Molecular Genetic Basis of Cancer Development

LASZL6 KOPPER," ISTVAN PETAK, AND

First Institute of Pathology and Experimental Cancer Research Semmelweis University of Medicine

Budapest, H-1085, Hungary

ANNA S E B E S ~ N

Life is based on the continuous flow of information among cells and inside a cell (inter- and intracellular communications). To maintain the structural andlor functional integrity of a multicellular organism, the cells should actively respond to physiological or pathological information by receiving and generating signals (e.g., the phosphorylation and dephosphorylation of hundreds of proteins by thousands of kinases and phosphatases) that can ultimately lead to a decision, that is, activation of a genetic program.

Although the signals are using seemingly independent pathways, there are many connections between them, forming a complex network, serving to fine-tune the signals (specificity, amplification, regulation), and, at the same time, making our effort to understand this complexity rather unrealistic. Nevertheless, these decision- making processes and genetic programs should be understood in order to identify the key genetic defects in human diseases, including cancer, and to design proper gene-based diagnosis and therapy.

The basic decisions at the cellular level are either do nothing (i.e., signals inhibit each other without activating genes) or (b) do something (i.e., activating programs for proliferation, cell death, or to exercise specific, differentiated functions).

If everything runs smoothly, the complexity mentioned above remains unnoticed. Problems can appear if the afferent or efferent components of the decision-making process, including the genes, acquire one (or more) defect, which could be caused either by exogenous (environmental), or endogenous factors (reflecting the extreme difficulty in completing these programs without mistakes as well as inherited de- fects). In reality the number of DNA defects per cell per day are about 80,000 (due to oxidation, depurination, depyrimidination, cytosine deamination, single-strand breaks, 06-methylguanine production). Most of these defects are without conse- quences (the defects are repaired, neutral, or recessive), but if not, their effect could be positive (contributing to the evolution of the species) or negative (contributing to the development of a disease in an individual).

Cancer is a failure of the regulated cell functions, mainly due to defects in the decision-making programs, resulting in a continuous accumulation of cells without obvious purpose, but with increasing autonomy by a selection of the most vital (or resistant) clone(s), and with the capacity to invade the host tissues. The continuous accumulation of cancerous cells clearly indicates that the balance between arising and dying cells has been destroyed by means of a multistep disorganization of the regulation of cell proliferation and/or cell death. The stepwise progression is caused

a Address for correspondence: Ldszl6 Kopper, M.D., Ph.D., D.Ac.S., m6i ht 26, Budapest, H-1085, Hungary; tellfax (36)(1) 117 0891; e-mail: kopper@korbl.sote.hu

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by the appearance of more and more defects in the genome with time (one defect, by creating genetic instability, can help the development of the next) and can be further supported by a decline in repair capacity. (Frequently, it is also the result of a gene defect.)

In cancer all kinds of genetic defectsterrors have been observed for example, DNA mutations (point mutations such as transition, transversion, and frame-shift, leading to missense, nonsense, or neutral mutations; deletion, insertion; duplication; inversion) and chromosomal mutations (accompanied by rearrangement due to deletion or translocation or as numerical aberrations). Obviously, many other factors can influence gene activity, for example, state of methylation.

These genetic defects have significant importance in tumor formation and pro- gression especially when the targets are those genes that have the key role in the regulation of cell proliferation and cell death as well as in the repair. These well- known genes and their products are (didactic classification with much overlap): (a) activated (defected) proto-oncogenes, that is, oncogenes; (b) suppressor genes; (c) growth factorslcytokines and their receptors; (d) signal-transducers, for example, receptors, protein kinases, transcription factors; and (e) repair enzymes. It seems that the accumulation of gene defects is generally more important than their exact order. It will lead to the selection of those clones which in that particular microenvi- ronment and essentially in that particular host have the greatest growth advantge. Further selection pressure(s) could be introduced by the therapy.

If one lists the frequent malignancies in adults and children the difference is obvious. In adults the most frequent tumor types are either epithelial (bronchial, colonic, gastric, oral) or hormone related (breast, prostate), whereas in childhood leukemias/lymphomas, brain tumors, quite unique types such as neuroblastoma, Wilms’ tumor, retinoblastoma, or rhabdomyosarcoma, bone tumors are the most frequent. The lists suggest that the difference in tumor types is probably due to different etiology: the genetic defects in adults are caused mainly (not exclusively) by exogenous factors, whereas the inherited genetic errors play a more important (again not exclusive) role in pediatric tumors. Similarly, in adults chronic physical or chemical irritations, inflammations, hormonal influences are seen, whereas in children inherited diseases and malformations are the most significant risk factors.

ERRORS IN REGULATORY GENES

Oncogenes are the activated forms of proto-oncogenes, which are a mixture of genes (and gene products) and represent widely different components of signal transduction. They could be growth factors (e.g., sk); growth factor receptors (e.g., EGFR); membrane-bound tyrosine kinases (e.g., src, abl), G-proteins (e.g., ras); cytoplasmic serinelthreonine kinases (e.g., raj mos), or transcription factors (e.g., myc, myb, fos, jun). Most proto-oncogenes support cell proliferation; therefore, mutations, amplifications, and translocations are common (deletion of a proto- oncogene is not “useful” for cancer). The genetic error is usually dominant and leads to the production of a continuous signal for proliferation without the need for the usual regulatory factors (e.g., truncated EGFR can miss EGF, mutated ras remains activated, fused abllbcr escapes from gene regulation, or translocated myc produces normal products but in an inappropriate time frame and quantity). Transcription factors, especially myc, can also act as cell death inducers (see below). Activated forms of known oncogenes were shown in about 15-20% of human malignancies, with a great variety among tumor types and oncogenes.

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Suppressor genes represent the negative arm in the regulation of cell prolifera- tion. Therefore, those defects are especially important that result in the loss of the activity (deletion, mutation, translocation; here, amplification provides no advantage for cancer). The defect is usually recessive, except the dominant p53 defect; that is, both alleles should be damaged. The loss of the activity of the second, still- working allele causes loss of heterozygosity (LOH). p53 is a very common suppressor gene, although it has been discovered to be an oncogene, because of the capacity of certain viral proteins to block suppressive gene products by complex formation. The main duty of p53 is to guard the genome integrity and stop the proliferation in case of DNA mutations, allowing time for the repair, usually via activating p21WAF’ and therefore inhibiting cyclin-dependent kinases. But if the defect is unrepairable, p53 should trigger apoptosis. If p53 is not working properly (the gene is mutated, deleted, or the normal p53 protein is inhibited by other proteins without regulatory reasons, e.g., by mutated mdm2 or viral proteins), the most severe consequence is the appearance of the gene defects in the daughter cells. (Mdm2 expression can also be induced by p53, suggesting the possibility of self-regulation.) The defect in p53 activity is one of the most common genetic errors (together with Rb) in human malignancies.I2 The retinoblastoma (Rb) gene is a prototype of the suppressor genes and normally is the main supervisor of the R (restriction) point in G1. pRb inhibits the traverse of cells through the cell cycle by complexing with the E2F transcription factor. Phosphorylation of pRb (e.g., by cyclinDkdk4) releases E2F, and the genes of the proliferation program are activated. The inactivation or loss of pRb can do the same, leaving the cell with uncontrolled proliferation.) (The regulation of p53 and Rb is much more complicated than described, with many exceptions such as p53- or pRb-independent pathways in different cell types.) There are many other suppressors with more or less defined functions: inhibitors of cyclin- dependent kinases (CKI); such as p21 (WAFZ), p16 (MTSZ; inhibitor of cdk4/6), p15 (MS72), p27 (kipl; TGFP responsive), p57 (kip2); neurofibromatosis genes (NFZ, G-protein modulator; and NE?), Wilm’s tumor gene (WTZ), APC, MCC, DCC, (mainly in colorectal cc), BRCA 1 and 2 (mainly in breast and ovarian cc), VHL, etc.

Transcription factors (TF, there are more than a hundred) form usually homo- or heterodimers (e.g., rnyc should dimerize with rnax to be activated; interestingly, rnax has no oncogenic form) and can bind to a specific segment of DNA and modulate gene expression. Certain TFs have a general function; they are needed for the expression of many genes, for example, TATA-factor, AP-1, and CREB proteins. Others are more cell type-specific: for example, steroid receptors, WT1, hepatocyte nuclear factors, MyoD, and myogenin. TFs frequently appear as part of translocations, especially in leukemias and lymphomas5 (TABLE 1). The new-either non-fused or fused-gene product has usually TF activity, too.

It has been suggested, more than 30 years ago, that cell loss rate has a profound effect on tumor growth. Obviously, cells could be killed “passively” by hypoxia or by other, partly therapeutic, cytotoxic effects, slowing down the increase in tumor volume (tumor-doubling time). However, the emphasis here is on active cell death, which is not an effective contributor to cell loss in tumors. On the contrary, the defect in the active cell death machinery (apoptosis) can lead to longer cell survival or even to the continuous accumulation of cells. The first clear example was given by showing the role of overexpression of bcl-2 in follicular non-Hodgkin’s lymphomas, due to translocation: t[14;18].6 Since then, apoptosis research has become very popular, proving that almost anything can switch on the apoptotic cascade, and if one trigger failed in a certain cell type, another can do the

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TABLE 1. Transcription Factors in Translocations of Hemopoietic Malignancies (Exatndes)

Translocation Involved Genes Diseases” tI8q2414q321 myc; Ig heavy chain

t[2p12;14q32] Ig K light chain, myc t[8q24;22qll] myc; Ig A light chain BL. B-ALL (L3)

t[9p21-22;14ql1] pl6INK4lMTSl and B-ALL

t[lq23;19p13] PBXl ;E12/47(E2A) pre-B-ALL t[17q22;19p13] HLF,E12/47 preB-ALL t[lp32;14ql1] tal-1 ;TCRdS T-ALL t[7q35;19p13] TCR&lyl-l T-ALL

TCRalS

t[10q24;14qlll HOXl1;TCRS T-ALL

t[15q22;17q12-21] PML;RARa (M3) t[6p23;9q34] DEK,CAN AML t[8q22;2 lq221 AMLllETO AML

t[llq23;19p13.3] MLLlENL (mainly ALL) or MLL/ELL (mainly AML)

a Abbreviations: BL, Burkitt’s lymphoma; ALL, acute lymphocytic leukemia; B, B-cell origin; T, T-cell origin; APL. acute promyelocytic leukemia; AML, acute myelogenous leukemia.

There is no question that apoptosis is an important, evolutionarily conserved process and that it has many distinct morphological and biochemical features. Strictly speaking, apoptosis is not equivalent to programmed cell death, which does not evoke apoptosis, for example, during physiological spermatogenesis; apoptosis is also not always triggered by nuclear activities but may be started by cytoplasmic constituents, for example, fas, UV, ceramide. It would be impossible in this space to count all apoptosis-inducing agents, so only a few of them will be mentioned.

The Bcl-2 family has several members, including inhibiting (bcl-2, bcl-xL, mcl-1, and bcl-w) and some viral components (adenovirus ElB19K, EBV BHRFl), as well as stimulating (bax, nbk/bikl, bad, bak, bcl-xs) apoptosis. Members of the bcl-2 family can form dimers, which usually require their BH1 and BH2 domains. In most eukaryotic cells, there is substantial redundancy in the expression of the bcl-2 family members, and the final decision depends on the regulation of the actual balance between anti- and pro-apoptotic molecules. However, some cells prefer to use one particular member as the survival factor (e.g., peripheral B cells: mcl-1; Reed-Stemberg cells: bcl-xL). In many studies the progression and prognosis of cancer patients has shown correlation with increased expression of bcl-2 or de- creased expression of bax. The function of bcl-2 is still unclear, but its cellular localization (mitochondria1 membrane) suggests it has a role in the regulation of oxygen tension. As previously mentioned, wild-type p53 can activate the apoptotic program (triggered by DNA mutations, caused by agents such as irradiation and cytotoxic drugs), but other stimuli do not require the presence of functional p53 to induce apoptosis, or even disregard whether p53 is wild-type or mutant. Myc, a nuclear transcriptional activator and powerful stimulator of cell proliferation, in certain circumstances can induce apoptosis, especially when the cells are confused by opposite signals, and when proliferative signal arrives but the survival factor is actually missing. Ligands like FasL and TNF can induce apoptosis by binding to the receptors FaslAPOI (CD95) and TNFRI, respectively. The signal is transmitted through the “death domain” of the cytoplasmic part of the receptors. The next step is, probably, the activation of the cysteine proteases, especially the ICE family

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(interleukin-lp-converting enzymes), whose potential targets besides ILlp are PARP (poli[ADP]ribose polymerase), lamins, UlsnRNP, and the proteases them- selves. P A W is also a guard of genomic integrity and at the same time is a potent regulator of the CaZ+, M$+-dependent endonucleases. If the proteases inactivate PARP, it is unable to work for the integrity of the cell, and apoptosis can start. ICE overproduction enhances, whereas inhibition prevents apoptosis.'

ERRORS IN OTHER GENES

The errors (mismatches) in the newly synthetized DNA should be repaired by the mismatch repair system (MMR). The insufficient repair capacity can be recog- nized by studying microsatellites (tandem-repeated short sequences, which are very polymorphous in a population but identical in one person). Errors in microsatellites are described as microsatellite instability (MIN), which reflects the decreased poten- tial of the whole genom to handle DNA errors (mutator phenotype). A defect in the MMR system is understood today to be related to the errors of four genes: hMSH2, hMLH1, hPMS1, and hPMS2. In HNPCC (hereditary non-polyposis colon cancer) families, the mutation of these genes appears in germ cells. Although MIN is a characteristic lesion in HNPCC, it occurs in many other tumor types. The lack of MMR activity can contribute to the resistance against akylating agents.

It has been recognized that in certain genes the expression of the alleles is related to their maternal or paternal origin (genetic imprinting), which is probably regulated by the methylation state of the allele.10 The loss of imprinting (LOI) could play a role in tumorigenesis. For example, in certain pediatric tumors-Wilms' tumor, rhabdomyosarcoma, hepatoblastoma-deletion appears in maternal chro- mosome 11; in AML in the paternal chromosome 7; in osteosarcoma in maternal chromosome 13; and in CML the translocation occurs between paternal chromo- some 9 and maternal chromosome 22.

SOME RECENT RESULTS ON PEDIATRIC TUMORS

Pediatric tumors can carry the prototype of certain, sometimes specific, genetic errors. Insulin-like growth factor 2 (IGF2) has recently been demonstrated to be maternally imprinted in both mice and humans. LO1 of IGF2 in rhabdomyosarcoma (RMS) has been reported, where IGF2 acts as an autocrine growth factor. (In Ewing sarcoma, however, LO1 of IGF2 was not associated with increased expression of IGF2 mRNA, suggesting that LO1 may not be involved in the regulation of IGF2 expression.) Embryonal and alveolar RMS showed c-met expression, in some cases amplification. In another study alveolar RMS cells had t[2;13][PAX3;FKHR], resulting in the tumor-specific expression of a transcriptional factor. PAX3 can stimulate c-met oncogene expression; the c-met oncogene is a receptor for the ligand HGF (hepatocyte growth factorkcatter factor). HGF controls cell motility and invasion.

The Wilms' tumor 1 gene (WT1) encodes a transcription factor of the zinc finger family. WT1 represses transcription of several growth factors and growth fac- tor receptors as well as transcription of bcl-2 and c-myc." WT1-induced apoptosis in an osteosarcoma cell line was accompanied by decreased synthesis of EGFR, but not of other postulated WT1-targeted genes. This effect, which was differen-

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tially mediated by the alternative splicing variants of WT1, was independent of

In 29% of medulloblastomas LOH of 17p (loss in the telomeric region) was observed without the involvement of the p53 gene and indicated a worse prognosis.'2 In the pathogenesis of ependymomas a-probably suppressor-gene on chromo- some 22 could be involved (it is not EWS or Nn). However, NF2 showed responsi- bility for autosomal, dominant appearance of multiple C N S tumors, including schwannomas, meningiomas, and ependymomas.

Neuroblastoma (NBL) is a tumor derived from cells of the neural crest, with a widely variable outcome. NBL was among the first tumors for which the relationship between the expression of an oncogene (N-myc) and the prognosis was discovered. Besides amplification of N-myc (increased expression without amplification was not correlated with poor prognosis), LOH of several chromosomal loci occur in NBL, representing genetic instability. However, microsatellite instability is infre- quent. The N-myc replicon is rather large (350 kb-1 Mb); therefore presence of co- amplified genes was suggested. The co-amplification and concomitant high level of expression of a DEAD box gene with N-myc was observed in human NBL. DDXI belongs to a family of genes that encode DEAD (Asp-Glu-Ala-Asp) box proteins, which are putative ATP-dependent RNA helicases implicated in several cellular processes involving alterations of RNA secondary structures." It was also found that NBLs express the neurotrophin receptors TrkA and TrkB. Expression of TrkA and TrkC correlates with favorable outcome, whereas expression of full-length TrkB is associated with unfavorable, more aggressive NBL.I4 Recently, a high level of nucleoside diphosphate kinase A (NDPK Alnm23-Hl) in NBL is associated with advanced stage disease.I5 There is no question that NBLs demonstrate both clinical and biological heterogeneity. Three genetically distinct subtypes can be separated? (1) hyperdiploid or triploid karyotypes; l p LOH and N-myc amplifica- tion absent, TrkA expression is high; usually infants are involved stage is I, 11, or IVS; prognosis is good (>90% cure); (2) near diploid or tetraploid karyotypes, no N-myc amplification; l p allelic loss, 1% allelic loss or other structural changes are present; TrkA expression is low; patients are older; in stage I11 or IV, prognosis is bad (25-30% cure); (3) near diploid or tetraploid karyotype; N-myc amplification and lp allelic loss are present; TrkA expression is low or absent; age 1-5 years; advanced stages (111, IV); very poor prognosis (-3% cure).

These are just examples. The molecular genetics of malignant tumors is a rapidly expanding area with a huge amount of new, sometimes conflicting information. We are now at the learning phase, and our real challenge is figuring out how to take advantage of the daily increasing knowledge and the opportunities offered by the molecular studies.

p53.

REFERENCES

1. GREENBLATT, M. S., W. P. BENNETT, M. HOLLSTEIN & C. C. HARRIS. 1994. Mutations in the p53 tumor suppressor gene: Clues to cancer etiology and molecular pathogenesis. Cancer Res. 54: 4855-4878.

2. GOITLIEB, T. M. & M. OREN. 1996. P53 in growth control and neoplasia. Biochim. Biophys. Acta 1287: 77-102.

3. BEISERSBERGEN, R. L. & R. BERNARDS. 1996. Cell cycle regulation by the retinoblastoma: A family of growth inhibitory proteins. BBA 1287: 103-120.

4. HALL, M., S. BATES & G. PETERS. 1995. Evidence for different modes of action of cyclin- dependent kinase inhibitors: p15 and p16 bind to kinases; p21 and p27 bind to cyclins. Oncogene 11: 1581-1588.

KOPPER ct ul: MOLECULAR GENETICS 7

5. 6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

RABBITS, T. H. 1994. Chromosomal translocation in human cancer. Nature 372: 143-148. KORSMEYER, S. J. 1992. Bcl-2 An antidote to programmed cell death. Cancer Surv.

KERR, J. F. R., A. H. WYLLIE & A. R. CURRIE. 1972. Apoptosk A basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer Me 239-257.

HALE, A. J., C. A. SMITH, L. C. SUTHERLAND, V. E. STONEMAN, V. L. LOGTHORNE, A. L. CWLHAME & G. T. WILLIAMS. 1996. Apoptosk Molecular regulation of cell death. Eur. J. Biochem. 236: 1-26.

PATEL, T., G. J. GORES & S. H. KAUFMANN. 1996. The role of proteases during apoptosis.

LATHAM, K. E., J. MCGRATH & D. SOLTER. 1995. Mechanistic and developmental aspects of genetic imprinting in mammals. Int. Rev. Cytol. 160.53-98.

HEWITT, S. M., M. C. HAMADAS, T. J. DONNELL, F. J. RAUSCHER 111 & G. F. SAUNDERS. Regulation of the proto-oncogenes bcl-2 and c-myc by the Wilms’ tumor suppressor gene WTl. Cancer Res. 55: 5386-5389.

BATRA, S. K., R. E. MCLENDON, J. S. Koo, S. CASTELINO-PRABHU, H. E. FUCHS, J. P. KRISCHER, H. S. FRIEDMAN, D. D. BIGNER & S. BIGNER. 1995. Prognostic implications of chromosome 17p deletions in human medulloblastomas. J. Neurooncol. a 39-45.

MANOHAR, C. F., H. R. SALWEN, G. M. BRODEUR & S. L. Corn. 1995. Co-amplification and concomitant high levels of expression of a DEAD box gene with Myc-N in human neuroblastoma. Genes Chromosomes Cancer 14: 1%-203.

YAMASHIRO, D. J., A. NAKAGAWARA, N. IKEGAKI, A. G. LIU & G. M. BRODEUR. 1996. Expression of TrkC in favorable human neuroblastoma. Oncogenes 12: 37-41.

CHANG, C. L., J. R. STRAHLER, D. H. PHORAVAL, M. QIAN, R. HIDERER & S. M. HANASH. 1996. A nucleoside &phosphate kinase A ( d - H l ) serine 12lkglycine substitution in advanced stage neuroblastoma affects enzyme stability and alters protein-protein interactions. Oncogene 12: 659-667.

BRODEUR, G. M. 1995. Molecular basis for heterogenity in human neuroblastomas. Eur.

15: 105-118.

FASEB J. 10: 587-597.

J. Cancer 31: 505-510.