Tumor suppressor genes

Several key tumor-suppressor pathways are fre-
quently inactivated in lung cancer. These include
the p53 and the p16INK4a
—CyclinD1-CDK4-RB
pathways.
The p53 pathway
The tumor suppressor gene p53 is the most fre-
quently mutated gene in human cancer, and p53
is inactivated by mutation in ∼90% of SCLCs and
∼50% of NSCLCs, respectively [26,46]. Most in-
activating mutations in p53 are caused by point
mutations in the DNA-binding domain (missense
mutation, 70–80%) of one parental allele and LOH
(deletion) of the other. Occasionally homozygous
deletions are observed. p53 is located at chromo-
some 17p13.1, and codes for a protein that functions
as a key transcription factor. The transcriptional tar-
gets of p53 include a number of cell cycle regula-
tory proteins such as p21 andMYC, as well as many
proteins involved in apoptosis such as BAX, 14-3-
3σ, and GADD45. p53 regulation occurs primarily atthe level of protein stability. p53 controls transcrip-
tion of MDM2, an E3 ubiquitin ligase, which in turn
regulates p53 stability in a feedback loop. This par-
ticular connection in the p53 pathway is a frequent
target of dysregulation in tumor cells.
The p53 pathway is activated in response cel-
lular stress and DNA damage induced by gamma-
irradiation, ultraviolet light, DNA damaging drugs,
and carcinogens. p53 stabilization results in the
expression of downstream genes, which induces
either cell cycle arrest to permit DNA repair, or
programmed cell death when there is too much
damage. Loss of p53 function allows cells to di-
vide in spite of genetic damage, which can result
the clonal expansion of premalignant cells. In most
cases, only mutant, missense p53 is present because
of LOH involving thewild-type p53 allele. However,
in some cases, mutant p53 proteins can form het-
erodimers with wild-type p53 inactivating its tumor
suppressive function even before LOH. These “gain-
of-function” mutations contribute to increased tu-
morigenicity and invasiveness of several types of
cancers [26,46]. However, despite large-scale stud-
ies, it is not clear whether NSCLCs with p53 muta-
tions have impaired survival compared to lung can-
cers with only wild-type p53.
There are two important upstream regulators
in the p53 pathway: MDM2 and p14ARF. MDM2
functions as an oncogene by reducing p53 levels
through enhancing proteasome-dependent degra-
dation. Amplifications of MDM2 were reported in
∼7% (2/30) of NSCLCs, resulting in loss of p53
function [46]. p14ARF
derives from the p16 locus
with an alternatively spliced 5-exon that results in
an alternative reading frame for translation. p14 en-
codes a protein that binds toMDM2 thereby inhibit-
ing its ubiquitination activity,which leads to the sta-
bilization of p53. Immunohistochemistry analyses
of p14ARF on lung cancers have shown that p14ARF
protein expression was lost in ∼65% of SCLCs and
∼40% of NSCLCs. Thus, through p53 mutation or
changes in MDM2 or p14, the p53 pathway is inac-
tivated in the majority of all lung cancers.
Lung cancer cells are addicted to loss of p53 func-
tion. When wild-type p53 is re-expressed in lung
cancer cells with mutant or deleted p53, the tumor
cells undergo apoptosis. These findings have led to
clinical trials of p53 gene replacement therapy. The
results frompreclinical and early-stage clinical trials
of p53 gene replacement therapy using a replication
incompetent retrovirus p53 expression vector in pa-
tients with NSCLCs, show evidence of antitumor
activity and the feasibility and safety of gene ther-
apy [47]. INGN 201 (Ad5CMV-p53, AdvexinTM),
a replication-impaired p53 adenoviral vector has
been evaluated in clinical trials, and is both safe and
effective for the treatment of several different types
of cancer [48]. This treatment has been approved in
China for the treatment of primary head and neck
cancers in combination with radiation therapy and
is currently undergoing phase III trials in head and
neck cancer in the United States.
The RB pathway
The RB pathway plays a central role in G1/S
cell transition. Hypophosphorylated RB exerts its
growth suppressive effect by binding to and inhibit-
ing the E2F transcription factor, which promotes
cells through the G1/S transition. RB is phosphory-
lated by the CyclinD1/CDK4 complex. Once these
kinases phosphorylate RB, it releases E2F, resulting
in transition from G1 to S. Thus, loss of RB function
though deletion ormutation leads to loss of theG1/S
checkpoint, and is a common event in lung cancer,
particularly SCLCs (>90%), while inactivation of
RB is found in 15–30% of NSCLCs [26].
The activity of the CDK4/Cyclin D1 complex is
regulated by p16. p16 keeps RB hypophosphory-
lated (and growth suppressingmode) by preventing
CDK4 from phosphorylating RB. Thus, loss of p16
function results in loss of function of the RB path-
way. By contrast to RB, p16 is more frequently in-
activated in NSCLCs (∼70%) than in SCLCs (10%)
[26]. Inactivation of p16 is caused by LOH coupled
with deletion, intragenic mutations or promoter
hypermethylation of the remaining allele. In lung
cancer, promoter methylation is the most frequent
method of inactivation of p16.
Overexpression of either CDK4 or Cyclin D1
inhibits RB pathway function by saturating the
growth suppressive activity of p16. CDK4 is am-
plified in some cases of NSCLCs, but cyclin D1 is
overexpressed in more than 40% of NSCLCs as as-
sessed by immunohistochemistry [26,49]. Recently,overexpression of Cyclin D1 in normal-appearing
bronchial epithelial of patients with NSCLCs has
been reported to be associated with smoking and to
predict shorter survival, suggesting the possible util-
ity of Cyclin D1 as a molecular marker to identify
high-risk individuals [50]. Thus through changes in
either RB, p16, CDK4, or cyclin D1, this important
growth regulatory pathway is inactivated and dis-
rupted in the large majority of lung cancers.