The Glucagon Receptor Functions as a Tumor Suppressor in Hepatocellular Carcinoma

Glucagon is an essential hormonal regulator of metabolic processes. Produced by pancreatic islet &agr; cells, glucagon temporarily increases blood glucose levels during fasting by stimulating hepatocytes to undergo glycogenolysis and gluconeogenesis, thereby releasing individual glucose molecules from glycogen stores or non-carbohydrate sources. Abnormally high circulating levels of plasma glucagon in patients are associated with a number of adverse effects including hyperglycemia, decreased amino acid production, and Type I or Type II diabetes. Interestingly, decreased expression of glucagon receptor (GR) protein has been observed in several malignant cancers, most notably hepatocellular carcinoma (HCC). In this study, a potential role for the GR in cancer progression was investigated to identify alternative therapeutic targets following its loss. More specifically, the hypothesis was tested that the GR can act as a tumor suppressor by maintaining strict transcriptional suppression of cationic channel genes linked to oncogenesis, and activation of anionic channel genes linked to tumor suppression. Consequently, GR loss and the corresponding derepression of cationic channel gene expression acts as an 'enabler' of HCC. A rapid decrease in GR protein expression was observed in primary patient samples beginning as early as inflammation and cirrhosis, with almost complete protein loss by advanced stages of HCC. Cell lines devoid of HCC expression were highly metastatic in vivo and caused significant tumor growth; however, stable reintroduction of the GR inhibited metastasis and tumor formation. GR signaling acted through a PKA-mediated mechanism to inhibit c-Raf activation and subsequent activation of the oncogenic mitogen-activated protein kinase (MAPK) pathway. GR loss resulted in a reduced inhibitory check on ERK1/2 and led to the up-regulation of cationic channel mRNA. These channels have been shown to contribute to behaviors required for metastasis, including epithelial-mesenchyme transition, growth factor secretion, and invasion. Conversely, mRNA expression of anionic channel genes was significantly reduced following GR loss. The voltage-gated sodium channel (VGSC) Nav1.5 and the gamma-amminobutyric acid type A (GABAA) receptor in particular were functionally active, demonstrating robust ion conductance in response to agonist stimulation. Pharmacological inhibition of Nav1.5 or GABAA receptor activation significantly inhibited HCC invasion. Moreover, transcriptional regulation of Nav1.5 was modulated by ERK1/2 and dependent on MAPK activation, whereas GABAA subunit beta3 gene expression was regulated by both the GR and PKA. Lastly, in vivo HCC metastasis was significantly reduced following treatment with ion channel modulators that selectively inhibit VGSCs or activate GABAA receptors, and the neuroactive steroid allopregnanolone significantly prolonged survival compared to control-treated animals. These findings demonstrate for the first time that the GR acts as a tumor suppressor that is lost in HCC, and that repurposing ion channel modulators in its absence may serve as an effective and novel cancer treatment.