Molecular Genetics Of Heritable Human Disorders

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

The hallmarks of GSD-Ia are impaired glucose homeostasis and long-term risk of hepatocellular adenoma and carcinoma (HCA/HCC). We have previously developed a gene transfer vector, rAAV-G6PC and shown that rAAV-G6PC-treated G6pc-/- mice expressing 3-63% of normal hepatic G6Pase-alpha activity maintain glucose homeostasis and do not develop HCA/HCC. However, the threshold of hepatic G6Pase-alpha activity required to prevent tumor formation remained unknown. To increase the efficacy of the gene transfer vector, we constructed rAAV-co-G6PC, a rAAV vector expressing a codon-optimized (co) G6PC and showed that rAAV-co-G6PC was more efficacious than rAAV-G6PC in directing hepatic G6Pase-alpha expression. Over an 88-week study, we showed that both rAAV-G6PC- and rAAV-co-G6PC-treated G6pc-/- mice expressing 3% of normal hepatic G6Pase-alpha activity maintained glucose homeostasis, lacked HCA/HCC, and were protected against age-related obesity and insulin resistance. Of the eleven rAAV-G6PC/rAAV-co-G6PC-treated G6pc-/- mice harboring 0.9-2.4% of normal hepatic G6Pase-alpha activity, 3 expressing 0.9-1.3% of normal hepatic G6Pase-alpha activity developed HCA/HCC, while 8 did not. Our data show that GSD-Ia mice harboring less than 2% of normal hepatic G6Pase-alpha activity are at risk of tumor development, establishing the threshold of hepatic G6Pase-alpha activity required to prevent HCA/HCC. The predominant subtypes of HCA in GSD-Ia are inflammatory HCA (IHCA, 52%) and -catenin-mutated HCA (bHCA, 28%). We have shown that the non-tumor-bearing (NT), rAAV-treated GSD-Ia mice (AAV-NT mice) expressing a wide range (0.9-63%) of normal hepatic G6Pase- activity maintain glucose homeostasis and display physiologic features mimicking animals living under calorie restriction. Here we show that in AAV-NT mice, the signaling pathways of the calorie restriction mediators, AMPK and SIRT1 were activated, leading to inhibition of the activity of STAT3 and NFB, the pro-inflammatory and cancer-promoting transcription factors. SIRT1 also inhibits cancer metastasis via increasing the expression of E-cadherin, a tumor suppressor, and decreasing the expression of mesenchymal markers. Consistently, in AAV-NT mice, hepatic levels of active STAT3 and NFB-p65 were reduced as were expression of mesenchymal markers, STAT3 targets, NFB targets, and -catenin targets. AAV-NT mice also expressed increased levels of E-cadherin and FGF21, targets of SIRT1, and -klotho, which can acts as a tumor suppressor. Importantly, treating AAV-NT mice with a SIRT1 inhibitor markedly reversed many of the observed anti-inflammatory/anti-tumorigenic signaling pathways. In summary, activation of hepatic AMPK/SIRT1 and FGF21/-klotho signaling pathways combined with down-regulation of STAT3/NFB-mediated inflammatory and tumorigenic signaling pathways can explain the absence of hepatic tumors in AAV-NT mice. The most severe long-term complication in GSD-Ia is HCA/HCC of unknown etiology. The global G6pc-/- mice die early, well before HCA/HCC can develop, making mechanism studies of HCA/HCC difficult. We therefore generated the liver-specific G6pc knock-out (L-G6pc-/-) mice that survive to adulthood and develop HCA. A recent report showed that G6Pase- deficiency causes impairment in autophagy, a recycling process important for cellular metabolism. However, the underlying mechanism is unclear. We showed that liver-specific knockout of G6Pase- led to downregulation of SIRT1 signaling that activated autophagy via deacetylation of autophagy-related (ATG) proteins and FoxO family of transcriptional factors which transactivate autophagy genes. Consistently, defective autophagy in G6Pase-alpha-deficient liver was characterized by attenuated expressions of autophagy components, increased acetylation of ATG5 and ATG7, decreased conjugation of ATG5 and ATG12, and reduced autophagic flux. We further showed that hepatic G6Pase-alpha deficiency resulted in activation of ChREBP, a lipogenic transcription factor, increased expression of PPAR-gamma, a lipid regulator, and suppressed expression of PPAR-alpha, a master regulator of fatty acid beta-oxidation, all contributing to hepatic steatosis and downregulation of SIRT1 expression. An adenovirus vector-mediated increase in hepatic SIRT1 expression corrected autophagy defects but failed to rectify metabolic abnormalities associated with G6Pase- deficiency. Importantly, a rAAV vector-mediated restoration of hepatic G6Pase-alpha expression corrected metabolic abnormalities, restored SIRT1-FoxO signaling, and normalized defective autophagy. Taken together, these data show that hepatic G6Pase-alpha deficiency-mediated down-regulation of SIRT1 signaling underlies defective hepatic autophagy in GSD-Ia. Mitochondrial dysfunction has been implicated in GSD-Ia but the underlying mechanism and its contribution to HCA/HCC development remain unclear. We have shown that hepatic G6Pase-alpha deficiency leads to downregulation of SIRT1 signaling that underlies defective hepatic autophagy in GSD-Ia. SIRT1 is a NAD+-dependent deacetylase that can deacetylate and PGC-1alpha, a master regulator of mitochondrial integrity, biogenesis, and function. We hypothesized that downregulation of hepatic SIRT1 signaling in G6Pase-alpha-deficient livers impairs PGC-1alpha activity, leading to mitochondrial dysfunction. Here we show that the G6Pase-alpha-deficient livers display defective PGC-1alpha signaling, reduced numbers of functional mitochondria, and impaired oxidative phosphorylation. Overexpression of hepatic SIRT1 restores PGC-1alpha activity, normalizes the expression of electron transport chain components, and increases mitochondrial complex IV activity. We have previously shown that restoration of hepatic G6Pase-alpha expression normalized SIRT1 signaling. We now show that restoration of hepatic G6Pase-alpha expression also restores PGC-1alpha activity and mitochondrial function. Finally, we show that HCA/HCC lesions found in G6Pase-alpha-deficient livers contain marked mitochondrial and oxidative DNA damage. Taken together, our study shows that downregulation of hepatic SIRT1/PGC-1alpha signaling underlies mitochondrial dysfunction and that oxidative DNA damage incurred by damaged mitochondria may contribute to HCA/HCC development in GSD-Ia. We have shown that hepatic G6Pase-alpha deficiency-mediated steatosis leads to defective autophagy that is frequently associated with carcinogenesis. We now show that hepatic G6Pase-alpha deficiency also leads to enhancement of hepatic glycolysis and hexose monophosphate shunt (HMS) that can contribute to hepatocarcinogenesis. The enhanced hepatic glycolysis is reflected by increased lactate accumulation, increased expression of many glycolytic enzymes, and elevated expression of c-Myc that stimulates glycolysis. The increased HMS is reflected by increased G6P dehydrogenase activity, elevated production of NADPH, and the reduced glutathione. We show that that restoration of hepatic G6Pase-alpha expression normalizes both glycolysis and HMS in GSD-Ia. Moreover, the HCA/HCC lesions in L-G6pc-/- mice exhibit elevated levels of hexokinase 2 and the M2 isoform of pyruvate kinase which play an important role in aerobic glycolysis and cancer cell proliferation. Taken together, hepatic G6Pase- deficiency causes metabolic reprogramming, leading to enhanced glycolysis and elevated HMS that along with impaired autophagy can contribute to HCA/HCC development in GSD-Ia. The G6pt-/- mice manifest both metabolic and myeloid dysfunction characteristic of human GSD-Ib. When left untreated, the G6pt-/- mice rarely survive weaning, reflecting the juvenile lethality seen in human GSD-Ib patients. Studies have shown that the choice of transgene promoter can impact targeting efficiency, tissue-specific expression, and the level of immune response or tolerance to