HCC 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
Department of Molecular Cell Biology, The Weizmann Institute, Rehovot, Israel
Division of Liver Diseases, Department of Medicine Mount Sinai School of Medicine, New York, NY
HCC 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
Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Catalonia, Spain
Potential conflict of interest: Nothing to report.
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.
Inactivation of KLF6 is common in hepatocellular carcinoma (HCC) associated with hepatitis 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, KLF6+/− 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 KLF6+/− mice closely recapitulated those associated with aggressive human HCCs. Expression microarray profiling also revealed an increase in Mdm2 mRNA in tumors from KLF6+/− compared with KLF6+/+ 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;)
Hepatocellular carcinoma (HCC) has a poor prognosis and is the third leading cause of cancer mortality worldwide. Development of preneoplastic lesions and their progression to HCC in patients with chronic liver disease reflect the convergence of genetic and epigenetic defects that provoke dysregulation of pathways controlling cell cycle, tissue repair, and regeneration. Loss of heterozygosity of tumor suppressor genes occurs commonly in HCC, but no single tumor suppressor inactivation predominates. For example, a loss of heterozygosity of p53 has been reported in only ≈25% of HCCs. For virtually all tumor suppressors, the presence of haploinsufficiency leads to an increased frequency of tumors in experimental models.1
Inactivation of the KLF6 tumor suppressor has been implicated in several human cancers, including HCC.2-5 KLF6 is a member of the Krüppel-like C2H2 zinc finger family, which regulate cell cycle, signal transduction, and differentiation. KLFs, in particular KLF6, can serve as either transactivators or transrepressors, depending on the cellular or developmental context.6 KLF6 mediates growth suppression through p53-independent p21 transactivation,2 sequestration of cyclin D1,7 and inhibition of the c-jun proto-oncogene.8
HCC can harbor a range of genomic alterations and somatic mutations. In a minority of HCCs, this includes structural defects in the p53 tumor suppressor,9 a nuclear phosphoprotein that regulates proliferation, maintenance of genomic stability, differentiation, apoptosis, and microRNA processing. However, other pathways affecting p53 activity may be implicated. A major pathway regulating p53 homeostasis is its interaction with the E3 ubiquitin ligase MDM2/HDM2 (herein referred to by the mouse homologue, MDM2). MDM2 directly binds p53, blocking the p53 transactivation domain and promoting its degradation.10 Conversely, p53 enhances Mdm2 transcription through its interaction with a pair of tandem p53-binding sites in the P2 intronic promoter of the Mdm2 gene.11 These interactions comprise the autoregulatory feedback loop controlling the steady state level and transcriptional function of p53 protein and the subsequent expression of the Mdm2 gene12; the amplification or enhanced translation of Mdm2 confers transforming activity by inappropriately hastening p53 degradation. Indeed, the MDM2 gene is an oncogene frequently amplified and overexpressed in human tumors.13
In characterizing the potential role of KLF6 in human HCC, our studies in a mouse model of the disease uncovered an inverse correlation between KLF6 and Mdm2 mRNA expression, leading us to examine whether a functional interaction exists between these two proteins. Moreover, whereas previous studies have indicated that KLF6 tumor suppressor activities are p53-independent,2 these new findings now directly link these two tumor suppressor pathways by demonstrating that KLF6 normally represses Mdm2 transcription, thereby stabilizing p53.
Correlation of KLF6 Messenger RNA Expression with Survival and Recurrence of Human HCC.
We analyzed KLF6 messenger (mRNA) expression in 149 hepatitis C virus (HCV)-infected human samples representing the full pathological spectrum from normal to advanced HCC based on previously established criteria: human samples were obtained with full institutional review board consent as previously detailed.14, 15
We next examined the impact of KLF6 mRNA expression levels on survival and recurrence in 56 patients with HCV-associated HCC by distinguishing between those tumors with very low expression (expression <10% that of normal livers) and low expression (>10% but <20% that of normal livers). We chose specific, stringent mRNA cutoffs based on a similar approach in an earlier study16 for two reasons: (1) to be sufficiently low to ensure biological relevance, because KLF6 expression in human HCC is extremely low, and (2) to preserve a sufficient number of patients in each group to allow meaningful comparisons (n = 16, ≈30% of the total 56 patients analyzed for clinical correlations). Clinical characteristics of patients included in the outcome analysis are shown in Supporting Table 1.
Diethyl Nitrosamine Model of Experimental Hepatocarcinogenesis.
Male KLF6+/− mice17 were bred with wild-type C57BL/6 to generate mixed litters of KLF6+/− and KLF6+/+ animals. At 2 weeks of age, mice were injected intraperitoneally with either a single dose of diethyl nitrosamine (DEN) (5 μg/g in 100 μL of saline) or vehicle alone. Mice were maintained on standard chow and then sacrificed following intraperitoneal injection of Avertin (250 mg/kg) 3, 6, or 9 months later. At the time of sacrifice, the animals were weighed, and blood and liver samples were harvested for analysis.
Macroscopic lesions were measured on the surface of the liver with a caliper at the time of sacrifice; the number and size of tumors were then quantified. Hepatic tissues were fixed in 10% paraformaldehyde and embedded in paraffin: liver sections of 5 μm were obtained from four different regions of the left lobe (at least 2 mm apart), stained with hematoxylin and eosin, and the surface area was quantified (BIOQUANT NOVA PRIME Measurement Software).
Quantitative Real-Time Polymerase Chain Reaction.
Total RNA was extracted using Trizol reagent (Invitrogen), followed by column purification (RNeasy mini kit, Qiagen) and treated with DNAse (Roche). A total of 0.5 μg of RNA was reverse-transcribed per reaction using Sprint RT Complete (Clontech Laboratories). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems). Primer sequences are listed in the Supporting Methods.
Western Blot Analysis.
Liver fragments were lysed in Lysis M buffer (Roche), then sonicated and pelleted. Supernatants were denatured; 40 μg of protein was separated by way of polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes (Invitrogen). Membrane blotting was performed using rabbit polyclonal antibody to KLF6 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology), Calnexin (Abcam), or mouse monoclonal antibody to MDM2 and p53 (Calbiochem) and β-tubulin (Sigma-Aldrich).
Chromatin Immunoprecipitation Assay.
HepG2 chromatin complexes were coimmunoprecipitated with KLF6 or immunoglobulin G control antibodies. The DNA resulting from the precipitation was detected using primers surrounding the putative KLF6-binding site (MDM2-P2) or another upstream region (MDM2-P1) of the Mdm2 promoter (primer sequences available in Supporting Methods).
Cell Culture and Transfection.
HepG2, Huh7, and Hep3B cells were cultured in standard conditions. Primary hepatocytes were isolated, cultured, and transfected as indicated in the Supporting Methods. Transient transfection of cell lines was performed with Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions. Cells were transfected with pCIneo vector, pCIneo-KLF6 vector (containing the full-length human KLF6 cDNA), pSuper-siControl, pSuper-siKLF6,18 pSN3-p53, different mdm2 promoter luciferase reporters (P1, P2, P1+P2),19 or P2-mut mdm2 promoter luciferase reporter (in which two KLF6 binding sites were mutated from CACCC to CACTC using Stratagene Quikchange XL) or cyclin G1-luciferase promoter.20 Renilla luciferase vector (Promega, WI) was used as internal control.
Gene expression analysis was performed using Affymetrix arrays (U133 Human Chip Plus 2.0) according to the manufacturer's instructions (see Supporting Methods). Human and mouse array data were deposited in NCBI's Gene Expression Omnibus (see Supporting Methods).
Data are expressed as the mean ± SEM or SD. Student t test, analysis of variance, Mann-Whitney test, and chi-square test (3-way contingency table) were calculated to compare experimental groups. The probability curves of survival and recurrence were calculated according to the Kaplan-Meier method and compared by way of log-rank test. P < 0.05 was considered statistically significant. Analyses were conducted using the R statistical package (www.r-project.org) and SPSS software (version 14).
Reduced Survival in Patients with HCCs Expressing Very Low Levels of KLF6 mRNA.
We previously reported that KLF6 mRNA expression was decreased in advanced HCC4; however, these findings did not indicate whether reduced KLF6 expression affected clinical outcomes. To address this issue, we correlated KLF6 mRNA with clinical outcomes in a cohort of 56 patients with HCV-related HCC following tumor resection in whom prospective data were available from among a larger cohort of 149 patients (Fig. 1A, Supporting Table 1). We selected a subgroup representing those with very low expression of KLF6 (defined by mRNA levels <10% of normal liver [n = 16]) and compared their clinical outcome to patients with low expression of KLF6 mRNA levels (defined by mRNA levels >10% but <20% of normal liver [n = 40]). As shown in Fig. 1B, very low KLF6 mRNA expression was correlated with reduced survival (P = 0.04) during a median follow-up of 32 months. Additionally, there was a nonsignificant trend toward higher recurrence rates in patients with very low KLF6 mRNA expression (P = 0.07) (Fig. 1C).
Reduced KLF6 mRNA Expression in Livers of KLF6+/− Mice.
Evidence that reduced KLF6 mRNA expression was associated with clinical outcomes in patients established a rationale to assess the impact of reduced KLF6 on tumor formation in mice following carcinogenic stress. Accordingly, male KLF6+/− mice were exposed to a single dose of DEN, a well-validated murine model of chemically induced HCC.21
Untreated KLF6+/− mice had reduced hepatic KLF6 mRNA and protein expression to 39%-65% of KLF6+/+ mice (Supporting Fig. 1). Interestingly, this expression level in KLF6+/− mice was similar to KLF6 mRNA expression in HCV-infected cirrhotic liver (Fig. 1A), further validating this model in assessing the impact of KLF6 loss on carcinogenic propensity in human HCC. Moreover, although KLF6 mRNA levels increased with age after DEN in both KLF6+/+ and KLF6 +/− animals, levels in the KLF6+/− animals remained significantly lower (Fig. 2A).
Increased Liver Size in KLF6+/− Mice After DEN Treatment.
Whereas body weight was not affected by KLF6 heterozygosity, liver weight in KLF6+/− mice after DEN administration increased relative to KLF6+/+ mice (P < 0.05), accounting for a significantly increased ratio of liver to body weight at 9 months (P < 0.05) (Fig. 2B-D).
Increased HCC Development in KLF6 +/− Mice After DEN Treatment.
We next assessed the impact of KLF6 allelic loss on tumor formation after DEN administration, measuring the number and size of tumors, as well as the total surface area of the liver occupied by tumors in DEN-treated animals. No macroscopic tumors or histologic abnormalities were visible at 3 months (Fig. 3A). At 6 months, macroscopic tumors were clearly evident in 20% of KLF6+/− mice, but none were present in KLF6+/+ animals, whereas at 9 months, tumor prevalence was 71% in KLF6+/− mice compared with 40% in KLF6+/+ animals (P < 0.01) (Fig. 3A, Table 1). Interestingly, the number, size, and relative surface area occupied by the tumors were significantly increased in KLF6+/− mice, compared with age- and sex-matched wild-type animals treated with DEN (Fig. 3B-D) (P < 0.05); in KLF6+/− mice, livers were largely replaced by tumors.
Table 1. Prevalence of Tumors in Diethyl Nitrosamine—Treated Mice
Prevalence of Lesions
KLF6+/+ (n = 39)
KLF6+/− (n = 39)
Macroscopic tumors (≥0.1 cm) were enumerated in the livers; 4 sections of the left lobe were analyzed after H&E staining at 20× magnification field to count microscopic lesions. The prevalence of tumors was compared by genotype with a modified chi-square test (3-way contingency table
Increased Liver Injury in KLF6+/− Mice After DEN Treatment.
There was evidence of increased liver injury associated with HCC progression in KLF6+/− mice compared with wild-type animals, as assessed by serum alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase measurements. These increased liver biochemistries were evident at 9 months (P < 0.05), indicating that loss of a KLF6 allele may amplify the inflammatory effect of DEN (Fig. 3E). As expected, the effects of KLF6 allelic loss on tumor propensity were present only in male mice. In contrast, in female KLF6+/− mice treated with DEN, there was no difference in tumor formation versus KLF6+/+ animals even at 12 and 15 months, although overall tumor frequency was significantly lower than in male mice (data not shown).
KLF6 Heterozygosity Generates a Poor-Prognosis Gene Expression Signature in Nonmalignant Livers Following DEN Treatment.
We hypothesized that carcinogenic signaling networks might be amplified in KLF6+/− livers following DEN treatment. To explore this possibility in an unbiased manner, we compared transcriptomic profiling of nontumoral and tumoral tissues between KLF6+/− and KLF6+/+ mice 6 months after DEN treatment, reasoning that changes in gene expression favoring tumorigenesis were likely to be present before HCC developed.
We previously reported a 186-gene expression signature predictive of poor survival following surgical resection of human HCC.22 The signature was identified in cirrhotic liver, not tumor tissues, and is thought to reflect a carcinogenic field effect present in the diseased liver prior to the development of HCC. We hypothesized that KLF6 deficiency might promote a comparably permissive microenvironment in murine liver. Indeed, in mice treated with DEN, loss of a single KLF6 allele generated a gene signature in nonmalignant liver that is highly predictive of poor survival in human HCC, compared with wild-type mice treated with DEN (FDR = 0.05) (Supporting Fig. 2). These findings support the notion that loss of KLF6 favors the emergence of HCC under carcinogenic stress, with molecular defects similar to the findings in human HCC.
HCCs in KLF6+/− Mice Reproduce the Gene Signatures of Highly Aggressive Human HCC.
Recent genomic profiling of human HCC tumor tissues has revealed distinct molecular subclasses of HCC that discriminate tumors with different behavior and outcomes.23 With these classifications in mind, we explored whether features of any of the HCC subclasses were present in the KLF6+/− tumors induced by DEN. Remarkably, signatures independently associated with aggressive human HCC behavior were overexpressed in KLF6+/− tumors compared with wild-type KLF6 tumors, whereas signatures associated with less aggressive tumor behavior were underexpressed (Supporting Fig. 3 and Supporting Table 2).
KLF6 Loss Is Linked to Increased mdm2 Expression and Reduced p53 Activity.
There was evidence of altered expression in KLF6+/− tumors of transcripts related to the cell cycle, the p53 pathway, and inflammation. In particular, Mdm2 mRNA expression was increased, reflecting dysregulation of a major pathway that controls p53 half-life and activity.24 Expression of Mdm2 mRNA at 6 months was increased 72% in KLF6+/− tumor tissue compared with tumors in KLF6+/+ mice (P < 0.01), and increased 40% in KLF6+/− tumors versus surrounding tissue (P = 0.05). We speculated that KLF6 might normally transrepress Mdm2 gene expression, whereas reduced KLF6 expression would derepress Mdm2, leading to increased degradation of p53 and attenuation of its tumor suppressive activity. Indeed, after DEN treatment, Mdm2 mRNA was significantly increased in KLF6+/− nontumoral livers compared with KLF6+/+ livers (Fig. 4A), associated with increased expression of MDM2 protein and reduced expression of p53 (Fig. 4B). The reduction of p53 was associated with down-regulation of p21 (both a p53 and KLF6 target gene), as well as p53-specific target genes PUMA, and cyclin G (Fig. 4C,D). To directly link KLF6 to p53 regulation, primary mouse hepatocytes were transfected with a KLF6 complementary DNA (cDNA), which led to an ≈50% decrease in MDM2 (the 72-kDa band, which derives from the KLF6-responsive Mdm2 transcript) and increased p53 protein levels (Fig. 4E).
Transcriptional Repression of mdm2 by KLF6 Leads to Increased p53.
We examined whether the Hdm2 gene (human homologue of mdm2) is a direct transcriptional target of KLF6 using chromatin immunoprecipitation. Sequence analysis uncovered two potential KLF6 target sites (CACCC Boxes)2 within the Hdm2 P2 promoter (Fig. 5A); on chromatin immunoprecipitation, KLF6 specifically interacted with this region upstream of exon 2. In contrast, no interaction was documented within the P1 promoter region.
To confirm that a reciprocal relationship exists between KLF6 and MDM2, we transfected a fixed amount of wild-type p53 into a p53 null hepatoma cell line (Hep3B) while modulating KLF6 expression; KLF6 overexpression led to decreased Mdm2 promoter activity, whereas KLF6 knockdown had the opposite effect (P < 0.05) (Fig. 5B). Moreover, KLF6's effect on Mdm2 expression was specifically correlated with the interaction of KLF6 with P2 promoter of Mdm2 (Fig. 5C). To examine whether KLF6-mediated repression of Mdm2 affects p53 activity, we cotransfected KLF6 cDNA into three HCC cell lines with a p53-responsive promoter derived from the cyclin G gene.20 As predicted, in HepG2 and Huh7 cells, which have partially functional p53,25 there was marked transactivation of cyclin G luciferase in the presence of KLF6. In contrast, Hep3B cells, which are p53 null, lacked transcriptional activity in response to KLF6 transfection (Fig. 5D).
Finally, because loss of KLF6 occurs in progressive hepatic carcinogenesis, we predicted that there should be a reciprocal relationship between progressively decreased KLF6 expression and rising levels of Mdm2 mRNA expression in human HCC. To test this possibility, we examined microarray data from a well-characterized cohort of HCV patients.15 As predicted, there was a progressive increase in Mdm2 mRNA expression during the progression from normal liver to dysplastic nodules, associated with declining KLF6 mRNA expression (Fig. 6A). Interestingly, Mdm2 mRNA levels did not correlate with survival (data not shown), indicating that the impact of KLF6 loss on survival is not mediated solely by its effects on mdm2 expression. To further evaluate this reciprocal correlation between KLF6 and MDM2, we cotransfected Hep3B with a fixed amount of p53 and KLF6: the overexpression of KLF6 within a physiological range (less than two-fold) was able to reduce Mdm2 mRNA expression levels by ≈50% (Fig. 6B). Although this finding does not establish that KLF6 loss is the only determinant of rising Mdm2 mRNA expression in human hepatocarcinogenesis, it nonetheless indicates that loss contributes to enhanced Mdm2 expression and loss of p53 function.
We have demonstrated that KLF6 deficiency promotes DEN-induced HCC in mice, which closely models gene signatures in both tumors and surrounding tissues that are associated with poor outcomes in human HCC. These findings reinforce the emerging importance of KLF6 as a tumor suppressor in HCC and suggest that KLF6 loss may contribute to a field effect in promoting a carcinogenic milieu during chronic liver injury. The association of KLF6 deficiency with more aggressive human HCC subclasses (S1 and S2)23, 26 implicates this gene in the stepwise malignant transformation and dedifferentiation associated with hepatic tumorigenesis. Moreover, these subclasses are associated with impaired p53 function (Supporting Fig. 3), consistent with the link we uncovered between KLF6 and p53.
Our findings also indicate that KLF6 functions as a tumor suppressor in part through its repression of MDM2, which negatively regulates p53 activity and induces p53 degradation. The data further suggest a potential prognostic link between reduced KLF6 expression in HCC and poor survival, underscoring its functional importance in this neoplasm. Progressive HCC is associated with reduced KLF6 mRNA expression in a large majority of HCCs associated with HCV infection.4 We modeled this condition in mice by analyzing the response of KLF6+/− animals to the carcinogen DEN, confirming a markedly increased tumor burden, and uncovering Mdm2 as a novel target gene of KLF6 using microarray and chromatin immunoprecipitation. Importantly, loss of KLF6 leads to reduced transrepression of Mdm2, thereby promoting loss of p53 through its accelerated degradation. The findings provide a novel pathway by which MDM2 activity is enhanced in human cancer, adding to other mechanisms that increase MDM2 expression, with or without gene amplification.13 Moreover, because KLF6 down-regulation is recognized in a growing number of human cancers, this pathway may contribute to enhanced MDM2 activity in a range of tumors. At least one mechanism by which down-regulation of KLF6 occurs is through MYC amplification27; however, other determinants are also likely. In particular, KLF6 down-regulation through promoter methylation occurs in esophageal cancer cells.28
Reciprocal expression of KLF6 and Mdm2 mRNAs was also demonstrated in human HCV-related HCC, in which there was a progressive increase of mdm2 mRNA that paralleled progressive diminution of KLF6 mRNA. Interestingly, this correlation was strongest in low-grade dysplastic nodules, which are critical premalignant stages from which the tumor phenotype emerges.29 Similarly, in mice the correlation was greatest at 6 months after DEN, before tumor formation was maximal. These findings are clinically important, because HCV-associated tumors arise in a stepwise fashion and almost exclusively develop once advanced fibrosis or cirrhosis is present.30 Additionally, a growing body of evidence implicates chronic inflammation in the pathogenesis of HCC.31-33 In that context, it is noteworthy that KLF6 haploinsufficiency led to increased elevations in enzymes indicative of liver injury, raising the possibility that part of the tumor-promoting effect of KLF6 depletion involves amplified inflammatory signaling.
The maximal impact of Mdm2 derepression through KLF6 deficiency would be expected only if p53 is not mutated. Indeed, inactivating mutations of p53 in HCC are relatively uncommon, and typically occur at late stages of the disease.34, 35 Moreover, the likelihood of p53 abnormalities varies considerably among different HCC etiologies36, 37 and has not been studied extensively in HCV,38 which is the most common etiology of the disease in the Western world and the sole cause among the tumors analyzed in our study. Other mechanisms of HCV pathogenesis have also been linked to p53,38 and it remains possible that KLF6 might also interact with HCV directly. Additionally, because all the HCCs in this study were derived from patients with HCV infection, a link between KLF6 and MDM2 cannot be assumed for other etiologies, in particular hepatitis B and fatty liver, even though the relationship was also documented in DEN-induced neoplasia.
In conclusion, our findings uncover a new pathway of tumor suppression in HCC, in which loss of KLF6 activity leads to increased MDM2 and accelerated degradation of p53. The convergence of these two tumor suppressor pathways underscores the highly interdependent molecular abnormalities that characterize this neoplasm.
We thank Rachel Schwartz and Romina Bromberg for assistance with human HCC sample analysis.