Hedgehog axis in cancer control

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Yin-Yang strands of PCAF/Hedgehog axis in cancer control Paola Infante1, Gianluca Canettieri2, Alberto Gulino1,2,3, and Lucia Di Marcotullio2 1

Center for Life Nanoscience (CLNS), Italian Institute of Technology-Sapienza, 00161 Rome, Italy Department of Molecular Medicine, ‘‘La Sapienza’’ University, 00161 Rome, Italy 3 IRCCS Neuromed, 86077 Pozzilli, Italy 2

PCAF (p300/CBP associated factor) harbors acetyltransferase and a recently identified ubiquitylation activity that regulates gene expression in response to genotoxic stress or mitogenic signals. We highlight the dual role of PCAF in the control of Hedgehog signaling, a master regulator of tissue development, stemness, and tumorigenesis. By promoting histone acetylation at Hedgehog/GLI1 target gene promoters or direct ubiquitylation and proteolysis of GLI1, the PCAF/GLI1 axis stands as a promising therapeutic target for Hedgehog-dependent tumors.

The PCAF/Hedgehog axis coordinates physiologic versus pathologic morphogenic outcomes Physiologic execution of morphogenic programs is the result of an appropriate balance of permissive (i.e., supply of growth factors, nutrients, and oxygen) and non-permissive microenvironmental signals (i.e., genotoxic or metabolic stress) that lead to the fine-tuning of cell replication or death. Morphogens coordinate this program by balancing mitogenic, differentiation, and death processes to shape appropriate tissue architecture, whereas subversion of these events may lead to cancer. The evolutionary conserved morphogen Hedgehog is a master regulator of tissue development and stem cell maintenance [1]. When hyperactivated by genetic and/or epigenetic alterations (PTCH1, SMO, SuFu mutations, GLI amplification/activation, ligand autocrine/paracrine mechanisms), Hedgehog signaling causes the activation of the oncogenic GLI1 transcription factor, which provides mitogenic and prosurvival properties, thus representing a leading cause of a wide variety of tumors [2]. Therefore, a lot of effort is being made to understand the mechanisms regulating GLI1 activity and to identify drugs that may limit its oncogenic potential. A growing amount of evidence reports that post-translational modifications, such as acetylation and ubiquitylation, play a crucial role in regulating the Hedgehog/GLI transcriptional output. In this regard, a key role has been recently attributed to PCAF (p300/CBP associated factor), a histone acetyltransferase (HAT) involved in development Corresponding authors: Gulino, A. ([email protected]); Di Marcotullio, L. ([email protected]). Keywords: PCAF; Hedgehog; acetyltransferase; E3-ubiquitin ligase; p53; cancer. 1471-4914/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/ j.molmed.2014.05.003

and tumorigenesis that regulates gene expression through chromatin remodeling and transcription factor acetylation (reviewed in [3]). The heterogeneous mechanisms whereby PCAF executes its programs mirror its ability to transduce opposite microenvironmental signals either: (i) growth factor receptor stimuli leading to cell replication and survival (chromatin remodeling of cell growth genes) or (ii) DNA damaging signals causing growth arrest and cell death (acetylated-p53- and acetylated-E2F1-dependent activation of proapoptotic genes) [3]. The duality of stimuli and functions of PCAF is confirmed by recent evidence showing that in addition to its acetyltransferase properties PCAF possesses an intrinsic ubiquitin E3 ligase activity against Mdm2, thus controlling p53 functions following genotoxic stress [4]. This dual HAT and ubiquitylation function reflects the very recently reported role of PCAF in Hedgehog signaling control. On the one hand, PCAF has been described to form a complex with GLI1 on target promoters that enhances transcription by increasing H3K9 acetylation. Therefore, PCAF is a positive cofactor of GLI1 and its downregulation leads to growth arrest of brain tumor cells [5]. On the other hand, PCAF also binds GLI1 and promotes its ubiquitylation and degradation via its E3 ligase activity, thus restraining the transcription factor function and oncogenic properties [6]. Such a mechanism is triggered by genotoxic stress that results in GLI1 suppression through the p53-dependent transcriptional activation and accumulation of PCAF [6]. Implications for tumor growth The dual molecular function of PCAF suggests that this molecule has opposite outcomes that are exploited to control Hedgehog-regulated cell activities, depending on microenvironmental conditions and/or p53 status. It acts as a HAT and transcriptional coactivator of GLI1-induced mitogenesis under permissive conditions [5], whereas it reverts to apoptotic activity via ubiquitylation-promoted degradation of GLI1 in non-permissive proapoptotic conditions occurring in response to DNA damage [6] (Figure 1A). In turn, p53 loss-of-function mutations perturbing the DNA damage response (frequently observed in cancer) would cause a failure of this balance, allowing the coactivating function of PCAF to prevail above negative signals that limit GLI1 activity, thus promoting Hedgehog-dependent cell survival and proliferation even in non-permissive microenvironmental conditions (Figure 1B). Trends in Molecular Medicine xx (2014) 1–3

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Figure 1. Model of PCAF function in the Hedgehog signaling control. (A) In normal cells, under permissive conditions, the transcriptional activity of the Hedgehog pathway effector GLI1 is regulated through a balance of GLI1 acetylation/deacetylation status, mediated by p300 and HDAC1/2, respectively, and by histone acetylation of target promoters, induced by PCAF HAT activity, thus promoting mitogenesis. In response to DNA damage (non-permissive conditions), functionally active p53 triggers the transcription of PCAF and promotes its accumulation. PCAF intrinsic E3 ligase activity is then responsible for the ubiquitin-dependent degradation of GLI1, thus restraining its oncogenic properties. (B) In cancer cells, the loss-of-function mutations of p53 cause a failure of the p53/PCAF inhibitory axis that favors the coactivator function of PCAF on GLI1 activity, thus promoting the growth of Hedgehog-dependent tumors. The fine-tuning of this mechanism makes the PCAF/p53/GLI1 axis a druggable target that can be exploited at the GLI effector level downstream SMO. Some small molecules that may regulate GLI1 transcriptional output are labeled in blue. These target aberrant acetylation/ubiquitylation mechanisms, involved in GLI1 control, that occur in cancer cells when non-permissive DNA damaging conditions are deregulated (i.e., loss of p53 function) inducing controlled cell growth and survival. Abbreviations: HAT, histone acetyltransferase; PCAF, p300/CBP associated factor.

Perspective for cancer therapy Recent evidence suggests that Hedgehog-sensitive tumors could be successfully treated with proper Hedgehog pathway inhibitors [2]. However, although anti-SMO inhibitors are effective in medulloblastoma treatment and have been approved by the FDA for the therapy of basal cell cancer, several clinical trials on a variety of solid tumors have failed [2]. The major hindrance is a result of acquired resistance to SMO inhibitors, frequently observed in Hedgehog-dependent tumor initiation and relapse, owing to alterations of downstream signals leading to GLI hyperactivation. Most of these GLI hyperactivation events (i.e., phosphorylation, amplification, deubiquitylation, and deacetylation) are caused by genetically or epigenetically determined enhancement of bypass oncogenic pathways (i.e., PI3K/AKT/mTOR/S6K1, RAS/ERK, p53 loss, SOX2/PKCi/l). This implies that targeting these pathways may revert anti-SMO resistance (i.e., PI3K/AKT inhibitors) or synergize with Hedgehog antagonists acting on either SMO or GLI in SMO-sensitive or resistant tumors, respectively [2]. However, the low number of small molecules available that inhibit GLI might be as a result of the poor understanding of the multiple mechanisms of regulation of GLI functions, including the way it controls transcription. HPI-1 and HPI-4 have been shown to target the 2

post-translational events of GLI processing and activation downstream of SMO, such as increase of the proteolytic cleavage of GLI2 to its repressor form or overall GLI1 degradation. Arsenic trioxide (ATO) has recently been shown to prevent GLI2 localization to the primary cilium, thus leading to its proteolytic degradation, whereas direct inactivation of GLI1 has not been yet characterized. Similarly, GANT61 inhibits GLI1/DNA binding only in living cells suggesting that it indirectly impairs its interaction with target gene promoters by as yet unelucidated mechanisms (reviewed in [2]). HDAC inhibitors have been shown to cause hyperacetylation of GLI1 (at K518) and GLI2 (at K757), thus preventing transcriptional activity [7,8] (Figure 1B). Therefore, when operating in Hedgehog-dependent cancer cells, the PCAF-mediated dual mechanisms may have therapeutic potential as a druggable checkpoint via the use of specific PCAF/HDAC modulators [9] and, by contrast, of DNA damaging agents or modifiers of p53 or ubiquitylation events [10] (Figure 1B). Acknowledgments This work was supported by AIRC (Associazione Italiana Ricerca Cancro) grants #IG10610 and #9979, and the Ministry of University and Research (FIRB and PRIN projects).

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6 Mazza`, D. et al. (2013) PCAF ubiquitin ligase activity inhibits Hedgehog/GLI1 signaling in p53-dependent response to genotoxic stress. Cell Death Differ. 20, 1688–1697 7 Canettieri, G. et al. (2010) Histone deacetylase and Cullin3– RENKCTD11 ubiquitin ligase interplay regulates Hedgehog signalling through GLI acetylation. Nat. Cell Biol. 12, 132–142 8 Di Marcotullio, L. et al. (2011) Protected from the inside: endogenous histone deacetylase inhibitors and the road to cancer. Biochim. Biophys. Acta 1815, 241–252 9 Ruthrotha Selvi, B. et al. (2010) Small molecule modulators of histone acetylation and methylation: a disease perspective. Biochim. Biophys. Acta 1799, 810–828 10 Hoe, K.K. et al. (2014) Drugging p53 pathway: understanding the route to clinical efficacy. Nat. Rev. Drug Discov. 13, 217–236

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