Gene Amplification and Activation of the Hedgehog Pathway
Curator: Larry H Bernstein, MD, FCAP
Hedgehog signaling pathway: an overview
Proteins of the Hedgehog (Hh) family are powerful signaling molecules that act as morphogens during development in both vertebrates and invertebrates.
Hh was first discovered in a genetic screen performed on cuticle embryo, that aimed to understand the body segmentation of Drosophila melanogaster (Nusslein-Volhard and Wieschaus, 1980). In this screen, mutant embryos for Hh developed as prickly little balls similar to a hedgehog (so the name of the protein).
The core components of the Hh pathway were initially identified in Drosophila and are conserved in vertebrates, where the pathway has maintained the same mechanisms of action through species (although with some exceptions). Most interesting, deregulation of the Hh pathway leads to developmental defects and cancer.
Hh signaling cascade in Drosophila
Hh maturation, release and movement
Hh is first synthesized as a precursor. It undergoes autoproteolytic cleavage where a cholesterol molecule (Porter et al., 1996), and a palmitic acid molecule (Ingham and McMahon, 2001) are added to the final product. The primary role of these modifications is to direct the mature signal to interact with a set of cellular components that are responsible of the Hh secretion, movement and reception. In particular, cholesterol is involved in Hh trafficking and movement (Gallet et al., 2003), whereas palmitoylation in Hh signaling (Chamoun et al., 2001; Liu et al., 2007).
Once Hh is modified, it is ready to be secreted from the cells (Burke et al., 1999). After secretion, Hh interacts with the extracellular matrix and has to find a way to move through it to reach the receiving cells, forming a concentration gradient.
Several models have been proposed to explain how Hh can move far from its source, such as its movement inside a special structures called lipoprotein particles (Bolanos-Garcia and Miguel, 2003; Olofsson et al., 1999) and through its interaction with heparan sulphate proteoglycans (HSPGs) (Jia et al., 2003; Nakato et al., 1995).
At the plasma membrane
Hh signal transduction is initiated at the plasma membrane where Hh interacts with its 12 transmembrane protein receptor Patched (Ptc) (Ingham and McMahon,2001). The interaction between Hh and Ptc is facilitated by the Ihog/Cdo family of coreceptors (Zhang et al., 2010). The binding between Ptc and Hh has two main important roles:
- Limiting the spreading of Hh: the binding between Hh and Ptc results in their internalization, targeting Hh to lysosomes for degradation (Gallet and Therond, 2005).
- Increase of Smoothened (Smo) expression and activation: (Chen and Struhl, 1996; Denef et al., 2000; Lum et al., 2003; Taipale et al., 2002) this gives rise to a cascade of signal transmission that function to regulate the transcription factor Cubitus interruputs (Ci) (Alexandre et al., 1996; Méthot and Basler, 1999).
Once Hh binds Ptc, the seven-pass transmembrane protein Smo undergoes several phosphorylation events (Hh dose-dependent) (Fan et al., 2012). Smo phosphorylation occurs at its cytoplasmic tail (C-tail) which contains several phosphorylation sites of PKA, CK1, GSK3 (Zhang et al., 2004). The main consequences of Smo phosphorylation are:
- Promoting Smo cell surface expression by inhibiting ubiquitation-mediated endocytosis and degradation (Fan et al., 2012).
- Controlling Smo conformation, which occurs on the C-tail itself of the Smo dimer that lead to an INACTIVE (C-tails far from each other in the absense of Hh) or ACTIVE (C-tails opening and approach in the presence of Hh). This conformation change is exclusively due to the phosphorylation events (Zhao et al., 2007).
Within the cytoplasm
The activation or inhibition of the Hh pathway is regulated by a multi-protein complex (Hh signaling complex, HSC) downstream of Smo. The components of the HSC complex are:
- The transcription factor Ci
- The serine/threonine kinase Fused (Fu)
- The kinesin-like molecule Costal 2 (Cos2), which also binds to PKA, CK1 and GSK3, all implicated in the Hh signaling pathway (Aza-Blanc et al., 1997).
- Suppressor of fused (Sufu)
The HSC complex is associated with microtubules in the absense of Hh (Robbins et al., 1997; Sisson et al., 1997; Stegman et al., 2000). In the presence of Hh, the complex dissociates from the microtubule and the Cos-Fu-Ci complex interacts with the C-tail of Smo (Hooper, 2003; Ingham et al., 1991; Lum et al., 2003; Ogden et al., 2003; Ruel et al., 2003) whereas the Sufu-Ci complex remains cytoplasmic.
Both Cos-Fu-Ci and Sufu-Ci complexes regulate the status of the transcription factor Ci. Ci is a 155 kDa protein (Ci-FL, full length) that contains a zinc finger domain responsible for its DNA binding (Slusarski et al., 1995). Ci is converted to an ACTIVE FORM (Ci-A, 155 kDa) responsible for target gene activation in the presence of Hh, or to a REPRESSOR FORM (Ci-R, 75 kDa), that still bind DNA but inhibit the pathway in the absence of Hh.
Control of the active/inactive form of Ci is mediated by phosphorylation events that are mainly under the control of Cos2. In the absense of Hh, Cos2-Fu-Ci and Sufu-Ci complexes promote Ci-R formation preventing its activation (Robbins et al., 1997; Sisson et al., 1997; Wang et al., 2000; Wang and Holmgren, 2000; Wang and Jiang, 2004; Zhang et al., 2004). In the presence of Hh, the Cos2-Fu-Ci complex interacts with the C-tail of Smo domains, which is regulated by Cos2 phosphorylation (Liu et al., 2007; Nybakken et al., 2002; Ranieri et al., 2012; Ranieri et al., 2014; Ruel et al., 2007), promoting Ci-A formation and consequent pathway activation.
Figure 1. Drosophila Hh signal transduction pathway (Chen and Jiang, 2013). The mature Hh molecule reaches Hh receiving cells by binding with HSPGs, such as Dally and Dally-like (Dlp). In the absense of Hh, Ptc inhibits Smo allowing Ci to be phosphorylated by PKA, CK1 and GSK3. These phosphorylation events target Ci to a partial proteolytic cleavage (mediated by Slimb/βTRCP) to generate the repressor form (Ci-R). Binding of Hh to its receptor Ptc and co-receptor Ihog releases Ptc inhibition on Smo, which undergoes phosphorylation mainly by PKA and CK1. Consequently, Smo accumulates at the cell surface recruiting the Cos2-Fu-Ci complex. Here, according to the amount of Hh received by the cell, phosphorylation events on Cos2 and Fu regulate the activation of Ci and therefore of the pathway itself.
Hh signaling orthologues in vertebrates
In mammals, there are three paralogous Hh genes: Sonic hedgehog (Shh, the most broadly expressed and best studied Hh molecule), Indian hedgehog (Ihh, primarily involved in bone differentiation) and Desert hedgehog (Dhh, involved in gonad differentiation).
The main difference between Hh signaling in Drosophila and vertebrates is the requirement for the vertebrate intraflaggular transport (IFT), which consists of large multisubunits complexes that are responsible for the bidirectional transport of proteins between the base and the tip cilia (Huangfu et al., 2003).
Both Ptc and Smo can localize to primary cilia in a mutually exclusive way, where the binding of Shh to Ptc allows Smo to move into the cilium, promoting pathway activation through the Gli transcription factors (Rohatgi et al., 2007).
Main similarities and differences between Drosophila and vertebrate Hh signaling are:
- The Smo structure is highly conserved between Drosophila and vertebrates. Interestingly, the phospho-sites on the Smo C-tail and their dimerization mechanism is conserved as well, though the kinases involved are slightly different (Chen et al., 2011).
- There are three Ci homologues known as Gli1, Gli2 and Gli3. Gli1 and Gli2 are transcriptional activators, whereas Gli3 functions as a transcriptional repressor (Ding et al., 1998; Matise et al., 1998; Park et al., 2000; Tempé et al., 2006).
- Unlike Drosophila Sufu, vertebrate Sufu has a central and very important role in the Shh pathway (Svärd et al., 2006). However, the two proteins share high sequence homology (Merchant et al., 2004; Stone et al., 1999).
- The Cos2 homologues, kif7 and kif27, have conserved their negative role within the pathway by controlling Gli’s function and abundance (Cheung et al., 2009; Tay et al., 2005; Wilson et al., 2009).
- Mammalian Fu can associate to kif27 and being involved in ciliogenesis, while a compensatory Fu kinase, associated with kif7, is necessary for Hh signaling (Wilson et al., 2009).
These suggest an evolutionary conservation in the Shh intracellular cascade, though further studies are necessary to better understand the molecular functions of the protein involved.
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Figure 2. Mammal Hh signal transduction pathway (Chen and Jiang, 2013). The mature Hh molecule reaches Hh receiving cells by binding with HSPGs (such as GPC3, GPC4 and GPC6). In the absence of Hh, Ptc inhibits Smo allowing Gli to be phosphorylated by PKA, CK1 and GSK3. These phosphorylation events target Gli to a partial proteolytic cleavage (mediated by β-TRCP) to generate the repressor form (Gli-R). In the presence of Hh, binding of Hh to its receptor Ptc and co-receptor Cdo releases Ptc inhibition on Smo, which undergoes phosphorylation by mainly CK1 and GRK2. Consequently, Smo accumulates at the cell surface (within the cilia). Sufu is the major negative regulator of the pathway (kif7 is a minor one). In the presence of Hh, Sufu destabilization and degradation allows the release of its repression on Gli, with consequent pathway activation.
References
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- Aza-Blanc P1, Ramírez-Weber FA, Laget MP, Schwartz C and Kornberg TB (1997). Proteolysis that is inhibited by hedgehog targets Cubitus interruptus protein to the nucleus and converts it to a repressor. Cell, 89, 1043–1053.
- Bolanos-Garcia VM and Miguel RN (2003). On the structure and function of apolipoproteins: more than a family of lipid-binding proteins. Prog Biophys Mol Biol, 83, 47–68.
- Burke R, Nellen D, Bellotto M, Hafen E, Senti KA, Dickson BJ and Basler K (1999). Dispatched, a novel sterol-sensing domain protein dedicated to the release of cholesterol-modified hedgehog from signaling cells. Cell, 99, 803–815.
- Chamoun Z, Mann RK, Nellen D, von Kessler DP, Bellotto M, Beachy PA and Basler K (2001). Skinny hedgehog, an acyltransferase required for palmitoylation and activity of the hedgehog signal. Science, 293, 2080–2084.
- Chen Y and Struhl G (1996). Dual roles for patched in sequestering and transducing Hedgehog. Cell, 1, 553–563.
- Chen Y, Sasai N, Ma G, Yue T, Jia J, Briscoe J and Jiang J (2011). Sonic Hedgehog dependent phosphorylation by CK1α and GRK2 is required for ciliary accumulation and activation of smoothened. PLoS Biol, 9, e1001083.
SMO Gene Amplification and Activation of the Hedgehog Pathway as Novel Mechanisms of Resistance to Anti-Epidermal Growth Factor Receptor Drugs in Human Lung Cancer
Carminia Maria Della Corte1, Claudio Bellevicine2, Giovanni Vicidomini3, Donata Vitagliano1, Umberto Malapelle2, Marina Accardo4, Alessio Fabozzi1, Alfonso Fiorelli3, Morena Fasano1, Federica Papaccio1, Erika Martinelli1, Teresa Troiani1, Giancarlo Troncone2, Mario Santini3, Roberto Bianco5, Fortunato Ciardiello1, and Floriana Morgillo1,*
Clin Cancer Res October 15, 201521; 4686 http://dx.doi.org:/ 10.1158/1078-0432.CCR-14-3319 http://clincancerres.aacrjournals.org/content/21/20/4686.full
Purpose: Resistance to tyrosine kinase inhibitors (TKI) of EGF receptor (EGFR) is often related to activation of other signaling pathways and evolution through a mesenchymal phenotype.
Experimental Design: Because the Hedgehog (Hh) pathway has emerged as an important mediator of epithelial-to-mesenchymal transition (EMT), we studied the activation of Hh signaling in models of EGFR-TKIs intrinsic or acquired resistance from both EGFR-mutated and wild-type (WT) non–small cell lung cancer (NSCLC) cell lines.
Results: Activation of the Hh pathway was found in both models of EGFR-mutated and EGFR-WT NSCLC cell line resistant to EGFR-TKIs. In EGFR-mutated HCC827-GR cells, we found SMO (the Hh receptor) gene amplification, MET activation, and the functional interaction of these two signaling pathways. In HCC827-GR cells, inhibition of SMO or downregulation of GLI1 (the most important Hh-induced transcription factor) expression in combination with MET inhibition exerted significant antitumor activity.
In EGFR-WT NSCLC cell lines resistant to EGFR inhibitors, the combined inhibition of SMO and EGFR exerted a strong antiproliferative activity with a complete inhibition of PI3K/Akt and MAPK phosphorylation. In addition, the inhibition of SMO by the use of LDE225 sensitizes EGFR-WT NSCLC cells to standard chemotherapy.
Conclusions:This result supports the role of the Hh pathway in mediating resistance to anti-EGFR-TKIs through the induction of EMT and suggests new opportunities to design new treatment strategies in lung cancer. Clin Cancer Res; 21(20); 4686–97. ©2015 AACR.
This article is featured in Highlights of This Issue, p. 4497
Translational Relevance
The amplification of SMO in non–small cell lung cancer (NSCLC) resistant to EGFR-TKIs opens new possibilities of treatment for those patients who failed first-line EGFR-targeted therapies. The synergistic interaction of the Hedgehog (Hh) and MET pathways further support the rationale for a combined therapy with specific inhibitors. In addition, Hh pathway activation is essential for the acquisition of mesenchymal properties and, as such, for the aggressiveness of the disease. Also, in EGFR wild-type NSCLC models, inhibition of Hh, along with inhibition of EGF receptor (EGFR), can revert the resistance to anti-EGFR targeted drugs. In addition, inhibition of the Hh pathway sensitizes EGFR wild-type NSCLC to standard chemotherapy. These data encourage further evaluation of Hh inhibitors as novel therapeutic agents to overcome tyrosine kinase inhibitor (TKI) resistance and to revert epithelial-to-mesenchymal transition (EMT) in NSCLC.
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Tyrosine kinase inhibitors (TKI) against the EGF receptor (EGFR) represent the first example of molecularly targeted agents developed in the treatment of non–small cell lung cancer (NSCLC) and are, currently, useful treatments after failure of first-line chemotherapy and, more importantly, for the first-line treatment of patients whose tumors have EGFR-activating gene mutations (1). However, after an initial response, all patients experience disease progression as a result of resistance occurrence. Recognized mechanisms of acquired resistance to anti-EGFR-TKIs in EGFR-mutated NSCLC are METgene amplification or the acquisition of secondary mutations such as the substitution of a threonine with a methionine (T790M) in exon 20 of the EGFR gene itself (2). However, these molecular changes are able to identify only a portion of patients with cancer defined as “non-responders” to EGFR-targeted agents. A number of molecular abnormalities in cancer cells may partly contribute to resistance to anti-EGFR agents (2, 3). Our group and others have shown that epithelial-to-mesenchymal transition (EMT) is a critical event in the metastatic switch and is generally associated with resistance to molecularly targeted agents in NSCLC models (4, 5). EMT is a process characterized by loss of polarity and dramatic remodeling of cell cytoskeleton through loss of epithelial cell junction proteins, such as E-cadherin, and gain of mesenchymal markers, such as vimentin (6). The clinical relevance of EMT and drug insensitivity comes from studies showing an association between epithelial markers and sensitivity to erlotinib in NSCLC cell lines, suggesting that EMT-type cells are resistant to erlotinib (7). In particular, recent data suggest that cancer cells with EMT phenotype demonstrate stem cell–like features and strategies reverting EMT could enhance the therapeutic efficacy of EGFR inhibitors (4, 5).
The Hedgehog (Hh) signaling cascade has emerged as an important mediator of cancer development and metastatic progression. The Hh signaling pathway is composed of the ligands sonic, Indian, and desert hedgehog (Shh, Ihh, Dhh, respectively) and the cell surface molecules Patched (PTCH) and Smoothened (SMO). In the absence of Hh ligands, PTCH causes suppression of SMO; however, upon ligand binding to PTCH, SMO protein leads to activation of the transcription factor GLI1, which in turn translocates into the nucleus, leading to the expression of Hh induced genes (8). The Hh signaling pathway is normally active in human embryogenesis and in tissue repair, as well as in cancer stem cell renewal and survival. This pathway is critical for lung development and its aberrant reactivation has been implicated in cellular response to injury and cancer growth (9–11). Indeed, increased Hh signaling has been demonstrated in bronchial epithelial cells exposed to cigarette smoke extraction. In particular, the activation of this pathway happens at an early stage of carcinogenesis when cells acquire the ability to growth in soft agar and as tumors when xenografted in immunocompromised mice. Treatment with Hh inhibitors at this stage can cause complete regression of tumors (12). Overexpression of Hh signaling molecules has been demonstrated in NSCLC compared with adjacent normal lung parenchyma, suggesting an involvement in the pathogenesis of this tumor (13, 14).
Reactivation of the Hh pathway with induction of EMT has been implicated in the carcinogenesis of several cancer types (15). Inhibition of the Hh pathway can reverse EMT and is associated with enhanced tumor sensitivity to cytotoxic agents (16). Recently, upregulation of the Hh pathway has been demonstrated in the NSCLC cell line A549, concomitantly with the acquisition of a TGFβ1-induced EMT phenotype with increased cell motility and invasion (17).
The aim of the present work was to study the role of the Hh signaling pathway as mechanism of resistance to EGFR-TKIs in different models of NSCLC.
Methods ….
Results
Activation of Hh signaling pathway in NSCLC cell lines with resistance to EGFR-TKIs
We established an in vitro model of acquired resistance to the EGFR-TKI gefitinib using the EGFR exon 19 deletion mutant (delE746-A750) HCC827 human NSCLC cell line by continuous culturing these cells in the presence of increasing doses of gefitinib. HCC827 cells, which were initially sensitive to gefitinib treatment (in vitro IC50 ∼ 80 nmol/L), became resistant (HCC827-GR cells) after 12 months of continuous treatment with IC50 > 20 μmol/L. This cell line was also cross-resistant to erlotinib and to the irreversible EGFR kinase inhibitor BIBW2992 (afatinib; data not shown). Sequencing of the EGFR gene in gefitinib-resistant HCC827-GR cells showed the absence of EGFRT790M mutation (data not shown). After the establishment of HCC827-GR cells, we characterized their resistant phenotype by protein expression analysis. While the activation of EGFR resulted efficiently inhibited by gefitinib treatment both in HCC827 and in HCC827-GR cells, phosphorylation of AKT and MAPK proteins persisted in HCC827-GR cells despite the inhibition of the upstream EGFR (Fig. 1A).
HCC827-GR cells exhibited a mesenchymal phenotype with increased ability to invade, to migrate, and to grow in an anchorage-independent manner (Fig. 2A–C). Therefore, we next examined whether HCC827-GR cell line exhibits molecular changes known to occur during the EMT. Indeed, we found expression of vimentin and SLUG proteins and loss of E-cadherin protein expression in gefitinib-resistant cells as compared with gefitinib-sensitive cells (Fig. 1B). Although activation of the AXL kinase and NF-κB (20–22) have been described as known mechanisms of EGFR-TKI resistance, the analysis of their activation status resulted not significantly different among our cell lines. However, further studies are needed to explore a potential cooperation of AXL and NF-κB with Hh signaling.
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Recently, expression of Shh and activation of the Hh pathway have been correlated to the TGFβ-induced EMT in A549 lung cancer cells (17). To investigate the expression profile of Hh signaling components in this in vitro model of acquired resistance to anti-EGFR–TKIs, we performed Western blot analysis for Shh, GLI1, 2, 3, SMO, and PTCH in HCC827-GR cells. While Shh levels did not differ between HCC827 and HCC827-GR cells, a significantly increased expression of SMO and GLI1 was found in HCC827-GR cells as compared with parental cells (Fig. 1B). No differences in the levels of GLI2 and 3 were observed (data not shown). Of interest, also PTCH protein levels resulted increased in HCC827-GR cells. This is of relevance, as PTCH is a target gene of GLI1 transcriptional activity and increased PTCH levels indicate activation of Hh signaling. We further analyzed expression and activation of MET, as a known mechanism of acquired resistance to anti-EGFR drugs in NSCLC. Indeed, MET phosphorylation resulted strongly activated in HCC827-GR cells (Fig. 1B). Analysis of the MET ligand levels, HGF, by ELISA assay, did not evidence any significant difference in conditioned media of our cells (data not shown). As previous studies have demonstrated MET gene amplification in NSCLC cell lines with acquired resistance to gefitinib (23), we evaluated MET gene copy number by FISH analysis and D-PCR in HCC827 and in HCC827-GR cell lines. The mean MET gene copy number was similar between gefitinib-sensitive and gefitinib-resistant HCC827 cell line (Fig. 1C).
Of interest, while we were working to these experiments, data on SMO gene amplification in EGFR-mutated NSCLC patients with acquired resistance to anti-EGFR targeted drugs were reported on rebiopsies performed at progression, revealing SMO amplification in 2 of 16 patients (12.5%; ref. 24). For this reason, we evaluated by FISH SMO gene copy number in HCC827-GR cells, in which the mean SMO gene copy number was 4-fold higher than that of parental HCC827 cells, indicating SMO gene amplification (Fig. 1C).
We further analyzed the expression and the activation of these molecules on a larger panel of EGFR-WT NSCLC cell lines, including NSCLC cells sensitive to EGFRTKIs, such as H322 and Calu-3 cells, NSCLC cell lines with intrinsic resistance to EGFR-TKIs, such as H1299 and H460 cells and Calu-3 ER (erlotinib-resistant) cells, which represents an in vitromodel of acquired resistance to erlotinib obtained from Calu-3 cells (refs. 4, 18; Supplementary Table S1). As shown in Fig. 1B, similarly to HCC827-GR cells, the Hh signaling pathway resulted in activation of these NSCLC models of intrinsic or acquired resistance to EGFR-TKI.
To further investigate the presence of specific mutations in the Hh pathway components, we sequenced DNA from our panel of NSCLC cell lines by Ion Torrent NGS; results indicated the absence of specific mutations in Hh-related genes (data not shown).
Because GLI1 is a transcription factor, we tested the functional significance of increased expression of this gene in the EGFR-sensitive and -resistant cell lines, using a GLI1-responsive promoter within a luciferase reporter expression vector (Fig. 1D). Analysis of luciferase activity of HCC827-GR cells revealed a 6- to 7-fold increase in GLI-responsive promoter activity as compared with HCC827 cells (P < 0.001), suggesting that transcriptional activity of GLI1 is significantly higher in gefitinib-resistant HCC827-GR cells. Furthermore, depletion of GLI1 protein expression by transfection with a GLI1-specific siRNA expression vector led to approximately 65% decrease in GLI1-driven promoter activity in HCC827-GR (P < 0.01; Fig. 1D). To determine whether SMO expression may promote resistance to gefitinib, 2 cell lines harboring the mutated EGFR gene, HCC827 and PC9 cells, and the sensitive EGFR-WT cell line Calu-3, were transiently transfected with an SMO expression plasmid. When treated with gefitinib, transfected cells exhibited a partial loss of sensitivity to the EGFR inhibition (Fig. 1E).
Activation of Hh signaling pathway mediates resistance to EGFR-TKIs in EGFR-dependent NSCLC cell lines
As previously mentioned, HCC827-GR cells acquired expression of vimentin and SLUG and loss of E-cadherin when compared with gefitinib-sensitive HCC827 cancer cells along with an increased ability to invade, migrate, and form colonies in semisolid medium (Fig. 2A–C). We next evaluated whether the Hh pathway activation was necessary for gefitinib acquired resistance by genetically or by pharmacologically inhibiting Hh components in the HCC827-GR cell line. Knockdown of GLI1 by a GLI1siRNA approach had a very little effect on HCC827-GR cells. However, when gefitinib treatment (1 μmol/L) was performed in HCC827-GR cells after GLI1 blockade, invasion, migration, and colony-forming capabilities were significantly inhibited (Fig. 2A–C). Next, we evaluated the effects of 2 small-molecule inhibitors of SMO, such as LDE225 and vismodegib. Treatment with LDE225 (1 μmol/L;Fig. 2A–D) or with vismodegib (1 μmol/L; data not shown) alone did not significantly affect the viability and the invasion and migration abilities of HCC827-GR cells. Combined treatment with gefitinib and LDE225 (1 μmol/L) or vismodegib (1 μmol/L) caused inhibition of these parameters in HCC827-GR cells (Fig. 2A–C).
Taken together, these data show that Hh activation is required for acquisition of gefitinib resistance in HCC827-GR cells.
As overexpression and activation of MET was found in HCC827-GR cells, we evaluated whether inhibition of MET phosphorylation by PHA-665752 could restore gefitinib sensitivity in this model. Although abrogation of MET signaling in combination with the inhibition of EGFR signaling marginally affected gefitinib sensitivity of HCC827-GR cells, surprisingly, inhibition of MET synergistically enhanced the effects of Hh inhibition in HCC827-GR cells (Fig. 2A–D) in terms of invasion, migration, colony-forming, and proliferation abilities, indicating a significant synergism between these 2 signaling pathways. The triple inhibition of EGFR, SMO, and MET did not result in any additional antiproliferative effects (data not shown).
Cooperation between Hh and MET signaling pathways in mediating resistance to EGFR-TKI in EGFR-dependent NSCLC cell lines
To study the role of Hh pathway in the regulation of key signaling mediators downstream of the EGFR and to explore the interaction between Hh and MET pathways, we further characterized the effects of Hh inhibition alone and in combination with EGFR or MET inhibitor on the intracellular signaling by Western blotting. As illustrated in Fig. 3A, treatment of HCC827-GR cells with the SMO inhibitor LDE225, gefitinib or with the MET inhibitor PHA-665772, for 72 hours, did not affect total MAPK and AKT protein levels and activation. A marked decrease of the activated form of both proteins was observed only when LDE225 was combined with PHA-665772, at greater level than inhibition of EGFR and MET, suggesting that the Hh pathway cooperates with MET to the activation of both MAPK and AKT signaling pathways. In addition, vimentin expression, induced during the acquisition of gefitinib resistance, was significantly decreased after Hh inhibition, suggesting that the Hh pathway represents a key mediator of EMT in this model. The combination of MET and Hh inhibitors strongly induced cleavage of the 113-kDa PARP to the 89-kDa fragment, indicating an enhanced programmed cell death.
Of interest, the inhibition of SMO by LDE225 also reduced the activated, phosphorylated form of MET (Fig. 3A), revealing an interaction between SMO and MET receptors. To address this issue, we hypothesized a direct interplay between both receptors. SMO immunoprecipitates from HCC827-GR cells showed greater MET binding than that from the parental HCC827 cells (Fig. 3B). As MET has been demonstrated to interact with HER3 to mediate resistance to EGFR inhibitors (25), we explored the expression of HER3 in SMO immunoprecipitates. Protein expression analysis did not show any association with HER3; similar results were obtained with EGFR protein expression analysis in the immunoprecipitates (data not shown).
The increased SMO/MET heterodimerization observed in HCC827-GR cells was partially reduced by the inhibition of SMO or MET with LDE225 or PHA-665752, respectively, and to a greater extent with the combined treatment (Fig. 3B). These results support the hypothesis that Hh and MET pathways interplay at level of their receptors.
To study whether the cooperation between these 2 pathways appears also at a downstream level, and considering that, as shown in Fig. 3A, MET inhibition partially reduces the levels of GLI1 and PTCH proteins, we analyzed luciferase expression of GLI1 reporter vector in HCC827-GR cells after treatment with LDE225, PHA-665752, or both. As shown in Fig. 3C, transcriptional activity of GLI1 resulted strongly decreased by the combined treatment. In particular, treatment with single-agent LDE225 did not abrogate the transcriptional activity of GLI1 suggesting a GLI1 noncanonical activation. In addition, single-agent PHA-665752 reduced GLI1-dependent signal, suggesting a role for MET in GLI1 regulation. To better investigate these findings, we hypothesized that MET can regulate GLI1 activity through its nuclear translocation. We, therefore, analyzed the binding ability of SUFU, a known cytoplasmic negative regulator of GLI1, following treatment of HCC827-GR cells with LDE225 and/or PHA-665752. Indeed, interaction between SUFU and GLI1 was markedly decreased in HCC827-GR cells as compared with HCC827 cells (Fig. 3D), which further confirmed the role of the activation of Hh pathway in this gefitinib-resistant NSCLC model. Furthermore, while combined treatment with LDE225 and PHA-665752 strongly increased the binding between GLI1 and SUFU, suggesting an inhibitory effect on GLI1 activity, also treatment with the MET inhibitor PHA-665752 alone favored the interaction of GLI1 with SUFU (Fig. 3D), indicating a role of MET on the activation of GLI1. This phenomenon could be a consequence of the decreased interplay between SMO and MET receptors or the effect of a direct regulation of GLI1 by MET.
Effects of the combined treatment with LDE225 and gefitinib or PHA-665752 on HCC827-GR tumor xenografts
We finally investigated the in vivo antitumor activity of Hh inhibition by LDE225, alone and in combination with gefitinib or with the MET inhibitor in nude mice bearing HCC827-GR cells. Treatment with gefitinib, as single agent, did not cause any change in tumor size as compared with control untreated mice, confirming that the in vitro model of gefitinib resistance is valid also in vivo. Treatment with LDE225 or with PHA-665752 as single agents caused a decrease in tumor size even stronger than that observed in vitro, suggesting a major role of these drugs on tumor microenvironment. However, combined treatments, such as LDE225 plus gefitinib or LDE225 plus PHA-665752, significantly suppressed HCC827-GR tumor growth with a major activity of LDE225 plus PHA-665752 combination. Indeed at 21 days from the starting of treatment, the mean tumor volumes in mice bearing HCC827-GR tumor xenografts and treated with LDE225 plus gefitinib or with LDE225 plus PHA-665752 were 24% and 2%, respectively, as compared with control untreated mice (Fig. 4A). Figure 4B shows changes in tumor size from baseline in the 6 groups of treatment. A total of eight mice for each treatment group were considered. Combined treatment of LDE225 plus gefitinib caused objective responses in 5 of 8 mice (62.5%). Of interest, the most active treatment combination was LDE225 plus PHA-665752 with complete responses in 8 of 8 mice (100%).
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We then studied the effects of gefitinib, LDE225, PHA-665752, and their combinations on the expression of PTCH, MET, and vimentin in tumor xenografts biopsies from mice of each group of treatment (Fig. 4C and Supplementary Table S2). We measured PTCH expression, as it represents a direct marker of Hh activation. While vimentin staining was particularly intense in control and gefitinib-treated tumors, treatment with LDE225 alone and in combination with PHA-665752 significantly reduced the intensity of the staining further confirming the role of Hh inhibition on the reversal of mesenchymal phenotype. Of interest, MET immunostaining resulted in a consistent nuclear positivity: this particular localization has been described as a marker of poor outcome and tendency to a mesenchymal phenotype (26). Although the combination of LDE225 and gefitinib resulted in a significant reduction of tumor growth with a concomitant reduction in staining intensity of vimentin, the combination of LDE225 and PHA-665752 was the most effective treatment, with 8 of 8 (100%) mice having a complete response in their tumors. In fact, histologic evaluations of these tumors found only fibrosis and no viable cancer cells. According to Western blot analysis of protein extracts harvested from the HCC827-GR xenograft tumors, the levels of phospho-EGFR, phospho-MET, and GLI1 resulted in a decrease after treatment with the respective inhibitor. Interestingly, the combined treatment with LDE225 and PHA-665752 resulted in a stronger inhibition of phospho-MAPK and phospho-AKT (Supplementary Fig. S1).
Role of the Hh pathway in mediating resistance to EGFR inhibitors in EGFR-WT NSCLC
As shown in Fig. 1B, although H1299, H460, and Calu-3 ER lacked SMO amplification (data not shown), these cells displayed Hh pathway activation. We further conducted luciferase expression analysis that showed a 8- to 9-fold increase in GLI1-dependent promoter activity in these lines as compared with EGFR inhibitor–sensitive H322 and Calu-3 cells, suggesting that transcriptional activity of GLI1 is higher in EGFR-TKI–resistant EGFR-WT NSCLC lines (Supplementary Fig. S2A). Similar to HCC827-GR cells, these cells showed also activation of MET. However, as reported in previous studies (4), MET inhibition alone or in combination with EGFR inhibition or with SMO inhibition resulted ineffective in inhibiting cancer cell proliferation and survival (data not shown).
We therefore tested the effects of Hh inhibition, by silencing GLI1 or by using LDE225, alone and/or in combination with erlotinib. Although knockdown of GLI1 or treatment with LDE225 (1 μmol/L) did not significantly affect NSCLC cell viability, combined treatment with erlotinib restored sensitivity to erlotinib (Supplementary Fig. S2B).
In addition, H1299, Calu-3 ER, and H460 cells exhibited significantly higher invasive and migratory abilities than H322 and Calu-3 cells and inhibition of Hh pathway significantly reduced these abilities. Collectively, these results suggest that Hh pathway activation mediates the acquisition of mesenchymal properties in EGFR-WT lung adenocarcinoma cells with erlotinib resistance (Supplementary Fig. S2B–S2D).
We next evaluated the effects of LDE225 alone and/or in combination with erlotinib on the activation of downstream pathways. Erlotinib treatment result was unable to decrease the phosphorylation levels of AKT and MAPK in H1299 and Calu-3 ER cells (Fig. 5A). However, when LDE225 was combined with erlotinib, a strong inhibition of AKT and MAPK activation was observed in these EGFR inhibitor–resistant cells (Fig. 5A). Furthermore, flow cytometric analysis revealed that combined treatment with both erlotinib and LDE225 significantly enhanced the apoptotic cell percentage to 65% and 70% (P < 0.001) in H1299 and Calu-3 ER cells, respectively (Fig. 5B), confirmed by the induction of PARP cleavage after the combined treatment (Fig. 5A). These findings suggest that Hh pathway drives proliferation and survival signals in NSCLC cells in which EGFR is blocked by erlotinib, and only the inhibition of both pathways can induce strong antiproliferative and proapoptotic effects. The in vitro synergism between EGFR and SMO was confirmed alsoin vivo. Combination of erlotinib and LDE225 significantly suppressed growth of Calu-3 ER xenografted tumors in nude mice (Supplementary Fig. S1F).
Hh pathway inhibition sensitizes EGFR-WT NSCLC cell lines to standard chemotherapy
To extend our preclinical observations, we further investigated the effects of Hh pathway inhibition on sensitivity of EGFR-WT NSCLC cells to standard chemotherapy used in this setting and mostly represented by cisplatin.
To investigate the role of the Hh pathway in mediating resistance also to chemotherapy, we evaluated the efficacy of cisplatin and Hh inhibition treatment alone or in combination on the colony-forming ability in semisolid medium of H1299 and H460 cell lines (Fig. 6). Treatment with cisplatin alone resulted in a dose-dependent inhibition of colony formation with an IC50 value of 13 and 11 μmol/L for H1299 and H460 cells, respectively. However, when combined with LDE225, the treatment resulted in a significant synergistic antiproliferative effect in both NSCLC cell lines (Fig. 6). Together, these results indicate that treatment of EGFR-WT NSCLC cells with Hh inhibitors could improve sensitivity of NSCLCs to standard chemotherapy.
Resistance to currently available anticancer drugs represents a major clinical challenge for the treatment of patients with advanced NSCLC. Our previous works (4, 18) reported that whereas EGFR-TKI–sensitive NSCLC cell lines express the well-established epithelial markers, cancer cell lines with intrinsic or acquired resistance to anti-EGFR drugs express mesenchymal characteristics, including the expression of vimentin and a fibroblastic scattered morphology. This transition plays a critical role in tumor invasion, metastatic dissemination, and the acquisition of resistance to therapies such as EGFR inhibitors. Among the various molecular pathways, the Hh signaling cascade has emerged as an important mediator of cancer development and progression (8). The Hh signaling pathway is active in human embryogenesis and tissue repair in cancer stem cell renewal and survival and is critical for lung development. Its aberrant reactivation has been implicated in cellular response to injury and cancer growth (9–11). Indeed, increased Hh signaling has been demonstrated by cigarette smoke extraction exposure in bronchial epithelial cells (12). In particular, the activation of this pathway correlated with the ability to growth in soft agar and in mice as xenograft and treatment with Hh inhibitors showed regression of tumors at this stage (12). Overexpression of Hh signaling molecules has been demonstrated in NSCLC compared with adjacent normal lung parenchyma, suggesting an involvement in the pathogenesis of this tumor (13, 14).
Recently, alterations of the SMO gene (mutation, amplification, mRNA overexpression) were found in 12.2% of tumors of The Cancer Genome Atlas (TCGA) lung adenocarcinomas by whole-exome sequencing (27). The incidence of SMO mutations was 2.6% and SMO gene amplifications were found in 5% of cases. SMO mutations and amplification strongly correlated with SHH gene dysregulation (P < 0.0001). In a small case report series, 3 patients with NSCLC with Hh pathway activation had been treated with the SMO inhibitor LDE225 with a significant reduction in tumor burden, suggesting that Hh pathway alterations occur in NSCLC and could be an actionable and valuable therapeutic target (27). Recently, upregulation of Shh, both at the mRNA and at the protein levels, was demonstrated in the A549 NSCLC cell line, concomitantly with the acquisition of a TGFβ1-induced EMT phenotype (17, 28, 29) and mediated increased cell motility, invasion, and tumor cell aggressiveness (30, 31).
In the present study, SMO gene amplification has been identified for the first time as a novel mechanism of acquired resistance to EGFR-TKI in EGFR-mutant HCC827-GR NSCLC cells. These data are in agreement with the results of a cohort of patients with EGFR-mutant NSCLC that were treated with EGFR-TKIs (24). Giannikopoulus and colleagues have demonstrated the presence of SMO gene amplification in tumor biopsies taken at occurrence of resistance to EGFR-TKIs in 2 of 16 patients (24). In both cases, theMET gene was also amplified. In this respect, although the MET gene was not amplified in HCC827-GR cells, we found a significant functional and structural interaction between MET and Hh pathways in these cells. In fact, the combined inhibition of both SMO and MET exerted a significant antiproliferative and proapoptotic effect in this model, demonstrated by tumor regressions with complete response in 100% of HCC827-GR tumors xenografted in nude mice.
Several MET inhibitors have been evaluated in phase II/III clinical studies in patients with NSCLC, with controversial results. Most probably, blocking MET receptor alone is not enough to revert the resistant phenotype, as it is implicated in several intracellular interactions, and the best way to overcome resistance to anti-EGFR-TKIs is a combined approach, with Hh pathway inhibitors.
In the context of EMT, Zhang and colleagues demonstrated that AXL activation drives resistance in erlotinib-resistant subclones derived from HCC827, independently from MET activation in the same subclone, and that its inhibition is sufficient to restore erlotinib sensitivity by inhibiting downstream signal MAPK, AKT, and NF-κB (21). In addition, Bivona and colleagues described in 3 HCC827 erlotinib-resistant subclones increased RELA phosphorylation, a marker of NF-κB activation, in the absence of MET upregulation, and demonstrated that NF-κB inhibition enhanced erlotinib sensitivity, independently from AKT or MAPK inhibition (22). Differently, we detected Hh and MET hyperactivation in our resistance model HCC827-GR without a clear increase in AXL and NF-κB activation.
Although the level of activation of AXL and NF-κB did not result in contribution to resistance in our model, further studies are needed to explore a potential cooperation of AXL and NF-κB with Hh signaling.
In a preclinical model, the evolution of resistance can depend strictly from the selective activation of specific pathways, whereas different mechanisms can occur simultaneously in patients with NSCLC, due to tumor heterogeneity. Thus, all data regarding EFGFR-TKIs resistance have to be considered equally valid.
We further extended the evaluation of the Hh pathway to NSCLC cell lines harboring the wild-type EGFR gene and demonstrated that Hh is selectively activated in NSCLC cells with intrinsic or acquired resistance to EGFR inhibition and occurred in the context of EMT.
To further validate these data, we blocked SMO or downregulated GLI1 RNA expression in NSCLC cells that had undergone EMT, and this resulted in resensitization of NSCLC cells to erlotinib and loss of vimentin expression, indicating an mesenchymal-to-epithelial transition promoted by the combined inhibition of EGFR and Hh. Inhibition of the Hh pathway alone was not sufficient to reverse drug resistance but required concomitant EGFR inhibition to block AKT and MAPK activation and to restore apoptosis, indicating that the prosurvival PI3K/AKT pathway and the mitogenic RAS/RAF/MEK/MAPK pathways likely represent the level of interaction of EGFR and Hh signals.
In EGFR-WT NSCLC models, the role of MET amplification/activation is less clear, and in our experience, its inhibition did not increase the antitumor activity of SMO inhibitors.
In addition, Hh inhibition contributed to increase the response to cisplatin treatment which is the standard chemotherapeutic option used in EGFR-WT NSCLC patients and in EGFR-mutated patients after progression on first-line EGFR-TKI, thus representing a valid contribution to achieve a better disease control in those patients without oncogenic activation or after progression on molecularly targeted agents.
Collectively, the results of the present study provide experimental evidence that activation of the Hh pathway, through SMO amplification, is a potential novel mechanism of acquired resistance in EGFR-mutated NSCLC patients that occurs concomitantly with MET activation, and the combined inhibition of these 2 pathways exerts a significant antitumor activity. In light of these results, screening of SMO alteration is strongly recommended in EGFR-mutated NSCLC patients with acquired resistance to EGFR-TKIs at first progression.
Hedgehog: functions and mechanisms
Markku Varjosalo and Jussi Taipale1
Genes & Dev. 2008. 22:2454-2472 Copyright © 2008, Cold Spring Harbor Laboratory Press http://dx.doi.org:/10.1101/gad.1693608
The Hedgehog (Hh) family of proteins control cell growth, survival, and fate, and pattern almost every aspect of the vertebrate body plan. The use of a single morphogen for such a wide variety of functions is possible because cellular responses to Hh depend on the type of responding cell, the dose of Hh received, and the time cells are exposed to Hh. The Hh gradient is shaped by several proteins that are specifically required for Hh processing, secretion, and transport through tissues. The mechanism of cellular response, in turn, incorporates multiple feedback loops that fine-tune the level of signal sensed by the responding cells. Germline mutations that subtly affect Hh pathway activity are associated with developmental disorders, whereas somatic mutations activating the pathway have been linked to multiple forms of human cancer. This review focuses broadly on our current understanding of Hh signaling, from mechanisms of action to cellular and developmental functions. In addition, we review the role of Hh in the pathogenesis of human disease and the possibilities for therapeutic intervention.
The origin of the name Hedgehog derives from the short and “spiked” phenotype of the cuticle of the Hh mutant Drosophila larvae. Mutations in the Hh gene were identified by Nusslein-Volhard and Wieschaus (1980) in their large-scale screen for mutations that impair or change the development of the fruit fly larval body plan. Drosophila Hh DNA was cloned in the early 1990s (Lee et al. 1992; Mohler and Vani 1992; Tabata et al. 1992; Tashiro et al. 1993). In addition to Drosophila,Hh genes have also been found in a range of other invertebrates including Hirudo medicinalis (leech) and Diadema antillarum (sea urchin) (Chang et al. 1994;Shimeld 1999; Inoue et al. 2002). It is important to note that the model organismCaenorhabditis elegans (roundworm) has no Hh ortholog, even though it has several proteins homologous to the Hh receptor Ptc (Kuwabara et al. 2000).
Hh orthologs from vertebrates—including Mus musculus (mouse), Danio rerio(zebrafish), and Gallus gallus (chicken)—were cloned in 1993 (Echelard et al. 1993;Krauss et al. 1993; Riddle et al. 1993; Chang et al. 1994). Cloning of the firstRattus rattus (rat) and human Hh genes were reported shortly thereafter, in 1994 and 1995, respectively (Roelink et al. 1994; Marigo et al. 1995). The vertebrate genome duplication (Wada and Makabe 2006) has resulted in expansion of the Hhgenes, which can be categorized into three subgroups: the Desert Hedgehog(Dhh), Indian Hedgehog (Ihh), and Sonic Hedgehog (Shh) groups (Echelard et al. 1993). The Shh and Ihh subgroups are more closely related to each other than to the Dhh subgroup, which in turn is closest to Drosophila Hh. Avians and mammals have one Hh gene in each of the three subgroups, but due to another whole-genome duplication (Jaillon et al. 2004) and further rearrangements, zebrafish has three extra Hh homologs, one in the Shh subgroup: tiggywinkle hedgehog (Twhh) (Ekker et al. 1995), and two others in the Ihh group; echidna hedgehog (Ehh) (Currie and Ingham 1996); and qiqihar hedgehog (Qhh) (Fig. 1A; Ingham and McMahon 2001).
Components of the Hh signal transduction pathway have been identified primarily using Drosophila genetics (for example, see Lee et al. 1992; Alcedo et al. 1996;van den Heuvel and Ingham 1996; Burke et al. 1999; Chamoun et al. 2001; Jacob and Lum 2007b). Mechanisms by which the Hh signal is transduced has been further characterized using Drosophila and mouse cell culture models (Fig. 1B,C; e.g., see Kinto et al. 1997; C.H. Chen et al. 1999; Chuang and McMahon 1999;Taipale et al. 2000; Lum et al. 2003a; Nybakken et al. 2005; Varjosalo et al. 2006). In both vertebrates and invertebrates, binding of Hh to its receptor Patched (Ptc) activates a signaling cascade that ultimately drives the activation of a zinc-finger transcription factor (Ci in Drosophila, GLI1–3 in mammals), leading to the expression of specific target genes (Huangfu and Anderson 2006; Jacob and Lum 2007a; Varjosalo and Taipale 2007).
Although many of the key components are conserved in vertebrates, the mammalian Hh signaling pathway is incompletely understood and harbors some differences and additional pathway components (see below). It was long thought that the main difference between Drosophila and mammalian Hh signaling was that mammals had multiple orthologs of many pathway components, including Hh, Ptc, and Ci. However, the roles of mammalian orthologs of two critical components of the Drosophila pathway, the protein kinase Fused (Fu) and the atypical kinesin Costal2 (Cos2), appear not to be conserved (Chen et al. 2005; Merchant et al. 2005; Svard et al. 2006; Varjosalo et al. 2006). This suggests that the mechanisms of Hh signal transduction from the receptor to the Ci/GLI transcription factors have evolved differentially after separation of the vertebrate and invertebrate lineages approximately 1 billion years ago (Hedges 2002; Varjosalo and Taipale 2007).
The Hh proteins act as morphogens controlling multiple different developmental processes (Fig. 2). All mammalian Hh proteins are thought to have similar physiological effects—the differences in their roles in development result from diverse pattern of expression (McMahon et al. 2003; Sagai et al. 2005).
Dhh expression is largely restricted to gonads, including sertoli cells of testis and granulosa cells of ovaries (Bitgood et al. 1996; Yao et al. 2002; Wijgerde et al. 2005). Consistent with its expression in a very narrow tissue range, Dhh-deficient mice do not show notable phenotypes is most tissues and are viable. However, males are infertile due to complete absence of mature sperm (Bitgood et al. 1996).
Ihh is specifically expressed in a limited number of tissues, including primitive endoderm (Dyer et al. 2001), gut (van den Brink 2007), and prehypertrophic chondrocytes in the growth plates of bones (Vortkamp et al. 1996; St-Jacques et al. 1999). Approximately 50% of Ihh−/− embryos die during early embryogenesis due to poor development of yolk-sac vasculature. Surviving embryos display cortical bone defects as well as aberrant chondrocyte development in the long bones (St-Jacques et al. 1999; Colnot et al. 2005). Homozygous hypomorphic mutations of IHH in humans cause acrocapitofemoral dysplasia, a congenital condition characterized by bone defects and short stature (Hellemans et al. 2003).
Shh is the most broadly expressed mammalian Hh signaling molecule. During early vertebrate embryogenesis, Shh expressed in midline tissues such as the node, notochord, and floor plate controls patterning of the left–right and dorso-ventral axes of the embryo (Sampath et al. 1997; Pagan-Westphal and Tabin 1998;Schilling et al. 1999; Watanabe and Nakamura 2000; Meyer and Roelink 2003). Shh expressed in the zone of polarizing activity (ZPA) of the limb bud is also critically involved in patterning of the distal elements of the limbs (Riddle et al. 1993;Chang et al. 1994; Johnson et al. 1994; Marti et al. 1995). Later in development, during organogenesis, Shh is expressed in and affects development of most epithelial tissues (Fig. 2).
Deletion of Shh leads to cyclopia, and defects in ventral neural tube, somite, and foregut patterning. Later defects include, but are not limited to, severe distal limb malformation, absence of vertebrae and most of the ribs, and failure of lung branching (Chiang et al. 1996; Litingtung et al. 1998; Pepicelli et al. 1998).
The different Hh ligands often act in the same tissues during development, and can function partially redundantly (Fig. 2). For example, Shh and Ihh act together in early embryonic development, and their combined loss phenocopies the loss of the Hh receptor component Smoothened (Smo), leading to early embryonic lethality due to defects in heart morphogenesis and extraembryonic vasculogenesis (Zhang et al. 2001; Astorga and Carlsson 2007).
Of the mammalian Hh genes, only the mechanisms controlling Shh expression have been studied in detail. The expression pattern of Shh is the result of the combined action of multiple enhancer-elements, which act independently to control Shh transcription in different tissues and expression domains. Both local-acting and very distal elements have been identified (Fig. 3).
Two independent enhancers—Shh floor plate enhancer 1 (SFPE1) and SFPE2, located at −8 kb and in intron 2, respectively—act to direct reporter expression exclusively to the floor plate of the hindbrain and spinal cord (Epstein et al. 1999). A third element in intron 2, Shh brain enhancer 1 (SBE1), directs reporter expression to the ventral midbrain and caudal diencephalon. The more distal elements SBE2, SBE3, and SBE4, which are located >400 kb upstream of the Shh transcription start site (TSS) drive reporter expression in the ventral forebrain. The combined activity of these enhancers appears to cover all regions of Shh transcription along the anterior-posterior axis of the mouse neural tube (Jeong et al. 2006).
The enhancer controlling Shh expression in the ZPA of limb buds, mammals–fish conserved sequence 1 (MFCS1), is located even further upstream of the start site, at −1 Mb in intron 5 of the Lmbr1 gene (Sharpe et al. 1999; Lettice et al. 2003;Sagai et al. 2004). This element is the only enhancer in Shh that has been analyzed also by loss-of-function studies (Sagai et al. 2005), which conclusively demonstrate that MFCS1 is necessary for Shh expression in mouse ZPA. Consistently in humans, germline mutations within the conserved MFCS1 element cause congenital limb malformations characterized by preaxial polydactyly (Lettice et al. 2003). Interestingly, the MFCS1 sequence is not conserved in limbless vertebrates such as snake, limbless lizard, and newt (Sagai et al. 2005). Although the SBE2–4 and MFCS1 elements are physically far from Shh, the TSS of the region upstream of Shh contains very few genes, and only one well-described TSS exists between the MFCS1 and the TSS of Shh (Fig. 3). Given the diverse expression pattern of Shh, it is likely that a number of other enhancer-elements remain to be identified in this “gene-poor” region.
Although many enhancers that drive Shh expression have been identified, very little is known about the specific transcription factors that control their activity. The temporal and spatial expression pattern of FoxA2 suggests that it could induce Shh expression (Chang et al. 1997; Epstein et al. 1999) in the midline. Consistently, conserved binding sites for FoxA2 and Nkx6 are required for SFPE2 activity (Jeong and Epstein 2003). The Nkx2.1 homeodomain protein has also been suggested as a likely candidate regulating Shh expression in ventral forebrain (Jeong et al. 2006).
No known consensus binding sites for transcription factors are affected by the mutations in the MFCS1 limb enhancer, and the mutations are not clustered close together. However, the severity of the polydactyly phenotype correlates negatively with the conservation of nucleotide at the mutation sites, suggesting that MFCS1 activity is controlled by conserved transcriptional regulators whose DNA-binding specificity is currently not known.
After translation, Hh undergoes multiple processing steps that are required for generation and release of the active ligand from the producing cell. The mechanisms involved in Hh processing and secretion are evolutionarily conserved (see Burke et al. 1999; Amanai and Jiang 2001; Chamoun et al. 2001; Ingham and McMahon 2001; Caspary et al. 2002; Dai et al. 2002; Ma et al. 2002).
After the signal sequence is removed, the Hh molecule undergoes a cleavage catalyzed by its own C-terminal domain that occurs between conserved glycine and cysteine residues (Fig. 4; Lee et al. 1994; Porter et al. 1996). First, the peptide bond between these residues is rearranged to form a thioester. Subsequently, a hydroxyl-oxygen of cholesterol attacks the carbonyl of the thioester, displacing the sulfur and cleaving the Hh protein into two parts, a C-terminal processing domain with no known signaling activity and an N-terminal Hh signaling domain (HhN) of ∼19 kDa that contains an ester-linked cholesterol at its C terminus (Porter et al. 1996). The cholesterol modification results in the association of HhN with the plasma membrane. Subsequently, a palmitic acid moiety (Pepinsky et al. 1998) that is required for HhN activity is added to N terminus of Hh by the acyltransferase Skinny hedgehog (Ski, HHAT in humans) (Chamoun et al. 2001; Lee et al. 2001; Buglino and Resh 2008). The resulting fully active HhN signaling molecule is thus modified by cholesterol at its C terminus and palmitate at its N terminus (Chamoun et al. 2001; Lee and Treisman 2001). For clarity, we refer to this protein as Hh hereafter.
Although Hh is tightly associated with the plasma membrane, it is able to act directly over a long range (Roelink et al. 1995; Briscoe et al. 2001; Wijgerde et al. 2002). In both Drosophila and vertebrates, the secretion of Hh from the producing cell requires the activity of the 12-span transmembrane protein, Dispatched (Disp). Disp, like Ptc, belongs to the bacterial RND (Resistance-Nodulation-Division) family of transport proteins. Loss of Disp leads to accumulation of Hh in the producing cells and failure of long-range signaling (Burke et al. 1999; Ma et al. 2002).
Distances over which Hh has been shown to act are ∼50 μm in Drosophila wing imaginal disc and ∼300 μm in vertebrate limb bud (Zhu and Scott 2004). How Hh moves over a such a long distance is still not clear, and could involve passive diffusion, active transport, and/or transcytosis. Genetic evidence points to a role of heparan sulfate proteoglycans in this process, as Hh cannot be transported across a field of cells lacking the heparan sulfate synthesizing enzymes of the EXT/tout velu (ttv)/brother of tout velu (botv)/sister of tout velu (sotv) family (Bellaiche et al. 1998; Lin et al. 2000; Bornemann et al. 2004; Han et al. 2004a;Koziel et al. 2004). The substrates of ttv involved in this process appear to be the glypicans (glycosylphosphatidylinositol-linked HSPGs) Dally and Dally-like (Han et al. 2004b). Dally and Dally-like also affect Hh signaling by facilitating binding of Hh to cell surfaces (Nakato et al. 1995; Lum et al. 2003a; Han et al. 2004b).
Whether Hh is transported as individual molecules or assembled into larger particles prior to transport is not clear. Several lines of evidence support the role of large lipid/protein particles in long-range Hh transport. First, Hh staining of receiving cells displays a punctate pattern (Panakova et al. 2005). In addition, soluble Shh multimers that contain lipids and that have strong signaling potency have been described in mammalian cells (Zeng et al. 2001), and it has been reported that Drosophila Hh is transported in lipoprotein particles (Panakova et al. 2005; Callejo et al. 2006). Recent genetic evidence also suggests that Hh may be secreted in two different forms, the first of which diffuses poorly and acts at a short range. The second form is “packaged” for long-range transport, and its formation requires the cytoplasmic membrane-scaffolding protein Reggie-1/flotillin-2 (Katanaev et al. 2008).
Multiple studies have analyzed the role of cholesterol modification in Hh transport in vivo, with conflicting results suggesting that cholesterol either aids or hinders Hh transport (for example, see Lewis et al. 2001; Dawber et al. 2005; Gallet et al. 2006; Li et al. 2006). These studies are complicated because the protein expression levels of the different mutant forms of Hh need to be constant in order to rule out dose effects. In addition, interpretation of the results is made even more difficult by the fact that Hh protein lacking cholesterol modification is soluble, and thus its secretion does not require Dispatched and it can escape the producing cell without being palmitoylated (Mann and Beachy 2004) and could even become palmitoylated later during transport or at the receiving cell. Thus, genetic experiments alone cannot conclusively determine the role of cholesterol modification in Hh activity and transport. In contrast, analysis of the role of the palmitate modification in Hh transport is more straightforward, as palmitoylation can be selectively prevented either by mutation of Ski, or mutation of the palmitoylated N-terminal cysteine of the Hh proteins. Such experiments indicate that palmitoylation is required for Hh activity in Drosophila (Burke et al. 1999), and for generation of soluble multimeric Hh protein complexes and long-range signaling in vertebrates (Chen et al. 2004).
Several mechanisms are used to control the shape and size of the Hh gradient (for review, see Teleman et al. 2001). Very high levels of Hh can induce Hh expression in responding cells both in Drosophila and in mammals (Tabata et al. 1992;Roelink et al. 1995; Methot and Basler 1999). This increases the local concentration of Hh near the original source. Hh also induces the expression of its receptor Ptc, which internalizes Hh and targets it to the lysosomes for degradation (Chen and Struhl 1996; Incardona et al. 2000; Gallet and Therond 2005). This negative feedback loop restricts the spreading of the Hh signal through tissues. Vertebrates also have an additional transmembrane protein, Hedgehog-interacting protein (HIP), which is also induced by Hh signaling and binds to and further reduces the range of movement of Hh (Chuang and McMahon 1999; Jeong and McMahon 2005).
In addition to the glypical dally-like, which acts both in Hh transport and as an accessory receptor, the binding of Hh to responding cells is facilitated by the transmembrane proteins Cdo and Boc (iHog and boi in Drosophila) (Lum et al. 2003a; Tenzen et al. 2006; Yao et al. 2006). These proteins act positively in the pathway, binding to Hh via conserved fibronectin repeats (Yao et al. 2006) and increasing Hh association with its signaling receptor Ptc (Tenzen et al. 2006; Yao et al. 2006). The expression levels of Cdo and Boc are down-regulated in response to Hh signaling, resulting in yet another negative feedback that limits pathway activity (Fig. 1C).
In the absence of Hh ligand, Ptc catalytically inhibits the activity of the seven-transmembrane-span receptor-like protein Smo (Taipale et al. 2002). Binding of Hh to Ptc results in loss of Ptc activity, and consequent activation of Smo. Smo then transduces the Hh signal to the cytoplasm (Stone et al. 1996; Taipale et al. 2002). This general model is based on the genetic observations that loss of Hh or Smo cause similar phenotypes, and that Ptc loss results in a phenotype that is similar to strong overexpression of Hh. Epistasis analyses in turn indicate that Ptc acts downstream from Hh and upstream of or parallel to Smo (Ingham et al. 1991;Alcedo et al. 1996; van den Heuvel and Ingham 1996). Binding of Hh to Ptc, in turn, was determined using purified Shh and cultured cells overexpressing Ptc (Stone et al. 1996; Fuse et al. 1999).
By inferring the protein levels of ligand-bound and unbound Ptc from gene expression, Casali and Struhl (2004) suggested that the activity of the pathway depends on the ratio between these two forms. However, the fact that increasing the level of Ptc protein decreases cellular responsiveness to Hh (see Bailey et al. 2002; Taipale et al. 2002) indicates that it is the absolute amount of unliganded Ptc in a cell that controls pathway activity. This mechanism, together with the induction of Ptc by Hh results in gradual desensitization of cells to Hh and allows cells to accurately interpret the wide range of Hh concentrations present in morphogenetic gradients.
In vertebrates, Ptc exists as two isoforms, Ptc and Ptc2. Mice deficient in Ptc2 are viable, but develop alopecia and epidermal hypoplasia and have increased tumor incidence in the presence of one mutant allele of Ptc (Lee et al. 2006; Nieuwenhuis et al. 2006). Loss of Ptc, in turn, results in complete activation of the Hh pathway (Goodrich et al. 1997), suggesting that Ptc is the functional ortholog of DrosophilaPtc. Ptc has been proposed to function as a permease to affect the transmembrane movement and/or concentration of small molecules that then either agonize or antagonize Smo (Taipale et al. 2002). Supporting this hypothesis, Smo activity can be modulated by many synthetic small molecules (Chen et al. 2002b; Frank-Kamenetsky et al. 2002) and natural products, including the steroidal alkaloids cyclopamine and jervine (Chen et al. 2002a). These compounds were identified byKeeler and Binns (1966) as active ingredients in Veratrum californicum, a plant whose ingestion by sheep led to an outbreak of cyclopia in US midwest in the 1950s. The clue that these compounds antagonize Shh signaling came from the observation that the stillborn lambs have a phenotype that is strikingly similar to that of Shh mutant mouse embryos (Chiang et al. 1996).
The structural similarity between cyclopamine and sterols (Cooper et al. 1998) suggests that endogenous sterols might regulate Smo activity. This hypothesis is also supported by genetic evidence, as disruption of embryonic cholesterol synthesis leads to developmental malformations that strikingly mimic Hh mutants (Kelley et al. 1996; Cooper et al. 1998). Oxysterols (Corcoran and Scott 2006) and vitamin D3 derivatives (Bijlsma et al. 2006) have been suggested to be the endogenous metabolites that modulate Smo activity. Of these, vitamin D3 appears to bind to Smo (Bijlsma et al. 2006) based on its ability to compete against binding of labeled cyclopamine (Chen et al. 2002a).
Based on the fact that increased activity of oncogenically activated Smo proteins correlates with their increased resistance to cyclopamine, it was suggested that Smo exists in active and inactive conformational states (Taipale et al. 2000). Similarly, experiments in Drosophila suggest that dSmo can exist in two conformational states (Zhao et al. 2007). However, the activity of all small molecules found to activate or inhibit Smo appear to be specific for vertebrate Smo proteins, suggesting that mechanisms of action of Drosophila and mammalian Smo may be different. Stronger evidence for this comes from both structural and functional analyses, which indicate that Smo C-terminal domain has evolved differentially in vertebrates and invertebrates.
Several lines of evidence suggest that the cytoplasmic components and the mechanism of Hh signal transduction have diverged between Drosophila and mammals. In the following section, we will first discuss the mechanism of intracellular Hh signal transduction in Drosophila, which is fairly well understood. We will then discuss the evidence suggesting that Drosophila and mammals appear to use different components and mechanisms in transducing the Hh signal between Smo and the Ci/GLI transcription factors.
Intracellular Hh signaling in Drosophila
In the absence of Hh, Ptc keeps Drosophila Smo in an unphosphorylated state. Unphosphorylated Smo is cleared from the cell surface via endocytosis and is degraded in lysosomes (Jia et al. 2004; Zhang et al. 2004). After Hh stimulation, Smo is hyperphosphorylated and its endocytosis and degradation are blocked. Phosphorylation can be mimicked by mutation of the phosphorylation sites to negatively charged residues or by mutating adjacent positively charged arginine clusters to alanine. Based on these observations, Zhao et al. (2007) suggested that phosphorylation neutralizes the positive charge of the dSmo C terminus and induces a conformational switch in the C-terminal cytoplasmic tail and consequent dimerization or multimerization of dSmo. How these events lead to activation of downstream signaling pathway components is not understood (Zhao et al. 2007).
dSmo C terminus binds directly to the kinesin-like protein Cos2, which acts as a scaffolding protein, bringing together multiple cytoplasmic components of the pathway (Jia et al. 2003; Lum et al. 2003b; Ogden et al. 2003; Ruel et al. 2003). These include the full-length transcriptional activator form of Ci, CiA (155 kDa) (Robbins et al. 1997), and multiple serine–threonine kinases, including a kinase that specifically acts on the Hh pathaway, Fused (Fu) (Therond et al. 1996) and the multifunction kinases PKA, GSK3β, CKIα, and CKIε (for review, see Aikin et al. 2008).
In the absence of Hh, CiA is hyperphosphorylated by the combined action of PKA, which acts as a priming kinase, and GSK3β and the casein kinases, which further phosphorylate the primed substrate (Fig. 1B). The hyperphosphorylation promotes recognition of CiA by the ubiquitin E3 ligase Slimb (β-TrCP in vertebrates) (Jiang and Struhl 1998), leading to the generation of a truncated transcriptional repressor form of Ci, CiR (75 kDa) (Y. Chen et al. 1999; Price and Kalderon 1999, 2002;Wang et al. 1999; Jia et al. 2002, 2005). In addition to promoting CiR formation, Cos2 regulates Ci by tethering it to the cytoplasm and preventing its nuclear translocation (C.H. Chen et al. 1999; G. Wang et al. 2000).
In the presence of Hh, Sno accumulates and the binding of Cos2 to Smo prevents conversion of CiA to CiR (Hooper 2003; Jia et al. 2003). However, this mechanism alone is not sufficient to fully activate the pathway, as some CiA is still retained in the cytoplasm by another protein, Supressor of Fused [Su(Fu)] (Pham et al. 1995;Methot and Basler 2000). Genetic evidence from Drosophila indicates that full activation of the pathway in response to Hh requires the Fu protein kinase, which blocks the negative influence of Su(Fu) on Ci (Ohlmeyer and Kalderon 1998; Lefers et al. 2001; Lum et al. 2003b). Upon entering the nucleus, CiA binds to specific sequences (Kinzler and Vogelstein 1990; Hallikas et al. 2006) in promoter and enhancer regions and controls the transcription of the Hh target gene(s).
In Drosophila, cellular responsiveness to Hh is controlled by modulating the expression of Ci. In the posterior compartment of the wing disc, Hh and its receptor components are expressed, but target genes are not activated, as Ci mRNA expression is repressed by Engrailed (Eaton and Kornberg 1990). Cells posterior to the morphogenetic furrow of Drosophila eye, in turn, fail to respond to Hh because Ci levels are post-transcriptionally down-regulated due to the expression of hib (Hh-induced MATH and BTB protein; SPOP in vertebrates), a protein that acts as a substrate recognition subunit for the Cul3 E3 ubiquitin ligase. In contrast to Slimb-mediated ubiquitinylation, which leads to partial Ci degradation, the hib/Cul3-mediated ubiquitinylation causes complete degradation of Ci (L. Zhang et al. 2006). Expression of hib increases in response to Hh, providing another negative feedback mechanism to this pathway (Fig. 1C; Kent et al. 2006; Q. Zhang et al. 2006).
Divergence of pathway components and mechanisms
Despite the conservation of the Hh signaling pathway and many of its roles in development between invertebrate and vertebrate species (Ingham and McMahon 2001; Taipale and Beachy 2001), the mechanisms by which Smo regulates the Ci/GLI transcription factors appears to be distinct between Drosophila and mammals (Huangfu and Anderson 2006; Varjosalo and Taipale 2007).
The relatively rapid evolution of some components of the Hh pathway, including Smo, Cos2, and Fu, is apparent at sequence level. The C-terminal domains of vertebrate Smo proteins are significantly shorter than those of invertebrates and lack the main phosphorylation regions described below. In addition, the two mammalian orthologs of Cos2, Kif27, and Kif7 have none of the unique sequence characteristics of Cos2 that differentiate Cos2 from the kinesin family of motor proteins. Based on sequence, Kif7 and Kif27 appear to be functional molecular motors, whereas Cos2 has apparently lost its ability to bind ATP and function as a motor protein. The closest mammalian homolog of Drosophila Fu is also highly diverged, and significant homology between these proteins can be seen only in the protein kinase domain itself (Murone et al. 2000).
Drosophila Smo activation is coupled to the hyperphosphorylation of 26 serine/threonine residues located within the C-terminal cytoplasmic tail by PKA and CKI (Jia et al. 2004; Zhang et al. 2004; Apionishev et al. 2005). None of these PKA or CKI phosphorylation sites are conserved in vertebrate Smo. The vertebrate Smo C termini lacks one of the two known Cos2-binding domains (Jia et al. 2003), and the region homologous to the other domain (Lum et al. 2003b) is dispensable for mouse Smo (mSmo) function (Varjosalo et al. 2006). Drosophila Cos2, or mouse Kif7 or Kif27 do not appear to bind to mSmo or GLI proteins or affect Shh signaling when overexpressed in NIH-3T3 cells (Varjosalo et al. 2006). Furthermore, loss of the Fu protein kinase—which forms a tight complex with Cos2 and is required for Hh signaling in Drosophila—appears not to impair Hh signaling in mice (Chen et al. 2005; Merchant et al. 2005). Taken together, this evidence suggests that the Cos2–Fu complex, which is centrally important inDrosophila, plays little or no role in mammalian Hh signaling. Instead, it appears that mammalian Hh signaling critically depends on Su(Fu) (Svard et al. 2006)—which has a minor role in Drosophila (Ohlmeyer and Kalderon 1998)—and on several components involved in formation of the primary cilia, which either do not have Drosophila orthologs or whose orthologs appear not to function on theDrosophila Hh pathway (Nybakken et al. 2005).
Primary cilium is an organelle that protrudes from the surface of most vertebrate cells. Genetic evidence suggesting a role for primary cilium in mammalian Hh signaling comes from studies that found that mutations of several proteins required for its formation, including Kif3a, Ift88, and Ift172, result in embryonic phenotypes characteristic of the loss of Shh signaling (Huangfu et al. 2003; Park et al. 2006; Caspary et al. 2007; Vierkotten et al. 2007). Subsequent studies have linked these proteins to the processing of the GLI transcription factors (May et al. 2005; Caspary et al. 2007). Some experiments suggest that primary cilium would act as a “signaling center” where the biochemical events of signal transduction take place. It has been reported that activated mammalian Smo accumulates to primary cilia in response to Shh treatment (Corbit et al. 2005); in the absence of Shh, this accumulation is prevented by Ptc (Rohatgi et al. 2007). Other components involved in Hh signaling, including Su(Fu) and unprocessed GLI proteins, have also been localized to the primary cilium (Haycraft et al. 2005).
Drosophila lacking centrioles, and all microtubule-based structures derived from them, including centrosomes, cilia, and flagella develop almost normally, indicating that cilia are not required for Drosophila Hh signaling (Basto et al. 2006). In contrast, the genetic studies described above have clearly established that mammalian Hh signaling depends on a process that requires components involved in formation of primary cilia. However, this evidence is also consistent with a model where some other microtubule-linked process that is critical for Hh signaling is disrupted by loss of these proteins. In addition, the fraction of cellular Hh pathway components found in the primary cilium at any given time appears small. Thus, it remains to be established what role cilia play in mammalian Hh signaling and whether localization of the pathway components to cilia is required for signaling.
The lack of effect of the closest mammalian homolog of Drosophila Fused on Hh signaling suggests that other—mammalian-specific—kinases act on this pathway. We recently identified two such kinases, DYRK2 and MAP3K10, which are required for Shh signaling in NIH-3T3 cells (Varjosalo et al. 2008). Of these, DYRK2 directly phosphorylates GLI2 and GLI3 and induces their degradation. MAP3K10, in turn, appears to act in a more indirect fashion, binding to and phosphorylating multiple other proteins that regulate the Hh pathway, including GSK3β, DYRK2, and Kif3a (Nagata et al. 1998; Varjosalo et al. 2008). Because of the many connections of MAP3K10 to different pathway components, its mechanism of action is likely to be complex, and requires further study. In addition to DYRK2 and MAP3K10, it has been reported that other vertebrate-specific kinases regulate Shh signaling. These include protein kinase C-δ (PKCδ), mitogen-activated protein/extracellular signal-regulated kinase-1 (MEK-1), Akt, and DYRK1 (Mao et al. 2002; Riobo et al. 2006a,b). From our studies and previous analyses of the Hh pathway, it appears that Hh does not regulate the activity of any known kinase toward a generic substrate. Thus, the mechanism by which Hh signal is transduced appears not to depend on activation of pathway-specific kinases, but on regulation of access of substrates to relatively generic multifunctional kinases.
In conclusion, the mechanisms of mammalian Hh signaling have clearly diverged from those of Drosophila. This suggests that even signal-transduction mechanisms of conserved signaling pathways have not been “locked” early in evolution, and that they can be subject to evolutionary change. The divergence of the Hh pathway—arguably the last major signaling pathway to evolve—is also relevant to the evolution of multicomponent signaling pathways. Some pathways, such as the Notch pathway, where the same protein (Notch) functions as a receptor and a transcriptional coactivator are relatively simple and consist of a small number of pathway-specific components (Artavanis-Tsakonas et al. 1999; Pires-daSilva and Sommer 2003). Other pathways, such as the Hh signaling pathway inDrosophila are more complex. In addition to many multifunctional proteins, the Hh pathway consists of 11 known specific components: Hh, Skinny hedgehog (Ski), Dispatched, iHog/boi, Ptc, Smo, Cos2, Fu, Su(Fu), and Ci (Burke et al. 1999;Chamoun et al. 2001; Lum and Beachy 2004). The emergence of the Cos2–Fu system in invertebrates suggests that such multicomponent pathways may evolve by insertion of novel proteins between existing pathway components.
Regulation of GLI activity
In contrast to the differences in signaling between Smo and GLI, the activities of the GLI proteins themselves are regulated similarly to Ci—with the added complexity that the activator and repressor functions of Ci are divided in mammals to three GLI proteins, GLI1–3 (Jacob and Briscoe 2003; Ruiz i Altaba et al. 2007). GLI1 and GLI2 are responsible for most activator functions and have similar activities at protein level (Bai and Joyner 2001). Whereas loss of GLI2 is embryonic lethal (Mo et al. 1997; Ding et al. 1998; Matise et al. 1998), GLI1 is dispensable for normal development (Park et al. 2000). GLI1 expression is induced by Hh ligands, and its function appears to be primarily to provide positive feedback and to prolong cellular responses to Hh. GLI3, in turn, functions primarily as a repressor (B. Wang et al. 2000; Litingtung et al. 2002), and its loss or mutation leads to limb malformations in mice and humans (Vortkamp et al. 1991; Schimmang et al. 1992).
GLI activity appears to be regulated by Hh in a way that is very similar to that observed in Drosophila. In the absence of Hh, GLI3 is phosphorylated, recognized by β-TrCP, and proteolytically processed to a truncated repressor form (B. Wang et al. 2000; Pan et al. 2006). Whether similar processing of GLI2 results in complete degradation or generation of a truncated repressor form is unclear (Pan et al. 2006; Wang and Li 2006). Addition of Shh leads to inhibition of processing and accumulation of full-length forms of both GLI2 and GLI3.
The developmental processes that the Drosophila and vertebrate Hh signaling pathways regulate appear remarkably conserved (Ingham and McMahon 2001). At the cellular level, the effects of Hh range from growth and self-renewal to cell survival (Liu et al. 1998; Rowitch et al. 1999), differentiation, and/or migration. During embryogenesis, the Hh cascade is used repeatedly and in different tissues to induce a large number of developmental processes. The ability of a single morphogen to affect almost every part of the vertebrate body plan is made possible by the fact that cellular responses to Hh depend on the type of responding cell, the dose of Hh received, and the time the cell is exposed to Hh (see below). At the molecular level, the diverse cellular responses are effected by induction of different sets of target genes. Among the genes regulated tissue specifically by Hh signaling are those encoding other secreted signaling proteins, including bone morphogenetic protein 4 (BMP4) (Astorga and Carlsson 2007),fibroblast growth factor 4 (FGF4) (Laufer et al. 1994), and vascular endothelial growth factor (VEGF)-A (Pola et al. 2001), genes involved in cell growth and division (e.g., N-Myc) (Oliver et al. 2003), and many transcription factors that are essential for animal development, including members of the Myod/Myf, Pax, Nkx, Dbx, and Irx families (Pierani et al. 1999; Gustafsson et al. 2002; Jacob and Briscoe 2003; Vokes et al. 2007). The total number of genes that Hh regulates is only beginning to be discovered: A number of expression profiling studies have identified several novel target genes (for example, see Xu et al. 2006; Vokes et al. 2007), and our genome-wide in silico analyses identified 42 conserved enhancer modules with two or more GLI sites in the human genome (Hallikas et al. 2006).
The genes that are induced by Hh in many tissues, in turn, are generally involved in positive and negative feedback to the pathway itself and include Hib, GLI1, Ptc, and HIP (Fig. 1C). As Ci and the GLI proteins act as repressors in the absence of Hh and activators in its presence, many of the target genes also behave similarly, being repressed in the absence of Hh and induced in its presence.
During the development of the Drosophila wing imaginal disc, posterior (P) compartment cells express and secrete the Hh protein (Fig. 5A). The secreted Hh then induces the expression of target genes in cells located in the anterior (A) compartment. Hh acts both directly at intermediate range to pattern the anterior wing tissues close to the A–P boundary and indirectly over long range by inducing the BMP family morphogen decapentaplegic (dpp) (Basler and Struhl 1994; Tabata and Kornberg 1994). Dpp diffuses bidirectionally into both A and P compartments and controls the growth and patterning of the entire wing. Dpp expression is normally repressed by CiR, and its activation only requires that this repression is lifted. Therefore, very low levels of Hh suffice to induce dpp expression (Methot and Basler 1999). The short and intermediate range effects of Hh require induction of target genes such as collier (col) and engrailed (en), whose expression require CiA function and higher levels of Hh (Methot and Basler 1999; Hooper 2003).
Shh has an analogous role in controlling vertebrate limb patterning. Shh expressed by the ZPA located at the posterior margin of developing limb buds diffuses to adjacent tissues, forming a temporal and spatial gradient that specifies the anterior–posterior pattern of the limbs (Fig. 5B).
The effect of Hh dose on target tissue responses is best characterized in the specification of cell identities in the ventral neural tube (Jessell 2000; Patten and Placzek 2000; Marti and Bovolenta 2002). During neural tube development, Shh protein diffuses from the notochord and floor plate, creating a concentration gradient across the ventral neural tube (Fig. 5C). Different doses of Shh within this gradient specify five neuronal subtypes at precise positions along the floor plate–roof plate axis. Initially, Shh induces Class II homeodomain (e.g., Nkx2.2, Nkx6.1) (Pierani et al. 1999; Jacob and Briscoe 2003) and represses Class I homeodomain (Pax6, Pax7, Irx3, and Dbx1/2) transcription factors. Cross-repressive interactions between these factors then act to sharpen the expression boundaries and to subsequently direct cells to differentiate into specific lineages (Briscoe and Ericson 2001).
The activity of Shh as a morphogen was initially thought to be due to concentration-dependent responses, but the duration of Shh signal seems also to critically affect cellular responses. Both during neural tube and limb development, the pattern of cellular differentiation is controlled not only by the amount but also by the time of Shh exposure (Briscoe and Ericson 2001; Ahn and Joyner 2004;Harfe et al. 2004). The changing of the concentration or duration of Shh seem to have an equivalent effect on intracellular signaling.
Chick neural cells convert different concentrations of Shh into time-limited periods of signal transduction, such that signal duration is proportional to Shh concentration (Dessaud et al. 2007). This depends on the gradual desensitization of cells to Shh caused by up-regulation of patched (Ptc) (Taipale et al. 2002). Thus, in addition to its role in shaping the Shh gradient (Chen and Struhl 1996; Briscoe et al. 2001; Jeong and McMahon 2005), Ptc participates cell-autonomously in gradient sensing. This mechanism integrates Shh signal strength over time, allowing cells to more accurately determine their distance from the Hh source—resulting in more robust patterning of the nervous system.
The multiple roles of Hh signaling in embryonic patterning are discussed above and reviewed in more detail in McMahon et al. (2003). Much less is known about the roles played by Hh in pupal development and in maintaining homeostasis of tissues during adult life.
During maturation of mouse pups, Ihh signaling is important for bone growth. Permanent deletion of Ihh in chondrocytes of mice carrying conditional and inducible null alleles of Ihh results in permanent defects in bone growth, inhibiting proliferation and promoting differentiation of chondrocytes, leading to dramatic expansion of the hypertrophic zone (Razzaque et al. 2005; Maeda et al. 2007) and truncation of long bones. Interestingly, similar phenotype was observed with treatment of young mice with Smo antagonist for just 48 h (Kimura et al. 2008). In adults, Hh pathway controls bone homeostasis; activation of the Hh pathway in osteoblasts leads to bone resorption, and conversely, Hh inhibition protects aging mice against bone loss (Mak et al. 2008; Ohba et al. 2008). Adult mice seem to tolerate Hh antagonists well, suggesting that short-term Hh pathway inhibition might not interfere with the possible role of Hh as a stem cell factor (Berman et al. 2002; Kimura et al. 2008).
The best-characterized role for Hh signaling in adults is in the reproductive system, and Hh proteins are expressed and required for maturation of the germ cells in multiple species. In Drosophila ovary, Hh acts as a somatic stem cell factor, directly controlling the proliferation and maintenance of ovarian somatic stem cells (Zhang and Kalderon 2001). In mammals, Ihh and Dhh produced by granulosa cells act as paracrine factors to induce target gene expression in the developing theca cell compartment. This suggests that hedgehog signaling regulates the theca cell development in growing follicles (Wijgerde et al. 2005). Dhh also has a role in the regulating the development and function of the somatic cells of the testis (Bitgood et al. 1996; Yao et al. 2002).
Loss of Hh signaling activity during vertebrate embryogenesis causes severe developmental disorders including holoprosencephaly, polydactyly, craniofacial defects, and skeletal malformations (Muenke and Beachy 2000; Hill et al. 2003;McMahon et al. 2003; L. Zhang et al. 2006). It is now also becoming evident that components required for the function of primary cilia are required in mammalian Shh signaling (Huangfu et al. 2003). It is therefore possible that Hh signaling may also be altered in human syndromes caused by defects in cilia, including Meckel, Bardet-Biedl and Kartagener syndromes, polycystic kidney disease, and retinal degeneration (Pan et al. 2005; Kyttala et al. 2006).
On the other hand, aberrant activation of Hh signaling can cause basal cell carcinoma (BCC, the most common type of skin cancer) (Hahn et al. 1996; Johnson et al. 1996), medulloblastoma (a childhood cancer with an invariably poor prognosis) (Goodrich et al. 1997; Berman et al. 2002), and rhabdomyosarcoma (Table 1; Kappler et al. 2004). These tumor types occur at an increased rate in patients or mice with germline mutations in Ptc, and sporadic cases are often associated with mutations in the Hh pathway components Ptc, Smo, or Su(Fu), or more rarely, the amplification of GLI1.
Aberrantly activated Shh signaling has also been suggested to play a role in other cancers, such as glioma, breast, esophageal, gastric, pancreatic, prostate, and small-cell lung carcinoma (see Table 1 for references). With the exception of rare GLI1 amplifications found in gliomas (Kinzler et al. 1987), the mutational basis of Hh pathway activation in these types of cancer has not been ascertained.
Multiple lines of evidence suggest that Hh acts to promote cancer by directly regulating cellular growth and/or survival. Loss of one ptc allele causes larger body size in mice (Goodrich et al. 1997) and in humans (Gorlin 1987). Several common human single nucleotide polymorphisms affecting body height map close to Hh pathway components, including Ihh, Ptc, and Hip (Lettre et al. 2008; Weedon et al. 2008), suggesting that individual variation in height is determined in part by the strength of negative feedback loops that fine-tune Ihh signaling during bone growth. Hh pathway controls growth also during embryonic development—transgenic mice that overexpress ptc are consistently smaller than control mice, but remarkably well proportioned, illustrating that Hh signaling controls growth in many tissues. However, whether this growth effect is direct or indirectly caused by altered placental or vascular development is unclear.
In development of midbrain and forebrain, Shh is crucial in driving the rapid, extensive expansion of the early brain vesicles. The action of Shh is mediated through coordination of cell proliferation and survival (Britto et al. 2002). In addition, Shh has been implicated in regulating cell proliferation and survival in a number of other cell types, including retinal precursor cells (Jensen and Wallace 1997), myoblasts (Duprez et al. 1998), optic nerve astrocytes (Wallace and Raff 1999), cerebellar granule cells (Dahmane and Ruiz i Altaba 1999), and neural crest cells (Ahlgren and Bronner-Fraser 1999).
The molecular mechanisms by which Shh controls growth are beginning to be unraveled. In vitro studies have shown that the Shh protein up-regulates N-myc expression in cerebellar granule neuron progenitor (CGNP) cultures and that N-myc overexpression promotes CGNP proliferation even in the absence of Shh (Kenney et al. 2003). N-myc is thought to promote proliferation of CGNPs synergistically with cyclins D and E (Knoepfler et al. 2002), whose expression is also regulated by Shh (Duman-Scheel et al. 2002).
Direct evidence for the role of N-myc in pathway-associated cancer comes from a study of Shh pathway-induced medulloblastoma formation in mice, where it was shown that the disruption of N-myc, but not c-myc, inhibits cellular proliferative responses to Shh (Hatton et al. 2006). This provides in vivo evidence that N-myc plays a central role in Shh-mediated proliferation in CGNPs and in medulloblastoma cells, which are thought to be derived from CGNPs (Hatton et al. 2006).
As the Hh pathway in BCC and medulloblastoma is often affected at the level of Ptc or Smo, small molecule antagonists should act at/or downstream from these components (Taipale et al. 2000). Furthermore, several studies have shown that Smo can be targeted by small molecule drugs, and that antagonizing Smo could provide a way to interfere with tumorigenesis and tumor progression. The most commonly used antagonist of the Hh pathway is the plant alkaloid cyclopamine (Taipale et al. 2000). Cell-based high-throughput screening has revealed several distinct classes of antagonists, which, like cyclopamine, bind to Smo. These include SANTs 1–4 (Chen et al. 2002b); KAAD-cyclopamine (Taipale et al. 2000), compound-5 and compound-Z (Borzillo and Lippa 2005), and Cur-61414 (Frank-Kamenetsky et al. 2002). Although one phase I clinical trial has already reported promising results of Hh pathway antagonist in advanced BCC (Garber 2008), further clinical studies are needed to establish which of these antagonists are suitable for therapeutic use. As it has been proposed that autocrine Shh expression is required for growth of some cancers (Dahmane et al. 1997;Karhadkar et al. 2004), and stromal cell-derived Shh can also activate the Hh pathway in tumors (Becher et al. 2008), it might also be possible to treat tumors with Shh-specific monoclonal antibodies. In fact, one such antibody, 5E1, has been shown to block the growth of some tumors, including small-cell lung carcinoma (Watkins et al. 2003). In addition to targeting tumors that themselves have hyperactive Hh pathways, antagonists of the Hh pathway could also affect growth of tumors that use Hh ligands to induce angiogenesis (Pola et al. 2001; Nagase et al. 2008) or recruit other types of stromal cells supporting tumor growth. Further studies are needed to characterize the role that Shh plays in such tumor–host interactions.
Because adults can tolerate inhibition of the Hh pathway (Berman et al. 2002;Kimura et al. 2008), specifically blocking Hh signaling offers an effective treatment for the various cancers originating from aberrant Hh pathway activation. However, systemic treatment of pediatric tumors such as medulloblastoma may not be feasible due to the severe effects that transient inhibition of the Hh pathway has on bone growth (Kimura et al. 2008).
The Hh signaling pathway was first identified in Drosophila 16 yr ago. Subsequently, it has taken its rightful place among the major signaling pathways controlling animal development, being found to regulate the morphogenesis of a variety of tissues and organs during the development of organisms ranging fromDrosophila to human (McMahon et al. 2003). In addition, the Hh pathway has been linked to multiple forms of human cancer, and the possibilities for therapeutic intervention are being actively pursued.
The synthesis and processing of the Hh ligand, its release and transport through tissues, and mechanisms of signal transduction in the receiving cells have been studied extensively. However, many aspects of Hh signaling remain incompletely understood. Further research is needed in multiple areas, including the study of Hh target gene responses, which is required to understand in detail how the graded Hh signals are converted to discrete cell-fate decisions, and to decipher the molecular mechanisms that underlie the exquisite specificity of cellular responses to Hh. In addition, the therapeutic potential of Hh pathway agonists and antagonists in human degenerative diseases and cancer should be further investigated.
Targeting the Hedgehog pathway in cancer
The Hedgehog (Hh) gene was initially discovered by Christiane Nusslein-Volhard and Eric F. Weischaus in 1980 in their screen for mutations that disrupt the Drosophila larval body plan [Nusslein-Volhard and Wieschaus, 1980]. The name Hedgehog originates from the short and ‘spiked’ phenotype of the cuticle of the Hh mutant Drosophila larvae, which resembled the spikes of a hedgehog [Varjosalo and Taipale, 2008;Ingham and McMahon, 2001]. The Hh family of proteins have since been recognized as key mediators of many fundamental processes in vertebrate embryonic development playing a crucial role in controlling cell fate, patterning, proliferation, survival and differentiation of many different regions. Hh signals have diverse functions in different contexts. They may act as morphogens in the dose-dependent induction of distinct cell fates within a target field, or may act as a mitogen in the regulation of cell proliferation controlling the form of developing organs [Ingham and McMahon, 2001]. The crucial developmental function of Hh signaling is illustrated by the dramatic consequences in human fetuses, with defects in the Hh signaling pathway resulting in fetuses with brain, facial and other midline defects such as holoprosencephaly (failure of forebrain development) or microencephaly, cyclopia, absent nose or cleft palate [Rubin and de Sauvage, 2006; Belloniet al. 1996; Roessler et al. 1996]. In adults, the Hh pathway remains active and is involved in regulation of tissue homeostasis, continuous renewal and repair of adult tissues, and stem cell maintenance [Hooper and Scott, 2005].
The Hh signaling pathway has also recently been recognized to be one of the most important signaling pathways and a therapeutic target in cancer. In adults, mutation or deregulation of this pathway plays a key role in both proliferation and differentiation leading to tumorigenesis or tumor growth acceleration in a wide variety of tissues. Basal cell carcinoma (BCC) and medulloblastoma are two well-recognized cancers with mutations in components of the Hh pathway [Tostar et al. 2006; Taylor et al. 2002; Dahmane et al. 1997]. Inappropriate activation of the Hh signaling pathway has been implicated in the development of several other types of cancer including lung, prostate, breast, and pancreas, as examples. In addition, some recent findings suggest that Hh might also promote tumorigenesis by signaling in a paracrine manner from the tumor to the surrounding stroma or in cancer stem cells (CSCs).
The first Hh pathway inhibitor to be identified was the naturally occurring plant alkaloid, cyclopamine. This was discovered as a teratogenic compound causing cyclopia and holoprosencephaly in lambs whose mothers had ingested corn lilies, a phenotype similar to Sonic Hedgehog (Shh) knockout mice [Bryden et al. 1971]. No untoward effect was seen in the adult sheep. The active chemical identified in the corn lily, cyclopamine, was subsequently shown to inhibit the Hh pathway by binding to and inactivating the Smoothened (SMO) transmembrane receptor protein [Chen et al. 2002; Cooper et al. 1998]. Cyclopamine is of low affinity, has poor oral bioavailability and suboptimal pharmacokinetics and thus more potent derivatives have been synthesized. Several synthetic, small-molecule SMO antagonists with higher potency than cyclopamine such as SANT1–SANT4, CUR-61414, HhAntag-691 and GDC-0449 are now available and have been tested in preclinical models against a variety of solid tumors [Rudin et al. 2009; Scales and de Sauvage, 2009; Von Hoff et al. 2009]. In this review, we provide a brief overview of Hh signaling, discuss the roles of this pathway in solid tumors, and summarize the clinical advances in using therapeutic agents targeting the Hh signaling cascade.
Hh proteins are secreted signaling proteins that were first discovered in Drosophila along with many other components of their signal transduction machinery [Nusslein-Volhard and Wieschaus, 1980]. The mechanism of Hh protein processing, secretion, and signaling appear to be more or less conserved in evolution between Drosophila and higher organisms, although some differences exist. Drosophila has only one Hh gene, whereas vertebrate Hh signal transduction involves three Hh homologues with different spatial and temporal distribution pattern: Sonic Hedgehog (Shh), Indian Hedgehog (Ihh) and Desert Hedgehog(Dhh) [Ingham and McMahon, 2001; McMahon, 2000]. The Hh proteins undergo multiple processing steps before signaling. The Hh protein is made as a precursor molecule, consisting of a C-terminal protease domain and an N-terminal signaling unit. The precursor Hh molecule is cleaved to release the active signaling domain called HhNp. Then, the C-terminal domain of the Hh polypeptide catalyzes an intramolecular cholesteroyl transfer resulting in a formation of a C-terminal cholesterol modified N-terminal Hh signaling domain. The cholesterol modification results in association of Hh with membranes, facilitating the final processing step in which a palmitoyl moiety is added to the N-terminus of Hh (acylation), generating the fully active HhN [Varjosalo and Taipale, 2007; Porter et al. 1996]. The gene Rasp encodes the enzyme, likely located at the endoplasmic reticulum, required for the Hh acylation and the production of active Hh [Micchelli et al. 2002]. Hh is then released from the secreting cell by a dedicated transmembrane transporterDispatched (Disp) protein. In embryonic development, the cells that synthesize Hh ligands are distinct from the responsive cells. These responsive cells may either be adjacent to, or at a significant distance from, the Hh secreting cell [Varjosalo and Taipale, 2007].
In humans, the Hh signaling cascade is initiated in the target cell by the Hh ligand binding to the Patched 1protein (PTCH), a 12-span transmembrane protein (Figure 1). In the absence of a Hh ligand, PTCH catalytically inhibits the activity of the seven-transmembrane-span receptor-like protein, SMO, potentially by affecting its localization to the cell surface. It is also proposed that an endogenous intracellular small molecule that acts as an agonist for SMO is transported outside the cell by PTCH, preventing its binding to SMO. Binding of Hh to PTCH results in the loss of PTCH activity and the consequent activation of SMO, which transduces the Hh signal to the cytoplasm [Taipale et al. 2002]. The Hh signal is transmitted via an alteration of the balance between the activator and repressor forms of the Ci (cubitus interruptus)/GLI family of zinc-finger transcription factors. In Drosophilia, the Hh signal is transmitted via a protein complex which includes the atypical kinesin-like protein, Costal 2 (Cos2), Fused (Fu) and Suppressor of Fused (SuFu) and the transcription factor, Ci. In higher organisms, the Cos2 and Fu are not conserved, although SuFu still seems to play an important role in signal transduction. In mammals, the Hh signaling takes place in the nonmotile cilia to which the SMO and other downstream pathway components must need to transit to activate the Ci ortholog in mammals, the GLI transcription factors [Rubin and de Sauvage, 2006; Corbit et al. 2005;Huangfu and Anderson, 2005; Huangfu et al. 2003]. The GLI transcription factors exist as three separate zinc-finger proteins, GLI 1 and GLI 2 functioning as transcriptional activators and GLI 3 as a transcriptional repressor [Ruiz i Altaba, 1997]. The expression of GLI 1 is highly dependent upon active Hh signaling and is thus often used as a readout of pathway activation. In the absence of a Hh ligand, PTCH blocks SMO activity and full length GLI proteins are proteolytically processed to generate the repressor GLIR, largely derived from GLI 3, which represses Hh target genes. Hh binding to PTCH relieves SMO inhibition, promotes generation of the activator GLIA, largely contributed by GLI 2 and the subsequent expression of the Hh target genes. Ubiquitous mammalian Hh target genes include GLI 1, PTCH1, Hh interacting protein (Hhip) and other cell-specific genes such as Cyclin D, Myc, Bmi1, Bcl-2, VEGF (vascular endothelial growth factor) and Snail depending upon the cell type [Scales and de Sauvage, 2009; Ferretti et al. 2005]. GLI activation is regulated at several different levels via phosphorylation by inhibitors such as SuFu, Ren, protein kinase A (PKA), glycogen synthase kinase 3β (GSK3β) and activators such as Dyrk1, Ras and Akt [Varjosalo and Taipale, 2007; Ferretti et al. 2005]. Hh and PTCH are subsequently internalized and degraded in the lysosomes.
Although the extent of Hh signaling is significantly lower in the adult compared with the embryo, it is still detectable at a few sites such as the central nervous system (CNS) neural stem cells [Palma et al. 2005;McMahon, 2000]. Hh also plays an important role in the maintenance and proliferation of continuously renewing tissues such as the gut epithelium [van den Brink et al. 2004] and is reactivated at sites of tissue damage and repair [Beachy et al. 2004; Mirsky et al. 1999; Parmantier et al. 1999].
In recent years, it has become increasingly clear that the aberrant activation of the Hh signaling pathway can lead to cancer. Three basic models have been proposed for Hh pathway activity in cancer (Figure 2A–C) [Scales and de Sauvage, 2009; Rubin and de Sauvage, 2006]. The first discovered were the type I cancers harboring Hh pathway-activating mutations which are Hh ligand independent, such as BCCs and medulloblastomas. Type II cancers are autocrine (or juxtacrine) ligand dependent, meaning that Hh is both produced and responded to by the same (or neighboring) tumor cells. Type III cancers, which are paracrine ligand dependent, have been described recently. In paracrine signaling, Hh produced by the tumor cells is received by the stroma, which feeds other signals back to the tumor to promote its growth or survival [Scales and de Sauvage, 2009; Rubin and de Sauvage, 2006].
The first hint to the involvement of the Hh pathway in human cancer was appreciated when inactivating mutations in PTCH were identified in the rare condition Gorlin’s syndrome [Hahn et al. 1996; Johnson et al. 1996]. Patients with Gorlin’s syndrome develop numerous BCCs during their lifetime and are at an increased risk of other tumors including medulloblastoma, a tumor of the cerebellar progenitor cells, and rhabdomyosarcoma, a muscle tumor. This link was further strengthened when ligand-independent activation of the Hh pathway was observed in a majority of sporadically occurring BCCs [Dahmane et al. 1997]. Most of these tumors either had inactivating mutations in PTCH (85%) or activating mutations in SMO (10%) [Xieet al. 1998]. Furthermore, about one third of all medulloblastomas and occasional rhabdomyosarcomas were shown to have inappropriate Hh pathway activation, often due to PTCH mutations and sometimes due to SuFu mutations [Tostar et al. 2006; Taylor et al. 2002]. Dysregulated Hh signaling led to increased cell proliferation and tumor formation. These observations have been confirmed in various mouse models as well. Mice that are heterozygous for a PTCH mutation have a higher frequency of developing medulloblastoma, and susceptible to formation of UV-induced BCC, similar to patients with the Gorlin’s syndrome [Aszterbaum et al. 1999]. Other mouse models with ectopic expression of various Hh signaling components have been shown to develop skin phenotypes with increased epidermal proliferation and BCC-like tumors as seen in Gorlin’s syndrome [Rubin and de Sauvage, 2006; Svard et al. 2006]. The first clinical trials of Hh pathway inhibitor therapy included several patients with recurrent or metastatic BCC. Since these tumors are ligand independent, Hh pathway inhibitors must act at or below the level of SMO to be effective.
Type II Hedgehog signaling: autocrine, ligand dependent
Constitutive activation of the Hh pathway has been detected in a broad variety of tumors including lung, stomach, esophagus, pancreas, prostate, breast, liver and brain [Clement et al. 2007; Sicklick et al. 2006;Karhadkar et al. 2004; Kubo et al. 2004; Berman et al. 2003; Thayer et al. 2003; Watkins et al. 2003b]. Most of these tumors are dissimilar to BCC or medulloblastomas in that they do not harbor any somatic mutations in the Hh signaling pathway. Rather, they demonstrate an autocrine, ligand-dependent, abnormal Hh pathway activation. Most of these tumors have an elevated expression of the Hh ligand (Shh or Ihh) and/or ectopic PTCH and GLI expression within the epithelial compartment. Ectopic Hh ligand production occurring in all tumor cells or in a small number of tumor stem cells, acts upon itself or the neighboring tumor cells to support tumor growth and survival. This autocrine tumor growth can be effectively suppressed by various pathway inhibitors such as Hh neutralizing antibodies or SMO antagonists.
Type III Hedgehog signaling: paracrine, ligand dependent
A recent report by Yauch and colleagues highlighted that tumor Hh signaling may occur via paracrine mechanisms and emphasized the importance of Hh signaling in promoting the tumor microenvironment [Jiang and Hui, 2008; Yauch et al. 2008]. Paracrine Hh signaling is critical during development and for the maintenance of various epithelial structures such as the small intestine [Theunissen and de Sauvage, 2009;Varjosalo and Taipale, 2008; Ingham and McMahon, 2001]. Hh ligand secreted by the epithelium is received by the mesenchymal stroma and directly affects and stimulates proliferation in the mesenchyme. Upon Hh target gene activation, the mesenchyme produces additional molecules that feed back to the epithelium.
Fan and colleagues first showed that at least one model of prostate cancer signals to the stroma through paracrine mechanisms, with an elevated expression of PTCH and GLI in the murine stroma in response to Hh production by human xenografts [Fan et al. 2004]. These results were extended recently by three reports which showed that the Hh ligand expressing cancers were refractory to the ligand, whereas the surrounding stroma was ligand responsive [Nolan-Stevaux et al. 2009; Theunissen and de Sauvage, 2009; Tian et al. 2009; Yauch et al. 2008]. Yauch and colleagues observed that the tumor-derived Hh from several naturally Hh overexpressing xenografts stimulated expression of GLI 1/GLI 2 and PTCH in the infiltrating stroma but not in the tumor itself. Treatment with both a Hh-blocking antibody 5E1 and a small-molecule SMO inhibitor downregulated these murine stromal genes and slowed tumor growth, implying that the stromal cells send growth or survival signals back to the tumor [Theunissen and de Sauvage, 2009; Yauch et al. 2008]. In addition, Nolan-Stevaux and colleagues recently showed that the genetic deletion of SMO from pancreatic cells did not substantially alter PTCH and GLI expression in the neoplastic ductal cells and more importantly did not affect the development or progression of Kras driven pancreatic adenocarcinoma [Nolan-Stevaux et al. 2009]. Conversely, Tian and colleagues showed that the epithelial expression of mutationally activated SMO, which triggers constitutive, ligand-independent activation of the Hh pathway, was not able to induce neoplastic transformation in murine pancreatic epithelium, nor affect tumor development and progression ofKras driven pancreatic ductal adenocarcinoma models [Theunissen and de Sauvage, 2009; Tian et al. 2009].
These studies support the paracrine model of Hh signaling in which tumor cells activate Hh signaling in the surrounding stroma, resulting in the expression of soluble factors and extracellular matrix components that act upon the tumor epithelium to ultimately promote tumor growth [Theunissen and de Sauvage, 2009]. The exact mechanism of stromal feedback to the tumor remains to be determined but could involve components of the molecular signaling pathways involving insulin-like growth factor (IGF) and Wnt pathways, as IGF and Wnt signaling molecules in the tumor stroma were modulated similar to GLI and other Hh target genes in xenograft tumor models treated with Hh pathway inhibitors [Scales and de Sauvage, 2009; Yauch et al. 2008]. Inhibition of this paracrine signaling in epithelial tumors may be of therapeutic value as specific inhibition of Hh signaling in the stroma did result in growth inhibition of tumor xenografts, although the most effective way of treating these tumors would possibly be to use a combination of a Hh pathway inhibitor to target the stroma and other drugs to target the tumor cells.
Reverse paracrine signaling
Very recently, a ‘reverse paracrine’ signaling model has also been recognized in which Hh is secreted from the stroma and is received by the tumor cells (Figure 2D) [Theunissen and de Sauvage, 2009]. So far, this has only been observed in hematological malignancies such as multiple myeloma, lymphoma and leukemia, in which the Hh secreted from the stroma seems to be essential for the survival of the cancerous B cells via upregulation of the antiapoptotic factor Bcl2 [Scales and de Sauvage, 2009; Hegde et al. 2008; Dierks et al. 2007]. Stromal Hh was also found in high-grade, platelet-derived growth factor (PDGF)-induced gliomas in endothelial cells [Becher et al. 2008]. In the reverse paracrine signaling model, stromal Hh is thought to provide the appropriate microenvironment for potentiating tumor growth and would thus be a suitable therapeutic target as well.
Most renewing tissues are maintained by small populations of stem cells that have the ability to both generate additional stem cells and give rise to all mature cell types of the tissue. Hh signaling is an important regulator of stem cell activity, stimulating self-renewal and proliferation of stem cells in various tissues (Figure 2E) [Taipale and Beachy, 2001; Zhang and Kalderon, 2001]. It is believed that tumor growth and propagation might be dependent on a small population of CSCs that are similar to normal tissue stem cells and are regulated by the same signaling molecules as the normal stem cells [Reya et al. 2001]. Growing evidence suggests that the abnormal formation and expansion of cancer is due to deregulation of the multiple signaling pathways in the stem cells including the Hh, Wnt, Notch and BMP pathways [Rubin and de Sauvage, 2006]. Hh signaling has been shown to regulate the self-renewal of CSCs in breast, glioma and multiple myeloma, and more convincingly in the maintenance of chronic myelogenous leukemia (CML) stem cells [Theunissen and de Sauvage, 2009; Dierks et al. 2008; Clement et al. 2007; Peacock et al. 2007; Liu et al. 2006]. Dierks and colleagues observed that CML stem cells (Bcr-Abl driven Lin−/Sca1+/c-Kit+) with SMO knockout had a reduced ability to form tumors in irradiated mice whereas SMOM2 expression enhanced it [Dierks et al. 2008; Peacock et al. 2007]. Furthermore, SMO antagonists such as cyclopamine and Hh blocking antibody 5E1 both inhibited growth of the CML CSCs in vitro and in vivo and enhanced time to relapse after the end of treatment. A recent report showing that Hh signaling is essential for maintenance of CSCs in CML lends further support for this concept. The loss of SMO in the mouse hematopoietic system resulted in decreased induction of CML by the Bcr-Abl oncoprotein and induced Numb, causing depletion of CML stem cells. Cyclopamine treatment inhibited the growth of imatinib-resistant mouse and human CML indicating that Hh signaling may be an important target to avoid induction of imatinib-resistant CML [Zhao et al. 2009].
Tumors contain only a minority of CSCs, which can give rise to more CSCs as well as nontumorigenic cancer cells [Al-Hajj and Clarke, 2004; Beachy et al. 2004]. CSCs are typically resistant to conventional chemotherapy and radiation as they are slow growing and are thought to be the cause of cancer relapse after tumor debulking by conventional therapy. The fact that active Hh signaling has been identified in several types of CSCs makes Hh inhibition a promising therapeutic target to deplete the tumor-forming CSCs, ideally in combination with other tumor debulking agents or radiation to remove the differentiated bulk of the tumor [Scales and de Sauvage, 2009]. Another recent finding that Hh positively regulates the expression of drug transport pumps in stem cells, enabling them to resist uptake of cytotoxic drugs [Sims-Mourtada et al. 2007], makes the strategy of using Hh inhibitors to target the CSCs more rational.
Hh signaling has also been shown to promote tumor metastasis by being actively involved in the epithelial–mesenchymal transition (EMT). EMT involves transforming polarized epithelial cells into motile mesenchymal cells facilitating invasive growth and ultimately causing metastasis. Hh exerts its effects on EMT via the upregulation of transcription factor SNAIL and downregulation of E-cadherin [Rubin and de Sauvage, 2006; Karhadkar et al. 2004]. This observation was first made by Karhadkar and colleagues in prostate cancer cell lines where they showed that the rarely metastasizing clone AT2.1 could be induced to metastasize by overexpression of GLI 1, and that the capacity of another cell line AT6.3 to metastasize to the lung was abrogated by cyclopamine [Karhadkar et al. 2004]. Similar observations in pancreatic cancer cell lines were made by Feldman and colleagues, who showed that ectopic expression of GLI led to increased invasiveness, whereas inhibition of the Hh pathway led to downregulation of Snail expression and reduction in invasive properties [Feldmann et al. 2007].
Aberrant Hh signaling can be activated in a variety of cancers through various mechanisms, as discussed earlier. Understanding the specific mechanism of Hh activation in a particular tumor might help in selecting the most appropriate agent and strategy for optimizing the therapeutic benefit to be obtained by Hh pathway inhibition. Tumors such as BCC or medulloblastoma, which have a constitutive, mutation-driven activation of the Hh pathway, may be best treated with single-agent Hh inhibitors acting downstream of the activating mutation. Tumors with predominant autocrine or paracrine Hh signaling and CSCs might be more effectively treated with a combination of Hh antagonists and cytotoxic drugs targeting tumor cells [Scales and de Sauvage, 2009].
The first Hh pathway inhibitor to be identified, cyclopamine, inhibited the Hh pathway by binding to, and inactivating, SMO [Chen et al. 2002; Cooper et al. 1998]. However, cyclopamine has low affinity, poor oral bioavailability and suboptimal pharmacokinetics, and more potent derivatives have been synthesized. Several synthetic, small-molecule SMO antagonists with higher potency than cyclopamine such as SANT1–SANT4, CUR-61414, HhAntag-691, GDC-0449, MK4101, IPI-926 and BMS-833923 as examples, are now available and have been tested in preclinical models [Scales and de Sauvage, 2009]. Hh-blocking antibodies, which act upstream of SMO by preventing the binding of Hh to PTCH like 5E1, are also available and have demonstrated good preclinical activity [Scales and de Sauvage, 2009]. Multiple other drugs targeting different points of the Hh pathway, such as the natural Hh inhibitor Hhip mimetic, SUFU mimetics and GLI activity/transcription blocking agents (Gant 61 and Gant 58) are in various phases of development, as well [Lauth et al. 2007; Lauth and Toftgard, 2007]. Recently, a small molecule that binds the extracellular Shh protein, robotnikin, was isolated from small-molecule microarray-based screens [Stanton et al. 2009]. Targeting Shh ligands may be an interesting approach since the tumor-derived Shh ligands directly activate signaling in stromal cells. So far, only the SMO antagonists have been tested in the humans, and of these the CUR-61414 and GDC-0449 compounds, IPI-926, and BMS-833923 (XL139) are in the most advanced phase of clinical evaluation.
Basal cell carcinoma
BCC is the most common skin cancer in the United States, with an annual incidence of approximately 1,000,000 new cases. BCC was the first group of cancers in which the tumorigenic potential of deregulated Hh signaling was identified. This was based on the identification that patients with Gorlin’s syndrome had a marked susceptibility to develop BCCs [Hahn et al. 1996; Johnson et al. 1996]. Using family-based linkage studies of kindred with Gorlin’s syndrome, the causative mutation was mapped to the Patched 1 gene (PTCH1) on chromosome 9 [Gailani et al. 1992]. It is believed that upregulation of Hh signaling is the sole and pivotal abnormality in all BCCs [Epstein, 2008; Hutchin et al. 2005]. Approximately 90% of the sporadic BCCs have an identifiable mutation in at least one allele of PTCH1 (loss-of-function mutation) and about 10% have activating mutations in SMO (gain-of-function mutation) [Epstein, 2008; Xie et al. 1998;Gailani et al. 1996]. These mutations cause constitutive Hh pathway signaling that mediate unrestrained proliferation of basal cells of the skin, which has been confirmed in various mouse models of BCC, as well [Grachtchouk et al. 2000; Aszterbaum et al. 1999; Xie et al. 1998]. With such strong evidence of dysregulated Hh ‘oncogene addiction’ in BCC, blocking the Hh pathway would theoretically be a useful therapeutic approach for patients with metastatic BCC not controllable by other local therapies.
The first discovered steroidal alkaloid cyclopamine was used as a topical application by one group to induce regression in four BCCs [Tabs and Avci, 2004]. Several other synthetic cyclopamine derivatives have subsequently been developed as Hh pathway inhibitors, with better pharmacological and inhibitory properties than cyclopamine. Cur-61414, one of the earlier synthetic SMO inhibitors, prevented the formation of BCC-like ‘basaloid nests’ in Shh-treated ex vivo skin punches from PTCH+/− mice and also eliminated preformed BCC-like lesions [Scales and de Sauvage, 2009; Athar et al. 2004]. Interestingly, Cur-61414 selectively induced apoptosis and decreased proliferation in the BCC-like lesions, without any deleterious effects on normal surrounding skin [Scales and de Sauvage, 2009; Athar et al. 2004]. Cur-61414 was safe and well tolerated in other preclinical models, as well, and was thus formulated as a topical agent [Scales and de Sauvage, 2009; Flagella, 2006]. It was the first class of Hh antagonists to enter phase I clinical trials for use in sporadic BCC patients. However, it did not produce any clinical changes or reduction in Hh target gene GLI1 transcription when applied topically to BCC lesions, possibly because the formulation did not adequately penetrate the human skin [Fretzin et al. 2006].
GDC-0449, a second Curis-Genentech novel SMO inhibitor, was discovered by high-throughput screening of a library of small-molecule compounds and subsequent optimization through medicinal chemistry. GDC-0449 is a selective Hh pathway inhibitor with greater potency and more favorable pharmaceutical properties than cyclopamine, with good antitumor activity seen in preclinical models [Rudin et al. 2009; Von Hoff et al. 2009; Yauch et al. 2008]. The results of the phase I study of GDC-0449 demonstrating antitumor activity in patients with BCC and medulloblastoma were published recently [Rudin et al. 2009; Von Hoff et al. 2009]. Thirty-three patients with metastatic or locally advanced BCC received oral GDC-0449 at one of three doses, 150, 270 or 540 mg daily for as long as the patients had clinical benefit. Of the 33 patients, 18 had an objective response to GDC-0449, with 2 complete responses and 16 partial responses. Eleven other patients had stable disease with 4 patients having progressive disease. GDC-0449 has an unusual pharmacokinetic profile with high, sustained micromolar plasma concentrations and long terminal half-life. The median time to steady state was 14 days (range, 7–22 days). A consistent steady-state total plasma level of GDC-0449 was maintained throughout the treatment period of the study, with no apparent decline at the time of disease progression. Pharmacodynamic downmodulation in the Hh pathway was shown by a decrease in GLI1 expression as compared with pretreatment biopsy-sample analysis. The extent of GLI1 downmodulation did not correlate with pharmacokinetic levels of GDC-0449 in individual patients. Grade 3 adverse events related to the study drug included fatigue, hyponatremia, muscle spasm and atrial fibrillation. Other milder side effects included hair loss or thinning, altered taste sensation, nausea and vomiting, dyspepsia and weight loss. Interestingly, some of these toxicities might be attributable to the on-target effects of Hh in taste bud papillae formation and hair growth [Scales and de Sauvage, 2009]. High levels of GLI1 mRNA expression were observed in the tumors from responding patients, consistent with constitutive activation of the Hh pathway. Based on these promising results, GDC-0449 has now entered phase II trials in advanced BCC.
Medulloblastoma
Medulloblastoma, an aggressive childhood tumor of cerebellar origin, is another malignancy with a well-recognized dependency on aberrant Hh signaling. The first indication that alteration in the Hh signaling pathway contributes to medulloblastoma was the discovery that patients with Gorlin’s syndrome, who have germline mutations in the PTCH-1 gene, have an increased incidence of medulloblastoma [Goodrich and Scott, 1998; Kimonis et al. 1997]. Although rare, the outcome of medulloblastomas is invariably poor. Primary management consists of surgical resection followed by radiation and chemotherapy, with serious treatment-related morbidity from these modalities. Patients with recurrent disease after primary therapy have a median survival of less than 6 months [Zeltzer et al. 1999].
Hh signaling has a critical role in the developing cerebellum. Shh released by the migrating Purkinje cells delays neuronal differentiation and induces proliferation of granular neuron precursors in the external germinal layer of the cerebellum [Berman et al. 2002; Wechsler-Reya and Scott, 2001; Wallace, 1999]. Although critical during embryogenesis, the Hh pathway is downregulated after early postnatal development in most tissues, including brain, and the constitutive activation of this pathway seems to give rise to medulloblastomas [Romer et al. 2004]. More than 30% of human medulloblastomas demonstrate high levels of GLI1 expression consistent with abnormal activation of the Hh pathway [Lee et al. 2003]. Hh pathway antagonists thus have potential therapeutic value in the treatment of medulloblastomas and have been tested successfully in preclinical models and most recently in the clinic as well.
Cyclopamine was shown to decrease the rate of growth of mouse medulloblastoma cells both in culture and in mouse allograft models [Berman et al. 2002; Dahmane et al. 2001]. Interestingly, cyclopamine inhibited the in vitro growth of all human medulloblastoma cell lines, although only about one third would be expected to harbor Hh pathway mutations, suggesting Hh antagonists could be broadly effective in treating all medulloblastomas [Scales and de Sauvage, 2009; Berman et al. 2002]. Romer and colleagues used another small-molecule SMO-binding Hh antagonist, Hh-Antag to treat endogenous medulloblastomas in PTCH1+/−p53−/− mice models, where tumors develop with 100% incidence [Romer et al. 2004]. Hh-Antag completely eliminated the medulloblastomas by blocking tumor cell proliferation and stimulating apoptosis, without adversely affecting the surrounding cerebellum [Romer et al. 2004]. Rudin and colleagues recently reported a patient with metastatic medulloblastoma, refractory to multiple therapies responding to the novel Hh pathway inhibitor, GDC-0449 [Rudin et al. 2009]. Treatment resulted in rapid regression of the tumor burden and reduction of symptoms, although resistance to drug developed rapidly. Molecular analyses of the patient’s tumor specimens obtained before treatment showed increased expression of Hh target genes including GLI1, PTCH1, PTCH2 and secreted frizzled-related protein 1 (SFRP1), suggesting activation of the Hh pathway. Genomic analysis of the PTCH1 locus in tumor cells showed loss of heterozygosity and somatic mutation with no such alterations seen in the normal skin tissue biopsies [Rudin et al. 2009]. There is currently an ongoing phase II trial evaluating the efficacy and safety of GDC-0449 in the treatment of adults with recurrent or refractory medulloblastoma (see www.clinicaltrials.gov). The use of Hh pathway inhibitors in the treatment of medulloblastomas may offer a more effective therapeutic option and may avoid some of the serious adverse effects of current treatments. Since the Hh pathway also regulates various developmental pathways, it is unclear what the adverse effects of Hh pathway blockade may be in prepubescent children.
Multiple other solid tumors that do not harbor any somatic mutations in the Hh signaling pathway, such as BCC or medulloblastoma, also demonstrate a ligand-dependent activation of the Hh pathway. Constitutive activation of the Hh pathway has been detected in a broad variety of tumors including lung, stomach, esophagus, pancreas, prostate, breast, liver and brain [Clement et al. 2007; Sicklick et al. 2006; Karhadkar et al. 2004; Kubo et al. 2004; Berman et al. 2003; Thayer et al. 2003; Watkins et al. 2003b]. Although preclinical xenograft and animal models of many of these Hh overexpressing tumors show tumor growth inhibition on treatment with cyclopamine [Karhadkar et al. 2004; Berman et al. 2003; Thayer et al. 2003;Watkins et al. 2003a; Watkins et al. 2003b], the potential usefulness of Hh pathway inhibitors have yet to be tested in a clinical setting.
In addition to the above effect of Shh signaling in cancer and stromal cells, inhibition of the Shh pathway seems to augment the formation of desmoplasia in pancreas cancer [Olive et al. 2009]. The expression of Shh was found to cause desmoplasia formation in pancreatic cancer [Bailey et al. 2008]. IPI-926, a synthetic, small-molecule SMO antagonist, combined with gemcitabine was shown to improve the gemcitabine delivery to this pancreatic tumor model by depleting tumor-associated stromal tissue.
There are multiple Hh pathway inhibitors in development, including SANT1–SANT4, CUR-61414, HhAntag-691, GDC-0449, MK4101, IPI-926, BMS-833923 and itraconazole [Kim, 2009; Scales and de Sauvage, 2009]. The orally available SMO inhibitor GDC-0449 is the farthest along in development and is the major Hh antagonist actively being tested for use in ligand-dependent cancers. Two trials utilized GDC-0449 as maintenance therapy, one in patients with ovarian cancer in a second or third complete remission and the other for first-line therapy for metastatic colorectal cancer in combination with concurrent chemotherapy and bevacizumab (see www.clinicaltrials.gov and Scales and de Sauvage, 2009). Two other trials evaluating the use of GDC-0449 for the treatment of extensive-stage small cell lung cancer in combination with chemotherapy and unresectable pancreatic cancer in combination with erlotinib have recently been opened and are actively recruiting patients (see www.clinicaltrials.gov).
The last decade has seen extraordinary progress in understanding the roles and mechanism of action of Hh proteins in development and cancer. Targeting the Hh signaling pathway provides a new and exciting therapeutic option for a broad variety of cancers. Novel associations with dysregulated Hh signaling and the formation of cancer continue to emerge. Although all mechanisms of the Hh signaling pathway are not completely understood, it is clear that aberrant Hh signaling causes tumor growth and proliferation, increases tumor aggressiveness and raises the frequency of metastasis. Inhibition of the Hh pathway is thus a promising new approach for the treatment of select advanced malignancies. These include cancers such as BCC and medulloblastoma, which have mutations leading to constitutive activation of the Hh pathway, as well as other tumors which are Hh ligand dependent for tumor growth either by autocrine or paracrine mechanisms. Initial clinical trials of the oral SMO antagonist GDC-0449 show good efficacy and safety in BCC and medulloblastoma [Rudin et al. 2009; Von Hoff et al. 2009]. Although Hh pathway inhibitors seem to be safe in adults, their safety in children, especially for the treatment of medulloblastoma, is yet to be ascertained. The use of Hh antagonists in the treatment of ligand-dependent cancers is also to be determined, with multiple ongoing clinical trials in other solid tumors (see www.clinicaltrials.gov). Hh signaling also seems to be important for regulating stem cells in various tissues and Hh pathway inhibition might represent another method to target these relatively resistant and slow-growing CSCs. Optimally this approach would warrant the combination of systemic Hh pathway inhibition with other cytotoxic inhibitors of tumor growth. To maximally exploit the Hh pathway for therapeutic purposes, a better understanding of the precise Hh signaling mechanisms in various tumors is required.
It has been exciting to follow the advances of Hh pathway inhibitors in the ongoing preclinical and clinical trials including the recently reported use in advanced and metastatic BCC. These preliminary studies have set the stage for using these inhibitors in other cancers. Hh pathway inhibitors truly represent an important new class of therapeutic agents, which are bound to have far-reaching implications in oncology.
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