Haploinsufficiency for tumour suppressor genes: when you don't need to go all the way

Biochimica et Biophysica Acta 1654 (2004) 105 – 122 www.bba-direct.com

Review

Haploinsufficiency for tumour suppressor genes: when you don’t need to go all the way Manuela Santarosa1, Alan Ashworth * The Breakthrough Breast Cancer Research Centre, Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK Received 20 October 2003; accepted 13 January 2004 Available online 26 January 2004

Abstract Classical tumour suppressor genes are thought to require mutation or loss of both alleles to facilitate tumour progression. However, it has become clear over the last few years that for some genes, haploinsufficiency, which is loss of only one allele, may contribute to carcinogenesis. These effects can either be directly attributable to the reduction in gene dosage or may act in concert with other oncogenic or haploinsufficient events. Here we describe the genes that undergo this phenomenon and discuss possible mechanisms that allow haploinsufficiency to display a phenotype and facilitate the pathogenesis of cancer. D 2004 Elsevier B.V. All rights reserved. Keywords: Haploinsufficiency; Tumour suppressor gene; Mutation; Breast cancer; Colorectal cancer; Prostate cancer; Leukemia

1. Introduction Multiple genetic and epigenetic changes are associated with the progression of a normal cell to a full-blown metastatic tumour. The genes affected by these changes have been classically defined as either proto-oncogenes, where gain-of-function mutations act dominantly, and tumour suppressor genes, which are recessive and are inactivated by mutation or loss. Over 30 years ago, Knudson [1,2] advanced a model of tumourigenesis that stipulated that inactivation of both alleles of a tumour suppressor gene was required to promote tumour progression. This model was developed by analysis of the age of onset of the hereditary form of retinoblastoma compared to that of the sporadic disease. As with several other hereditary cancer syndromes, the predisposition to develop cancer arises as a consequence of a heterozygous germ-line mutation of one tumour suppressor gene allele. It was the observation that hereditary cancer developed earlier than sporadic cancer that prompted Knudson to postulate that the inactivation of * Corresponding author. Tel.: +44-20-7153-5333; fax: +44-20-71535340. E-mail address: [email protected] (A. Ashworth). 1 Present address: Division of Experimental Oncology I, Centro di Rifermento Onclogico, Aviano 33081 PN, Italy. 0304-419X/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbcan.2004.01.001

both tumour suppressor gene alleles was required to develop a cancer phenotype. The early development of cancer in hereditary cancer syndromes is because individuals carry a constitutional germ-line mutation in a tumour suppressor gene and therefore inactivation of the other allele is the rate-limiting step. Sporadic cancer requires a considerably longer time to develop because two mutational events, the inactivation of both alleles of the same gene, have to occur. Knudson’s hypothesis not only suggested the mechanisms through which inherited and somatic mutations might collaborate in tumourigenesis, but also linked the notion of recessive genetic determinants for cancer susceptibility to previous findings from somatic cell genetic studies [1,2]. Tumour suppressor genes were originally thought to be directly involved in the regulation of cellular proliferation, hence their name. However, it has become clear in the last few years that many do not appear to have such a direct role but rather have involvement in a variety of other cellular pathways in the cell. In particular, there is an important class of tumour suppressor genes that is involved in the maintenance of the integrity of the genome. Kinzler and Vogelstein [3] have proposed the designation of Gatekeeper genes for those involved in the control of cell proliferation and Caretaker genes involved in the control of DNA integrity. The distinction between these two classes of genes may be

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important in considering how loss of a tumour suppressor might contribute to tumourigenic progression. For example, the inactivation of a Caretaker gene may not initiate tumour formation in itself but could foster transformation by rendering the cell genetically unstable and therefore more likely to sustain other mutations. Although the two-hit paradigm of tumour suppressor gene mutation has held sway for many years, recently there have been indications that this mechanism may not be universal. Although originally thought to be of little functional significance, there is now considerable evidence suggesting that loss of only one allele of a tumour suppressor might contribute to the tumourigenic process. Indeed, there are now numerous examples of functional effects of heterozygous loss-of-function mutations in genes and these have been suggested to be of considerable importance in tumour development [4]. Here we will discuss the evidence for the phenomenon of gene dosage sensitivity and discuss possible mechanisms whereby this haploinsufficiency can facilitate cancer progression.

2. Potential mechanisms for the generation of phenotypes after mutation of a single allele of a gene A variety of potential mechanisms could explain a phenotype arising as a result of mutation in one allele of a gene. Some of these are essentially trivial and due to having only one allele of a gene constitutionally (hemizygous genes present on the X-or Y chromosomes in males) or having only one allele of a gene expressed (monoallelic expression, for example due to imprinting, X-inactivation in females or other mechanism). Another possibility is a dominant negative mutation blocking function of the product of the remaining allele. There are a number of examples of this latter phenomenon [5,6]. A further potential mechanism to explain a heterozygous effect is that of haploinsufficiency, i.e. that a reduction in gene dosage brings about a phenotypic change that contributes to tumourigenesis. In this review we will focus on this particular subset of heterozygous effects. It is important to note that for some genes more than one mechanism may apply. For example, certain mutations in P53 may act as dominant negatives whereas others are lossof-function (see Fig. 1). In the case of the PML-RARa translocation protein in Acute Promyelocytic Leukaemia (APL), the translocation results in reduction of gene dosage for PML but also produces a chimeric PML-RARa protein that may act in a dominant negative fashion (see Section 7.4). Furthermore, in many cases haploinsufficiency for a gene may be viewed as partial penetrance for a classical two-hit tumour suppressor gene. For example, heterozygous mutations in the p53 and Dmp1 genes clearly contribute to tumourigenic progression but have less of an effect than homozygous loss of gene function. In the case of Smad4, heterozygous loss facilitates the early stages of carcinogen-

Fig. 1. Possible modes of tumour formation by tumour suppressor mutation, exemplified by p53. Dominant negative mutations act by producing proteins that block the action of the wild-type allele. Gain-of-function mutations result in inappropriate activity. Heterozygous loss-of-function mutation followed by wild-type allele loss results in a null genotype and rapid tumour onset. Heterozygous loss-of-function mutation and a haploinsufficient effect leads to tumourigenesis with a slower onset.

esis but loss of the remaining wild-type allele is required for continued progression.

3. Dosage sensitivity for cell cycle-regulatory genes implicated in tumourigenesis In normal cells, entry into the cell cycle from a quiescent state and progression around the cycle are precisely controlled. This ensures that cell growth, DNA synthesis and cytokinesis are co-ordinated appropriately with cell size increases. Cyclins, cyclin-dependent kinases and the Retinoblastoma protein are all involved in regulating passage through the restriction point late in G1 [7]. It is not surprising therefore that loss of cell cycle checkpoint function by mutation is a common feature of most if not all cancers. Considerable evidence now suggests that reduction in the gene dosage of critical cell cycle regulators could also play an important role in inactivating these checkpoints. 3.1. p27Kip1 One of the first examples of definitive evidence for haploinsufficiency for tumour suppression was provided for the p27Kip1 gene. p27Kip1 binds to and inhibits cyclindependent kinases and therefore acts to arrest the cell cycle at G1 [7,8]. Low levels of p27Kip1 have been documented in a variety of human carcinomas and these have been correlated with both aggressiveness of the tumour and mortality [9,10].

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However, conclusive evidence for a role for this gene in human cancer is lacking because homozygous mutations are not common in tumours. Evidence for dosage sensitivity for the p27Kip1 gene in tumour suppression has come from the analysis of mice carrying mutations in this gene [11]. Mice homozygous for loss-of-function mutations in p27Kip1 suffer from multi-organ hyperplasia. As with mice carrying mutations in the Rb gene, these mice develop melanotroph tumours of the pituitary [12]. While phenotypically normal, mice heterozygous for this mutation also developed the same tumour type with a penetrance of 32% [11]. Furthermore, when treated with a carcinogen (N-ethyl-N-nitrosourea, ENU) or X-irradiated, heterozygous p27Kip1 mice developed many more tumours than wild-type mice but fewer than homozygous mutants. Analysis of the expression of p27Kip1 in these tumours indicated that the wild-type allele continued to be expressed. This provides clear evidence of haploinsufficiency for tumour suppression in mice. Although definitive evidence for haploinsufficiency in human tumourigenesis is still lacking, it is of note that hemizygous deletions at chromosome 12p13, where p27Kip1 is located, have been observed in human tumours [13]. 3.2. P53 The p53 protein is essential for the checkpoint control that arrests cells with damaged DNA in G1. P53 is a transcription factor that regulates a number of genes involved in cell cycle regulation and apoptosis. One of the most important p53 targets is the cyclin-kinase inhibitor p21, which binds to and inhibits G1 Cdk-cyclin complexes. p53 is stabilised by DNA damage which results in p21 induction and arrest in G1. Once the DNA damage is repaired, p53 and p21 levels fall and the cells enter S phase. p53 induction may also in some circumstances lead to apoptosis. Therefore, loss of p53 function may lead to inappropriate cell cycle progression and resistance to apoptosis. Inherited mutations in the P53 gene result in the Li – Fraumeni syndrome which strongly predisposes to cancer; half of those individuals inheriting a single mutant P53 allele develop cancer by the age of 30 [14]. Tumours in these individuals often display loss-of-heterozygosity at the P53 gene, which is consistent with the Knudson ‘‘two-hit’’ model. P53 is also mutated somatically and is thought to be the most frequently mutated gene in cancer and, overall, about half of all human tumours have P53 mutations [15]. P53 mutation appears to contribute to tumourigenesis by several different mechanisms. In many cancers, the most frequent events are P53 mutation accompanied by complete loss of the wild-type allele [15]. This situation mimics that in Li – Fraumeni syndrome. However, in some tumours the wild-type P53 allele appears to remain intact and is still expressed. A possible explanation could be that the mutant form has a dominant gain-of-function activity or that it may block the wild-type protein by acting as a dominant negative. The possibility that reduction in gene dosage in P53+/ cells

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might also have a pathogenic effect has also been addressed [16]. Venkatachalam et al. [16] analysed mice carrying one mutant allele of p53. Nearly all these p53+/ mice develop tumours or die prematurely by 2 years of age whereas in wild-type mice only 20% of wild-type mice die or have tumours by this age. The wild-type p53 allele is retained in many of tumours occurring in the p53+/ mice. Intriguingly, the proportion of mice retaining the wild-type allele varies depending on age of tumour onset. About half of the tumours occurring before 18 months of age retained the wild-type allele whereas this figure increased to more than 85% when tumours from mice over 18 months were examined. In addition to retention of the wild-type allele, a considerable amount of additional evidence strongly suggested that haploinsufficiency contributed to tumourigenesis in p53+/ animals. First, p53 transcripts were sequenced and found to be unmutated. Second, intact p53 protein expression was observed. Third, treatment with ionising radiation increased apoptosis in p53+/ tumours but not in those arising in p53 / animals. Fourth, transcriptional responses dependent on p53 were still present in p53+/ tumours. Finally there were about fivefold fewer chromosomal abnormalities in p53+/ tumours compared to those arising in p53 / animals. What might be the implications of this study for the pathogenesis of human tumours? In the case of Li– Fraumeni syndrome the available evidence may not fit the Knudson model as well as generally thought. A detailed study of P53 allele loss in tumours arising in Li –Fraumeni patients showed that over half the tumours retained the wildtype allele. Although it is possible that other mechanisms were in play, this result is consistent with the mouse model described above. There may also be implications for the role of P53 in sporadic tumours. It is clear that many, perhaps most, tumours harbouring a P53 mutation lose the wild-type allele. However, a considerable number do not and it has been suggested that the reduction in P53 dosage leads to a growth advantage [17]. A synthesis of the evidence suggests a model where mutation in both P53 alleles, or loss of the wild-type allele, leads to rapid tumourigenesis. Haploinsufficiency of P53 is in itself likely to contribute to tumour progression but lead to a later onset of disease (see Fig. 1). The mechanism for the tumour-promoting effect of P53 haploinsufficiency is not clear. It has been noted that p53+/ cells have an intermediate phenotype to that of wild-type and p53 / cells for a number of biological properties such as apoptotic responses and cellular proliferation [18,19]. A possible reason for this is that p53 functions as a tetramer. Reduction in the dosage of wildtype p53 by 50% may result in a disproportionate reduction in active tetramer concentration. Hence, this could result in a situation similar to that elucidated for Nkx3.1 (see Section 6) where reduction in the concentration of a transcription factor leads to an all-or-none stochastic effect or an increase in transcriptional noise (see Discussion).

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This model is supported by evidence that mice heterozygous for a p53 mutation are only marginally more effective at activating a p53-responsive gene than homozygous mutant mice [20]. Recently p21, the key P53 target gene involved in mediating G1 cell cycle arrest mentioned above, has also been found to be haploinsufficient for tumour development [21]. Mice heterozygous and homozygous for a mutation in p21 are more prone to tumour formation after irradiation and tumours arising in the heterozygous animals retained expression of the wild-type allele. However, only homozygosity for p21 mutation affected the propensity of a tumour to metastasise, whereas heterozygosity had no effect. This is consistent with the model for P53 gene mutation where homozygous mutation has a more penetrant phenotype, i.e. generates earlier-onset tumours than heterozygous mice. 3.3. Dmp1, a regulator of Arf As discussed above (Section 3.2), p53 is a critical regulator of the response of the cell to genotoxic stress. The best-characterised negative regulator of p53 is Mdm2, which causes p53 ubiquitination, targeting it for degradation by the proteasome. Arf is the product of a tumour suppressor gene that antagonises Mdm2 function and stabilises p53 and enhances its activity. The transcriptional regulation of the Arf gene is complex but Dmp1 is thought to be an important player in this. Dmp1 is a transcription factor and binding to the Arf promoter induces Arf transcription [22]; conversely in Dmp1 / cells Arf transcription is dampened [23]. The effects of Dmp1 haploinsufficiency have been investigated in Dmp1 mutant mice [24]. Both homozygous and heterozygous Dmp1 mutant mice are significantly more tumour prone than wild-type mice both spontaneously and after treatment with a carcinogen or after g-irradiation. Tumours in the Dmp1+/ animals retain the wild-type allele, which appears to be expressed providing evidence for haploinsufficiency. Analysis of Dmp1+/ mice carrying an El-myc transgene provides further insight into the haploinsufficient phenotype. El-myc mice are prone to lymphoid malignancies [25] and this is accelerated by the loss of a single Dmp1 allele [24]. The retention and expression of the wild-type Dmp1 allele in these tumours further supported the suggestion of haploinsufficiency. Loss of function of the p53 pathway either by p53 mutation or biallelic Arf mutation is commonly found in lymphomas in El-myc. However, these events were much less frequent in El-myc mice with reduced Dmp1 gene dosage. This suggests that Dmp1 haploinsufficiency can substitute for p53 mutation or Arf loss, and this strengthens the case for the contribution of a haploid Dmp1 phenotype to tumourigenesis. Interestingly, evidence has also been provided for Arf haploinsufficiency in melanoma development in certain contexts [26].

4. Dosage sensitivity of signalling molecules implicated in gastro-intestinal cancer Haploinsufficiency for several genes involved in cancer of the gastrointestinal tract has been described. Intriguingly these have the common feature that when mutated they may give rise to the precursor lesions hamartomas (hyperplastic disorganised growth) in the digestive tract and these lesions frequently progress into carcinomas. This may reflect a role for gene dosage reduction in the early stages of carcinogenesis. 4.1. PTEN PTEN (for ‘phosphatase and tensin homologue deleted from chromosome 10’) is a tumour suppressor gene somatically inactivated in a wide variety of human malignant neoplasms [27]. Furthermore, individuals with germline mutations in PTEN are prone to the development of hamartomatous polyps in the gastrointestinal tract, developmental defects and cancer-predisposition syndromes such as Cowden’s disease (thyroid cancer and breast cancer), Juvenile polyposis (gastrointestinal tumours), and other very rare syndromes [28,29]. PTEN somatic mutations or deletions have been observed in a variety of human malignancies, including those of the brain, breast, endometrium, kidney and prostate [28]. PTEN can dephosphorylate phosphatidylinositol 3,4,5-trisphosphate, the product of PI-3 kinase. Cells lacking PTEN have elevated levels of phosphatidylinositol 3,4,5-trisphosphate and protein kinase B, which results in an inhibition of apoptosis and stimulation of cell cycle progression [27]. Insight into mechanisms of tumourigenesis caused by PTEN mutation has been provided by analysed of Pten mutant mice. Three different Pten heterozygous knockout mice have been generated with somewhat distinct phenotypes. Two groups observed features reminiscent of Cowden’s Syndrome such as skin hyperkeratinosis, papillary or papillary-like thyroid carcinoma atypia and hyperplasia of the endometrium. They also observed a low incidence of loss-of-heterozygosity at the PTEN locus [30,31]. The other group observed mainly lymphomas which, when spontaneous, retained in most cases the wild-type allele, whereas when induced by g-irradiation had lost the wild-type allele [32]. After long latency these mice developed mammary gland and endometrial cancer both of which had lost the wild-type allele [33]. It seems possible that in some tumours or genetic backgrounds PTEN is haploinsufficient but for others both alleles need to be lost. 4.2. SMAD4 TGFh is a secreted growth factor that has the ability to inhibit the growth of a variety of cell types such as epithelial and immune cells [34]. Smad proteins are key intracellular signal transducers downstream from TGFh receptors. Phos-

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phorylation of Smad2 or Smad3 by the type I TGFh receptor induces association with Smad4. These Smad complexes enter the nucleus and activate transcription of a number of genes. One critical gene appears to be p15, encoding a G1 cyclin-kinase inhibitor, which mediates the cell cycle arrest caused by TGFh. Disruption of components of this pathway results in the promotion of cell proliferation. SMAD4 is located within human chromosome 18q21.1, which frequently shows loss-of-heterozygosity in a variety of tumour types, such as pancreatic, colon and gastric adenocarcinomas [35 –37]. SMAD4 was first identified as a tumour suppressor under the guise of DPC4, which is deleted or mutated in 40 – 50% of pancreatic cancers [35]. Furthermore, the gene is mutated in 30% of colorectal cancers, but less than 10% of other cancer types [38,39]. Heterozygous germline mutations in SMAD4 are also responsible for a subset of Familial Juvenile Polyposis (FJP), a disorder characterised by predisposition to hamartomatous polyps and gastrointestinal cancer [40,41]. Evidence for a role of haploinsufficiency of SMAD4 in this disease was provided by the finding that tumours in FJP individuals almost always retained the wild-type allele of SMAD4 [41]. Although continued expression of SMAD4 was not demonstrated in this study, this prompted Xu et al. [42] to investigate the effects of Smad4 haploinsufficiency in a mouse model. All the Smad4+/ mice studied developed hyperplasia of the fundus and antrum. Although the fundal hyperplasias only rarely progressed, the polyps in the antrum eventually developed into andenocarcinoma. Polyps were also found at lower frequency in the duodenum and caecum. Taken together these results suggest that Smad4 is a tumour suppressor in mice in the GI tract, especially in the stomach. The development of these tumours was analysed in some detail. The pathogenesis of gastric cancer in Smad4+/ mice showed a multi-step process of hyperplasia, dysplasia, insitu and then invasive carcinoma. However, loss of the wildtype allele was not detected until the later stages of cancer progression. This suggests that haploid loss of Smad4 is responsible for the initiation of polyps and tumours. However, the loss of the wild-type allele in more advanced tumours suggests that this may be a rate-limiting step for tumour progression [42]. Therefore, Smad4 represents an interesting example of a gene that is haploinsufficient early in carcinogenesis but later needs to be homozygously mutated to allow further cancer progression, at least in the mouse model studied. In human tumours, mutation of other genes either dominantly, heterozygously or homozygously could give similar effects to loss of the second SMAD4 allele. It is interesting given the role of Smad4 in TGFh signalling that haploinsufficiency for TGFh1 has also been shown to influence tumour progression in certain circumstances [43] (see Section 7.7). In a caveat to the study described above by Xu et al. [42], it should be pointed out that Takaku et al. [44] have observed a complete loss of the wild-type allele in gastric

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and duodenal polyps in Smad4+/ mice although they reported only a few cases. This could reflect a difference in the specific mutation in the Smad4 gene studied in the two different systems. 4.3. LKB1 Peutz –Jeghers syndrome (PJS) is a dominantly inherited condition characterised by multiple gastrointestinal hamartomatous polyps and mucocutaneous pigmented spots on the lips, digits and buccal mucosa [45]. Patients with PJS are at least 10 times more likely to develop cancer than the general population [46]. Malignant neoplasms may occur in a variety of tissues including colon (possibly by malignant transformation of the hamartomas), breast, cervix, ovary, testis and pancreas. Multiple independent mutations were found in the gene LKB1 (STK11) in affected members of PJS families [47]. Loss of the presumptive wild-type allele in the epithelial component of the gastrointestinal hamartomas suggested that the PJS gene was a tumour suppressor and that there may be a hamartoma –adenoma –carcinoma sequence in neoplastic transformation [48]. Multiple independent mutations were found in the gene LKB1 (STK11) in affected members of PJS families [47]. LKB1 encodes a serine/threonine kinase and PJS mutations have been shown to cause loss or severe abrogation of autokinase activity [49]. To investigate the role of LKB1 in PJS, several groups have generated mice that carry mutated versions of Lkb1 gene [50 –52]. In all cases homozygous mutant Lkb1 mice died during embryogenesis. Heterozygous mice were superficially normal but developed gastrointestinal hamartomatous polyps with features similar to those observed in patients with PJS. In particular the stomach is particularly prone to polyp formation in these mice. One group showed loss of the wild-type allele in 25% of hamartomas and the absence of Lkb1 protein in 57%. In contrast, two other groups showed that the wild-type Lkb1 allele was not lost or mutated in any of the polyps analysed. Furthermore, the presence in the hamartomous epithelia of Lkb1 mRNA and the Lkb1 protein at levels of 50% of Lkb1+/ + mice suggested that haploinsufficiency was the cause of the early steps of progression. However, after long latency (>50 weeks), most Lkb1+/ mice develop hepatocellular carcinoma in which the expression of Lkb1 was virtually abolished suggesting biallelic inactivation of Lkb1 [53].

5. The Nk x3.1 gene: a model for dosage sensitive gene regulation during tumourigenesis Loss of NKX3.1 protein expression has been observed in about 40% of human prostate tumours and in 20% of prostatic intraepithelial neoplasia (PIN) suggesting a tumour suppressor role for this gene [54]. Haploid deletions of the NKX3.1 gene are also commonly found in human

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prostate cancers and PIN [55 –57]. The gene encodes a transcription factor homeodomain protein specifically expressed in the luminal epithelia of the prostate [57,58]. Consistent with these observations, mice in which a single Nkx3.1 allele is deleted develop prostatic hyperplasia and PIN [59]. The hyperplastic lesions of these mice, as well as those constitutionally heterozygous, retain Nkx3.1 protein expression from the remaining wild-type allele suggesting that haploinsufficiency contributes to tumourigenic progression [59,60]. The molecular basis of dosage-sensitive gene regulation by Nkx3.1 has recently been investigated [61]. At the cellular level, the reduction of Nkx3.1 protein expression in Nkx3.1+/ mice extends the proliferative stage of regenerating luminal cells, via regulating cell cycle exit, leading to epithelial hyperplasia [61] (see Fig. 2). This occurs because of the differential regulation of androgen target genes in Nkx3.1+/ prostate epithelial cells compared to wild-type. Androgens play an important role in the growth and maintenance of the prostate and this might explain the haploinsufficient hyperplastic phenotypes. Interestingly, the response of Nkx3.1-target genes to the reduction in Nkx3.1 dosage was variable, with a spectrum of dosage sensitivity. Some target genes such as intelectin were very sensitive to Nkx3.1 dosage reduction; expression of this gene was almost absent in the heterozygous prostate. Other target genes such as probasin were hardly affected at all. In addition to having differential effects on the levels of expression, reduction in Nkx3.1 gene dosage also affected the proportion of cells expressing target genes. These results are consistent with a model where Nkx3.1 protein levels affect the probability of target gene activation. This stochastic effect would result in the abolition of expression of some target genes and therefore potentially generate the equivalent of a null phenotype for these genes. In the case of Nkx3.1, this appears to manifest as an extended prolifera-

tive phase that could provide the clonal expansion necessary for tumourigenic progression [61]. This model of stochastically regulated target genes could be a widely applicable paradigm for haploinsufficiency of a transcription factor.

6. Haploinsufficiency of genes implicated in the maintenance of genomic stability As we discussed above, the inactivation of genes involved in the maintenance of genome integrity may not initiate tumour formation in itself. Rather, this may foster transformation by rendering the cell genetically unstable and more likely to sustain other mutations. Therefore, a reduction in the ability of a cell to repair DNA efficiently might be expected to be tumourigenic, and there are a number of examples of genes involved in DNA repair which have been suggested to show haploinsufficiency. 6.1. MSH2 Hereditary Non-Polyposis Colorectal Cancer (HNPCC) is a condition characterised by a high incidence of tumours particularly of the gastrointestinal and female reproductive tract [62]. The causative defect in this disease is in the DNA mismatch repair pathway and instability of simple-sequence (microsatellite) repeats (the replication error (RER+) phenotype) is a hallmark of HNPCC. Heterozygous mutations in four genes MSH2, MLH1, PMS1 and PMS2 have been found in affected members of HNPCC families. However, the underlying cause of tumour progression in HNPCC patients is controversial. Loss of the wild-type allele of these MMR genes seems to be common in HNPCC tumours but whether this is random loss at the ‘normal’ rate or is at and elevated rate due to heterozygosity is not clear. Support for the latter hypothesis comes from studies of mice het-

Fig. 2. A model depicting the effects of haploinsufficiency at Nkx3.1 on prostate tumour initiation. Heterozygous mutation of the Nkx3.1 gene in an amplifying cell results in the stochastic inactivation of certain Nkx3.1-target genes and continued inappropriate proliferation. This results in amplification of this population. Further genetic alterations result in neoplasia (modified from J.A. Magee et al. [61]).

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erozygous for mutations in Msh2 [63 – 65]. Msh2 / mice develop lymphomas and tumours of the small bowel [63]. Heterozygous Msh2 mutant mice are more tumour prone than wild-type animals but this does not appear to affect survival. Investigation of Msh2+/ mouse cells has revealed two distinct phenotypes that might contribute to tumourigenic progression. Bone marrow cells from Msh2+/ mice do not show any increase in spontaneous or X-ray induced chromosomal aberrations, whereas they do have a decrease in sister-chromatid exchange induced by methyl-nitrosourea, suggesting an impaired activity of DNA repair relative to wild-type Msh2 cells [65]. Furthermore, exposure of Msh2+/ embryonic stem cells with low-energy radiation resulted in the accumulation of more oxidised DNA bases than arose in wild-type cells and they were less likely to undergo radiation-induced apoptosis [64]. These results suggest that haploinsufficiency for Msh2 might have phenotypic effects that could contribute to progression in HNPCC individuals but this needs further investigation. 6.2. MAD2 MAD2 functions in the mitotic checkpoint to arrest cells in mitosis when chromosomes unattached to the mitotic spindle are present [66]. When activated this checkpoint causes a delay at the metaphase-anaphase transition until all chromosomes are attached to the spindle. Defects in the mitotic checkpoint have been correlated with chromosomal instability (CIN) in human cancer cells [67]. MAD2 arrests cells in metaphase by associating with the anaphase-promoting complex (APC), thereby inhibiting its ubiquitin ligase activity [68 –70]. The effects of mutation of MAD2 in mice and cell lines have been examined by Michel et al. [71]. Mice heterozygous for a Mad2 mutation are phenotypically normal but develop late-onset papillary lung adenocarcinomas at high frequency (28%), whereas this kind of tumour is usually very rare in mice. However, it remains to be proven whether this is a true haploinsufficiency as retention of the wild-type Mad2 allele was not demonstrated. To investigate a possible cellular phenotype for MAD2 haploinsufficiency, two cellular systems were examined: primary mouse embryonic fibroblasts harbouring a Mad2 mutation and human HCT116 tumour cells in which a single allele of MAD2 has been disrupted by targeted mutation [71]. In both systems several of the hallmarks of CIN were observed. In particular, these cells showed premature sisterchromatid separation in the presence of spindle inhibitors and an elevated rate of chromosome mis-segregation events, resulting in aneuploidy, in the absence of these agents. It was suggested that these phenotypes were as a result of a defective mitotic checkpoint caused by mutation of a single MAD2 allele. The strong cellular phenotype of CIN conferred by MAD2 gene dosage reduction suggests that this gene may play a role in human cancer progression. The presence of the human MAD2 gene at chromosome 4q27, a

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frequent site of loss-of-heterozygosity in human tumours, adds weight to this argument. 6.3. Haploinsufficiency for the breast cancer susceptibility genes BRCA1 and BRCA2: the jury still out About 5– 10% of breast cancers are thought to result from an inherited predisposition to the disease [72] and two highly penetrant breast cancer-associated genes, BRCA1 and BRCA2, have been isolated [72]. Inheritance of one defective BRCA1 or BRCA2 allele is associated with a lifetime risk of up to 85% for breast cancer and also confers a considerably enhanced risk of ovarian cancer, plus an elevated risk of male breast cancer, prostate cancer and pancreatic cancer [72]. In these individuals, loss or mutation of the wild-type allele of the BRCA1 and BRCA2 genes is frequently seen within tumours, suggesting they act as tumour suppressor genes. The normal functions of the BRCA1 and BRCA2 proteins are not yet completely understood. However, there is evidence for roles in transcriptional regulation and DNA repair for both proteins [73]. Their potential function in DNA repair has perhaps been best characterised. BRCA1 and BRCA2 are increasingly recognised to occupy a central role in DNA repair by homologous recombination, a process that is used by mammalian cells to repair DNA double-strand breaks and interstrand cross-links [74]. Unexpected confirmation of the role of BRCA2 in the repair of DNA interstrand cross-links in humans has come from studies on the genetics of Fanconi anaemia (FA) [75,76]. FA is an autosomal recessive disorder characterised by anaemia, bone marrow failure, birth defects and progression to myelodysplasia and acute myeloid leukaemia [77]. Lymphoid cells from FA carriers show a characteristic hypersensitivity to the DNA cross-linking agents Mitomycin C (MMC) and diepoxybutane, which forms the basis for clinical screening of affected individuals [77]. FA is a genetically heterogeneous disorder and is caused by homozygosity for mutations in at least eight different genes, FA-A to FA-G [77]. It has been shown recently that the FANCD1 gene is in fact BRCA2 due to the presence of biallelic BRCA2 mutations in individuals from the FANCD1 complementation group [15,16]. Furthermore, cells from the FANCD1 complementation group, like cells with loss of BRCA2 function, are defective in the formation of nuclear RAD51 foci after X-irradiation or MMC treatment [18]. BRCA1 may also play a role in the FA pathway as it can interact with FANCD2 [78]. It is clear that heterozygosity for loss-of-function mutations in BRCA1 and BRCA2 confers high susceptibility to breast and other cancers. However, it is unclear whether heterozygosity itself carries a phenotype leading to increased loss of the wild-type allele or that this loss occurs stochastically. As has been suggested for the Rb gene, it seems possible that haploinsufficiency could accelerate loss of the wild-type allele [79]. A number of studies have claimed or dismissed the existence of haploinsufficiency for BRCA1 and

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BRCA2. Studies utilising human dermal fibroblasts and lymphocytes from BRCA1 and BRCA2 mutation carriers suggested defects in DNA repair pathways but these were not conclusive as they involved small numbers of samples and were subject to interindividual variation [80 – 82]. Bucholz et al. [80] compared fibroblast and lymphoid cells from BRCA1 and BRCA2 mutation carriers, and suggested that they exhibited reduced colony forming capacity and increased chromatid breaks in response to gamma irradiation compared to cells from healthy controls. Foray et al. demonstrated that EBV-immortalised lymphoblasts from a small number of BRCA1 and 2 mutation carriers also exhibited lower clonogenic survival, increased micronucleus formation and a DNA repair defect following treatment with gamma irradiation [81]. However, both these studies were limited by the small number of samples and the fact that the cells compared are not isogenic. Furthermore, contradicting evidence has been provided [83] that failed to show a difference using a similar approach. In a study of mice heterozygous for a Brca2 mutation [84], distinct mammary phenotypes were observed in response to treatment with the estrogenic compound diethylstilbestrol (DES). Mice with a heterozygous Brca2 genetic alteration on a 129/SvEv genetic background when treated with DES displayed abnormalities of mammary gland branching as a result of growth inhibition [84]. Although this effect was not investigated at the molecular level, it is of interest that DES has been shown to stimulate sister chromatid exchange which is partially defective in mouse cells carrying two mutant Brca2 alleles [85]. In contrast, most studies using mouse cells heterozygous for Brca1 and Brca2 have failed to provide conclusive evidence for a haploinsufficient phenotype. In cell-based assays, mouse cells heterozygous for Brca1 and Brca2 mutations had the same radio- and chemo-sensitivity as wild-type cells both in terms of DNA repair ability and in clonogenic survival assays [86 – 88]. Unlike humans, mice heterozygous for Brca1 or Brca2 mutations are not any more tumour-prone than wild-type animals [89 – 91]. Furthermore, heterozygosity of Brca1 mutation in ApcMin/ + genetic background does not affect tumorigenesis in this colon cancer model [92]. In contrast, mice heterozygous for both Brca1 and p53 did show a slight increase in mammary carcinoma incidence over p53 heterozygotes and all tumours retained Brca1 expression [93]. This difference was exacerbated after treatment with ionising radiation. Similarly, mice in which Brca2 was deleted specifically in the mammary gland developed tumours only in a p53 deficient background [94]. Some of these tumours retained a wild-type Brca2 allele but this may have been inactivated by methylation or mutation and the possibility that this continued to be expressed was not addressed in this study. The effects of disruption of one allele of the BRCA2 gene in the chicken B cell line DT40 have recently been described [95]. This results in specific phenotypes including a reduced growth rate and sensitivity to specific genotoxic agents producing DNA double-strand cross-links, providing

strong evidence for a phenotype associated with heterozygosity for a mutation in BRCA2 in vertebrate cells. However, although it was established that the wild-type BRCA2 continues to be expressed in these cells, the possibility that a dominant negative allele had inadvertently been created cannot be ruled out. In summary, therefore, it is not yet clear whether loss of one wild-type allele confers a phenotype which is relevant to human carcinogenesis. As the chromosomal regions in which both BRCA1 and BRCA2 are located are frequently sites of loss-of-heterozygosity in cancer, this is an important issue to resolve.

7. Compound haploinsufficiency effects In some cases haploinsufficiency for a gene only manifests itself on a background of other genetic changes. These additional mutations may be heterozygous or homozygous loss of function mutations or dominantly acting oncogenic changes. These cryptic effects can be revealed in a defined way by introducing specific additional mutations onto the genetic background. Alternatively they can be induced by treatment with chemical carcinogens or by treatment with ionising radiation. We have already discussed some compound effects where the gene of interest already displays an overt haploinsufficient phenotype (Section 3.3). In this section, we provide examples of where the haploinsufficiency is silent in itself, but is revealed under genetic or chemical challenge (see Table 1). 7.1. Cooperation in tumourigenesis between the Blm and Apc genes Bloom’s syndrome is an autosomal recessive disorder characterised by small stature, immunodeficiency, male infertility and a strong predisposition to cancer [96]. Cells from Bloom’s syndrome individuals show increased somatic recombination and chromosome breakage which is thought to underlie the disease. Mice have been generated carrying a putative null mutation in the Blm gene [97]. Although the prevalence of tumours in these mice was not reported, other knock-out Blm mice did not appear to be tumour-prone [97 – 99]. When challenged by infection with murine leukaemia virus, both wild-type and heterozygous mice developed metastatic Tcell lymphoma. However, the mutant mice died at a faster rate despite the tumour pathology being similar. To compare rates of colon carcinogenesis, the Blm heterozygous mutant mice were crossed with mice heterozygous for the min mutation in the Apc gene. These Apcmin develop gastrointestinal tumours and are a model of familial adenomatous polyposis coli. Twice the number of intestinal tumours developed in the Apcmin carrying heterozygous Blm mutations than those wild-type at Blm. These tumours retained the wild-type Blm allele and continued to express the protein. A

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Table 1 Genes for which a haploinsufficient contribution to tumourigenesis has been ascribed Gene

Function

Cancer syndrome

Human tumour types

References

APC

Regulation of h-catenin; microtubule binding

FAP

[150]

Arf ATM

Stabilises P53 DNA integrity/repair control

BML BRCA1

RecQ DNA helicase Involved in DNA repair, transcription Involved in DNA repair

Colorectal cancer, colorectal adenomas, duodenal and gastric tumour; osteomas and desmoid cancer; medulloblastoma Melanoma Lymphoma, cerebellar ataxia, immunodeficiency, breast cancer Solid tumours Breast, ovarian and other cancers Female and male breast cancer, ovarian, prostate, pancreatic and other cancers

[73,80,81,87]

BRCA2

Dmp1 FEN1 H2AX Lig4 LKB1

Transcription factor Not known, probably involved in MMR Non-homologous end-joining DNA repair Non-homologous end-joining DNA repair Serine-threonine kinase of unknown function

MAD2 MSH2

Mitotic checkpoint DNA mismatch repair

NF1

GAP for p21 ras proteins; microtubule binding? Transcription factor Cyclin-dependent kinase inhibitor (INK4 family) Cell cycle control checkpoint Transcription factor; response to DNA damage and stress; apoptosis Transmembrane receptor for hedgehog signalling molecule Dual-specificity phosphatase with similarity to tensin Modulating cell cycle progression Signal transduction Growth factor

Nkx3.1 P18INK4c p27 p53 PTCH PTEN RB SMAD4 TGFb1

Ataxia telangectasia syndrome Bloom’s syndrome HBOC HBOC

Gastrointestinal cancer

[26] [151 – 154] [97,100] [80,81,86,88]

[24] [103] [115] [112]

Peutz – Jeghers syndrome

Hamartomatous polyps mucocutaneous pigmentations of the lips, bucal, mucosa, and digits

HNPCC

Colorectal, endometrial, ovarian; hepatobilliary and urinary tract cancer, glioblastoma Neurofibrosarcoma, AML, brain tumours

Neurofibromatosis type 1

Li – Fraumeni Syndrome NBCCS Cowden disease. Juvenile polyposis Retinoblastoma Juvenile polyposis

[50 – 52]

[71] [63 – 65]

[121,128]

Prostate cancer

[61] [105]

AML Soft tissue sarcoma, osteosarcomas, breast and other carcinomas Basal cell skin cancer, medulloblastomas, ovarian fibromas Breast cancer, thyroid cancer; endometrial cancer, intestinal hamartomous polyps Retinoblastoma, osteosarcoma Pancreatic and colorectal cancers

[11] [16] [155] [27] [79] [42,44,156] [43]

HBOC, hereditary breast and ovarian cancer syndrome; HNPCC, hereditary non-polyposis colorectal cancer; FAP, familial adenomatous polyposis; NBCCS, nevoid basal cell carcinoma syndrome.

possible explanation of the promotion of tumourigenicity is provided by the observation that heterozygous Blm cells have a subtle increase in genomic stability. Analysis of the mechanism of loss of the wild-type Apc allele in Apcmin/ mice suggested an increase in loss by somatic recombination in mice heterozygous for Blm mutation compared to wildtype. Therefore, the increase in genomic stability combined with an increase in somatic recombination caused by loss of Blm protein seems to result in increased frequency of the penetrance of a tumour suppressor mutation. These results may also have important implications for human cancer predisposition. About 1% of Ashkenazi Jewish individuals carry a loss-of-function mutation in the BLM gene and these individuals are at an approximately twofold increased risk of developing colorectal cancer

[100]. It remains to be determined if the wild-type BLM allele is retained in these tumours. 7.2. Fen1 The Flap endonuclease1 (Fen1) gene encodes a protein that functions in DNA replication in the processing of the 5V ends of Okazaki fragments in lagging strand synthesis and in DNA repair in long patch-base excision repair [101,102]. A complex series of phenotypes result from null mutations in the Saccharomyces cerevisiae Fen1 gene (also known as rad27). These display an increased frequency of frame-shift mutations and this has led to the suggestion that rad27 mutation may cause a partial DNA mismatch repair defect [103].

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Mice have been generated carrying a null mutation in the Fen1 gene [103]. Heterozygous Fen1+/ mice appeared superficially normal and had a similar lifespan to wild-type mice but based on small numbers may have a mild susceptibility to B cell lymphomas. To investigate whether heterozygosity for a null mutation in Fen1 can influence tumourigenic progression, mice were generated that were doubly heterozygous for Fen1 and a loss-of-function mutation in the Apc gene (Apc1638N). As described above, heterozygous mutations in Apc confer susceptibility to gastrointestinal tumours. Additional heterozygosity for the Fen1 mutation results in a shortened median survival. Although there was no difference in overall tumour burden, tumours in Apc1638N/ + /Fen1+/ mice were likely to be much more advanced than those in Apc1638N/ + /Fen1+/ + mice. Evidence for a role for haploinsufficiency of Fen1 in tumour progression was provided by retention of the wild-type allele of Fen1 and the absence of point mutations. However, continued expression of this allele was not directly demonstrated. Insight into how Fen1 mutation contributes to increased tumour incidence was provided by the demonstration that tumours in the Apc1638N/ + /Fen1+/ mice showed microsatellite instability whereas normal tissues did not. It was suggested that the tumour selectivity of the reduction in dose of Fen1 was due to the high rates of cell division in tumour cells compared to normal cells [103]. 7.3. Haploinsufficiency for p18Ink4c in tumourigenesis after challenge with chemical carcinogens p18Ink4c is a member of the INK4 family of cyclindependent kinase inhibitors that negatively regulate CDK4 and CDK6 by binding to their regulatory cyclin D subunits [7]. Mice homozygous for mutations in the p18Ink4c gene are viable but display widespread organomegaly and their T and B cells have a higher proliferative rate after mitogenic stimulation [104]. This suggested a role for p18Ink4c in maintaining cellular homeostasis and proliferation, in particular in mediating the cell cycle regulatory activity of the RB gene product. Mice lacking p18Ink4c spontaneously develop intermediate lobe pituitary tumours late in life but heterozygotes for the p18Ink4c null mutation do not appear to be tumour prone. However, a haploinsufficiency phenotype for p18Ink4c was revealed when mice were challenged with chemical carcinogens [105]. In this study, wild-type, heterozygous and homozygous p18Ink4c mutant mice were treated with the carcinogen dimethylnitrosamine. The homozygous mutant animals had the shortest mean survival time and developed most tumours. However, the heterozygous mice also developed significantly more tumours and fewer survived on average than the wild-type mice. Tumours in the heterozygous p18Ink4c mice retained the wild-type allele in an unmutated form and p18Ink4c protein continued to be expressed. Therefore, this provides good evidence for a gene dosage-sensitive effect but only after treatment with a carcinogenic agent. Although there is as yet

no strong evidence implicating p18Ink4c in human carcinogenesis, the gene maps to chromosome 1p32, a region that frequently shows loss-of-heterozygosity in human tumours [106]. 7.4. The role of Pml haploinsufficiency in the pathogenesis of APL The majority of cases of APL are associated with the presence of a chimeric fusion oncoprotein PML-RARa composed of portions of two distinct proteins, PML and RARa [107]. This is usually generated by a reciprocal translocation, t(15;17)(q22;q11.2), between human chromosomes 15 and 17. This creates not only a PML-RARa fusion but also the reciprocal fusion protein RARa-PML, and the result is haploinsufficiency for both PML and RARa. Mice carrying homozygous mutations in Pml are not significantly tumour-prone, so this gene does not fall into the category of ‘classical’ tumour suppressor genes. However, when mice carrying a leukaemia-inducing PMLRARa transgene were crossed with mice deficient for Pml, it was clear that reduction in Pml gene dosage could act to decrease latency and increase the penetrance of acute leukaemia [108]. Although Pml haploinsufficiency was demonstrated in this study, it was clear that this was not the only factor in play. Increasing the dosage of the leukaemogenic PML-RARa transgene also anatagonised wildtype Pml function presumably via a dominant negative mechanism. Loss of Pml function appears to contribute to leukaemogenesis by enhancing the survival of immature myeloid cells and making them more resistant to differentiation signals [108]. Whether haploinsufficiency of PML is relevant to the pathogenesis of human leukaemia remains to be demonstrated but may depend on the relative expression levels of PML-RARa and PML in the appropriate cell type in humans. 7.5. Dosage sensitivity of genes involved in non-homologous end-joining (NHEJ) in cooperation with inactivation of the cell cycle checkpoint regulators Mammalian cells have two important pathways for the repair of DNA double-strand breaks: homologous recombination, predominant in the S and G2 cell cycle phases, and NHEJ which predominates in G1 [109]. Mutation of components of NHEJ in mice causes sensitivity to ionising radiation, genomic instability, impaired lymphocyte development and dramatically increased lymphoma development in the context of P53 pathway deficiency [110]. DNA ligase IV, encoded by the Lig4 gene, is a key component of the NHEJ [109]. Homozygous mutant Lig4 mice die during embryogenesis probably from the effects of massive neuronal apoptosis, but this can be rescued by P53 deficiency [111]. Sharpless et al. [112] have examined the effects of Lig4 gene dosage reduction on carcinogenesis in tumour-prone ink4a/arf / mice. Haploinsuffi-

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ciency for Lig4 sensitised ink4a/arf deficient mice to DNA damage and resulted in increased tumourigenesis. In particular, the Lig4 heterozygous mice were prone to sarcoma formation [112]. Interestingly, an increased number of cytogenetic abnormalities have been noted in mouse cells heterozygous for Lig4 mutations [113]. Recently haploinsufficiency for another gene implicated in NHEJ, H2AX, has been reported [114,115]. In concert with homozygosity p53 mutation, H2AX gene dosage reduction results in an increased predisposition to B and T cell lymphomas and solid tumours. 7.6. Haploinsufficiency of Pten and the TRAMP model of prostate cancer Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mice express the SV40 T antigen in prostate epithelial cells under the control of the probasin promoter. These animals exhibit PIN and can display well-differentiated adenocarcinomas within 12 weeks of age. These subsequently progress to a poorly differentiated state followed by metastatic spread around 24 weeks [116]. As discussed above (Section 4.1), Pten+/ mice develop hyperplasia and dysplasia of several tissues but invasive prostate carcinoma is not observed [30, 31]. Given the well-documented loss-of-heterozygosity at PTEN in human prostate cancers (see Section 4.1), Kwabi-Addo et al. [117] generated TRAMP model mice carrying Pten mutations. They were able to show that losing one wild-type allele of Pten significantly accelerated tumourigenesis in TRAMP mice over that seen in mice that retained two wild-type Pten alleles. Furthermore, a significant number of these tumours retained Pten protein expression in the tumour. Heterozygosity for mutations in Pten has also been shown to generate invasive prostate cancer when on a p27 / background [30]. However, the physiological relevance of this finding has been questioned [117,118]. 7.7. Haploinsufficiency for TGFb1 cooperates with chemical carcinogens The TGFhs are potent inhibitors of epithelial cell proliferation and the TGFb1 response pathway has been implicated in tumour suppression in several organ systems (see Section 4.2). The role of the TGFb1 gene as a potential tumour suppressor in mice has been examined [43]. TGFb1 / mice die soon after weaning of a multifocal inflammatory syndrome. In contrast, heterozygous TGFb1 +/ mice are viable and grossly normal but have a subtle phenotype displaying increased cell turnover in the lung and liver. When challenged with a chemical carcinogen, diethylnitrosamine, the heterozygous mice were much more likely to develop liver and lung tumours than wild-type mice. The expression of the wild-type allele was maintained in the tumours but surprisingly the

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heterozygous mice expressed only 10 – 30% of the level of TGFh1 in wild-type mice.

8. Gene dosage effects in tumour stroma affecting tumourigenesis Over the last few years there has a growing realisation of the key importance of stromal components in tumour development [119]. A tumour is composed of several other cell types apart from the tumour including fibroblasts, endothelial cells and immune cells such as macrophages and mast cells. It is clear that these ‘non-tumour’ cells are profoundly affected by the proximity of the tumour. These in turn can also influence tumour progression and genetic changes in this compartment can result in tumour progression [120]. Strong evidence that haploinsufficiency for a tumour suppressor in stromal components can influence tumour progression has recently been provided for the Neurofibromatosis-1 (NF1) gene [121]. Heterozygous carriers of mutations in NF1 develop neurofibromas, which are benign tumours of the peripheral nerve sheath [122,123]. One of the unusual features of neurofibromas is their cellular heterogeneity—they contain all of the cell types usually found in normal peripheral nerves : axonal processes, Schwann cells, fibroblasts, mast cells and perineural cells. In NF1 mutation carriers a neurofibroma is thought to initiate when the wild-type allele becomes lost or otherwise inactivated in a Schwann cell [121]. NF1 encodes Neurofibromin, a large protein containing a Ras-GAPdomain that regulates Ras a critical component of many growth factor signalling pathways [42,124]. Loss of NF1 function results in constitutive activation of Ras causing increased proliferative capacity or insensitivity to apoptotic signals [123]. To develop a physiologically relevant mouse model of neurofibromatosis Zhu et al. [121] used a gene targeting strategy to allow the elimination of the Nf1 gene specifically in Schwann cells. They developed a conditionally inactivatable (flox) allele of Nf1 that was functionally wild type but becomes deleted when Cre recombinase is expressed under the control of the Schwann-cell-expressed Krox20 promoter. Mice with deletion of Nf1 develop neurofibromas at high frequency that were highly reminiscent of these seen in human NF1 mutation carriers demonstrating that Schwann cells are the cell type of origin of this disease. However, neurofibroma development was dependent on genetic background. Deletion of Nf1 on a heterozygous Nf1f lox/ background gave frank neurofibroma formation, but on an essentially wild-type Nf1flox/flox background no neurofibromas formed and pathological examination revealed only small infrequent hyperplasias. This result indicates that tumour environment is critical for tumour development and that haploinsufficiency can play a permissive role in facilitating this. There may be significant implications of this work for other cancer susceptibility syndromes such as those conferred by BRCA1 and BRCA2 mutation (see Discussion).

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It is presently unclear which are the critical cell types affected by haploinsufficiency for Nf1. Zhu et al. observed increased infiltration of mast cells in the peripheral nerves of heterozygous mice compared to wild type. Indeed, others have shown that Nf1+/ mast cells hyper-proliferate in vitro and in vivo in Nf1+/ mice [125]. Mast cells have also been shown to be able to contribute to tumourigenesis in a model of squamous carcinoma [120,126]. Nevertheless, the contribution of other cell types such as fibroblasts may also be important [127]. The mechanism by which haploinsufficiency of Nf1 leads to altered behaviour of stroma is not clear but mathematical modelling studies have suggested that reduced levels of Neurofibromin leads to a reduction in signal-to-noise ratio in signalling pathways caused by increased noise [128] (see Discussion).

9. Discussion 9.1. Cancer as a statistically unlikely event Most human cancers develop slowly and there may be 20 or more years between carcinogen exposure and clinical presentation. It is unclear exactly how many genetic changes are rate-limiting for tumour development. However, estimates from epidemiological studies suggest that the minimum number of events required is between 4 and 7 [129,130]. Similarly, in vitro transformation of human cells indicates that at least three genes have to be introduced before cells acquire the ability to form tumours [131,132]. Finally, consideration of the biochemical pathways that need to be inactivated or augmented for tumour development yields a similar number [119]. It is suggested that some of the key requirements are self-sufficiency for growth signals, insensitivity to antigrowth signals, evasion of apoptosis, limitless replicative potential, sustained angiogenesis and tissue invasion and metastasis [119]. However, the estimates of the number of genetic events required for tumour formation do not square with what is known about the mutation rate in normal human somatic cells. The exact rate is not easy to estimate because of the diversity of potential changes including point mutations, large deletions, amplifications, etc. Nevertheless, estimates of between 2.0  10 7 and 1  10 6 mutations/gene/cell division have been made [133,134]. Based on these data, it has been suggested that each cell might accumulate only one mutant gene during the life span of an individual [135]. However, because this figure encompasses all genes, this means that the overall frequency of mutations that might contribute to cancer progression may be considerably lower than 1/cell/lifetime. A further complication is that solid tumours are thought to arise in stem cells. Although the mutation rate of these cells is not known, mounting evidence suggests that it is lower than that of somatic cells [136]. Stem cells are less

effective at DNA repair and tend to die when they suffer DNA damage. Furthermore, they divide more slowly with a cycle time of approximately twice than that seen in their daughters [136,137]. Finally, it has been suggested that stem cells might contain ‘immortal’ strands of DNA protecting them from mutation [138]. How normal cells can accumulate enough mutations to become a tumour is controversial. On the one hand, genetic changes that increase mutation rates have been proposed to be essential to account for the large numbers of mutations observed in human tumours and cells might acquire a mutator phenotype in the early steps of tumorigenesis [130]. Direct evidence of this has come from hereditary non-polyposis cancer (HNPCC) in which tumours, developed as a consequence of a mutation in mismatch repair genes, are characterised by thousands of changes in the length of repetitive nucleotide tracts [139]. On the other hand, it has been postulated that tumour progression involves successive waves of clonal selection [140]. In this model, each mutation, occurring at a normal rate, imparts a proliferative advantage and is selected for in the successive clonal lineages. The actual events are likely to involve an interplay between these two scenarios. Here we consider how inactivation of a single allele of a tumour suppressor gene may contribute to this process. 9.2. Haploinsufficiency: increasing acceptance of the phenomenon but still difficulties in validation There is now considerable evidence supporting the concept of haploinsufficiency. However, acceptance has been held back by claims of haploinsufficiency supported by inadequate evidence. This should normally include evidence for retention of the wild-type allele, absence of deleterious mutations and continued expression of the protein product. Nevertheless, this in itself is insufficient. It is also necessary to demonstrate a phenotypic effect that can make a plausible contribution to tumourigenesis. Some studies of haploinsufficiency in human systems are confounded by inter-individual variation. For example, studies purporting to show BRCA1/2 haploinsufficiency have failed to the phenomenon conclusively. Studies on gene dosage effects in the mouse are not subject to this criticism but mouse models of cancer are not always directly relevant to the human disease. For example, mouse models of tumour suppressor loss often either fail to develop tumours or develop tumours in different tissues than humans carrying similar mutations. Other major problems in interpretation have come from the possibility of other mechanisms contributing to the tumourigenic process. For example, mutations in the ATM gene have been claimed in some cases to be haploinsufficient and in others to be dominant negative [141]. For both missense and truncating mutation, both mechanisms are formally possible. It is only with truly

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null mutations or those that lead to absence of transcript by, for example, nonsense-mediated decay that a dominant negative mutation can be ruled out. In some cases both haploinsufficiency and dominant negative affects may operate simultaneously as with PML-RAR (Section 7.3). Despite this, there are now many convincing examples of haploinsufficiency. 9.3. Modifying Knudson’s model The two-hit model of inactivation of tumour suppressor genes has held sway for many years and there are a considerable number of examples where this simple paradigma seems to apply. However, the situation may be more complex in the case of some tumour suppressor genes. A key issue is whether loss of the second allele is at the normal cellular rate for such events or whether it is facilitated by haploinsufficiency. Knudson’s model was originally built around models of retinoblastoma progression and it seems even here that haploinsufficiency might play a role. It has been shown in embryonic stem cell that mutation in a single allele of the Rb gene increased the frequency of loss of a marker chromosome over that of cells that carry two wildtype alleles [79]. Likewise, it has been claimed that heterozygosity for Msh2 confers a phenotype that could facilitate chromosomal loss. It is not yet known in what proportion of classical tumour suppressor genes haploinsufficiency makes a contribution to loss of the wild-type allele and in what proportion it is random. Another possibility is that the tumorigenic advantage conferred by an heterozygous mutation in a tumour suppressor gene is too low to be selected for but a second mutation in another tumour suppressor gene can synergise with the first. As a result, this cell could gain a phenotype either of proliferative increase or genomic instability and be selected for and advance down the tumourigenic path (Fig. 2). Therefore, it seems probable that modifications of the original Knudson hypothesis are necessary to take account of haploinsufficient effects.

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population for mutagenesis. As well as haploinsufficiencies that cause small proliferative advantages, relatively modest impairments in DNA repair could have a similar effect; instead of increasing the target population, this would result in increasing the mutation rate. If, as is thought, cancer arises in stem cells then phenotypes caused by haploinsufficiency could result in ‘field cancerization’ [143] (Fig. 3). As well as predisposing cells in the field to tumourigenesis, this effect could alter the behaviour of surrounding tissue as may be the case with Nf1 (see Section 8). Haploinsufficiencies can sometimes in themselves not have a measurable contribution to tumourigenesis. However, in combination with other genetic changes they may no longer remain cryptic but foster a more penetrant phenotype. An excellent example is the potential for haploinsufficiency of the Blooms syndrome gene Blm to contribute to tumourigenesis mediated by classical tumour suppressor genes. Loss-of-function mutations in Blm lead to a hyper-recombinogenic phenotype. As tumourigenesis by classical tumour suppressor genes relies on loss of the wild-type allele by missegregation or mitotic recombination, these defects in the pathway in which Blm is involved might be expected to enhance significantly the effects of mutation of classical tumour suppressor genes. Likewise, haploinsufficiency for defects in DNA repair pathways could also accelerate tumourigenesis by making mutation of the wild-type allele of a tumour suppressor more likely. Evidence that haploinsufficiency effects may be pathway-specific has recently come from analysis of the cooperation of dosage reduction in the p27 gene with models of colorectal cancer [8]. Haploinsufficiency of p27 can accelerate tumourigenesis induced by the carcinogen 1,2-dimethylhydrazine and by heterozygosity for an Apc mutation but not by homozygosity for a Smad3 mutation, another model of colorectal cancer. This is probably due to the requirement for the reduction in levels of p27 for tumourigenesis induced chemically or by loss of Apc but not for that induced by Smad3 mutation. This latter notion is supported by the continued expression of normal levels of p27 protein in tumours arising in Smad3 mutant mice.

9.4. Strong, weak and cooperative haploinsufficient effects A general theme in haploinsufficient genes is that tumours generated in a haploinsufficient context are frequently of later onset and less severe than the corresponding tumour carrying the homozygously mutated gene. This is true for p27, p53 and Dmp1. Similarly, in the case of some haploinsufficient tumour suppressors, haploinsufficiency is associated with the early stage of disease such as hamartomatous polyp formation associated with PTEN, Smad4, and Lkb1 haploinsufficiency. Development of frank carcinoma formation requires loss of both alleles. These observations have led to the suggestion that haploinsufficiency effects can be strong or weak [4,142] (Fig. 3). As pointed out by Quon and Berns, even a small proliferative advantage could produce a large ‘‘sensitised’’ target cell

9.5. The implications of haploinsufficiency for tumour progression in carriers of mutations in cancer susceptibility genes The contribution of genetic or epigenetic changes in nontumour cells to tumourigenesis has only begun to be appreciated recently. This phenomenon has perhaps been best exemplified by the Nf1 gene (Section 8). As described above, haploinsufficiency of the Nf1 gene is required for tumour formation by Nf1 homozygous mutant cells. This might be a phenomenon of general applicability. Some tumour suppressor genes such as BRCA1 and BRCA2 are only rarely, if at all, mutated in sporadic cancer. It is possible that this is because the events required for mutation or loss of both alleles of these genes occurs at too low a frequency. However, it seems

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Fig. 3. Strong and partial haploinsufficient effects. (A) A strongly haploinsufficient effect results in tumour formation in the absence of loss of the wild-type allele. (B) In a partial haploinsufficient effect, after a proliferative phase, the wild-type allele needs to be lost for tumour formation. (C, D) Familial cancer, where the entire body is heterozygous and the situation where there is no haploinsufficient effect are shown for comparison (modified from Quon and Berns [4]).

possible that like Nf1, tumour formation requires constitutional heterozygosity for BRCA1 and BRCA2. There has been considerable discussion as to why mutations in genes such as BRCA1 and BRCA2, which are ubiquitously expressed and involved in a generic pathway of DNA repair, should show such a restricted pattern of cancer predisposition [72,144,145]. Redundant pathways that rescue loss of BRCA2 in unaffected tissues and the high proliferative index of susceptible tissues such as breast and ovary have been invoked to explain this tropism. Recently, Elledge and Amos [145] have proposed a new hypothesis for tumour development by BRCA1, which

shows a similar restricted pattern of cancer predisposition to BRCA2. This involves the supposition that complete loss of function of BRCA1 or BRCA2 is lethal to most cell types but can be rescued by specific survival factors in certain tissues or environments such as the breast. However, tissuespecific haploinsufficiency suggests another possibility. Perhaps certain cell types, particularly those susceptible to tumourigenesis in human BRCA mutation carriers, might be particularly sensitive to BRCA dosage leading to impaired fidelity of DNA repair. In combination with other genetic or environmental factors, this could lead to further mutation, including loss of the wild-type BRCA allele,

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concluded that the phenotypic differences in NF1 heterozygotes could be interpreted as an alteration in the signal-tonoise ratio as a consequence of increased noise. These studies are in their infancy but are likely to have widespread implications for understanding the effects of the reduced dosage of a tumour suppressor gene.

10. Summary Fig. 4. A model of the variance in gene expression in a two-allele system (Diploid) and a single-allele system (Haploid). The stochastic nature of gene expression means that there is considerably more variance in the haploid than in the diploid system. Frequently in the haploid system levels approach 10% (indicated by arrows) of wild-type, which could mimic a null mutation in many systems. This rarely, if ever, happens in the diploid system; see text (adapted from Cook et al. [149]).

perhaps as a later event, such as pancreatic cancer [146]. This may trigger further genome instability and tumourigenic progression in the susceptible tissues. 9.6. Stochastic behaviour and the role of noise in the generation of a haploinsufficient phenotype Haploinsufficiency of genes implicated in tumourigenesis is generally thought to indicate the requirement for more than half the normal diploid product level of a given protein. However, this takes little account of the reality of the regulation of genes or pathways. There is an increasing recognition that changes in processes such as gene expression and signal processing within cells have a strong probabilistic or stochastic component [147,148]. Mathematical modelling of the effects of reducing gene dosage from diploid to haploid indicates that haploinsufficiency phenotypes might result from increased susceptibility to stochastic delays in the initiation of a response or in interruptions in processes such as gene transcription [149]. This is illustrated in Fig. 4. In this model, based on an assumption of stochastic gene expression, steady-state gene product levels fluctuate around a mean. In the case of haploid gene expression, this mean is 50% of the diploid state. However, transient levels significantly below 50% could occur. In some cases this could reach a critical level causing in effect a null phenotype (in this model 10% of the diploid product level) for the genes or pathway being regulated. Because of a positive feedback loop, this could lead to prolonged effects. Diploid cells essentially never reach the 10% level of product in this model. If the gene product were involved in cell-cycle checkpoint regulation, this might mean that an inappropriate cell division occurs. Other scenarios disastrous for the cell could be envisioned for many gene products. The increased variance in expression levels seen in the haploid system has been investigated for the NF1 gene by Kemkemer et al. [128]. These authors examined the consequences of NF1 haploinsufficiency on the morphology of cultured melanocytes from NF1 mutation carriers. They

Haploinsufficiency appears to be widespread among tumour suppressor genes. It seems possible that under the correct circumstances, the majority of these genes will exhibit the phenomenon. The phenotypes involved include enhanced proliferation, absence of checkpoint function, inability to respond to inhibiting signals and loss of genomic stability. Haploinsufficiency in non-tumour cells may also have a permissive effect on tumour development. Haploinsufficient effects are likely to vary from gene to gene, depending on the inherent noise in the system. Acting together or alone, haploid gene loss seems likely to play a major role in tumourigenesis.

Acknowledgements We thank Breakthrough Breast Cancer for generous funding, and Suzy Small for her patient and skilled preparation of manuscript and figures.

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