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