The two-hit hypothesis clearly explained the differences in tumour number and age of onset of RB in hereditary vs. sporadic cases of RB. Moreover, it led to the eventual cloning of RB1 as the first bona fide human TSG. The hypothesis has been useful in cloning genes for hereditary cancers and for their non-hereditary counterparts. However, this success has led to a questionable dogma in the field that all important tumour suppressors should behave according to the “two-hit” model.
Investigators sought to apply the principles learned from the two-hit model to sporadic cancers in order to identify novel TSGs. Many recurring regions of chromosomal deletion have been identified in sporadic cancers, suggestive of the presence of a TSG. Unfortunately, it has become apparent that not all regions of consistent loss are accompanied by obvious aberrations on the other allele, raising the question of whether these deletions may represent “passenger” events not relevant to carcinogenesis. Indeed, very few of the identified regions of chromosomal losses have yielded clear TSGs in those loci that conform to the two-hit hypothesis. This finding has led many to conclude that the relevant tumour suppressors in those regions have not been identified or do not exist, when in fact many genes in those loci are known to have tumour suppressive properties in vitro or in vivo.
Alternatively, these regions could harbour bona fide TSGs but may represent regions/genes where single copy mutation or loss plays a role in tumourigenesis. These single copy events may be even be selected for during tumourigenesis instead of biallelic TSG loss. One possibility is that the initial lesion, when reduced to homozygosity, may lead to cell death or senescence. The lethality could be due to homozygosity of the disease gene itself or of other distinct genes included in the initial “hit,” since recombination is a principal “second hit” in tumourigenesis26
. Such a scenario would result in an “obligate haploinsufficiency” in which selection pressure during tumourigenesis favours partial, but not complete, loss of the TSG.
A second possibility is that the single copy mutation of a TSG functions as a dominant-negative towards the wild-type gene/protein. After mutation of one allele, the mutant protein product interferes with the normal wild-type protein produced from the remaining wild-type allele. Because the complete normal function of the TSG is already impaired with only one hit, there is no selection pressure by the tumour for loss or mutation of the wild-type allele.
A third possibility is that a gene or genes in the region of consistent deletion exhibits haploinsufficiency for tumour suppressor function and single-copy loss of the TSG is sufficient for aberrant TSG function and promotion of cancer (). In contrast with classical TSGs that are insensitive to large reductions in expression or activity, the function of haploinsufficient TSGs is impaired by a 50% partial reduction in expression or activity, or sometimes by subtler changes, as in a “quasi-insufficiency” of TSG function that we discuss below.
The role of haploinsufficiency in cancer has been met with scepticism, despite the well-established role of haploinsufficiency in numerous developmental disorders such as, for instance, aniridia (caused by haploinsufficiency for PAX6
) and Grieg syndrome (caused by haploinsufficiency of GLI3
. Just as increased dosage of genes can result in developmental syndromes, such as Down syndrome, increased dosage or activity of oncogenes is an established genetic mechanism of malignancy28
, exemplified by the role of MYC
amplification or RAS
hyperactivity in cancer. However, the notion that subtle decreases in gene dosage or protein activity can be relevant to cancer has gained only limited acceptance in the scientific community.
One reason for this scepticism stems from the difficulty in definitively proving a haploinsufficient TSG is involved in tumourigenesis. Whereas the rare TSGs that fully conform to the two-hit hypothesis can be identified by their homozygous deletion or mutation in cancer, haploinsufficient TSGs cannot be identified in such a manner. Moreover, large regions of the genome, encompassing many genes, can be targeted by allelic loss and likewise, many genes may acquire somatic mutations during the course of tumour development. It is assumed that only a portion of these genes is responsible for the cancer, while the rest are passenger mutations. What then could be the gold standard by which to determine which gene or genes in the region are causative and which are non-involved bystanders? Unlike the analysis of traditional TSGs, no one assay or approach offers definitive proof that a haploinsufficient TSG is causally involved in tumourigenesis. Instead, an integrated approach involving human genetic analysis, in vitro and ex vivo functional studies, and murine cancer genetic modelling is critical to ascertain whether a gene may function as a haploinsufficient gene in cancer. Although there are caveats of each analysis on its own, a body of evidence from each of these analyses can strongly implicate a gene in dosage-sensitive tumour suppression.
Throughout this review, we use PTEN
as a model dosage-sensitive TSG, but numerous other TSGs exhibit haploinsufficiency and dosage-sensitivity. Notably, TP53
, commonly referred to as p53
, exhibits haploinsufficiency. Mice with heterozygous (+/−) p53
mutation show an intermediate survival to that of p53
homozygous mutants and wild-type mice, and tumours that develop in the +/− animals do not always display loss of the remaining wild-type allele29
. Similarly, tumours in patients with Li-Fraumeni syndrome, a cancer susceptibility syndrome caused by germline mutation of p53
, do not always exhibit loss of the wild-type p53
allele, suggesting that haploinsufficiency of p53
may be sufficient for tumour initiation in humans as well30
. A caveat with these analyses is that the effect of p53
haploinsufficiency on cancer initiation in mouse models and Li-Fraumeni patients may be due to complex non-cell autonomous effects stemming from the partial loss of p53
throughout the body. However, ex vivo
analysis of murine p53
heterozygous thymocytes showed that these cells have an impaired apoptotic response to ionizing radiation or etoposide31
. Similarly, a p53
-deleted HCT116 isogenic cell line expressed only 25% the normal level of p53
mRNA and exhibited an impairment in induction of p53-responsive genes and apoptosis after exposure to UV radiation32
. These studies demonstrate that reductions in p53
dosage and function can impact on a cells' ability to respond to oncogenic stimuli and strengthen the notion that p53 haploinsufficiency may drive tumourigenesis.
Similarly, other classic cancer susceptibility genes such as BRCA1
may exhibit haploinsufficiency and/or dosage effects. Primary breast and ovarian cells from patients with heterozygous germline mutation of BRCA1
have altered mRNA profiles compared to BRCA
wild-type cells, suggesting that single hits in these genes can confer phenotypic differences that could in principle impact on breast and/or ovarian tumourigenesis33
. Indeed, ex vivo
studies of human BRCA1
mutant breast cells have found that these cells exhibit enhanced colony formation potential34
and show impaired lineage commitment in differentiation assays35
. Although tumour formation in BRCA1
carriers does appear to require the “second hit,” these “one-hit” premalignant changes could impact tumourigenesis by either promoting loss of the second TSG allele or of other TSGs, or by altering the cellular phenotype to predispose to certain tumour subtypes.
Another haploinsufficient TSG is the transcription factor PAX5. PAX5
is found mutated, deleted, or translocated in approximately 30% of acute lymphoblastic leukaemia cases, but the aberrant mutations/deletions are almost invariably mono-allelic and appear to function as hypomorphs, not dominant negative alleles36
. Similarly, the E3 ligase FBW7
is mutated in approximately 10% of human colorectal cancers37
. In 70% of these cases, the mutations are mono-allelic, suggestive of haploinsufficiency. Conditional mono-allelic deletion of Fbw7
in the murine intestine cooperated with the tumourigenic APCmin
allele in intestinal tumourigenesis, albeit to a lesser degree than biallelic deletion of Fbw7
, suggesting that Fbw7
Like complete loss of TSGs, the effect of TSG haploinsufficiency can be highly tissue-specific and context dependent (). Thus, the cellular and molecular context in which TSG function is altered will determine the outcome of such impairment of TSG function. The differing outcomes of TSG expression level in differing situations may reflect distinct thresholds of protein expression or activity needed for certain processes or in differing cell contexts. For example, in one cell type there may be other compensatory proteins that mask the potential phenotype caused by TSG haploinsufficiency whereas in other cells these proteins are not expressed and so the haploinsufficiency of the TSG manifests as tissue-specific cancer susceptibility.
Tissue specificity and context dependency of tumour suppression