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  • Several layers of regulation for this canonical


    Several layers of regulation for this canonical activation of the HH pathway exist. First, the protein kinase A (PKA), the casein kinase 1α (CK1α) and GSK3β can phosphorylate and mark for proteosomal degradation the GLI transcription factors. [36] Second, Suppressor of Fused (SUFU) binds to GLI and sequesters GLI in the [Ala92]-p16 (84-103) synthesis in the absence of hedgehog ligands [37]. Third, genes encoding for negative regulators of the HH pathway, such as PTCH 1 and 2 and hedgehog inhibitory protein (HHIP) are GLI target genes, therefore they engage in a negative feedback loop with GLI. [38,39] Also, neuropilin 1 and 2 (NRP1 and NRP2), activate a positive feedback loop with GLI as described recently [40,41]. Neuropilins enhance HH signaling in both a PKA dependent41] and independent [42] way. In the mouse, non-canonical Hedgehog pathway also exists, where astrocyte derived Shh activates nestin in a medulloblastoma model. Importantly, this paracrine loop is Ptc1 and Gli independent but Smo dependent [43]. Last but not least, pathways other than the HH pathway, for example RAS, TGFβ and PI3K, can induce GLI expression in cancer in a number of ways. [44,45] First, RAS, as well as PI3K induce the nuclear localization and activate GLI1 in melanoma models [46]. Further, PI3K/AKT signaling inhibits GLI phosphorylation by PKA and prevents its degradation [47]. Third, signaling through EGFR activates c-JUN via MAPK which functions as a co-transcription factor with GLI for certain GLI targets [48]. Fourth, SMAD transcription factors are regulated by TGFβ and synergize with GLI1 to induce TGFβ and HH dependent CCND1 expression. [49] Finally, TGFβ signaling increases transcription of the GLI genes. [50] Fig. 1 illustrates the canonical and non-canonical HH pathway and its regulation.
    HH pathway inhibitors There are currently several strategies to inhibit the hedgehog pathway in the clinic. Interfering directly with SMO activity is a well-studied means to inhibit the HH pathway. Cyclopamine belongs to Veratrum alkaloids, plant derived compounds known to cause teratogenesis including cyclopia. [51,52] Cyclopamine binds to the extracellular loops of the transmembrane domain of SMO and is a SMO inhibitor [51,53]. Vismodegib, saridegib and sonidegib are more potent inhibitors of SMO which also bind to the transmembrane domain [54]. On the contrary, oxysterol and other oxidized derivatives of cholesterol activate SMO by binding to the cysteine rich domain located in the extracellular domain of SMO. [[55], [56], [57]], Indeed, statins which are known to block cholesterol synthesis by inhibiting 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) inhibit medulloblastoma growth in vivo [58]. Additionally, cholesterol is necessary and sufficient to activate SMO and might represent the missing link between PTCH1 and SMO [30,31]. In this model, PTCH1 negatively regulates plasma membrane cholesterol in the vicinity of SMO further supporting the rationale for cholesterol synthesis inhibition as a HH targeting strategy. Itraconazole, a triazol antifungal agent was identified as an inhibitor of HH signaling in a library screen of 2,400 drugs with FDA approval or in post phase I drug development process. [59] Despite the well-established target of itraconazole, 14-α-lanosterole demethylase which is necessary for ergosterol synthesis in fungi and cholesterol in mammals with higher potency for the fungal enzyme, the inhibitory effects of itraconazole on the HH pathway result from direct binding and inhibition of SMO at a site different from the binding site for oxysterols or cyclopamine [59]. Itraconazole effectively prevents the accumulation of SMO in the primary [Ala92]-p16 (84-103) synthesis cilium and inhibits the growth of Hh dependent medulloblastoma in vivo [59]. The itraconazole doses necessary to inhibit SMO are higher compared to doses used to inhibit ergosterol synthesis in fungi but still clinically feasible. Another approach to inhibit the HH pathway is to target the transcription factors GLI. The active form of GLI2 is the most significant mediator of HH activity in mammals, GLI3 is mostly a suppressor and GLI1 serves as a pathway output amplifier. [34,35,60] Arsenic trioxide (ATO) is used clinically for the treatment of acute promyelocytic leukemia because it degrades PML-RARA [61]. ATO inhibits GLI1 in a primary cilium independent manner, blocks Gli2 accumulation in the primary cilium and exerts anti-tumorigenic effects in a wide array of cancer cell lines and HH dependent in vivo mouse models. [62,63] GANT-58 and GANT-61 were identified in a drug screen as inhibitors of GLI1 as they prevent binding of GLI1 transcription factor to DNA [64]. SSTC3 is an agonist for CK1α, which along with GSK3β phosphorylates the GLI transcription factors marking them for degradation [65]. SSTC3 inhibits TRP53 mutated, MYCN amplified medulloblastoma mouse models by promoting GLI degradation. [65]