Although many signalling pathways are dysregulated in hepatocellular carcinoma (HCC), molecular changes which are causal during the early stages of cellular transformation, have yet to be identified. Liver progenitor cells (LPCs) have been implicated in the development of HCC and were therefore used as a model to identify molecular changes that occur during their transformation. LPC lines were generated from embryonic livers and livers of adult mice fed a choline-deficient ethionine-supplemented diet.
Embryonic (BMEL) cell lines isolated by the “plate and wait” method were initially non-tumorigenic. However, their continuous culture with repeated passaging produced tumorigenic LPCs. Serial selection of cells grown in soft agar was necessary to derive a tumorigenic counterpart for the non-tumorigenic adult (BMOL) LPC lines. Transformed BMEL and BMOL cells were capable of anchorage independent growth in soft-agar and resulted in tumours when implanted into nude mice.
To identify molecular changes associated with transformation, we examined protein levels of the tumour suppressor p53, the YAP oncogene and the E2F1 regulatory pathway. Comparison of the embryonic cell lines revealed loss of p53 and p19ARF expression and increased YAP and E2F1 protein levels. Upon transformation in adult cell lines, p19ARF expression was decreased but p53 expression and activity were unaffected. E2F1 and YAP protein levels were also unaltered.
This study shows there are clear differences in the protein expression profiles of several major signalling pathways following the transformation of both adult and embryonic LPC lines. However, the consistent loss of p19ARF indicates there may be a common mechanism underlying LPC transformation independent of the p53 tumour suppressor and YAP oncogene. Future studies will test our hypothesis that p19ARF loss is a causal event in the transformation of LPCs.