Sporadic somatic mutations in EpCAM-positive hepatocellular carcinoma.

BACKGROUND

The relationship between specific genome alterations and hepatocellular carcinoma (HCC) cancer stem cells (CSCs) remains unclear. In this study, we evaluated the relationship between somatic mutations and epithelial cell adhesion molecule positive (EpCAM) CSCs.

METHODS

Two patient-derived HCC samples (HCC1 and HCC2) were sorted by EpCAM expression and analyzed by whole exome sequence. We measured PCDH18 expression level in eight HCC cell lines as well as HCC1 and HCC2 by real-time quantitative RT-PCR. We validated the identified gene mutations in 57 paired of HCC and matched non-cancerous liver tissues by Sanger sequence.

RESULTS

Whole exome sequencing on the sorted EpCAM and EpCAM HCC1 and HCC2 cells revealed 19,263 nonsynonymous mutations in the cording region. We selected mutations that potentially impair the function of the encoded protein. Ultimately, 60 mutations including 13 novel nonsense and frameshift mutations were identified. Among them, mutation was more frequently detected in sorted EpCAM cells than in EpCAM cells in HCC1 by whole exome sequences. However, we could not confirm the difference of mutation frequency between sorted EpCAM and EpCAM cells by Sanger sequencing, indicating that mutation could not explain intracellular heterogeneity. In contrast, we found novel mutations, including c.2556_2557delTG, c.1474C>G, c.2337A>G, and c.2976G>T, were detected in HCC1 and 3/57 (5.3%) additional HCC surgical specimens. All four HCCs with mutations were EpCAM-positive, suggesting that somatic mutations might explain the intertumor heterogeneity of HCCs in terms of the expression status of EpCAM. Furthermore, EpCAM-positive cell lines (Huh1, Huh7, HepG2, and Hep3B) had lower expression than EpCAM-negative cell lines (PLC/PRL/5, HLE, HLF, and SK-Hep-1), and knockdown in HCC2 cells slightly enhanced cell proliferation.

CONCLUSIONS

Our data suggest that is functionally suppressed in a subset of EpCAM-positive HCCs through somatic mutations, and may play a role in the development of EpCAM-positive HCCs.