Analysis of the Role of Glucagon in Hepatocellular Carcinoma Cell Growth, Invasion, and Migration and the Regulation of Glucagon Receptor Signaling

Liver cancer is one of the leading causes of cancer-related mortality. Since few approved therapeutics are available, there is an urgent need for novel therapies. This study sought to characterize the role of glucagon in hepatocellular carcinoma (HCC) through an analysis of the proliferative, migratory, invasive, and anti-apoptotic/survival effects of glucagon on HCC cells. In addition, regulation of glucagon receptor (GR) signaling was examined in HEK cells and hepatocytes.;Glucagon (100 nM; 96 hr) had an inhibitory effect on proliferation in Hep3B HCC cells stably expressing GR (Hep3BGR). EGF (10 nM; 96 hr) increased proliferation, consistent with previous reports, and glucagon (or forskolin or 8-bromo cAMP) blocked this effect. Glucagon inhibited migration, but did not affect the invasiveness or survival of Hep3BGR cells. Glucagon (or forskolin) inhibited EGF-mediated ERK1/2 phosphorylation. Ser 338 Raf-1 phosphorylation was increased by EGF, and reduced by cotreatment with glucagon.;GR internalized maximally by 30 min of glucagon stimulation in surface binding experiments and co-localized with early endosomes in confocal microscopy experiments. PKC-alpha translocated to the membrane and co-immunoprecipitated with GR with glucagon stimulation. The last 70 amino acids of the COOH terminus were critical for GR signaling/cAMP accumulation and recycling, while the third intracellular loop was essential for GR internalization, signaling/cAMP accumulation, and recycling.;In conclusion, glucagon inhibits HCC cell proliferation and migration, and the effects on proliferation occur through cAMP signaling events that inhibit ERK1/2 phosphorylation, possibly through Raf-1. The results of this study are critical in understanding how glucagon might block HCC cell proliferation, particularly growth factor-mediated proliferation, a target of some cancer therapies. In addition, the GR is an important potential target of therapies for diabetes and other diseases; the current study provides key information for understanding the regulation of GR signaling, which will be important for the success of such therapies.