KLF4 was identified as a tumor suppressor with loss of expression in a series of cancers 
. However, KLF4 is also one of four transcription factors required for the development of induced pluripotent stem cells. The other three transcript factors have elevated expression and/or displayed oncogenic properties in HCC cells. Moreover, high KLF4 expression has been shown in primary breast ductal carcinoma and oral squamous cell carcinoma 
. Together, these studies suggest that the effect of KLF4 is tissue specific, and likely depends on the target genes regulated in a given cell type. Before the study reported herein, it was unknown whether KLF4 displayed tumor-suppressive or oncogenic properties in HCC.
Several pieces of data presented in this manuscript strongly support the hypothesis that KLF4 acts as a tumor suppressor in HCC. Ectopic Klf4 expression decreased anchorage-independent growth of HCC cells in culture, as well as their tumorigenic growth in vivo. This reduced tumor growth was associated with decreased staining for the proliferation marker Ki-67. More recently, KLF4 has been shown to inhibit the migration and invasion activities in several cancer models, suggesting its potential role as a metastasis suppressor 
. Similar to these previous findings, our data of in vitro
and in vivo
functional analyses simultaneously supported that KLF4 functions as a suppressor of HCC cell migration, invasion and metastasis.
Based on data mining using Oncomine and validation by qRT-PCR using a small collection of HCC samples, we have demonstrated that KLF4 mRNA is down-regulated in most of HCC tissues compared with normal liver tissues 
. Similar to the findings in other types of cancers 
, our data suggested a possible role of KLF4 as a tumor suppressor in HCC. Importantly, a gradual decrease in KLF4 transcript in Wurmbach’s data set containing 75 liver samples representing the stepwise carcinogenic process from preneoplastic lesions to HCC, indicated that KLF4 might participate in the initiation as well as progression of HCC.
While we did not observe a consistent effect of Klf4/KLF4 expression on cell cycle progression in HCC cell lines (Figure S6
), we consistently observed that enforced Klf4/KLF4 expression reduced HCC cell migration and invasion. EMT is associated with increased cell motility. Consistent with its inhibition of migration and invasion, we also observed that ectopic Klf4 inhibited mesenchymal phenotypes in HCC cells, illustrated by changes in cell morphology and reduction of the mesenchymal markers N-cadherin and Vimentin. Previous studies suggested that KLF4 regulates E-cadherin gene expression by binding a GC-rich/E-box region in its promoter, and further demonstrated that enhanced levels of KLF4 resulted in the restoration of E-cadherin expression in breast cancer cells 
. Consistent with this prior work, we observed that E-cadherin mRNA levels were increased by ectopic Klf4/KLF4 expression and inhibited by Klf4/KLF4 knockdown in several HCC cell lines (Figure S5
). However, we did not observe a concomitant increase in E-cadherin protein by immunoblot assay, although we were able to show faint E-cadherin staining in tumor sections from MM189 PB-Klf4 tumors (Figure S7
). These results suggest that there may be multiple levels of regulation of E-cadherin protein levels in HCC cells. Based on down-regulation of E-cadherin in the process of EMT, a previous study demonstrated that Twist and Snail, but not Slug, are major EMT regulators in HCC as shown by the correlation of over-expression of Snail and/or Twist, down-regulation of E-cadherin, and nonmembranous localization of β-catenin 
. Our data suggest that Klf4 promotes an epithelial phenotype in HCC cells, and that Klf4 suppresses the expression of Slug, but not Twist or Snail, indicating that there was a specific regulation between Slug and Klf4. Also, our data showed that forced expression of Slug induced features associated with EMT- morphological change, increased expression of mesenchymal proteins, enhanced migration as well as increased lung colonization- in HCC cells with ectopic Klf4 expression. These data suggest that regulation of Slug expression is a key mechanism underlying Klf4-mediated MET in HCC cells. In agreement with our finding, Liu recently reported the reciprocal regulation of KLF4 and SLUG in TGF-β initiated prostate cancer EMT and demonstrated that TGF-β induced loss of KLF4 was sufficient to initiate SLUG induction and EMT 
The reduction of Slug mRNA observed with ectopic Klf4 expression led us to examine the ability of Klf4 to transcriptionally regulate Slug gene expression. Using both ChIP and luciferase reporter assays, we found that endogenous Klf4-containing transcription complex binds to and represses the Slug promoter. Our current results suggest the possibility that Klf4 indirectly repress the promoter through interactions with other transcriptional repressor. This location (−300 bp) was different from that by software prediction, indicating the possibility that the down-regulated Slug by Klf4 was mediated by interaction with other transcription repressors but not by directly binding to the Slug promoter. Moreover, our data did not rule out the possibility that unpredicted Klf4 binding site was located within −300 bp. Additional studies will be needed to provide these mechanistic details.
In KLF family, down-regulation of KLF6, an early event of hepatocarcinogenesis, was also demonstrated to contribute to pathogenesis of HCC 
. KLF6 was shown to be frequently inactivated either by LOH or inactivating somatic mutations 
. Similar to the inactivation of KLF6, KLF4 was shown to undergo promoter methylation and LOH in several cancer types 
. In this study, our data demonstrated that down-regulated KLF4 was frequently detected in HCC tissues. To verify whether methylation led to down-regulation of KLF4 in HCC cell lines, we treated HCC cells with the methylation inhibitor, 5-Aza-dC and found that KLF4 expression could be re-activated after treatment (Z-S. L. and Y-W. C., unpublished data). These results indicated that down-regulation of KLF4 might be caused by gene methylation. However, alternative mechanisms for KLF4 transcriptional inactivation may occur in other KLF4 deficient cancers that don’t exhibit genetic loss and promoter methylation. In colon cancers, KLF4 could be down-regulated by caudal type homeobox 2 (CDX2) 
, notch signaling 
, transcription factor 4 (TCF4) 
or sex determining region Y-box 9 (Sox9) 
. Furthermore, KLF4 could also be regulated post-transcriptionally by microRNA targeting, as found in human esophageal cancer cell lines 
In summary, our data demonstrate that KLF4 acts as a tumor suppressor in HCC, at least in part by repressing SLUG expression. Whereas further studies are required to characterize the reciprocal regulation between KLF4 and SLUG as well as the mechanisms leading to down-regulation of KLF4 in HCC, our findings provide new insights into a potential role and mechanism by which KLF4 inhibits tumorigenesis and metastasis of HCC.