Hedgehog signalling in foregut malignancy

Abstract

Hedgehog (Hh) signalling mediates axial patterning and stem cell fate in development. This is mediated by Sonic, Desert and Indian Hedgehogs whose morphogen gradients determine the level of signalling in recipient tissues. Aberrant, cell autonomous, ligand-dependent Hh signalling has recently been demonstrated in small cell lung cancer (SCLC), as well as in upper gastrointestinal malignancies arising from pancreas, esophagus and stomach. These tumors lack mutations in the Hh receptor PATCHED, identifying a mechanism of pathway activation distinct from Gorlin’s syndrome associated neural and skin tumors. We believe that this phenomenon represents a conserved mechanism for establishing niche-independent stem cell fates in cancer which is essential for malignant transformation and metastasis. Specific inhibition of Hh signalling by the naturally occurring plant alkaloid cyclopamine provides the opportunity for pharmacologic assessment of the role of Hh signalling in these tumors. Cyclopamine inhibits growth of SCLC and a wide range of foregut derived malignancies both in vitro and in vivo. This demonstrates an ongoing requirement for Hh signalling in these highly lethal and aggressive tumors. A novel therapeutic strategy is proposed using pharmacologic targeting of Hh dependent tumors with high potency pathway antagonists.

Introduction

Originally identified as a mediator of segment polarity in the fly [1], the Hedgehog (Hh) pathway is essential for normal animal development [2]. The three mammalian orthologues of the Drosophila gene Hedgehog, Sonic (Shh), Indian (Ihh) and Desert (Dhh) Hedgehog establish morphogenic gradients essential for axial patterning of the mammalian embryo [2], [3], [4]. Shh is the predominant signalling molecule in lung, brain and limb development and is the most extensively studied Hh protein in vertebrates [5], [6], [7], [8].

The active Shh signalling peptide is formed by an auto-processing reaction that converts a 45 kDa precursor into a 19 kDa signalling peptide that is doubly lipid modified, with palmitate and cholesterol residues at the N and C termini, respectively [9], [10]. While cholesterol modification occurs during auto-processing, palmitylation of Hh protein is mediated in flies by the acyl transferase Skinny Hedgehog [11]. Efficient generation and reception of the Hh ligand signal requires these lipid modifications [9], [10], the transporter-like function of the transmembrane protein Dispatched [12], and an appropriate heparin sulfate peptidoglycan environment [13], [14].

The Hh receptor Patched (Ptch) is a 12-transmembrane protein with homology to the resistance, nodulation, division (RND) bacterial transporter family [15]. Ptch acts catalytically to inhibit the 7-transmembrane protein Smoothened (Smo), rendering the pathway inactive in the absence of Hh ligand [15]. Evidence points to Ptch acting as a transporter of a small molecule which regulates Smo activity, although the identity of such a regulatory molecule remains elusive [15]. Binding of Hh ligand inactivates Ptch, de-repressing Smo and resulting in positive Hh pathway signalling [2], [7], [16].

Recent elucidation of events downstream of Smo in Drosophila cells has suggested potential mechanisms in mammalian cells. Activation of pathway involves a multiprotein cytoplasmic complex scaffolded by the atypical kinesin Costal2 [17], [18], [19]. This complex contains the serine/threonine kinase Fused (Fu), and a novel protein Suppressor of Fused (SuFu) [18]. Hh signalling promotes association of this complex with the C terminus of Smo, which in turn regulates the activity of the latent zinc finger transcription factor Cubitus interruptus (Ci) through proteolytic processing and nuclear translocation events [17], [20], [21], [22], [23]. Such processing is enhanced by Ci phosphorylation, which can mediated by protein kinase A, shaggy/GSK3β and casein kinsase 1, all of which represent important negative Hh pathway regulators [14], [24], [25], [26], [27], [28]. The processed form of Ci acts as a transcriptional repressor of Hh pathway genes. Hh signalling results in the accumulation of unprocessed, full length Ci which acts a transcriptional activator. Following nuclear translocation, the full length Ci induces transcriptional activation of Hh target genes, which include Ptc itself [2], [16].

In vertebrate cells, Ci function has diverged into three Gli proteins which vary in their level of processing, and in their transcriptional activity [7], [29]. The oncoprotein Gli1, itself a transcriptional target of mammalian Hh signalling, is a strong positive regulator of Hh pathway targets [30], [31], [32], and is thought not to be regulated by processing. Gli2 and Gli3 possess both transcriptional activation and repression properties [8], [30], [31], [32], [33], [34], [35], [36], [37], and Gli3 is significantly processed in response to Hh signalling in vertebrates [37]. A simplified diagram illustrating the mammalian Hh pathway is shown in Fig. 1.