Abstract B40: Derangement HNF4α gene expression as a candidate marker of hepatocellular carcinoma progression
Authors: Shavochkina Daria A. , Fleishman Daria I. , Morozova Olga V. , Chuchuev Evgeniy S. , Patyutko Yuriy I. , Lazarevich Natalia L. 
Published: January 2011

Hepatocellular carcinoma (HCC) is one of the world's most common cancers. The development of malignant phenotype is considered to be a multistep process that results from the accumulation of genetic alterations. Mechanisms of epithelial carcinogenesis are determined by the specific properties of tumor progenitor tissue. According to our investigations, HCC progression is associated with the dysfunction of hepatocyte nuclear factors (HNFs), which control liver-specific gene expression, and particularly HNF4α, a nuclear receptor playing a key role in hepatic differentiation. This gene is transcribed from two distinct promoters, which activity results in producing two groups of isoforms and reflects the level of cell differentiation. HNF4αP2 isoforms are synthesized in embryonic liver and normally disappear after birth, while HNF4αP1 become dominant in mature hepatocytes. Reexpression of HNF4αP1 reversed the malignant phenotype of dedifferentiated mouse HCC variants. Studies on the collection of chemically induced mouse HCCs of independent origin revealed strict correlation of HNF4α expression with tumor differentiation state.

Using semiquantitative RT-PCR we have analyzed the expression of 46 genes in 21 samples of human HCCs. 17 of studied cases were not associated with Hepatitis viruses infections, while 4 of patients were infected with hepatitis B virus. The most frequent event revealed in HCC samples compared to corresponding non-tumor tissues was the complete loss (in 7 of 17 non-hepatitis HCCs) or significant decrease (in 5 of 17 non-hepatitis HCCs) of HNF4αP1 transcription. Decrease of HNF4α expression appeared to be a poor prognostic factor: patients with highly expressed HNF4αP1 had 100% 3-years survival, and 83% of them didn't have any disease progression, while only 33% of patients with low HNF4αP1 expression showed no disease progression. HCCs which were associated with hepatitis infection didn't show relevant alterations in HNF4αP1 expression. We suppose that hepatitis-associated hepatocarcinogenesis might develop through HNF4α1-independent mechanism.

Intracellular localization and protein expression level of HNF4α isoforms on the paraffine sections of human HCCs was investigated immunohistochemically using specific mouse monoclonal antibodies for HNF4αP1 (K9218; R&D systems; 1:6000) or HNF4αP2 (H6939; R&D systems; 1:3000) groups of isoforms. HNF4α isoforms immunoreactivity was detectable in all studied tumors except severely dedifferentiated variants. Intense nuclear staining for HNF4αP2 was observed in hepatocytes, but nuclear staining for HNF4αP1 showed decreased intensity. We haven't revealed synthesis of any HNF4α isoforms in dedifferentiated tumors. All this data corroborate with RT-PCR analysis. We suppose that HCC progression is accompanied with activation of HNF4αP2 isoform, decreased synthesis of HNF4αP1 isoform while in severely dedifferentiated tumors expression of both groups of HNF4α isoforms is repressed. We assume that induction of HNF4αP2 expression might serve a marker of early hepatocarcinogenesis, while decrease and subsequent loss of HNF4αP1 and lately HNF4αP2 marks HCC progression. This provides a new data important for further development of approaches for HCC diagnostics and prognosis.

Expanded analysis of human HCCs archival samples and further investigation of correlation between tumor differentiation state and patients postoperative survival rate with the pattern of HNF4α isoforms expression would allow to estimate the usefulness of the analysis of HNF4α isoforms expression for the long-term outlook for this tumor type.

Citation Information: Clin Cancer Res 2010;16(7 Suppl):B40