Overview of the HEDGEHOG-GLI signalling

The Hh signalling has been initially identified in Drosophila melanogaster, where it is required for determining proper embryonic patterning (Ref. Reference Nusslein-Volhard and Wieschaus8). Smo protein is conserved and maintains its function in mammals, whereas there are two Ptch proteins in vertebrates. Hh ligand has diversified into Sonic (SHH), Indian (IHH) and Desert (DHH) Hedgehog, and the function of the downstream transcription factor Cubitus interruptus (Ci) has evolved into three GLI proteins: GLI1, GLI2 and GLI3. Here we focus on the function and regulation of the three GLI transcription factors and we present only a brief introduction of the key steps and components of vertebrate HH-GLI signalling upstream of GLI.

The initiation of the HH signalling begins with the binding of one of the three HH ligands, each with distinct spatial and temporal expression patterns, to the 12-pass transmembrane protein receptor PTCH, which resides in the primary cilium, a non-motile structure that functions as a sensor and coordinator centre for the HH signalling (Refs Reference Rohatgi, Milenkovic and Scott9, Reference Eggenschwiler and Anderson10, Reference Wong and Reiter11). Binding of HH ligands to PTCH relieves its inhibitory effect on the G-protein-coupled receptor-like SMO, which moves into the tip of the cilium and triggers a cascade of events that promote the formation of GLI activator forms (GLI-A). GLI2/3-A translocate into the nucleus and induce HH pathway target genes, including GLI1 (Refs Reference Rohatgi and Scott12, Reference Jiang and Hui13, Reference Varjosalo and Taipale14) (Fig. 1). In absence of HH ligands, PTCH inhibits pathway activation by preventing SMO to enter the cilium. This results in the phosphorylation and proteasome-mediated carboxyl cleavage of GLI3 and, to a lesser extent, of GLI2 to their repressor forms (GLI2/3-R; Refs Reference Wang, Fallon and Beachy15, Reference Pan16). GLI1 is degraded by the proteasome and is transcriptionally repressed, with consequent silencing of the pathway. GLI1 acts exclusively as an activator, whereas GLI2 and GLI3 display both positive and negative transcriptional functions (Refs Reference Wang, Fallon and Beachy15, Reference Dai17, Reference Bai, Stephen and Joyner18) (Fig. 1).

Figure 1. Key components of the mammalian HH signalling pathway. In absence of HH ligands (a), PTCH inhibits SMO by preventing its entry into the primary cilium. GLI proteins are phosphorylated by PKA, GSK3β and CK1, which create binding sites for the E3 ubiquitin ligase β-TrCP. GLI3 and, to a lesser extent, GLI2 undergo partial proteasome degradation, leading to the formation of repressor forms (GLI3/2^R, red), that translocate into the nucleus where they inhibit the transcription of HH target genes. Full-length GLI may also be completely degraded by the proteasome. This process can be mediated by Spop and Cullin 3-based E3 ligase for GLI2 and GLI3, whereas GLI1 can be degraded by β-TrCP, the Numb-activated Itch E3 ubiquitin ligase and by PCAF (see text for details). Upon HH ligand binding (b), PTCH is displaced from the primary cilium, allowing accumulation and activation of SMO. Active SMO promotes a signalling cascade that ultimately leads to translocation of full length (FL) activated forms of GLI (GLI^A, green) into the nucleus, where they induce transcription of HH target genes. Abbreviations: CK1, casein kinase 1; GSK3β, glycogen synthase kinase 3β; HH, Hedgehog; PCAF, p300/CREB-binding protein (CBP)-associated factor; PKA, protein kinase A; PTCH, Patched; SMO, Smoothened; Spop, speckle-type POZ protein; SUFU, Suppressor of Fused; β-TrCP, β-transducin repeat-containing protein.

The HH target genes include GLI1, which further amplifies the initial HH signalling at transcriptional level and, therefore, is a reliable and robust read-out of an active pathway (Ref. Reference Lee19). Other HH target genes are PTCH1 and HH interacting protein (HHIP1), which both mediate negative feedback by limiting the extent of HH signalling. The outcome of the HH signalling varies according to the receiving cell type, and includes a number of cell-specific targets mediating a variety of cellular responses: proliferation and differentiation (Cyclin D1 and D2, E2F1, N-Myc, FOXM1, PDGFRα, IGFBP3 and IGFBP6, Hes1, Neogenin), cell survival (BCL-2), self-renewal (Bmi1, Nanog, Sox2), angiogenesis (Vegf, Cyr61), cardiomyogenesis (MEF2C), epithelial–mesenchymal transition (Snail1, Sip1, Elk1 and Msx2) and invasiveness (Osteopontin). (Ref. Reference Milla, Gonzalez-Ramirez and Palma20). The strength of HH signalling is tuned by a number of post-transcriptional and post-translational modifications of the three GLI transcription factors.

Modes of action of HH-GLI signalling in cancer

Multiple mechanisms of HH pathway activation have been proposed in cancer (reviewed in Ref. Reference Scales and de Sauvage57). The mode of action of HH-GLI signalling has important implications for the design of therapeutic antagonists, therefore it is important to dissect the cellular and molecular mechanisms of HH activation in human cancers.

Ligand-independent activation (Type I) was the first type of aberrant HH pathway activation recognised in cancer, with the finding that patients with Gorlin syndrome (Ref. Reference Gorlin58) harbour mutations in PTCH1. Tumours with ligand-independent activation of HH pathway carry genetic aberrations that confer cell-intrinsic growth properties to the tumour. The most frequent alterations found are inactivating mutations of pathway repressors, such as PTCH1 (Refs Reference Hahn59, Reference Johnson60), SUFU (Refs Reference Taylor61, Reference Sheng62) or REN (Ref. Reference Di Marcotullio63), mutations leading to constitutive activation of SMO (Ref. Reference Xie64), or gene amplifications of GLI1 and GLI2 (Refs Reference Kinzler65, Reference Northcott66), that result in constitutive HH pathway activation. Defining the molecular mechanisms of ligand-independent activation of the signalling is crucial to determine whether a tumour might respond to the treatment with a HH inhibitor acting at the level of SMO or, in case the genetic alteration affects downstream components of the pathway, at the level of the GLI proteins.

Ligand-dependent autocrine/juxtacrine activation of the pathway (Type II) has been identified in the last few years in different types of cancers, including lung, pancreas, gastrointestinal tract, prostate and colon cancers, glioma and melanoma (Refs Reference Sheng62, Reference Varnat67, Reference Watkins68, Reference Yuan69, Reference Thayer70, Reference Feldmann71, Reference Berman72, Reference Karhadkar73, Reference Sanchez74, Reference Clement75, Reference Bar76, Reference Ehtesham77, Reference Stecca78). In this case, tumours show increased HH ligand expression, in absence of genetic aberrations of HH pathway components, and respond to HH stimulation in cell-autonomous manner. This concept is supported by a number of experimental data showing that: (i) tumour cells, but not the surrounding stroma, express HH ligands and downstream HH signalling components (e.g. PTCH1, GLI1) (e.g. Refs Reference Varnat67, Reference Sanchez74, Reference Stecca78); (ii) tumour cell growth could be inhibited by RNAi-mediated knockdown of SMO or GLI1 and by treatment with cyclopamine (a SMO antagonist) in vitro and in xenograft models in vivo; (iii) metastatic growth could be prevented in vivo upon RNAi-mediated knockdown of SMO or GLI1 (Ref. Reference Varnat67). These effects appear to be specific, because GLI1 epistatically rescues the inhibition of metastatic colonies obtained with SMO silencing (Ref. Reference Varnat67).

Ligand-dependent paracrine activation of HH pathway (Type IIIa) is a mode of action that resembles the physiological HH signalling occurring during embryo development. In this case, HH ligands secreted by cancer cells activate HH signalling in the surrounding stroma rather than in the tumour itself. The mechanisms by which the HH signalling pathway and the tumour stroma interact during paracrine signalling are not completely understood. However, activation of HH signalling in the tumour-associated stroma might lead to the production of growth factors (e.g. VEGF, IGF) and stimulation of other signalling pathways (e.g. Wnt, Interleukin-6) that in turn create a favourable microenvironment sustaining the growth and progression of the tumour (Ref. Reference Yauch79). Evidence supporting this mechanism has accumulated from studies in human tumour xenograft models of pancreatic and colorectal cancers that express high levels of HH ligands, in which increased expression of HH targets is detected specifically in tumour-infiltrating mouse stromal cells (Ref. Reference Yauch79). Interestingly, growth of mutant Kras-driven tumours is reduced in mice lacking Gli1 in the pancreatic microenvironment compared to wild-type mice (Ref. Reference Mills80).

Similarly, the reverse paracrine HH pathway activation (Type IIIb) has been described in an experimental model of glioma (Ref. Reference Becher81) and in haematological malignancies such as B-cell lymphoma and mantle cell lymphoma (MCL; Refs Reference Dierks82, Reference Hegde83). According to this modality, HH ligands are secreted by the tumour microenvironment (bone marrow stromal cells or endothelial cells) and activate the pathway on tumour cells, thus affecting its growth.

Activation of HH-GLI signalling in human cancers

The initial link between HH signalling and cancer came from the finding that loss of function mutations in PTCH1 gene are associated with a rare and hereditary form of BCC, basal cell nevus syndrome (BCNS) (also known as Gorlin syndrome) (Refs Reference Hahn59, Reference Johnson60, Reference Gailani99). BCNS is an autosomal dominant disorder with two distinct sets of phenotypes; increased risk of developing cancers such as BCC, medulloblastoma, rhabdomyosarcoma and meningioma, as well as developmental defects, including bifid ribs and ectopic calcifications (Ref. Reference Gorlin58), that reflect the involvement of HH pathway in many developmental processes.

Consistent with the risk for specific cancers in Gorlin syndrome, sporadic BCCs and at least a subset of medulloblastomas (MBs), are the tumour types that show the strongest association with aberrant HH pathway activation, both in humans and in experimental mouse models. Activation of HH pathway in BCC and MB occurs through direct genetic alterations of HH pathway genes. Sporadic BCC and MB, a malignant brain tumour in children, harbour high frequency of inactivating mutations in PTCH1 (Refs Reference Gailani99, Reference Reifenberger100, Reference Pietsch101, Reference Raffel102, Reference Wolter103) or, to a lesser extent, activating mutations in SMO (Refs Reference Xie64, Reference Reifenberger104), both leading to the constitutive activation of HH pathway. In addition, MBs also show mutations in SUFU (Ref. Reference Taylor61) and GLI1 and GLI2 amplifications (Ref. Reference Northcott105). Deletion of 17p region, which produces loss of the negative HH modulator REN(KCTD11), also leads to unrestrained HH signalling and uncontrolled proliferation of immature cerebellar granule neuron precursors cells (Ref. Reference Ferretti106).

The genetic equivalent mouse model of BCNS, is a mouse heterozygous for a loss-of-function allele of Ptch1. Many of the BCNS features are recapitulated in this model, including occurrence of MB (Ref. Reference Goodrich107), rhabdomyosarcoma (Ref. Reference Hahn108) and developmental aberrations. Notably, full-blown BCCs are rarely seen in Ptch1^+/− mice maintained in normal conditions, but lesions resembling BCCs develop when mice are exposed to ultraviolet (UV) or ionising radiations (Ref. Reference Aszterbaum109). This observation is in agreement with the clinical course of BCC in BCNS patients, where BCCs occur preferentially on sun-exposed areas of the body (Ref. Reference Epstein110). BCNS patients are predisposed to BCC, MB and rhabdomyosarcoma, but they are not at increased risk to develop other cancer types, such as glioma, breast or prostate cancers. Genetic mouse models and identification of genetic mutations in BCC and MB have suggested that aberrant activation of HH signalling is required and sufficient for the development of these cancers. In other types of cancer activation of HH signalling might require additional alterations/mutations in other signalling pathways to contribute to tumour development.

Glioma is the most frequent tumour of the central nervous system and can be classified into four grades, with glioblastoma multiforme (GBM) being the most aggressive. GLI1 was originally identified as a gene amplified in malignant GBM (Ref. Reference Kinzler65), although its amplification is detected in a small fraction of gliomas (Refs Reference Hui111, Reference Mao and Hamoudi112). The landscape of driver genomic alterations in glioblastoma has been recently revealed, suggesting that ligand-independent activation of the HH pathway is not frequent (Ref. Reference Frattini113). Nevertheless, several reports support a role for HH signalling in gliomas. For instance, expression of components of HH signalling is observed in gliomas of different grades, with SHH expression mostly confined to the surrounding endothelial cells and astrocytes. Activation of the pathway sustains growth, survival and stemness of glioma cells and progenitors (Refs Reference Clement75, Reference Ehtesham77, Reference Bar84). Consistently, inhibition of HH signalling by cyclopamine treatment or by overexpression of miR-326, which targets SMO, decreases glioma growth, stemness and tumourigenicity (Refs Reference Clement75, Reference Bar84, Reference Du114).

There are strong indications that the HH pathway is involved also in human breast cancer (BC), the leading cause of cancer death among women. High expression of components of HH pathway, including GLI1, is associated with a higher risk of recurrence after surgery and poorer prognosis (Refs Reference O'Toole115, Reference Jeng116, Reference ten Haaf117). Consistently, transgenic mice that conditionally express GLI1 in the mammary epithelium develop mammary tumours (Ref. Reference Fiaschi118). Activation of HH signalling in BC results from genetic alterations, such as loss of PTCH1 or GLI1 amplification (Refs Reference Naylor119, Reference Nessling120) or from ligand-dependent stimulation. Indeed, invasive BC, but not normal breast epithelium, shows high expression of SHH, PTCH1 and GLI1 (Ref. Reference Kubo121). The elevated expression of HH ligands is associated with the development of a basal-like BC phenotype and to a poor prognosis (Ref. Reference O'Toole115), and may result from hypomethylation of SHH promoter (Refs Reference Wolf122, Reference Cui123) or from HH up-regulation mediated by transcription factors, such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) (Ref. Reference Cui123), p63 (Ref. Reference Caserta124) or Runx2 (Ref. Reference Pratap125). In addition, other cellular pathways contribute to directly activate the downstream effectors of the pathway. In oestrogen receptor (ER)-positive BC cells, oestrogen stimulation induces GLI1, which promotes CSC self-renewal and invasiveness (Ref. Reference Sun126). In tamoxifen-resistant ER positive BC cells, ligand-independent activation of HH pathway results from phosphoinositide 3-kinase (PI3K)/AKT pathway (Ref. Reference Ramaswamy127). Several reports point out the importance of paracrine HH signalling in BC, whose occurrence is associated to poor prognosis (Ref. Reference O'Toole115). HH ligands are often expressed by the tumour epithelium, whereas the highest levels of SMO, GLI1 and GLI2 are found in the stroma (Ref. Reference Mukherjee128). GLI1 promotes vascularisation by inducing the pro-angiogenic factor CYR61 (cysteine-rich angiogenic inducer 61) (Ref. Reference Harris129), which influences the tumour microenvironment. In addition, the alternatively spliced, truncated tGLI1 that is frequently expressed in BC, but not in normal tissue, induces the migration-associated genes VEGF-A and CD24 (Ref. Reference Cao130).

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive tumour that develops from pancreatic intraepithelial neoplasia (PanIN), characterised by frequent mutations of KRAS, CDKN2A, TP53 and SMAD4 (Ref. Reference Fokas131). A comprehensive genomic analysis revealed few missense mutations in GLI1 and GLI3, whose oncogenic function remains to be determined (Ref. Reference Jones132). Nevertheless, HH signalling is involved in pancreatic development (Ref. Reference Hebrok133) and cooperates with oncogenic KRAS during the early stages of PDAC formation (Ref. Reference Morton134). The ligands SHH and IHH are expressed in the duct epithelium of PanIN lesions and in PDAC, but not in normal human pancreas (Refs Reference Thayer70, Reference Berman72). Human pancreatic cell lines produce SHH, IHH and show detectable level of the target genes GLI1, PTCH1 and HHIP, indicating HH pathway activity. Moreover, proliferation and metastatic behaviour of some of these cell lines can be blocked by cyclopamine both in vitro and in vivo (Refs Reference Thayer70, Reference Feldmann71, Reference Berman72), supporting a ligand-dependent autocrine mode of action. Multiple evidence indicates the presence of paracrine HH signalling in pancreatic cancer. HH ligands produced by tumour cells activate HH pathway in the surrounding stroma, thus inducing the expression of HH targets that promote perineural invasion and metastasis (Refs Reference Yauch79, Reference Tian135, Reference Li136, Reference Bailey, Mohr and Hollingsworth137). Paracrine HH signalling also promotes the formation of desmoplasia, which contributes to the failure of the standard therapy (Ref. Reference Bailey138); indeed, chemical inhibition of HH pathway enhances the efficacy of chemotherapy (Ref. Reference Olive139). However, recent data obtained in murine models of PDAC propose a controversial role for HH signalling in PDAC. In fact, activation of HH signalling has been shown to induce stromal hyperplasia and reduce epithelial growth, thus restraining tumour. Conversely, HH pathway inhibition accelerates tumour progression because, although reducing desmoplasia, it promotes proliferation and vascularisation of the tumoural epithelium, which exhibits a more undifferentiated phenotype (Refs Reference Rhim140, Reference Lee141).

HH signalling is involved in prostate cancer (PC). Aberrant activation of HH signalling in PC might result from loss of SUFU or by ligand-dependent activation of the pathway due to high expression of SHH (Ref. Reference Sheng62). However, it is not clear whether HH activation occurs in a paracrine and/or autocrine/juxtacrine manner. Evidence suggests that the PC cells secrete HH ligands that activate the pathway in the surrounding stromal cells, which in turn produce factors promoting cancer cells proliferation (Refs Reference Fan142, Reference Wilkinson143, Reference Shigemura144). Conversely, other reports indicate the presence of a cell-autonomous activation of HH signalling in PC cells, whose proliferation is greatly decreased by cyclopamine treatment. The expression of HH ligands and of target genes in the tumour epithelium is higher than in the normal adjacent tissue and correlates with Gleason score, metastasis and poor prognosis (Refs Reference Sheng62, Reference Karhadkar73, Reference Sanchez74, Reference Azoulay145). The HH effector GLI2 is highly expressed in PC where it enhances proliferation, cell survival and tumourigenicity (Refs Reference Thiyagarajan146, Reference Narita147). Multiple evidence suggests an interplay between HH and androgen signalling. Long-term androgen deprivation in PC leads to a strong up-regulation of HH signalling, which is also observed in androgen-independent (AI) PC cells (Refs Reference Azoulay145, Reference Efstathiou148, Reference Chen149). Overexpression of GLI1 and GLI2 enhances androgen-specific gene expression, indicating that HH signalling supports androgen signalling even in absence of androgen and in AI prostate cancer cells (Ref. Reference Chen150).

HH pathway plays a role also in the most lethal form of skin cancer, malignant melanoma. A recent global genomic screening of 100 melanomas revealed few missense mutations in the core genes of the HH pathway (PTCH1, SMO, SUFU, GLI1, GLI2 and GLI3) (Ref. Reference Krauthammer151), although their potential oncogenic function remains to be determined. Several studies report an active role for HH signalling in melanoma. Human melanomas express components of HH pathway (Ref. Reference Stecca78) and about half of melanoma cell lines express high levels of SMO, GLI2 and PTCH1 and low levels of the negative regulators PKA and DYRK2 compared to melanocytes (Ref. Reference O'Reilly152). Interestingly, high HH pathway activity is associated with decreased post-recurrence survival in metastatic melanoma patients (Ref. Reference O'Reilly152). Moreover, we previously showed that growth and metastasis of human melanomas xenografts in nude mice can be blocked by local or systemic treatment with cyclopamine. Cyclopamine treatment drastically reduces tumour growth also in melanomas induced by oncogenic NRAS in a Tyrosinase-NRAS^Q61K; Ink4a^−/− mouse model (Ref. Reference Stecca78). Two recent studies confirmed and extended these findings; the SMO antagonist sonidegib has shown to reduce proliferation of human melanoma cell lines and to decrease human melanoma xenograft growth in nude mice (Refs Reference O'Reilly152, Reference Jalili153). Interestingly, one of the two studies showed a stronger inhibition of proliferation in BRAF mutant cell lines than in BRAF wild-type cells and a modest but significant effect combining BRAF and Hedgehog inhibitors (Ref. Reference O'Reilly152), suggesting that a combined therapy targeting both mutant BRAF and HH pathway could be beneficial in patients with mutated BRAF and activated HH signalling. Activation of HH pathway might also play a role in melanoma progression, by contributing to the acquisition of an invasive behaviour. Melanoma cells with high GLI2 expression are characterised by an invasive and metastatic phenotype, associated with loss of E-cadherin and secretion of metalloproteases, and metastasise to bone more quickly than cells with low GLI2 expression (Ref. Reference Alexaki154). Furthermore, GLI1-mediated induction of Osteopontin correlates with tumour progression and metastasis of human melanomas (Ref. Reference Das155).

High expression of components of HH pathway is observed in colon cancer, where it correlates with poor prognosis and overall survival (Refs Reference Xu156, Reference Wang157). Autocrine HH signalling in colon cancer promotes cell growth, self-renewal of CSCs and metastatic behaviour (Ref. Reference Varnat67). Consistently, inhibition of GLI1 and GLI2 function induces apoptosis and DNA damage response in colon cancer cell lines (Ref. Reference Mazumdar158). Gastric cancers express high levels of PTCH1 and SHH (Refs Reference Berman72, Reference Lee159) and active HH signalling correlates with metastatic behaviour and poor prognosis (Refs Reference Yoo160, Reference Saze161, Reference Niu162, Reference Wang163). Activation of the pathway results from SMO and PTCH1 mutations (Ref. Reference Wang164) or methylation of the promoter of the negative regulators PTCH1 and HHIP (Refs Reference Zuo and Song165, Reference Song166). Direct activation of GLI1 may also result from MAPK signalling, which leads to the induction of HH targets (Ref. Reference Seto167), such as Bcl-2 (Ref. Reference Han168). HH signalling promotes gastric cancer cell growth and proliferation in vitro and in vivo (Ref. Reference Wan169) and is highly active in gastric CSCs, where it is required for their self-renewal and resistance to chemotherapy (Refs Reference Yoon170, Reference Song171).

Lung cancer is the malignancy with the highest mortality and includes small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC). In a fraction of SCLC, ligand-dependent activation of HH signalling drives tumour growth in vivo and in vitro (Ref. Reference Watkins68). In NSCLC the expression of HH signalling components is higher than in the non-tumoural parenchyma and associates with high grade, poor survival and metastases (Refs Reference Gialmanidis172, Reference Hong173). In NSCLC, GLI1 regulates cell proliferation in cell-autonomous manner. Moreover, increased production of SHH by tumour cells leads to activation of fibroblasts in the tumour-associated stroma, indicating the presence of paracrine HH signalling (Refs Reference Yuan69, Reference Bermudez174).

Aberrant activation of HH signalling has been observed in ovarian cancer (Refs Reference Chen175, Reference Bhattacharya176, Reference Schmid177) and high expression of its components correlates with poor clinical outcome (Refs Reference Chen175, Reference Liao178, Reference Ciucci179). HH pathway has been shown to be involved in different aspects of ovarian carcinogenesis, by controlling proliferation and survival of ovarian carcinoma (Ref. Reference Chen175), growth of cancer spheroid forming cells (Ref. Reference Ray180), cell migration and invasion, through integrin β4-mediated activation of focal adhesion kinase (FAK; Ref. Reference Chen181), and drug sensitivity, through regulation of the ATP-binding cassette transporter ABCB1 and ABCG2 (Ref. Reference Chen, Bieber and Teng182).

HH signalling is involved also in haematological malignancies. Increased HH activity has been reported in different haematological diseases, including CML (Ref. Reference Sengupta183), acute myeloid leukaemia (AML) (Ref. Reference Kobune184), acute lymphocytic leukaemia (ALL) (Ref. Reference Lin91), MM (Ref. Reference Peacock88), chronic lymphocytic leukaemia (CLL; Ref. Reference Desch185), Hodgkin's lymphoma (Ref. Reference Greaves186), MCL (Ref. Reference Hegde83), diffuse large B-cell lymphoma (DLBCL) (Ref. Reference Singh187) and ALK+ anaplastic large cell lymphoma (ALCL) (Ref. Reference Singh188). The activation of HH signalling in these diseases likely results from the integration of deregulated oncogenic inputs that contribute to the direct activation of the GLI proteins. Different haematological malignancies also show different modalities of HH signalling activation, which has been proposed to be paracrine mainly in CLL and plasma cell myeloma, both paracrine and autocrine in DLBCL and autocrine in ALL, AML and ALK+ALCL.

Modulation of HH-GLI signalling by oncogenic pathways

The activity of HH-GLI signalling observed in human cancer is the result of its functional interaction with other pathways and of the direct or indirect regulation of the final effectors of the HH signalling by oncogenes and tumour suppressors (Fig. 2). Multiple lines of evidence support an interplay between HH-GLI and PI3K/AKT or RAS/RAF/MEK signalling. PI3K/AKT negatively regulates the degradation of GLI2 by interfering with PKA/GSK3β-mediated phosphorylation of GLI2, which targets the protein to proteasome-mediated degradation (Ref. Reference Riobo189). In zebrafish, a constitutively active form of Akt1 synergises with activated Smo in tumour formation (Ref. Reference Ju190). AKT1 potentiates GLI1 transcriptional activity and nuclear localisation in melanoma cells (Ref. Reference Stecca78). In contrast, GLI1 function is inhibited by PI3K/AKT2 signalling in neuroblastoma; AKT2 phosphorylates GSK3β and prevents the destabilisation of SUFU, resulting in reduced GLI1 nuclear localisation and transcriptional activity (Ref. Reference Paul191).

Figure 2. Cooperative integration between HH-GLI signalling and other oncogenic pathways. (a) Schematic diagram of the basic components of the HH-GLI signalling (filled circles) and their positive (in green) and negative regulators (in red) (unfilled circles). (b) Direct transcriptional regulators of GLI1, GLI2 and SHH. See text for further details. Abbreviations: AKT, v-akt murine thymoma viral oncogene homologue; aPKCι/λ, atypical protein kinase C-ι/λ; β-CAT, β-catenin; DYRK1/2, dual specificity Yak-1 related kinase 1/2; ERα, oestrogen receptor α; EWS/FLI1, Ewing's sarcoma/friend leukaemia integration 1 transcription factor fusion gene; HES1, hairy and enhancer of split-1; HH, Hedgehog; mTOR, mammalian target of rapamycin; MEF2C, myocyte enhancer factor 2C; MEK, mitogen-activated protein/extracellular signal-regulated kinase; NF-kB, nuclear factor kappa-light-chain-enhancer of activated B cells; NRP1/2, neuropilin; PI3K, phosphoinositide-3-kinase; PKA, protein kinase A; PTCH, Patched; PTEN, phosphatase and tensin homologue; RACK1, receptor for activated C kinase 1; RTK, receptor tyrosine kinase; S6K1, ribosomal protein S6 kinase 1; SHH, Sonic hedgehog; SMO, Smoothened; SUFU, Suppressor of Fused; TNFα, tumour necrosis factor α; TSC1/2, tuberous sclerosis 1/2; WIP1, wild-type p53-induced phosphatase 1.

An interplay between RAS/RAF/MEK and HH signalling has been described in various systems. For instance, oncogenic H- or N-Ras increase GLI1 function in melanoma cells and HH-GLI signalling is required for N-Ras-induced mouse melanoma growth (Ref. Reference Stecca78). Active K-Ras potentiates GLI1 activity in gastric cancer (Ref. Reference Seto167) and in PDAC contributing to tumour progression (Ref. Reference Ji192). K-Ras and activated HH signalling cooperate in vivo to initiate PDAC development (Ref. Reference Pasca di Magliano193). An additional mouse model of K-Ras-induced PDAC shows that Smo-independent Gli1 activation is required for survival of tumour cells and K-Ras-mediated transformation. Interestingly, K-Ras and TGF-β were shown to regulate Gli1 expression in absence of Smo (Ref. Reference Nolan-Stevaux194). K-Ras also contributes to a shift from autocrine-to-paracrine signalling in PDAC: it induces SHH expression, thus leading to HH stimulation of adjacent cells, and negatively modulates canonical HH signalling through its effector DYRK1B (Ref. Reference Lauth195).

The ERK pathway positively modulates HH-GLI signalling. MEK1 increases GLI1 and GLI2 transcriptional activity (Ref. Reference Riobo, Haines and Emerson196) and the ERK5 target myocyte enhancer factor 2C (MEF2C) directly regulates the expression of and cooperates with GLI2 during cardiomyogenesis in vitro (Ref. Reference Voronova197). Biochemical studies identified GLI1 and GLI3 as new MAPK substrates, because they can be phosphorylated in vitro by JNK1/2 and ERK2 (Ref. Reference Whisenant198).

A crosstalk between epidermal growth factor receptor (EGFR) signalling and HH-GLI pathway has also been reported. In normal keratinocytes, EGFR signalling modulates HH-GLI target gene expression (Ref. Reference Kasper199) and during their transformation it induces activation of JUN/AP1, which cooperates with GLI1 and GLI2 (Ref. Reference Schnidar200). Interestingly, a group of HH-EGFR cooperation response genes – SOX2, SOX9, JUN, CXCR4 and FGF19 – has been shown to determine the oncogenic phenotype of BCC and pancreatic CSCs (Ref. Reference Eberl201).

HH signalling is differently modulated by distinct members of the PKC family. Upregulation of aPKC ι/λ potentiates HH signalling by directly phosphorylating and activating GLI1. Because aPKC ι/λ is also an HH target gene, it sustains a positive feedback loop contributing to HH activation (Ref. Reference Atwood40). Similarly, PKCα increases GLI1 transcriptional activity in a MEK/ERK-dependent manner (Ref. Reference Cai202). Conversely, PKCδ reduces GLI1 nuclear localisation and transcriptional activity, leading to suppression of HH signalling (Ref. Reference Cai202).

Receptor for activated C kinase 1 (RACK1) interacts with and activates SMO, enhancing GLI1 function and increasing cell proliferation and survival in NSCLC (Ref. Reference Shi203). A positive feedback regulation fuelling Hh signalling activation involves Neuropilin1 (Nrp1) and 2 (Nrp2). Activated Hh pathway induces Nrp1 and Nrp2, which in turn potentiate Hh signalling transduction acting between Smo and SuFu (Ref. Reference Hillman204). Activation of tumour necrosis factor alpha  (TNF-α)/mammalian target of rapamycin (mTOR) pathway in oesophageal carcinoma activates HH-GLI signalling through phosphorylation of GLI1 by S6K1, which induces its release from SUFU (Ref. Reference Wang41).

Activation of HH-GLI signalling due to direct induction of GLI1 expression is observed after activation of WNT/β-catenin signalling (Ref. Reference Nakamura205) and in Ewing Sarcoma Family Tumours (ESFT), where the oncogenic transcription factor EWS/FLI1, resulting from the chromosomal translocation t(11;22), directly induces GLI1 expression (Refs Reference Zwerner206, Reference Beauchamp207) (Fig. 2b). Likewise, transforming growth factor β (TGF-β) stimulation leads to a SMAD3-dependent induction of GLI2, which in turn increases GLI1 expression (Ref. Reference Dennler208). TGF-β also induces Kindlin-2, which increases GLI1 protein levels by inhibiting GSK3β. GLI1, in turn, represses Kindlin-2 creating a regulatory loop (Ref. Reference Gao209) (Fig. 2b). Activation of HH pathway in some tumours results from the increase of HH ligands. For instance, ERα pathway in gastric cancer (Ref. Reference Kameda210) or NF-kB in pancreatic cancer cells (Refs Reference Kasperczyk211, Reference Nakashima212) directly increase SHH expression, leading to enhanced proliferation and resistance to apoptosis. Direct induction of SHH is also mediated by p63β, p63γ and TAp73β, which bind to SHH promoter (Ref. Reference Caserta124) (Fig. 2b).

Although HH signalling activation is regulated by many phosphorylation events, only few phosphatases have been described to modulate the pathway. In Drosophila PP4 and PP2A act as negative and positive modulators of HH signalling, acting at the level of Smo and Ci, respectively (Ref. Reference Jia213). Recently, the oncogenic wild-type p53-induced phosphatase 1 (WIP1) has been described to cooperate with SHH to enhance tumour formation in SHH-dependent medulloblastoma (Ref. Reference Doucette214). Our group showed that WIP1 phosphatase activity enhances GLI1 function in melanoma by increasing GLI1 nuclear localisation, protein stability and transcriptional activity, whereas its inhibition reduces self-renewal and tumourigenicity of melanoma cells with activated HH signalling (Ref. Reference Pandolfi215).

A negative reciprocal regulation is observed between GLI1 and the tumour suppressor p53. p53 inhibits the activity, nuclear localisation and protein levels of GLI1 in neural stem cells and glioblastoma cells (Ref. Reference Stecca and Ruiz i Altaba216). Conversely, HH signalling inhibits p53 by inducing activating phosphorylations on MDM2, thus enhancing p53 degradation (Ref. Reference Abe217). Inhibition of HH signalling results from activation of NOTCH pathway observed in glioblastoma and melanoma. The NOTCH target hairy and enhancer of split-1 (HES1) binds to the first intron of GLI1, repressing its expression (Ref. Reference Schreck218) (Fig. 2b). In BC, high levels of liver kinase B1 (LKB1) are associated with low levels of HH signalling activation (Ref. Reference Zhuang219). Another suppressor of HH signalling is REN^KCTD11, which is often deleted in medulloblastoma and it has been shown to retain GLI1 in the cytoplasm, reducing its transcriptional activity (Ref. Reference Di Marcotullio63).

Regulation of HH signalling occurs also at epigenetic level. Menin, the gene mutated in multiple endocrine neoplasia type 1, recruits the protein arginine methyltransferase 5 (PRMT5) to growth arrest-specific 1 (Gas1) promoter. The consequent Gas1 repression prevents the binding of Shh to Ptch1, thus resulting in reduced HH pathway activity (Ref. Reference Gurung220). Different components of HH signalling are also targets of micro-RNAs (miR). miR-125b and miR-326, which target SMO, and miR-324-5p, which targets both SMO and GLI1, are downregulated in HH-driven MB and contribute to sustain tumour growth (Ref. Reference Ferretti221). In glioblastoma the miR-302-367 cluster inhibits clonogenicity and stemness of glioblastoma stem cells, through downregulation of CXCR4/SDF1 and consequent reduction of SHH, GLI1 and NANOG levels (Ref. Reference Fareh222).

Inhibitors of HH-GLI signalling

Current HH pathway antagonists can be classified according to what level of the pathway they modulate: (i) HH/PTCH interaction; (ii) SMO translocation and activation; (iii) GLI nuclear translocation and transcriptional activation (Fig. 3).

Figure 3. Targeting aberrant HH-GLI pathway. HH-GLI antagonists, classified according to what level of the pathway they inhibit: SMO translocation and activation (blue); HH/PTCH interaction (orange); GLI nuclear translocation and transcriptional activity (red). Abbreviations: aPKC-i, atypical protein kinase C-inhibitor; ATO, arsenic trioxide; BET-i, BET bromodomain inhibitor; HDAC-i, histone deacetylase-inhibitors; HH, hedgehog; HPI-1/4, hedgehog pathway inhibitors 1–4; mTOR-i, mammalian target of rapamycin inhibitors; PTCH, Patched; SMO, Smoothened; SUFU, Suppressor of Fused; WIP1-i, wild-type p53-induced phosphatase 1-inhibitors. See the main text for details.

Acting at the level of SMO

The development of strategies targeting the HH signalling pathway began with the discovery that cyclopamine, a steroidal alkaloid derived from Veratrum californicum with teratogenic properties (Ref. Reference Keeler223), inhibits SMO (Refs Reference Incardona224, Reference Cooper225). Cyclopamine has been extensively used to study HH signalling and found to inhibit tumour growth in multiple in vitro and in vivo models. For instance, oral cyclopamine can block the growth of UV-induced BCCs in Ptch^+/− mice by 50%, as well as inhibit the formation of new tumours (Ref. Reference Athar226). Cyclopamine also reduces medulloblastoma development in Ptch^+/− mice (Ref. Reference Sanchez and Ruiz i Altaba227) and decreases growth of many human cancer cell lines in xenotransplantation (Refs Reference Thayer70, Reference Karhadkar73, Reference Clement75, Reference Stecca78, Reference Berman228). However, cyclopamine is not suitable for clinical development because of its poor oral solubility. Efforts to improve the specificity, potency, and pharmacologic profile of cyclopamine have led to the synthesis of novel derivatives such as KAAD-cyclopamine (Ref. Reference Taipale229), IPI-609 (Ref. Reference Feldmann87) and saridegib (IPI-926) (Ref. Reference Tremblay230).

Additional SMO inhibitors are currently available and many, including vismodegib (GDC-0449), sonidegib (LDE-225), BMS-833923, PF-04449913 and LY2940680 are being investigated in clinical trials in a number of advanced cancers (Ref. Reference Amakye, Jagani and Dorsch231) (Table 1). Among these, vismodegib is the first Hedgehog signalling antagonist approved by U.S. Food and Drug Administration (FDA) for treatment of advanced or metastatic BCC. Two SMO inhibitors, saridegib and TAK-441, have been discontinued for lack of efficacy (Refs Reference Williams232, Reference Williams233). A number of additional SMO antagonists have been used in preclinical studies; they include Cur-61414 (HhAntag; Refs Reference Williams234, Reference Romer235), provitamin D3 (Ref. Reference Bijlsma236), Sant1-4 (Ref. Reference Chen237), Sant-75 (Ref. Reference Yang238), bis-amide compound 5 (Ref. Reference Dijkgraaf239) and desmethylveramiline (Ref. Reference Guerlet240). Glucocorticoids have recently been proposed as modifiers of HH signalling and SMO ciliary translocation; one class promotes ciliary accumulation resulting in enhanced Hh ligands response, whereas a second class inhibits SMO ciliary accumulation and is active against oncogenic and resistant SMO mutations (Ref. Reference Wang241). Similarly, itraconazole, a common antifungal agent, has been identified as a potent inhibitor of the HH pathway by preventing ciliary translocation of SMO (Ref. Reference Kim242). Systemic administration of itraconazole inhibits the growth of HH-dependent MB and BCC in mice and it is also active against drug-resistant mutant SMO D473H and Gli2 overexpression (Ref. Reference Kim243).

Table 1. Selected clinical trials of SMO inhibitors in cancer

Abbreviations: AEs, adverse events; ALT, alanine transaminase; AST, aspartate transaminase; BCC, basal cell carcinoma; BCNS, basal cell nevus syndrome; DLT, dose-limiting toxicity; ER, oestrogen receptor; HER2, human epidermal growth factor receptor 2; MB, medulloblastoma; MTD, maximum tolerated dose; ORR, overall response rate; OS, overall survival; PFS, progression free survival.

^aStatus assessed on December 10th 2014; all ongoing trials are not recruiting.

^bNCT # Identifier at http://clinicaltrials.gov.

SMO inhibitors in clinical development

SMO inhibitors are being investigated in clinical trials in a range of advanced cancers (Table 1). Several of these agents have induced tumour response in patients with tumours that harbour mutations in SMO and PTCH1, such as BCC and MB. Vismodegib drastically reduces the rate of appearance of new BCCs in patients with BCNS, without signs of resistance during treatment, in contrast with HH-dependent MBs. However, most BCCs have been shown to regrow after the drug is stopped (Ref. Reference Tang244). In sporadic cases, 58% of patients with late advanced or metastatic BCC showed tumour regression in phase I clinical trials (Refs Reference Von Hoff245, Reference LoRusso246) and 30% of metastatic and 43% of locally advanced BCC responded in phase II clinical trials (Ref. Reference Sekulic247). These results suggest that tumours with low mutation rate such as in BCNS patients are predicted to respond well to SMO inhibition, whereas metastatic BCCs with high mutation rate have a higher likelihood to develop acquired resistance during treatment. Similar responses have been observed in BCC with sonidegib (Ref. Reference Rodon248). A phase II study evaluated vismodegib after chemotherapy in patients with ovarian cancer in second or third remission. However, the trial did not meet the primary endpoint and only a modest improvement in progression free survival was observed for vismodegib compared to placebo (7.5 versus 5.8 months). In addition, more than half of the patients discontinued treatment for disease progression and adverse effects (Ref. Reference Kaye249). Similarly, a phase II study of vismodegib in patients with advanced chondrosarcoma did not meet the primary endpoint (Ref. Reference Italiano250).

Tumour responses in MB have been reported with vismodegib and sonidegib (Refs Reference Rodon248, Reference Gajjar251). Sonidegib has shown anti-tumoural activity in relapsed MBs associated with activated HH pathway, with dose- and exposure-dependent inhibition of GLI1 expression (Ref. Reference Rodon248). A recent study showed the usefulness of a five-gene HH signature in formalin-fixed, paraffin-embedded tumour samples as a preselection tool for HH inhibitor therapy in MB patients (Ref. Reference Shou252).

The use of SMO inhibitors has been associated with the acquisition of resistance to SMO inhibitors, mostly described in medulloblastoma, as a consequence of (i) mutations in human SMO (D473H) and the matching mutation in mouse (D477G), observed during vismodegib treatment (Ref. Reference Yauch253); (ii) amplification of downstream HH target genes, such as GLI2 and CyclinD1 (Refs Reference Dijkgraaf239, Reference Buonamici254), reported for both vismodegib and sonidegib; (iii) upregulation of other oncogenic signalling, such as PI3K/AKT pathway (Ref. Reference Buonamici254), observed during LDE-225 treatment; (iv) increased expression of adenosine triphosphate (ATP)-binding cassette transporter (ABC) such as P-glycoprotein, leading to increased drug efflux (Ref. Reference Lee255), observed during saridegib treatment.

In studies investigating systemic treatments with SMO inhibitors, a common set of adverse effects has been observed, including muscle spasms, loss of taste (dysgeusia), hair loss (alopecia), fatigue, nausea, diarrhoea, decreased appetite, weight loss and hyponatraemia (summarised in Table 1). It is likely that hair loss, altered taste and diarrhoea are directly related to the inhibition of the intended molecular target (SMO), since HH signalling is known to be active in hair follicle, taste buds and gastrointestinal tract (Refs Reference Hall, Bell and Finger256, Reference St-Jacques257, Reference Ramalho-Santos, Melton and McMahon258). Therefore, these effects are unlikely to be avoided by modifying the molecular structure of the agents. Possible strategies to lessen these effects would be to perform interval dosing of single agent or lower doses in combination with other agents (see later). Although most of the side effects of SMO inhibitors are mild to moderate (grade 1/2, Table 1), in some cases their severity has caused 50% of dropouts (Ref. Reference Tang244) and raised concerns about long-term treatment in patients with BCC, typically a non-life-threatening cancer. One way to avoid or reduce such effects in BCC might be to use these inhibitors topically, limiting systemic exposure. A study employing topical treatment of LDE-225 for 4 weeks documented an effective reduction in tumour size or clinical clearing that correlated with effective inhibition of HH signalling (Ref. Reference Skvara259).

Evidence for rational combinations

Combination of SMO inhibitors and other agents in preclinical studies

Support for combinatorial strategies is derived from the increasing amount of experimental data showing evidence of non-canonical HH signalling activation in tumours (summarised in Table 2). Combined inhibition of HH and MEK or AKT has been shown to yield additive/synergistic effects in reducing melanoma and cholangiocarcinoma cell proliferation in vitro (Refs Reference Stecca78, Reference Jinawath275). Combination of EGFR and SMO inhibitors has been described in several preclinical models. In pancreatic cancer cells, treatment with cyclopamine and EGFR inhibitor gefinitib decreased tumour growth rate and increased apoptosis (Ref. Reference Chitkara276). Treatment of prostate cancer cells with cyclopamine in combination with gefitinib and docetaxel cooperatively inhibited proliferation and invasiveness (Ref. Reference Mimeault277). Combination of cyclopamine with erlotinib or sequential treatment with erlotinib followed by cyclopamine inhibited tumour-initiating potential in glioblastoma cells (Ref. Reference Eimer278). Similarly, combination of the SMO inhibitor saridegib and EGFR inhibitor cetuximab drastically decreased head and neck squamous cell carcinoma tumour growth in vivo (Ref. Reference Keysar279).

Table 2. Examples of preclinical combination studies of SMO inhibitors and other agents

Abbreviations: CCT, CCT007093; CML: chronic myeloid leukaemia; CSC, cancer stem cells; GBM: glioblastoma; HNSCC, head and neck squamous cell cancer; MB, medulloblastoma.

Several preclinical studies have evaluated HH pathway inhibitors in combination with PI3K and mTOR inhibitors. Simultaneous treatment with sonidegib and the PI3K inhibitor buparsilib (BKM120) or the dual mTOR/PI3K inhibitor BEZ235 led to a significant delay in resistance development (Ref. Reference Buonamici254). Similarly, HH and PI3K pathways have been shown to synergise in promoting tumour growth in PTEN-deficient glioblastomas, and combined inhibition of the two pathways resulted in improved efficacy compared with inhibition of either pathway alone (Ref. Reference Gruber Filbin280). In pancreatic cancer, combination of chemotherapy, cyclopamine and the mTOR inhibitor rapamycin led to a near complete elimination of CSCs and increased long-term survival in mouse model (Ref. Reference Mueller281). Combination of vismodegib and the mTOR inhibitor everolimus resulted into a better response than each treatment alone in EAC xenografts (Ref. Reference Wang41). Recently, multimodal treatment with the novel HH pathway inhibitor SIBI-C1, the mTOR inhibitor rapamycin and gemcitabine was shown to eliminate pancreatic CSCs and to increase survival of primary human pancreatic cancer tissue xenografts (Ref. Reference Hermann282).

Combination of HH and Notch inhibitors has also proved potential therapeutic efficacy in preclinical studies. For instance, cyclopamine and a γ-secretase inhibitor showed additive growth suppression in leukaemia cell lines (Ref. Reference Okuhashi283). Combined inhibition of cyclopamine and the γ-secretase inhibitor MRK-003 led to decreased glioblastoma cell growth, increased apoptosis and decreased colony formation compared with either agent alone (Ref. Reference Schreck218). Similarly, treatment of CD133+ glioblastoma stem cells with cyclopamine and a γ-secretase inhibitor enhanced the therapeutic effect of temozolomide (Ref. Reference Ulasov284). Inhibition of Notch and Hedgehog signalling were also shown to affect docetaxel-resistant hormone-refractory prostate cancer cells, which have a high tumour-initiating potential. Treatment with the γ-secretase inhibitor DBZ or compound E and with cyclopamine or vismodegib reduced growth of docetaxel-resistant hormone-refractory prostate cancer cells in vitro and in vivo through inhibition of the survival molecules AKT and Bcl-2 (Ref. Reference Domingo-Domenech285).

BCR-ABL tyrosinase kinase inhibitors (TKI) are effective against CML; however, these agents are unable to eliminate quiescent leukaemia stem cells (Ref. Reference Chomel and Turhan286). Therefore, combination therapies with HH inhibitors are being explored. First of all, it was shown that imatinib-sensitive and -resistant CML cell lines express components of HH signalling, and genetic silencing of GLI1 reduced BCR-ABL protein expression, effect that is reversed by SMO agonist treatment (Ref. Reference Liao287). Cyclopamine enhanced the effect of the BCR-ABL inhibitor nilotinib and prolonged the survival of mice by acting on leukaemic stem cells in a mouse model of CML (Ref. Reference Dierks89). SMO inhibition impaired propagation not only of wild-type BCR-ABL, but also of imatinib-resistant mouse and human CML (Ref. Reference Zhao90). In the BCR-ABL-positive cell line OM9;22, the combination of vismodegib with the BCR-ABL TKI dasatinib resulted in enhanced cytotoxicity compared with each drug alone (Ref. Reference Okabe288). Similarly, simultaneous treatment with vismodegib and the pan-ABL kinase inhibitor ponatinib reduced the percentage of CD19-positive leukaemia cells and overall tumour burden, and increased survival compared with treatment with either compound alone (Ref. Reference Katagiri289).

Another example of combination drug for SMO inhibitors is gemcitabine for the treatment of pancreatic cancer. The activity of SMO inhibitor as a single agent in a pancreatic cancer xenograft model is modest and seems to be mediated by stromal pathway inhibition (Ref. Reference Yauch79). The combination of cyclopamine with gemcitabine completely abrogated metastases and significantly reduced the size of primary tumours in an orthotopic model of pancreatic cancer (Ref. Reference Feldmann71). In a mouse model of pancreatic cancer (Trp53^R172H and Kras^G12D) saridegib (IPI-926) was proposed to sensitise tumours to gemcitabine treatment through depletion of the tumour stroma (Ref. Reference Olive139).

In light of the role of HH signalling in the maintenance of CSCs, combinatorial therapy with SMO antagonists and debulking chemotherapeutic agents has attracted interest, particularly with the respect to preventing relapse or resistance to standard treatments. For instance, SMO inhibitors have also shown to increase the effects of the alkylating agent temozolomide in glioblastoma xenograft models, mostly acting on the CSC population that is spared by temozolomide alone (Refs Reference Clement75, Reference Ferruzzi290).

Similarly, inhibition of Aurora kinase and Polo-like kinase, two important G₂–M cell cycle regulators (Ref. Reference Lens, Voest and Medema291), has shown to enhance the effect of SMO antagonist LDE-225 in blocking tumour cell proliferation in vitro and tumour growth in vivo and to increase sensitivity to conventional chemotherapy in murine PTCH1 mutant cells and in human MB cell lines (Ref. Reference Markant292).

Recently, cyclopamine has been shown to act synergistically with WIP1 inhibitor CCT007093 in reducing in vitro growth of patient-derived melanoma cells and BC cell lines (Ref. Reference Pandolfi215). These data suggest a possible novel therapeutic approach for tumours expressing high levels of WIP1 and with activated HH pathway, such as a subset of MB, gliomas and melanomas (Refs Reference Pandolfi215, Reference Castellino293, Reference Buss294, Reference Liang295). Targeting WIP1 in tumours with wild type p53 would lead not only to restoration of p53 tumour suppressor activity (Ref. Reference Lu, Nannenga and Donehower296), which in turn might inhibit GLI1 (Ref. Reference Stecca and Ruiz i Altaba216), but also to a direct attenuation of GLI1 function (Ref. Reference Pandolfi215), resulting in a stronger inhibition of the HH pathway. This is particularly relevant to melanoma, as nearly 90% of human melanomas express functionally defective wild-type p53 and restoration of p53 function has recently been suggested as an alternative for melanoma therapy (Ref. Reference Lu297). Moreover, this approach based on WIP1-p53-GLI1 axis might inhibit not only the growth of tumour bulk, but also that of putative CSCs (Ref. Reference Pandolfi215).

Clinical trials of SMO inhibitors in combination with other targets

Based on the crosstalk between HH signalling and other pathways, several combinations with SMO inhibitors are being evaluated in clinical trials (Table 3). Most of these trials are still recruiting and do not have published data. In a clinical phase 2 study, 199 patients with metastatic colorectal cancer were treated with vismodegib or placebo in combination with VEGF inhibitor bevacizumab and chemotherapy. The study failed to show clinical benefit in vismodegib compared with placebo (Ref. Reference Berlin298) (Table 3). Interestingly, the placebo group had a slightly better overall response than vismodegib-treated group (51% versus 46%), probably reflecting differences in safety and tolerability, as vismodegib-chemotherapy combination is less well tolerated compared with placebo-chemotherapy combination. In a pilot study, 25 patients with metastatic pancreatic adenocarcinoma were treated with a combination of vismodegib and gemcitabine. Vismodegib treatment for 3 weeks led to downregulation of GLI1 and PTCH1 in post-treatment biopsies in the majority of patients, without significant changes in the CSC compartment compared with baseline. However, vismodegib and gemcitabine were not better than gemcitabine alone in the treatment of metastatic pancreatic cancer (Ref. Reference Kim299).

Table 3. Clinical trials investigating SMO inhibitors in combination with other agents in cancer

AEs, adverse events; AML, acute myeloid leukaemia; BCR-ABL, breakpoint cluster region-Abelson; CML, chronic myeloid leukaemia; EGFR, epidermal growth factor receptor; FOLFIRI, folinic acid, fluorouracil and irinotecan; FOLFOX, folinic acid, fluorouracil and oxaliplatin; FOLFIRINOX, folinic acid, fluorouracil, irinotecan and oxaliplatin; GnRH, gonadotropin-releasing hormone; JAK, janus kinase; mTOR, mammalian target of rapamycin; MB, medulloblastoma; MDS, myelodysplastic syndrome; ORR, overall response rate; PI3K, phosphatidylinositol-3-kinases; SCLC, small cell lung carcinoma; VEGF, vascular endothelial growth factor.

^aOther than SMO.

^bStatus assessed on 10 December 2014; all ongoing trials are not recruiting.

^cNCT # Identifier at http://clinicaltrials.gov.

Vismodegib is also being tested in combination with the mTOR inhibitor sirolimus, and in combination with the gonadotropin-releasing hormone agonist leuprolide or goserelin in metastatic pancreatic cancer and locally advanced prostate cancer, respectively (Table 3). In addition, clinical studies combining vismodegib with the Notch pathway inhibitor RO4929097 in advanced BC and sarcoma are ongoing. Multiple combination studies with sonidegib and BMS-833923 are either recruiting or ongoing. For instance, phase 1 studies of sonidegib in combination with PI3K inhibitor buparlisib in several types of advanced solid tumours, or in combination with BCR-ABL inhibitor nilotinib in patients with chronic myeloid leukaemia are recruiting (see Table 3 for details). Results from these clinical trials will address the applicability of SMO inhibitors in combination with other targets in multiple cancer types.

Perspectives

Over the last decade, knowledge of the HH-GLI signalling has greatly increased, enabling a better understanding of the interaction of the major oncogenic pathways during tumourigenesis. Despite these advances, our understanding of this signalling pathway is far from complete and many important questions remain to be answered. For example, which are the mechanisms of gene regulation by GLI protein and how are cell type-specific responses determined? Are there co-factors that play a role in determining the HH transcriptional response? What is the evolutionary role of cilia in the HH signalling and do cilia play a crucial role in regulating HH signalling in human cancers?

Besides answering these questions, it will be important to develop sensitive biomarkers of HH-GLI pathway activation to identify the subset of cancers that will respond to HH inhibitors, sparing patients who are unlikely to benefit from a potentially toxic treatment. This is particularly true for MB, where only 25% harbour mutations in HH pathway genes. A reliable read-out of an active HH signalling is the expression of GLI1; however, the use of GLI1 as a biomarker by immunohistochemistry is hampered by the lack of specific GLI1 antibodies for diagnostic purposes. Equally important is to differentiate cancers with canonical and non-canonical HH pathway activation, and among the latter SMO-dependent from SMO-independent cancers. Only a clear understanding of the mechanisms leading to GLI activation in each tumour will allow for selection of the appropriate HH pathway inhibitor and, in cases where crosstalk between HH and other oncogenic pathways occurs, the optimal combinatorial partner. The prevalence of cancers with non-canonical HH activation strongly argues for the development of molecules able to target the final effectors of the HH signalling. This would provide a good approach to block ligand-independent and ligand-dependent HH pathway activation and perhaps overcome anti-SMO drug resistance.