We previously reported that human carboxylesterase 1 (CES1), a serine esterase containing a unique N-linked glycosyl group at Asn79 (N79 CES1), is a candidate serological marker of hepatocellular carcinoma (HCC). CES1 is normally present at low-to-undetectable levels in normal human plasma, HCC tumors, and major liver cancer cell lines. To investigate the potential mechanism underlying the suppression of CES1 expression in liver cancer cells, we took advantage of the low detectability of this marker in tumors by overexpressing CES1 in multiple HCC cell lines, including stable Hep3B cells. We found that the population of CES1-overexpressing (OE) cells decreased and that their doubling time was longer compared with mock control liver cancer cells. Using interactive transcriptome, proteome, and subsequent Gene Ontology enrichment analysis of CES1-OE cells, we found substantial decreases in the expression levels of genes involved in cell cycle regulation and proliferation. This antiproliferative function of the N79 glycan of CES1 was further supported by quantitative real-time polymerase chain reaction, flow cytometry, and an apoptosis protein array assay. An analysis of the levels of key signaling target proteins via Western blotting suggested that CES1 overexpression exerted an antiproliferative effect via the PKD1/PKCμ signaling pathway. Similar results were also seen in another HCC cell line (PLC/RFP/5) after transient transfection with CES1 but not in similarly treated non-HCC cell lines (e.g., HeLa and Tera-1 cells), suggesting that CES1 likely exerts a liver cell-type-specific suppressive effect. Given that the N-linked glycosyl group at Asn79 (N79 glycan) of CES1 is known to influence CES1 enzyme activity, we hypothesized that the post-translational modification of CES1 at N79 may be linked to its antiproliferative activity. To investigate the regulatory effect of the N79 glycan on cellular growth, we mutated the single N-glycosylation site in CES1 from Asn to Gln (CES1-N79Q) via site-directed mutagenesis. Fluorescence 2-D difference gel electrophoresis protein expression analysis of cell lysates revealed an increase in cell growth and a decrease in doubling time in cells carrying the N79Q mutation. Thus our results suggest that CES1 exerts an antiproliferative effect in liver cancer cells and that the single N-linked glycosylation at Asn79 plays a potential regulatory role. These functions may underlie the undetectability of CES1 in human HCC tumors and liver cancer cell lines. Mass spectrometry data are available via ProteomeXchange under the identifier PXD021573.
Bibliographical noteFunding Information:
This work was supported by a grant from the Korean Ministry of Health and Welfare (to Y.-K.P., HI16C0257) and a grant from the Science Research Center (2015R1A5A1009024 to J.-H.B.). We also acknowledge the support from the Severance Hospital Gene Bank and the Liver Cancer Specimen Bank funded by the National Research Foundation (2015M3A9B8028339 to H.K.) and the Faculty Research Grant of Yonsei University College of Medicine (6-2016-0122 to D.H.H.). We thank the staff members of the Yonsei Proteome Research Center for their assistance with this project. We thank F. Hoffmann-La Roche, Ltd. for its generous funding of the collaborative research for this work.
Clinical samples (nontumor liver and HCC tissue) were obtained from the archives of the Department of Pathology, Yonsei University (Seoul, Korea) and the Liver Cancer Specimen Bank of the National Research Resource Bank Program affiliated with the Korea Research Foundation of the Ministry of Science and Technology. All HCC diagnoses ( n = 8) were made by pathologists at Severance Hospital, Yonsei University. The clinical information on patients is provided in Supplementary Table 2 . This research was approved by the Institutional Review Board (IRB, 4-2015-0474) of the Yonsei University College of Medicine. Human hepatocyte lysates were purchased from ScienCell Research Laboratories.
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