % to
60 % of primary NSCLCs. Some studies show that silencing
occurs via promoter hypermethylation (11) and that this gene
is inactivated mainly by cigarette smoking (16).
SEMA3F. The semaphorin SEMA3F gene was originally
cloned from a recurrent homozygous deletion involving the
chromosomal region 3p21.3 (60). This gene is s a member
of the semaphoring/collapsing family, a group of secreted or
membrane-associated proteins that contain a characteristic
500-amino-acid SEMA domain. SEMA3F was identified in
SCLC cell lines from a homozygous deletion. SEMA3F and
vascular endothelial growth factor (VEGF) isoforms compete
for the same receptor (45). In lung tumors and their premalignant lesions, an adverse correlation between SEMA3F
and VEGF has been demonstrated, with low levels of SEMA3F
and high levels of VEGF, leading to a possible growth
advantage. In lung cancer, reduced ß-catenin is associated with
lymph node metastasis and poor prognosis (11).
Loss of heterozygosity (LOH)
LOH is a common genetic feature of lung cancer and is a
significant mechanism by which critical genes involved in
growth regulation and homeostasis become inactivated, or
silenced, during disease evolution (105). The molecular
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basis of allelic loss in lung cancer has been suggested to be
DNA adducts (16). In a normal individual’s DNA, every
genetic locus is composed of two alleles. Most genetic loci
are composed of two homozygous alleles. A limited number
(only about 10 %) of genetic loci are heterozygous (the two
copies of that gene are different) (14). The loss of both copies
is called homozygous deletion and causes silencing of the
gene; the loss of one of them is defined as LOH (17). Allelic
loss may result in functional loss only when the retained allele
is deleted or silenced through methylation. Allelic loss occurs
at a homozygous site followed by a point mutation or gene
silencing (via promoter methylation in the remaining allele).
Loss or silencing of one allele can occur, usually through mitotic
recombination, at any locus, homozygous or heterozygous
(105). The result of deleterious genetic variations that have the
potential to cause disease depends on which tumor suppressor
genes are lost during the formation and progression of many
types of cancer, including lung cancer. LOH occurs in both
types of lung cancer, SCLC and SCLS (26).
Zander et al. (105) showed LOH on chromosomes 3p,
13q, and 17p frequently, in both SCLC (<90 %) and NSCLC
(<70%), which include (FHIT), p53, and (RB). Some studies
have linked exposure to asbestos to allelic deletion of the
FHIT gene on chromosome 3p14 and a significant reduction in
Fhit protein expression. In SCLC, LOH occurs at a relatively
high frequency on chromosomes 3p (>90 %), 5q (>50 %),
4q (>40 %), 10q (>80 %), 13q (>90 %), 15q (>40 %), and
17p (>90 %). LOH in NCSLC occurs at a high frequency
on chromosomes 1p (>60 %), 3p (>50 %), 8p (>70 %), 9p
(>70 %), 13q (>60 %), 17p (>80 %), 19p (>70 %), Xp (>60 %),
and Xq (>60 %) (105).
In general, more LOH alterations are detected in NSCLCs
than in SCLCs, and are a more common feature of squamous
cell carcinomas, with <90 % of them exhibiting LOH
(compared with >70 % of adenocarcinomas. LOHs at 8p, 9p,
11q, and 13q are early lesions that occur upon exposure to
tobacco smoke (105).
Loss of heterozygosity at 1p. Loss of heterozygosity
at 1p36 occurs in NSCLC more often than in SCLC, and
encompasses several candidate tumor suppressor genes,
including p73 and TNFR2. p73 is a member of the p53 family
and is capable of mimicking some of the effector functions
of p53, including induction of permanent growth arrest and
promotion of apoptosis. TNFR2 binds TNF and mediates
signals, regulating the induction of cell death (105).
LOH at 9p13 is an early event observed in the normal
appearing epithelium of smokers and in pre-invasive lesions
and is reversible after smoking cessation (40). The gene most
frequently identified in LOH (9p21) in NSCLC is p16INK4A.
It encodes a protein that inhibits Cdk4, Cdk6, and cyclindependent phosphorylation of the Rb gene product (84).
A study of the relation between smoking duration and the
mechanisms of gene silencing shows that LOH of 9p13 and
homozygous deletion is linearly correlated with smoking, and
is determined by innate susceptibility and smoking exposure
4056
(21). Clinical observations show that individuals with 9p21
LOH have better survival rates than those with homozygous
deletion (16). Interestingly, experimental evidence suggests
that the silencing of the remaining p16INK4A allele after
allelic loss is predominantly due to epigenetic methylation
rather than mutation (3).
LOH at 3p. The best-described and best-documented gene
targeted for LOH in nearly all lung cancers is FHIT on 3p14,
which we mentioned above in TSG. TSG candidates targeted
by LOH in the same region (3p12–3p22) as the FHIT gene
include (TGFRb2), MLH/HNPCC2, DLC1, RASSF1A, RARb,
and BRCA1-associated protein 1 (BAP1) (100, 105). LOH at
3p25, which includes the von Hippel–Lindau (VHL) locus,
has also been reported as a frequent event in both SCLC and
NSCLC (105).
LOH at 4q. In both of SCLC and NSCLC, chromosome 4q
is frequently targeted at two regions (4q21–28 and 4q34-ter).
Despite consistent observations of LOH in these areas, to
date clear tumor suppressor gene candidates have not been
identified (105).
LOH at 5q. In SCLC high frequency of LOH is
characterized at 5q32-ter. The SPARC gene maps to 5q32 and
has been associated with LOH SPARC protein (20), which is
involved in the regulation of cell adhesion and growth, but has
also been correlated with metastasis based on changes in cell
shape which can promote tumor cell invasion (61). Although
NSCLC has not been associated with LOH at 5q32, there is
often LOH at 5q21.3–31, which suggests that it may be an
early genetic change (105). The 5q21.3–31 region contains
several key TSG, including MCC, APC, and IRF, and changes
in this region are more prevalent in squamous cell carcinomas
than in adenocarcinomas (102). However, as for other LOH
events, studies have failed to provide a strong correlation
between these events and patient prognosis. In lung cancer, it
appears that LOH at the APC locus is relatively frequent but
the mechanism by which the remaining allele is silenced is
usually promoter methylation, rather than mutation (9).
LOH at 8p. LOH on 8p21–23 is a frequent and early
occurrence in NSCLC, and is believed to occur after LOH
events at 3p and 9p (93).
LOH at 10q. LOH at 10q22–23 is a frequent observation in
SCLC. Although no tumor suppressor genes in this region have
been definitively associated with lung cancer, multiple studies
have identified LOH targeted to the PTEN/MMAC locus at
10q23. The encoded PTEN protein is a lipid phosphatase that
negatively regulates the phosphatidylinositol 3-kinase/Akt
pathway. Loss of PTEN function results in reduced apoptosis
and stimulation of cellular proliferation and migration (93,
105).
LOH at 13q. Chromosome 13q12–14 is a prevalent
hotspot for LOH in both SCLC and NSCLC. The RB1 gene is
a well-characterized tumor suppressor gene at 13q12. LOH on
13q12.1–13.1 is proposed as one of a set of markers that could
© Biotechnol. & Biotechnol. Eq. 27/2013/5
be used for early detection of lung cancer in both SCLC and
NSCLC. Another gene targeted to 13q12–14 is BRCA2 (105).
LOH at 15q. LOH at 15q is a common occurrence in
SCLC but no tumor suppressor gene candidate has been
indicated (105).
LOH at 17p. The TP53 gene at 17p13 is the most frequently
altered tumor suppressor gene in human cancers, including
lung cancers. TP53 is targeted by mutation, methylation,
and homozygous deletion. TP53 mutations are found in
precancerous lesions, but allelic loss of TP53 occurs during
disease progression, after LOH events on chromosome 3p (93,
105).
LOH at 19p. LOH at 19p13.3 is a very frequent event in
NSCLC but is not targeted in SCLC. One tumor suppressor
gene candidate residing at this locus is STK11/LKB1, a gene
implicated in Peutz–Jeghers syndrome (105).
LOH at the X Chromosome. The X Chromosome is
targeted for LOH at Xp–q21 and Xq22.1 in NSCLC. Specific
tumor suppressor genes have not been mapped to these regions
or implicated in lung cancer (105).
DNA methylation in human lung cancer
DNA methylation is an epigenetic event whose pattern changes
frequently in a wide variety of human cancers, including
promoter-specific hypermethylation as well as genome-wide
hypomethylation. Normally, methylation of cytosines occurs
at distinct sites of the genome containing stretches of repeated
CpG (CpG islands) often found within promoter areas of
transcribed genes (10). Aberrant DNA methylation within CpG
islands is among the earliest and most common alterations in
human cancers, leading to abnormal expression of a broad
spectrum of genes (91). DNA methylation changes have been
reported in various genes implicated in lung cancer (13). DNA
methylation could provide one or two of the hits necessary
for inactivation of tumor suppressor genes, as postulated by
Knudson’s two-hit hypothesis for oncogenic transformation
(43). Over 100 genes, many of them tumor suppressor genes
such as RASSF1A, p16 (CDKN2A), MLH1, MGMT, BCL2,
DAPK, TCF21 and BMP3B among others, have been described
as aberrantly methylated and silenced in human lung cancer.
One of the first genes shown to be systematically methylated
in lung cancer was the p16INK4a tumor-suppressor gene. This
gene is mapped in Chromosome 9p, and might affect the early
stages of tumor development (10).
Toyooka et al. (89) observed differences in the
methylation status of lung cancer: the SCLS tumor had a
higher frequency of methylation of RASSF1A (TSG located
on 3p.21), compared to NSCLC tumors, while the frequency
of methylation of p16, APC, CDH13 were higher in NCSLC
than in SCLC. Squamous cancers had the highest frequency of
p16 methylation in the NSCLC group, while adenocarcinomas
had the highest frequency of APC and CDH13 methylation.
The APC methylation was associated with non-squamous
histology (89).
© Biotechnol. & Biotechnol. Eq. 27/2013/5
Vassière et al. (91) determine a high frequency of aberrant
hypermethylation in MTHFR, RASSF1A, and CDKN2A in lung
tumors, but no significant increase in the methylation levels
of GSTP1 and CDH1. These results are consistent with the
notion that epigenetic changes mediated by hypermethylation
are tumor-specific events (91). Tobacco smoking, sex and
alcohol intake have a strong influence on the methylation
levels of distinct genes (RASSF1A and MTHFR), whereas
age and histologic subtype have no significant influence on
methylation states (10). MTHFR hypermethylation in lung
cancer seems to be strongly associated with tobacco smoking,
whereas hypermethylation in CDH1, CDKN2A, GSTP1, and
RASSF1A is not associated with smoking, indicating that
tobacco smoke targets specific genes for hypermethylation
(91).
Lamy et al. (47) also demonstrate methylation of p16 in 19 %
of pre-invasive lesions of high-risk individuals, the frequency
increasing with the histological grade. The determinants of
aberrant methylation are not well characterized. Gilliand et al.
(31) hypothesize that the functional polymorphism of NADPH,
glutathione-S-transferase P1 and M1 myeloperoxidase and
XRCC1 genes is associated with p16 and MGMT methylation
in sputum. In humans, p16 methylation is found in situ in 75 %
of carcinoma lesions adjacent to squamous cell carcinoma
and the frequency of p16 methylation increases with the
progression of the disease (16).
Vassière et al. (91) suggest that various environmental
and lifestyle exposures, such as tobacco, Helicobacter pylori,
plutonium, or radon exposure, are suspected to be implicated
in the development of a wide range of human cancers by
eliciting DNA methylation changes.
Biomarkers in human lung cancer. Several research
groups investigated the possibility of utilizing DNA methylation
as a biomarker for early detection of lung cancer. This line of
research showed great potential, since for over two decades
now it has been known that metaplastic cells can be detected
in the sputum of patients with squamous cell carcinoma of
the lung (85). The outcome of these investigations has been
promising: several assays have been developed to detect
aberrant DNA methylation at the p16 locus (9), among others,
from bronchial lavage, sputum and serum of patients at risk of
developing lung cancer (current or former smokers). Notably,
it has been well-documented that aberrant p16 methylation can
be detected in patients several years before the onset of lung
cancer (91). This raises hopes that DNA methylation could
become a pan-cancer biomarker. Moreover, Shames et al. (73)
were able to identify aberrant DNA methylation signatures
common not only to lung, but also to breast, colon and prostate
cancers.
Telomerase. Telomerase is not expressed in normal
somatic tissues but is highly expressed in over 90 % of human
tumor cell lines and tumor tissues (42). It has been shown that
activation of telomerase is an essential step to the bypass of
replicative senescence and to the transformation of human
cells (33). Analysis of lung samples showed that telomerase
4057
activation was observed in 100 % of SCLCs and 85 % of
NSCLCs (36). In addition, reactivation of telomerase activity
may be an early event in lung tumorigenesis (42, 102). Since
the enzyme is not present in normal tissues (except germ cells),
it may become a good target for therapeutic intervention (94).
New candidate genes associated with lung cancer
Researchers suggest there are many candidate genes which
play an important role in the development of lung tumors.
More studies are needed, however, to better understand the
relations with tumors.
The study of Yang et al. (99) revealed that about 30 % of
patients who never smoked but developed lung cancer had the
same uncommon variant, or allele, of the GPC5 gene, which is
located on Chromosome 13 (13q32), The authors suggest that
the gene has an important tumor suppressor-like function and
that insufficient function can promote lung cancer development;
GPC5 may be a critical gene in lung cancer development and
genetic variations of this gene may significantly contribute to
increased risk of lung cancer. What is known about the GPC5
gene is that it can be overexpressed in multiple sclerosis, and
that alterations in the genome where GPC5 is located are
commonly found in different human tumors. Possibly, GPC5
might play different roles depending on the tissue type, during
various disease development and progression (99).
Hsu et al. (37) and Kendal et al. (41) have identified three
adjacent genes: TTF1, NKX2-8, and PAX9, that interact with
cancerous results in 20 % of lung cancers. These genes are
located on human chromosome 14 and two of them are known
to play key roles in fetal lung development. The three genes
apparently interact to reactivate an early fetal gene expression
pattern that leads to cancer tumor growth. The research team
attempt to explain why this mutation is so common in lung
cancer with the collaboration of the three genes and the
fact that they are very close together on the chromosome.
According to Kendal et al. (41), mutations in this region are
more prevalent in late stage lung cancer and are possibly a risk
factor for recurrence (41).
Inoue et al. (38) show the involvement of a gene known
as DMP1 in human lung cancer. The DMP1 gene normally
works to suppress tumor formation and is non-functional
in about 35 % of human lung cancers. According to earlier
studies in mice, the gene is involved in activating the tumor
suppressors p53 and Arf (51). When the DMP1 gene is not
functional, these tumor suppressors are probably not available
to stop tumor growth by killing cancer cells (38). Deletion of
one copy of DMP1 has been identified in 30 % to 40 % of mice
lung tumors as well as in human NSCLCs (51). Inoue et al.
(38) suggest that the gene may also be a target for future drug
development, since high expression of DMP1 significantly
inhibits the growth of some lung cancer cells.
The study of Tong et al. (88) on TIP30 expression in paired
cancerous and non-cancerous lung tissue suggests that TIP30
is a putative tumor suppressor gene with decreased expression
in numerous cancers including melanoma, breast cancer, and
4058
colon cancer. TIP30 expression was decreased in a third of
NSCLCs compared with normal controls, and reduced TIP30
expression correlated with lymph node metastasis. The authors
propose that TIP30 may function as a tumor suppressor gene
and may play important roles in suppressing the progression
and metastasis of lung cancer. These findings highlight TIP30
as a potential new therapeutic for metastatic lung cancer (88).
Carretero et al. (13) recently showed that loss of the LKB1
tumor suppressor gene in a significant population of lung
tumors results in metastasis in mice. The LKB1 gene has also
been linked with about 30 % of human lung cancers (13). Loss
of Lkb1 in mouse and human lung cancer cells (progression
of NSCLC) is associated with an increase in the activity of
proteins that are known to modulate cell motility and adhesion.
Importantly, combined pharmacological inhibition of these
key regulatory proteins in Lkb1-deficient cells decreases cell
migration and induces tumor regression (13).
Dooley et al. (23), using a new technique called wholegenome profiling, managed to pinpoint a gene that appears to
drive progression of SCLC, from 15 % of lung cancer cases.
The authors found extra copies of a few short stretches of
DNA, including a segment of chromosome 4 that includes the
Nuclear Factor I/B (NFIB) gene. This is the first time NFIB
has been implicated in small cell lung cancer, although it has
been seen in a mouse study of prostate cancer. Little is known
about the gene’s exact function, except that it is involved in the
development of lung cells. It codes for a transcription factor,
i.e. it controls the expression of other genes, which remain to
be identified in the hope of providing new targets for SCLC
therapy (23).
Conclusions
Cytogenetic and molecular studies indicate that multiple
genetic lesions occur during lung cancer pathogenesis.
Chromosome instability can be observed early in cancer
development and is associated with poor prognosis. In
fact, chromosomal abnormalities, activation of dominant
oncogenes, and inactivation of tumor suppressor genes have
been described. The chromosomal aberrations present in
NSCLC and SCLC cells include aneuploidy, chromosomal
translocations, and amplifications. Aberrations and genetic
changes play a role in different histologic types. Oncogene
genetic defects occur in NSCLC and SCLC cells. The cellular
pathway involving RAS oncogenes appears to be activated in
all NSCLCs, but not in SCLC or very rarely. Amplification
and overexpression of MYC family genes seems to be more
prevalent in tumor cell lines than in tumor tissues, and in
SCLCs than in NSCLCs. The ERBB2 and the EGFR genes are
overexpressed in all NSCLCs. The tumor suppressor genes RB
and TP53 are almost universally abnormal, with mutations in
SCLC, but less commonly so in NSCLC. Loss of heterozygosity
(LOH) is another common genetic feature of lung cancer
and is a key mechanism by which critical genes involved
in growth regulation and homeostasis become inactivated,
or silenced, during tumor evolution. LOH is slightly more
© Biotechnol. & Biotechnol. Eq. 27/2013/5
frequent in SCLC than in NSCLC. DNA methylation is an
epigenetic aberration event whose pattern changes frequently
in different lung cancers. There are many candidate genes
which play an important role in the development of lung
tumors. The significance of these patterns is unclear as yet,
and more studies are needed to better understand the relations
with tumors. In the future, it may become possible to define
unique molecular patterns and pathways responsible for the
development of distinct malignancies of the lung, and thus,
to assemble rational, molecularly based therapies. Although
gene replacement therapy may be impractical for tumor
suppression, better understanding of the underlying molecular
mechanisms may make it possible to intervene at other steps
along the pathway, or to target the mutant oncogene products
themselves by means of immunotherapy.
REFERENCES 1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
Ahrendt S.A., Decker P.A., Alawi E.A., Zhu Yr Y.R., et al.
(2001) Cancer, 92, 1525-1530.
Al Zeyadi M. (2008) Tissue Microarray Analysis of EGFR
Copy Number Changes In Lung Cancer, MSc. Thesis, Sofia
University, Bulgaria.
Awaya H., Takeshima Y., Amatya V.J., Furonaka O., et al.
(2004) Pathol. Int., 54(7), 486-489.
Barnes L.D., Garrison P.N., Siprashvili Z., Guranowski A.,
et al. (1996) Biochemistry, 35, 11529-11535.
Belinsky S.A., Nikula K.J., Palmisano W.A., Michels R., et
al. (1998) P. Natl. Acad. Sci. USA, 95, 11891-11896.
Bennett W.P., Colby T.V., Travis W.D., Borkowski A., et al.
(1993) Cancer Res., 53, 4817-4822.
Bergh J.C. (1990) Am. Rev. Respir. Dis., 142(6 Pt 2), S20-26.
Berrieman H.K., Ashman J.N., Cowen M.E., Greenman J.,
et al. (2004) Brit. J. Cancer, 90, 900-905.
Brabender J., Usadel H., Danenberg K.D., Metzger R., et al.
(2001) Oncogene, 20(27), 3528-3532.
Brena R.M. (2007), Aberrant DNA, PhD Dissertation, Ohio
State University, pp. 6-60.
Breuer R.H., Postmus P.E., Smit E.F. (2005) Respiration,72(3),
313-330.
Cairns P., Polascik T.J., Eby Y., et al. (1995) Nat. Genet., 11,
210-212.
Carretero J., Shimamura T., Rikova K., Jackson A.L., et al.
(2010) Cancer Cell, 17, 547-559.
Cecil R.L., Goldman L., Ausiello D.A. (2007) Cecil Textbook
of Medicine, 23rd Ed., W.B. Saunders Elsevier, Philadelphia.
Chang P.M., Yeh Y.C., Chen T.C., Wu Y.C., et al.(2013) Ann.
Surg. Oncol., (in press).
Cherneva R., Dimova I. (2006) Balk. J. Med. Gen., 9(1-2),
9-17.
Chung G.T., Sundaresan V., Hasleton P., Rudd R., et al.
(1995) Oncogene, 11, 2591-2598.
Cooper D.N. (2005) The Molecular Genetics of Lung Cancer,
Springer, Berlin, Heidelberg, NY.
Dammann R., Takahashi T., Pfeifer G.P. (2001) Oncogene,
20, 3563-3567.
© Biotechnol. & Biotechnol. Eq. 27/2013/5
20. Demopoulos K., Arvanitis D.A., Vassilakis D.A., Siafakas
N.M., Spandidos D.A. (2002) J. Cell Mol. Med., 6(2), 215-222.
21. Denissenko M.F., Pao A., Pfeifer G.P., Tang M. (1998)
Oncogene, 16(10), 1241-1249.
22. Devereux T.R., Taylor J.A., Barrett J.C. (1996) Chest, 109(3
Suppl.), 14S-19S.
23. Dooley A.L., Winslow M.M., Chiang D.Y., Banerji S., et al.
(2011) Gene. Dev., 25(14), 1470-1475.
24. Dosaka-Akita H., Hu S.X., Fujino M., Harada M., et al.
(1997) Cancer, 79, 1329-1337.
25. Engelman J.A., Zejnullahu K., Mitsudomi T., Song Y., et
al.(2007) Science, 316, 1039-1043.
26. Fong K.M., Sekido Y., Minna J.D. (1999) J. Thorac. Cardiov.
Sur., 118(6), 1136-1152.
27. Frame S., Balmain A. (2000) Curr Opin Genet Dev, 10, 106-113.
28. Frederick L., Eley G., Wang X.Y., James C.D. (2000) NeuroOncology, 2, 159-163.
29. Fröhling S., Döhner H. (2008) New Engl. J. Med., 359, 722-734.
30. Gene Cards (2013) GPC5 TP53 gene <http://www.genecards.
org/cgi-bin/carddisp.pl?gene=GPC5>
31. Gilliland F.D., Harms H.J., Crowel R.E., Li Y.F., Willink
R., Belinsky S.A. (2002) Cancer Res., 62(8), 2248-2252.
32. Gschwind A, Fischer O.M., Ullric A. (2004) Nat. Rev. Cancer,
4, 361-370.
33. Hahn W.C., Counter C.M., Lundberg A.S., Beijersbergen
R.L,, et al. (1999) Nature, 400(6743), 464-468.
34. Hibi K., Takahashi T., Yamakawa K., Ueda R., et al. (1992)
Oncogene,7, 445–449.
35. Hirsch F.R., Varella-Garcia M., Bunn P.A. Jr., Di Maria
M.V., et al. (2003) J. Clin. Oncol., 21(20), 3798-3807.
36. Hiyama K., Hiyama E., Ishioka S., Yamakido M., et al.
(1995) J. Natl. Cancer I., 87, 895-902.
37. Hsu D.S., Acharya C.R., Balakumaran B.S., Riedel R. F., et
al. (2009) P. Natl. Acad. Sci. USA, 106(13), 5312-5317.
38. Inoue K., Sugiyama T., Taneja P., Morgan R.L., Frazier
D.P. (2008) Cancer Res., 68(12), 4487-4490.
39. Jemal A., Bray F., Center M.M., Ferlay J., et al. (2011) CA
Cancer J. Clin., 61(2), 69-90.
40. Kalomenidis I., Orphanidou D., Papamichalis G.,
Vassilakopoulos T., et al. (2001) Lung, 179(5), 265-278.
41. Kendall J., Liu Q., Bakleh A., Krasnitz A., et al. (2007) P.
Natl. Acad. Sci. USA, 104(42), 16663-16668.
42. Kim N.W., Piatyszek M.A., Prowse K.R., Harley C.B., et al.
(1994) Science, 266, 2011-2015.
43. Knudson A.G. (2001) Nat. Rev. Cancer, 1, 157-162.
44. Kok K., Osinga J., Carritt B., Davis M.B., et al. (1987)
Nature, 330(6148), 578-581.
45. Kolodkin A.L., Levengood D.V., Rowe E.G., Tai Y.T., Giger
R.J., Ginty D.D. (1997) Cell, 90, 753-762.
46. Krystal G., Birrer M., Way J., Nau M., et al. (1988) Mol. Cell
Biol., 8, 3373-3381.
47. Lamy A., Sesboue R., Bourgignon J., Datur B., et al. (2002)
Int. J. Cancer, 100(2), 189-193
48. Lechner J.F., Fugaro J.M. (2000) In: Lung Cancer. Principles
and Practice, 2nd Ed. (H.I. Pass, J.B. Mitchell, et al. Eds.),
Lippincott Williams & Williams, Philadelphia, 89-97.
49. Lingfei K., Pingzhang Y., Zhengguo L., Jianhua G., Yaowu
Z.A. (1998) Cancer Lett., 130, 93-101.
4059
50. Ma P.C., Kijima T., Maulik G., Fox E.A., et al. (2003) Cancer
Res., 63, 6272-6281.
51. Mallakin A., Sugiyama T., Taneja P., Matise L.A., et al.
(2007) Cancer Cell, 12(4), 381-394.
52. Marsit C.J., Zheng S., Aldape K., Hinds P.W., et al. (2005)
Hum Pathol., 36(7), 768-776.
53. Matakidou A., Eisen T., Houlston R.S. (2005) Br. J. Cancer.
93(7), 825-833
54. Merlo A., Herman J.G., Mao L., Lee D.J. , et al. (1995) Nat.
Med., 1, 686-692.
55. Mori N., Yokota J., Akiyama T., Sameshima Y. (1990)
Oncogene, 5, 1713-1717.
56. Morstyn G., Brown J., Novak U., Gardner J., et al. (1987)
Cancer Res., 47, 3322-3327.
57. Murphy D.J., Junttila M.R., Pouyet L., Karnezis A., et al.
(2008) Cancer Cell, 14(6), 447-457.
58. National Center for Biotechnology (2012) UniGene
entry for cyclin-dependent kinase inhibitor 1A (p21, Cip1)
<http://www.ncbi.nlm.nih.gov/UniGene/clust.cgi?ORG
=Hs&MAXEST=999999&CID=370771> .
59. Pao W., Mller V.A. (2005) J. Clin. Oncol., 23(11), 2556-2568.
60. Potiron V.A., Sharma G., Nasarre P., Clarhaut J.A, et al.
(2007) Cancer Res., 67, 8708-8715.
61. Puisieux A., Lim S., Groopman J., Ozturk M. (1991) Cancer
Res., 51, 6185-6189.
62. Puisieux A, Valsesia-Wittmann S, Ansieau S.(2006), A twist
for survival ,Br J Cancer, 16;94(1):13-7.
63. Richardson G.E., Johnson B.E.(1993) Semin. Oncol., 20,
105-127.
64. Robles A.I., Linke S.P., Harris C.C. (2002) Oncogene, 21,
6898-6907.
65. Roengvoraphoj M., Tsongalis G.J., Dragnev K.H., Rigas
J.R. (2013) Cancer Treat. Rev., (in press).
66. Rom W.N., Hay J.G., Lee T.C., Jiang Y., Tchou-Wong K.M.
(2000), Molecular and genetic. Am. J. Resp.ir Crit Care Med ,
161, :1355–-1367.
67. Rom W.N., Tchou-Wong K.-M. (2003) Methods in Molecular
Medicine, 75, 3-28.
68. Rusin M.R., Okamoto A., Chorazy M., Czyzewski K. (1996)
Int. J. Cancer, 65(6), 734-739.
69. Schauer I.E., Siriwardana S., Langan T.A., Sclafani R.A.
(1994) P. Natl. Acad. Sci. USA, 91, 7827-7831.
70. Sekido Y., Bader S., Latif F., Gnarra J.R., et al. (1994)
Oncogene, 9, 1599-1604.
71. Sen S. (2000) Curr. Opin. Oncol., 12(1), 82-88.
72. Sequist L.V., Joshi V.A., Janne P.A., Muzikansky A., et al.
(2007) Oncologist, 12(1), 90-98.
73. Shames D., Girard L., Gao B., Sato M., et al. (2006) PLoS
Med., 3(12), e486.
74. Shapiro G.I., Park J.E., Edwards C.D., Mao L., et al. (1995)
Cancer Res., 55, 6200-6209.
75. Sharpless N.E., DePinho R.A. (2002) Cell, 110, 9-12.
76. Shepherd F.A., Bunn P.A., Paz-Ares L. (2013) Am Soc Clin
Oncol Educ Book,339-46.
77. Sherr C.J. (2004) Cell, 116, 235-246.
78. Shigematsu H., Lin L., Takahashi T., Nomura M., et al.
(2005) J. Natl. Cancer I., 97(5), 339-346.
4060
79. SjalanderA., Birgander R., Rannug A., Alexandrie A., et al.
(1996) Hum. Hered., 46, 221-225.
80. Skaug V., Ryberg D., Kure E., Arab M., et al. (2000) Clin.
Cancer Res., 6(3), 1031-1037.
81. Smith D.I., Huang H., and Wang L. (1998) Int. J. Oncol., 12,
187-196.
82. Song L., Turkson J., Karras J.G., Jove R., Haura E.B.
(2003) Oncogene, 22, 4150-4165.
83. Sozzi G., Veronese M.L., Negrini M., Baffa R., et al. (1996)
Cell, 85, 17-26.
84. Sumitomo K., Shimizu E., Shinohara A., Yokota J., Sone S.
(1999) Int. J. Oncol.,14(6), 1075-1080.
85. Swank P.R., Greenberg S.D., Hunter et al. (1989) Diagn.
Cytopathol, 5, 98-103.
86. Tal M., Wetzler M., Josefberg Z., Gutman M., et al. (1988)
Cancer Res., 48, 1517- 1520.
87. Tomizawa Y., Kohno T., Kondo H., Otsuka A., et al. (2002)
Clin Cancer Res., 8, 2362-2368.
88. Tong X., Li K., Luo Z., Lu B., et al. (2009) Am. J. Pathol.,
174(5), 1931-1939.
89. Toyooka S., Toyooka K.O., Maruyama R., Virmani A., et al.
(2001) Mol. Cancer Ther., 1(1), 61-67.
90. Tsai C.M., Levitzki A., Wu L.H., Chang K.T., et al. (1996)
Cancer Res., 56, 1068-1074.
91. Vaissiere T., Hung R.J., Zaridze D., Moukeria A., et al.
(2009) Cancer Res., 69, 243-252.
92. Vuillemenot B.R., Pulling L.C., Palmisano W.A., Hutt J.A.,
Belinsky S.A. (2004) Carcinogenesis, 25(4), 623-629.
93. Wagner B.J., Presnell S.P. (2009) In: Basic Concepts of
Molecular Biology (T.C. Allen, P.T. Cagle, Eds.), Springer,
Dodrecht, 97-108.
94. Wang M. (2003) Fine Mapping and Candidate Gene Analyses
of Murine Lung Tumor Susceptibility Genes, PhD Dissertation,
Ohio State University, USA, pp. 2-73.
95. Weiner D.B., Kern J.A., Schwartz D.A., Nordberg J.E., et
al. (1990) Cancer Res., 50, 5184-5187.
96. Whang-Peng J., Kao-Shan C.S., Lee E.C., Bunn P.A., et al.
(1992) Science, 215, 181-182.
97. Wistuba I.I., Behrens C., Virmani A.K., Mele G., et al.
(2000) Cancer Res., 60, 1949-1960.
98. Yanagisawa K., Kondo M., Osada H., Uchida K., et al.
(1996) Cancer Res., 56, 5579-5582.
99. Yang P. (2011) Sem. Resp. Crit. Care M., 32(1), 10-21.
100. Yang T.L., Su Y.R., Huang C.S., Yu J.C., et al. (2004) Gene.
Chromosome. Canc., 41(3), 250-256.
101. Yarden Y., Sliwkowski M.X. (2001) Nat. Rev. Mol. Cell Biol.,
2(2), 127-137.
102. Yashima K., Litzky L.A., Kaiser L., Rogers T., et al. (1997)
Cancer Res., 57, 2373-2377.
103. Yen C.C., Liang S.C., Jong Y.J., Chen Y.J., et al. (2007)
Lung Cancer, 57(3), 292-301.
104. Yuen K.W.Y. (2010) In: eLS, John Wiley & Sons Ltd,
Chichester <http://www.els.net>
105. Zander D.S., Popper H.H., Jagirdar J., Haque A., Barrios R.
(Eds.) (2008) Molecular Pathology of Lung Diseases, Springer,
New York.
106. Zhang Z. (2010) Study of Molecular Mechanisms, PhD
Dissertation, Case Western Reserve University.
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