SLC-0111, an inhibitor of carbonic anhydrase IX, attenuates hepatoblastoma cell viability and migration.

Background: In response to hypoxia, tumor cells undergo transcriptional reprogramming including upregulation of carbonic anhydrase (CA) IX, a metalloenzyme that maintains acid-base balance. CAIX overexpression has been shown to correlate with poor prognosis in various cancers, but the role of this CA isoform in hepatoblastoma (HB) has not been examined. Methods: We surveyed the expression of CAIX in HB specimens and assessed the impact of SLC-0111, a CAIX inhibitor, on cultured HB cells in normoxic and hypoxic conditions. Results: CAIX immunoreactivity was detected in 15 out of 21 archival pathology HB specimens. The CAIX-positive cells clustered in the middle of viable tumor tissue or next to necrotic areas. Tissue expression of CAIX mRNA was associated with metastasis and poor clinical outcome of HB. Hypoxia induced a striking upregulation of CAIX mRNA and protein in three HB cell models: the immortalized human HB cell line HUH6 and patient xenograft-derived lines HB-295 and HB-303. Administration of SLC-0111 abrogated the hypoxia-induced upregulation of CAIX and decreased HB cell viability, both in monolayer and spheroid cultures. In addition, SLC-0111 reduced HB cell motility in a wound healing assay. Transcriptomic changes triggered by SLC-0111 administration differed under normoxic vs. hypoxic conditions, although SLC-0111 elicited upregulation of several tumor suppressor genes under both conditions. Conclusion: Hypoxia induces CAIX expression in HB cells, and the CAIX inhibitor SLC-0111 has in vitro activity against these malignant cells.

GATA6 modulates the ductular reaction to bile duct ligation.

BACKGROUND: GATA6, a transcription factor expressed in cholangiocytes, has been implicated in the response to liver injury. In biliary atresia, a disease characterized by extrahepatic bile duct obstruction, liver expression of GATA6 increases with pathological bile duct expansion and decreases after successful Kasai portoenterostomy. The aim of this study was to garner genetic evidence that GATA6 is involved in ductular formation/expansion. METHODS: The murine Gata6 gene was conditionally deleted using Alb-cre, a transgene expressed in hepatoblasts (the precursors of hepatocytes and cholangiocytes) and mature hepatocytes. Bile duct ligation (BDL) was used to model biliary obstruction. RESULTS: Alb-Cre;Gata6flox/flox mice were viable and fertile. Cre-mediated recombination of Gata6 in hepatocytes had little impact on cellular structure or function. GATA6 immunoreactivity was retained in a majority of biliary epithelial cells in adult Alb-Cre;Gata6flox/flox mice, implying that surviving cholangiocytes were derived from hepatoblasts that had escaped biallelic Cre-mediated recombination. Although GATA6 immunoreactivity was preserved in cholangiocytes, Alb-cre;Gata6flox/flox mice had a demonstrable biliary phenotype. A neutrophil-rich infiltrate surrounded newly formed bile ducts in neonatal Alb-Cre;Gata6flox/flox mice. Foci of fibrosis/necrosis, presumed to reflect patchy defects in bile duct formation, were observed in the livers of 37% of adult Alb-cre;Gata6flox/flox mice and 0% of controls (p < 0.05). Most notably, Alb-cre;Gata6flox/flox mice had an altered response to BDL manifest as reduced survival, impaired bile ductule proliferation, increased parenchymal necrosis, reduced fibrosis, and enhanced macrophage accumulation in the portal space. CONCLUSIONS: GATA6 orchestrates intrahepatic biliary remodeling and mitigates liver injury following extrahepatic bile duct obstruction.

Chloroquine Triggers Cell Death and Inhibits PARPs in Cell Models of Aggressive Hepatoblastoma.

Background: Hepatoblastoma (HB) is the most common pediatric liver malignancy. Despite advances in chemotherapeutic regimens and surgical techniques, the survival of patients with advanced HB remains poor, underscoring the need for new therapeutic approaches. Chloroquine (CQ), a drug used to treat malaria and rheumatologic diseases, has been shown to inhibit the growth and survival of various cancer types. We examined the antineoplastic activity of CQ in cell models of aggressive HB. Methods: Seven human HB cell models, all derived from chemoresistant tumors, were cultured as spheroids in the presence of relevant concentrations of CQ. Morphology, viability, and induction of apoptosis were assessed after 48 and 96 h of CQ treatment. Metabolomic analysis and RT-qPCR based Death Pathway Finder array were used to elucidate the molecular mechanisms underlying the CQ effect in a 2-dimensional cell culture format. Quantitative western blotting was performed to validate findings at the protein level. Results: CQ had a significant dose and time dependent effect on HB cell viability both in spheroids and in 2-dimensional cell cultures. Following CQ treatment HB spheroids exhibited increased caspase 3/7 activity indicating the induction of apoptotic cell death. Metabolomic profiling demonstrated significant decreases in the concentrations of NAD+ and aspartate in CQ treated cells. In further investigations, oxidation of NAD+ decreased as consequence of CQ treatment and NAD+/NADH balance shifted toward NADH. Aspartate supplementation rescued cells from CQ induced cell death. Additionally, downregulated expression of PARP1 and PARP2 was observed. Conclusions: CQ treatment inhibits cell survival in cell models of aggressive HB, presumably by perturbing NAD+ levels, impairing aspartate bioavailability, and inhibiting PARP expression. CQ thus holds potential as a new agent in the management of HB.