Mutation of PRSS23 promotes HCC development via activation of AKT/PI3K/mTOR signaling

Human hepatocarcinogenesis represents a step-wise process, in which premalignant Dysplastic Nodules precede the development of hepatocellular carcinoma (HCC). During this process, hepatocellular tumors showing a nodule-in-nodule appearance may develop, suggesting the evolution of a faster growing neoplasm from its precursor lesion. In this study we aimed at the identification of clonally expanded driver mutations in a well-differentiated HCC evolving from a Dysplastic Nodule (DN).

Exome sequencing was performed from surrounding non-neoplastic cirrhotic liver, DN, and HCC. The clonally expanded mutations were identified using the ANNOVAR software tool. Candidate genes were validated by Sanger sequencing. Each three shRNAs were designed against each candidate gene and an in vivo shRNA screen was performed in p53 heterozygous mice. In brief, transposon vectors allowing for the combined expression of c-myc-MyrAKT1 and the shRNA library were hydrodynamically injected into the tail vein of mice. ShRNAs accelerating the tumor development were identified by next generation sequencing. Single gene validation was performed using either the mutant or the wildtype allele together with a c-myc transposon vector in p53 heterozygous mice. Stable cell lines expressing either a wildtype or a mutated candidate gene were generated and expression profiling was performed to exploit the mutation-mediated effects in more detail. Biochemical and functional assays were used for further characterization.

In total, 17 clonally expanded mutations were identified by sequencing. A mutation of the PRSS23 gene was validated to promote hepatocarcinogenesis in the in vivo RNAi screen and subsequently confirmed by combined hydrodynamic tail vein injection of PRSS23mut and c-myc-GFP into p53 heterozygous mice. In line, murine HCC cells (CaMCaA) expressing PRSS23mut showed significantly higher proliferation compared to PRSS23wt cells. Interestingly, we noted that PRSS23mut expressing murine HCC cells used more nutrients compared to Prss23wt cells as indicated by a color change of the culture medium, leading us to hypothesize that PRSS23 may affect cell metabolism. Expression profiles of PRSS23mut, PRSS23wt, and parental cells revealed that PRSS23 is involved in the regulation of the PI3K/mTOR and AMPK pathways, which was experimentally confirmed by increased phosphorylation of AKT both in PRSS23mut cells in vitro and PRSS23mut HCC in vivo.

Integration of exome sequencing and in vivo RNAi screening allows for the identification of new clonally expanded driver mutations in human HCC. The identified PRSS23 mutations provides a growth advantage to hepatocytes in context of activated myc- and AKT1-signalling and promotes HCC development in c-myc-overexpressing, p53 heterozygous mice. PRSS23mut allows for a more efficient use of nutrients as showed by activation of the AKT/PI3K/mTOR signaling pathway.