Reduced Klf6 Expression Correlates With Poor Prognosis In Human Hepatocellular Carcinoma (hcc) and Enhanced Chemical Carcinogenesis In Klf6+/- Mice

Background: The KLF6 tumor suppressor is functionally inacti- vated in HCC by allelic loss, somatic mutation, and/or increased expression of the dominant negative alternatively spliced form, KLF6-SV1 (J Hepatol. 2007; 645). Loss of KLF6 is associated with enhanced cell cycle progression and reduced p21 expression. Aims: we explored the clinical impact of reduced KLF6 and enhanced mRNA splicing in human HCC, and modeled these changes in KLF6 +/- mice. Methods: KLF6 full length and SV1 splice variant mRNAs were quantitated by real time PCR in 149 patients from the full range of clinical dis- ease stages [normal liver, (n=9); cirrhosis (n=9); dysplasia (n=27); early to advanced HCC (n=116)] in whom clinical out- comes were all available. Concurrently, we compared the fre- quency and characteristics of HCC in 39 KLF6 +/- and 39 KLF6 +/+ male mice following a single IP administration of DEN (5 μg/g) in 2 week-old mice. Results: The expression of KLF6 mRNA was significantly reduced in cirrhosis compared to normal liver and markedly reduced in dysplasia and in all stages of HCC, compared to surrounding tissues (p<0.01 for all cases). Reduced expression of KLF6 was significantly correlated with poor survival (p<0.01), while increased KLF6-SV1 was associated with increased disease recurrence. Livers of KLF6 +/- mice had reduced levels of KLF6 mRNA expression to 40- 60% of KLF6 +/+ mice, which is comparable to reduced expression in human HCCs. Following one injection of DEN, macroscopic HCCs first appeared at 6 months in KLF6 +/-, but not in KLF6 +/+ mice. At 9 months there were significant dif- ferences in the prevalence of HCC (40% KLF6 +/+ mice vs 71% in KLF6 +/-), the mean number of macroscopic lesions per animal (p<0.05), percent replacement of normal liver by neo- plastic tissue (p<0.05) and biochemical abnormalities (Alk phos, AST, ALT; p≤0.05). PCNA staining confirmed increased proliferation in KLF6+/- mice but no difference in apoptosis by TUNEL. Microarray analysis identified a range of differentially expressed genes in tumors and surrounding tissues of KLF6 +/- vs KLF6 +/+ mice regulating metabolism, as well as signalling by interferon, TLR, STAT and NFKB. Conclusions: These data unveil a comprehensive portrait of KLF6 dysregulation in HCV- related HCC, in which reduced expression of full length KLF6 confers reduced survival whereas increased of the KLF6-SV1 splice variant promotes recurrence. KLF6 +/- mice faithfully recapitulate the changes in gene expression seen in human HCCs. These findings identify dysregulation of KLF6 as a prog- nostic marker and highlight an important role for the KLF6 tumor suppressor gene in HCC development and progression.