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.