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Hedgehog Signaling Promotes the Degradation of Tumor Suppressor Sufu Through the Ubiquitin–Proteasome Pathway

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Hedgehog Signaling Promotes the Degradation of Tumor Suppressor Sufu Through the Ubiquitin–Proteasome Pathway Oncogene (2009) 28, 492–499 & 2009 Macmillan Publishers Limited All rights reserved 0950-9232/09 $32.00 www.nature.com/onc ORIGINAL ARTICLE Hedgehog signaling promotes the degradation of tumor suppressor Sufu through the ubiquitin–proteasome pathway S Yue1,2, Y Chen1 and SY Cheng1,2 1Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, Jiangsu, PR China and 2The Cancer Center, Nanjing Medical University, Nanjing, Jiangsu, PR China Sustained Sonic hedgehog (Shh) pathway activity is that binding of the ligand to itsreceptor, Patched (Ptch), associated with tumorigenesis in a wide variety of tissues. alleviatesan inhibition from Ptch on a downstream Mutational inactivation of Shh receptor Patched (Ptch) membrane protein, Smoothened (Smo), which ulti- and a downstream gene Suppressor of fused (Sufu), both mately activatestarget genesthrough a family of three of which are negative regulators of the pathway, increases Gli transcription factors (Jacob and Lum, 2007; Ruiz i susceptibility to cerebellum cancer in humans and mice. Altaba et al., 2007; Wang et al., 2007). The Gli proteins Sufu is a binding partner of Shh pathway transcription recognize a common cis-element via a zinc-finger DNA factor Gli. Recent data indicate that inactivation of Sufu, binding domain, and can either activate or repress through either gene targeting in mice or RNAi-mediated transcription depending on whether Gli2 and Gli3 are silencing in cultured fibroblasts, is sufficient to turn on Shh processed by a proteolytic cleavage that converts them target gene expression. Here, we report that Sufu is into repressors. Gli1, a target of Shh signaling itself, degraded rapidly in certain cancer cells and we show that doesnot undergo thiscleavage event, and therefore Shh signaling promotes ubiquitination of Sufu, which always acts as an activator. Expression of Gli1 is leads to its destruction in the proteasomes. We identified elevated in a variety of tumorsasthe consequence of an ubiquitin attachment site on K257 of Sufu, and showed Shh pathway activation, and ectopic expression of Gli1 that Sufu-K257R mutant is more potent as a transcription and Gli2 results in skin cancers in mice (Ruiz i Altaba repressor and cell growth inhibitor because of increased et al., 2007). stability. These results indicate that Shh signaling Suppressor of Fused (Sufu), identified as a genetic regulates Sufu activity by inducing its turnover via the suppressor of fused mutantsin Drosophila (Preat et al., ubiquitin–proteasome system. 1993), isan important regulator of Gli. Genetic and Oncogene (2009) 28, 492–499; doi:10.1038/onc.2008.403; biochemical studies indicated that Drosophila Sufu published online 10 November 2008 binds, stabilizes and retains Ci (Cubitus interruptus) in the cytoplasm (Monnier et al., 1998; Ohlmeyer and Keywords: hedgehog; Sufu; ubiquitin; lung cancer Kalderon, 1998; Ingham and McMahon, 2001). These biochemical activitiesof Sufu are conservedin mammals (Kogerman et al., 1999; Jacob and Lum, 2007), and mammalian Sufu was also found to possess a nuclear activity in repressing Gli-mediated transcription via its Introduction interaction with SAP18 (Cheng and Bishop, 2002), a component of the Sin3-HDAC corepressor complex. Sonic hedgehog (Shh) isone of the primary morpho- Sufu mutations are present in 9% of medulloblastoma genic signals that specify patterns of cell growth and patients(Taylor et al., 2002), and mice that carry one differentiation during vertebrate development (Ingham target-inactivated Sufu allele exhibited heightened risk and McMahon, 2001). Mutationsin variouscompo- of developing medulloblastoma in p53 null background nentsof itssignalingpathway frequently occur in (Lee et al., 2007). Although Drosophila Sufu is not tumors originated from the skin, cerebellum and skeletal essential for embryonic development, mouse embryos muscle (Ruiz i Altaba et al., 2002), and abnormal devoid of Sufu die early because of multiple develop- pathway activity is associated with a subset of lung, mental anomaliesincluding failure to closethe neural digestive tract, pancreatic and prostate cancers (Beachy tube (Cooper et al., 2005; Svard et al., 2006), a et al., 2004). Shh signaling follows an unusual logic, in phenotype shared by inactivation of Ptch1. RNAi- mediated gene silencing experiment in NIH3T3 cells showed that attenuation of Sufu activity is sufficient to Correspondence: Dr SY Cheng, Department of Developmental activate Gli-mediated transcription (Varjosalo et al., Genetics, Nanjing Medical University, 140 Hanzhong Road, Xianzhi 2006). Lou 1908, Nanjing, Jiangsu, PR China. E-mail: [email protected] Given the important rolesof Sufu in regulating Gli Received 4 June 2008; revised 24 September 2008; accepted 1 October activities, we set out to investigate how Sufu itself is 2008; published online 10 November 2008 regulated in response to Shh signaling. Because Sufu is a Ubiquitination-mediated degradation of Sufu SYueet al 493 unique protein with few recognizable featuresexcept an To determine whether the low level of Sufu in NCI- N-terminal globular domain, we decided to examine H322M cellswasindeed becauseof reduced protein whether itsprotein turnover issubjectto control. Our stability, we treated the cells with MG132, a specific results show that Shh promotes Sufu degradation inhibitor of proteasomes, and observed accumulation through the ubiquitin–proteasome pathway, and this of Sufu starting at 6 h and continued up to 12 h Shh-induced degradation may account for reduced (Figure 1d). Blocking proteasomes also led to accumu- stability of Sufu in certain tumor cell lines. lation of Sufu in two prostate (PC3 and DU145) and a breast (MD-MBA-231) cancer cell lines (Figure 1d). Because the above cancer cell lines all have been reported to have an elevated Shh pathway (Beachy et al., Results 2004; Yuan et al., 2007), we decided to test whether Sufu turnover in NCI-H322M cells is subject to Shh signaling We began our analyses by assessing Sufu levels in a control. To that end, we measured the rate of Sufu decay panel of lung cancer cell lines, and found that the after blocking protein synthesis with cycloheximide. steady-state level of Sufu protein was drastically reduced Under thiscondition, the level of Sufu rapidly decreased in NCI-H322M cellswhereassignificant levelsof Sufu to below half of itsoriginal amount in 6 h, but failed to were expressed in the rest of lung cancer cell lines change significantly even after 24 h when the cells were examined and in human HEK293 and mouse NIH3T3 also treated with cyclopamine, a specific inhibitor of cells(Figure 1a). The reduction of Sufu in NCI-H322M Smo (Figure 2a). To determine whether Sufu turnover is cells is likely because of post-translational regulation normally controlled by Shh signaling, we repeated the because the level of Sufu transcript in these cells is comparable with that of HEK293 and NIH3T3 cellsas determined by standard and real-time reverse transcrip- tion PCR (RT–PCR) experiments(Figures1b and c). Figure 2 Shh signaling promotes turnover of Sufu protein in vivo. (a) Western analyses of Sufu in NCI-H322M cells following cycloheximide (CHX) treatment in the absence or presence of 1 mM KAAD-cyclopamine (CPA). (b) Western analysis of Sufu turnover in primary mouse embryonic fibroblasts (MEFs) in the presence of various Shh agonists/antagonist and CHX. CHX, 20 mM; purmor- Figure 1 Expression of Sufu is regulated by the proteasome- phamine, 10 mM; KAAD-CPA, 1 mM; Shh-N-conditioned medium, mediated degradation in several cancer cell lines. (a) Western blot 50%. In each of the above conditions, MG132 (20 mM) wasalso analysis of Sufu protein level in total lysates of human lung cancer added to one cell sample for 6 h. (c) Time course of Sufu protein cellsA549, NCI-H522, NCI-H322M, HOP62, EKVX, HOP92, turnover. Line graphsrepresenttwo independent experimentsas NCI-H226, NCI-H460, mouse fibroblast NIH3T3 and human described in (b). The intensity of Sufu bands relative to that of embryonic kidney fibroblast HEK293 with antibodies against Sufu. GAPDH from each time point wasquantified by NIH ImageJ1.34s The level of GAPDH (glyceraldehyde 3-phosphate dehydrogenase) software package and plotted against the incubation time. Values wasusedasa loading control. ( b) Standard and (c) real-time RT– are the mean±s.d. (d) Western analysis of Sufu protein in torso PCR analyses of Sufu mRNA levels in NCI-H322M, A549 and tissues of e9.5 embryos showing reduced Sufu protein stability in HEK293 cellswith b-actin asthe loading control. ( d) Effect of Ptch1À/À homozygousembryoscompared with heterozygous proteasome inhibitor MG132 (20 mM, Calbiochem) on the level of littermate controls. Genotyping was carried out by PCR using Sufu protein in NCI-H322M, prostate cell lines PC3 and DU145, primers specific for neomycin resistance (knockout allele) and exon and breast cancer cell line MD-MBA-231. 2 (wild-type allele). Oncogene Ubiquitination-mediated degradation of Sufu SYueet al 494 stability measurement in freshly isolated mouse em- examined whether activation of Shh signaling promotes bryonic fibroblasts (MEFs), which have low Shh path- ubiquitination of the endogenousSufu in primary way activity (Cooper et al., 2005; Svard et al., 2006). MEFs. In this experiment, we transfected MEFs with Our results indicated that Sufu was quite stable in the HA-tagged ubiquitin to enhance ubiquitin signal, MEFs, with an estimated half-life of 24 h (Figures 2b and treated the cellswith MG132 to let poly-ubiquiti- and c). Upon treating
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