Tumor Suppressor Genes

Tumor Suppressor Genes: A Historical Perspective

From a historical point of view, the articulation of the modern concept of tumor suppressor gene (TSG) was possible through the convergence of three major lines of research: somatic cell hybridization experiments, the detection of loss of heterozygosity (LOH) in tumors, and the study of highly penetrant familial cancers. It is worth mentioning, however, that the existence of TSGs had already been anticipated early in the 20th century by Theodor Boveri (1862-1915), one of the founders of the chromosomal theory of inheritance (the modern concept of the gene had not been developed at the time). Boveri suggested that the uncontrolled growth characteristic of tumors arises as a result of an incorrect chromosomal dosage, which could be explained by an abnormal segregation of chromosomes during cell division. Thus, “growth inhibitory chromosomes” are removed from cells during the process of tumorigenesis. As a corollary, Boveri suggested that these inhibitory chromosomes were part of a mechanism that kept normal cells in a proliferation-arrested state unless they were stimulated to divide. It was not until the mid-20th century, however, that more sensitive cytogenetic techniques allowed the identification of LOH (loss of heterozygosity), indicative of chromosomal loss, in human tumors.

The next piece of evidence linking loss of function mutations and tumorigenesis came from somatic cell hybridization experiments performed in the early 1970s. The crucial observation here was that cell hybrids generated through fusion of normal somatic cells with tumor-derived cells were usually nontumorigenic. The dominant effect of normal traits over the malignant phenotype seemed to indicate that loss of growth-regulatory genetic information had contributed to the transformation of the parental tumor cell line in the first place. Conversely, the neoplastic phenotype could be reversed following the reacquisition of the normal complement of genetic information.

Ultimately, however, it was the study of familial cases of cancer predisposition that led to the identification, and subsequent cloning and characterization, of the first TSGs. Highly penetrant cancer susceptibility syndromes constitute a small group of inherited disorders in which the affected individuals develop a unique type of tumor (or a narrow set of tumors) with an unusual high incidence and at a younger age compared with sporadic (noninherited) cases. Although cancer-associated syndromes display a dominant mode of inheritance, the experimental evidence gathered from in vitro cell fusion experiments and LOH analyses seemed to indicate that TSGs acted in a recessive manner at the cellular level. A theoretical explanation for this paradox was first provided by Alfred Knudson and became later known as the “two-hit” hypothesis.

In 1971, Knudson was studying the epidemiology of retinoblastoma, a relatively rare pediatric tumor that originates in the fetal retina. Retinoblastoma is associated with an inherited predisposition in approximately 40% of the cases. Most children with an affected parent develop multiple retinoblastomas in both eyes, which are diagnosed at a younger age compared with the sporadic forms of the disease. In contrast, children diagnosed with sporadic retinoblastoma (children with no family history of the disease, about 60% of the cases) show unilateral involvement and typically a single tumor in the affected eye. Based on these differences, Knudson postulated that the cell of origin of retinoblastoma (the retinoblast) must undergo two critical genetic events at a single locus in order to initiate a tumor. Because the first mutation or “hit” is already present in every somatic cell (including retinoblasts) of an individual with hereditary retinoblastoma, an inactivating mutation of the remaining allele (the second “hit”) would be sufficient to drive tumor formation. The increased probability of tumor initiation in the context of a large population of already mutated retinoblasts helps explain the multiplicity of tumors per retina and the characteristic early onset of hereditary retinoblastoma. In cases of sporadic retinoblastoma, on the other hand, the hypothesis postulated that both hits must take place in a single somatic cell, itself a much less probable event, thus explaining the unilateral involvement and the lower number of tumors per retina.

Evidence for the “two-hit” model was later provided by cytogenetic analyses of blood samples from patients with inherited retinoblastoma. These studies identified germline deletions of chromosomal band 13q14 in these patients, and similar changes were subsequently found in sporadic tumors, providing strong support for the notion that the same genetic locus was affected in both variants of the disease. Although several mechanisms were initially proposed to explain the second hit, their final documentation was only possible through DNA restriction fragment length polymorphism (RFLP) analysis. RFLP-based studies demonstrated that LOH achieved through deletion, mutation, or recombination can all occur as second events in retinoblastomas. Finally, these studies provided the necessary molecular clues that led to the cloning of the retinoblastoma susceptibility gene ( RB-1 ) in 1986.

The protein encoded by RB-1 (pRB) is now recognized as a key suppressor of cell cycle progression. Evidence for pRB’s function dates back to studies on cellular immortalization mediated by viral oncoproteins, including the simian virus 40 (SV40) large T antigen. These studies demonstrated that cellular immortalization was in part a result of the direct inactivation of pRB by the SV40 large T antigen. Because immortalized cells were especially sensitive to transformation by several oncogenes, the natural conclusion was that inactivation of pRB might represent a common requirement for the initiation of most cancers. However, it soon became evident that inactivating mutations in RB-1 are found in a rather narrow group of human malignancies, most typically in small-cell lung carcinomas and osteosarcomas. As mentioned later in this chapter, we know now that deregulation of numerous cancer-associated genes can lead to the inactivation of pRB in more indirect ways, a hallmark shared by virtually all cancers.

At around the same time, the status of TP53 (also known as p53 ) as a TSG was also established. Its gene product, the transcription factor p53, was originally identified as an interacting partner of the SV40 large T antigen. Although the mechanisms of tumorigenesis involving p53-large T antigen interaction initially suggested that TP53 acted as an oncogene, further experimental evidence challenged this notion. For example, wild-type p53 was shown to act as a suppressor of transformation in cultured cells, and genetic rearrangements at the TP53 locus, which resulted in loss of function instead of activation, were discovered in some leukemia cell lines. In addition, it was suggested that the interaction between p53 and the SV40 large T antigen resulted in loss of p53 function, in a manner reminiscent of pRB inactivation. Nevertheless, it was not until TP53 was mapped within a region consistently deleted in human tumors that the gene gained its final recognition as a bona fide TSG. Seminal among these studies was the discovery that TP53 was biallelically deleted in human colorectal cancers, which was soon followed by the identification of mutations in other malignancies. Loss-of-function mutations in TP53 were then linked to Li-Fraumeni syndrome, a known dominantly inherited condition that predisposes individuals to several cancers, most typically breast cancer. Over the following 40 years, an explosion of research has confirmed TP53 as one of the most frequently mutated genes in human tumors. As mentioned later, p53 is involved in the orchestration of a variety of tumor-suppressive processes, including cell cycle arrest, apoptosis, and senescence. Deregulation of each one of these processes following p53 inactivation can thus increase the probability that a cell will become malignant.

In summary, the RB-1 and p53 paradigms defined three important properties of “classic” TSGs: (1) TSGs are recessive at the cellular level, with biallelic inactivation typically found in tumors; (2) inheritance of a single mutant allele increases tumor susceptibility because only one additional inactivating event is required for complete loss of gene function; and (3) the same gene is frequently inactivated in sporadic cancers. Theoretically, reversion of the tumorigenic phenotype following the reintroduction of the relevant TSG into a cancer cell may also serve as a functional criterion to classify a gene as tumor suppressor. However, this requirement is not always met experimentally, presumably because loss of a TSG can allow further genetic changes that may confer resistance to its restoration at a later time.

These principles served as guidance for the identification and cloning of other cancer susceptibility loci. Table 3-1 lists selected TSGs along with the function of their encoded proteins, the cancer syndrome they are associated with, and examples of sporadic cancers associated with their loss of function.

Moderate and Low Penetrance Cancer Susceptibility Loci

Despite their importance in the discovery of TSGs, highly penetrant cancer syndromes account for a relatively small proportion of human malignancies (typically less than 0.1%). In fact, the excess in familial risk for some types of cancer has remained, to a large extent, unexplained. For example, breast cancer shows a pattern in which relative risk increases by two- to threefold in first-degree relatives of early-onset cancer cases. However, mutations in BRCA1 and BRCA2 account for only ∼20% of this overall risk increase. It has been proposed that much of the inherited risk may result from a polygenic mode of cancer predisposition. In this scenario, multiple loci, each one having a modest individual effect, may ultimately dictate the relative risk of an individual to develop cancer. In support for this idea, recent reports have identified susceptibility alleles associated with a wide range of risk in human populations. For example, the screening of genes associated with BRCA1- and BRCA2- dependent pathways demonstrated “moderate” breast cancer risk in individuals who are heterozygous for allelic variants of CHEK2 . These rare alleles function dominantly, each conferring a moderate but detectable increase in the relative risk of developing breast cancer. More recently, genome-wide association (GWA) studies based on differences in single-nucleotide polymorphism (SNP) across human populations have begun to identify “low-penetrance” cancer susceptibility loci for the most common types of cancer.

Haploinsufficiency

The two-hit hypothesis has been challenged by recent studies indicating that many chromosomal deletions in cancer cells consistently affect a single allele. Although these monoallelic deletions were initially considered to be mere “passenger events” with no actual causal role, numerous genes that behave as TSGs in vitro have been identified in these regions. This observation implies that a single-copy mutation or loss of these loci might be sufficient to explain their tumorigenic effect, a genetic property known as haploinsufficiency . For some alleles, haploinsufficiency may even confer a relative advantage to cells, most typically in situations where complete loss of function leads to apoptosis or senescence. For example, whereas a monoallelic deletion of the tumor suppressor PTEN is sufficient to produce prostate cancer in mice, loss of both parental alleles triggers a p53-dependent senescence program. Alternatively, a monoallelic mutation may confer dominant-negative capabilities on TSG products. In this modality, the mutant protein may negatively interfere with the function of the wild-type protein produced by the unaffected allele. Because dominant-negative mutations can result in considerable loss of function, there is no selective pressure in tumors to inactivate or delete the wild-type allele. A classic example of a dominant-negative effect is provided by mutant p53. As part of its function as a transcription factor, wild-type p53 binds DNA as a tetramer, a capacity impaired in mutated p53 because of missense mutations affecting the DNA binding domain. Although the wild-type and the mutant p53 proteins are still able to form hetero-oligomers in cells harboring monoallelic mutations, these complexes show impaired DNA association and transcriptional activity, resulting in loss of p53 function.

A third interpretation of the role of haploinsufficiency in tumorigenesis is based on the exquisite sensitivity of cells to even small changes in the levels of some cancer-associated proteins. Thus, a 50% functional reduction in a TSG product may be sufficient to endow a cell with a relative advantage for proliferation. Experimental evidence for this dosage-sensitivity effect has been shown for several TSGs, including TP53 , BRCA1 , BRCA2 , and PTEN . For example, a subset of tumors arising in p53 ^+/− mice, or in patients with Li-Fraumeni syndrome, retain the wild-type allele, suggesting that haploinsufficiency of TP53 may be sufficient for tumor initiation. Finally, the pro-tumorigenic effect imparted by haploinsufficiency might also be dependent on the loss or gain of function of other alleles. An example of this interaction is illustrated in mouse models of Pten and Tp53 deficiency. Haploinsufficiency of Pten in the context of wild-type p53 enhances proliferation and subsequent transformation of prostate epithelial cells. In contrast, complete loss of Pten in this tissue triggers a p53-dependent senescence program, with tumors arising only after Tp53 inactivation.