Klf6 Deficiency Promotes Hepatocellular Carcinoma In Mice and Humans By Increasing Mdm2 Expression and Accelerating P53 Degradation

Background & Aims: Dysregulation of the KLF6 tumor suppres- sor is frequent in many human cancers including HCC, and occurs through allelic loss, somatic mutation or increased expression of dominant negative splice variants. KLF6 has anti- proliferative activity in HCC-derived cell lines and in KLF6-over- expressing hepatocytes in vivo; this effect is due to p53-independent transactivation of p21 and consequent inhi- bition of cdk/cyclin activity. The aim of this study explored whether KLF6, like other tumor suppressors, mediates additional pathways of growth suppression apart from its effects on p21. Methods: We induced experimental HCC by a single IP injec- tion of DEN(5μg/g) in 2 week-old C57Bl/6 KLF6+/+ and KLF6+/- mice. The animals were sacrificed at 3, 6 and 9 months. Macro and microscopic lesions in the liver were char- acterized. Expression of candidate growth regulatory molecules was compared between KLF6+/+ and KLF6+/- mice by real time PCR and Western blot. Candidate pathways uncovered in mouse models were compared to findings in 149 HCC samples from patients with chronic HCV, and correlated with clinical out- comes. Results: KLF6+/- mice developed significantly more HCCs than KLF6+/+ animals after DEN: at 9 months, hepatic tumors were macroscopically evident in 71% of KLF6+/- vs.40% of KLF6+/+ mice (p≤0.01), with 13.9 visible tumors vs. 1.2 (p≤0.05), and a total percent replacement of normal liver by tumor of 19.7% vs. 1.4% (p≤0.05). Increased tumor burden in KLF6+/- mice correlated with progressively reduced expres- sion of the p53 tumor suppressor over 9 months, as assessed by Western blot. Importantly, loss of p53 was accompanied by a 10-fold increase in mdm2/hdm2, which normally enhances proteosomal degradation of p53 (p≤0.05). Transactivation and Co IP assays confirmed that KLF6 directly transrepresses the mdm2 promoter. Moreover, transfection of Hep3B cells with siRNAs targeting KLF6 induced a 2.4-fold (p≤0.05) increase in mdm2 promoter activity. Finally, in human livers there was a direct correlation between reduced KLF6 mRNA expression, increased mdm2 and the progression from cirrhosis to dyspla- sia. In these patients, reduced KLF6 mRNA expression in HCCs correlated with decreased survival (p≤0.01). Conclusion: These findings uncover a novel pathway of tumor suppression by KLF6 through transrepression of mdm2, leading to p53 stabilization. Both experimental and human HCC are associated with pro- gressively reduced KLF6 expression, leading to increased mdm2 and accelerated p53 loss. These are the first data link- ing these two major tumor suppressor pathways, p53 and KLF6, and they uncover an important new therapeutic target in the pathogenesis of HCC.