36_01_03

Other Biologic Abnormalities for Lung Cancer Development

Telomerase Activity

During normal cell division, telomere shortening leads to cell
senescence and thus governs normal cell "mortality." Telomeres are
maintained in normal stem cells by the enzyme telomerase. However, with
abnormal expression of the enzyme in, for example, tumors, telomerase
has been implicated in contributing to human cell immortalization and
cancer cell pathogenesis. Telomerase is a ribonucleocomplex, and
ectopic expression in tumors of its catalytic subunit, human telomerase
reverse transcriptase, appears critical for the cellular
immortalization typical of cancer cells. Using the highly sensitive
telomere replication amplification protocol assay, approximately 100%
of SCLCs and 80% to 85% of NSCLCs were demonstrated to express high
levels of telomerase activity. High telomerase activity was associated
with increased cell proliferation rates and advanced stage in NSCLCs.
[ref: 48] Further tests must be performed to conclusively demonstrate
that true telomerase-negative NSCLCs exist. If these telomerase-
negative tumors truly exist, debulking therapy with surgery and
radiotherapy should be considered, even for metastatic disease. It
would be predicted that eventually such tumors would "senesce" and stop
growing when their telomeres got too short. Telomerase activity and
expression of its RNA component are also dysregulated in carcinoma in
situ lesions associated with lung cancer, indicating that the timing of
telomerase activation for lung cancer development can occur in
preneoplasia. [ref: 49] Because of the nearly ubiquitous expression of
telomerase in human tumors, including lung cancer, there is much
interest in developing anti-telomerase drugs as new therapeutics.

Apoptosis

Unlike normal cells, tumors have acquired the ability to escape from
programmed cell death (apoptosis), which usually occurs under adverse
conditions such as DNA damage. In addition to p53, other molecules of
the complex apoptotic signaling pathways are abnormal in lung cancer
cells. For example, the anti-apoptotic gene, BCL2, can be abnormally
overexpressed in SCLCs (75% to 95%) and some NSCLCs (25% to 35% of
squamous cell carcinoma and an approximately 10% of adenocarcinoma).
Because of the potent role BCL2 plays in suppressing apoptosis and thus
also in inhibiting responses to chemotherapy and radiotherapy, there is
considerable ongoing effort to develop antisense BCL2 therapeutics,
which are entering clinical trials. Also, extracellular matrix proteins
may protect SCLC against chemotherapy-induced apoptosis via beta(1)
integrin-stimulated tyrosine kinase activation.[ref: 50] In addition,
Fas (CD95) and its ligand (FasL), which play key roles in the
initiation of one apoptotic pathway, also have been implicated in lung
cancer.[ref: 51] In this case, lung cancers express Fas ligand but not
the receptor, whereas T cells express the receptor. In this way, lung
cancers could cause the clonal deletion of immune T cells that were
directed against lung cancer antigens and thus provide a mechanism for
escape from immune surveillance.

Metastasis and Angiogenesis

Many potential factors influencing metastasis from primary lung cancers
have been studied, including cell adhesion molecules. For instance,
reduced E-cadherin expression, which can occur by promoter
hypermethylation, was associated with tumor dedifferentiation,
increased lymph node metastasis, and poor survival in NSCLC patients.
[ref: 52] Reduced alpha(3) integrin expression correlated with a poor
prognosis of patients with lung adenocarcinoma.[ref: 53] Specific CD44
isoforms may be associated with lung cancer metastasis. Meanwhile,
matrix metalloproteinases inducing stromal degradation may also be
involved in lung cancer invasion: Gelatinase A expression was observed
in approximately 50% of SCLCs and 65% of NSCLCs, and stromelysin-3
overexpression was detected in stromal elements of primary NSCLCs.
Because of this expression, several ongoing clinical trials of matrix
metalloproteinase inhibitors in the treatment of lung cancer are
ongoing. Finally, multiple other as yet unidentified genes may also be
involved in lung tumor progression and metastases, as evidenced by the
development of additional allelic losses (at 2q, 9p, 18q, and 22q) in
brain metastases compared to the primary NSCLCs in the same
patients.[ref: 54]
Tumor angiogenesis is necessary for a tumor mass to grow beyond a few
millimeters in size. Currently, angiogenesis is thought to be regulated
by the balance of inducers and inhibitors that are released by both
tumor cells and host cells. Vascular endothelial growth factor (VEGF)
and basic fibroblast growth factor are two major angiogenesis inducers
produced by human lung cancers. [ref: 55] Angiogenic CXC chemokines,
such as interleukin-8, also have been implicated in lung cancer.
Mutations in p53 lead to decreased expression of thrombospondin, a
negative regulator of angiogenesis. Overall it is thought that lung
cancers produce factors that stimulate angiogenesis and stop producing
others that would inhibit this process. Thus, tumor angiogenesis has
become a major new therapeutic target for lung cancer. Clinical trials
with humanized recombinant anti-VEGF monoclonal antibody combined with
chemotherapy in NSCLC are already ongoing. In addition, several new
small molecules that inhibit the tyrosine kinase activity of the VEGF
receptor(s) are entering clinical trials as new drugs.

Carcinogens in Tobacco Smoke and Genetic Susceptibility to Lung Cancer
(Genetic Epidemiology)

The major cause of lung cancer, of course, comes from smoking, and
tobacco smoke contains many substances, including carcinogens, co-
carcinogens, and tumor promoters. Among them, 20 carcinogens
convincingly cause lung tumors in laboratory animals or humans and are
likely to be involved in lung cancer induction. [ref: 56] The
carcinogenic effects of tobacco smoke in the lung involve the induction
of carcinogen-activating and inactivating enzymes, as well as covalent
DNA adduct formation, which may cause DNA misreplication and mutation.
DNA adducts have been identified in the bronchial tissue of lung cancer
patients, and adduct levels correlate with the amount of tobacco smoke
exposure. Of great importance is preventing children from starting to
smoke. In this regard, it was found that, in former smokers, age at
smoking initiation was inversely associated with DNA adduct
levels.[ref: 57] Thus, after controlling for the amount of smoking, the
earlier one started smoking, the worse the long-term damage. In
addition, for reasons that are not yet clear, it appears that women are
more susceptible to developing lung cancer from cigarette smoking than
men. [ref: 58] Of the three major classes of carcinogens in tobacco
smoke (polycyclic aromatic hydrocarbons, such as benzo[a]pyrene;
nitrosamines; and aromatic amines), much interest focuses on the
nitrosamines, especially 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone
(NNK), partly because it induces tumors of the lung--primarily adenomas
and adenocarcinomas--independent of the route of administration in
mice.[ref: 56]
The finding that not every heavy smoker develops lung cancer has led
to the concept of interindividual variation and the hypothesis that
individuals may exhibit genetic polymorphisms in carcinogen-
metabolizing pathways that determine individual lung cancer risk.
Clearly, such genetic susceptibility operates in close interaction with
smoking and other external carcinogenic factors ("gene-environment"
interaction, with smoking being the primary environment factor). Among
genes for carcinogen-metabolizing enzymes, polymorphisms in the
cytochrome P-450 genes CYP1A1, CYP2D6, CYP2E1 and in mu-class
glutathione S-transferase (GSTM1) have received the most attention.
Although studies have suggested that there may be a modest association
of GSTM1 null polymorphism with lung cancer,[ref: 59] studying single
candidate genes may not be adequate to predict lung cancer risk due to
complexity of carcinogen metabolism and gene-gene and gene-environment
interactions. [ref: 56] In addition to carcinogens, it also appears
that persons may inherit different susceptibility to become addicted to
nicotine, for example, through polymorphisms in one of the dopamine
receptors. Overall, molecular epidemiology, aided by DNA microarray
technology together with the Human Genome Project, should in the near
future help to identify individuals at highest risk of developing lung
cancer. Such information will be of great value in new lung cancer
screening trials (for example with spiral CT scans) and in
chemoprevention trials.

Molecular Changes in Preneoplasia

Before clinically recognizable lung cancer develops, a series of
morphologically distinct changes (hyperplasia, metaplasia, dysplasia,
and carcinoma in situ) can be observed in the bronchial epithelium of
smokers. It is felt that dysplasia and carcinoma in situ represent true
preneoplastic (precancerous) changes. These sequential changes found
with squamous cell cancers arising from central bronchi have long been
recognized, whereas other changes in peripheral bronchioles and alveoli
(adeno- and large cell cancers), such as adenomatous and alveolar
hyperplasia, are more recently described.
It is now clear that preneoplastic cells contain several genetic
abnormalities identical to some of the abnormalities found in overt
lung cancer cells. Immunohistochemical analysis has confirmed abnormal
expression of protooncogenes (cyclin D1) and TSGs (p53) in these
lesions.[ref: 60] Allelotyping of precisely microdissected,
preneoplastic foci of cells shows that 3p allele loss is currently the
earliest known change, suggesting that one or more 3p TSGs may act as
"gatekeepers" for lung cancer pathogenesis. This loss is followed by 9p
allele loss, 8p allele loss, and 17p allele loss (and p53 mutation)
(Fig. 31.1_2). [ref: 61] Even the histologically normal bronchial
epithelium adjacent to cancers has been shown to have genetic losses.
Similarly, atypical alveolar hyperplasia, the potential precursor
lesion of adenocarcinomas, also harbors KRAS mutations. [ref: 62] These
observations are also consistent with the multistep model of
carcinogenesis and a "field cancerization" process, whereby the whole
tissue region is repeatedly exposed to carcinogenic damage (tobacco
smoke) and is at risk for developing multiple separate foci of
neoplasia. Although all types of lung cancers have associated molecular
abnormalities in their normal and preneoplastic lung epithelium, small
cell lung cancer patients in particular appear to have multiple genetic
alterations occurring in their histologically normal-appearing
respiratory epithelium. Molecular changes have been found not only in
the lungs of patients with lung cancer but also in the lungs of current
and former smokers without lung cancer.[ref: 63,64] These molecular
alterations are thus important targets for use in the early detection
of lung cancer and for use as surrogate biomarkers in following the
efficacy of lung cancer chemoprevention. In this regard, it appears
that the smoke-damaged lung has thousands of multiple clonal or
subclonal patches of approximately 90,000 cells each in the respiratory
epithelium containing clones of cells with 3p and other allele loss
abnormalities. [ref: 65]

Molecular Tools in the Lung Cancer Clinic

Our understanding of the molecular genetic changes in lung cancer
pathogenesis is advancing rapidly. Some abnormalities also occur in
other human cancers, whereas others appear more specific for lung
cancer. Where their biochemical function is known, the proteins
rendered abnormal appear to fall into several growth regulatory
pathways. Thus, the "wiring" diagram of the lung cancer cell is
becoming clear. There is a substantial effort to translate this current
scientific knowledge of these abnormalities from the bench to the
bedside. These approaches fall into four general categories:
1. Identification of persons at highest risk of developing lung
cancer to enable chemoprevention and intensified smoking cessation
efforts. In this regards, with improved methods of molecular
identification of true precancerous lesions, our paradigm will become
"treatment" of precancerous lesions rather than "chemoprevention."
Obviously, because this treatment would occur in individuals without
clinically evident cancer, the treatments must have low toxicity and
high risk-benefit ratios.
2. Early detection tools to identify primary and recurrent disease
(e.g., polymerase chain reaction-based molecular methods for testing
body fluids). [ref: 5,66] Again, such "early detection" of invasive but
clinically occult disease would require careful analysis of risk-
benefit ratios. Because only one out of ten cigarette smokers
eventually develops lung cancer, the identification of persons with a
genetic susceptibility to lung cancer should allow targeting and
intensification of smoking cessation, early detection, and
chemoprevention efforts. In this regard, the encouraging new
information on spiral CT scanning for the early detection of lung
cancer[ref: 67] should be greatly targeted and enhanced by combining
radiologic screening with identification of genetic epidemiology
markers and acquired respiratory genetic alterations to identify the
individuals at highest risk.
3. Identification of prognostic biomarkers [ref: 68] that would also
include markers that would predict the response to various therapies
such as chemo- and radiotherapy.
4. The designing of new cancer-specific therapies based on knowledge
of genetic abnormalities. This includes replacing mutant TSGs;
developing drugs targeted against activated protooncogenes; interfering
with autocrine or paracrine loops; and inhibiting angiogenesis,
metastasis, and apoptotic pathways in cancer cells. Although new
therapies may be dramatically effective, it is probably more reasonable
to assume that they would complement rather than replace existing
therapies.

36_01_02

Protooncogenes and Growth Stimulation

The activation of protooncogenes requires mechanisms such as gene
amplification or point mutation of a single allele leading to
constitutive overexpression. In some cases, however, the mechanisms
causing overexpression are still unclear. Protooncogene products
include several growth factor receptors, such as epidermal growth
factor receptor (EGFR), ERBB2, KIT, and MET. Indeed, many growth
factor/receptor systems are expressed by either the lung tumor or
adjacent normal cells, thus providing autocrine or paracrine growth
stimulatory loops (Fig. 31.1_1). For example, overexpression of the
EGFR encoded by the ERBB1 gene is more common in NSCLC than SCLC and
may be related to tumor stage and differentiation. Coexpression of EGFR
and their ligands, especially transforming growth factor-alpha (TGF-
alpha), by lung cancer cells indicates the presence of an autocrine
(self-stimulatory) growth factor loop. Overexpression of EGFR occurred
in 70% of NSCLCs, and overexpression of both EGFR and TGF-alpha
occurred in 38%.[ref: 6] Clinically, the presence of this loop had no
significant impact on overall survival in early-stage NSCLCs and thus
seems to play a role in lung tumor formation rather than tumor
progression. [ref: 6] ERBB2 (HER2/neu) is highly expressed in more than
one-third of NSCLCs, especially adenocarcinomas. Its expression
correlates with a shorter survival in lung adenocarcinomas and may also
be a marker for intrinsic multidrug resistance in NSCLC cell lines.
[ref: 7] KIT and its ligand, stem cell factor (SCF), are both
preferentially expressed in many SCLCs. Activation of this putative
autocrine loop may provide a growth advantage, or it may mediate
chemoattraction. The SCF/KIT signal transduction pathway has been shown
to be associated with Lck, a src-related tyrosine kinase. [ref: 8] MET
and its ligand, hepatocyte growth factor (HGF), are involved in fetal
lung development. Coexpression of this putative loop is observed in
most NSCLCs, and high HGF levels were associated with a poor outcome in
resectable NSCLC patients. [ref: 9] There are several clinical
applications of these growth factor receptor abnormalities that have
occurred in other cancers, which also need to be explored as new
treatments for lung cancer. These include treatment with humanized
monoclonal anti-HER-2/neu receptor antibody Herceptin alone or in
combination with chemotherapy, treatment with monoclonal anti-EGF
receptor antibody combined with radiotherapy, and new drugs that would
inhibit the tyrosine kinase activity of these receptors.
Apart from protooncogene products, other growth stimulatory loops are
found in lung cancer. The best known is that governed by gastrin-
releasing peptide (GRP) and other bombesin-like peptides, together with
their receptors, which participates in lung development and repair as
well as promoting SCLC growth via an autocrine loop. This loop
represents a future therapeutic target because a clinical trial of an
anti-GRP monoclonal antibody showed some antitumor activity in patients
with previously treated SCLC. [ref: 10]
Because downstream effectors are needed to transduce incoming growth
factor/receptor signals to the nucleus, it is not surprising that
cytoplasmic signal transduction cascades are also implicated in
carcinogenesis. For instance, the receptor tyrosine kinases initially
signal the guanosine triphosphate-binding RAS protein. The RAS gene
family (KRAS, HRAS, and NRAS) can be activated by point mutations at
codons 12, 13, or 61, and one member of this family is mutated in
approximately 20% to 30% of NSCLC (particularly adenocarcinomas) but
probably never in SCLC.[ref: 11] KRAS accounts for 90% of the RAS
mutations in lung adenocarcinomas, with approximately 85% of the KRAS
mutations affecting codon 12. Characteristically, approximately 70% of
KRAS mutation are G to T transversions, with the substitution of the
normal glycine (GGT) with either cysteine (TGT) or valine (GTT).
Similar G to T transversions also affect the P53 gene in lung cancer
and represent the type of DNA damage expected from bulky DNA adducts
caused by the polycyclic hydrocarbons and nitrosamines in tobacco
smoke. Further evidence for a causative role for tobacco smoke is the
correlation of KRAS mutations with cigarette consumption. The presence
of KRAS mutations portends a poor prognosis in both early- and late-
stage NSCLCs, [ref: 12] although the data conflict.[ref: 13]
Nonetheless, a metaanalysis of eight studies of 217 (of 881) NSCLC
patients positive for KRAS mutations suggested a negative prognostic
role.[ref: 14] A prospective study has shown that neither
chemosensitivity or survival correlated with KRAS mutation in advanced
lung adenocarcinomas.[ref: 15] New drugs, farnesyltransferase
inhibitors, were developed to specifically kill or inhibit the growth
of tumor cells with RAS mutations. However, these drugs appear to be
active against tumors with and without RAS mutations and are coming
into clinical trials against lung cancer.
A direct downstream effector of RAS is the RAF1 protooncogene
product. Unexpectedly, the experimental growth arrest of SCLC by
activated RAF1 suggests that it has more of a TSG function. [ref: 16]
Although one copy of RAF1 is frequently lost; however, mutations in the
RAF1 gene have not been detected in human lung cancers. Molecules
downstream of RAF1 in the signal transduction pathway, such as MEKK,
MEK, and mitogen-activated protein kinase/extracellular-regulated
kinase (MAPK/ERK), may also be involved occasionally in lung
carcinogenesis.[ref: 3] The PPP2R1B gene, encoding the beta isoform of
protein phosphatase 2A (PP2A), which regulates the RAS/MAPK cascade, is
also infrequently mutated in lung cancers.[ref: 17]
Ultimately, signal transduction cascades result in the activation of
nuclear protooncogene products such as those encoded by the myc family
genes (MYC, MYCN, MYCL). MYC, when heterodimerized with a protein
called MAX, functions as a transcription factor, necessary for normal
cell-cycle progression, differentiation, and programmed cell death. MYC
is most frequently activated via gene amplification or transcriptional
dysregulation in both SCLC and NSCLC, whereas abnormalities of MYCN and
MYCL generally occur in SCLC. Indeed, MYCL was originally isolated from
a SCLC cell line. The myc family gene amplification has been
differentially observed in the major lung cancer subtypes. In SCLC, one
member of the MYC family was amplified in 18% of tumors and 31% of cell
lines, compared to 8% of NSCLC tumors and 20% of cell lines,
respectively. [ref: 11] Amplification appears more frequent in patients
previously treated with chemotherapy, the "variant" subtype of SCLC,
and its presence correlates with adverse survival. In terms of therapy,
all-trans-retinoic acid (RA) treatment inhibited the in vitro growth of
a SCLC cell line overexpressing MYC, a process associated with
increased neuroendocrine differentiation and increased MYCL and
decreased MYC expression. [ref: 18] Lastly, there also have been
reports of MYCL amplification with rearrangement in which MYCL fuses to
the RLF gene causing a chimeric protein.[ref: 19]
Occasional reports have implicated other oncogenes, such as ERBA,
MYB, JUN, and FOS in lung cancer, but data are relatively few.

Tumor Suppressor Genes and Growth Suppression

p53 Pathway

p53 (or TP53) maintains genomic integrity in the face of cellular
stress from DNA damage (for example caused by gamma- and UV
irradiation, carcinogens, or chemotherapy). It functions as a
transcription factor to activate the expression of genes that control
cell-cycle checkpoints (e.g., p21**WAF1/CIP1), apoptosis (BAX), DNA
repair (GADD45), and angiogenesis (thrombospondin). The p53 gene is the
most frequently mutated TSG in human malignancies, and mutations affect
approximately 90% of SCLCs and 50% of NSCLCs. [ref: 20,21] Most
mutations occur in the evolutionarily conserved p53 exons 5 to 8. In
NSCLCs, p53 alterations occurred more frequently in squamous cell
(51.2%) and large cell (53.7%) carcinomas than adenocarcinomas (38.8%).
[ref: 21] p53 mutations correlate with cigarette smoking and most are
of the type of G to T transversions expected from tobacco smoke
carcinogens. More evidence linking smoking damage with p53 mutations is
the finding that a major cigarette smoke carcinogen, benzo(a)pyrene,
selectively forms adducts at the p53 mutational hot spots.[ref: 22] The
types of p53 mutations are varied and include missense, nonsense, and
splicing abnormalities as well as larger deletions. Missense mutations
(the most common type of mutation) often prolong the half-life of the
p53 protein to several hours, leading to increased levels detectable by
immunohistochemistry and thus the use of immunohistochemistry as a
surrogate assay for p53 mutation. [ref: 23] Whether the occurrence of
p53 mutations (detected by either immunohistochemistry or molecular
analysis) in a patient's tumor affect survival is controversial. [ref:
24] Approximately 15% of lung cancer patients develop antibodies to the
p53 protein, raising the possibility that mutant p53 protein
overexpression can lead to a humoral immune response. Although p53
antibodies have been proposed as a marker for early diagnosis [ref: 25]
and chemoresponsiveness,[ref: 26] the development of these antibodies
does not appear to improve prognosis in lung cancer.[ref: 27] There
have been several promising gene therapy clinical trials with objective
response rates of approximately 10% to 15% in which lung cancers are
treated by intratumoral injection [endobronchially, or by computed
tomography (CT)-guided needle injection] with a normal (wild-type) p53
gene using retroviral [ref: 28] or adenoviral vectors.[ref: 29] These
local injections are now being combined with chemo- and radiotherapy to
test if gene therapy increases sensitivity to conventional treatments.
Also, systemic methods (such as with lipid vesicles) of delivering p53
gene therapy to disseminated tumors are being developed. In another
approach, clinical trials are being conducted to immunize patients
against either mutant p53 or RAS proteins occurring in patients' tumors
in attempts to generate a tumor-specific cytotoxic T-cell response.
p53 functions in a biochemical pathway; thus it is reasonable to
consider that other components of this pathway may be mutated in lung
tumors that are wild-type for p53. One of the upstream components is
the kinase that phosphorylates p53 encoded by the ataxia telangiectasia
(ATM) gene. However, this gene has not yet been found to be mutated in
lung cancer. Other components are the MDM2 and p14**ARF genes (see
p16**INK4A, later in this chapter), which regulate the levels of p53
protein, but so far they have not been implicated in lung cancer. Two
proteins homologous to p53--p51 and p73--have been discovered, leading
to the hypothesis that they may be mutated in p53 wild-type tumors.
However, they are only infrequently, if ever, mutated in lung cancers.
Finally, the Li-Fraumeni syndrome of inherited susceptibility to cancer
determined by an inherited germline mutation of p53 may also lead to
increased susceptibility to lung cancer in adults in these pedigrees.

The p16**INK4A-Cyclin D1-Cyclin-Dependent Kinase-4-Retinoblastoma
Protein Pathway

p16**INK4A

The p16**INK4A-cyclin D1-cyclin-dependent kinase-4 (CDK4)-
retinoblastoma (RB) protein pathway is a key cell-cycle regulator at
the G(1)/S phase transition, and one of the components of this pathway
is abnormal in the majority of lung cancers. The activation of CDKs by
cyclins eventually leads to the phosphorylation (inactivation) of the
growth-suppressive RB protein (see Fig. 31.1_1). As p16**INK4A is an
inhibitor of CDKs, especially CDK4 or CDK6 (which phosphorylate and
keep RB in an "inactive" state), its normal role is to positively
regulate RB's growth-controlling function by keeping RB
unphosphorylated. However, if p16**INK4A is inactivated by mutation, RB
remains chronically phosphorylated and thus cannot function to regulate
growth (see Fig. 31.1_1).
The p16**INK4A (also called CDKN2) gene locus on chromosome 9p21 is
frequently abnormal in human malignancies. In lung cancer, p16**INK4A
abnormalities are frequent in NSCLC but rare in SCLC (in which, by
contrast, RB is mutated in 90% of cases). Thus, these two major
histologic lung cancer types have this pathway inactivated by mutation
of one or the other gene, and double mutants in the same tumor are very
rare. A summary of 20 studies showed that p16**INK4A point mutations in
NSCLCs were observed in only 14% of primary tumors. [ref: 3] However,
homozygous deletions or aberrant promoter methylation can also down-
regulate p16**INK4A. Indeed, aberrant methylation of p16**INK4A may be
the most frequent as well as an early preneoplastic event in the
pathogenesis of squamous cell carcinomas. [ref: 4] Taken together,
these mechanisms ultimately result in absent p16**INK4A expression in
approximately 40% of primary NSCLCs, [ref: 3] indicating that this may
be the most common way to inactivate the p16**INK4A-cyclin D1-CDK4-RB
pathway in NSCLC. Because of the frequency of the abnormalities,
p16**INK4A is an attractive clinical trials candidate for replacement
gene therapy or induction of re-expression with antimethylation drug
therapy.
Complicating matters is the discovery of an alternative reading frame
at the p16**INK4A locus, p14**ARF, which encodes a protein that binds
p53/MDM2 complex, leading to p53 protein stabilization. If p14**ARF is
missing because of mutations in the p16 locus, p53 is less stable and
its function diminished. It thus emerges that inactivation of the p53
pathway may also be triggered by abnormalities of the p16**INK4A gene
locus. Another CDK inhibitor gene, p15**INK4B is situated close to
p16**INK4A and can be co-deleted with p16**INK4A in NSCLC. However, it
appears that the majority of lung cancer abnormalities focus on
p16**INK4A and not on p15**INK4B.

Cyclin D1 and Cyclin-Dependent Kinase-4

Because cyclin D1/CDK4 complex inhibits RB activity by stimulating its
phosphorylation, cyclin D1 or CDK4 overexpression is an alternative way
to disrupt this pathway. Immunohistochemically, cyclin D1 is
overexpressed in 12% to 47% of primary NSCLCs and, in some cases,
cyclin D1 overexpression is associated with a poor prognosis.[ref: 30]
How this overexpression occurs in lung cancer is unknown.

Retinoblastoma Protein

The RB gene (RB), located at chromosomal region 13q14, encodes a
growth-suppressive nuclear phosphoprotein. When active (i.e.,
hypophosphorylated), RB binds and inactivates proteins such as
transcription factor E2F-1, which is essential for G(1)/S transition of
the cell cycle (see Fig. 31.1_1). RB mutations (truncation by
deletion, nonsense mutation, or splicing abnormalities), together with
loss of the wild-type RB allele, have been demonstrated in lung
cancers, particularly SCLC. The RB protein is absent or structurally
abnormal in more than 90% of SCLCs and 15% to 30% of NSCLCs. [ref: 31]
Absent RB expression may be associated with poor prognosis in NSCLCs,
although this is not a consistent finding. The relatively low frequency
of RB abnormalities in NSCLC is consistent with the frequent disruption
of the p16**INK4A-cyclin D1-CDK4-RB pathway in these histologic types.
Essentially, lung cancers can be characterized as having either RB
mutation (mostly SCLC) or p16**INK4A inactivation (mostly NSCLC). Gene
therapy with replacement of RB function has been considered, but
because of the large size of the RB coding region and the necessity for
systemic delivery in, for example, typical widely metastatic SCLCs,
such therapy has not been actively pursued. Mutations of two other RB
related genes, p107 and RB2/p130, also have been implicated in lung
cancer. In the case of RB2, loss of protein expression and restoration
of growth control by genetically re-introducing a normal copy of RB2
have been demonstrated. [ref: 32] Finally, retinoblastoma patients or
their relatives who carry a mutant RB in the germline have an excess
risk of developing small cell lung cancer if they survive into adult
life. [ref: 33]

PTEN, FHIT, RAR-beta, Putative 3p Tumor Suppressor Genes, and Other
Tumor and Growth-Suppressive Gene Sites in Lung Cancer Pathogenesis

Besides the p53, p16**INK4A, RB, and loci, cytogenetic and allelotyping
studies show nonrandom, hemiallelic loss at many other chromosome
regions in lung cancer. Such tumor-specific somatically acquired loss
of heterozygosity is a hallmark feature of traditional TSG
inactivation. In other words, the consistent identification of multiple
sites of loss of heterozygosity at various chromosomal regions suggests
the existence of underlying TSGs in these regions. Usually the
remaining allele is silenced by point or small mutations, epigenetic
hypermethylation of the promoter region or, less frequently, by a
larger deletion. These sites of allele loss have been defined at more
than 30 regions dispersed on 21 different chromosomal arms, although
the molecular targets of most of these sites is not known. [ref: 34]
Although several of these chromosomal arms contain known TSGs,
including VHL (3p25), APC (5q21), WT1 (11p13), DCC (18q21), and NF2
(22q12), these genes are not known to be mutated in lung cancer. The
TSG PTEN, which encodes a phosphatase, is located at chromosome region
10q23, another common area of allele loss in lung cancer. However, PTEN
is mutated in only a subset of lung cancers. [ref: 35] Frequent loss
(60% to 80%) of several 4p and 4q regions were found in thoracic
malignancies (particularly SCLC and mesotheliomas); however, the genes
involved are not yet known. [ref: 36]
Among these chromosomal locations, chromosome 3p allele loss
(occurring at more than four different 3p regions) stands out as a very
frequent and early event in lung cancer pathogenesis. 3p loss occurs in
more than 90% of SCLCs and more than 80% of NSCLCs. In addition, it
appears to be the earliest genetic change found in lung cancer
development, occurring at great frequency in patches of normal
epithelium accompanying lung cancer or in smokers, as well as in sites
of hyperplasia, dysplasia, and carcinoma in situ of respiratory
epithelium (see Molecular Changes in Preneoplasia, later in this
chapter). Multiple distinct 3p regions have been identified by
allelotyping, including 3p25-26, 3p21.3-22, 3p14, and 3p12.
Furthermore, homozygous deletions are found in several lung cancer cell
lines (at several 3p21.3 sites, as well as at 3p12 and 3p14.2). Several
candidate TSGs have been identified in an approximately 600-kilobase
3p21.3 region homozygously deleted in three SCLCs, and another 800-
kilobase deletion region at 3p21 also has been described. [ref: 37,38]
The FHIT (fragile histidine triad) gene is found in the 3p14.2
homozygous deletion region. Forty percent to 80% of lung cancer cells
express abnormal FHIT messenger RNA transcripts but paradoxically
almost always also express wild-type FHIT transcripts. [ref: 39]
Regardless of these molecular complexities, the FHIT protein is absent
in many lung cancers, particularly in the squamous cell type (87%)
compared to adenocarcinoma (57%) and may also be lost in some
preneoplastic lesions. The loss of FHIT protein expression is also
strongly associated with smoking.[ref: 40,41] Functionally, FHIT may be
involved in the regulation of apoptosis and in cell-cycle control.[ref:
42] Transfection of wild-type FHIT into lung cancers induces apoptosis
and blocks tumor formation in vivo in mouse models. [ref: 43,44]
Because of the occurrence of FHIT abnormalities early in lung cancer
pathogenesis (e.g., in preneoplastic stages), it is possible to
consider delivering FHIT gene therapy to airways containing multiple
preneoplastic lesions by using aerosols.
TGF-beta1 is a potent inhibitor of proliferation of most epithelial
and hematopoietic cells, and its signal is mediated through TGF-beta
receptors and subsequently SMAD proteins. In SCLC cells, down-
regulation of the type II receptor (TGFbeta-RII) located in chromosome
region 3p, has been shown to correlate with the resistance to growth
inhibition by TGF-beta1. [ref: 45] However, the TGFbeta-RII or SMAD
family genes are rarely mutated in lung cancer.
There is considerable evidence of dysfunction of retinoic acid
receptor beta (RAR-beta), located in chromosome region 3p24, in lung
cancers, leading to resistance of lung cancer cells to retinoids and
making it an excellent candidate 3p TSG. [ref: 46] Although initial
studies did not find RAR-beta mutations, more recent studies have shown
loss of expression of RAR-beta protein in approximately 50% of
clinically overt lung cancers. [ref: 47] It is quite likely that this
loss of expression without genomic changes occurs because of
methylation of the promoter region. Because of the widespread testing
of retinoids as chemoprevention agents, it will be important to
characterize the timing of loss of RAR-beta function in lung cancer
preneoplasia. It is possible that loss of expression of RAR-beta may
occur at such an early stage that chemoprevention with retinoids cannot
succeed.

31_01_01

Chapter 31: Cancer of the Lung
31.1: Molecular Biology of Lung Cancer

Yoshitaka Sekido
Kwun M. Fong
John D. Minna

Cancer: Principles and Practice of Oncology, 6th Edition
Published by Lippincott Williams & Wilkins, Copyright 2001

Lung cancer cells have accumulated a number of molecular genetic and
epigenetic lesions, which appear necessary to transform normal
bronchial epithelium to an overt lung cancer. There is complex
interaction between the various molecular changes that ultimately
result in the abrogation of key cellular regulatory and growth control
pathways. Of the three major classes of human "cancer" genes, the
protooncogenes and tumor suppressor genes (TSGs) are involved in lung
carcinogenesis, whereas evidence implicating DNA repair genes is not
yet conclusive. Many of the protooncogene and TSG changes are present
in both major lung cancer subtypes: small cell lung cancer (SCLC) and
non-small cell lung cancer (NSCLC), although certain mutations have
subtype specificity (Table 31.1_1). Protooncogenes generally encode
proteins that are positive effectors of the transformed phenotype and
can simplistically be considered positive growth regulators. Their
"activation" results in their functional deregulation, leading to a
gain in function or "dominant" effect. Conversely, TSG products are
negative growth regulators and their "inactivation" results in a loss
of function that contributes to malignancy. Interacting with yet other
biologic changes, these fundamental molecular events appear to underlie
the characteristics of dysregulated growth, clonal expansion, and
immortality, which are typical of overt lung cancers. In addition,
these, and yet other to be discovered molecular changes, may affect the
processes of invasion, metastasis, and resistance against cancer
therapy. In translating these laboratory discoveries into the clinic,
it is important to identify these various changes, determine the
frequency of occurrence, and test whether they have clinically
important associations (e.g., with histologic type, stage, survival,
response to therapy), as well as to determine if they could be used for
early diagnosis, to monitor prevention and treatment efforts, and as
targets for the development of new treatments. In addition, these
abnormalities will probably also give us important understanding about
lung development and differentiation.

Genetic and Epigenetic Alterations in Lung Cancers

Chromosomal Abnormalities

Lung cancer cells display numerical abnormalities (aneuploidy) of
chromosomes, which are suggestive of allele loss or gain, as well as
structural cytogenetic abnormalities. The latter include nonreciprocal
translocations and deletions, whereas the presence of double minutes
and homogeneously staining regions indicate gene amplification, such as
for the MYC gene family. [ref: 1] In SCLCs, losses from chromosomes 3p,
5q, 13q, and 17p predominate, but double minutes may be common late in
disease. In NSCLCs, deletions of 3p, 9p, and 17p, together with +7,
i(5)(p10), and i(8)(q10) are often seen. Molecular cytogenetic analysis
with comparative genomic hybridization has identified hitherto
unrecognized abnormalities, including deletions at 10q26, 16p11.2, and
22q12.1-13.1 and amplification at 1q24, 3q, 5p, 17q, and Xq26.
It has been proposed that human tumors may be genetically unstable at
two levels: at the chromosomal level, including losses and gains
(amplification), and at the DNA nucleotide level, including single or
several base changes. [ref: 2] It will thus be important to determine
if aneuploidy and structural cytogenetic abnormalities, apart from
targeting key genes, actually represent the phenomenon of chromosomal
instability in lung cancers.

Microsatellite Instability

A genetic change that manifests itself as a mutator phenotype (often
called the replication error repair phenotype) in human cancers results
in widespread microsatellite instability. The result of microsatellite
instability is a "laddering" of short-tandem DNA repeat sequences at
multiple loci seen on high-resolution polyacrylamide electrophoretic
gels. This phenotype is usually due to mutational inactivation of DNA
mismatch repair enzymes, resulting in marked instability of these
polymorphic DNA repeat sequences. This phenotype was initially reported
in hereditary nonpolyposis colon cancers. Lung cancer frequently
exhibits microsatellite instability; however, this occurs at only a few
loci and results only in "shifts" of individual allelic bands
("microsatellite alterations") compared to normal DNA in the same
patient. The abnormal mechanism underlying this phenotype is currently
unknown, and apparently mutations in DNA mismatch repair enzymes are
very uncommon in lung cancer. The human 8-oxoguanine DNA glycosylase
(hOGG1) gene, involved in the repair of oxidative DNA damage, is
another candidate for involvement in generating multiple lung cancer
mutations. However, abnormalities in this gene only rarely occur in
lung cancer, and mutations in other DNA repair genes have not yet been
reported in lung cancer. Overall, approximately 35% (37 of 106) of
SCLCs and 22% (160 of 727) of NSCLCs showed some examples of
microsatellite alterations at individual loci. [ref: 3] Microsatellite
alterations in lung cancer have been reported to be associated with
younger age, reduced survival, and advanced tumor stage. Regardless of
the underlying mechanism, many groups are testing the possibility of
using this microsatellite alteration phenotype for the early diagnosis
of lung cancer by detecting these shifted DNA bands in sputum,
bronchial washings, or blood.

Aberrant DNA Methylation

DNA methylation involves covalent modification at the fifth carbon
position of cytosine residues within CpG nucleotides of DNA, which tend
to be clustered around the 5' ends of many housekeeping genes (CpG
islands). Hypermethylation in the 5' promoter region of genes is
associated with transcriptional silencing and is an alternative
mechanism for down-regulating TSG expression rather than gene deletion
or mutation.
Hypermethylation of the promoter region of the p16**INK4A gene in a
subset of NSCLCs results in its down-regulation and may be an early
event in lung cancer pathogenesis. [ref: 4] Other genes also have been
found to undergo aberrant promoter methylation in lung cancer but are
not found in the normal lung associated with these tumors, including
DAP (death associated protein) kinase, GSTP1 (glutathione S-
transferase), and MGMT (O**6-methylguanine-DNA-methyltransferase).
[ref: 5] In addition, DNA containing these methylated sequences could
be detected in the corresponding blood samples from the same patients,
indicating that the tumor cells had shed DNA into the peripheral blood.
Thus, aberrantly methylated DNA sequences, which can be sensitively
detected among a background of normal DNA, represent an attractive
strategy for early molecular detection. Other sites of
hypermethylation, including 3p, 4q34, 10q26, and 17p13, have been
implicated in lung cancer pathogenesis, although the precise gene
targets are uncertain. In addition to use as an early detection target,
it may be possible to reverse methylation pharmacologically. In tissue
culture systems, this is routinely done with the demethylating agent 5-
aza-2'-deoxycytidine. Clinical trials with such agents have been
attempted in other diseases, and agents with less toxicity need to be
developed and tested in lung cancer.
Another acquired tumor abnormality is loss of imprinting (loss of
methylation) to allow the expression of genes in lung cancer.
Methylation plays a role in mediating genomic imprinting, which is a
gamete-specific modification causing differential expression of the two
alleles of a gene in somatic cells. Loss of genomic imprinting of the
insulin-like growth factor-2 (IGF-2) gene and the H19 gene (associated
with hypomethylation of its promoter region) also occurs in lung
cancer.