JANUARY /FEBRUARY 1989 VOLUME 2, NUMBER 1 @Copyright 1989 by the American Chemical Society
Invited Review Loss of Cancer Suppressors, a Driving Force in Carcinogenesis Noel P. Bouck* and Benjamin K. Benton Department of Microbiology-Immunology and Cancer Center, Northwestern University Medical and Dental Schools, Chicago, Illinois 60611 Received December 9, 1988 Cancer has been a toxicology problem since the identification of the first carcinogens in the 1700s. Today one out of three individuals faces a diagnosis of cancer, and the majority of these lesions are thought to be environmentally induced (1). A vast number of structurally diverse compounds are able to induce and promote the development of tumors. Understanding the roles and relative importance of this array of carcinogens and promoters not only requires an understanding of their chemistry but also demands both a knowledge of exactly what types of damage actually contribute to the conversion of a cell from normal to tumorigenic and an estimate of the relative frequency with which each type of damage occurs in the development of a single tumor. Some of the most useful information on these points is currently coming from studies in cancer genetics. Cancer in humans and other organisms is fundamentally a genetic disease resulting from stable, heritable changes in the cellular genome. It is a genetic disease in the classic sense in that almost all the different types of tumors can occur not only sporadically but also in a hereditary form whose passage from generation to generation can be followed in classic family studies (2-4). It is a genetic disease a t the cellular level as tumors themselves are clonal outgrowths of calls that differ genetically from adjacent normal tissue (5). In addition, a number of diseases whose primary defect results in increased sensitivity to DNA damage have cancer as their major outcome (4),and most carcinogens and promoters are capable of damaging DNA or changing the chromosome complements of cells (6-11). The classification of cancer as a genetic disease does not mean that tumor development is immune to epigenetic influences that alter gene expression without altering the *Address correspondence to this author a t the Department of Microbiology-Immunology, 303 East Chicago Ave., Chicago, IL 60611.
nucleotide sequence of the gene. Regulatory toggle switches sensitive to extracellular controls operate in higher organisms (12) and are thought to orchestrate the normal process of development (13) Genes that can contribute to tumorigenicity are active during development (14). The sensitivity of such cancer genes to epigenetic changes can be demonstrated experimentally by the ability of a variety of agents, including tumor promoters, to induce whole populations of normal cells to temporarily display the phenotype of transformed cells or vice versa (15-19). Phenotypic shifts like this taking place in vivo can provide opportunities for setting up stable regulatory changes, for selection, and for further genetic changes, any or all of which might be crucial to the development of a tumor. Genes involved in neoplasia are also sensitive to changes in expression that are due to modifications of DNA rather than to changes in primary sequence. The potent demethylating agent 5-aza-2-deoxycytidine can induce tumors (20). It transforms cultured BHK hamster cells in the absence of detectable mutations by reversibly altering a gene(s) in the same functional complementation group as a suppressor gene that is mutated when transformation is induced by mutagenic carcinogens like nitrosomethylurea (NMU) and 4-nitroquinoline N-oxide (4-NQO) (21). Fascinating arguments can be made that methylation changes play a role in carcinogenesis (22), but as there is little hard data giving an indication of how frequent or how effective these or other epigenetic influences are, this discussion will deal primarily with tumor-specific changes in gene structure. Genes that have been identified so far that are found to be altered when tumor cells are compared to normal cells fall into two general classes: oncogenes and cancer suppressor genes. Oncogenes are derived from normal cellular genes usually involved in mitogenic stimulation ( 1 4 ) . In tumor cells these cellular genes are activated by mutations, amplifications, or rearrangements of DNA that 0 1989 American Chemical Society
2 Chem. Res. Toxicol., Vol. 2, No. I , 1989
Bouck and Benton
OPERATIONAL DETECTION OF CANCER SUPPRESSOR GENES
C.
5.
By Cell Fusion.
By Transfection.
- \
/
Transformed Tumorigenic Segregant
Normal Somatic Cells
4.
\i-i
Tumorcell
-
By Family Studies Linking Dominantly inherited Cancen to Specilic Chromosomes. lst Generation
A Figure 1. Operational detection of cancer suppressor genes. In part 4, = male; 0 = female; and and 0 = individuals with tumors, all of whom have inherited the same specific chromosome, presumably one carrying a defective suppressor gene.
result in an increase in the amount or the activity of their protein product (14, 23, 24). Weinberg has effectively classified oncogenes by the location of their protein products and by their ability to perform complementary functions into nuclear oncogenes and cytoplasmic oncogenes (25). While some oncogenes can function in both categories when overexpressed or when present in cells isolated from their neighbors, most fit in well and it is generally found that primary rodent cells can be transformed to tumorigenicity by transfection with a combination of one activated oncogene from each category. Cooperating oncogenes also occur together in some transforming viruses and a number of human tumors (14, 23-25). Cancer suppressor genes, also called tumor suppressor genes, antioncogenes, or emerogenes, are genes that suppress or block the expression of malignancy or of transformation (parameters of malignancy measured in cultured cells; 26). Unlike oncogenes whose activity increases in tumor cells, suppressor genes are most active in normal cells; their function is lost in tumor cells. Their existence can be experimentally demonstrated in several ways, illustrated in Figure l. Cell fusion studies pioneered by Harris and Klein have shown that, with few exceptions, when a normal cell is fused to a tumor cell the hybrid cell containing all of the chromosomes from each parent has a normal, nonmalignant phenotype (27-30). As such hybrids grow and lose chromosomes from the normal parent, malignancy is reexpressed (Figure 1, part 1). The imposition of the normal phenotype in hybrids is due to trans suppression of the transformed, malignant phenotype for (i) the type of transformation that reemerges in hybrid segregants (for example, temperature sensitivity for anchorage independence) matches that present in the original transformed parent (31, 32) and (ii) the malignancy of a human tumor line can be sequentially blocked, restored,
and blocked again by the addition, loss, and readdition of a single human chromosome (33). Perceptive interpretation of epidemiological studies of sporadic and inherited childhood tumors by Alfred Knudson led to a second independent line of evidence supporting the existence of suppressor genes. He concluded in 1971 (34) that children at high risk for such tumors inherited one defective allele from their affected parent and that tumors arose when the functional, wildtype allele at this same locus, inherited from the unaffected parent, was also inactivated. Rare spontaneous tumors on the other hand required inactivation of both wild-type alleles of this gene in a single clone of somatic cells. The suppressor gene whose loss can be responsible for one of these tumors, retinoblastoma, has now been cloned (35-37) and Knudson’s hypothesis validated by the demonstration that a defective allele, RB-1, is indeed inherited with disease susceptibility (37,38). In both spontaneous and inherited retinoblastoma tumors the wild-type RB-1 alleles are often lost, either physically or functionally, leaving a hemizygous (top tumor cell, Figure 1,part 2) or homozygous (middle tumor cell, Figure 1, part 2) defective gene at the DNA level that results in the absence of the protein product of the suppressor gene in the tumor tissue (35-37, 39). Replacement of the lost RB-1 gene in tumor cell lines via a retroviral vector slows growth and delays tumorigenicity (39a). Transfection of normal DNA into transformed cells is a third way to identify suppressor genes (Figure 1, part 3). Dominant tumor suppressor genes have recently been isolated both by Noda (40a,b) and by Schaefer (41) who transfected normal human cDNA, or DNA, into rodent cells transformed by multiple copies of an active ras oncogene, selected for those that reverted to normal upon uptake of human DNA, and rescued the active human sequences. Fourth, genes that dominantly predispose to a particular tumor type or subset of types (see, for example, reference 42) that have been mapped to specific chromosomes by their segregation in family pedigrees may also be suppressor genes (Figure 1, part 4). Chromosomes inherited along with tumor susceptibility are thought to carry an inactivated suppressor gene that predisposes the carrier to cancer by virtue of the fact that only one allele, present on the homologous chromosome inherited from the unaffected parent, must be inactivated for loss of suppression gene function. Such genes may be quite common in some tumor types. A recent kindred analysis suggests that over half of common colorectal cancers are associated with a dominantly inherited susceptibility (43)that may result from the inheritance of a defective suppressor gene. Since the intellectual framework for the concept of suppressor genes was laid by Ohno (44)and Comings (45) in the 1970s and the field defined in its present form by reviews in 1985-6 (26, 46-48), summaries from a variety of points of view have been published (4,30,49-55). Data contained in these reviews indicate that multiple suppressor genes exist in the genomes of humans and rodents as they do in Drosophila (56)and in the fish Xiphophorus (57). Fifteen putative suppressor genes scattered over twelve different chromosomes have been identified so far in the human genome by detection of tumor-specific hemior homozygosities similar to those that accompany the loss of the RB-1 suppressor gene during the development of retinoblastomas. Verification that an actual suppressor function is associated with these chromosomes has been obtained for a total of six different chromosomes and seven genes: four genes by cell fusion or chromosome transfer [on chromosomes 1 (58-60), 11,which appears to carry two suppressor genes (33,58,61,62),and 13 (58)]and six genes
Invited Review by their linkage to an inherited predisposition for the same tumor type [on chromosomes 1 ( 4 ) ,3 (63-65),5 (66,67), 11 ( 4 , 42), 13 (4, 38),and 22 (68)l. The consistent recessiveness of the malignant phenotype in cell fusion indicates that most if not all tumors lose both alleles of a t least one suppressor gene during their development. Even cells transformed in vitro by myc plus ras or expressing both oncogenes in vivo need additional changes to become fully tumorigenic (25, 71, 70),changes that in hamster cells are satisfied by loss of what is presumably a suppressor on hamster chromosome 15 (71). Loss of both alleles of a wild-type suppressor gene is required for the development of retinoblastoma. This is likely true for many other suppressor genes, although a failure to detect the expected allelic deletions on chromosome 5 in colonic adenomas of patients with familial polyposis coli, a predisposing condition inherited on chromosome 5, suggests this may not always be the case (72-75). Loss of the activity of both alleles of a single suppressor gene can promote development of tumors in a wide but not unlimited range of different tissue types (76, 77). A number of functions have been envisioned as suitable for suppressor genes on the basis of the fact that their presence could block and/or their loss could contribute to a neoplastic phenotype (26). There is some evidence available to support the possibilities that different suppressor genes may act to do the following: (1) Block Oncogene Action. In HeLa cells carrying human papilloma virus 18, a suppressor on chromosome 11 seems to mediate the sensitivity of viral oncogene transcription to methylation control (78)as well as to block tumorigenicity (33). Fibroblasts cultured from individuals with a deletion of the region of chromosome 11 associated with suppression of Wilms’ tumor are sensitive to partial transformation by oncogenic proteins from the human papova virus BK (79) and from HPV-16 (80) whereas normal human fibroblasts lacking this deletion are not. Sensitivity correlates with high levels of HPV-16 RNA expression. In mouse cells a dominant function has been identified that is able to block transformation by ras and a subset of other oncogenes by actively interfering with either a necessary modification of the oncogene product or with a proximal or distal ras target (81, 82; see also below), and DNAs and cDNAs able to block ras transformation have been derived from normal human cells (40, 41). It seems doubtful that the normal, nonactivated allele of oncogenes like H-ras routinely serves as a suppressor of their activated allele as high expression of the normal allele does not limit the efficiency of transformation in vitro by the activated allele (83,83a),and in several tumors where a mutated ras allele occurs with a very high frequency and hence probably plays a causative role, it coexists with the unaltered wild-type allele (84-87). ( 2 ) Allow normal growth control, for example, by mediating the receipt and processing of negative growthregulating signals. There are a number of inhibitory growth factors (see reference 14) and several retinoblastoma lines have been shown to be deficient in receptors for one of them, TGF-0 (88). Other growth-controlling signals are able to block expression of transformation in vitro or tumorigenicity in vivo (89-94). Some of these seem to be passed directly from cell to cell, suggesting genes required for the synthesis of such compounds or for the integrity of the junctions through which they pass would be able to suppress malignancy. One cell variant deficient in such junctions is unusually sensitive to in vitro transformation (91). Maintenance of the proper
Chem. Res. Toxicol., Vol. 2, No. 1, 1989 3 intracellular pH may also be required to maintain normal growth control (95). (3) Mediate Normal Differentiation. Leukemias lacking the ability to respond to normal differentiation signals have been extensively characterized by Sachs and others (96-98), and tumor suppressor genes in flies and fish have clear defects in differentiation (56,57,99).The failure of some suppressed hybrids to form tumors has been attributed to their restored ability to differentiate in vivo (93, 100). (4) Enable Immune Rejection. Transplantation antigens have been suggested by Sager to be suppressor genes (26),and their functional loss can be shown to enable cells that would otherwise be rejected to grow into tumors in an immune-competent animal (101-103). Whether such loss is helpful or harmful to tumor growth may vary with the experimental setting (104). ( 5 ) Promote Senescence. Senescence, the limited capacity for cell division exhibited by mammalian cells, is a dominant cellular trait mediated in some cells by sensitivity to differentiation factors (98). It can be shown to block tumorigenicity (105) as cells stop dividing. (6) Prevent Angiogenesis. All solid tumors require neovascularization to grow progressively (106),and in BHK hamster cells a suppressor gene controls the extracellular activity of an inhibitor able to block angiogenesis (107). With the broad outlines of the concept of suppressor genes firmly established, it is now possible to look beyond verification of their existence and to begin to ask how much influence these genes have on the overall process of carcinogenesis. Evidence rapidly accumulating on two different fronts suggests that the influence of suppressor genes will be startlingly wider than originally suspected. This evidence and some of its implications are summarized below.
Loss or Inactlvatlon of Multiple Suppressor Genes Contributes to the Development of a Single Tumor Recent studies suggest that individual tumors from various tissues lose suppressor gene alleles from several different specific chromosomal locations during their progression to malignancy. Although such secondary losses may not be frequent in tumors of childhood and adolescence ( 2 ) ,they are being seen with increasing frequency in a number of common adult tumors. In the case of the retinoblastoma suppressor it is possible to define the actual locus that is lost, but most other suggestions of suppressor gene loss in human material are based on detecting alleles lost in tumor tissue but present in normal cells of the same individual using anonymous DNA probes defined only by their general location on a specific chromosome. Except for chromosomes 1,3, 5,11, 13, and 22 (see above), arguments that tumor-specific homozygosities detected in this way really represent loss of tumor suppressor genes linked to these probes rest on observations that these tumorspecific changes occur with high frequency and considerable tumor-type specificity and consistently represent a loss of genetic material. Vogelstein has cataloged losses from multiple chromosomes occurring a t high frequency in individual colon tumors (72). Allelic deletions present in the DNA of colon tumor tissue but absent in DNA prepared from adjacent normal mucosa were detected on chromosome 17p (p indicates the petite, short arm of the chromosome, q the long arm) in 75% of sporadic carcinomas and 83% of these also had deletions on 18q. About 50% of these had an activated ras and 36% had lost material on chromosome 5q
4 Chem. Res. Toxicol., Vol. 2, No. 1, 1989 where the autosomal locus for familial adenomatous polyposis resides. Multiple loss in colon tumors was also seen by Monpezat et al. (73). These data on colon tumors are particularly compelling because almost 200 individual tumors were analyzed and because it is one of the few studies to minimize the contribution to tumor DNA preparations of stromal cells, which can obscure tumor-specific losses. It raises the possibility that modest frequencies of allele loss seen in some other studies and other tumor types might in reality be much higher. In addition, correlating the various changes in individual tumors with histopathological staging suggested that sequential ras activation/5q loss, 18q loss, and 17p loss often occurred as colon tumors progressed clinically from benign class I adenomas to malignant carcinomas (72). In a smaller study of uncultured tumor tissue from small cell carcinoma of the lung, Yokota (108) found typical 3p deletions in tumors from 7 / 7 patients and in addition saw deletions of 13q in 10/11 individuals and 17p in 5/5. These changes also appeared sequential as 3p loss was frequent in adenocarcinomas and both 3p and 13q loss occurred prior to the amplification of the N-myc oncogene that is characteristic of the majority of these tumors (109). The loss of chromosome 13q may reflect loss of the retinoblastoma suppressor as in one study 80% of cell lines derived from small cell lung cancers expressed little or no mRNA specific for the Rb gene and apparently homozygous structural rearrangements of this specific locus could be detected in DNA from 1/8 primary tumors examined (110).
Provocative if not yet thoroughly convincing data suggest that multiple suppressor genes may be lost from many other tumors. Predisposition to multiple endocrine neoplasia type 2, presumably due to inheritance of a suppressor gene, is linked to chromosome 10 (111,112),yet in 7/14 tumor samples genes are deleted from the short arm of chromosome 1 (113). Twenty (114)to twenty-seven (115)percent of primary breast tumors have tumor-specific homozygosities on chromosome 11,and 4/6 lines and 3/41 tumors have homozygous rearrangements detectable by a retinoblastoma probe from chromosome 13 (116). Only one of these tumors seems to derive from the subset of ductal breast tumors associated with a loss of DNA from chromosome 13 rather than from chromosome 11 (117). Heterozygous deletions have been seen in an additional three tumors, suggesting loss of a gene near to but not identical with the Rb gene (77). Karyotypes of eight primary anal canal carcinomas obtained prior to treatment showed seven had lost chromosomal material from chromosome 11,and five from 3p (118). Loss of chromosome 10 in 28/29 glioblastomas was accompanied by nonrandom loss of sequences from one of three other chromosomes (119). In one family where Wilms’ tumor susceptibility was not linked to chromosome 11,a tumor-specific loss of alleles on chromosome l l p was seen (120). Seventy-seven percent of osteosarcomas, a tumor associated by a number of criteria with loss of the retinoblastoma gene, also show loss of alleles on chromosome 17 (121),and 12/19 meningiomas with losses on chromosome 22 showed additional loss of genetic material on one to three other chromosomes (122). So far all of the osteosarcomas and meningiomas with secondary losses fall into the subset of such tumors that have already lost some other (presumed) suppressor allele. This raises the possibility that secondary losses in these cases could represent random segregation from a tetraploid stem line, but at least in the case of osteosarcoma, the high frequency with which stem lines with a loss of chromosome 17 are maintained
Bouck and Benton
in tumors suggests its loss confers a genuine growth advantage. Interpreting those multiple tumor-specific allele losses that are seen with high frequency in human tumors as indicative of multiple suppressor gene loss seems reasonable in light of data on the in vitro transformation of cultured cells, which clearly lose multiple suppressors as they progress to malignancy. Cultured cells transform in vitro (123),as do developing tumors in vivo (5),by stepwise changes. Acquisition of immortality is frequently a first step. Immortality itself is a recessive trait (124, 125) achieved by the loss of alleles with a dominant phenotype that assure senescence and that can be classified as cancer suppressor genes (26,105,126). Immortal lines that have lost these genes are not yet tumorigenic but must undergo further changes, which in the case of Syrian hamster BHK (31) and SHE cells (127),as well as various mouse lines (27), involve loss of another suppressor gene detectable by cell fusion. Even abl-transformed lymphoid cells require additional changes, possibly suppressor loss, to become tumorigenic (128). Tumorigenic Chinese hamster cells, CHEF-16, have acquired three independent recessive traits, immortality, anchorage independence, and the ability to grow in low serum, suggesting loss of three suppressor genes (129). Even the immortal mouse line NIH/3T3, that is so exquisitely sensitive to spontaneous transformation that it is thought to have progressed to a stage very close to tumorigenicity, loses a suppressor function when transformed by ras oncogenes. Transformed clones can be reverted to a normal phenotype when they are fused to parental NIH/3T3 (see Table I), although mutated ras genes are retained in the transformants and hybrids (Cohen, Tolsma, and Bouck, unpublished data). A DMBA-transformed mouse line and NIH/3T3 transformed by polyoma virus middle t, both known to behave dominantly in cell fusions (130),serve as controls, showing the ability of this protocol to detect lack of suppression. Additional evidence that ras transformants of NIH/3T3 cells have lost suppressor activity comes from (i) Harris’ demonstration that NIH/3T3 but not ras-transformed NIH/3T3 can suppress the high malignancy of mouse melanoma PG19 (100);(ii) his finding that the tumorigenicity of NIH/3T3 ras transformants evolves in vivo to become independent of the ras gene product (131);and (iii) the ability of Noda to clone from normal cells genes that dominantly block the transformation of Ki-ras-transformed NIH/3T3 (40). It can also explain the observations that differentiated functions of thyroid cells transformed by a temperature-sensitive Ki-ras remain blocked at transcription when cells are shifted to the temperature that inactivates ras and that such a block can be alleviated by fusion of the transformed cell to a nontransformed thyroid cell (132, 133). One can speculate that the other immortal cells that are not converted to malignancy solely by an active ras oncogene, for example, the rat line REF 32 (134) and hamster CHEF-18 (135), may also require suppressor gene inactivation. The observation that SV-40 large T antigen, a protein that complexes with the retinoblastoma suppressor gene protein, will convert ras-transformed REF 32 cells to tumorigenicity supports this interpretation (136). Ras-transformed CHEF-18 cells, on the other hand, seem to become tumorigenic upon duplication of DNA of chromosome 3q, not suggestive of suppressor loss (135).
Oncogenes Can Interact with Suppressor Genes and Mimic Phenotypes of Suppressor Loss The oncogenic proteins of several DNA viruses have
Chem. Res. Toxicol., Vol. 2, No. 1, 1989 5
Invited Review
Table 1. Suppression of Oncogene Transformed NIH/3T3" fusions
NIH/3T3 X NIH/3T3 X A31 DMBA x polyoma m T transformed NIH NIH/3T3 transfected with pEJ clone S2 X NIH/3T3 clone S l b X NIH/3T3 x polyoma mT transformed NIH clone B X NIH/3T3 NIH/3T3 transfected with HT1080 DNA X NIH/3T3 X polyoma m T transformed NIH NIH/3T3 transfected with T24 DNA clone 12 X NIH/3T3 X polyoma m T transformed NIH clone 2 1 X NIH/3T3 X polyoma m T transformed NIH
agar plating eff. of parents, %
