Elsevier

Journal of Hepatology

Volume 46, Issue 4, April 2007, Pages 645-654
Journal of Hepatology

Downregulation of KLF6 is an early event in hepatocarcinogenesis, and stimulates proliferation while reducing differentiation

https://doi.org/10.1016/j.jhep.2006.10.012Get rights and content

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 stage (p < 0.05). 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 beta-catenin, 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 HCV-related HCCs and occurs at critical stages during cancer progression. Effects of KLF6 are attributable to regulation of genes controlling hepatocyte growth and differentiation.

Introduction

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death in the world with an increasing incidence in the US, mainly due to HCV, HBV and alcoholic liver disease [1]. While new therapeutic strategies have significantly improved survival for tumors detected at early stages, the majority of patients are still diagnosed at an advanced stage and their prognosis remains poor [2]. These findings highlight the need for improved diagnosis and treatment of HCC.

Many molecular pathways have been implicated in the pathogenesis of HCC, which usually develops in a diseased liver with chronic hepatitis and/or cirrhosis [3]. Hepatocyte turnover is markedly increased in this setting, leading to clonal selection of cells with growth advantage through the accumulation of genetic and epigenetic events disrupting key regulatory pathways. These events include chromosomal instability, activation of oncogenes, as well as inactivation of tumor suppressor genes through either mutation or promoter methylation [3]. Typically, HCC associated with viral hepatitis occurs in a step-wise progression from dysplastic nodules to early, advanced and very advanced stages [4]. Little is known about the specific regulatory derangements occurring at each stage of the cancer’s development, although a clearer understanding of these events could lead to advances in diagnosis, treatment and defining prognosis.

KLF6 is a tumor suppressor gene that is functionally inactivated in several types of cancer, including HCC, through a range of mechanisms. Inactivation by loss of heterozygosity (LOH) and/or mutation occurs in prostate cancer [5], [6], colorectal carcinoma [7], astrocytic glioma [8], nasopharyngeal carcinoma [9] and gastric cancer [10]. Furthermore, there is decreased KLF6 expression in primary lung cancer [11] and prostate cancer cell lines [5] and gene silencing through promoter hypermethylation has been reported in esophageal cancer cells [12]. Recently, a unique mechanism of KLF6 inactivation has been identified, involving the generation of KLF6 alternatively spliced isoforms that antagonize the tumor suppressing functions of the full-length, wtKLF6 protein [13], [14].

We and others have previously established KLF6’s role in HCC by demonstrating frequent loss and/or mutation in the gene [15], [16], but stage-specific changes in KLF6 biology during hepatocarcinogenesis have yet to be characterized. Here we investigate the functional deregulation of KLF6 in HCC by assessing its mRNA levels in HBV- and HCV-related tumors, and thereby establish a role for KLF6 in hepatocyte growth and differentiation. Furthermore, we have defined the behavior of KLF6 in the initiation and progression of HCC in HCV-related disease from pre-malignant lesions through successive histological stages of the tumor.

Section snippets

HBV

HCC samples from 33 patients and matched surrounding tissue (ST), as well as 10 control livers (CL), were obtained, all with the approval of the Institutional Review Board (IRB) of all institutions involved, as described previously [17]. These were mostly HBV-related HCC, all with Edmondson’s grade II or III. ST were either cirrhotic or non-cirrhotic patient samples.

HCV

An HCV-related sample set in which histological progression was carefully characterized was analyzed for KLF6 mRNA expression.

Reduced KLF6 mRNA expression in HCC from patients with HBV, compared to surrounding tissue (ST)

KLF6 mRNA levels were evaluated in 33 HBV-related HCCs and matched surrounding tissue (ST) patient samples, in addition to 10 control livers (CL). This specific subset of patient samples was previously used to identify gene signatures associated with patient survival [17]. In that study, consistent downregulation of the KLF6 mRNA was also noted in the microarray analysis comparing HCCs to surrounding tissue [17]. Based on these findings, we performed QRT-PCR on a subset of these same samples to

Discussion

We have previously shown that KLF6 is involved in HCC by LOH and mutation [15]. Here we demonstrate that KLF6 mRNA levels are reduced in the majority of tumors compared to matched surrounding tissue in HBV-related HCC. In addition, we demonstrate a very early decrease in KLF6 mRNA levels in HCV-related preneoplastic lesions compared to expression levels in cirrhotic livers, with an even further decrease in metastatic tumors, compared to focal HCC. The data provide a more comprehensive picture

Acknowledgement

Financial support: SLF: NIH Grants DK37340 and DK56621; Department of Defense DAMD17-03-1-0100; The Bendheim Foundation, Samuel Waxman Foundation; JAM: DAMD17-02-1-0720 and DAMD17-03-10129; JML: AGAUR (2004BE00226, Generalitat de Catalunya, Spain), Instituto de Salud Carlos III (Fondo de Investigaciones Sanitarias 2002-2005, PI02/0596) and by the Institut Catala de Recerca Avançada (ICREA); STK: NIH Training Grant T32 DK 07792-01; MS: NIH (NIDDK), 1 K24 DK 60498-03; JSL: The Intramural Research

References (43)

  • S. Lee et al.

    Aberrant CpG island hypermethylation along multistep hepatocarcinogenesis

    Am J Pathol

    (2003)
  • B. Yang et al.

    Aberrant promoter methylation profiles of tumor suppressor genes in hepatocellular carcinoma

    Am J Pathol

    (2003)
  • D. Li et al.

    Kruppel-like factor-6 promotes preadipocyte differentiation through histone deacetylase 3-dependent repression of DLK1

    J Biol Chem

    (2005)
  • N. Matsumoto et al.

    Developmental regulation of yolk sac hematopoiesis by Kruppel-like factor 6

    Blood

    (2006)
  • S.S. Thorgeirsson et al.

    Molecular pathogenesis of human hepatocellular carcinoma

    Nat Genet

    (2002)
  • S. Tal-Kremer et al.

    The genetic basis of hepatocellular cancer

  • G. Narla et al.

    KLF6, a candidate tumor suppressor gene mutated in prostate cancer

    Science

    (2001)
  • Y.M. Jeng et al.

    KLF6, a putative tumor suppressor gene, is mutated in astrocytic gliomas

    Int J Cancer

    (2003)
  • H.K. Chen et al.

    Mutation analysis of KLF6 gene in human nasopharyngeal carcinomas

    Ai Zheng

    (2002)
  • Y.G. Cho et al.

    Genetic alterations of the KLF6 gene in gastric cancer

    Oncogene

    (2005)
  • G. Ito et al.

    Kruppel-like factor 6 is frequently down-regulated and induces apoptosis in non-small cell lung cancer cells

    Cancer Res

    (2004)
  • Cited by (0)

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