Activation of the Wnt/β-catenin pathway has been observed in at least 1/3 of hepatocellular carcinomas (HCC), and a significant number of these have mutations in the β-catenin gene. Therefore, effective inhibition of this pathway could provide a novel method to treat HCC. The purposed of this study was to determine whether FH535, which was previously shown to block the β-catenin pathway, could inhibit β-catenin activation of target genes and inhibit proliferation of Liver Cancer Stem Cells (LCSC) and HCC cell lines. Using β-catenin responsive reporter genes, our data indicates that FH535 can inhibit target gene activation by endogenous and exogenously expressed β-catenin, including the constitutively active form of β-catenin that contains a Serine37Alanine mutation. Our data also indicate that proliferation of LCSC and HCC lines is inhibited by FH535 in a dose-dependent manner, and that this correlates with a decrease in the percentage of cells in S phase. Finally, we also show that expression of two well-characterized targets of β-catenin, Cyclin D1 and Survivin, is reduced by FH535. Taken together, this data indicates that FH535 has potential therapeutic value in treatment of liver cancer. Importantly, these results suggest that this therapy may be effective at several levels by targeting both HCC and LCSC.
Digital Object Identifier (DOI)
Gedaly, Roberto; Galuppo, Roberto; Daily, Michael F.; Shah, Malay B.; Maynard, Erin Colleen; Chen, Changguo; Zhang, Xiping; Esser, Karyn A.; Cohen, Donald A.; Evers, B. Mark; Jiang, Jieyun; and Spear, Brett T., "Targeting the Wnt/β-Catenin Signaling Pathway in Liver Cancer Stem Cells and Hepatocellular Carcinoma Cell Lines with FH535" (2014). Surgery Faculty Publications. 11.
Female NOD/SCID mice (NOD.CB17-prkdc∧SCID/NCrSD, 4–5 week old) were purchased from Harlan Animal Research Laboratory (Indianapolis, IN, USA), housed and maintained in our Division of Laboratory Animal Resources animal facility. Mice received filtered air, sterile water and irradiated food ad libitum. Tumors were generated by harvesting first passage of LCSC cells (CelProgen Catalog number 36116-43, San Pedro, CA) that were cultured in CelProgen Liver Cancer Stem Cell Growth Media with Serum, from mid-log growth phase and trypsinized with 0.05% Trypsin/EDTA (Invitrogen). Cells were then washed and resuspended in a 50% mixture of Matrigel (BD Biosceince, San Diego, CA, USA) in CelProgen Liver Cancer Stem Cell Growth Media (serum free) to final cell number of 20,000 cells/ml. A volume of 0.1 ml of the cell suspension (2000 cells) was injected subcutaneously at the right flank of each mouse. The mice were checked for tumor growth every other week and mouse weight was measured. Tumors were found 28 days after inoculation in all the 3 tested mice. When the tumor sizes reached 940–1020 mm3, the mice were euthanized by CO2. The tumors were isolated and fixed in 10% Formalin for 48 h and subsequently changed to 70% ethanol. The tumors were paraffin embedded, cut to 5 µm sections and hematoxylin and eosin stained for histological analysis. A: H&E 200× This photomicrograph depicts a tumor growing in sheets of disorganized, haphazardly-oriented and pleomorphic cells that attest to its poor differentiation. Brisk mitotic activity is present and areas of necrosis are seen, implying rapid growth. B: H&E 400× This tumor is composed of cells with pleomorphic, vesicular nuclei that have prominent nucleoli with occasional macronucleoli. There are large numbers of mitoses, including abnormal forms with quadripolar spindles, reflecting very high proliferative activity.
Figure_S3.tif (118 kB)
Huh7 cells were plated to 96 well plates at 2500 cells/well in 0.1 ml culture medium and cultured for 24 h. The following day, 3H-thymidine and varying concentrations of FH535 were added simultaneously to the designated wells and cultured for 18 h. 3H-thymidine incorporation was assayed as indicated in Materials and Methods. *: p = 0.007 as compared to control; **: p<0.001 as compared to control (n = 6).
Figure_S4.tif (268 kB)
Flow cytometry indicates that a sub-G1 peak is not observed in Huh7 cells treated with FH535, indicating that FH535 does not increased apoptosis as judged by DNA fragmentation. A. Control (DMSO alone). B. FH535 at 7.5 µM. C. FH535 at 15 µM.