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GLI Activation by Atypical Protein Kinase C Ι/Λ Regulates the Growth Of LETTER doi:10.1038/nature11889 GLI activation by atypical protein kinase C i/l regulates the growth of basal cell carcinomas Scott X. Atwood1, Mischa Li1, Alex Lee1, Jean Y. Tang1 & Anthony E. Oro1 Growth of basal cell carcinomas (BCCs) requires high levels of To identify new druggable targets in the HH pathway, we used the hedgehog (HH) signalling through the transcription factor GLI1. scaffold protein MIM, which potentiates GLI-dependent activation Although inhibitors of membrane protein smoothened (SMO) effec- downstream of SMO (ref. 9), as bait in a biased proteomics screen of tively suppress HH signalling, early tumour resistance illustrates the factors involved in HH signalling and ciliogenesis. Two of the hits were need for additional downstream targets for therapy1–6.Herewe polarity proteins not previously linked to the HH pathway: aPKC-i/l, identify atypical protein kinase C i/l (aPKC-i/l) as a novel GLI regu- a serine/threonine kinase, and PARD3, a scaffold protein and aPKC-i/ lator in mammals. aPKC-i/l and its polarity signalling partners7 l substrate (Supplementary Fig. 1a). Reciprocal immunoprecipitation co-localize at the centrosome and form a complex with missing-in- of aPKC-i/l and PARD3 pulled down MIM, suggesting a specific metastasis (MIM), a scaffolding protein that potentiates HH signal- interaction (Supplementary Fig. 1b). Because MIM is a centrosome- ling8,9. Genetic or pharmacological loss of aPKC-i/l function blocks associated protein that promotes ciliogenesis8, we fractionated cen- HH signalling and proliferation of BCC cells. Prkci is a HH target trosomes and found that aPKC-i/l, along with PARD3 and PARD6A, gene that forms a positive feedback loop with GLI and exists at co-fractionated and co-immunoprecipitated with MIM in c-tubulin- increased levels in BCCs. Genome-wide transcriptional profiling positive fractions that mark centrosomes (Fig. 1a and Supplementary shows that aPKC-i/l and SMO control the expression of similar Fig. 1c). MIM partly co-localizes with aPKC-i/l-complex members genes in tumour cells. aPKC-i/l functions downstream of SMO to at the basal body in dermal fibroblasts, keratinocytes and the well- phosphorylate and activate GLI1, resulting in maximal DNA bind- characterized mouse BCC cell line ASZ00110 (Fig. 1b), where aPKC- ing and transcriptional activation. Activated aPKC-i/l is upregu- i/l and MIM interact through co-immunoprecipitation (Fig. 1c). Loss lated in SMO-inhibitor-resistant tumours and targeting aPKC-i/l of aPKC-i/l or MIM protein suppressed HH signalling, because mes- suppresses signalling and growth of resistant BCC cell lines. These senger RNA (mRNA) levels of the HH target gene Gli1 were reduced results demonstrate that aPKC-i/l is critical for HH-dependent and ciliogenesis was inhibited (Fig. 1d, e and Supplementary Fig. 1d, e). processes and implicates aPKC-i/l as a new, tumour-selective thera- Because the kinase activity is necessary for many of the cellular peutic target for the treatment of SMO-inhibitor-resistant cancers. functions of aPKC-i/l (refs 7, 11), we used a myristoylated aPKC ab Input ChickenMIM IgY IP 180 kD Primary mDC PARD3 150 kD 100 kD aPKC 76 kD PARD6A 43 kD Purified centrosomes Keratinocyte c Input ProteinMIM G IPaPKC IP aPKC 76 kD mBCC MIM 102 kD aPKC γ-tub/Actub PARD3 γ-tub/Actub CDC42 γ-tub PARD6A γ-tub MIM γ-tub/Actub dge f ASZ001 mBCC ASZ001 mBCC ASZ001 mBCC ASZ001 mBCC 1.5 60 2.0 1.0 Scramble 1.5 PSI 0.8 1.0 40 Cyclopamine 0.6 Scramble > 100 cells) 1.0 PSI n 0.4 Cyclopamine 0.5 20 (570 nm) 0.5 MTT assay mRNA level (dR) mRNA level (dR) 0.2 0.0 0 0.0 0.0 Gli1 Gli1 Relative absorbance Cilia (%, 0 5 10 15 20 0 10203040 M) μ Drug (μM) Drug (μM) MIM KD MIM KD Control sh aPKC KD Control sh aPKC KD PSI (10 Figure 1 | aPKC-i/l is a centrosome-associated protein that regulates HH and aPKC-i/l interact in BCC cells. d–f, Gli1 mRNA levels (n 5 3) or cilia signalling. a, MIM and aPKC-i/l interact in purified centrosomes. IgY, percentage (n 5 3) after MIM or aPKC-i/l short hairpin (sh) RNA knockdown, immunoglobulin-Y. b, MIM and aPKC-i/l complexes localize at the centrosome or aPKC or SMO inhibition in BCC cells. KD, knockdown. g,Cellproliferation (c-tub, c-tubulin) versus primary cilia (Actub, acetylated tubulin) of mouse was decreased in BCC cells (n 5 3) after PSI or cyclopamine treatment, but not dermal cells (mDC), mouse keratinocytes and mouse BCC cells (mBCC). c,MIM after treatment with myristoylated scrambled peptide. Error bars, s.e.m. 1Program in Epithelial Biology, Stanford University School of Medicine, Stanford, California 94305, USA. 484 | NATURE | VOL 494 | 28 FEBRUARY 2013 ©2013 Macmillan Publishers Limited. All rights reserved LETTER RESEARCH 12 peptide inhibitor (PSI) to suppress kinase activity (Supplementary abASZ001 mBCC Primary hBCC 20 1,000 Fig. 1f). PSI, but not a myristoylated scrambled peptide, inhibited 15 HH signalling in BCC cells in a dose-dependent manner similar to 10 100 5 the SMO antagonist cyclopamine (Fig. 1f). PSI, the pan-PKC inhibitor 3 10 i l 2 Go6983 and genetic loss of aPKC- / expression also resulted in dose- 1 dependent inhibition of cell growth in BCC cells, leading to cell death 1 Relative mRNA level Relative mRNA level according to MTT assay (Fig. 1g and Supplementary Fig. 1g, h). PSI 0 0.1 inhibited BCC cell growth at a concentration similar to that of cyclo- Gli1; mBCC Gli1; hBCC pamine, with a half-maximal inhibitory concentration (IC50)of5mM. Prkci; mBCC Prkci; hBCCPrkcz; hBCC Prkcz; mBCC /Prkci; normal Primary cilia were reduced by 50% in PSI-treated BCC cells (Fig. 1e), Gli1/Prkc; normal Gli1 indicating that aPKC activity is critical to both HH signalling and c Normal human epithelia Primary human BCCs ciliogenesis in BCC cells. Interestingly, PSI did not affect proliferation H&E in several non-tumorigenic cells (Supplementary Fig. 1i). PSI speci- fically inhibited aPKC, because loss of aPKC-i/l in BCC cells in com- DAPI bination with PSI treatment possessed no additional activity to reduce levels of Gli1 or Prkci mRNA (Supplementary Fig. 1j). aPKC To address whether the effects of aPKC-i/l on the HH pathway are direct, we assayed aPKC-i/l function in several nonpolar cell lines (Supplementary Fig. 1k, l and data not shown). These cells maintained their primary cilia or developed more after loss of aPKC-i/l; how- DAPI ever, aPKC-i/l removal still blocked HH activation, reducing target gene induction. We conclude that the effects of aPKC-i/l on HH signalling are cilia independent and are required for maximal sus- P-aPKC tained signalling. aPKC-i/l is necessary for maximal HH signalling, and so we next d e f FLAG:GLI1 investigated whether aPKC-i/l is overexpressed in BCCs. Indeed, Primary mDC TSS 80 GLI1-binding sites 10 ChIP in mBCCs Prkci expression, but not Prkcz expression, is specifically upregulated 60 with Gli1 in BCC cells (Fig. 2a). Similar results are found using freshly 40 Prkci 8 20 3,610 bp 6 isolated human BCCs compared with primary human keratinocytes p1 3 3,585 bp p2 2,952 bp 4 (Fig. 2b). Immunohistochemical staining of human BCCs and nor- 2 KRCCNCMMA canonical GLI1-binding site 2 mal skin with antibodies recognizing both total and activated aPKC 1 K = G/T; R = A/G; M = A/C; N = any base pair Relative enrichment (P-aPKC) show higher levels in invasive and nodular tumours, with Relative mRNA level 0 0 P-aPKC showing greater overexpression (Fig. 2c). Loss of aPKC-i/l Actin removes both aPKC and P-aPKC staining in primary mouse dermal Prkci-p1Prkci-p2 ; SHH-N CM Gli1; SHH-N CM cells, indicating the specificity of the antibody (Supplementary Fig. 2). Prkci Prkcz; SHH-N CM Because aPKC-i/l and HH signalling are required for BCC cell Gli1/Prkc; control CM ghi growth, we then asked whether Prkci is a HH target gene. Activation ASZ001 mBCC Primary mBCC ASZ001 mBCC 2.0 3 2.5 of HH signalling in both polarized and non-polarized primary and 1.5 2.0 immortalized cells using SHH-N ligand (the amino-terminal fragment 2 1.5 of SHH) induces both Gli1 and Prkci transcripts, but not Prkcz 1.0 1.0 (Fig. 2d). Additionally, when we block HH signalling by treating BCC 1 0.5 0.5 Relative mRNA level cells with cyclopamine, Gli1 and Prkci transcripts, but not Prkcz, are Relative mRNA level Relative mRNA level specifically suppressed (Fig. 2g). Three putative GLI1-binding sites13 0.0 0 0.0 are present in the promoter region within 5 kilobases of the Prkci μM) μM) transcriptional start site in mice (Fig. 2e). Chromatin immunopreci- Cdc42; DMSO /Cdc42; normalPard6a; mBCCCdc42; mBCC / Gli1/Prkc; DMSO ; SANT-1 (60 nM) pitation (ChIP) with BCC cells overexpressing FLAG:GLI1 enrich in Gli1; cyclo (10 Prkci; cycloPrkcz (10; cyclo (10 μM) Pard6a Pard6a regions containing the three GLI1-binding sites (Fig. 2f), indicating Pard6a Cdc42; SANT-1 (60 nM) that Prkci is a direct target of GLI1. The first two GLI1-binding sites Figure 2 | aPKC-i/l and HH form a positive-feedback loop in BCCs. promote expression of luciferase when expressed in BCC cells, sug- a, b, Gli1, Prkci, and Prkcz mRNA levels in mouse BCC cells (a)(n 5 3) and gesting that these sites are functional (Supplementary Fig.
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