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TP53 AND HEPATOCELLULAR CARCINOMA
PUISIEUX A., OZTURK M. - TP53 and hepatocellular carcinoma.
Path Biol, 1997, 45, n° 10, 864-870.
SUMMARY: TP53 gene mutations occur in 30 to 55 %
hepatocellular carcinomas. Both the frequency and the type of p53 mutations in HCC vary according to geographical location of tumors. A specific mutation at codon 249 (AGG---. AGT) was found at high frequency in tumors from high aflatoxin-arcas. TP53 mutations in
other geographic locations are less frequent and scattered on the exons encoding the central region of the protein. TP53 mutations observed in hepatocellular
carcinoma are accompanied by a loss of wild-type p53 function. Moreover, the p53-249scr mutant appears to display a gain of function at some degree. In addition to p53 inactivation by gene mutation, there is growing evidence that the wild-type p53 functions can be inactivated by the HBx protein of Hepatitis B Virus. The hepatocellular functions of wild-type p53 protein are not entirely known. The present data suggest that the DNA damaging agents induce p53-dependent cell cycle arrest or apoptosis in cell lines derived from normal liver or hepatocellular carcinoma. In contrast, the exposure of mice to genotoxic agents does not induce p53-dependent changes in normal adult liver. This could be due to the fact that the hepatocytes of the adult liver are quiescent cells.
KEY-WORDS: TP53. -Tumor sup_pressor gene. - Mutational hotspot. - Carcinogenesis. - Hepatitis B virus.
Manuscript received on February 25, 1997.
A. PUISIEUXl,2, M. OZTURKl,3
PUISIEUX A., OZTURK M. - TP53 et carcinome hepatocellulaire. (En Angfafa).
Path Biol, 1997, 45, n° 10, 864-870.
RESUME: Trente
a
55 % des carcinomeshepato-cellulaires presentent une mutation du gene TP53. La
frequence et le type de mutations varicnt selon I' origine geographique des patients. Une mutation specifique au niveau du codon 249 (AGG ---. AGT) a ete identifiee avec une grande frequence dans Jes tumeurs provenant de regions hautement contaminees par l'aflatoxine Bl. Les mutations de TP53 dans les
autres regions sont moins frequentes et sont reparties au niveau de la region centrale de la proteine. Les mutations de TP53 observees dans Jes carcinomes
hepatocellulaires provoquent une perte de l'activite de la protcinc p53. En plus de !'inactivation de p53 par mutation genique, la proteine p53 sauvage pourrait etre inactivee par interaction avec la proteine x du virus de l'hepatite B. Les fonctions biologiques de la proteine p53 dans Jes hepatocytes ne sont pas entierement connues. Les donnees actuclles suggcrent que les agents genotoxiques induisent un arret du cycle cellulaire ou une mort cellulaire par apoptose dependants de p53 dans les lignees cellulaires derivees de foie normal ou de carcinome hepatocellulaire. Cependant, !'exposition de souris aux agents genotoxiques ne semble pas induire p53 dans le foie adulte normal. Ceci pourrait etre lie au fait que Jes hepatocytes normaux sont des cellules quiescentes.
MOTS-CLES: TP53. - Gene supprcsseur de tumeur. - Foyer de mutations. -Careinogcnese. - Virus de l'Hepatite B.
I. Unite INSERM U453, 2. Unite d'Oncologie Moleculaire,
Centre Leon Berard, 28, rue Laennec. 69008 LYON (France). 3. Bilkent University, Department of Molecular Biology cmd Genetics, 06533 Bilkelll, ANKARA (Turkey).
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TP53 INACTIVATION
IN HEPATOCELLULAR CARCINOMAS During the past decade, systematic analysis of genetic changes in different human tumors has made it possible to uncover molecular pathways leading to malignant cellular transformation. Like many other tumors, human hepatocellular carcinoma (HCC) is a multistep disease, in which many genomic changes occur as a result of uncontrolled proliferation of cells. It is estimated that it takes at least 30 years for an HCC to develop as a clinically detectable disease. Both epidemiologic and molecular studies indicate that five to six independent events are necessary for a normal hepatocyte to develop a fully malignant tumor. It is therefore assumed that several oncogenes and tumor suppressor genes are involved in HCC. Up to now, there is no known oncogene shown to be consistently activated in these tumors. In contrast, analysis of chromosomal changes has permitted the identification of several loci deleted in HCCs. This observation suggests that different tumor suppressor genes are involved in the development of these tumors. Genomic loss on chromosomes 16 ( l 6p and l 6q), 5p, 4q, l p, 5q, 8q and I 3q was found in 20 to 45 % of HCCs [36]. However, the short arm of the chromosome I 7 appears to be the most frequently deleted ( 51 % ) [ 13, 50]. A common region of deletion is I 7p13, where the TP53 tumor suppressor gene is located. TP53 gene aberrations have been described for many tumors, and, overall constitute the most common genetic change detected in human malignancies. Although gene deletions and rearrangements occur, the most frequent alterations are single point mutations in one allele and subsequent aminoacid substitutions, usually in the evolutionary conserved regions of the gene. By examining the spectrum of TP53 mutations in a series of tumors, hypotheses can be generated regarding the environmental and endogenous molecular processes that contribute to the development of the cancer.
Genetic alterations of TP53 in hepatocellular
carcinomas
Important etiological factors of HCC arc chronic infection with Hepatitis B virus (HBV) or hepatitis C virus (HCV) and dietary exposure to aflatoxin B 1. The relative risk of HCC is elevated in viral hepatitis carriers with chronic active hepatitis. Overall, it is estimated that about 80 % of HCC cases are attributable to HBV [2]. Aflatoxin B 1 is considered to be a significant etiological factor in certain areas (southern Africa and Asia), where this mycotoxin is consumed in food contaminated by Aspergillus flavus [62]. Aflatoxin Bl is a potent chemical carcinogen and mutagen in many animal species, which forms deoxyguanosine adducts in rodent and human hepatocytes and frequently causes G to T transversions in mutation assays [ 12]. The first studies of TP 53
mutations in hepatocellular carcinoma were undertaken in patients residing in high risk regions of the world (Qidong, China; Mozambique), where HBV infection and aflatoxin Bl intake are synergistic risk factor of HCC [3, 20]. Fifty percent of tumors studied displayed TP 53 mutations. Strikingly, 10 of the 13 mutations described were guanine to thymine tranversions that occurred at a unique site in the gene coding sequence, the third base pair of codon 249. This mutation converted an arginine residue to serine residue. Specific mutations might reflect exposure to specific carcinogens. This hypothesis was tested by studying the presence of the hotspot mutation at codon 249 of the TP53 gene in HCC samples from 14 different geographic locations in the world [3 7]. A mutation was detected in 17 % (12/72) from four countries, namely, Mozambique, Transkei, China, and Vietnam, clustered in southern Africa and the southeast Asia. There was no codon 249 mutation in 95 HCCs from other geographic locations, including North America, Europe, the Middle East, and Japan. Hence, worldwide, the presence of this codon 249 mutation in HCCs correlated with high risk of exposure to aflatoxins and the hepatitis B virus. Further studies were performed in two groups of HBV infected patients at different risks of exposure to aflatoxins. Fifty-five percent of patients (8/15) from Mozambique at high risk of aflatoxin exposure had a tumor with a codon 249 mutation, in contrast to 8 % of patients (1/12) at low risk from Transkei. These studies suggested that a codon 249 mutation of the TP53 gene identifies an endemic form of HCC strongly associated to dietary aflatoxin intake. Subsequent reports on different groups of patients with HCC around the world have strengthened this hypothesis. Fujimoto et al. studied TP 53 mutations in HCCs from Chinese patients. The HCCs were obtained from two different areas in China: Qidong, where exposure to hepatitis B virus and aflatoxin B 1 is high, and Beijing, where exposure to HBV is high but that of aflatoxin Bl is low [14]. The frequency ofG to T tansvcrsion at codon 249 from Qidong and Beijing were 52 % and O %, respectively.
Analysis of TP 53 mutations in HCC, in the database [ 18] yields statistically significant conclusions consistent with previous data. The frequency of TP53 mutations is correlated with exposure of aflatoxin B 1. If we take into account studies that reported TP 53 mutations at least from exons 5 to 8 of the TP53 gene, 312 mutations have been described in HCCs [3, 4, 5, 7, 14, 17, 19,20,24,25,28,31,32,33,34,35,43,45,47, 48, 51, 52, 58]. One hundred sixty nine of these genetic alterations were from tumors from populations not exposed to aflatoxin contamination (mostly Europe, North America and Japan) and 143 from patients coming from aflatoxin exposed areas, although at various degrees (southern Africa and Asia, except Japan). In contrast with HBV infection, aflatoxin
PATHOLOGJF. BIOLOGIF.
866 TP53 AND HEPATOCELLULAR CilRC/NOMA DECEMBRE 1997
G:AtoG:C 1% G:CtoC:G 6% • I ' .
.
.
'..''
'
· ..
r
G:CtoA:T 9% . A:TtoT:A 12% G:C toA:T 25% G:CtoC:G 7% G:C toT:A 60%Fig. 1. - Spectrum of TP53 mutations in hepatocellular carcinomas. (n) = the number of mutations for which exact base change, insertion or deletion was confirmed by DNA sequencing. A, the mutational spectrum of TP53 in hepatocarcinomas from high aflatoxin areas (n = 143). B, the mutational spectrum of TP53 in hepatocarcinomas from low aflatoxin areas (n = 169). Del.+ Ins.: deletions and insertions. Fig. 1. - Eventail des mutations de TP53 observees dans les carcinomes hepatocellulaires (n) = nombre de mutations pour lesquelles le sequern;:age a permis de determiner la base exacte substituee, inseree ou eliminee. A: eventail de mutations de TP53
observees dans les hepatocarcinomes dans les zones d'exposition
a
de fortes concentrations d'aflatoxine (n = 143). B: eventail de mutations de TP53 dans les hepatocarcinomes dans les zones de faible expositiona
l'aflatoxine (n = 169). Del. et Ins.: deletions et insertions.exposure appears to influence the rate of mutation. Indeed, 55 % of HCCs from atlatoxin exposure areas display TP53 mutations for only 28% in low exposure
areas. In populations not considered to be at risk for dietary contamination, the pattern of mutation is heterogenous with only 23 % (39/169) of G to T transversions on the non transcribed DNA strand. The mutations are scattered throughout the genome, with 79 different codons involved. Only 6 % of the mutations ( 11/ 169) are G to T transversions located at the third base of codon 249 (figs. 1 and 2).
In
contrast,A 80 249 60 40 20 0 B 80 60 40 20
Fig. 2. - Location of TP53 mutations in hepatocellular carcinomas. (n) = the number of mutations for which exact base change, insertion or deletion was confirmed by DNA sequencing. A, location of TP53 mutations in hepato
-carcinomas from high aflatoxin areas (n = 143). B, location of TP53 mutations in hepatocarcinomas from low aflatoxin areas (n = 169).
Fig. 2. - Siege des mutations de TP53 dans les carcinomes hepatocellulaires. (n) = nombre de mutations pour lesquelles le sequem;:age a permis de determiner la base exacte substituee, inseree ou eliminee. A: siege des mutations de TP53 observees dans les hepatocarcinomes dans les zones d'exposition
a
de fortes concentrations d'aflatoxine (n = 143). B: siege de mutations de TP53 dans les hepatocarcinomes de zones de faible expositiona
l'aflatoxine (n = 169).in aflatoxin exposed areas, the pattern of mutations is highly homogenous. Sixty percent of mutations (85/143) are G to T transversions on the non transcribed DNA strand, 72 of them occurring at the third base of codon 249 (50 % of all mutations). In well documented high aflatoxin areas (mostly Province of Qidong in China and Mozambique in southern Africa), tumors contain an overwhelming preponderance of G to T transversion at codon 249 (82 %, 41/50).
The study of the worldwide occurrence of the TP53
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N° 10 TP53 AND HEPATOCELLULAR CARCINOMA 867
this somatic mutation and an endemic form of HCC. Striking differences between continents and even between neighboring countries strongly associate this mutation with high aflatoxin intake. The frequency of G: C to T: A transversion in human hepatocellular carcinomas in these regions may be due to the high mutability of the third base of codon 249 by aflatoxin B 1 and/or a selective growth advantage of hepatocyte clones carrying this specific p53 mutant. Experimental models of aflatoxin exposure have provided evidence for the specific mutational effect of aflatoxin B 1 on this
TP53 codon. Using an assay based on DNA
polymerase fingerprint analysis, it has been shown that the activated form of aflatoxin BI forms adducts with the third base of codon 249 [41 ]. In a human liver cell line exposed to aflatoxin BI, this guanine residue is preferentially but not exclusively mutated compared to immediately adjacent codons, suggesting that both preferential mutability and clonal selection are involved in human hepatocellular carcinogenesis [l]. Different observations suggest that indeed the p53-249ser mutant protein displays intrinsic activities [29]. In confirmation of this hypothesis, the human p53-249ser was shown to increase the mitotic activity of p53-null hepatocarcinoma cells [39]. Moreover, the introduction of the murine p53-246ser mutant, equivalent to human p53-249ser [ 15], into a murine hepatoma cell line confers growth advantage [9]. Functional inactivation of p53 in hepatocellular carcinomas?
Most of HCCs from areas with low aflatoxin exposure do not display TP53 genetic alteration. This observation may imply the involvement of alternative mechanisms leading to p53 protein inactivation. The p53 protein is a common target of transforming proteins encoded by several viruses, including simian virus 40, adenovirus and papillomavirus [26, 30, 44, 61]. All these viral proteins bind p53 and presumably alter its biological functions. As previously mentioned, HBV is the major risk factor associated with hepatocellular carcinoma. This virus has no acute transforming activity, but it has been found to be integrated into host genome in most HBV-related hepatocellular carcinoma. Integrated viral DNA sequences might act in cis to modify host gene expression or encode viral proteins that may interfere with normal cellular functions either directly or indirectly. Accordingly, the inactivation of cellular proteins by viral proteins has been proposed as a potential mechanism of malignant transformation of hepatocytes. Several studies indicate a possible role of the HBV-encoded x protein in tumor development in chronically infected patients. The HBx gene is frequently integrated into the genome [57] and expressed in malignant hepatocytes [38]. The HBx product can transactivate cellular and viral genes
through various cis elements present in the promoters of genes transcribed by RNA polymerases II and Ill [55], and it can transform rodent cells in vitro [49].
Recent studies, using exogenous expression vectors, have shown that the x protein binds to cellular p53 [ 11, 59, 53]. This interaction leads, in vitro, to a decrease of the p53 specific DNA binding activity. HBx expression also inhibits p53-mediated transcriptional activation and the in vitro association of p53 and ERCC3, a general transcription factor involved in nucleotide excision repair. Furthermore, using a microinjection technique, Wang et al. reported that HBx efficiently blocks p53-mediated apoptosis [60]. Based on these data, it was suggested that HBV may affect a wide range of p53 functions. As a transgene, x induces hepatocellular carcinoma in mice expressing the transgene under its own promoter [23]. Interestingly, tumor development was correlated with p53 binding to x in the cytoplasm and complete blockage of p53 entry in the nucleus. This led to the suggestion that the association ofx with p53 was an important mechanism for hepatocarcinogenesis in these mice through p53 inactivation by quantitative sequestration in the cytoplasm [56]. However, this observation was not reported in human hepatocellular carcinoma [ 16]. Furthermore, it was of interest that in similar experimental conditions, using the cx.-1-antitrypsin promoter to express x in transgenic mice, no hepatic tumors were obtained [27]. It has been argued that the absence of tumors in that case may be due to the lack of sustained expression of x in mice after birth. If this hypothesis was correct, it would predict that fairly high amounts of x should be present in most tumors, in which it should be bound to p53. At last, the ability ofx protein to bind both wild type and mutant forms ofp53 [11, 59] questions the relevance of x protein binding to p53 functions. We reported studies on wild-type p53 activity in cell lines reproducing conditions of HBV replication [ 40]. The 2215 cell line was obtained by Sells et al., after transfection of the hepatocarcinoma cell line HepG2 with a plasmid containing the HBV genome [ 46]. This cell line produces high levels of Hbe and Hbs antigens and HBV-specific particles morphologically identical to infectious Dane paiticles. All known steps of p53 participation in the cellular response to DNA damage were unaffected in 2215 cells, demonstrating that HBV replication does not interfere with known biological functions of p53. Viral replication occurs during acute and chronic hepatitis. During this early stage of the dis~ase, the expression of x transcripts is low. In HBV-related HCCs, the malignant cells display integrated forms instead of free replicating forms of the virus. 1n these cells, incomplete and rearranged forms of integrated viral DNA sequences may encode full length or truncated forms of several proteins including HBV sand x proteins. At this step, viral proteins may interfere with p53 protein function, consequently allowing tumor progression.
868 TP53 AND HEPATOCELLULAR CARCINOMA PATHOLOGIE BIOLOGIE 0~:CEMIJRE 1997
IMPLICATIONS OF p53
INACTIVATION IN THE PATHOGENESIS OF HEPATOCELLULAR CARCINOMA The role of wild-type p53 was linked to the protection of normal cells from DNA-damage induced mutations. p53 protein levels increase in cells exposed to DNA damaging agents as a result of post-translational stabilization. Under these conditions p53 migrates into the nucleus and functions as a transcriptional regulator in two ways. Genes that contain a consensus p53 binding site are induced, whereas genes containing a TATA box but not a p53 binding site arc repressed. Most cells exposed to DNA damage undergo a p53-dependent cell cycle arrest at the G 1 phase and possibly at the G2 phase. However, certain cells (mainly hematopoietic cells) undergo programmed cell death (apoptosis) rather than cell cycle arrest when exposed to DNA damage. Both of these two types of response are abrogated in cells which have lost wild-type TP53 as a result of either miss-sense mutation or gene deletion. Data about p53-mediated changes in liver cells are quite limited and sometimes contradictory. Apoptosis as well as cell cycle arrest were observed in the liver or hepatocyte-like cells. For example, the expression of TP 53 mRNA was shown to be increased during the apoptosis induced by partial liver ischaemic atrophy due to distal portal vein ligation [6]. In the other hand, ultraviolet-induced growth arrest of primary rat hepatocytes was shown to be relieved by TP 53 antisense oligonucleotide treatment, suggesting that wild0type p53 induces growth arrest in these cells [54]. The only known p53 target directly involved in cell cycle regulation is the cyclin-dependent kinase inhibitor p2IWAFI/CIPI [10]. The expression of p2JWAFI/C!Pl is induced by wild-type p53 in hepatocarcinoma cells [ 40]. Targeted in vivo expression p2 l WAFIICJPJ halts hepatocyte cell-cycle progression, postnatal liver development, and regeneration [63]. It still remains unknown whether p53 is activated in vivo in the liver after DNA damage. The activation of wild-type p53 after DNA damage was shown in vitro, in normal hepatocytes as well as hepatoma-derived cell lines [ 42, 54, 21]. However, as most cells in the adult liver are quiescent, the p53-mediated response to DNA damage in normal and tumorous liver may be related to the proliferative stage of cells. The tissue-specific effects of wild-type p53 have been addressed using TP53 knock-out mice mice. These transgenic mice spontaneously develop tumors (mostly lymphomas and sarcomas) in different tissues, but not in the liver [8]. Intriguingly, the response of TP53 null mice to carcinogen-induced hepato-carcinogenesis was similar to the response of normal mice [22]. However, it is noteworthy that the liver is not the only tissue that is resistant to tumor occurrence in TP53 knock-out mice. In general, hepatocellular carcinomas develop after a
long period of chronic liver disease. Cellular and molecular changes associated with chronic liver disease may render hepatocytcs sensitive to a p53-related transformation mechanism.
A COMPREHENSIVE MODEL FOR p53 IMPLICATIONS
TN NORMAL LIVER
AND HEPATOCELLULAR CARCINOMA Based on the present status of our knowledge on p53 in hepatocytes and hepatoccllular carcinoma cells, we would like to propose the following model.
Hepatocytes of adult liver arc exposed to both viral and chemical agents that induce an acute or chronic liver disease. Such a disease condition is accompanied by cell loss due to the hepatotoxic effects of these agents. Any significant loss of hepatocyte mass in the liver results in a regenerative process involving hepatocellular proliferation. If such response occurs during the exposure to a genotoxic agent such as an aflatoxin derivative, p53 protein is activated to stop cell growth either by cell cycle arrest or apoptosis. Both responses are important for the inhibition of abnormal replication of damaged DNA. However, some cells may escape the p53-mediated growth arrest and give rise to daughter cells with mutated DNA. If these alterations are not corrected by the post replicative repair systems, they become permanent. It is possible that most of such mutations occur in the non coding regions of the DNA with no direct effect on the cellular phenotype. However, mutations may also hit critical genes involved in the control of normal phenotype, resulting in the initiation of progression of malignant phenotype. Interestingly, the best candidate of such mutations is the G-T transversion occuring on codon 249 of TP 53 gene. This particular mutation gives rise to the synthesis of a mutant p53 protein (p53-249ser) which has lost wild-type p53 activity. Moreover, the p53-249ser mutant protein displays a gain of function resulting in a direct involvement in cellular transformation. Any cell with such a mutation will lose the wild-type p53-mediated cellular response to DNA damage and become senstitive to mutagenic effects of genotoxic agents. Additional mutations affecting the other oncogenes and tumor suppressor genes will again increase the chances of malignant transformation. Hepatocellular carcinomas not-related to aflatoxins also display TP53 mutations albeit at lower frequency. The occurrence of these mutations is independent of hepatitis B and hepatitis C virus status. They are scattered throughout the TP53 gene and appear to result from random mutations due to increased cell proliferation during chronic liver disease. However, some of such mutations could be due to yet unknown carcinogens. Finally, there is growing evidence that the
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HBx protein of the hepatitis B virus is able to form complexes with p53 protein. Such complexes arc able to inhibit p53-dcpcndent transcriptional activation under experimental conditions. The HBx protein encoded by the freely replicating virus or by integrated viral sequences could interfere with p53-dependent
cellular functions in normal liver as well as hepatocellular carcinoma cells.
ACKNOWLEDGEMENT
Authors would like to thank N.Borel for her careful preparation of the manuscript.
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870 TP53 AND HEPATOCELLULAR CARCINOMA PA THO LOGIE BIO LOGIE
DECEMBRE 1997
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