Tumor suppressors in hepatocellular carcinoma: Many are called, but few are chosen
Authors: Marie Annick Buendia
Journal: Journal of Hepatology (2007)

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 [1El-Serag H.B. Hepatocellular carcinoma: recent trends in the United States.Gastroenterology. 2004; 127: S27-S34Abstract Full Text Full Text PDF PubMed Scopus (898) Google Scholar, 2Parkin D.M. The global health burden of infection-associated cancers in the year 2002.Int J Cancer. 2006; 118: 3030-3044Crossref PubMed Scopus (2308) Google Scholar]. 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]Bruix J. Boix L. Sala M. Llovet J.M. Focus on hepatocellular carcinoma.Cancer Cell. 2004; 5: 215-219Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar]. 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 [4Bruix J. Hessheimer A.J. Forner A. Boix L. Vilana R. Llovet J.M. New aspects of diagnosis and therapy of hepatocellular carcinoma.Oncogene. 2006; 25: 3848-3856Crossref PubMed Scopus (145) Google Scholar, 5Avila M.A. Berasain C. Sangro B. Prieto J. New therapies for hepatocellular carcinoma.Oncogene. 2006; 25: 3866-3884Crossref PubMed Scopus (356) Google Scholar]. 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 [6Buendia M.A. Genetics of hepatocellular carcinoma.Semin Cancer Biol. 2000; 10: 185-200Crossref PubMed Scopus (286) Google Scholar, 7Laurent-Puig P. Zucman-Rossi J. Genetics of hepatocellular tumors.Oncogene. 2006; 25: 3778-3786Crossref PubMed Scopus (306) Google Scholar, 8Lee J.S. Thorgeirsson S.S. Comparative and integrative functional genomics of HCC.Oncogene. 2006; 25: 3801-3809Crossref PubMed Scopus (101) Google Scholar]. 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]Laurent-Puig P. Zucman-Rossi J. Genetics of hepatocellular tumors.Oncogene. 2006; 25: 3778-3786Crossref PubMed Scopus (306) Google Scholar]). 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]Breuhahn K. Longerich T. Schirmacher P. Dysregulation of growth factor signaling in human hepatocellular carcinoma.Oncogene. 2006; 25: 3787-3800Crossref PubMed Scopus (348) Google Scholar]). 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]Zucman-Rossi J, Benhamouche S, Godard C, Boyault S, Grimber G, Balabaud C, et al. Differential effects of inactivated Axin1 and activated beta-catenin mutations in human hepatocellular carcinomas. Oncogene 2007, in press.Google Scholar]. 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]Bluteau O. Jeannot E. Bioulac-Sage P. Marques J.M. Blanc J.F. Bui H. et al.Bi-allelic inactivation of TCF1 in hepatic adenomas.Nat Genet. 2002; 32: 312-315Crossref PubMed Scopus (295) Google Scholar]. 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]Calvisi D.F. Ladu S. Gorden A. Farina M. Conner E.A. Lee J.S. et al.Ubiquitous activation of Ras and Jak/Stat pathways in human HCC.Gastroenterology. 2006; 130: 1117-1128Abstract Full Text Full Text PDF PubMed Scopus (607) Google Scholar]. 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]Pineau P. Marchio A. Nagamori S. Seki S. Tiollais P. Dejean A. Homozygous deletion scanning in hepatobiliary tumor cell lines reveals alternative pathways for liver carcinogenesis.Hepatology. 2003; 37: 852-861Crossref PubMed Scopus (48) Google Scholar]. 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]Murakami Y. Saigo K. Takashima H. Minami M. Okanoue T. Brechot C. et al.Large scaled analysis of hepatitis B virus (HBV) DNA integration in HBV related hepatocellular carcinomas.Gut. 2005; 54: 1162-1168Crossref PubMed Scopus (272) Google Scholar]. 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]Kremer-Tal S. Narla G. Chen Y. Hod E. DiFeo A. Yea S. et al.Downregulation of KLF6 is an early event in hepatocarcinogenesis, and stimulates proliferation while reducing differentiation.J Hepatol. 2007; 46: 645-654Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar] 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 [16Narla G. Heath K.E. Reeves H.L. Li D. Giono L.E. Kimmelman A.C. et al.KLF6, a candidate tumor suppressor gene mutated in prostate cancer.Science. 2001; 294: 2563-2566Crossref PubMed Scopus (386) Google Scholar, 17Kremer-Tal S. Reeves H.L. Narla G. Thung S.N. Schwartz M. Difeo A. et al.Frequent inactivation of the tumor suppressor Kruppel-like factor 6 (KLF6) in hepatocellular carcinoma.Hepatology. 2004; 40: 1047-1052Crossref PubMed Scopus (123) Google Scholar]. 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 [18Boyault S. Herault A. Balabaud C. Zucman-Rossi J. Absence of KLF6 gene mutation in 71 hepatocellular carcinomas.Hepatology. 2005; 41 (author reply 682–683): 681-682Crossref PubMed Scopus (29) Google Scholar, 19Song J. Kim C.J. Cho Y.G. Kim S.Y. Nam S.W. Lee S.H. et al.Genetic and epigenetic alterations of the KLF6 gene in hepatocellular carcinoma.J Gastroenterol Hepatol. 2006; 21: 1286-1289Crossref PubMed Scopus (36) Google Scholar]. Notwithstanding, KLF6 activities in inhibition of cell proliferation and induction of apoptosis, as evidenced in earlier functional studies [16Narla G. Heath K.E. Reeves H.L. Li D. Giono L.E. Kimmelman A.C. et al.KLF6, a candidate tumor suppressor gene mutated in prostate cancer.Science. 2001; 294: 2563-2566Crossref PubMed Scopus (386) Google Scholar, 20Ito G. Uchiyama M. Kondo M. Mori S. Usami N. Maeda O. et al.Kruppel-like factor 6 is frequently down-regulated and induces apoptosis in non-small cell lung cancer cells.Cancer Res. 2004; 64: 3838-3843Crossref PubMed Scopus (139) Google Scholar], 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]Ratziu V. Lalazar A. Wong L. Dang Q. Collins C. Shaulian E. et al.Zf9, a Kruppel-like transcription factor up-regulated in vivo during early hepatic fibrosis.Proc Natl Acad Sci USA. 1998; 95: 9500-9505Crossref PubMed Scopus (238) Google Scholar]. 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]Starkel P. Sempoux C. Leclercq I. Herin M. Deby C. Desager J.P. et al.Oxidative stress, KLF6 and transforming growth factor-beta up-regulation differentiate non-alcoholic steatohepatitis progressing to fibrosis from uncomplicated steatosis in rats.J Hepatol. 2003; 39: 538-546Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar]. 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]Botella L.M. Sanchez-Elsner T. Sanz-Rodriguez F. Kojima S. Shimada J. Guerrero-Esteo M. et al.Transcriptional activation of endoglin and transforming growth factor-beta signaling components by cooperative interaction between Sp1 and KLF6: their potential role in the response to vascular injury.Blood. 2002; 100: 4001-4010Crossref PubMed Scopus (155) Google Scholar]. 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 [16Narla G. Heath K.E. Reeves H.L. Li D. Giono L.E. Kimmelman A.C. et al.KLF6, a candidate tumor suppressor gene mutated in prostate cancer.Science. 2001; 294: 2563-2566Crossref PubMed Scopus (386) Google Scholar, 21Ratziu V. Lalazar A. Wong L. Dang Q. Collins C. Shaulian E. et al.Zf9, a Kruppel-like transcription factor up-regulated in vivo during early hepatic fibrosis.Proc Natl Acad Sci USA. 1998; 95: 9500-9505Crossref PubMed Scopus (238) Google Scholar, 24Kojima S. Hayashi S. Shimokado K. Suzuki Y. Shimada J. Crippa M.P. et al.Transcriptional activation of urokinase by the Kruppel-like factor Zf9/COPEB activates latent TGF-beta1 in vascular endothelial cells.Blood. 2000; 95: 1309-1316PubMed Google Scholar, 25Warke V.G. Nambiar M.P. Krishnan S. Tenbrock K. Geller D.A. Koritschoner N.P. et al.Transcriptional activation of the human inducible nitric-oxide synthase promoter by Kruppel-like factor 6.J Biol Chem. 2003; 278: 14812-14819Crossref PubMed Scopus (65) Google Scholar, 26DiFeo A. Narla G. Camacho-Vanegas O. Nishio H. Rose S.L. Buller R.E. et al.E-cadherin is a novel transcriptional target of the KLF6 tumor suppressor.Oncogene. 2006; 25: 6026-6031Crossref PubMed Scopus (68) Google Scholar]. 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 [27Li D. Yea S. Li S. Chen Z. Narla G. Banck M. et al.Kruppel-like factor-6 promotes preadipocyte differentiation through histone deacetylase 3-dependent repression of DLK1.J Biol Chem. 2005; 280: 26941-26952Crossref PubMed Scopus (144) Google Scholar, 28Das A. Fernandez-Zapico M.E. Cao S. Yao J. Fiorucci S. Hebbel R.P. et al.Disruption of an SP2/KLF6 repression complex by SHP is required for Farnesoid X receptor-induced endothelial cell migration.J Biol Chem. 2006; Google Scholar]. 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]Slavin D.A. Koritschoner N.P. Prieto C.C. Lopez-Diaz F.J. Chatton B. Bocco J.L. A new role for the Kruppel-like transcription factor KLF6 as an inhibitor of c-Jun proto-oncoprotein function.Oncogene. 2004; 23: 8196-8205Crossref PubMed Scopus (74) Google Scholar]. 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]Narla G. Difeo A. Reeves H.L. Schaid D.J. Hirshfeld J. Hod E. et al.A germline DNA polymorphism enhances alternative splicing of the KLF6 tumor suppressor gene and is associated with increased prostate cancer risk.Cancer Res. 2005; 65: 1213-1222Crossref PubMed Scopus (192) Google Scholar]. 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]Narla G. DiFeo A. Yao S. Banno A. Hod E. Reeves H.L. et al.Targeted inhibition of the KLF6 splice variant, KLF6 SV1, suppresses prostate cancer cell growth and spread.Cancer Res. 2005; 65: 5761-5768Crossref PubMed Scopus (137) Google Scholar]. The paper by Kremer-Tal et al. [[15]Kremer-Tal S. Narla G. Chen Y. Hod E. DiFeo A. Yea S. et al.Downregulation of KLF6 is an early event in hepatocarcinogenesis, and stimulates proliferation while reducing differentiation.J Hepatol. 2007; 46: 645-654Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar] 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]Martin-Caballero J. Flores J.M. Garcia-Palencia P. Serrano M. Tumor susceptibility of p21(Waf1/Cip1)-deficient mice.Cancer Res. 2001; 61: 6234-6238PubMed Google Scholar]. 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.