Tumor and Stem Cell Biology Histone Acetyltransferase PCAF Is Required for Hedgehog – Gli-Dependent Transcription and Cancer Cell Proliferation

The Hedgehog (Hh) signaling pathway plays an important role in embryonic patterning and development of many tissues and organs as well as inmaintaining and repairingmature tissues in adults. Uncontrolled activation of theHh–Gli pathway has been implicated indevelopmental abnormalities aswell as in several cancers, including brain tumors like medulloblastoma and glioblastoma. Inhibition of aberrant Hh–Gli signaling has, thus, emerged as an attractive approach for anticancer therapy; however, the mechanisms that mediate Hh–Gli signaling in vertebrates remain poorly understood. Here, we show that the histone acetyltransferase PCAF/KAT2B is an important factor of the Hh pathway. Specifically, we show that PCAF depletion impairs Hh activity and reduces expression of Hh target genes. Consequently, PCAF downregulation in medulloblastoma and glioblastoma cells leads to decreased proliferation and increased apoptosis. In addition, we found that PCAF interacts with GLI1, the downstream effector in theHh–Gli pathway, and that PCAF or GLI1 loss reduces the levels of H3K9 acetylation on Hh target gene promoters. Finally, we observed that PCAF silencing reduces the tumor-forming potential of neural stem cells in vivo. In summary, our study identified the acetyltransferase PCAF as a positive cofactor of the Hh–Gli signaling pathway, leading us to propose PCAF as a candidate therapeutic target for the treatment of patients with medulloblastoma and glioblastoma. Cancer Res; 73(20); 6323–33. 2013 AACR. Introduction The evolutionary conserved Hedgehog (Hh) signaling pathway plays an important role in development, proliferation, and stem cell maintenance (1, 2). In agreement with such role, deregulation of the Hh pathway leads to several developmental syndromes and tumors of different tissues (3, 4). In mammals, Hh signaling is initiated when the secreted Hh proteins bind to and inhibit the transmembrane receptor PTCH. The interaction between Hh and PTCH releases Smoothened (SMO), a second transmembrane protein, which in turn induces the downstream components of the Hh signaling pathway and leads to the activation of the glioma-associated oncogene (GLI) transcription factor family (5). When activated, the GLI proteins induce the expression of genes that regulate multiple cellular functions such as cell-cycle progression, proliferation, and apoptosis (6, 7). In addition, GLI1 and PTCH are activated by Hh signaling, suggesting a regulation of the pathway through both a positive and a negative feedback (8). The importance of the Hh pathway in tumorigenesis was first discovered in patients of Gorlin syndrome. This rare pathology is caused by an inactivating mutation in PTCH, which leads to the development of tumors like basal cell carcinoma, medulloblastoma, and rhabdomyosarcoma (9). In addition, inappropriate activation of Hh signaling has been shown to lead to the development of tumors in the lung, gastrointestinal tract, and pancreas (10–12). GLI1 was originally isolated as a highly amplified gene in human malignant glioma and subsequently found amplified in other tumor types, including liposarcoma, rhabdomyosarcoma, and osteosarcoma (9, 13, 14). GLI1 is a key regulator of glioma growth and of cancer stem cell self-renewal. Moreover studies in transgenic mice have shown that ectopic expression of Gli1 is sufficient to induce basal cell carcinoma (15–17). Despite the importance of the Hh pathway in development and disease, themolecularmechanisms bywhich the Hh signal leads to the activation of GLI-regulated transcription is still not completely understood. Here, we show that transcriptional activation by the Hh pathway requires the histone acetyltransferase (HAT) PCAF. Materials and Methods Cell culture and reagents Human cell lines (U87, U118, T98G, and Daoy) were purchased from the American Type Culture Collection (ATCC) and used at low passage numbers. A short tandem Authors' Affiliations: Biotech Research and Innovation Centre (BRIC) and Centre for Epigenetics, University of Copenhagen; The Danish Stem Cell Center (Danstem), Copenhagen, Denmark; and F-BBVA Cancer Cell Biology Programme, Centro Nacional de Investigaciones Oncol ogicas (CNIO), Madrid, Spain Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Author: Kristian Helin, BRIC, University of Copenhagen, Ole Maaløes Vej 5, DK-2200 Copenhagen N. Phone: 45-35-32-56-66; Fax: 45-35-32-56-69; E-mail: kristian.helin@bric.ku. dk doi: 10.1158/0008-5472.CAN-12-466