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

Abstract

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.

Keywords: Notch; Wnt/β-catenin; cancer stem cells; hepatocellular carcinoma.

Figure 1. Expression of CD90, CD24, CD13 and CD133 in liver cancer cells correlated with poor prognosis in patients with HCC

A. and B. Kaplan-Meier analyses for overall and disease-free survival were compared according to the CD133 and CD13 expression in tumor tissues. C. Patients who had higher advanced TNM Stages HCC (p = 0.0155, One-way ANOVA analysis) presented significantly higher CD13 expression. D. Patients with high CD90 expression had a significantly poorer differentiation status (p < 0.05, t test). E. Kaplan-Meier survival analysis of patients demonstrated that CD90CD24CD13CD133+ primary tumors displayed worse overall and disease-free survival. (log-rank test, p = 0.0062 and 0.0095).

Figure 2. Sphere-forming HCC cells possessed characteristics of cancer stem cells and capacity to metastasize in vivo and in vitro

A. The PLC/PRF/5 sphere-formation cells express higher cancer stem cells markers (CD90, CD24, CD13, CD133) (p < 0.05, t test). The representative spheres from HCC cell lines also shown on the top. B. The PLC/PRF/5 sphere-formation process higher colony formation efficiency (p = 0.044, t test). C. Efficiency of tumor formation and tumor volume of sphere-forming HCC cells (right black arrows) from PLC/PRF/5 and their parental cells (left white arrows) in NOD/SCID mice. D. RT-PCR and western blotting for the detection of stemness-associated genes over-expressed in PLC/PRF/5 sphere-formation cells. E. Transwell migration assay in parental cells and sphere-forming HCC cells (magnification times, 100X and 200X, respectively). F. Representative pulmonary metastasis of sphere-forming SMMC-7721 cells in NOD/SCID mice. Hematoxylin and eosin staining of a pulmonary metastasis tumor (right parts). G. The mRNA and protein expression level of EMT-related genes in sphere-forming SMCC-7721 cells.

Figure 3. Notch and Wnt/β-catenin signaling pathway components are up-regulated in sphere-forming liver cancer stem cells

A. and B. The mRNA and protein expression levels of Notch and Wnt/β-catenin signaling pathway components are up-regulated in sphere-forming liver cancer stem cells. C. and D. Notch and Wnt/β-catenin signaling pathway were inhibited by 20 μM of DAPT and 20 μM XAV939, respetctively.

Figure 4. Notch and Wnt/β-catenin signaling pathways promoted stem-ness characteristics and metastasis potential in sphere-forming liver cancer stem cells

A. and B. Sphere formation and colony formation ability (A) and efficiency of tumor formation (B) in sphere-forming PLC/PRF/5 decreased when inhibiting Notch and Wnt/β-catenin signaling pathway by 20 μM of DAPT and 20 μM XAV939. C. Efficiency of tumor formation and tumor volume of sphere-forming LCSCs were decreased by the blocking of Notch and Wnt/β-catenin. D. Stemness-associated genes (NANOG and SOX2) were significantly down-regulated upon Notch or/and Wnt/β-catenin inhibited (p < 0.05, t test). E. The cancer stem cells surface markers phenotype, CD90, CD44, CD133, were diminished by DAPT or XAV-939. But the decrease observed with DATP and XAV939 combined was no more than that observed by either individual treatment. F. and G. The metastasis capacity in transwell assay and EMT-related genes (Vimentin, Snail and Twist) were decreased in sphere-forming PLC/PRF/5 after inhibition of Notch and Wnt/β-catenin signaling pathway. Error bars represent standard deviation (SD) from at least three independent experiments.

Figure 5. Notch1 was downstream of Wnt/β-catenin signaling in liver cancer stem cells

A. Notch1 signaling pathway was dramatically down-regulated in set of the inhibition of Wnt/β-catenin by XAV939. B. and C. Relative β-catenin/TCF luciferase activity of sphere-forming LCSCs increased depending on the dosage of Wnt3a and BIO. D. IF Staining of Active β-catenin accumulated in sphere-forming LCSCs when treat with BIO (2 μM). E. Notch1 signaling pathway was up-regulated upon the activation of Wnt/β-catenin by Wnt3a (100 ng/ml) and BIO (2 μM). All luciferase values were normalized to Renilla activity (mean ± SD; P < 0:01). P values were determined using a two-tailed Student t-test, type II (see Materials and Methods). Gapdh antibody was used as a loading control. Numbers on western blots correspond to relative quantification.

Figure 6. β-catenin protein levels were diminished by Notch1 in liver cancer stem cells

A. Relative β-catenin/TCF luciferase activity of LCSCs decreased when treated with increasing doses of DAPT for 48 h. B. The activation of Wnt/β-catenin signaling by BIO (2 μM) rescue γ-secretase inhibitor (BMS-708163)-induced suppression of β-catenin/TCF dependent luciferase activity. C. Knockdown of Notch1 by LV-N1ShRNA. D. Reduced Notch1 levels which knockout by LV-N1ShRNA increased the transcriptionally active form of β-catenin protein. E. Relative β-catenin/TCF luciferase activity of sphere-forming LCSCs transfected with the control or LV-N1ShRNA in the presence or absence of BIO (2 μM) or Wnt3a (100 ng/ml; mean ± SD; P < 0:01). F. Notch1 was activited by LV-Notch1 after infected 48 h. G. Relative β-catenin/TCF luciferase activity of sphere-forming LCSCs transfected with the control or actived Notch1 (NICD) construct, LV-Notch1. H. active β-catenin protein levels increased after the over-expression of NICD by LV-Notch1.

Figure 7. A non-proteasome mediated feedback loop between Notch1 and Wnt/β-catenin signaling in LCSCs

The destruction complex in Wnt/β-catenin signaling is composed of Axin, APC and GSK3β. When the destruction complex of is inactivated by Wnt (Wnt3a) or BIO, dephosphorylated (active) β-catenin functions as a transcriptional activator with LEF/TCF. We show that Notch1 may be the downstream of Wnt/β-catenin. Active β-catenin protein levels can be negatively regulated by interaction with Notch. NECD, Notch extracellular domain; NICD, Notch intracellular domain.