Identification of a Novel Candidate to Promote Hepatic Maturation in HepG2 Cells In Vitro.

Drug-induced cholestasis (DIC) is characterized by impaired bile flow caused by exogenous compounds and remains a major culprit of attrition of drug candidates, as illustrated by liver toxicity representing 40% of all clinical trial terminations. Strategies for earlier (preclinical) detection of DIC-inducing drug candidates has thus been an area of extensive research. Nevertheless, the currently available in vitro models for DIC suffer from major knowledge gaps in terms of underlying mechanisms. The crux is the inability to fully mature hepatic phenotypes. Previous studies show a different transcriptome, proteome and epigenetic profile between iPSC-derived hepatocyte-like cells (HLCs) and primary human hepatocytes (PHHs), with low levels of hepatic transcription factors (TFs) and biotransformation CYP450 enzymes in HLCs. A recent mechanistic study revealed that maintenance of mature phenotypes of PHHs and their (glucose) metabolism in vitro, at least partially, depends on blocking epithelial-mesenchymal transition (EMT) and subsequently identified TGF-β, Wnt, BMP, Notch, and cAMP signaling as master regulators of this process. However, 5 different chemical compounds, each corresponding to a specific pathway, had to be administered into the culture medium for sufficient EMT inhibition. Building further on the previous knowledge that AKT/mTORC1 signaling is a strong inducer of gluconeogenesis and CYP450 activity, we hypothesized that an upstream pathway and/or common protein(s) could simultaneously activate AKT/mTORC1 and inhibit EMT. Not only would it lead to more mature hepatic phenotypes (and thus better models) in vitro, but it would also broaden the mechanistic understanding underlying hepatic maturation. Preliminary data suggests that this goal may be accomplished. After targeted inhibition with a small molecule in HepG2 cells, expression patterns were consistent with (i) an upregulation of AKT/mTORC1 as well as estrogen biosynthesis (indicative for strong CYP450 activation), and (ii) a shift in cell metabolism from glucose fueling to β-oxidation and amino acid consumption with induction of gluconeogenesis (indicative for strong EMT inhibition). Furthermore, the epithelial marker CDH1 (encoding for E-cadherin) was sharply increased and AKT/cAMP signaling together with typical hepatic TFs that serve as maturation biosensor (HNF4A, FOXO1 and FOXO3) were predicted to be significantly activated. Consistent with this expression data, mitochondrial functionality was confirmed to be improved by quantifying urea production and measuring mitochondrial membrane potentials. Taken together, gathered preliminary data points towards a novel link between (loss of) the identified target, inhibition of EMT and the enhancement of hepatic maturation TFs as well as CYP450 expression and activation. Generation of knock-out cells and elaborate bile acid profiling is on-going and will be applied in further mechanistic studies of DIC.