Of mice and men: The nonrandom genomic instability in hepatocarcinogenesis
Authors: Diego F. Calvisi
Journal: Hepatology (2011)

CGH, comparative genomic hybridization; CIN, chromosomal instability; DEN, diethylnitrosamine; DN, dysplastic nodules; HCC, hepatocellular carcinoma; LOH, loss of heterozygosity; MSI, microsatellite instability, PB, phenobarbital. The stepwise progression from early dysplastic lesions to fully malignant and metastatic tumors is associated with an increased occurrence of genomic alterations, a condition known as genomic instability.1 Most tumor types exhibit a form of genomic instability called chromosomal instability (CIN), referring to the high rate by which chromosome structure (amplification, duplication, deletion, translocation, etc.) and number (aneuploidy, polyploidy) change over time in cancer cells compared with normal cells.1 Some of these chromosomal abnormalities are detected in all tumor cells but others are not, suggesting that tumor cells initially derive from a genetically unstable single cell, which continues to acquire chromosomal abnormalities over time. The latter assumption has been substantiated by the observation that in vitro growth stimulates the generation of CIN in cancer cells.1 Other forms of genomic instability have also been discovered. These include microsatellite instability (MSI), in which the expansion or contraction of the number of oligonucleotide repeats present in microsatellite sequences occurs,2 and forms of genomic instability that are characterized by increased frequencies of basepair mutations.3 In hepatocellular carcinoma (HCC), mounting evidence indicates that CIN is the predominant form of genomic instability, whereas MSI seems to be a rare event.4 Investigations have been performed on this issue both in rodent models of liver cancer and in human HCC. In transgenic mouse models, two distinct patterns of genomic instability were found to be associated with HCC development. Transgenic mice overexpressing the c-Myc and E2F1 protooncogenes either alone or in combination display a low rate of genomic instability, whereas a high rate of genomic instability accompanies the accelerated and more aggressive hepatocarcinogenesis driven by coexpression of c-Myc and transforming growth factor alpha (TGF-α) transgenes.5 Strikingly, an opposite pattern was detected when analyzing the activation of Wnt/β-catenin pathway in the same models, with the latter cascade being frequently activated in preneoplastic and neoplastic lesions developed in c-Myc and c-Myc/E2F1 transgenic mice but rarely in c-Myc/TGF-α corresponding lesions.5 Similarly, human HCCs have been divided into two major subgroups, one displaying β-catenin mutations and chromosome 8p losses in a context of genomic stability, and the other exhibiting widespread chromosomal instability, with frequent allelic losses on chromosomes 1p, 4q, 6q, 9p, 13q, 16p, 16q, and 17p associated with p53 and Axin1 mutations.6 Therefore, extensive genomic instability and activation of the Wnt/β-catenin pathway seem to represent two mutually exclusive oncogenic mechanisms in hepatocarcinogenesis. In this issue of HEPATOLOGY, the report by Aleksic et al.7 provides new significant insights into the genomic changes occurring along multistep liver carcinogenesis. The authors established the chronological order of genetic alterations occurring in liver tumors developed in mice subjected to diethylnitrosamine (DEN) initiation and phenobarbital (PB) feeding by analyzing the neoplastic lesions via array-comparative genomic hybridization (array-CGH) at different timepoints. To avoid contamination from normal liver tissue, tumor samples were isolated by laser microdissection from their nontumorous surrounding counterparts. The results show that chromosomal gains and losses were already observed in tumors by week 32 and increased significantly by week 56. In particular, loss of distal chromosome 4q was a frequent, early event and persisted during all tumor stages, suggesting a crucial role of this locus both in liver cancer development and progression. Consistent with the authors' hypothesis, the distal chromosome 4q is syntenic to human chromosome 1p, which is frequently lost in human HCC.4 In particular, the distal part of chromosome 4q contains several putative tumor suppressor genes, such as Runx3, Nr0b2/Shp, Nbl1, Alpl, Rap1gap, and Ephb2.7 Among them, Runx3 and Nr0b2/Shp have been supposed to be tumor suppressors due to their recurrent downregulation in human HCC. Runx3 (runt related transcription factor 3), belonging to the Runt family of transcriptional factors that can activate or repress target gene transcription, is a frequently hypermethylated gene in HCC8 and might exert its antineoplastic properties both via suppression of the Notch signaling cascade and functioning as coactivator of the p53 tumor suppressor.9, 10 Nr0b2/Shp (nuclear receptor subfamily 0, group B, member 2 gene/small heterodimer partner), a member of the nuclear receptor superfamily that participates in the biological regulation of several major liver functions, is also frequently epigenetically silenced in human HCC and its genetic disruption results in spontaneous development of HCC in Shp−/− mice.11 Additional alterations were found to consistently occur in frankly malignant HCC from older mice, including the loss of chromosome 6 and chromosome 9 material, and the gain of chromosome 15 material.7 Based on these data, the authors envisage the possibility that nonrandom, recurrent chromosomal alterations are necessary for liver tumor development and progression in this mouse model. A similar phenomenon has been previously described in c-Myc/TGF-α transgenic mice. In these mice, the specific sites of breakages occurring during early stages of hepatocarcinogenesis were later involved in stable rearrangements in the tumors. In particular, the breakage of chromosomes 1, 5, 6, 7, and 12 may represent an early event during hepatocarcinogenesis, while the deletions of chromosomes 4, 12, 14, and X appear during HCC progression.12 Of note, the breakpoints described on c-myc/TGF-α chromosomes 1, 4, 7, and 12 correspond to human 1q, 1p, 11p, and 14q that are also rearranged in human HCC.4, 13 In human liver specimens, loss of heterozygosity (LOH) was found to be uncommon in cirrhosis, focal nodular hyperplasia, and hepatocellular adenomas, but detectable in high-grade dysplastic nodules (DN), which are putative precancerous lesions.13 Importantly, the frequency and pattern of genetic alterations detected in DNs highly resembled those in HCCs. Gains of DNA were found to cluster in chromosome arms 1p, 1q, 7q, 15q, 16p, 17q, and 20q and losses of DNA at 3p, 4q, 9p, and 11q in both lesion types. Also, chromosomal alterations responsible for HCC progression and metastasis were found to be located on chromosome arms 4q, 6q, 8p, 13q, 16q, and 17p.14 Thus, the data from transgenic mice and human HCC together with those obtained by Aleksic et al. strongly support the hypothesis of the need of specific genomic alterations "dictating," more than simply accompanying, the histopathological and molecular changes along hepatocarcinogenesis. The authors' findings also have important clinical implications. Indeed, the data from Aleksic et al. further substantiate the unfavorable prognostic role of genomic instability in HCC. In this regard, it is worthwhile noting that gene expression patterns from DEN-initiated/PB-fed mice and c-Myc/TGF-α transgenic mice, both exhibiting elevated genomic instability,5, 7, 12 were highly similar to those of human HCCs characterized by a short survival of the patients.15 The latter human HCC subgroup also displays elevated genomic instability, thus linking a specific gene signature to genomic instability and poor prognosis. A chromosomal instability gene signature predicting the patient's outcome has been previously described in a vast collection of lung, breast, and brain tumors.16 Noticeably, among the genes that were positively correlated with genomic instability was the forkhead box M1b (FoxM1b) transcription factor and its target genes CYCLIN B1 and B2, CDC2, NEK2, KIF20A, TOP2A, CDC25B, AURORA KINASE A, and AURORA KINASE B.16 Given that FoxM1b is overexpressed in HCC and directly correlates with patient survival,17 it would be significant to determine whether the same chromosomal instability gene signature predicts the prognosis of HCC patients as well. Also, since preliminary data show that FoxM1b can be inhibited pharmacologically,18 it would be of high importance to assess whether HCC with elevated genomic instability would benefit from therapies aimed at suppressing FoxM1b expression. Aleksic et al. also investigated the rate of mutations and nuclear accumulation of β-catenin (as a sign of its activation) in DEN-initiated/PB-fed mice. Unexpectedly, mutations and activation of β-catenin occurred at late stage of tumor progression and were preceded by chromosomal instability in DEN-initiated/PB-fed mice. Thus, mutations and activation of the Wnt/β-catenin pathway are not involved in tumor initiation in this model of hepatocarcinogenesis. Furthermore, these findings add complexity to the previous assumption5, 6 that genomic instability and activation of β-catenin characterize two distinct and mutually exclusive groups of liver tumors. Although numerous studies, including the one by Aleksic et al., significantly improved our knowledge of the extent and significance of genomic instability in hepatocarcinogenesis, many questions on this topic remain unanswered. Indeed, while mutations of DNA repair genes are responsible for the generation of genomic instability in hereditary cancers, the molecular bases of genomic instability in sporadic tumors (including HCC) are still poorly delineated.1 Telomere shortening, changes in global DNA methylation status, oncogene-induced DNA replication stress, generation of reactive oxidative, and nitroxidative species by the tumor microenvironment and the viral etiological factors as well as inactivation of detoxification systems have been proposed as the "primum movens" of genomic instability in HCC.19, 20 It is plausible to believe that many of these mechanisms act in combination to disrupt the hepatocytes genome integrity. Additional investigations are required to better elucidate the interaction of the aforementioned mechanisms in liver carcinogenesis. Also, systematic in vivo and in vitro studies are needed to define the oncogenic and oncosuppressive roles of the genes located in the chromosomal loci affected by genomic alterations in order to identify the key genes responsible for liver cancer development and progression.