title: Induction of tumor initiation is dependent on CD44s in c-Met⁺ hepatocellular carcinoma.
abstract: BACKGROUND
  Hepatocellular carcinoma (HCC) patients with active hepatocyte growth factor (HGF)/c-Met signaling have a significantly worse prognosis. c-Met, a high affinity receptor for HGF, plays a critical role in cancer growth, invasion and metastasis. c-Met and CD44 have been utilized as cell surface markers to identify mesenchymal tumor-initiating stem-like cells (TISC) in several cancers including HCC. In this work, we examine the complex relationship between c-Met and CD44s (standard form), and investigate the specific role of CD44s as a tumor initiator and stemness marker in HCC.
  
  
  METHODS
  Gene and protein expression assays were utilized to investigate the relationship between CD44s and c-Met in HCC cell lines. Tumor-sphere assays and in vivo tumor assays were performed to investigate the role of CD44+ cells as TISCs. Student's t-test or one-way ANOVA with Tukeys post-hoc test was performed to test for differences amongst groups with a p < .05 as significant.
  
  
  RESULTS
  In an immunohistochemical and immunoblot analysis of human HCC samples, we observed that more than 39% of human HCC samples express c-Met and CD44. To study the relationship between c-Met and CD44, we used MHCC97-H cells, which are CD44(+)/c-Met(+). The knockdown of c-Met in MHCC97-H cells decreased CD44s, reduced TISC characteristics and decreased tumorsphere formation. Furthermore, we demonstrate that the inhibition of PI3K/AKT signaling decreased CD44s expression and subsequently decreased tumorsphere formation. The down-regulation of CD44s leads to a significant loss of a TISC and mesenchymal phenotype. Finally, the down-regulation of CD44s in MHCC97-H cells decreased tumor initiation in vivo compared with the scrambled control.
  
  
  CONCLUSIONS
  In summary, our data suggest that CD44s is modulated by the c-Met-PI3K-AKT signaling cascade to support a mesenchymal and TISC phenotype in HCC cells. Moreover, c-Met could be a potential therapeutic drug for targeting HCC cells with TISC and mesenchymal phenotypes.
authors.affiliation: Department of Pediatrics and Pharmacology, The Pennsylvania State University, College of Medicine, Penn State Children's Hospital, Hershey, PA, USA. danghi@mail.nih.gov.|Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, 37 Convent Drive, Bethesda, MD, 20892, USA. danghi@mail.nih.gov.
authors.forename: Hien
authors.initials: H
authors.lastname: Dang
authors.affiliation: Department of Pediatrics and Pharmacology, The Pennsylvania State University, College of Medicine, Penn State Children's Hospital, Hershey, PA, USA. ssteinway@hmc.psu.edu.
authors.forename: Steven N
authors.initials: SN
authors.lastname: Steinway
authors.affiliation: Department of Pediatrics and Pharmacology, The Pennsylvania State University, College of Medicine, Penn State Children's Hospital, Hershey, PA, USA. wud1@psu.edu.
authors.forename: Wei
authors.initials: W
authors.lastname: Ding
authors.affiliation: Department of Pediatrics and Pharmacology, The Pennsylvania State University, College of Medicine, Penn State Children's Hospital, Hershey, PA, USA. carl_rountree@bshsi.org.|Bon Secours St. Mary's Hospital, 5875 Bremo Road, MOB South Suite 303, Richmond, VA, 23226, USA. carl_rountree@bshsi.org.
authors.forename: Carl B
authors.initials: CB
authors.lastname: Rountree
mesh_terms: D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D002471:Cell Transformation, Neoplastic / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D018960:Hyaluronan Receptors / Q000096:biosynthesis*; D008113:Liver Neoplasms / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D008819:Mice, Nude; D014411:Neoplastic Stem Cells / Q000378:metabolism* / Q000473:pathology; D020794:Receptor Protein-Tyrosine Kinases / Q000096:biosynthesis*
chemical_list: C473647:CD44S antigen; D018960:Hyaluronan Receptors; C080835:RON protein; D020794:Receptor Protein-Tyrosine Kinases