Notch And Wnt/β-catenin Signaling Pathway Play Important Roles In Activating Liver Cancer Stem Cells

Objectives:Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third most frequent cause of cancer-related death worldwide. Despite advances in its diagnosis and treatment, the prognosis of patients with advanced HCC remains unfavorable. Recent advances in stem cell biology and associated technologies have enabled the identification of minor components of tumorigenic cells, termed cancer stem cells(CSC) or tumor-initiating cells, in cancers such as HCC. There is a small portion named Liver cancer stem cell(LCSCs) has a "stem-ness" potential, such as the capacity of unlimited proliferation, self-renewal, tumorigenicity in immunodeficient mice, differentiation and biological characteristics of tumorigenic capacity cells. Recently research has demonstrated that CD133, CD90, CD13, CD24, CD44 and epithelial cell adhesion molecule (EpCAM), can be used as liver cancer stem cells (LCSCs) surface markers. Notch, Wnt/β-catenin and Hedgehog signaling pathways play important roles in stem cells self-renewal and differentiation development. So we first observed the associations between HCC patients’ long term prognosis and clinical pathological features and the expression of LCSC these surface markers expressed in samples. And we identified the sphere-forming culture enriched liver cancer stem cells by detecting if they obtain the "stem-ness" characteristic. Furthermore, we investigate Notch and Wnt/β-catenin signaling pathway in maintaining of liver cancer stem cells (LCSCs) through a set of intervention studies.Methods:We first used immunohistochemistry (IHC) to detect the expression of liver cancer stem cell surface markers (CD90, CD44, CD133, CD13 and CD24) in 61 HCC patients. And then we analyzed the correlation between them with the clinicopathological prognosis by multivariate analysis. In addition, we used 3D hepatoma cell culture conditions to screening active cells, and detecting the four liver cancer stem cell surface marker molecules (CD90, CD24, CD133 and CD13) by flow cytometry. We detected the sphere-forming culture enriched stem cells self-renewal and metastasis capacity in vitro and in vivo using cloning experiments, NOD/SCID immunodeficient mice tumor formation. And RT-PCR and Western blot showed the expression of "stem-ness" genes (NANOG, OCT3/4, SOX2, and BMI-1) and EMT related genes (Vimentin, E-cadherin, Snail and Twist). Furthermore, XAV939 was utilized to suppress liver cancer stem cells Wnt/ P-catenin, and Wnt ligands Wnt3a or BIO were used to active Wnt/P-catenin signaling pathway. We detected the intervention of Notch pathway activity afterwards; Conversely, we tested the Wnt signaling pathway activity after y-secretase inhibitors-BMS-708163, DAPT or lentivirus LV-ShRNA Notch were used respectively to inhibit the Notchl signaling pathway. Finally, with lentivirus LV-Notchl over-expression of Notchl, Wnt/ P-catenin pathway was explored, and we explored the molecular mechanisms of crosstalk between them.Results:(1) Kaplan-Meier survival analysis showed that overall survival and disease-free survival in CD90CD24CD13CD133+/ high patients was significantly reduced, compared with CD90CD24CD13CD133-/low counterparts; (2) The CD90CD24CD13CD133+ expression in hepatocellular carcinoma not only correlated with the advanced stage of HCC, but also positively correlated with tumor size. (3) Logistic regression analysis showed that the expression of CD90CD24CD13CD133+HCC patients negatively correlated with TNM of tumor; (4) the expression of CD90, CD24, CD133 and CD13 were enriched in liver cancer stem cells (LCSCs); (5) Colony formations in LCSCs from the PLC/PRF/5 and Huh7 was significantly higher than the parental cell. Tumors from LCSCs in NOD/SCID mice is larger than the parent tumor formation, and they higher incidence of metastasis, "stern-ness" genes (NANOG, OCT3/4, SOX2, and BMI-1) and EMT-related genes (Vimentin, E-cadherin, Snail and Twist) expressed higher in LCSCs. (6) Notch signaling pathway and Wnt/β-catenin signaling pathway expressed increased significantly in liver cancer stem cell; (7) DAPT, XAV939 inhibiting Notch and Wnt/β-catenin respectively, inhibited the clone formation, tumorigenic and metastatic ability in NOD/SCID mice and expression of genes (NANOG and SOX2); (8) Expression Notch ligand Jaggedl, intracellular domain receptor NICD, target gene Hesl were significantly reduced after XAV939 inhibiting Wnt in liver cancer stem cells; (9) The application Wnt ligands Wnt3a or BIO activate Wnt/β-catenin signaling pathway and Notchl as well; (10) Conversely, Wnt/β-catenin signal was actived when we use y-secretase inhibitors-BMS-708163 or lentivirus LV-ShRNA Notch inhibit Notchl signaling pathway; (11) Overexpression of Notchl signaling pathway by lentivirus LV-Notchl increased expression of P-catenin.Conclusions: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 Notchl was downstream of Wnt/β-catenin. The active form of Notchl intracellular domain (NICD) expression depended on Wnt/β-catenin pathway activation. Moreover, Notchl negatively contributed to Wnt/β-catenin signaling modulation. Knock down of Notchl with lentivirus NIShRNA up-regulated the active form of β-catenin. Ectopic expression of NICD with LV-Notchl in LCSCs attenuated β-catenin/TCF dependent luciferase activity significantly. In addition, there was a non-proteasome mediated feedback loop between Notchl 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.