Hepatocellular carcinoma (HCC) is a leading cause of cancer death worldwide, reflecting incomplete characterization of underlying mechanisms and lack of early detection. Krüppel-like factor 6 (KLF6) is a ubiquitously expressed zinc finger transcription factor that is deregulated in multiple cancers through loss of heterozygosity (LOH) and/or inactivating somatic mutation. We analyzed the potential role of the KLF6 tumor suppressor gene in 41 patients who had HCC associated with hepatitis C virus (16 patients), hepatitis B virus (12 patients, one of whom was coinfected with hepatitis C virus), and other etiologies (14 patients) by determining the presence of LOH and mutations. Overall, LOH and/or mutations were present in 20 (49%) of 41 tumors. LOH of the KLF6 gene locus was present in 39% of primary HCCs, and the mutational frequency was 15%. LOH and/or mutations were distributed across all etiologies of HCC evaluated, including patients who did not have cirrhosis. Functionally, wild-type KLF6 decreased cellular proliferation of HepG2 cells, while patient-derived mutants did not. In conclusion, we propose that KLF6 is deregulated by loss and/or mutation in HCC, and its inactivation may contribute to pathogenesis in a significant number of these tumors.

Background/Aims-Hepatocellular carcinoma (HCC) has the most rapidly rising cancer incidence in the US and Europe. The KLF6 tumor suppressor is frequently inactivated in HCC by loss-of-heterozygosity (LOH) and/or mutation.Methods-Here we have analyzed 33 HBV-and 40 HCV-related HCCs for mRNA expression of wildtype KLF6 (wtKLF6) as well as the KLF6 variant 1 (SV1), a truncated, growth-promoting variant that antagonizes wtKLF6 function. The HCV-related tumors analyzed represented the full histologic spectrum from cirrhosis and dysplasia to metastatic cancer.Results-Expression of KLF6 mRNA is decreased in 73% of HBV-associated HCCs compared to matched surrounding tissue (ST), with reductions of ~80% in one-third of the patients. KLF6 mRNA expression is also reduced in dysplastic nodules from patients with HCV compared to cirrhotic livers (p < 0.005), with an additional, marked decrease in the very advanced, metastatic ). An increased ratio of KLF6SV1/wt KLF6 is present in a subset (6/33, 18%) of the HBV-related HCCs compared to matched ST. Reconstituting KLF6 in HepG2 cells by retroviral infection decreased proliferation and related markers including cyclin D1 and betacatenin, increased cellular differentiation based on induction of albumin, E-cadherin, and decreased alpha fetoprotein.Conclusions-We conclude that reduced KLF6 expression is common in both HBV-and HCVrelated HCCs and occurs at critical stages during cancer progression. Effects of KLF6 are attributable to regulation of genes controlling hepatocyte growth and differentiation.

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...

Gastric cancer is the second most common cancer and a leading cause of cancer-related death worldwide. The KLF6 tumour suppressor gene has been previously shown to be inactivated in a number of human cancers through loss of heterozygosity (LOH), somatic mutation, decreased expression, and increased alternative splicing into a dominant negative oncogenic splice variant, KLF6-SV1. In this present study, thirty seven gastric cancer samples were analysed for the presence of loss of heterozygosity (LOH) of the KLF6 locus and somatic mutation. In total, 18 of 34 (53%) of the gastric cancer samples analysed demonstrated KLF6 locus specific loss. Four missense mutations, T179I, R198G, R71Q, and S180L were detected. Interestingly, two of these mutations R71Q and S180L have been identified independently by several groups in various malignancies including prostate, colorectal and gastric cancer. In addition, decreased wtKLF6 expression was associated with loss of the KLF6 locus and was present in 48% of primary gastric tumour samples analysed. Functional studies confirmed that wtKLF6 suppressed proliferation of gastric cancer cells via transcriptional regulation of the cyclin dependent kinase inhibitor p21 and the oncogene c-myc. Functional characterization of the common tumour-derived mutants demonstrated that the mutant proteins fail to suppress proliferation and function as dominant negative regulators of wtKLF6 function. Furthermore, stable overexpression of the R71Q and S180L tumour derived mutants in the gastric cancer cell line, Hs746T resulted in increased tumourigenicity in vivo. Combined, these findings suggest an important role for the KLF6 tumour suppressor gene in gastric cancer development and progression and identify several highly cancer-relevant signaling pathways regulated by the KLF6 tumour suppressor gene.