Towards the cellular functions of tumour suppressors

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The basic model of how TSG loss leads to tumour formation was first proposed by Knudson I from a statistical analysis of retinoblastoma, a childhood cancer of the developing retina that occurs in both sporadic and familial forms. Knudson proposed that two inactivating mutations were necessary to induce a retinoblastoma. In the sporadic form both mutations occurred somatically, whereas in the familial form one mutation was inherited and the second occurred in somatic cells. This model accounted for the earlier onset and more frequent bilateral cases in the familial form of the disease. Comings 2 then pointed out that the two mutations might occur in the two alleles of one gene. Thus in sporadic cases (Fig. la) both copies of the dominant, normal gene are lost or mutated (probably at different developmental stages) in the same cell clone during development, and the progeny of the defective cell become tumourous. In familial cases, one mutant gene is inherited and the second allele undergoes somatic loss or mutation (Fig. lb), leading to a tumour. This theory has been well supported by experimental evidence 3-s, with the gene whose loss is associated with the development of retinoblastomas identified as RB-1.

Towards the cellular functions of tumour suppressors Many of the documented changes in cellular DNA that occur during tumour development involve activation of proto-oncogenes, but newer evidence has shown that oncogenesis can involve loss or inactivation of a different group of genes, called tumour suppressor genes (TSGs). Molecular analysis of TSGs is revealing that their protein products are involved in cell adhesion, signal transduction, transcription, translation and cell cycle control. Surprisingly, most of the TSG products had not been previously identified in studies

Human TSGs The Wilms' tumour gene WT1, the neurofibromatosis gene NF1, the familial adenomatous polyposis gene APC, and the p53 gene all show patterns of inheritance and allele loss associated with tumours that are similar to those seen with RB-1 (Ref. 6), and they are therefore considered as TSGs (Table la). Furthermore, the common finding of nonrandom loss of heterozygosity of genetic markers in tumours 7 suggests that loss of TSGs, both known and unknown, may contribute to oncogenesis in many, and perhaps all, human cancers. The genetics of Wilms' tumour is more complex than retinoblastoma in that different genetic loci appear to be associated with the sporadic and familial forms s, and the situation with p53 is complicated by the existence of dominant negative alleles whose products appear to interfere with the fimction of the normal protein 9. This unusual mode of action accounts for some of the initial confusion over whether the p53 gene is an oncogene or an antioncogene 10. Dominant negative alleles of WT1 have also been reported 11,12. Six additional genes (Table lb) have been identified as candidate TSGs based on different kinds of evidence. In some cases (PT~, DCC, MCC and nm23), the primary evidence is an association between gene loss or inactivation and tumour development, with the causal connection being only inferred. In other cases [K-revl(=rapl) and prohibitin] the primary evidence is the ability of the cloned gene or its RNA to suppress transformation or block proliferation in vitro, while the potential role in vivo remains to be established.

Drosophila TSGs The ease of genetic manipulation in Drosophila makes this animal a favourable system for identTRENDS IN CELL BIOLOGY VOL. 3 FEBRUARY1993

of normal cells, so their analysis is contributing not only to our understanding of oncogenesis, but also to basic cell biology. The 'comment' articles in this issue discuss progress towards understanding the cellular functions of TSG products.

ifying and characterizing new TSGs that may have mammalian homologues. Mutagenesis screens and the analysis of spontaneously occurring mutations have led to the identification and genetic mapping of TSGs that function at a variety of stages and in different tissues 13-1a. Mutations in most of these genes are recessive lethals that lead to either hyperplastic or neoplastic overgrowth of specific tissues, followed by death of the animal during the embryonic, late larval or early pupal stage. The gonadal tumour mutations are different, in that they allow survival to the adult stage but then cause infertility. At the molecular level, the first of the Drosophila TSGs (lgi) was characterized in 1985 (Ref. 19) and seven more have since been characterized (Table 2). Three of the Drosophila TSGs (fat, dig and air8) show clear homology to human genes, none of which had previously been recognized as a TSG. TSG products from the cell membrane to the nucleus Tumour development involves many changes in cell behaviour, but its most characteristic feature is increased cell proliferation. Cell proliferation is controlled by a variety of mechanisms, both extrinsic and intrinsic to the cell. The extrinsic factors, including both diffusible factors and signals coming from adjacent cells, must act on the cell © 1993 ElsevierScience Publishers Ltd (UK)0962-8924/93/$06.00

Peter Bryant is at the Developmental Biology Center, University o,~ California, Irvine, CA 92717, USA. 31

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(b) Inheritance and somatic loss

(a) Somatic loss

candidate TSG2°. The phosphatase could act by counteracting the actions of growth-promoting receptor and non-receptor tyrosine kinases in the same part of the cell 20. Proteins involved in the signal transduction pathways mediated by GTP-binding proteins may also be tumour suppressors; these are disAllele loss, Constitutional @ A l l e l e loss cussed in the article by Rey and ~11 mutation or Hall (page 39). / ~ or mutation recombination Another group of TSG products carry out their functions in the nucleus. Both p53 and RB-1 encode Cells 0 ~ Allele loss, nuclear phosphoproteins (p53 and / ~ rmUtoafi~in°trion pRB) that are expressed ubiquitously in mammalian cells, show phosTumour phorylation during specific phases of the cell cycle and have been implicated in nuclear functions including cell cycle control and transcriptional controlZl, 22. pRB is Tumour discussed in the article by Helin and FIGURE I Harlow (page 43) and pS3 in the Two ways in which cells can lose both copiesof the normal alleleof a TSG. (a) The first allele is article by Rotter, Foord and Navot lost by mutation, and the second is lost by a second mutation or by mitotic recombination (page 46). WT1 encodes a zincbetween the two homologous chromosomes. (b) One mutant allele is inherited from a parent, finger DNA-binding protein 8 of the and the second is lost by a second mutation or by mitotic recombination between the two early growth response family of homologous chromosomes. transcriptional activators, but unlike the other members of this family WT1 functions as a transcriptional through its membrane, and the signals must be repressor rather than an activator. One of its tartransduced to the nucleus where they ultimately gets may be the insulin-like growth factor 2 (IGF-2) control replication. Tumour suppressors may be gene, a finding that is consistent with the overexpected to act as negative control elements at any expression of IGF-2 in Wilms' turnouts. IGF-2 is point in the cascade, so it is not surprising to find abundantly expressed in blastemal cells of the kidsome whose products are secreted or membrane asney 23 but this expression is later turned off and sociated, others that encode membrane.associated WT1 is expressed when the cells differentiate into signal transduction proteins, and others that funcan epithelium24, 2s. It has therefore been sugtion in the nucleus to control transcription and gested 26 that IGF-2 is an autocrine growth regureplication. The TSGs and candidate TSGs for lator in normal kidney development and that its which the molecular nature of the gene product production is limited by WT1. When the latter has been deduced are shown in Tables 1 (human) function is lost, unrestrained production of IGF-2 and 2 (Drosophila). might cause or at least contribute to the tumourous Cell adhesion molecules may be necessary to growth. One of the target genes whose expression bring cells into close enough contact for cell-cell is downregulated by pS3 and pRB encodes the autosignalling events to occur normally, or in some crine growth factor interleukin 6 (Ref. 27), sugcases they may have intrinsic signalling functions gesting transcriptional repression of growth factor in addition to their roles in adhesion. Cell-cell and genes may be a common mode of action for TSGs. cell-substrate contacts are often disrupted in Even genes that encode what appear to be housemalignant cells, and during metastasis there must keeping proteins, essential for basic cell functions, be a loss of normal cell contacts and formation of can behave as TSGs; the aberrant immune response 8 new ones. It is therefore not surprising that some (air8) locus of Drosophila is an example. Mutations tumour suppressor genes predict protein products that block expression of this gene cause massive related to known cell adhesion molecules. These overgrowth of the lymph gland, which is the are discussed in the article by Hedrick, Cho and major haemopoietic organ of the Drosophila larva. Vogelstein (page 36). Surprisingly, the air8 gene encodes the Drosophila Signal transduction pathways at the inner face of homologue of the human $6 ribosomal protein 28. the cell membrane are the next level at which TSG Although S6 has not been considered as the prodproducts have been found. The PTP? gene, which uct of a TSG, there are several indications that it encodes a receptor-type protein tyrosine phosphamay play a role in cell proliferation control and tase, maps to a region of chromosome 3 that is fre- tumourigenesis. It shows developmentally regulated quently deleted in renal cell carcinoma and lung phosphorylation of a cluster of serine residues at carcinoma, and has therefore been suggested as a the C-terminus, and this phosphorylation is stimu-

arents e i e

•

Q ®

1

32

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TABLE 1A - CLONED TSGS AND THEIR PRODUCTS IN MAN

lated in quiescent mammalian cells by treatments that increase protein synthesis and cell proliferation 29. In the air8 mutant the growth of most tissues is slowed, as expected; why the haemopoietic system should respond so differently to the loss of $6 expression is not yet understood. D o TSGs c o n t r o l d e v e l o p m e n t , or suppress t u m o u r s ?

Mutations in the Drosophila TSGs are almost invariably lethal in the homozygote, indicating that these genes encode proteins that have important functions in normal development. The role of roammalian TSGs during development has been less clear. In the case of WT1, mutations that are thought to be dominant negative cause abnormal urogenital development 11, suggesting that the normal gene might function in the development of this organ system. Other human TSGs might exist specifically to guard the body against neoplasia, rather than function in normal development. One way that they could do this is by inhibiting the action of onco-

Gene

Protein or predicted protein

Tumour

Refs

NF1

GTPase-activatingprotein

Neurofibromatosis

41

APC

Novel

Adenosis polyposis

42

RB-1

Cell.cycle-regulatednuclear transcriptional repressor

Retinoblastoma,osteosarcoma

43

p53.

Cell-cycle-regulated nuclear transcription factor

Lung,brain, breast tumours, chronic myelogenous leukaemia, others

44

WT1

Zinc-finger transcription factor Wilms' (kidney) tumour

TABLE 1B - CLONED CANDIDATE TSGS AND THEIR PRODUCTS IN MAN

Gene

Protein or predicted protein

PTP¢

Receptor-type protein tyrosine Renalcell carcinoma, lung carcinoma phosphatase

45

DCC

NCAM-like cell adhesion molecule

Colorectalcarcinoma

46

MCC nm23

Novel

Colorectal carcinoma

47

Nucleoside diphosphate kinase

Breast and colorectal carcinoma

48

K.revl

GTP-binding protein

None reported

49

Tumour

Refs

Breast cancer genes. In fact, TSGs have sometimes Prohibitin Novel been called anti-oncogenes, implying that they act by antagonizing the action of oncogenes. There is little expression in adult tissues might suggest a more evidence for such a mode of action 3o, although the general function at late stages. The abnormalities possibility has not been excluded. The converse, in mutant homozygotes do appear at the expected however, does occur, in that some oncogene proddevelopmental stage, but the details are quite unanucts abrogate cell division control by antagonizing ticipated. The mutants develop normally through the function of TSG products. The best examples

50,51

are provided by the transforming proteins of DNA the major stages of embryogenesis and organogenesis (up to day lO.S) before showing (after day tumour viruses such as SV40, adenovims and papillomavims, which bind to and inactivate pS3 and pRB10. Tumours in TABLE 2 - CLONED TSGS AND THEIR PRODUCTS IN DROSOPHILA many different tissues can be produced by tissue-specific expression Gene Protein or predicted Tumour Refs of the SV40 T antigen 31. protein New technologies of mouse genetics are now being used to assess Giant caclherin-likecell Imaginal disc hyperplasia 52 tat the functions of mammalian TSGs adhesion molecule in vivo: TSGs can be inactivated in Imaginal disc hyperplasia 53; A. Shearn, Novel cultured embryonic stem cells, and hyd these cells then used to derive mice

(-1(3)c43)

homozygous for TSG mutations. This method has recently been used in three laboratories to produce mice lacking function of the Rb.1 gene 32-34. Severe developmental abnormalities were expected, given the many indications of a fundamental role of pRB in cell proliferation control. Rb-1 is not expressed in the mouse until day 11, so it cannot function in regulating early cell cycles, but its almost ubiquitous

Igl

pers. commun.

Membrane.associated Ser/Thr kinase component

Imaginal disc neoplasia

19; D. Strand, pers. commun.

Septate-junction-associated guanylate kinase homologue

Imaginal disc neoplasia

54

Brain tumour

55; G. Hankins, pers. commun.

$6 ribosomal protein

Blood cell neoplasia

28

otu

Novel

Ovarian tumours

56

barn

Novel

Ovarian tumours

57

dig

Novel brat (--1(2)37C0 air8

TRENDS IN CELLBIOLOGYVOL. 3 FEBRUARY1993

33

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¸

11.5) defective haemopoiesis leading to an abnormally high fraction of nucleated erythrocytes in circulation, defective cell proliferation control leading to ectopic mitoses in areas of the nervous system normally occupied by migrating or differentiating cells, and massive cell death throughout the central nervous system. The embryos die by day 16. The phenotypes indicate that pRB may be required to prevent cell proliferation from occurring in terminally differentiating cells in both the nervous and haemopoietic systems, so that in its absence there is unscheduled division of cells at inappropriate stages in their development. The incompatibility between division and differentiation may lead to cell death, at least in the central nervous system. Another surprising feature of the results is that none of the heterozygotes has shown any signs of retinoblastoma. This might be due to the smaller number of target cells or shorter development time compared to h u m a n embryos; however, the mice do show a high incidence of pituitary and other brain tumours, suggesting that the difference between mice and humans reflects a real difference in the tissue specificity of pRB requirement rather than a quantitative difference in susceptibility. However, if so, this raises questions about the mode of action of SV40 T antigen in the retina of transgenic mice, which produces ocular tumours with similar characteristics to those of h u m a n retinoblastoma 3s. The results of knock-out experiments on the p53 gene are even more surprising than those with Rb-1. As with pRB, previous work on p53 had suggested a role in cell cycle regulation, leading to the expectation that homozygous mutants would show aberrant cell proliferation control during development. However, the homozygotes develop completely normally to birth, showing that the p53 protein has no essential function during prenatal development2L The homozygotes do show an extremely high frequency of tumours, including lymphomas and sarcomas, by six months of age. Thus the pS3 protein appears not to have a role in normal prenatal development, but instead functions mainly to prevent tumour development in later life. These experiments are the clearest demonstration so far of the tumour-suppressing function of normal pS3, and they show that loss, rather than oncogenic mutation, of p53 is sufficient to predispose m a n y tissues to neoplasia. An interesting hypothesis, consistent with most results, is that the pS3 protein is not required in normal, healthy cells, but is a component of a check point in the cell cycle that causes cell cycle arrest and/or suicide (apoptosis36) of cells that have sustained DNA damage37-39. Treatment of cells with UV light or radiomimetic drugs leads to accumulation of normal p53, which causes G1 arrest 40. In myeloid leukaemia cells, p53 goes further and induces apoptotic cell death 39. Since p53 acts as a transcription factor 9, the G1 arrest and/or apoptosis is presumably brought about by changes in the expression of other genes in response to the elevated pS3 level. Loss of pS3 function could 34

therefore contribute indirectly to oncogenesis by allowing the survival and reproduction of cells in which DNA damage has caused activation of oncogenes and/or loss of TSGs. This kind of relationship might be extremely important to understanding oncogenesis, in that it suggests functional links between some of the changes that occur during multistep tumourigenesis. The many genes that encode DNA repair enzymes and proteins involved in cell cycle check points may emerge in the future as a new category of TSG. A mouse knockout experiment has recently provided the first evidence of a TSG encoding a secreted protein. Mice homozygous for a deletion of the ainhibin gene develop normally but eventually suffer from gonadal stromal tumours sS. Thus ainhibin, a member of the transforming growth factar [~ family of transforming growth factors, is essential for normal regulation of gonadal stromal cell proliferation. The rare cases of gonadal stromal tunours in man can now be checked to determine whether the h u m a n a-inhibin gene also acts as a TSG. Prospects The next few years will almost certainly see the identification of several new human TSGs - geneticists are fast closing in on multiple endocrine neoplasia, astrocytoma and other cancers. Many new TSGs can be expected from work on Drosophila, and perhaps from mouse, Caenorhabditis and zebrafish. The human genome project and related work can be expected to contribute enormously to our understanding of the genetic changes underlying cancer. The collection and sequencing of expressed sequence tags from human cells have already led to the identification of a human homologue of a Drosophila tumour suppressor gene, /ind the accelerating pace of these projects makes it likely that more such examples will be found. The field is steadily progressing from the gene collection stage to analytical studies of the functions of TSG products in the cell, and we hope that this collection of articles wil~ stimulate further work and thought on the subject. References 1 KNUDSON,A. G. (1971) Proc. NatlAcad. Sci. USA 68, 820-823 2 COMINGS,D. E. (1973) Proc.Natl Acad. Sci. USA 70, 3324--3328 3 CAVENEE,W. K. et al. (1983) Nature 305, 779-784 4 FRIEND,S. H, et al. (1986) Nature 323, 643-646 $ LEE,W-H., _ROOKSTEIN,R., HANG, F., YOUNG,L-]., SHEW,]-Y. and LEE,E. Y-H. P. (1987) Science235, 1394-1399 6 MARSHALL,C. I. (1991) Ce1164, 313-326 7 LASKO,D., CAVENEE,W. and NORDENSKJOLO,M. (1991) Annu. Rev. Genet. 25, 281-314 8 HEYNINGEN,V. V. and HASTE,N. D. (1992) Trends Genet.8, 16-21 9 VOGELSTEIN,B. and KINZLER,K. W. (1992) Cell 70, 523-526 10 LANE,D. P. and BENCHIMOL,S. (1990) GenesDev. 4, 1-8 11 PELLETIER,I. et al. (1991) Cell 67, 437-447 12 HABER,D. A., TIMMERS,H. T., PELLETIER,I., SHARP,P. A. and HOUSMAN,D. E. (1992) Proc. NatlAcad. Sci. USA 89,

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In the next Issue of TCB: Potocytosis of small molecules and ions by caveolae Richard Anderson A class of membrane proteins with a C-terminal anchor Uirike Kutay, Enno Hartrnann and TornRapoport Transcription and translation factors in Xenopusoocyte RNA storage particles Sherrie Tafuriand Alan Wolffe

Bidirectional membrane traffic between the endoplasmic reticulum and the Golgi apparatus

Jennifer Lippincott.Schwartz Genetic approaches to studying peroxisome biogenesis Paul Lazarow

TRENDS IN CELL BIOLOGY VOL. 3 FEBRUARY1993

35