Article Figures & Data
Figures
- Figure 1.
Pyrvinium suppresses HH signaling in a CK1α-dependent manner. A, Light-II cells treated with SHH, or the Smo agonist SAG, were subsequently incubated with the indicated doses of pyrvinium, or vehicle (DMSO), and luciferase activity was determined. B, SHH-treated NIH-3T3 cells were treated with DMSO, pyrvinium, or the inactive pyrvinium analog VU211. Twenty-four hours later, RNA was harvested from these cells and the expression of the HH target genes Gli1 and Ptch1 determined relative to that of the housekeeping gene Gapdh. C, primary GCP cells were incubated with BrdUrd and the indicated drugs (cyclopamine, 5 μmol/L; pyrvinium, 100 nmol/L) for 48 hours, followed by quantitation of BrdUrd incorporation using microscopy. D, Light-II cells were infected with lentivirus expressing the indicated shRNA. Seventy-two hours after infection, cells were treated with the indicated agents (SAG, 100 nmol/L; pyrvinium, 1, 10, 100 nmol/L) for 48 hours. RNA was harvested from these cells and the expression of Gli1 determined relative to the expression of Gapdh. E, Light-II cells were treated with the indicated agents in the presence or absence of the Wnt inhibitor IWR-1 (10 μmol/L) and luciferase activity was determined. F, HH or Wnt signaling activity in Gli2−/−; Gli3−/− MEFs was induced with either SAG or Wnt3a conditioned media, in the absence or presence of 10 nmol/L pyrvinium. The expression of the Wnt target gene Dkk1, normalized to the expression of Gapdh, was analyzed as a readout of Wnt activity. Error bars, SEM (n = 3); *, P < 0.05.
- Figure 2.
Pyrvinium inhibits HH signaling downstream of Smo. A, cells expressing a vismodegib-resistant, oncogenic Smo mutant (D473-M2 Smo) were treated with the indicated drugs and then assayed for luciferase activity (left). The effectiveness of vismodegib was validated on Light-II cells treated with the Smo agonist SAG (right). B, NIH-3T3 cells stably expressing Smo–GFP were treated with vehicle, 100 nmol/L SAG, 100 nmol/L pyrvinium, or SAG plus pyrvinium, for 24 hours. Primary cilia localization of Smo was revealed by immunocytochemistry. Smo localization was manually quantitated over five random images. Representative images are shown at the bottom (green, Smo-GFP; blue, DAPI; red, primary cilia). C, Sufu−/− or Sufu+/− MEFs were treated with the indicated agents (5 μmol/L cyclopamine) for 3 days. The expression of Gli1, Ptch1, and Hip was determined and normalized to that of Gapdh. D, the indicated MEFs were treated with the Smo agonist SAG in the presence or absence of pyrvinium (10 nmol/L). Twenty-four hours later, the expression of Gli1 (left) or Ptch1 (right) was determined. E, Light-II cells expressing Myc-Gli1 were treated with the indicated drugs, followed by quantitation of luciferase activity a day later. Error bars, SEM (n = 3); *, P < 0.05.
- Figure 3.
Pyrvinium enhances the degradation of Gli transcription factors. A, NIH-3T3 cells expressing Flag-Gli1 were treated with the indicated agents (vismodegib, 100 nmol/L; cyclopamine, 5 μmol/L), followed by immunoblotting of these cellular lysates. B, NIH-3T3 cells expressing HA-Gli2 were treated with the indicated agents (SAG, 50 nmol/L; pyrvinium, 100 nmol/L), followed by immunoblotting of immunoprecipitated Gli2. C, NIH-3T3 were treated with indicated agents (SAG, 50 nmol/L; pyrvinium, 100 nmol/L), followed by immunoblotting of endogenous Gli3. Quantification of multiple replicates (left) and a representative immunoblot (right) are shown for A–C. Error bars, SEM; NS, not statistically different; *, P < 0.05. D, NIH-3T3 cells stably expressing the indicated shRNA were transfected with a plasmid expressing Myc-Gli1 (1 μg in control shRNA–infected cells and 0.5 μg in CK1α-knockdown cells). These cells were subsequently treated with 10 or 100 nmol/L of pyrvinium, lysed, and then analyzed by immunoblotting. E, HEK 293T cells expressing Flag-Gli1 were treated with 200 nmol/L pyrvinium and 5 μmol/L of MG132 overnight. Anti-Flag immunoprecipitates, from lysates of these cells, were immunoblotted using the indicated antibodies. F, NIH-3T3 cells expressing Myc-Gli1 were treated with 100 μg/mL cycloheximide (CHX) at the indicated time points, in the presence or absence of 100 nmol/L pyrvinium. Immunoblotting was then performed to detect Gli1 and Gapdh levels. Error bars, SEM (n = 3); *, P < 0.05.
- Figure 4.
CK1α associates with Gli transcription factors. A and B, HEK 293T cells transfected with pcDNA3.1-Flag, Flag-Gli1 (A) or pcDNA3, Myc-Gli2 (B) were subjected to immunoprecipitation and immunoblotting with the indicated antibodies. C, pcDNA3.1-Flag (Ctrl) or Flag-Gli1–transfected HEK 293T cells were treated with vehicle or 200 nmol/L pyrvinium for 1 hour, before any detectable changes in Gli1 protein levels. Samples were immunoprecipitated with Flag M2 beads and subject to immunoblot analysis for CK1α or Flag-Gli1. D, immunohistologic staining of CK1α and Gli1 proteins in Ptch+/−-derived medulloblastoma tissue. Scale bar, 100 μm. E, endogenous CK1α (left) or Gli1 (right) was immunoprecipitated from homogenized medulloblastoma tissue using the indicated antisera (2 μg). These immunoprecipitates were then immunoblotted for CK1α or Gli1.
- Figure 5.
Pyrvinium attenuates the growth of a Ptch+/−-derived medulloblastoma allograft. A, RNA was extracted from normal mouse brains (N1 or N2) or Ptch+/−-derived medulloblastoma tissue (T1 or T2). Real-time RT-PCR was then used to detect the expression of the indicated HH or Wnt target genes, normalized to 18S ribosomal RNA. B, allografts of Ptch+/−-derived medulloblastoma were grown in nude mice until they were approximately 100 mm3 in size. These mice were subsequently treated with 5 mg/kg pyrvinium, or vehicle, for the indicated times. The expression of Gli1 or Ptch2 was then quantitated by real-time RT-PCR. Error bars, SEM (n = 5); *, P < 0.05. C, allografts of similar medulloblastoma were grown in nude mice until they were approximately 100 mm3 in size. These mice were subsequently treated with vehicle or pyrvinium (0.8 mg/kg) by subcutaneous injection every 2 days. Tumor volumes were measured at the indicated time points. Error bars, SEM (n = 6); *, P < 0.05. D, representative H&E staining of tumors from vehicle and pyrvinium-treated mice in C. Scale bar, 100 μm. E, following the chronic administration of pyrvinium in mice harboring Ptch+/− medulloblastoma allografts in C, tumors were harvested and subjected to real time RT-PCR analysis for HH or Wnt target genes. Error bars, SEM (n = 6); *, P < 0.05.
Additional Files
Supplementary Figures S1-S3
Files in this Data Supplement:
- Data Supplement - Pyrvinium attenuates SHH signaling via CK1a (Figure S1); Validation of CK1a shRNA (Figure S2); Increased CK1a activity induces Gli1 degradation (Figure S3).
- Data Supplement - Legend for Supplementary Figures S1-S3.
Volume 74, Issue 17
