GLI activation by atypical protein kinase C ι/λ regulates the growth of basal cell carcinomas

Abstract

Growth of basal cell carcinomas (BCCs) requires high levels of hedgehog (HH) signalling through the transcription factor GLI. Although inhibitors of membrane protein smoothened (SMO) effectively suppress HH signalling, early tumour resistance illustrates the need for additional downstream targets for therapy. Here we identify atypical protein kinase C ι/λ (aPKC-ι/λ) as a novel GLI regulator in mammals. aPKC-ι/λ and its polarity signalling partners co-localize at the centrosome and form a complex with missing-in-metastasis (MIM), a scaffolding protein that potentiates HH signalling. Genetic or pharmacological loss of aPKC-ι/λ function blocks HH signalling and proliferation of BCC cells. Prkci is a HH target gene that forms a positive feedback loop with GLI and exists at increased levels in BCCs. Genome-wide transcriptional profiling shows that aPKC-ι/λ and SMO control the expression of similar genes in tumour cells. aPKC-ι/λ functions downstream of SMO to phosphorylate and activate GLI1, resulting in maximal DNA binding and transcriptional activation. Activated aPKC-ι/λ is upregulated in SMO-inhibitor-resistant tumours and targeting aPKC-ι/λ suppresses signalling and growth of resistant BCC cell lines. These results demonstrate that aPKC-ι/λ is critical for HH-dependent processes and implicates aPKC-ι/λ as a new, tumour-selective therapeutic target for the treatment of SMO-inhibitor-resistant cancers.

Figure 1. aPKC is a centrosome-associated protein that regulates Hh signaling

a, MIM and aPKC interact in purified centrosomes. b, MIM and aPKC complexes localize at the centrosome (γ-tub) versus primary cilia (Actub) of mouse dermal cells (mDC), mouse keratinocytes, and mouse BCC cells. Actub, acetylated tubulin. γ-tub, γ-tubulin. c, MIM and aPKC interact in BCC cells. d–f, gli1 mRNA levels (n=3) or cilia percentage (n=3) after MIM or aPKC shRNA, or aPKC or Smo inhibition in BCC cells. sh, short-hairpin. KD, knockdown. g, Cell proliferation reduced in BCC cells (n=3) after PSI or cyclopamine treatment, but not myristoylated scrambled peptide. Error bars, s.e.m.

Figure 2. aPKC and Hh form a positive feedback loop in BCCs

a, b, gli1, aokciota, or apkczeta mRNA levels in (a) mouse BCC cells (n=3) and (b) primary human BCC tumors (n=7). c, Total and activated aPKC (P-aPKC) overexpressed in primary human BCC tumors. d, apkciota is upregulated in Shh-N-treated mouse dermal cells (n=3). CM, conditioned media. e, Gli1 binding sites within the promoter region of aPKC. TSS, transcriptional start site. f, Flag:Gli1 ChIP of aPKC promoter Gli1 sites. g, Cyclopamine suppresses apkciota expression in BCC cells (n=3). h, pard6a expression is reduced and cdc42 is upregulated in mouse BCC tumors (n=3). i, Sant-1-treatment of BCC cells increases pard6a and decreases cdc42 mRNA expression (n=3). Error bars, s.e.m.

Figure 3. aPKC phosphorylates and activates Gli1

a, Venn diagram and (b) heatmap of significantly changed transcripts upon Sant-1 or PSI treatment in BCC cells. c, Gene ontology terms of commonly altered transcripts. d, PSI does not affect nuclear Gli1 in BCC cells. e, aPKC interacts with in vitro translated (IVT) human Gli1 or Gli1 DNA-binding domain. f, aPKC phosphorylates human Gli1 DNA-binding domain. CBB, coomassie brilliant blue. g, PSI reduces phosphorylated serine/threonine levels of immunoprecipitated Gli1 from BCC cells. h, aPKC promotes DNA binding of IVT human Gli1 at S243 and T304 in an electrophoretic mobility shift assay (EMSA). NS, non-specific binding. i, Densitometry of aPKC rescue, phosphomimetic versus phosphodeficient Gli1 EMSA (n=3). j, BCC cells expressing phosphomimetic Gli1 show reduced PSI sensitivity (n=3). Error bars, s.e.m. k, Flag:Gli1 ChIP showing PSI and Sant-1 inhibit Gli1 binding to target chromatin sites in mouse BCC cells.

Figure 4. Topical aPKC inhibitor suppresses primary tumor growth

a, PSI inhibits Hh signaling in allografted mouse BCC tumors from Ptch1 +/−, K14CreER2, p53flox/flox mice (n=7). b, Topical treatment of allografted BCC tumors slows tumor growth (DMSO n=10, PSI n=9). c, Intermediate levels of PSI (n=8) compared to intermediate concentrations of Itraconazole (n=8) and arsenic trioxide (n=5). d, Five independently derived Smo-resistant BCC cell lines that amplify apkc or apkc and gli1 mRNA levels (e) are sensitive to PSI treatment. f, g, Vismodegib-treated resistant human BCC tumors (n=6) display elevated levels of active aPKC compared to vismodegib-treated sensitive tumors (n=8), non-drug treated tumors (n=17), and normal skin (n=7). Error bars, s.e.m.