The Hedgehog pathway effector smoothened exhibits signaling competency in the absence of ciliary accumulation

Abstract

Misactivation of the seven-transmembrane protein Smoothened (Smo) is frequently associated with basal cell carcinoma and medulloblastoma. Cellular exposure to secreted Hedgehog (Hh) protein or oncogenic mutations in Hh pathway components induces Smo accumulation in the primary cilium, an antenna-like organelle with mostly unknown cellular functions. Despite the data supporting an indispensable role of the primary cilium in Smo activation, the mechanistic underpinnings of this dependency remain unclear. Using a cell-membrane-impermeable Smo antagonist (IHR-1), we demonstrate that Smo supplied with a synthetic agonist or activated with oncogenic mutations can signal without ciliary accumulation. Similarly, cells with compromised ciliary Smo trafficking due to loss of the phosphatidylinositol-4-phosphate 3-kinase (PI3K)-C2α retain transcriptional response to an exogenously supplied Smo agonist. These observations suggest that assembly of a Smo-signaling complex in the primary cilium is not a prerequisite for Hh pathway activation driven by Smo agonists or oncogenic Smo molecules.

Figure 1. Identification of IHR-1, an Hh pathway inhibitor that targets the heptahelical domain of Smo

(A) Schematic of the screening platform used to identify novel Hh pathway antagonists. Compounds that induced loss of firefly luciferase (FL) activity, which reports cell autonomous Hh-dependent Gli transcriptional activity (GliBS reporter), without changing levels of a control Renilla luciferase (RL) in 3T3-ShhFL cells were identified as potential Hh pathway inhibitors. (B) IC50 of the seven most potent IHR compounds as determined using the 3T3-ShhFL cells. Cyclopamine and SANT1 are established Smo inhibitors. (C) IHR compounds directly target Smo. Cells transfected with either Smo or Frizzled 4 (Fzd4, a Smo-related molecule that functions in Wnt-mediated signaling) were treated with a fluorescently labeled Smo antagonist (BD-cyclopamine) in the presence of one of seven IHR compounds identified from the chemical screen or a control compound. Percent of Bodipy-labeled cells were then scored using FACS analysis to determine the ability of unlabeled compounds (1μM) to compete with BD-cyclopamine (5nM) for Smo binding. Cyclopamine, SANT1, and SAG directly bind to Smo. IWR-1 is a Wnt/β-catenin pathway inhibitor and serves as a negative control. (D) IHR-1 prevents Smo from accumulating in the primary cilium. % of NIH-3T3 cells with Smo-GFP co-localizing with acetylated tubulin (labeling the primary cilium) were quantified (n= 100). Conditioned medium containing ShhN protein (ShhN CM). Vismodegib is a FDA-approved Smo antagonist. Data are mean+SEM of three fields. (E) IHR-1 blocks Hh-induced suppression of Gli processing into repressor molecules. Gli3F= full-length Gli3; Gli3R= Gli3 repressor. (F) Structures of IHR-1 and a fluorophore-labeled IHR-1 compound (IHR-Cy3). (G) IHR-1 and SAG target the Smo heptahelical pocket. SAG targeting the Smo 7TM domain but not 20(S)-hydroxylcholesterol [20(S)-OHC] targeting the extracellular Smo cysteine rich domain competes with IHR-Cy3 for Smo binding. Cells transfected with either Smo or Fzd4 were treated with 5μM IHR-Cy3 with and without 10μM SAG or 20(S)-OHC. Percent of Cy3-labeled cells were then scored using FACS analysis.

Figure 2. Markedly different cell membrane permeability of IHR-1 and its derivative IHR-NAc correlate with their ability to inhibit SAG-induced Hh pathway activation

(A) IHR-1 is incapable of blocking SAG-induced Hh pathway response. IC50s of IHR-1 against Hh pathway response induced by either expression of Shh or SAG (0.1μM) treatment were obtained in NIH-3T3 cells transfected with the GliBS reporter. Data are mean+SEM from triplicate experiments. (B) The IHR-1 derivative IHR-NAc exhibits greater maximal inhibitory activity against SAG-induced pathway response as compared to IHR-1. IHR-NAc activity in Hh-induced response is similar to that observed for IHR-1. Data are mean+SEM from triplicate experiments. (C) Basal wild-type Smo activity is equally sensitive to IHR-1 and IHR-NAc. Data are mean+SEM from triplicate experiments. (D) Schematic of a transwell assay to measure cellular permeability of small molecule modulators of Smo activity. Smo antagonists are deposited in a growth chamber that is separated into two chambers by a monolayer of Caco-2 cells and a porous membrane (0.4μm). (E) IHR-NAc exhibits improved ability to transverse a cell monolayer as compared to IHR-1. Mass spectrometric analysis of compound levels that have traversed the Caco-2 cell monolayer for IHR-1 and IHR-NAc. Indicated compounds (10μM) were deposited in the donor well and concentration of compound found in receiver well determined by mass spectrometry. Propranolol and nadolol are typical positive and negative controls for permeability, respectively. Data are mean+SEM from triplicate experiments with two separate studies reported. (F) Schematic of parallel artificial membrane permeability assay (PAMPA). A phospholipid membrane separates the donor and receiving chambers. (G) IHR-NAc exhibits improved ability to transverse a lipid bilayer as compared to IHR-1. Verapamil and atenolol represent permeable and impermeable reference molecules, respectively. IHR-NAc exhibits ~100 fold greater cell membrane permeability than IHR-1. N=3 in each experiment. (H) Model of IHR-1 and IHR-NAc action in SAG-stimulated cells. Extracellularly exposed Smo is accessible to both IHR-1 and IHR-NAc. However, intracellularly localized activated Smo is accessible only to the cell permeable IHR-NAc.

Figure 3. IHR-1 blocks Smo accumulation in the primary cilium without inhibiting SAG-induced pathway response

(A) IHR-NAc but not IHR-1 blocks SAG-induced depletion of Gpr161 from the primary cilium. % of IMCD3 cells with Gpr161 localized to the primary cilium (labeled with an acetylated tubulin antibody) is indicated for each chemical condition (N=100). Compound concentrations: 0.1 μM SAG, 10μM IHR-1, 10μM IHR-NAc. Data are mean+SEM of three fields. (B) IHR-NAc but not IHR-1 blocks Smo-mediated disengagement of Gli3 proteolytic processing and Sufu/Gli interaction. NIH-3T3 cells were treated with SAG and IHR-1 or IHR-NAc. Cellular lysates isolated 24hrs later were either directly subjected to Western blot analysis or immunoprecipitation with a Sufu antibody. Controls for Sufu immunoprecipitation are provided in Figure S5. (C) SAG-induced Gli2 accumulation at the ciliary tip is blocked by IHR-NAc but not IHR-1. The % of NIH-3T3 cells with Gli2 at the primary cilium tip was quantified (N=50) at each time point subsequent to addition of SAG (0.1μM) with or without IHR-1 or IHR-NAc (each 10μM). Data are mean+SEM of three fields. Representative images are shown on the right. (D) IHR-1 and IHR-NAc both inhibit Smo accumulation in the primary cilium. % of NIH-3T3 cells with endogenous Smo in primary cilia were quantified at each time point as described in Figure 3C. Data are mean+SEM of three fields.

Figure 4. Loss of PI3K-C2α uncouples Smo accumulation and activity induced by SAG

(A) Validation of on-target effect of a lentivirally delivered Pik3c2α shRNA construct in NIH-3T3 cells. (B) Representative images of primary cilia and Smo in cells harboring a Pik3c2α shRNA construct in the presence of SAG. (C) Quantification of cilia with Smo accumulation in the presence of SAG in cells treated with a control or Pik3c2α shRNA construct. Data are mean+SEM of three fields. (D) SAG induces similar transcription of Ptch1 in cells harboring a control or Pik3c2α shRNA construct. Data are mean+SEM from triplicate experiments.

Figure 5. Extraciliary oncogenic Smo contributes to deviant Gli activation

(A) Location of two cancer-associated Smo mutations superimposed on a crystal structure of the Smo heptahelical domain [modified from (Wang et al., 2013)]. (B) SmoM2 and SmoL412F exhibit similar levels of activity. The GliBS reporter system was used to monitor Gli activity in cells. Data are mean+SEM from triplicate experiments. (C) IHR-NAc but not IHR-1 is capable of inhibiting Hh pathway response induced by SmoM2 and SmoL412F. The GliBS reporter system was used to monitor Gli activity in cells overexpressing SmoM2 or SmoL412F. Data are mean+SEM from triplicate experiments. (D) IHR-1 and IHR-NAc both block SmoM2 and SmoL412F accumulation in the primary cilia. % of cells with SmoM2 localized to the primary cilium was quantified as before (N=100). Data are mean+SEM of three fields.

Figure 6. Model of Smo signaling in response to SAG, oncogenic mutations, and Hh

In the presence of the synthetic Smo agonist SAG or in cancers expressing a constitutively active Smo protein, Gli is predominantly regulated by Smo found in an extraciliary compartment. On the other hand, Hh-induced Smo activation in the primary cilium may be dependent upon Smo interaction with an endogenous small molecule.