title: Hepatic stellate cells promote upregulation of epithelial cell adhesion molecule and epithelial-mesenchymal transition in hepatic cancer cells.
abstract: Microenvironment plays an important role in epithelial-mesenchymal transition (EMT) and stemness of cells in hepatocellular carcinoma (HCC). Epithelial cell adhesion molecule (EpCAM) is known as a tumor stemness marker of HCC. To investigate the relationship between microenvironment and stemness, we performed an in vitro co-culture assay. Four HCC cell lines (HepG2, Hep3B, HuH-7 and PLC/PRF/5) were co-cultured with the TWNT-1 immortalized hepatic stellate cells (HSCs), which create a microenvironment with HCC. Cell proliferation ability was analyzed by flow cytometry (FCM) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, while migration ability was assessed by a wound healing assay. Expression of EpCAM was analyzed by immunoblotting and FCM. HCC cell lines were co-cultured with TWNT-1 treated with small interfering RNA (siRNA) for TGF-β and HB-EGF; we then analyzed proliferation, migration ability and protein expression using the methods described above. Proliferation ability was unchanged in HCC cell lines co-cultured with TWNT-1. Migration ability was increased in HCC cell lines (HepG2, Hep3B, HuH-7 and PLC/PRF/5) directly (216.2±67.0, 61.0±22.0, 124.0±66.2 and 51.5±40.3%) and indirectly (102.5±22.0, 84.6±30.9, 86.1±25.7 and 73.9±29.7%) co-cultured with TWNT-1 compared with the HCC uni-culture. Immunoblot analysis revealed increased EpCAM expression in the HCC cell lines co-cultured with TWNT-1. Flow cytometry revealed that the population of E-cadherin-/N-cadherin+ and EpCAM-positive cells increased and accordingly, EMT and stemness in the HCC cell line were activated. These results were similar in the directly and indirectly co-cultured samples, indicating that humoral factors were at play. Conversely, HCC cell lines co-cultured with siRNA‑treated TWNT-1 showed decreased migration ability, a decreased population of EpCAM-positive and E-cadherin-/N-cadherin+ cells. Taken together, humoral factors secreted from TWNT-1 promote upregulation of EpCAM and EMT in hepatic cancer cells.
authors.affiliation: Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Shikata-cho, Kita-ku, Okayama 700-8558, Japan.
authors.forename: Teruya
authors.initials: T
authors.lastname: Nagahara
authors.affiliation: Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Shikata-cho, Kita-ku, Okayama 700-8558, Japan.
authors.forename: Hidenori
authors.initials: H
authors.lastname: Shiraha
authors.affiliation: Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Shikata-cho, Kita-ku, Okayama 700-8558, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Sawahara
authors.affiliation: Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Shikata-cho, Kita-ku, Okayama 700-8558, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Uchida
authors.affiliation: Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Shikata-cho, Kita-ku, Okayama 700-8558, Japan.
authors.forename: Yasuto
authors.initials: Y
authors.lastname: Takeuchi
authors.affiliation: Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Shikata-cho, Kita-ku, Okayama 700-8558, Japan.
authors.forename: Masaya
authors.initials: M
authors.lastname: Iwamuro
authors.affiliation: Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Shikata-cho, Kita-ku, Okayama 700-8558, Japan.
authors.forename: Junro
authors.initials: J
authors.lastname: Kataoka
authors.affiliation: Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Shikata-cho, Kita-ku, Okayama 700-8558, Japan.
authors.forename: Shigeru
authors.initials: S
authors.lastname: Horiguchi
authors.affiliation: Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Shikata-cho, Kita-ku, Okayama 700-8558, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kuwaki
authors.affiliation: Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Shikata-cho, Kita-ku, Okayama 700-8558, Japan.
authors.forename: Hideki
authors.initials: H
authors.lastname: Onishi
authors.affiliation: Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Shikata-cho, Kita-ku, Okayama 700-8558, Japan.
authors.forename: Shinichiro
authors.initials: S
authors.lastname: Nakamura
authors.affiliation: Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Shikata-cho, Kita-ku, Okayama 700-8558, Japan.
authors.forename: Akinobu
authors.initials: A
authors.lastname: Takaki
authors.affiliation: Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Shikata-cho, Kita-ku, Okayama 700-8558, Japan.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Nouso
authors.affiliation: Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Shikata-cho, Kita-ku, Okayama 700-8558, Japan.
authors.forename: Kazuhide
authors.initials: K
authors.lastname: Yamamoto
mesh_terms: D000951:Antigens, Neoplasm / Q000096:biosynthesis*; D006528:Carcinoma, Hepatocellular / Q000473:pathology*; D015815:Cell Adhesion Molecules / Q000096:biosynthesis*; D045744:Cell Line, Tumor; D002465:Cell Movement; D018920:Coculture Techniques; D000071858:Epithelial Cell Adhesion Molecule; D058750:Epithelial-Mesenchymal Transition / Q000502:physiology*; D005434:Flow Cytometry; D055785:Gene Knockdown Techniques; D055166:Hepatic Stellate Cells / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D008113:Liver Neoplasms / Q000473:pathology*; D034741:RNA, Small Interfering; D014162:Transfection; D059016:Tumor Microenvironment / Q000502:physiology; D015854:Up-Regulation
chemical_list: D000951:Antigens, Neoplasm; D015815:Cell Adhesion Molecules; C499379:EPCAM protein, human; D000071858:Epithelial Cell Adhesion Molecule; D034741:RNA, Small Interfering