Understanding Molecular Heterogeneity in Hepatocellular Carcinoma

Abstract

Hepatocellular carcinoma (HCC) is the 5th most common cause of cancer‐related death in the United States with an estimated 32,000 annual deaths. Roughly 30% of HCC cases demonstrate mutations in the CTNNB1 oncogene that encodes a form of the ꞵ‐catenin protein that cannot be degraded. Recently, we developed a mouse model of HCC that genetically represents ~9‐12% of all human HCCs. Here, we combine hydrodynamic tail vein injections with the Sleeping Beauty Transposase – a non‐viral method of expressing genes of interest. Our model uses a clinically observed mutant of ꞵ‐catenin‐(T41A) that is co‐expressed with a mutant form of the nuclear factor erythroid 2–related factor 2 (Nrf2‐G31A) in the livers of wild‐type mice (B+N). This system results in microscopic HCC within 3 weeks and evident macroscopic disease by 7 weeks after induction.ꞵ‐catenin activation can be identified by the expression of its downstream target gene Glutamine Synthetase (GS) in the liver. Patient HCCs with ꞵ‐catenin mutations strongly correlate with GS expression. The evolution and function of GS in b‐catenin‐mutated HCC, remains unclear. Also, there is a gap in the knowledge whether elevated GS expression correlates with HCC biology, which were all subjects of the current study. B+N mouse model of HCC was examined temporally for development of HCC along with GS expression. B+N mice develop HCCs with heterogeneous GS expression in the form of GS‐high and GS‐low to GS‐negative foci. We next asked whether GS expression is associated with cell proliferation or death in the B+N model. We stained tissues for TUNEL and PCNA to mark cell apoptosis and proliferation, respectively in 7‐ and 9.5 weeks post‐injection livers, which were then correlated to GS staining of HCC nodules. While we found no correlation between apoptosis and GS, we found nodules with lower GS expression to have higher proliferation and vice versa. Finally, we asked if eliminating GS expression from B+N HCCs will impact tumor burden. Five weeks after B+N tumor induction in GS‐floxed mice, these animals were injected with AAV8‐TBG‐Cre virus to delete Glul,and mice were harvested after 2‐ or 4.5‐weeks. We found B+N mice with Glul deletion show increased liver weight to body weight ratio, a measurement of HCC burden, compared to controls. The livers of these mice were enriched for GS‐negative tumors. Together, our data suggests that HCC nodules with lower GS expression may outperform GS‐positive foci, and thus are more aggressive compared to the nodules with higher GS expression, which may be more differentiated. The mechanisms of GS heterogeneity and elucidation of its functional significance may help select the most effective targeted therapies for HCC patients with ꞵ‐catenin mutations.