For people who received their introduction to cancer genetics in college in the first
half of the 1990s, everything looked simple and straightforward. It was the stuff you could
explain to sincerely interested relatives who wanted to know what you were spending your
time on. There were oncogenes and there were tumour suppressor genes. Oncogenes were
overactive genes and proteins that somehow caused cancer because they were overactive;
therefore, they were dominant. Tumour suppressor genes were genes that would normally
prevent a tumour from happening and that needed to be inactivated for a tumour to start to
form; both copies of a tumour suppressor gene had to be inactivated, and the mutation was
recessive. If inactivation of these genes is a random process, it was understandable that
people who inherit an inactivated copy of a tumour suppressor gene had a higher risk of
developing the associated form(s) of cancer than people born with two normal copies, as
postulated in Alfred Knudson's (1971) two-hit model. And, indeed, it was shown that in the
tumours in these predisposed patients, the remaining wild-type copy of the tumour
suppressor gene was lost, a process referred to as loss of heterozygosity.
For me, in 1998 things started to change. Venkatachalam et al. (1998) published a paper
in the
EMBO Journal describing a detailed study of tumours in mice lacking one
copy of the p53 tumour suppressor gene (
Trp53 ). This gene is known to be the most mutated gene in human cancer
and its function to be central to many processes that are involved in the cellular
prevention of cancer. Mice lacking both copies of this gene are for the most part viable,
but succumb to cancer (mainly thymic lymphomas) at three to five months of age (Donehower
et al. 1992). Mice born with one copy of the
Trp53 gene start to develop cancer at around nine months, and incidence
increases with age.
In their study, Venkatachalam and colleagues analysed an impressive group of 217
Trp53
+/− mice of controlled genetic background and followed the fate of the
Trp53 wild-type allele in the tumours. According to the two-hit model, it
was expected that in these tumours this copy would have been lost or inactivated. However,
this turned out not to be the case. Half of the tumours from mice younger than 18 months
were found to have retained the wild-type copy of
Trp53 , a number that increased to 85% in mice older than 18 months. In
two tumours, the researchers sequenced the complete coding region of the remaining
wild-type allele and showed it was structurally intact. To exclude the possibility of
downregulation or inactivation at the level of protein expression, they irradiated
tumour-bearing mice prior to sacrifice, a treatment known to increase p53 protein levels
via posttranslational mechanisms. Their data showed the retained wild-type allele in these
tumours was expressed normally and suggested it had a normal wild-type conformation.
Next, the authors did a rigorous test of different functions of the p53 protein. They
first tested whether the tumours showed amplification of Mdm2. This protein, whose
expression is regulated by p53, stimulates breakdown of p53, thereby forming a negative
feedback mechanism that keeps p53 levels low. Some tumours therefore amplify the
Mdm2 gene as a means of inactivating p53. However, this was not found in
the tumours from the
Trp53
+/− mice. Subsequently, the researchers tested to what extent the
retained
Trp53 copy behaved normally. Irradiation of many tissues leads to
p53-dependent apoptosis, and, indeed, in tumours that had retained the wild-type allele,
irradiation did lead to an increase in apoptosis, whereas in tumours that had lost the
wild-type allele, it did not.
The p53 protein is known to function as a transcriptional regulator by either up- or
down regulating target genes in response to different forms of cellular stress, including
irradiation-induced DNA damage. The authors studied the expression of two p53-upregulated
genes (
Cdkn1a , which encodes p21, and
Mdm2 ) and one downregulated gene (
Pcna ) in p53-positive tumours after irradiation and showed responses
indicative of normal p53 function. Furthermore, it was shown that the p53 protein from the
tumours was able to bind to a p53-binding DNA sequence in an in vitro setting. Finally,
since it is known that p53 absence in tumours is correlated with chromosomal instability,
Venkatachalam et al. (1998) used comparative genome hybridisation to compare this feature
between p53-negative and p53-positive tumours and found a 5-fold greater stability in the
latter.
In short, this paper clearly showed that, at least in mice, in many
Trp53
+/− tumours the wild-type allele of
Trp53 is not only retained, but also appears to function normally. This
strongly suggested that a decrease of dosage in p53 is already sufficient for
tumourigenesis, a phenomenon referred to as haploinsufficiency. Shortly before, the group
of Moshe Oren (Gottlieb et al. 1997) had shown that a
Trp53
+/− background leads to a greater than 50% reduction in p53 activity
using a p53-responsive
lacZ reporter gene in transgenic mice. Venkatachalam and colleagues
suggested the strong concentration dependence of p53 function could be explained by the
fact that p53 functions as a tetramer. A 50% decrease in p53 monomers can easily be
imagined to result in a greater than 50% decrease in functional tetramers, which in turn
increases the chances of these cells becoming cancerous.
This paper by Venkatachalam et al. (1998) made me realise how important it is to remain
critical, even of long-established theories and models. Since then, haploinsufficient
mechanisms have been described in more tumour suppressor genes in humans and mice (reviewed
in Fodde and Smits 2002). For instance, in a recent paper in
PLoS Biology , Trotman et al. (2003) used mouse models to describe how
the dosage of the
Pten tumour suppressor gene influences the occurrence of prostate cancer.
Further genes have been described with other unexpected roles in the tumourigenic process.
There is a long-standing debate in the literature about the number and role of mutations in
a tumour, and without going into the details, it is clear that haploinsufficient mechanisms
for tumour suppressor genes will greatly influence the statistics on which these
discussions are based. At a time when microarray analysis has become a standard experiment
and the many thousands of changes in tumour cells are analysed across the whole genome, it
is important to keep in mind that the correct interpretation of this wealth of information
might be more complicated than the widely accepted models would have us believe.
