Disruption of proteome by an oncogenic fusion kinase alters metabolism in fibrolamellar hepatocellular carcinoma

Abstract

Fibrolamellar hepatocellular carcinoma (FLC) is a usually lethal primary liver cancer driven by a somatic dysregulation of protein kinase A. We show that the proteome of FLC tumors is distinct from that of adjacent nontransformed tissue. These changes can account for some of the cell biological and pathological alterations in FLC cells, including their drug sensitivity and glycolysis. Hyperammonemic encephalopathy is a recurrent problem in these patients, and established treatments based on the assumption of liver failure are unsuccessful. We show that many of the enzymes that produce ammonia are increased and those that consume ammonia are decreased. We also demonstrate that the metabolites of these enzymes change as expected. Thus, hyperammonemic encephalopathy in FLC may require alternative therapeutics.

Fig. 1.. Proteomics of FLC.

Hierarchical clustering of proteins in FLC and normal samples; each row represents the sample labeled to the right of the row. The top cluster are the normal samples; the bottom are the tumor samples. (A) Unsupervised hierarchical clustering of all proteins that are statistically up-regulated or down-regulated in the TMT detection. (B and C) UMAP analysis indicates two clusters of the proteome by LFQ (B) and TMT (C) and tSNE clustering by LFQ (D) and TMT (E). Sample 4T in the TMT plots is marked at the arrow in the proteome by TMT. (F) RT-PCR to test whether a tissue sample had the DNAJB1::PRKACA fusion. Chim is RT-PCR with primers probing for chimeric DNAJB1::PRKACA (expected amplicon: 160 bp), and WT is RT-PCR with primers probing for wild-type PRKACA (expected amplicon: 184 bp). From left to right: T is the tumor sample, LN is a metastatic lymph node, N is the adjacent normal tissue, (+)ctl is RNA from a tumor of patient with FLC, (−)ctl is RNA from a non-neoplastic liver sample from a non-FLC patient, NTC is a no RNA template control, and 4T is the tumor sample from patient #4.

Fig. 2.. Analysis of the proteome.

(A to B) Volcano plots of differential protein expression. Each point represents a protein assessed by (A) LFQ or (B) TMT. The x axis is the log₂ fold change in the tumor relative to the adjacent normal. The y axis is the −log(FDR). Points above the dotted line are those that are significant at an FDR of ≤ 0.05. The proteins on the right have a higher expression in FLC tumor; those on the left have a higher expression in normal liver. (C) The differential expression of the transcriptome plotted as a function of the differential protein expression, as assessed by LFQ. Dots in red are those where the proteome is increased relative to the transcriptome, and dots in green are those where the proteins are decreased relative to the transcriptome (|log₂| fold change ≥ 0.585).

Fig. 3.. Pathways involved in metabolism of ammonia.

Proteins are in ovals and metabolites are in rectangles. Those in green are proteins or metabolites increased in FLC cells, and those in red are decreased.

Fig. 4.. Immunofluorescence of mitochondrial enzymes.

Probing of levels of PYCR1, PRODH, OAT, PRKACA, and GLS in FLC tumor and adjacent normal tissue. As is typical for FLC, the tumor cells are much larger than the normal hepatocytes. In the merged figure, the stromal cells, which are easily resolved by their smaller nuclei, are marked with an arrow. Each field is 212 μm on a side.