Abstract
BackgroundThe 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.MethodsTwo 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.ResultsWhole 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, PCDH18 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 PCDH18 mutation frequency between sorted EpCAM+ and EpCAM− cells by Sanger sequencing, indicating that PCDH18 mutation could not explain intracellular heterogeneity. In contrast, we found novel PCDH18 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 PCDH18 mutations were EpCAM-positive, suggesting that PCDH18 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 PCDH18 expression than EpCAM-negative cell lines (PLC/PRL/5, HLE, HLF, and SK-Hep-1), and PCDH18 knockdown in HCC2 cells slightly enhanced cell proliferation.ConclusionsOur data suggest that PCDH18 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.
Highlights
The relationship between specific genome alterations and hepatocellular carcinoma (HCC) cancer stem cells (CSCs) remains unclear
Recent evidence has suggested that HCC may conform to the cancer stem cell (CSC) hypothesis; this hypothesis proposes that a subset of cells with stem cell-like features divide asymmetrically to generate a heterogeneous cell population, and these stem cell-like cells play a fundamental role in tumor maintenance, chemoresistance, and metastasis [14]
Our aim was to examine whether epithelial cell adhesion molecule (EpCAM) expression is associated with specific genetic mutations in E pCAM+ CSCs or EpCAM-positive HCCs, and to determine whether HCC conforms to the clonal evolution or CSC model
Summary
The relationship between specific genome alterations and hepatocellular carcinoma (HCC) cancer stem cells (CSCs) remains unclear. We evaluated the relationship between somatic mutations and epithelial cell adhesion molecule positive (EpCAM+) CSCs. Hepatocellular carcinoma (HCC) is a leading cause of cancer death worldwide [1, 2], and it is usually associated with specific risk factors including hepatitis B or C virus infection, high alcohol intake, hemochromatosis, and Hayashi et al Cancer Cell Int (2017) 17:94 whole genome analysis of the HCC genome has indicated that one of the most frequent mutations identified in HCC is TERT promoter region mutation [12]. HCC is a heterogeneous disease in terms of its morphology, biological behavior, response to treatment, and clinical outcome. This heterogeneity has been explained by cancer cell clonal evolution, and the step-wise acquisition of genetic mutations [13]. Our previous data suggested that EpCAM is a marker of liver CSCs, and might be used to enrich a highly tumorigenic and chemoresistant cell population
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