This framework will be used to organize the material presented in this chapter.
Basics of Genetics and Molecular Biology Understanding oncology requires an integrated knowledge of the basics of molecular biology and genetics. While a general overview is provided here, more detailed descriptions should be sought in genetics textbooks. The central dogma of molecular biology holds that
cellular genetic information fl ows from DNA which undergoes replication, to RNA by the process of transcription and fi nally to proteins by the process of translation (Crick 1958). All of these steps are highly coordinated into a sequence of events known as the cell cycle. Of importance in lung cancer are alterations in the structure and transcription of DNA and subsequent disruption of critical processes associated with the cell cycle. DNA is a linear polymer of the four bases adenine (A), guanine (G), cytosine (C), and thymine (T) which defi ne the genetic code. These bases, which differ in their ring structure, are attached to an invariant backbone of deoxyribose sugars connected by phosphodiester bonds. Two strands of DNA hybridize to form a double helix through hydrogen bonding between bases, A to T and G to C (Watson
and Crick 1953). The double stranded DNA associates with accessory proteins such as histones which
package the long polymer into a stable form called chromatin (Laskey and Earnshaw 1980). For the
processes of replication and transcription to take place, the DNA must fi rst be uncoiled from the histones
to allow the appropriate molecular machinery to bind.
Genes, the most basic unit of inheritance, are coded by DNA. The linear sequence of the bases, in sets of
three, define each amino acid to be translated and hence, the structure of proteins. While there are over
three billion base pairs in the human genome, only approximately 1%–2% are coding, resulting in an
estimated 30,000–40,000 genes (Lander et al. 2001).
The structure of genes can be simplifi ed conceptually into two components, a coding region and a promoter
region. The promoter is a section of DNA upstream of the coding region which, in concert with other “enhancer” and “silencing” regions of DNA and numerous associated proteins, controls gene transcription. This regulation depends on a number of factors including cell type, extracellular signals, and stresses.
A particularly important method by which gene transcription is regulated in cancer is methylation
of the promoter region leading to gene silencing (Herman and Baylin 2003). In this process, termed
“epigenetics”, a cytosine that precedes a guanosine (CpG dinucleotide) in the DNA sequence is methylated.
While this can be a normal process utilized by the cell to inhibit transcription, abnormal levels of methyl cytosines have been observed in lung cancer cells. This aberrant transcriptional inhibition appears to play a signifi cant role in disruption of tumor suppressor genes and can act as one or both hits in Knudson’s (1971) two-hit hypothesis. The actual inhibition of transcription occurs as a result of the complex interplay of histones and proteins binding the methyl cytosines.
Another mechanism of gene alteration is inherited or de novo mutations in the DNA code. DNA
is damaged from a variety of sources including inherent instability, exposure to environmental and
toxic stresses, and a natural limit to its replicative accuracy, necessitating repair mechanisms to maintain
genetic integrity. The responsible DNA repair genes can be altered early in carcinogenesis leading
to a greater propensity for mutations (Ronen and Glickman 2001). Chromosomal rearrangements
also alter genes and are frequently seen in lung cancers. This process involves the exchange of
DNA from one chromosome to another and can lead to abnormal gene activation or aberrant coding regions. A target of many of the genetic changes noted in lung cancer is the cell cycle. The cell cycle is the
discrete states through which cells must pass for replication and is normally tightly regulated from
external and internal signaling. Lung cancer cells frequently acquire genetic changes which disrupt
the normal balance of positive and negative signals resulting in a variety of growth abnormalities. This
deregulation represents a fundamental change from normal cells.
The Hanahan and Weinberg framework is helpful in understanding how the current body of knowledge
regarding the molecular biology and genetics of lung cancer fi t into the observed disease process. Many
of the abnormalities and a summary of the different expression levels between lung cancer types is provided.
Self-Suffi cient Growth Signaling
In cancer, the tight growth control of normal cells is lost, allowing for continuous proliferation. The regular
homeostasis is disrupted as cells acquire the ability to both produce their own growth factors and increase
their sensitivity to exogenous ones. Key factors in these paracrine and autocrine loops are encoded by
proto-oncogenes, many of which are activated in lung cancer. Proto-oncogenes encode proteins important
for normal cell growth and are called oncogenes only after becoming abnormally activated. This activation,
usually a result of point mutations or chromosomal translocations, leads to gain-of-function effects for
the cell. Several well studied families of oncogenes have been identifi ed in lung cancer including RAS,
MYC, and ERB-B. Ras:
The RAS family of oncogenes, including H-, K-, and N-RAS, encode a 21-kDa protein acting at the
cytoplasmic cell membrane as a guanosine-associated switch. The protein is associated with receptor
tyrosine kinases (RTKs) and plays a pivotal role in transducing extracellular signals to numerous growth
signaling pathways. Ras is activated by binding guanosine triphosphate (GTP), a process accomplished
by associated proteins; hydrolysis of this GTP to guanosine diphosphate inactivates Ras. Once active, Ras
activates multiple effector molecules including components of the following pathways: Raf-MAPK, PI3KAkt, and Rac-Rho (Shields et al. 2000).
K-RAS is mutated in 25% of non-small cell lung cancer (NSCLC), with rates highest in adenocarcinoma
at 30%–50%, and lowest in squamous cell at 0%–5% (Graziano et al. 1999). The mutations in
K-RAS are usually in codons 12, 13, and 61 and have been associated with frequent G-T transformations
linked to polycyclic hydrocarbons found in cigarette smoke (Rodenhuis and Slebos 1992). While a common
occurrence in NSCLC, mutations in RAS are not seen in small cell lung cancer (SCLC) (Wistuba et
al. 2001). Although results have been mixed, K-RAS mutational status appears to be related to prognosis
in NSCLC. Early studies found shortened diseasefree and overall survival for patients with K-RAS
point mutations (Slebos et al. 1990). Subsequent studies did not consistently fi nd this relationship but
on meta-analysis an increased risk of worsened 2-year survival was noted (Huncharek et al. 1999). A
possible explanation for this relationship is that mutated RAS appears to confer treatment resistance to
cancer cells. Its role in chemotherapeutic resistance is unclear but there is a growing body of evidence
showing the importance of the Ras pathway in radiation resistance. In vitro studies have demonstrated
increased radiation resistance in cell lines expressing mutant RAS (Sklar 1988). Therapeutics have been
developed which inhibit the activation of Ras and lead to reversal of radiation resistance in studies in
vivo (Cohen-Jonathan et al. 2000). The mechanism for the radiation resistance is still unclear but may
relate to activation of signals downstream of Ras such as phosphoinositide 3-kinase (PI3K) or Rho
(Lebowitz and Prendergast 1998).
Akt: Akt is a protein kinase downstream of PI3K in a growth signaling pathway. It is activated by many
growth signals including insulin-like growth factor (IGF) and Ras activation. Once activated, Akt plays
a role in progression through the cell cycle and cell survival. Akt is inactivated by PTEN, a protein frequently
mutated or epigenetically inhibited in lung cancer (Soria et al. 2002). Akt is constitutively activated
at high rates in both NSCLC (70%–90%) and Molecular Abnormalities in Lung Cancer.
SCLC (65%) and is associated with chemotherapeutic and radiation resistance in SCLC cell lines (Kraus et
al. 2002). Myc: The MYC oncogenes, c-, N-, and L-, encode DNA-binding proteins associated with transcriptional regulation. The activity of the Myc protein is regulated through homo- and heterodimerization
(Henriksson and Luscher 1996). When Myc is bound to the protein Max for example, it activates
transcription of cell cycle checkpoint proteins such as Cdc25A which promote cell replication (Santoni-
Rugiu et al. 2000). Similarly, inhibition of Myc occurs through heterodimerization with proteins such
as Mad. MYC activation occurs through dysregulated expression of the normal gene (Krystal et al. 1988).
Overexpression is seen in approximately 20%–60% of NSCLC and 30% of SCLC (Gazzeri et al. 1994).
In SCLC, Myc overexpression has been linked to cell lines treated with chemotherapeutics suggesting a
response mechanism. Additionally, overexpression of MYC is associated with worsened prognosis in SCLC
but not NSCLC. In vitro studies indicate that while v-Myc expression alone does not affect radiation resistance, when coexpressed with H-Ras, there is synergistically increased radioresistance compared to Ras expression alone (McKenna et al. 1990).
Receptor tyrosine kinases: The ERB-B family of transmembrane RTKs include epidermal growth
factor receptor (EGFR or ERB-B1) and HER2/neu (ERB-B2). When bound to ligands, these proteins
homo- or heterodimerize, becoming activated. The downstream effectors of the receptors include Ras
and mitogen-activated protein kinase (MAPK) leading to various processes including cell growth and
proliferation. Ligands are produced exogenously as well as from the cancer cells themselves, creating selfactivating loops.
Overexpression of EGFR is seen in 50% of NSCLC, with the highest rate (80%) noted in squamous cell.
Higher expression appears to predict a slightly worsened survival for those with NSCLC (Meert et al.
2002). Increased expression of HER2/neu is seen in 30% of both NSCLC and SCLC and appears in both
cases to predict worsened survival (Meert et al. 2003; Potti et al. 2002). The worsened prognosis may be a
result of chemotherapeutic resistance but this is still unclear. EGFR is known to be an upstream regulator
of the PI3K-Akt pathway, possibly through Ras, and thus may play a role in radioresistance (Gupta
et al. 2002). Similarly, cells overexpressing HER2/neu have been shown to be radioresistant (Pietras et al.
1999).
Another RTK highly expressed (50%) in SCLC is c-Kit. This receptor is frequently coexpressed with its
ligand, stem cell factor, leading to stimulated growth. As with EGFR and HER2/neu, c-Kit represents a potential target for therapy.
Other factors: Neuropeptides act as both neurotransmitters in the central nervous system and
as endocrine factors in non-neurologic tissue. The family of bombesin-like peptides includes gastrin-
releasing peptide (GRP) and neuromedin B (NMB). SCLC cells have been shown to synthesize
and secrete these factors which function in a complex system of neuropeptide induced cell growth
(Heasley 2001). Other growth factors such as IGF, found to be elevated in ~90% of NSCLC and SCLC,
have been shown to play a role in carcinogenesis and are associated with an increased risk of acquiring
lung cancer (Yu et al. 1999). Interestingly, overexpression of the IGF receptor has been shown to induce radiation resistance in vitro (Macaulay et al. 2001).
Insensitivity to Antigrowth Signals
The growth of normal cells is kept in check by antigrowth signals, many of which are encoded by tumor
suppressor genes (TSGs). The loss of one allele either through inheritance or damage and the second
through damage from mutation or epigenetics, leads to complete loss of function of these factors. When
intact, many of the proteins encoded by these genes exert their control through regulation of the cell cycle.
The ability to evade the inherent checkpoints of this system gives the cell the capacity to grow without
inhibition. While important antigrowth pathways such as p16(INK4A)-RB have been studied in lung
cancer, other TSGs and their roles are just beginning to be evaluated.
RB: The RB1 gene located on chromosome 13q14.11 was identifi ed initially in retinoblastoma
but has been subsequently identifi ed in many human cancers including lung. The RB protein plays a pivotal
role in inhibiting G1/S transition via the E2F family of transcription factors. RB inhibits transcriptional
activation by binding E2F. As the cell progresses from G1 to the S phase, RB becomes increasingly hyperphosphorylated in which state it disassociates from the E2F. Once unbound, the E2F can induce transcription of genes necessary for normal DNA synthesis.
Evasion of Programmed Cell Death
The process of programmed cell death, apoptosis, occurs in cells throughout the body in response to
various signals. This stereotyped process involves a cascade of signals from cell surface receptors and
internal monitoring processes to effector proteins which act on the mitochondria and nucleus to kill the
cell. Signals that induce apoptosis include activation of oncogenes, DNA damage, absence of stroma–cell
and cell–cell interactions, and hypoxia. Apoptosis is important in cancer because the tumor’s rate of
growth is determined not only by the constituent cells’ ability to replicate but also the attrition rate of
those same cells. In addition, the end result of many cancer therapies is apoptosis and treatment resistant
cells have frequently developed mechanisms to evade this fate.
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