GENOMIC INSTABILITY, TELOMERES, AND DNA DAMAGE IN LUNG CANCER

Malignant transformation is characterized by genetic instability
that can exist at the chromosomal level (with loss or gain
of genomic material, translocations, and microsatellite instability);
at the nucleotide level (with single or several nucleotide base
changes); or in the transcriptome (with altered gene expression).
Abnormalities are typically targeted to proto- oncogenes, TSGs,
DNA repair genes, and other genes that can promote outgrowth
of affected cells. 10 The erosion of telomeres at the end of chromosomes
is also associated with genomic instability leading to
chromosomal abnormalities. Telomere length regulates the replicative
capacity of a cell, where progressive telomere shortening
occurs with each replication. Once the telomere becomes
too short, the cell will undergo cellular senescence or apoptosis.
Activation of telomerase, the telomere-lengthening enzyme, in
premalignant cells prevents loss of telomere ends beyond critical
points and is essential for cell immortality. Although silenced in
normal cells, telomerase is activated in 80% of NSCLCs and
almost uniformly in SCLCs. 11–13 In normal cells, the presence
of DNA damage engenders a DNA repair response, and if this
is not successful, the apoptosis program is activated to remove
the damaged cell. However, in premalignant and cancer cells the
apoptosis program is often itself damaged, thus allowing unrepaired
or misrepaired DNA damage to persist in clones of cells.
ONCOGENES AND GROWTH-STIMULATORY
PATHWAYS
Many oncogenes and TSGs have been identified by mapping of
copy number changes throughout the cancer genome. 14–23 Earlier
genomic analysis technology such as karyotyping and comparative
genomic hybridization (CGH) enabled low-resolution characterization
of the lung cancer genome identifying whole-arm or
large-scale gain or loss on nearly every chromosomal arm, but
most commonly 3p, 4q, 9p, and 17p loss and 1q, 3q, 5p, and
17q gain 24,25 (Table 5.1). However, high-resolution microarray
analyses can now narrow in on these aberrant regions to detect
focal amplifications and deletions often spanning only a handful
of genes (Table 5.1).
Oncogenic activation typically occurs by gene amplification,
point mutation, rearrangement, or through gene overexpression
by other mechanisms including those mediated by microRNAs
(miRNAs). These changes can result in persistent upregulation
of mitogenic growth signals that induce cell growth. Although
promoting the malignant transformation of a cell, persistent upregulation
of a particular growth signal or pathway can also result
in “oncogenic addiction”—whereby the cell becomes dependent
on this aberrant oncogenic signaling for survival. 26 This presents
an obvious target for therapeutics; remove or inhibit the oncogenic
signal and an addicted tumor cell will die, whereas normal
“nonaddicted” cells will be unaffected. Signaling pathways commonly
involved in lung cancer are shown in Figure 5.1.
Epidermal Growth Factor Receptor Signaling The
ErbB family of tyrosine kinase receptors includes four
members—EGFR, ErbB-2 (HER-2), ErbB-3, and ErbB-4. 27
Although the intracellular tyrosine kinase domains of the four
receptors are highly conserved, the extracellular domain is not
so conserved, enabling the receptors to bind different ligands.
Following ligand binding, the ErbB receptors form homodimers
or heterodimers that results in receptor activation and
subsequent activation of various signaling pathways.
Activation of EGFR through the binding of EGF and
EGF-like binding growth factors such as transforming growth
factor- (TGF- ) enables the regulation of epithelial cell behavior
and the initiation of tumors from epithelial cell origin
through multiple signaling pathways. These include the
RAS/RAF/MEK/MAPK pathway (cell proliferation), the
PI3K/AKT pathway, and signal transduction and activator
of transcription (STAT) 3 and STAT5 pathways (cell survival
through the evasion of apoptosis) 28 (Fig. 5.1). EGFR exhibits
overexpression or aberrant activation in approximately 50%
to 90% of NSCLCs with activating mutations occurring
with or without amplification. 29–32 Activating mutations,
which are found with increased frequency in certain subsets
of lung cancer patients, occur as three different types of
somatic mutations—deletions, insertions, and missense point
mutations—and are located in exons 19 to 21 that code for the
tyrosine kinase domain of EGFR. 17–19 Mutant EGFRs (either
by exon 19 deletion or exon 21 L858R mutation) show an
increased amount and duration of EGFR activation compared
with wild-type receptors, 17 and have preferential activation of
the PI3K/AKT and STAT3/STAT5 pathways rather than the
RAS/RAF/MEK/MAPK pathway. 33 EGFR mutant tumors
(primarily adenocarcinomas) are initially highly sensitive to
EGFR tyrosine kinase inhibitors (TKIs). 17–19 This represents
an example of oncogene addiction in lung cancer where tumors
initiated through EGFR mutation-activation of EGF signaling
rely on continued EGF signaling for survival. However, despite
an initial response, patients treated with EGFR TKIs eventually
develop resistance to TKIs, which is linked (in approximately
50% tumors) to the acquiring of a second mutation at
T790M in exon 20. 34–39 The presence of the T790M mutation
in a primary lung cancer that had not been treated with
EGFR TKIs, however, suggests that this resistance mutation
may develop with tumor progression and not necessarily as a
response to treatment. 40 Recently, amplification of the mesenchymal–
epithelial transition (MET) proto-oncogene has been
associated with acquired resistance to EGFR TKIs in 20% of
resistant cases 36,41 with MET activating the PI3K pathway
through phosphorylation of ERBB3, independent of EGFR
and ERBB2. 41 Importantly, inhibition of MET signaling was
able to restore sensitivity to TKIs.
The RAS/RAF/MEK/MAPK/MYC Pathway The
RAS proto-oncogene family (KRAS, HRAS, NRAS, and RRAS)
encode four highly homologous 21kDa membrane-bound
proteins involved in signal transduction. Proteins encoded by
the RAS genes exist in two states: an active state, in which
guanosine triphosphate (GTP) is bound to the molecule and
an inactive state, where the GTP has been cleaved to guanosine
diphosphate (GDP). 42 Activating point mutations can confer
oncogenic potential through a loss of intrinsic GTPase activity
resulting in an inability to cleave GTP to GDP. This can initiate
unchecked cell proliferation through the RAS/RAF/MEK/
MAPK pathway, downstream of the EGFR signaling pathway.
43 Activating RAS mutations occur in approximately 15%
to 20% of NSCLCs and, in particular, 30% to 50% of adenocarcinomas.
44 In lung cancer, 90% of mutations are located
in KRAS (80% in codon 12, and the remainder in codons 13
and 61) with HRAS and NRAS mutations only occasionally
documented. 44 KRAS mutations are mutually exclusive with
EGFR and ERBB2 mutations, and confer resistance to EGFR
TKIs and chemotherapy. 45–47 Additionally, whereas KRAS
mutations are primarily observed in lung adenocarcinomas of
smokers, EGFR mutations are primarily observed in lung adenocarcinomas
of never-smokers. 4 These data demonstrate how
lung adenocarcinoma can develop through different pathways,
and it is likely, given the importance of EGFR targeted therapy
that determination of EGFR and KRAS mutations in tumors
will soon become part of standard care.
BRAF mutations occur in 1% to 5% of lung cancers, and
mutant BRAF mouse models can develop lung adenocarcinomas.
48 The MYC proto-oncogene members are targets of RAS
signaling and key regulators of numerous downstream pathways
such as cell proliferation. 49 Activation of MYC members often
occurs through gene amplification. MYC is most frequently
activated in NSCLC, 50 with the other two members, MYCN
and MYCL along with MYC , usually activated in SCLC. 24,51
The PI3K/AKT Pathway The PI3K/AKT pathway that
lies downstream of several receptor tyrosine kinases (RTKs; such
as EGFR) is a key regulator of cell proliferation, cell growth,
and cell survival and is commonly activated in lung cancer
through changes in several of its components, including PI3K,
PTEN, AKT, or EGFR or KRAS . In lung tumorigenesis, activation
of the PI3K/AKT pathway is thought to occur early 52
and results in cell survival through inhibition of apoptosis.
Activation can occur through the binding of the SH2-domains
of p85, the regulatory subunit of PI3K, to phosphotyrosine residues
of activated RTKs. 53 Alternatively, activation can occur
via binding of PI3K to activated RAS. Mutation and more
commonly amplification of PIK3CA , which encodes the catalytic
subunit of phosphatidylinositol 3-kinase (PI3K), occur
most commonly in squamous cell carcinomas. 20,54–56 AKT, a
serine/threonine kinase that acts downstream from PI3K can
also have mutations that lead to pathway activation. One of
the primary effectors of AKT is mTOR, a serine/threonine kinase
involved in regulating proliferation, cell cycle progression,
mRNA translation, cytoskeletal organization, and survival. 57
The tumor suppressor PTEN, which negatively regulates the
PI3K/AKT pathway via phosphatase activity on phosphatidylinositol
3,4,5-trisphosphate (PIP3), a product of PI3K, 58
is commonly suppressed in lung cancer by inactivating mutations
or loss of expression. 59,60
NKX2-1 (TITF1): A Lung Cancer Lineage–Dependent
Oncogene Genome-wide screens for DNA copy number
changes in primary NSCLCs found multiple examples of amplification
at 14q13.3—and subsequent functional analysis
(siRNA knockdowns in NSCLCs) identified NKX2-1 (also
termed TITF1 ) as the most likely target of amplification in lung
cancer. 14,15,61 NKX2-1 encodes a lineage-specific transcription
factor essential for branching morphogenesis in lung development
and the formation of type II pneumocytes, the cells lining
lung alveoli. 62,63 Amplification of tissue-specific transcription
factors in cancer has been observed in AR in prostate cancer, 64
MITF in melanoma, 65 and ESR1 in breast cancer. 66 These findings
have led to the development of a “lineage-dependency”
concept in tumors 67 whose survival and progression of a tumor
is dependent upon continued signaling through a specific lineage
pathways (i.e., abnormal expression of pathways involved
in normal cell development) rather than continued signaling
through the pathway of oncogenic transformation as seen with
oncogene addiction. 26
EML4-ALK Fusion Proteins Oncogenic fusion proteins
created by recurrent chromosomal translocations are generally
not common in solid tumors such as lung cancer; however, recent
studies indicate that this infrequency may be attributable to the
difficulties in detection. The fusion of PTK echinoderm microtubule-
associated protein like-4 (EML4)-anaplastic lymphoma
kinase (ALK) was recently associated with lung cancer 68 and occurs
in approximately 7% of NSCLCs. 68–70 Fusing with EML4
induces a significant transforming potential in ALK. Whereas
wild-type ALK is thought to undergo transient homodimerization
in response with specific ligand binding, EML4-ALK is
constitutively oligomerized resulting in persistent mitogenic signaling
and ultimately malignant transformation. 71 Additionally,
EML4-ALK generally appears to be mutually exclusive to that
of EGFR or KRAS mutations in NSCLC and is more common
in never or former smokers