Frequent deregulations in the hedgehog signaling network and cross-talks with the epidermal growth factor receptor pathway involved in cancer progression and targeted therapies

Abstract

The hedgehog (Hh)/glioma-associated oncogene (GLI) signaling network is among the most important and fascinating signal transduction systems that provide critical functions in the regulation of many developmental and physiological processes. The coordinated spatiotemporal interplay of the Hh ligands and other growth factors is necessary for the stringent control of the behavior of diverse types of tissue-resident stem/progenitor cells and their progenies. The activation of the Hh cascade might promote the tissue regeneration and repair after severe injury in numerous organs, insulin production in pancreatic beta-cells, and neovascularization. Consequently, the stimulation of the Hh pathway constitutes a potential therapeutic strategy to treat diverse human disorders, including severe tissue injuries; diabetes mellitus; and brain, skin, and cardiovascular disorders. In counterbalance, a deregulation of the Hh signaling network might lead to major tissular disorders and the development of a wide variety of aggressive and metastatic cancers. The target gene products induced through the persistent Hh activation can contribute to the self-renewal, survival, migration, and metastasis of cancer stem/progenitor cells and their progenies. Moreover, the pivotal role mediated through the Hh/GLI cascade during cancer progression also implicates the cooperation with other oncogenic products, such as mutated K-RAS and complex cross-talk with different growth factor pathways, including tyrosine kinase receptors, such as epidermal growth factor receptor (EGFR), Wnt/beta-catenin, and transforming growth factor-beta (TGF-beta)/TGF-beta receptors. Therefore, the molecular targeting of distinct deregulated gene products, including Hh and EGFR signaling components and other signaling elements that are frequently deregulated in highly tumorigenic cancer-initiating cells and their progenies, might constitute a potential therapeutic strategy to eradicate the total cancer cell mass. Of clinical interest is that these multitargeted approaches offer great promise as adjuvant treatments for improving the current antihormonal therapies, radiotherapies, and/or chemotherapies against locally advanced and metastatic cancers, thereby preventing disease relapse and the death of patients with cancer.

Fig. 1.

Schematic representation of the molecular events associated with cellular processing, lipid modification, and secretion of the SHH protein and the autocrine and paracrine actions of mature and secreted SHH protein. The scheme shows the molecular mechanisms associated with the cellular processing of the SHH protein precursor, including the cleavage of its N-terminal signal peptide fragment. The autocatalytic cleavage at the position 198 of SHH protein precursor catalyzed by its C-terminal fragment, which results in the release of cleaved N-terminal and C-terminal products, is also shown. Moreover, the lipid modifications of cleaved N-terminal SHH fragment via the attachment of a cholesterol moiety at the C-terminal position and a palmitate molecule at the N-terminal position catalyzed by the palmitoylacyltransferase Hhat are also indicated. The secretion of the mature and lipid-modified SHH protein into extracellular space as well as its diffusion and potential autocrine or paracrine action on secreting and neighboring responsive cells are also illustrated. In addition, the function of dispatched (DISP) transmembrane protein in the formation of large SHH oligomers and their secretion in extracellular compartment is illustrated. Moreover, the inhibitory effect on the SHH actions induced through the sequestration of cell surface-associated SHH molecules by HHIP is shown.

Fig. 2.

Proposed models of the molecular mechanisms involved in the regulation of ligand-dependent and independent-SMO activation and modulation by the pharmacological agents. a, the binding of SHH protein to the PTCH1 transmembrane receptor might lead to either membrane changes or activation of an endogenous SMO agonist. These molecular events, in turn, may result in the adoption of an active conformation by the SMO transmembrane protein and the stimulation of SMO-mediated cellular response. b, in the absence of SHH ligand, the PTCH1 receptor can act as a transporter and pump the endogenous cholesterol derivatives, such as oxysterols, out of the cells. The binding of the SHH protein to PTCH1 receptor, however, might inhibit the efflux of cholesterol derivatives such as oxysterols, and thereby promote the adoption of an active conformation by the SMO protein and SMO-induced cellular response. The occurrence of activating mutations in the SMO oncoprotein (c) or inactivating mutations in the PTCH1 tumor suppressor protein (d) might result in the adoption of an active conformation by SMO protein in the absence of SHH ligand and a sustained induction of a cellular response. In the same way, the exposure of cells to a pharmacological agent acting as a SMO agonist (e) also can induce the adoption of an active conformation by the SMO protein and a cellular response. In contrast, the exposure of cells to a chemical compound acting as a SMO antagonist (f), such as cyclopamine, KAAD-cyclopamine, IPI-269609, or GDC-0449, can inhibit the SHH protein-induced SMO activation and cellular response.

Fig. 3.

Schematic representation of molecular events associated with the repressive effect induced by PTCH1 receptor on the SMO activity and the activation of the Hh signaling pathway mediated by the SHH protein in the primary cilium. a, in the absence of the SHH ligand, PTCH1 is localized at the base of the ciliary structure and inhibits SMO protein translocation to the primary cilium and its activation. In the absence of activated SMO protein, the negative modulator of Hh cascade SUFU sequesters full-length GLI proteins in the cytoplasm and prevents their nuclear translocation, Hh target gene expression, and induction of a cellular response. Moreover, the cytoplasmic GLI proteins also may be degraded through the proteasomal pathway, and the GLI3 protein cleaved into a C-terminal fragment (GLI3R) that acts as a nuclear transcriptional repressor of Hh gene expression. b, the binding of the mature and lipid-modified SHH protein to the PTCH1 receptor in the primary cilium leads to its translocation out of the ciliary structure and retrieves its repressive effect on the SMO protein localized in intracellular vesicles or plasma membrane out of the primary cilium. These molecular events culminate in SMO translocation into the primary cilium and activation of downstream signaling elements, GLI proteins localized in the primary cilium. The negative modulator of GLI proteins, SUFU protein, is then degraded by the proteasomal pathway, and the activated GLI zinc-finger transcriptional activators, GLI1 or GLI2 molecules, are translocated to nucleus and participate to the up-regulation of Hh target gene expression, including GLI1 and PTCH1, and induction of a cellular response.

Fig. 4.

Schematic representation of structural features of human GLI3 protein and molecular events associated with its processing into a transcriptional repressor. The scheme shows the positions of the zinc finger DNA-binding domain (ZF), the six potential sites of the phosphorylation by protein kinase A (PKA) (asterisks) identified by mutagenesis analyses, and the intracellular cleavage site of the full-length GLI3 protein. The processing of the full-length 190-kDa GLI3 protein, which implicates its phosphorylation by PKA followed by its intracellular proteolytic cleavage, yielding an N-terminal fragment of GLI3, is also illustrated. The cleaved N-terminal fragment of GLI3 of approximately 83 kDa (GLI3R) can act as a transcriptional repressor and inhibit the Hh target gene expression.

Fig. 5.

Cellular events and signaling elements involved in the regulation of SHH expression, GLI activation, and mediation of the Hh activation-induced cellular response. The increase of the SHH expression, which might be induced during tissue regeneration in homeostatic conditions, and after an adaptive response to tissue injury, ischemia and hypoxia, chronic inflammation, and cancer progression, is indicated. The potential cellular signaling elements involved in the regulation of the SHH expression are also indicated. These intracellular signaling components include NF-κB, PI₃K/Akt, and K-RAS, which might be induced through the stimulation of different growth factor and cytokine signaling pathways in normal and cancer cells. The possibility of SHH-dependent and -independent activation of GLI1 and GLI2 transcriptional activators by different growth factor pathways is also indicated. In addition, the potential biological effects induced through stimulation of GLI protein-induced Hh target gene expression in normal and cancer cells are also indicated. ER-α, estrogen receptor α; PDGF, platelet-derived growth factor; RORα, retinoid-related orphan receptor α; TNF-α, tumor necrosis factor-α.

Fig. 6.

Scheme showing the signaling elements and frequent deregulations in the Hh signaling network and potential cross-talk with the EGFR signaling pathway involved in the malignant behavior of cancer cells. The molecular events associated with the cellular processing of the SHH precursor into a biologically active form via autocatalytic cleavage and lipid modifications are illustrated. Autocrine and paracrine stimulation of the cancer cells by the monomeric and multimeric SHH molecules is also illustrated. The repressive effect of SUFU on the GLI activity is shown. Moreover, the frequent deregulations, including overexpression of the SHH ligand; inactivating mutations in HHIP, PTCH, or SUFU; or activating mutations in SMO coreceptor, which may contribute to cancer development, are also indicated. The potential stimulatory effect induced by the activation of EGFR pathway and oncogenic mutations in K-RAS^mut and B-RAF^mut on the GLI transcriptional activity is indicated. The target gene products induced through the activation of Hh and EGFR signaling pathways are also described. Moreover, the stimulatory effect induced through the activation of TGF-β/TGF-βR/Smad3-Smad4 and Wnt/β-catenin on the GLI2 expression is illustrated. In addition, the potential inhibitory effect induced by diverse pharmacological agents, such as a mAb directed against SHH ligand, EGF, EGFR, and Wnt, selective inhibitors of SMO (cyclopamine, KAAD-cyclopamine, IPI-269609, or GDC-0449), EGFR tyrosine kinase activity (gefitinib and erlotinib), TGF-β type I activin receptor-like kinase, and ALK5 (SB431542) are also indicated. COX2, cyclooxygenase 2; CXCR4, CXC chemokine receptor 4; FOXM1, forkhead box M1 transcription factor; MMP, matrix metalloproteinase; VEGF, vascular endothelial growth factor.