Cleavage of Endoplasmic Reticulum Proteins in Hepatocellular Carcinoma: Detection of Generated Fragments in Patient Sera

Background & Aims: In the past decade, there has been a rising incidence of hepatocellular carcinoma (HCC) and a progressive increase in HCC-related mortality in the United States and Western Europe. The poor survival of patients with HCC is largely related to the lack of reliable tools for early diagnosis. Methods: We have applied proteomics tools to the comparative analysis of protein profiles between HCC and adjacent nontumor tissues as a means for discovering novel molecular markers. Results: Forty-seven protein spots that showed reproducible variation were identified by mass spectrometry, corresponding to 23 distinct genes. A positive correlation between transcript and protein level variations was observed for only 7 out of the 23 genes. Proteolytic cleavage accounted for the discrepancies between messenger RNA and protein level changes for 7 genes including calreticulin, PDIA3, PDI, and GRP78. We detected a fragment of each of these 4 endoplasmic reticulum proteins in the culture supernatant of the PLC-PRF5 hepatoma cell line, suggesting that their cleavage leads to release of selected cleaved products in the extracellular compartment. We also detected calreticulin and PDIA3 cleavage products in sera of patients with HCC. A statistically highly significant difference in calreticulin and PDIA3 fragment serum levels between patients with HCC and healthy individuals was observed. Amounts of calreticulin and PDIA3 fragments were also significantly different between patients with HCC and at-risk patients (patients with chronic hepatitis or cirrhosis). Conclusions: Specific isoforms in general and cleavage products in particular should therefore be further evaluated as new markers for HCC.

Abbreviations used in this paper: AFP, α-fetoprotein, ER, endoplasmic reticulum, ESI/Q-TOF MS/MS, electrospray ionization quadrupole time of flight tandem mass spectrometry, HCC, hepatocellular carcinoma, MW, molecular weight, PDI, protein disulfide isomerase