MDM4 and eEF1A2, new oncogene candidates in human HCC, revealed by high-resolution array-bases CGH

Although the etiology of HCC is well characterized (e.g. HBV, HCV, alcohol, and NASH), the host molecular alterations preceding HCC development are less well understood. In order to identify novel host driver genes of human hepatocarcinogenesis, we performed a combination of high-resolution fine-mapping of genomic imbalances using array-based CGH (aCGH) with extensive expression and functional analyses. Using aCGH (n=63) we identified 36 common loci exhibiting small amplifications or putative homozygous deletions. Among them, we detected a novel single amplicon at 1q32.1 that contained the p53-regulating oncogene MDM4. Further novel amplicons were detected at 10p11.21-q11.21, 17q21.33, and 18p11.32–11.31, and 20q13.3 as well as a homozygous deletion at 12p13.33. By defining minimally overlapping regions of all sample data, we were able to fine-map the size of the commonly gained region at 20q13 to a region of 2.5 Mb at 20q13.3, containing the potential oncogene EEF1A2. Expression analyses revealed upregulation of MDM4 and EEF1A2 on the mRNA- and protein level in about 50% of the HCCs (n=24). Immunohistochemistry (n=216) showed an increase of EEF1A2 protein expression from normal liver over chronic hepatitis/liver cirrhosis to HCC (p<0.01). siRNA-mediated silencing of MDM4 in Hep3B and HepG2 cells resulted in 74% and 83% (p<0.01) reduction of cell viability, respectively. This effect was associated with a reduction of cell proliferation in both cell lines, but seemed to be mediated mainly by induction of apoptosis. Reduction of EEF1A2 expression resulted in a comparable decrease of cell viability in both cell lines (p<0.01) due to reduced proliferation and induction of apoptosis.

In conclusion, using a combination of high-resolution array-CGH with expression and functional analyses, we identified two novel oncogenes in human hepatocarcinogenesis. Since knock-down of the p53-regulating oncogene MDM4 resulted in similar effects in both p53-wildtype (HepG2) and-deficient cells (Hep3B), it seems likely that MDM4 may also exhibit p53-independent protumorigenic effects in human HCCs.

CGH - HCC - chromosomal instability - oncogene