Arsenic antagonizes the Hedgehog pathway by preventing ciliary accumulation and reducing stability of the Gli2 transcriptional effector

Abstract

Aberrant Hedgehog (Hh) pathway activation has been implicated in cancers of diverse tissues and organs, and the tumor growth-inhibiting effects of pathway antagonists in animal models have stimulated efforts to develop pathway antagonists for human therapeutic purposes. These efforts have focused largely on cyclopamine derivatives or other compounds that mimic cyclopamine action in binding to and antagonizing Smoothened, a membrane transductory component. We report here that arsenicals, in contrast, antagonize the Hh pathway by targeting Gli transcriptional effectors; in the short term, arsenic blocks Hh-induced ciliary accumulation of Gli2, the primary activator of Hh-dependent transcription, and with prolonged incubation arsenic reduces steady-state levels of Gli2. Arsenicals active in Hh pathway antagonism include arsenic trioxide (ATO), a curative agent in clinical use for acute promyelocytic leukemia (APL); in our studies, ATO inhibited growth of Hh pathway-driven medulloblastoma allografts derived from Ptch+/-p53-/- mice within a range of serum levels comparable to those achieved in treatment of human APL. Arsenic thus could be tested rapidly as a therapeutic agent in malignant diseases associated with Hh pathway activation and could be particularly useful in such diseases that are inherently resistant or have acquired resistance to cyclopamine mimics.

Fig. 1.

Arsenicals as potent and specific antagonists of the Hh pathway. (A) Sodium arsenite, ATO, and PAO inhibited Hh reporter activity. NIH 3T3 cells transfected with an Hh-responsive reporter (Gli-luciferase) were treated with the indicated concentrations of arsenicals in the presence of ShhN. Reporter activities in the presence of arsenicals were normalized to activity in the vehicle control. (B) ATO inhibited Gli-luciferase reporter activity in a dose-dependent manner with an IC₅₀ of 0.7 μM. (C and D) Arsenic inhibits the Hh pathway but not the Ras or Wnt pathways. (C) Ras pathway activity was assayed with 4× SRE-luciferase reporter in NIH 3T3 cells cotransfected with YFP or constitutively active K-rasG12V in the presence or absence of 10 μM of sodium arsenite. As controls, Gli-luciferase reporter activities in the presence or absence of sodium arsenite were included also. (D) For Wnt assay, NIH 3T3 cells were transfected with a Wnt reporter (7× TCF/LEF-luciferase) and treated with increasing concentrations of ATO in the presence or absence of Wnt3a. In A–D, specific reporter activities were normalized to activities of a cotransfected, constitutively active Renilla luciferase reporter. All experiments were repeated more than three times. Error bars indicate SD.

Fig. 2.

ATO targets Gli transcription factors. (A) ATO inhibited ShhN- or SAG-induced reporter activation, whereas cyclopamine blocked ShhN-induced reporter activity and was less effective in blocking SAG-induced reporter activity. (B) ATO but not cyclopamine inhibited constitutive reporter activity produced by transfection of mSmoA1 (corresponding to human SMOM2). (C) ATO but not cyclopamine inhibited high constitutive Gli-luciferase reporter activity in Sufu^−/− MEFs. Note that cyclopamine also blocked ShhN-inducible reporter activity in Sufu^−/− MEFs cotransfected with Sufu. (D) ATO but not cyclopamine inhibited reporter activity produced by Gli1 or Gli2 cotransfection. All experiments were repeated more than three times. Error bars indicate SD.

Fig. 3.

ATO affects ciliary trafficking and stability of Gli2. (A and B) ATO blocks ShhN-induced ciliary accumulation of Gli2 but not Smo. NIH 3T3/HA-Gli2 cells were incubated in the presence or absence of ShhN and ATO for 20 h, as indicated. (A) Cells were stained to visualize Smo, Gli2, acetylated tubulin or detyrosinated tubulin, and DNA (DAPI). (B) The levels of ciliary Smo and Gli2 were quantified from immunofluorescence images. Error bars indicate SD. (C) Prolonged treatment with ATO destabilized Gli2 protein. NIH 3T3/HA-Gli2 cells were incubated in the presence or absence of ShhN and ATO for up to 30 h, as indicated. Gli2 repressor, present in the resting state, was dramatically reduced by 6 h of ShhN treatment, whereas the levels of full-length Gli2 remained stable. In the presence of ATO, Gli2 full-length protein levels began to decrease by 20 h. IP, immunoprecipitation; WB, Western blot.

Fig. 4.

Arsenic inhibits growth of Hh activity-induced medulloblastoma allografts in nude mice. (A and B) Primary medulloblastomas from Ptch^+/−p53^−/− mice were dissected and grafted into athymic nude mice. The mice were injected i.p. daily with PBS (control) or three different doses of ATO. Representative mice photographed at day 23 are shown in A. ATO suppressed tumor growth in a dose-dependent manner. The changes in average tumor volumes are shown as a function of time in B. (n = 4 per group; *P < 0.05; **P < 0.01). Error bars show SD.

Fig. 5.

Arsenic suppresses ShhN-induced pathway activity mediated by the cyclopamine- and GDC-0449–resistant mutant SmoD477G. The effects of GDC-0449 (A), cyclopamine (Cyc) (B), and ATO (C) on ShhN-induced Gli-luciferase reporter activity were measured in Smo^−/− cells transfected with Smo or SmoD477G. Although both Smo and SmoD477G rescued ShhN-inducible reporter activity in Smo^−/− cells (Fig. S6), this activity was differentially sensitive to inhibition. GDC-0449 thus did not significantly suppress SmoD477G-mediated reporter activity at concentrations 40-fold above its IC₅₀ for Smo (A), whereas cyclopamine suppressed SmoD477G-mediated activity with an IC₅₀ 8.4-fold higher than that of Smo (B). ATO suppression of reporter activity mediated by Smo and SmoD477G was indistinguishable (C). Error bars indicate SD.