HEPATOBILIARY MALIGNANCIES Carcinogen-Induced Hepatic Tumors in KLF61/2 Mice Recapitulate Aggressive Human Hepatocellular Carcinoma Associated with p53 Pathway Deregulation Mirko Tarocchi,1,2 Rebekka Hannivoort,1,3 Yujin Hoshida,4 Ursula E. Lee,1 Diana Vetter,1 Goutham Narla,5,6 Augusto Villanueva,7,8 Moshe Oren,8 Josep M. Llovet,1,7,9 and Scott L. Friedman1 Inactivation of KLF6 is common in hepatocellular carcinoma (HCC) associated with hepa- titis C virus (HCV) infection, thereby abrogating its normal antiproliferative activity in liver cells. The aim of the study was to evaluate the impact of KLF6 depletion on human HCC and experimental hepatocarcinogenesis in vivo. In patients with surgically resected HCC, reduced tumor expression of KLF6 was associated with decreased survival. Consistent with its role as a tumor suppressor, KLF61/2 mice developed significantly more tumors in response to the chemical carcinogen diethyl nitrosamine (DEN) than wild-type animals. Gene expression signatures in both surrounding tissue and tumors of KLF61/2 mice closely recapitulated those associated with aggressive human HCCs. Expression microarray profiling also revealed an increase in Mdm2 mRNA in tumors from KLF61/2 compared with KLF61/1 mice, which was validated by way of quantitative real-time polymerase chain reaction and western blot analysis in both human HCC and DEN-induced murine tumors. Moreover, chromatin immunoprecipitation and cotransfection assays established the P2 intronic promoter of Mdm2 as a bona fide transcriptional target repressed by KLF6. Whereas KLF6 overexpression in HCC cell lines and primary hepatocytes led to reduced MDM2 levels and increased p53 protein and transcriptional activity, reduction in KLF6 by small interfering RNA led to increased MDM2 and reduced p53. Conclusion: Our findings indicate that KLF6 deficiency contributes significantly to the carcinogenic milieu in human and murine HCC and uncover a novel tumor suppressor activity of KLF6 in HCC by linking its transcriptional repression of Mdm2 to stabilizing p53. (HEPATOLOGY 2011;54:522-531) epatocellular carcinoma (HCC) has a poor preneoplastic lesions and their progression to HCC in prognosis and is the third leading cause of patients with chronic liver disease reflect the conver- Hcancer mortality worldwide. Development of gence of genetic and epigenetic defects that provoke Abbreviations: cDNA, complementary DNA; DEN, diethyl nitrosamine; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; mRNA, messenger RNA; qRT-PCR, quantitative real-time polymerase chain reaction. From the 1Division of Liver Diseases, Department of Medicine Mount Sinai School of Medicine, New York, NY; the 2Department of Clinical Pathophysiology/ Gastroenterology Unit, University of Florence, Florence, Italy; the 3Department of Gastroenterology and Hepatology, University of Groningen, Groningen, Netherlands; the 4Cancer Program, Broad Institute of Massachusetts Institute of Technology and Harvard University, Cambridge, MA; the 5Division of Hematology/Oncology, Department of Medicine, and the 6Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York, NY; the 7HCC Translational Research Laboratory, Barcelona-Clinic Liver Cancer Group, Liver Unit, Institut d’Investigacions Biomediques August Pi i Sunyer (IDIBAPS), Liver Unit, Hospital Clinic, Barcelona, Catalonia, Spain; 8Department of Molecular Cell Biology, The Weizmann Institute, Rehovot, Israel; and the 9Institucio´ Catalana de Recerca i Estudis Avanc¸ats (ICREA), Barcelona, Catalonia, Spain. Received December 15, 2010; accepted April 25, 2011. Supported by the National Institutes of Health (Grants RO1DK37340 and RO1DK56621 to S. L. F., Grant R01DK076986 to J. M. L.). R. H. was funded by the Graduate School for Drug Exploration and the Stichting Nicolaas Muleriusfonds, Groningen, Netherlands. A. V. is a recipient of a Sheila Sherlock fellowship from the European Association for the Study of the Liver. D. V. is a recipient of a research fellowship from the Swiss National Fund. G. N. is a recipient of the Howard Hughes Medical Institute Physician-Scientist Early Career Award. Address reprint requests to: Scott L. Friedman, M.D., Division of Liver Diseases, Box 1123, Mount Sinai School of Medicine, 1425 Madison Ave., Room 11-70C, New York, NY 10029-6574. E-mail: [email protected]; fax: 212-849-2574. Copyright VC 2011 by the American Association for the Study of Liver Diseases. View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.24413 Potential conflict of interest: Nothing to report. Additional Supporting Information may be found in the online version of this article. 522 HEPATOLOGY, Vol. 54, No. 2, 2011 TAROCCHI ET AL. 523 dysregulation of pathways controlling cell cycle, tissue Materials and Methods repair, and regeneration. Loss of heterozygosity of tu- mor suppressor genes occurs commonly in HCC, but Correlation of KLF6 Messenger RNA Expression no single tumor suppressor inactivation predominates. with Survival and Recurrence of Human HCC. We For example, a loss of heterozygosity of p53 has been analyzed KLF6 messenger (mRNA) expression in 149 reported in only 25% of HCCs. For virtually all tu- hepatitis C virus (HCV)-infected human samples rep- mor suppressors, the presence of haploinsufficiency resenting the full pathological spectrum from normal leads to an increased frequency of tumors in experi- to advanced HCC based on previously established cri- mental models.1 teria: human samples were obtained with full institu- Inactivation of the KLF6 tumor suppressor has been tional review board consent as previously detailed.14,15 implicated in several human cancers, including HCC.2- We next examined the impact of KLF6 mRNA 5 KLF6 is a member of the Kru¨ppel-like C2H2 zinc expression levels on survival and recurrence in 56 finger family, which regulate cell cycle, signal transduc- patients with HCV-associated HCC by distinguishing tion, and differentiation. KLFs, in particular KLF6, can between those tumors with very low expression serve as either transactivators or transrepressors, depend- (expression <10% that of normal livers) and low ing on the cellular or developmental context.6 KLF6 expression (>10% but <20% that of normal livers). We mediates growth suppression through p53-independent chose specific, stringent mRNA cutoffs based on a simi- p21 transactivation,2 sequestration of cyclin D1,7 and larapproachinanearlierstudy16 for two reasons: (1) to inhibition of the c-jun proto-oncogene.8 be sufficiently low to ensure biological relevance, because HCC can harbor a range of genomic alterations and so- KLF6 expression in human HCC is extremely low, and matic mutations. In a minority of HCCs, this includes (2) to preserve a sufficient number of patients in each structural defects in the p53 tumor suppressor,9 anuclear group to allow meaningful comparisons (n ¼ 16, 30% phosphoprotein that regulates proliferation, maintenance of the total 56 patients analyzed for clinical correlations). of genomic stability, differentiation, apoptosis, and micro- Clinical characteristics of patients included in the out- RNA processing. However, other pathways affecting p53 come analysis are shown in Supporting Table 1. activity may be implicated. A major pathway regulating Diethyl Nitrosamine Model of Experimental Hep- p53 homeostasis is its interaction with the E3 ubiquitin atocarcinogenesis. Male KLF6þ/À mice17 were bred ligase MDM2/HDM2 (herein referred to by the mouse with wild-type C57BL/6 to generate mixed litters of homologue, MDM2). MDM2 directly binds p53, block- KLF6þ/À and KLF6þ/þ animals. At 2 weeks of age, ing the p53 transactivation domain and promoting its deg- mice were injected intraperitoneally with either a single radation.10 Conversely, p53 enhances Mdm2 transcription dose of diethyl nitrosamine (DEN) (5 lg/g in 100 lL through its interaction with a pair of tandem p53-binding of saline) or vehicle alone. Mice were maintained on sites in the P2 intronic promoter of the Mdm2 gene.11 standard chow and then sacrificed following intraperi- These interactions comprise the autoregulatory feedback toneal injection of Avertin (250 mg/kg) 3, 6, or 9 loop controlling the steady state level and transcriptional months later. At the time of sacrifice, the animals were function of p53 protein and the subsequent expression of weighed, and blood and liver samples were harvested the Mdm2 gene12; the amplification or enhanced transla- for analysis. tion of Mdm2 confers transforming activity by inappropri- Tumor Quantification. Macroscopic lesions were ately hastening p53 degradation. Indeed, the MDM2 gene measured on the surface of the liver with a caliper at is an oncogene frequently amplified and overexpressed in the time of sacrifice; the number and size of tumors human tumors.13 were then quantified. Hepatic tissues were fixed in In characterizing the potential role of KLF6 in 10% paraformaldehyde and embedded in paraffin: human HCC, our studies in a mouse model of the liver sections of 5 lm were obtained from four differ- disease uncovered an inverse correlation between KLF6 ent regions of the left lobe (at least 2 mm apart), and Mdm2 mRNA expression, leading us to examine stained with hematoxylin and eosin, and the surface whether a functional interaction exists between these area was quantified (BIOQUANT NOVA PRIME two proteins. Moreover, whereas previous studies have Measurement Software). indicated that KLF6 tumor suppressor activities are Quantitative