(164ax) FOXA1/2 Depletion Drives Global Reprogramming of Differentiation State and Metabolism in a Human Hepatic Cell Line and Human Stem Cell-Derived Hepatic Progenitor Cells

FOXA factors are critical members of the developmental gene regulatory network (GRN) composed of master transcription factors (TF) that regulates murine cell fate and metabolism in the gut and liver. Nonetheless, how FOXA factors individually and collectively (through changes in interconnected GRN TF) dictate cell fate during human gut and liver differentiation and simultaneously regulate metabolic pathways, is poorly understood. If these molecular mechanisms can be elucidated, FOXA factors could be targeted for human liver diseases, which exhibit altered metabolism and differentiation, like cancer and fibrosis, and for controlling key gene regulatory pathways during human stem cell differentiation, maturation, and organogenesis. In this study, we aimed to determine the role of FOXA2 (and FOXA1 which is believed to compensate for FOXA2) in the regulation of hepatic differentiation and cell metabolism in a human hepatic cell line and during human stem cell differentiation. We employed (siRNA and shRNA), qRT-PCR, Western Blot, RNA-seq and bioinformatics, metabolic pathway maps, and functional metabolic analysis (Seahorse XF Analyzer, lipid accumulation) to determine the effects of FOXA1/2 on liver differentiation and metabolism. Depletion of both FOXA1 and FOXA2 via RNAi in human hepatic (HepG2) cells and during hepatic differentiation significantly downregulated albumin (p < 0.05) and GRN TF gene expression (HNF4A, HEX, HNF1B, TBX3) (p<0.05) and significantly upregulated endoderm/gut/hepatic endoderm markers (goosecoid (GSC), FOXA3, and GATA4), gut TF (CDX2), pluripotent TF (NANOG), and neuroectodermal TF (PAX6)(p<0.05), all consistent with a partial reprogramming of cell fate towards earlier and alternate lineages. Conditional, dox-sensitive shFOXA1/2 lines resulted in similar findings and demonstrated evidence of reversibility. RNA-seq and bioinformatic analysis of shFOXA1/2 knockdown in human liver (HepG2) cell line including principal component analysis, volcano plot, and heatmap analysis demonstrated distinct control (scrambled-shRNA) and shFOXA1/2 knockdown cell populations, and 235 significant downregulated genes and 448 upregulated genes. Gene set enrichment analysis employing normalized enrichment scores and false discovery rate cut offs demonstrated hundreds of downregulated genes associated with differentiation, and surprisingly, several hundreds of genes upregulated that are associated with alternate germ layers lineages (cardiac, endothelial, muscle) and neurectoderm (eye, neural) (FDR-qval<0.1). Interestingly, the same bioinformatic analysis approach demonstrated downregulation of genes associated with mitochondria, and hundreds of genes associated with metabolic reactions which were visualized with metabolic pathway maps, including widespread downregulation of glycolysis, citric acid cycle, mitochondrial genes, and alterations in lipid metabolism, pentose phosphate pathway, and ketogenesis (FDR-qval<0.1). Functional metabolic analysis agreed with these findings, demonstrating significantly diminished glycolysis and mitochondrial respiration, and accumulation of lipid droplets. These data demonstrate that FOXA1/2 depletion results impartial de-differentiation and activation of alternate lineages, and disruption of glycolysis, mitochondrial respiration, lipid metabolism, and other key metabolic pathways in human liver (HepG2) cells, together with evidence of de-differentiation of human pluripotent-derived hepatic progenitor cells.