Volume 131, Issue 4, Pages 1218-1227 (October 2006) Prickle-1 Negatively Regulates Wnt/β-Catenin Pathway by Promoting Dishevelled Ubiquitination/Degradation in Liver Cancer  David W. Chan, Chung–Yiu Chan, Judy W.P. Yam, Yick–Pang Ching, Irene O.L. Ng  Gastroenterology  Volume 131, Issue 4, Pages 1218-1227 (October 2006) DOI: 10.1053/j.gastro.2006.07.020 Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

Figure 1 Prickle-1 inhibits Wnt/β-catenin activity in human HEK293 cells and SMMC7721 HCC cells. Activation of TCF/LEF-1–dependent luciferase activity as induced by Wnt-1 was inhibited by Prickle-1 in a dose-dependent manner in (A) HEK293 cells and (B) SMMC7721 HCC cells. The relative luciferase activities were the ratios of TOP/FOP normalized to the Renilla luciferase activities. Experiments were performed in triplicate, and error bars represent standard deviation. *P = .043; **P = .001. Gastroenterology 2006 131, 1218-1227DOI: (10.1053/j.gastro.2006.07.020) Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

Figure 2 Prickle-1 reduces the levels of Dvl3 and downstream targets of the Wnt/β-catenin pathway in HCC cells. (A) Transient transfection of pCS-Myc/Prickle-1 reduced the endogenous levels of Dvl3, β-catenin, c-myc, and cyclin D1 in SMMC7721 and BEL7402 cells. β-actin was used as a loading control. (B) Myc/Prickle-1 also reduced the mRNA levels of 2 liver-specific TCF-dependent downstream targets, GS and EGFR. GAPDH was used as a loading control. (C) Prickle-1 reduced the level of β-catenin via the GSK3β/APC/Axin/β-catenin pathway. Treatment with lithium chloride (20 mmol/L, 24 hours), a GSK3β inhibitor, was capable of inhibiting the activity of GSK3β and stabilizing β-catenin. Gastroenterology 2006 131, 1218-1227DOI: (10.1053/j.gastro.2006.07.020) Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

Figure 3 Prickle-1 interacts with and promotes degradation of Dvl3 via the proteasome/ubiquitination pathway. (A) The pCMV2-Flag/Prickle-1– and Dvl3-expressing constructs were cotransfected into HEK293 cells. Cell lysates were immunoprecipitated with the anti-Flag or anti–immunoglobulin G control and revealed by probing with anti-Dvl3 and anti-Flag. The input cell lysate was used as control. (B) The proteasome/ubiquitination of both Dvl3 and β-catenin was inhibited by MG132 in a dose-dependent manner (0, 5, and 10 μmol/L) in PLC/PRF/5 cells. (C) In vivo ubiquitination assay showed that Dvl3 was degraded via the proteasome/ubiquitination pathway. HEK293 cells were transfected with pCMV2-Flag/Dvl3 and pcDNA-HA(Ub)8. Cell lysates were incubated with anti-Flag, and the immunoprecipitates were probed with anti-HA. The positions of polyubiquitinated Dvl3 protein ((Ub)n-Flag/Dvl3) are indicated. (D) In vivo ubiquitination assay showed that Prickle-1 promoted degradation of Dvl3. HEK293 cells were transfected with pCMV2-Flag/Dvl3, pCS-Myc/Prickle-1, and pcDNA-HA(Ub)8 plasmids Cell lysates were incubated with anti-Flag, and the immunoprecipitates were probed with anti-HA. The positions of polyubiquitinated Dvl3 protein ((Ub)n-Flag/Dvl3) are indicated. The same membrane was reprobed with anti-Flag and anti-Myc to detect the expression of Flag/Dvl3 and Myc/Prickle-1, respectively. Gastroenterology 2006 131, 1218-1227DOI: (10.1053/j.gastro.2006.07.020) Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

Figure 4 Prickle-1 mediates ubiquitination of Dvl3 and inhibits Wnt/β-catenin activity through its D-box motifs. (A) A schematic diagram showing the positions of point mutations at D-box 1 (R557M and L560M) and D-box 2 (R699I and L702M). The expression of Prickle-1 and Prickle-1 mutant was defined by Western blot analysis. (B) Interaction of Dvl3 with Prickle-1 and Prickle-1 mutant. pCMV2-Flag/Dvl3 was cotransfected with either pCS-Myc/Prickle-1 or pCS-Myc/Prickle-1 mutant in HEK293 cells. Cell lysates were immunoprecipitated by anti-Flag and probed with anti-Myc (left) or by anti-Myc and probed with anti-Flag (right). The cell lysate immunoprecipitated by anti–immunoglobulin G was used as a negative control. (C) In vivo ubiquitination assay showed that Prickle-1 mutant could not promote Dvl3 ubiquitination as compared with Prickle-1. pCMV2-Flag/Dvl3 was cotransfected with pCS-Myc/Prickle-1 or pCS-Myc/Prickle-1 mutant and pcDNA-HA(Ub8) in HEK293 cells. Cell lysates were incubated with anti-Flag, and the immunoprecipitates were probed with anti-HA. The positions of polyubiquitinated Dvl3 protein ((Ub)n-Flag/Dvl3) are indicated. The same membrane was reprobed with anti-Flag and anti-Myc for the detection of the expression of Flag/Dvl3 and Myc/Prickle-1 or Myc/Prickle-1 mutant, respectively. (D) Prickle-1 inhibited Wnt-1–induced activation of TCF/LEF-1–dependent luciferase activity in HEK293 cells in a dose-dependent manner, whereas Prickle-1 mutant could not. (E) Prickle-1 mutant had less efficiency on reducing the levels of Dvl3, β-catenin, and downstream targets. Western blot analysis revealed that transient transfection of pCS-Myc/Prickle-1 or pCS-Myc/Prickle-1 mutant construct in SMMC7721 (left) or BEL7402 (right) resulted in reduction in the protein level of Dvl3, β-catenin, c-myc, and cyclin D1 by Prickle-1, but not by Prickle-1 mutant, in a dose-dependent manner. β-actin was used as a loading control. Gastroenterology 2006 131, 1218-1227DOI: (10.1053/j.gastro.2006.07.020) Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

Figure 5 Prickle-1 inhibits the cell growth of HCC cells partly via its D-box motifs and reduction of β-catenin level. The cell growth of HCC cells was evaluated by colony formation assay. (A) To examine the function of D-box motifs of Prickle-1 in HCC cells, pCMV2-Flag/Prickle-1, pCMV2-Flag/Prickle-1 mutant, and pCMV2-Flag plasmids were cotransfected with pGK/puromycin vector in a 10:1 ratio into SMMC7721 (left) and BEL7402 (right) cells. (B) To show that the cell growth retardation mediated by Prickle-1 was in a β-catenin–dependent manner, BEL7402 cells with stable expression of β-catenin mutant were generated by transfecting pcDNA3-MycHisA-β-catenin (SA mutant) followed by selection with G418 at 500 μg for 2 weeks. The expression of β-catenin mutant (SA) was verified by Western blot analysis using anti-myc antibody (inset: lane C, BEL7402 parental cells; lane SA, pool of G418-resistant BEL7402 clones). pCMV2-Flag/Prickle-1 or pCMV2-Flag plasmid was then cotransfected with pGK/puromycin vector into the pool of BEL7402 clones with stable expression of β-catenin mutant (SA) as above. After puromycin selection for 14 days, the resistant colonies were stained by Giemsa and scored. Data were obtained from at least 3 independent experiments, and the values are represented as mean ± SD. *P < .001. Gastroenterology 2006 131, 1218-1227DOI: (10.1053/j.gastro.2006.07.020) Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

Figure 6 The Prickle-1 expression level inversely correlates with Dvl3 expression level in human HCCs. (A) Expression level of Prickle-1 by semiquantitative reverse-transcription polymerase chain reaction and Dvl3 level by Western blot analysis in HCC cell lines. (B) The expression level of Prickle-1 inversely correlated with the levels of Dvl3 and β-catenin in human HCC samples t, tumorous tissue. nt, corresponding nontumorous tissue. The expression levels of Prickle-1 were analyzed by semiquantitative reverse-transcription polymerase chain reaction, while Dvl3, β-catenin, c-myc, and cyclin D1 were analyzed by Western blot analysis. GAPDH and β-actin were used as controls in the semiquantitative reverse-transcription polymerase chain reaction and Western blot analyses, respectively. Gastroenterology 2006 131, 1218-1227DOI: (10.1053/j.gastro.2006.07.020) Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions