ELUCIDATING THE ROLE OF AUTOPHAGY IN ZEBRAFISH MODELS OF LIVER CANCER.

Abstract

Autophagy is an evolutionarily conserved cellular process that involves autophagosomal sequestration of cytoplasm, long-lived proteins and organelles, and subsequent lysosomal degradation, in response to cellular stress such as starvation. It is also involved in the degradation of superfluous organelles, proteins and protein aggregates. In addition to its main role in regulating basal cellular homeostasis, it has also been implicated in a number of diseases. Presently, there are evidences implicating autophagy induction and inhibition during carcinogenesis; however the exact role of autophagy in carcinogenesis remains to be elucidated. With the zebrafish gaining popularity as a model organism in the study of human diseases including cancers, we aim to investigate the role of autophagy in liver cancer using our established zebrafish liver cancer models. As little study is so far conducted on autophagy in zebrafish, we first characterized autophagy in zebrafish, and found the autophagic machinery and function are well-conserved between human and zebrafish. We also established and characterized a transgenic line, Tg(fabp10:egfp-lc3), with constitutively liver-specific egfp-lc3 expression, in order to visualize the autophagic flux in the liver. We found that this transgenic line is able to produce EGFP-LC3 puncta under starvation and everolimus treatment, both processes inducing autophagy. Preliminary studies using our liver cancer transgenic zebrafish with inducible expression of cmyc or Xmrk oncogene in the liver suggested that autophagy may be inhibited during liver carcinogenesis. To further study this phenomenon, we crossed Tg(fabp10:egfp-lc3) with these oncogene transgenic lines in order to visualize the autophagic flux during tumour initiation, progression and regression in vivo, and we obtained evidences suggesting autophagy inhibition during liver carcinogenesis. We further observed that autophagy may have been de-repressed during tumour regression, although more studies need to be performed to validate our observations. Finally, we also developed a transgenic line with constitutively liver-specific mrfp-egfp-lc3 expression. This transgenic line was developed based on the concept of lysosomal quenching of GFP fluorescence, with the mRFP-EGFP-LC3 reporter labelling autophagosomes yellow and autolysosomes red, thus allowing autophagic flux to be measured by comparing the ratio between yellow and red puncta. This will be the first attempt in generating a transgenic organism expressing this novel reporter. We hope that this second transgenic line will be useful and complement the first transgenic line in elucidating autophagic flux during liver carcinogenesis as well as other liver diseases.