Introduction
Notch3 receptor is involved in different aspects of hepatocellular carcinoma (HCC). Nevertheless, to unlock Notch3 therapeutic/diagnostic/prognostic potential a deeper understanding of its role in HCC onset/progression is needed. KDM2A demethylase epigenetically regulates gene expression. Its levels increase with HCC grading.
Aim
To investigate the involvement of KDM2A in controlling Notch3 expression in HCC.
Material and Methods
An expression analysis of Notch3 and KDM2A was conducted by Real-Time PCR on mRNA from formalin-fixed paraffin-embedded (FFPE) HCCs and peritumoral tissue (PT). Huh7 cells were transiently silenced for KDM2A using siRNAs for a first evaluation of Notch3/KDM2A association. KDM2A and Notch3 levels after silencing were assessed by Real-Time PCR and Western-Blotting (WB). The stem-cell marker CD133, associated with Epithelial Mesenchymal Transition, was evaluated in Notch3-silenced cells. Immunohistochemistry (IHC) was conducted using anti-Notch3 and anti-KDM2A antibodies.
Results
Notch3 and KDM2A in FFPE samples were higher in HCCs compared to PT (p<0.001 and p<0.01, respectively) and increased from G1 to G3 HCC. In well differentiated HCC the staining was mainly localized in the vascular endothelium while in G3 HCC it involved clusters of tumoral hepatocytes, sometimes invading portal areas. CD34 staining showed that the Notch3 positive blood vessels were consequences of neo-angiogenesis. The transient KDM2A silencing resulted in Notch3 transcript downregulation (p≤0.001), confirmed by WB (p≤0.01). CD133 was downregulated in Notch3-silenced Huh7 (p≤0.0001).
Conclusions
An increasing Notch3 expression was observed during HCC progression. IHC revealed the involvement of Notch3 in neo-angiogenesis in early HCC and a role in invasiveness of stromal portal areas in G3 tumors. This latter, together with Notch3/CD133 association, suggests an involvement of Notch3 in local invasiveness. Furthermore, we found an association between Notch3 and KDM2A suggesting a possible mechanism of epigenetic regulation that could be responsible for the higher Notch3 expression in poorly differentiated HCC with high KDM2A levels.