In vivo regulation of p21 by the Kruppel-like factor 6 tumor-suppressor gene in mouse liver and human hepatocellular carcinoma

Abstract

Kruppel-like factor (KLF) 6 is a tumor-suppressor gene functionally inactivated by loss of heterozygosity, somatic mutation and/or alternative splicing that generates a dominant-negative splice form, KLF6-SV1. Wild-type KLF6 (wtKLF6) expression is decreased in many human malignancies, which correlates with reduced patient survival. Additionally, loss of the KLF6 locus in the absence of somatic mutation in the remaining allele occurs in a number of human cancers, raising the possibility that haploinsufficiency of the KLF6 gene alone contributes to cellular growth dysregulation and tumorigenesis. Our earlier studies identified the cyclin-dependent kinase inhibitor p21 as a transcriptional target of the KLF6 gene in cultured cells, but not in vivo. To address this issue, we have generated two genetic mouse models to define the in vivo role of KLF6 in regulating cell proliferation and p21 expression. Transgenic overexpression of KLF6 in the liver resulted in a runted phenotype with decreased body and liver size, with evidence of decreased hepatocyte proliferation, increased p21 and reduced proliferating cell nuclear antigen expression. In contrast, mice with targeted deletion of one KLF6 allele (KLF6 þ /À) display increased liver mass with reduced p21 expression, compared to wild type littermates. Moreover, in primary hepatocellular carcinoma samples, there is a significant correlation between wtKLF6 and p21 mRNA expression. Combined, these data suggest that haploinsufficiency of the KLF6 gene may regulate cellular proliferation in vivo through decreased transcriptional activation of the cyclin-dependent kinase inhibitor p21. Oncogene (2007) 26, 4428-4434; doi:10.1038/sj.onc.1210223; published online 5 February 2007 Keywords: KLF6; Kruppel-like factor; tumor-suppressor gene; p21; haploinsufficiency Kruppel-like factor 6 (KLF6) belongs to the Kruppellike family of transcription factors, which play roles in the regulation of diverse cellular processes including development, differentiation, proliferation and apoptosis (Bieker, 2001). Functional inactivation of the KLF6 gene occurs through several mechanisms, including loss of heterozygosity (LOH), somatic mutation and/or increased alternative splicing that yields a dominantnegative splice isoform, KLF6-SV1. KLF6 dysregulation has been demonstrated in a number of human cancers including prostate (Narla et al., 2001;Chen et al., 2003), colorectal , non-smallcell lung (Ito et al., 2004), gastric (Cho et al., 2005), nasopharyngeal (Chen et al., 2002), hepatocellular (Kremer-Tal et al., 2004) and ovarian carcinomas (DiFeo et al., 2006b) as well as glioma (Jeng et al., 2003). Furthermore, decreased KLF6 mRNA expression is associated with reduced patient survival in prostate (Singh et al., 2002;Glinsky et al., 2004) and lung cancers (Kettunen et al., 2004). Interestingly, reconstitution of KLF6 decreases cell proliferation and reverts tumorigenicity in glioblastoma cell lines (Kimmelman et al., 2004).Depending on the cell type and context, KLF6's growth-suppressive prop...

Biology of Krüppel‐like factor 6 transcriptional regulator in cell life and death

“…, reduction of roughly 80% in proliferating cell nuclear antigen expression and 50% decrease in DNA synthesis, consistent with reduced cell proliferation of hepatocytes from KLF6 transgenic mice (32). Additionally, there was a significant correlation between low levels of KLF6 and p21 CIP1/WAF1 mRNA expression in primary hepatocellular carcinoma, suggesting that decreased expression of p21 CIP1/WAF1 in primary tumors results, at least in part, from the downregulation of the KLF6 tumor-suppressor gene (32).…”

“…Additionally, there was a significant correlation between low levels of KLF6 and p21 CIP1/WAF1 mRNA expression in primary hepatocellular carcinoma, suggesting that decreased expression of p21 CIP1/WAF1 in primary tumors results, at least in part, from the downregulation of the KLF6 tumor-suppressor gene (32).…”

“…, a transcriptional target gene for KLF6 in differentiated cells (19,32), was not downregulated in klf62/2 embryos (15). Most importantly, in hepatoma cell lines, the situation is particularly interesting as KLF6 knockdown led to de-phosphorylation of Rb together with downregulation of cyclin D 1 and cdk4 expression, which in turn strongly impaired cell proliferation involving G 1 -S arrest (35).…”

KLF6 and TP53 mutations are a rare event in prostate cancer: distinguishing between Taq polymerase artifacts and true mutations

“…They suggested that only the first and third mechanisms would be involved. Recent reports by Narla et al 7 support the notion that the loss of one KLF6 allele, even in the absence of somatic mutations in the remaining one, could be enough to contribute to cell proliferation and tumor development. The exact mechanism by which this would be effective is still unknown.…”

“…6 This has been proven in several in vitro 5 and in vivo assays. 7 KLF6 is believed to regulate cancer development and progression through the downregulation of the c-Jun oncoprotein 8 and the activation of E-cadherin. 9 The reported frequency of KLF6 mutations in different types of human cancer varies in the different published studies.…”