Genotype–Phenotype Relationships in Hepatocellular Carcinoma: p53 Inactivation Promotes Tumors With Stem Cell Features

See “Disruption of Trp53 in livers of mice induces formation of carcinomas with bilineal differentiation,” by Katz S–F, Lechel A, Obenauf AC, et al, on page 1229. Cancer is a disease of the genome caused by an accumulation of genetic and epigenetic alterations in oncogene and tumor suppressor genes drawing the landscape of human tumors. In liver tumors, like in other human cancers, TP53 (for tumor protein 53) is the most frequently inactivated tumor suppressor gene. TP53 mutations are identified in 20%–50% of hepatocellular carcinoma (HCC)1Hsu I.C. Metcalf R.A. Sun T. et al.Mutational hotspot in the p53 gene in human hepatocellular carcinomas.Nature. 1991; 350: 427-428Crossref PubMed Scopus (1407) Google Scholar with a highest frequency of mutations in Asia and Africa owing to chronic hepatitis B infection and aflatoxin B1 (a mycotoxin contaminating peanut, rice, and corn) exposure.2Nault J.C. Zucman-Rossi J. Genetics of hepatobiliary carcinogenesis.Semin Liver Dis. 2011; 31: 173-187Crossref PubMed Scopus (126) Google Scholar In cells, p53 plays its tumor suppression function at several levels such as control of G1/S checkpoint, apoptosis, and DNA repair. It is also considered as the guardian of chromosomal stability.3Levine A.J. Oren M. The first 30 years of p53: growing ever more complex.Nat Rev Cancer. 2009; 9: 749-758Crossref PubMed Scopus (1314) Google Scholar In this issue of Gastroenterology, Rudolph et al addressed the role of p53 deletion alone in liver carcinogenesis.4Katz S.F. Lechel A. Obenauf A.C. et al.Disruption of Trp53 in livers of mice induces formation of carcinomas with bilineal differentiation.Gastroenterology. 2012; 142: 1229-1239Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar To this end, they constructed a mouse model harboring liver specific deletion of p53 using a chimeric albumin/alpha-fetoprotein enhancer promoter leading to early p53 inactivation in “fetal” hepatocytes. Organ-specific deletion of p53 was required because mice with germline deletion of p53 developed sarcoma and lymphoma at a very early age (4–5 months) and died around 10 months of age.5Donehower L.A. Harvey M. Slagle B.L. et al.Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours.Nature. 1992; 356: 215-221Crossref PubMed Scopus (4016) Google Scholar In this new model, liver p53−/− mice spontaneously developed HCC at 14–20 months of age, thereby showing that specific deletion of p53 during embryonic development was able to induce spontaneous HCC in old age. The late occurrence of HCC in this model also suggested that other genetic or epigenetic alterations could be necessary for tumor development together with p53 inactivation. These results reinforced the central role of p53 as a tumor suppressor gene in liver carcinogenesis (Figure 1) . Strikingly, in the study presented in this issue of Gastroenterology, HCC in the liver of p53−/− mice exhibited features of bilineal differentiation. Bilineal differentiation is defined by co-expression of hepatocyte (albumin) and cholangiocytes (keratin 19 [K19]) markers, which reflect a stem cell-like features. The first evidence of the role of p53 in stem cell homeostasis was identified in p53-null mice in the hematopoietic system that harbored stem cells with an increased ability of self-renewal and proliferation.6Nguyen L.V. Vanner R. Dirks P. et al.Cancer stem cells: an evolving concept.Nat Rev Cancer. 2012; 12: 133-143Crossref PubMed Scopus (860) Google Scholar In the liver of rodent models, stem cells have been identified near the canal of herring.7Roskams T. Liver stem cells and their implication in hepatocellular and cholangiocarcinoma.Oncogene. 2006; 25: 3818-3822Crossref PubMed Scopus (340) Google Scholar Chronic liver disease impaired the ability of mature hepatocytes to divide; in response, aberrant activation of liver stem cells increased with the severity of underlying disease. In the liver cancer stem cell theory, a subset of these cells could give rise to HCC harboring bilineal markers. Accordingly, in advanced HCC, a subgroup of cancer cells could conserve stem cell features and give rise to new HCC when transplanted in immunocompromised mice.8Shackleton M. Quintana E. Fearon E.R. et al.Heterogeneity in cancer: cancer stem cells versus clonal evolution.Cell. 2009; 138: 822-829Abstract Full Text Full Text PDF PubMed Scopus (874) Google Scholar In contrast, some data also showed that mature hepatocytes could dedifferentiate and lead to tumor formation harboring stem cells markers.9Cerec V. Glaise D. Garnier D. et al.Transdifferentiation of hepatocyte-like cells from the human hepatoma HepaRG cell line through bipotent progenitor.Hepatology. 2007; 45: 957-967Crossref PubMed Scopus (252) Google Scholar, 10Michalopoulos G.K. Barua L. Bowen W.C. Transdifferentiation of rat hepatocytes into biliary cells after bile duct ligation and toxic biliary injury.Hepatology. 2005; 41: 535-544Crossref PubMed Scopus (236) Google Scholar In the present model, Rudolph et al sought to elucidate the origin of the tumor stem cells in a p53−/− context. First, they purified cells with bilineal features and tumor hepatocytes without stem cell-like features (the “side population”) from HCC. They showed that both kinds of cells are able to reinduce HCC when transplanted in immunocompromised mice. In a second experiment, liver stem cells purified from p53−/− nontransformed liver demonstrated an age-independent increase in the ability to proliferate and form colonies with bipotent features. Bipotent and cholangiocytic stem cells, as well as mature hepatocytes isolated from nontransformed p53−/− liver, also showed an increased capacity to form cell lines. These 3 cell types were able to induce HCC with stem cell markers and chromosomal instability when transplanted in immunocompromised mice. In contrast, p21 knockout mice did not develop cancer and purified cells from their liver did not demonstrate tumorigenic potential. Together these data showed that, when deleted for p53, both mature hepatocytes and stem cells are tumorigenic, independent of p21, and they could both lead to the formation of HCC with stem cells features. Consequently, liver stem cells and dedifferentiation of mature hepatocytes theories are not mutually exclusive in a p53−/− context. This mouse model with liver-specific p53 deletion also demonstrated an interesting link with human liver tumorigenesis. In human HCC, a subset of tumors (10%–20%) exhibited stem cells markers (like K19 or EPCAM). These tumors have close similarity with hepatoblasts in their gene expression profile, and typically have a poor prognosis.11Lee J.S. Heo J. Libbrecht L. et al.A novel prognostic subtype of human hepatocellular carcinoma derived from hepatic progenitor cells.Nat Med. 2006; 12: 410-416Crossref PubMed Scopus (783) Google Scholar Another study from Woo et al12Woo H.G. Wang X.W. Budhu A. et al.Association of TP53 mutations with stem cell-like gene expression and survival of patients with hepatocellular carcinoma.Gastroenterology. 2011; 140: 1063-1070Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar showed that HCC with inactivating mutations of TP53 exhibited stem cell-like features and was associated with high risk of tumor relapse and death. Another strikingly similar feature observed in HCC arising on liver p53 knockout mouse and in human HCC mutated for TP53, was chromosome instability (gain or loss of fractions of chromosome or whole chromosome).13Laurent-Puig P. Legoix P. Bluteau O. et al.Genetic alterations associated with hepatocellular carcinomas define distinct pathways of hepatocarcinogenesis.Gastroenterology. 2001; 120: 1763-1773Abstract Full Text Full Text PDF PubMed Scopus (485) Google Scholar Rudolph et al14Begus-Nahrmann Y. Lechel A. Obenauf A.C. et al.p53 deletion impairs clearance of chromosomal-instable stem cells in aging telomere-dysfunctional mice.Nat Genet. 2009; 41: 1138-1143Crossref PubMed Scopus (87) Google Scholar showed in a previous study that intestinal p53 deletion in telomerase knockout mice leads to aberrant proliferation of chromosome-instable intestinal stem cells. Telomerase dysfunction leads to telomere shortening, which induced chromosome instability and DNA damage. When p53 is deleted, stem cells deficient for telomerase accumulate and harbor chromosomal instability. These data link telomerase dysfunction and TP53 deletion with chromosomal instability and aberrant stem cells activation, which could be translated in mouse model of liver cancer.15Liu G. Parant J.M. Lang G. et al.Chromosome stability, in the absence of apoptosis, is critical for suppression of tumorigenesis in Trp53 mutant mice.Nat Genet. 2004; 36: 63-68Crossref PubMed Scopus (264) Google Scholar Carcinogenesis is clearly the consequence of accumulation of genetic alterations that cooperate mutually. Whole genome sequencing of human tumors has shown that multiple mutations in driver and passenger genes exist in every kind of solid and hematopoietic tumors. In a study recently published in Gastroenterology, McClendon et al16McClendon A.K. Dean J.L. Ertel A. et al.RB and p53 cooperate to prevent liver tumorigenesis in response to tissue damage.Gastroenterology. 2011; 141: 1439-1450Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar showed cooperation between inactivation of p53 and the retinoblastoma gene (Rb, a major cell cycle regulator) in the development of HCC in mice. The 2 mouse models are not directly comparable because (i) genetic backgrounds were different (C57BL/6J vs mixed FVB;129), (ii) promoters to induce gene inactivation (albumin-cre vs albumin/alpha-fetoprotein-cre) are active at different stages of development and can affect different types of cells (hepatocytes for albumin-cre and both hepatocytes and liver stem cells for albumin/alpha-fetoprotein-cre), and (iii) follow-up times are different. However, HCC arising on p53−/− mice were characterized by significant dysregulation of the Rb pathway compared with HCC developed on diethylnitrosamine-treated mice.4Katz S.F. Lechel A. Obenauf A.C. et al.Disruption of Trp53 in livers of mice induces formation of carcinomas with bilineal differentiation.Gastroenterology. 2012; 142: 1229-1239Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar These results demonstrated novel evidence for a complex cooperation in liver tumorigenesis between the p53 and Rb pathways, 2 major checkpoints of the cell cycle (Figure 1). Interestingly, simultaneous disruption of the p53 and Rb pathways has been frequently linked with more aggressive tumors. In human HCC tumors, RB is rarely mutated but CDKN2A (p16), a gene of the retinoblastoma pathway, is frequently found inactivated through mutation, homozygous deletion, or promoter hypermethylation.2Nault J.C. Zucman-Rossi J. Genetics of hepatobiliary carcinogenesis.Semin Liver Dis. 2011; 31: 173-187Crossref PubMed Scopus (126) Google Scholar In our transcriptomic classification of HCC in 6 subgroups (G1–G6) associated with genetic and clinical features,17Boyault S. Rickman D.S. de Reynies A. et al.Transcriptome classification of HCC is related to gene alterations and to new therapeutic targets.Hepatology. 2007; 45: 42-52Crossref PubMed Scopus (848) Google Scholar we showed that HCC classified in the G3 subgroup are characterized by both mutation of TP53 and inactivation of CDKN2A. G3 tumors were associated with the worst prognosis. Recently, the prognostic value of this G3 molecular subclass was confirmed in a large series of resected HCC by Llovet et al.18Villanueva A. Hoshida Y. Battiston C. et al.Combining clinical, pathology, and gene expression data to predict recurrence of hepatocellular carcinoma.Gastroenterology. 2011; 140: 1501-1512Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar All these results underlined that inactivation of TP53 and RB pathways in liver carcinogenesis cooperate to induce an aggressive tumor phenotype both in mice and humans (Figure 1). Finally, questions about preneoplastic stages arose from this new study because, according to different tumor types, p53 inactivation could act early in tumorigenesis, at the initiation of tumor development, or latter during progression of cancer. In the liver p53−/− mice, Rudolph et al did not find any preneoplastic lesions in liver; the earliest tumors demonstrated the same features as advanced HCC. In human tumorigenesis, aberrant expression of stem cells markers has been described in a subset of preneoplastic lesions like dysplastic macronodules.7Roskams T. Liver stem cells and their implication in hepatocellular and cholangiocarcinoma.Oncogene. 2006; 25: 3818-3822Crossref PubMed Scopus (340) Google Scholar Moreover, aberrant expression of stem cells markers (K19, K7, and EPCAM) in hepatocytes has been linked with an increased incidence of HCC in hepatitis C-related cirrhosis.19Ziol M. Nault J.C. Aout M. et al.Intermediate hepatobiliary cells predict an increased risk of hepatocarcinogenesis in patients with hepatitis C virus-related cirrhosis.Gastroenterology. 2010; 139: 335-343Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar Consequently, this mouse model could be very useful to study more precisely the early stages of liver tumorigenesis in relation with exposure to different carcinogens and of fibrosis development. All these data underlined that a liver-specific p53−/− mice model at embryonic stage mimics classical features (chromosomal instability, stem cell-like markers) of human HCC mutated for TP53. This mouse model constitutes a new tool that recapitulates p53-related carcinogenesis and will be useful to dissect the role of chromosomal instability and stem cell activation in liver tumorigenesis. Finally, it could also be used to test new therapeutics targeting the p53 pathway and liver cancer stem cells before translation in humans. The authors are very grateful to Karine Poussin for critical reading of this manuscript. This work was supported by the Labex Immuno-Oncology, Université Paris Descartes and the BioIntelligence collaborative program (OSEO). J.C.N is supported by a fellowship from INCa. Disruption of Trp53 in Livers of Mice Induces Formation of Carcinomas With Bilineal DifferentiationGastroenterologyVol. 142Issue 5Previewp53 limits the self-renewal of stem cells from various tissues. Loss of p53, in combination with other oncogenic events, results in aberrant self-renewal and transformation of progenitor cells. It is not known whether loss of p53 is sufficient to induce tumor formation in liver. Full-Text PDF

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