Tumor suppressors in hepatocellular carcinoma: Many are called, but few are chosen
Article Outline
Recent epidemiological data have demonstrated that liver cancer incidence is continuously rising and will continue to do so for more than one decade, not only in developing countries but also in America and Europe [1], [2]. Moreover, hepatocellular carcinoma (HCC) is frequently diagnosed when advanced stage of the disease precludes local ablative or surgical interventions that could improve patient outcome, and HCC remains refractory to current chemotherapeutic regimens [3]. In this context, advances in our understanding of the molecular basis of HCC are needed to promote the clinical development of early tumor markers and novel targeted agents with improved therapeutic efficiency [4], [5]. This challenge is complicated by the tumor background: chronic liver disease giving rise to frequent tumor recurrence, these therapies should be associated to treatments leading to improve the hepatic functions.
For HCC as for other human cancers, the recent years have seen an explosion of data providing global insights into genetic alterations and molecular profiles in large sets of tumors. Most of these studies, showing a large variety of genetic alterations and striking heterogeneity of gene expression profiles between individual HCC cases, have supported the notion that HCC ranks probably among the most complex and heterogeneous types of human solid tumors [6], [7], [8]. This idea also fits with the multiple etiologies of HCC and the long period of chronic inflammatory disease that fosters the accumulation of genetic and epigenetic defects. As a consequence of the activation of cellular oncogenes or the inactivation of tumor suppressor genes, deregulation of various signaling pathways has been reported in HCC subsets, such as Wnt/β-catenin signaling, the p14ARF/p53, p16INK4A/RB, TGF-β, and PTEN/Akt pathways (reviewed in [7]). Additionally, altered expression of growth factors such as HGF, IGFs, Amphiregulin and their receptors, as well as genes involved in angiogenesis might participate in the development and progression of HCC (reviewed in [9]).
Familial cancer genes with high-penetrance mutations have not been identified so far in HCC, and different strategies have been developed to search for tumor suppressor genes implicated in liver tumorigenesis. Besides mutational screening of selected candidates, systematic scan of loss of heterozygosity (LOH) has been extensively used to define major chromosomal aberrations and minimally deleted regions for cloning candidate suppressor genes. However, although high LOH rates were found at various chromosomal loci, this strategy did not yield conclusive data in HCC and the number of candidate genes harboring loss-of-function mutations has remained low. Unambiguous data in a significant percentage of tumors were obtained for two genes: TP53 (17q13.1) for which mutations that compromise p53 function were found in about 25% of primary HCCs, and the AXIN1 gene (16p13.3) in 7–10% of cases, leading to aberrant activation of Wnt signaling and deserving also probably other functions [10]. In this context, it is worth noticing that the finding of frequent LOH at chromosome 12q24.2 in hepatocellular adenomas has led to identify mutations and bi-allelic inactivation of the HNF1-α gene, linking MODY 3 diabetes to benign liver tumors, but also occasionally to HCC [11]. By contrast, mutations of RB, CDKN2A, PTEN or CDH1 were found only at rare occasions, and epigenetic silencing mechanisms such as the methylation of promoter sites may be responsible for inhibition of tumor suppressor functions, as also recently found for activation of the Ras and Jak/Stat pathways in HCC [12]. Other valuable attempts using homozygous deletion scanning in hepatobiliary tumor cell lines have uncovered new candidates that included NF2, Bax, FHIT, LKB1 and LRP1B, albeit with low frequency (2–4% cases) [13]. It is also interesting to reconsider the search for cancer-related genes in hepatitis B virus (HBV)-related HCC by large scale analysis of HBV integration sites, which recently provided evidence for preferential viral integration into transcribed regions and for recurrent insertion sites in the human telomerase (hTERT) and mixed lineage leukemia (MLL) genes [14]. It may be anticipated that new tools provided by sequencing of the human genome and high-throughput screening of DNA and RNA alterations in HCC will permit a comprehensive view of the signaling networks operating in liver cell transformation.
In this issue of the Journal, Kremer-Tal et al. [15] examined the functional deregulation of the potential tumor suppressor Krüppel-like factor 6 (KLF6) at different steps of liver tumorigenesis associated with viral infections. This work follows several reports from the group of Scott Friedman showing that KLF6 is deregulated in multiple cancers, notably in prostate and liver cancers, by LOH and/or inactivating somatic mutations [16], [17]. Controversial issues have been raised, since KLF6 maps to the telomere of chromosome 10p where LOH has been reported infrequently in HCC, and somatic or germline mutations of KLF6 were not detected in large HCC series from Europe and Asia [18], [19]. Notwithstanding, KLF6 activities in inhibition of cell proliferation and induction of apoptosis, as evidenced in earlier functional studies [16], [20], should encourage further investigations in the liver cancer context.
KLF6 (formerly called Zf9 or CPBP) is a ubiquitously expressed zinc finger transcription factor. Interestingly, KLF6 was originally cloned from rat stellate cells activated in vivo by experimental acute liver injury and it has been shown that KLF6 expression and its nuclear accumulation are strongly upregulated in activated stellate cells [21]. Furthermore, lipid peroxidation, stellate cell activation and inflammation in a rat model of steatosis have been associated with concomitant increase of KLF6 expression and TGF-β1 production [22]. The ability of KLF6 to activate transcription of endoglin and related members of the TGF-β signaling complex was reported in independent studies, in which KLF6 was also implicated in vascular repair [23].
KLF6 belongs to the Sp/KLF family of Krüppel-like transcription factors that contains 25 identified members so far. These factors bind to GC-rich DNA sequences in target gene promoters and regulate the expression of a large number of genes involved in crucial cellular functions such as differentiation, proliferation and apoptosis. First described as a transcriptional activator, KLF6 has been shown to stimulate transcription from the SV40 promoter, and from various cellular gene promoters, including p21 (WAF1/CIP1), the insulin-like growth factor I receptor (IGF-1R), E-cadherin, human inducible nitric-oxide synthase (hINOS), collagen α1, and urokinase in vascular endothelial cells [16], [21], [24], [25], [26]. These studies point to the importance of the cellular context and the cooperative effect of Sp1 for KLF6 transcriptional activity. Conversely, a transcriptional repressor function has also been documented for KLF6 in more recent reports providing evidence that the genes encoding delta-like homolog-1 (DLK1) and matrix metalloproteinase-9 (MMP9) are downregulated by KLF6 [27], [28]. It has been shown that KLF6 forms with Sp2 a repressor complex that maintains the MMP9 gene in a silenced state, thereby regulating endothelial cell motility, which plays an important role in vascular remodeling and tissue repair. Furthermore, it has been reported that KLF6 overexpression can downregulate c-Jun-dependent transcription [29]. Thus, variations in KLF6 expression may profoundly affect a variety of cellular processes related to cancer. Another intriguing aspect of KLF6 biology is the production of alternatively spliced variants that was found to be associated with a DNA polymorphism in patients with increased prostate cancer risk [30]. Interestingly, whereas the wild-type KLF6 displays growth suppressor functions, the KLF6 SV1 isoform has gained proliferation-promoting activity, and its targeted inhibition is able to suppress the growth and spread of prostate tumor cells, showing that it plays a crucial role in prostate cancer [31].
The paper by Kremer-Tal et al. [15] shows that the mRNA levels of KLF6 are markedly reduced in a majority of HBV- and HCV-related HCCs compared to surrounding tissues. Moreover, close inspection of liver samples at different stages of HCV-related tumorigenesis indicated that downregulation of KLF6 expression might be implicated both at early, preneoplastic steps and at late stages of tumor progression, whereas the contribution of the splice variant KLF6 SV1 did not appear to be predominant. Considering KLF6 activities as a potential tumor-suppressor gene, functional assays conducted in HepG2 cells provided evidence that KLF6 overexpression can reduce cellular growth, antagonize Wnt/β-catenin signaling, and activate several markers of hepatic differentiation.
In conclusion, these studies describe a novel, interesting player in liver cancer and give rise to many additional questions on the downstream targets and the oncogenic network that implicate KLF6 in liver tumorigenesis. In particular, potential interactions between KLF6 and p53, and between KLF6 and the TGF-β pathway, warrant further investigations. The role of p21WAF1 has not been extensively evaluated in HCC and it might be determinant in the regulation of cell cycle and liver cell proliferation [32]. Finally, because KLF6 shares some common properties with other members of the Sp/KLF family, compound mouse models will be instrumental in defining the specific contribution of KLF6 in liver tumorigenesis. Most importantly, better understanding of the molecular pathogenesis of HCC has considerable implication for cancer prevention and therapy.
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PII: S0168-8278(07)00041-4
doi:10.1016/j.jhep.2007.01.005
© 2007 European Association for the Study of the Liver. Published by Elsevier Inc. All rights reserved.
