Fig. 6: Targeted expression of a non-phosphorylatable mutant of NUMB reduces HCC development induced by alcohol-HCV synergism mouse model.

a Non-phosphorylatable mutant of NUMB (named as NUMB-3A) was generated by serine-to-alanine substitutions of three NUMB phosphorylation sites. b The experimental strategy of tamoxifen-inducible Cre-mediated NUMB-3A expression using Alb::CreERT2; NUMB3A^LSL;NS5A mice. c Incidence of liver tumor (upper left panel), ratio of tumor mass/liver weight (%) (upper middle panel) and liver tumor pictures (upper panel, right) of the four groups of mice was decreased in Alb::CreERT2; NUMB3A^LSL;NS5A mice with Ethanol WD. The visible tumors were measured at the indicated days. Error bars represent ±SEM (n = 3). p Values are shown from a chi-square test *p = 0.0003773 (chi-square test). H&E staining for mouse tumors (bottom left panel). Immunofluorescence staining of Vimentin and AFP in tumor tissues (bottom right panel). Scale bar, 30.32 μm. The representative pictures are shown from three independent experiments. d (top) The percentage of CD133+ TICs in tumor cells is compared among the four different genotypes of mice. (bottom) Tumor volume kinetics of TIC-derived tumors. The percentage of TICs of tumor cells were calculated as mean ± SD (n = 3). p-Values by two-tailed paired t test. *p = 0.022474 (Student’s t test). e (left) Immunoblots of tumor cell lysates isolated from hepatocyte-specific expression of NUMB-3A mice. (right) Schematic presentations of LPS-activated TLR4 inducing NANOG mediated NUMB phosphorylation for p53 loss and TIC self-renewal, and of NUMB-3A-antagonism of this pathway. f Micro-CT imaging and texture-based VRT were reduced tumor size and tumor volume. The bar graph shows changes in tumor volume before and after the aPKCζ inhibitor. Error bars represent ±SEM. p-Values by two-tailed paired t test. *p = 0.00654 (aPKCζ inhibitor; before vs after treatment), *p = 0.00095 (after treatment; vehicle vs aPKCζ inhibitor) (Student’s t test). g Τhe aPKCζ inhibitor treatment reduced liver tumors spontaneously developed in HCV NS5A Tg mice fed alcohol Western diet as shown by ultrasound sonography. h Immunoblots of tumor cell lysates isolated from NS5A Tg mice fed alcohol WD of the aPKCζ inhibitor vs vehicle. i Summary of oncogenic pathways. NUMB phosphorylation and TBC1D15 are mutually required for liver tumorigenesis. Source data are provided as a Source Data file.