K. M. Riggle1, R. S. Yeung1, H. L. Kenerson1, K. J. Riehle2 1University Of Washington,Surgery,Seattle, WA, USA 2Seattle Children’s Hospital,General And Thoracic Surgery,Seattle, WA, USA
Introduction: Hepatocellular carcinoma (HCC) is a heterogenous disease that commonly arises in a background of cirrhosis. Fibrolamellar HCC (FL-HCC) is a subtype of HCC occurring in children and young adults in the absence of known liver disease. Currently, there is no effective therapy for unresectable or metastatic FL-HCC. Recent genomic analysis identified a recurrent mutation in FL-HCC involving a deletion on chromosome 19. The mutation results in a common chimeric transcript containing the 5’-region of a heat shock protein (DNAJB1) fused to the catalytic subunit of protein kinase A (PRKACA). We sought to characterize this chimeric protein and its effects on PKA activity in human FL-HCC.
Methods: We prepared tissue lysates from four snap-frozen FL-HCC samples along with paired, non-tumor liver tissues. PRKACA expression was determined by immunoblot analysis. PKA activity was determined via a radioactive kinase assay in the presence of cAMP, a known activator of PKA, with and without PKI, a specific inhibitor of PKA accounting for background activity. RNA was extracted using TRIZOL reagent, and used to create cDNA for qRT-PCR analysis of the mutant transcript.
Results: We confirmed that all tumor samples expressed a 46-kDa fusion gene product in addition to the wild-type 41-kDa PKA protein. The paired normal liver samples only expressed the wild-type protein. Further, the mutant protein was not detected in ‘classic’ HCCs nor cancer-associated fibroblasts isolated from a case of FL-HCC. Using qPCR we found that the FL-HCC tumors expressed the chimeric transcript at levels that were 10.59±4.2 fold higher than normal liver (p = 0.016). Basal PKA activities from freshly lysed tumors and paired livers were not significantly different, but cAMP-stimulated PKA activity was significantly higher in FL-HCC tumors when compared to normal liver. In a dose response experiment, the PKA activity in FL-HCC was 3.62, 5.52, and 6.41 pmol/min/mg (vs. normal liver with PKA activity of 2.60, 3.14, and 4.95 pmol/min/mg) at cAMP concentrations of 0.05, 0.5, and 5 uM respectively.
Conclusion: Our data verify the unique expression of the DNAJB1-PRKACA fusion protein in all FL-HCC samples tested, but absent in the adjacent non-tumor liver and non-FL-HCCs. Further, the lack of mutant protein expression in fibroblasts derived from FL-HCC highlights the primary effects of the mutation on transformed hepatocytes and not the stromal component. The expression of the mutant transcript was significantly greater than that of the native PKA indicative of higher intrinsic promoter activity of DNAJB1 compared to that of PRKACA. Importantly, PKA activity in the FL-HCCs remains cAMP-dependent but with increased sensitivity to cAMP without evidence of enhanced basal activity. These findings suggest that the expression of DNAJB1-PRKACA in FL-HCC leads to over-activation of PKA under conditions of cAMP production, which may contribute to tumor development.