Notch and Wnt/β-catenin signaling pathway play important roles in activating liver cancer stem cells

Human hepatocellular carcinoma (HCC) is driven and maintained by liver cancer stem cells (LCSCs) that display stem cell properties. These LCSCs are promoted by the intersecting of Notch and Wnt/β-Catenin signaling pathways. In this study, we demonstrate that LCSCs with markers CD90, CD24, CD13, and CD133 possess stem properties of self-renewal and tumorigenicity in NOD/SCID mice. The increased expression of these markers was correlated with advanced disease stage, larger tumors, and worse overall survival in 61 HCC cases. We also found that both Notch and Wnt/β-catenin signaling pathways played important roles in increasing the stem-ness characteristics of LCSCs. Our data suggested that Notch1 was downstream of Wnt/β-catenin. The active form of Notch1 intracellular domain (NICD) expression depended on Wnt/β-catenin pathway activation. Moreover, Notch1 negatively contributed to Wnt/β-catenin signaling modulation. Knock down of Notch1 with lentivirus N1ShRNA up-regulated the active form of β-catenin. Ectopic expression of NICD with LV-Notch1 in LCSCs attenuated β-catenin/TCF dependent luciferase activity significantly. In addition, there was a non-proteasome mediated feedback loop between Notch1 and Wnt/β-catenin signaling in LCSCs. The central role of Notch and the Wnt/β-catenin signaling pathway in LCSCs may provide an attractive therapeutic strategy against HCC.


Colony formation assay
The PLC/PRF/5 and Huh7 spheres were dissociated as described above. Trypan blue staining was used to determine cell viability, and more than 95% of cells with viability were acceptable for the following experiments. The single cells were seeded in DMEM with 10% FBS at a density of 2000 cells/well on 6-well plates that were pre-coated with Matrigel (BD Biosciences, San Jose, CA, USA). After 2 weeks, the colony formation ability was assessed by counting the number of colonies (> 70 cells) under a microscope after crystal violet staining (Sigma-Aldrich, St. Louis, MO, USA). Representative views were photographed. The parental cells were plated at the same density as the control.
Briefly, the parental adherent monolayer, tumor sphere SMMC-7721, PLC/PRF/5 and Huh7 cells or 20 uM DATP/15 uM XAV939 treated sphere cells were resuspended in serum-free DMEM. The upper chamber was loaded with 2 × 10 4 cells/100 µl suspension and the lower chamber was loaded with 500 µl DMEM with 15% FBS. Following culture for 36 h, the cells in the upper chamber were removed and the lower chamber filter was fixed with 4% paraformaldehyde and stained with crystal violet. The number of cells that migrated to the undersurface of the membrane was counted and six randomly selected fields were analyzed. Three independent experiments were performed.

In vivo tumorigenicity experiments
All animal experiments were approved by the Institutional Animal Care and Use Committee of the Huazhong University of Science and Technology, which in according with the U.S. Public Health Service Policy on Use of Laboratory Animals. The PLC/PRF/5 parental and the third passage of sphere-forming cells with or without 10 μM DMSO, 20 μM DATP or 15 μM XAV939 pretreated for 7 days were used in tumorigenicity experiments. various numbers of viable single cells were subcutaneously injected into 5-week-old NOD/SCID male mice (animal centre of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China) in serum-free DMEM/Matrigel (1:1) using 100 μl microsyringe. Tumor-bearing mice and controls housed in the Animal Institute of Tongji Medical College in laminar flow cabinets for 45 days and sacrificed. The length (L) and width (W) of the tumors were measured externally with a vernier caliper every 3 days. Tumor volume was calculated with the formula: V = (L × W 2 )/2. Then the tumors were harvested for further examination.

Flow cytometry
We've analyzed the expression of all markers in 3rd passage of sphere-forming cells simultaneously by flow cytometry on IMM LSRII (Starzl Transplantation Institute, University of Pittsburgh School of Medicine,

RNA isolation, quantitative real-time PCR (qRT-PCR)
Total RNA was isolated from cultured cells using TRIzol reagent (Invitrogen) and cDNA was synthesized from 100 ng RNA using PrimeScript RT reagent Kit (Takara, Japan). Real-time quantitative PCR using SYBRP remix (TaKaRa) was performed as described previously [6]. Amplifications were performed in a Step One Real-Time PCR system (Applied Biosystems, USA) following the manufacturer's instructions. The expression of RNA was determined from the threshold cycle (Ct) and the relative expression levels were calculated by the 2-∆∆Ct method. All samples were assayed in triplicate. The primer sequences used to amplify specific target genes are listed in Supplementary Table S5.