Smoothened Receptors

The signaling by lipid-modified secreted glycoproteins of the Hedgehog (Hh) family plays fundamental roles during pattern formation in animal development. Mature Hh proteins are dually modified by cholesteryl and palmitoyl adducts.126 127 Dysfunction of Hh pathway components is frequently associated with a variety of congenital abnormalities and cancers. Transcriptional regulation of Hh target genes is mediated by the Gli zinc finger transcription factors. Activation of Gli leads, for example, to tissue-specific cell proliferation during embryogenesis, but Gli activity is restricted in adult tissues.

The smoothened GPCR (Smo) is kept in an inactive state by the 12-transmem-brane Patched (Ptc) protein, the receptor for Hh. Binding of Hh to Ptc activates Ptc to release the catalytic suppression128 of the activity of the Smo receptor to induce downstream signaling in a constitutively active manner, i.e., in the absence of ligands bound to Smo (although there may still be a hypothetical endogenous ligand whose access to Smo is controlled by Ptc in a Hh-dependent manner). Downregu-lation of Ptc, as observed in many cancers, thus results in constitutive activation of the Hh pathway.

In addition to the interaction with Ptc, in Drosophila, Smo has also been shown to interact with a protein complex consisting of the zinc finger transcription factor Cubitus interrruptus (Ci), the serine-threonine kinase Fused (Fu), and the kinesin-like Costal2 (Cos2)129 recently suggested to serve as a catalytic subunit of protein kinase A (PKA).130 The protein complex is associated with Smo in membrane fractions both in vitro and in vivo, and Cos2 binding on Smo is necessary for the Hh-dependent dissociation of Ci from this complex.129 Activated Ci then translocates to the nucleus to modulate gene expression.

Consistent with the notions that phosphorylation of PKA renders Ci in Droso-phila susceptible to proteolytic cleavage and that Hh signaling reduces PKA-depen-dent cleavage of Ci,131 Gai proteins are suggested to participate in Smo-mediated signaling upon stimulation of Ptc with Sonic Hedgehog (SHh).132 However, intracellular cAMP levels are not altered in mammalian cells stimulated with SHh,133 suggesting additional signaling pathways are involved in mediating SHh actions.

Members of the ubiquitously expressed G12 family of heterotrimeric G proteins that include Ga12 and Ga13 have been shown to mediate the actions of SHh to activate RhoA and its downstream effector Rho-associated kinase (Rho-kinase) to activate the Gli transcription factors and inhibit neurite outgrowth of neuroblastoma cells.134 The signaling pathways activated by Smo/Ptc are depicted in Figure 5.2.129

In analogy to the Wnt/Frizzled signaling, the Hh signaling controls many aspects of development and also regulates cell growth and differentiation. Hh and Wnt signaling often act together to control cell growth and tissue morphogenesis. Hh/Smoothened signaling is active in a variety of tumors and is also involved in the maintenance of normal stem cells in vivo. Several SHh signal transducers including Ptc, Smo, and the Gli transcription factors are upregulated in basal cell carcinomas

(BCCs).135 The mRNAs, for example, of Hh, Ptc, and Smo are upregulated in pancreatic cancer tissues.136

Despite the prominent role of Smo in activating Hh signaling, experiments using transgenic mice indicated that gain-of-function mutations in Smo are insufficient for the development and/or maintenance of basal cell carcinomas.137 In contrast, the magnitude of Hh pathway activation appears to be the determinant of skin tumor phenotype.137

Altered SHh signaling is crucial for the development of BCCs, the most common human cancers. Mutations in SHh, Ptc, and Smo have been identified — in particular loss-of-function mutations of Ptc, and gain-of-function mutations of Smo.138 Mutations in SHh are rare,139 and the identified mutations do not result in SHh proteins with altered activities compared to the wild-type proteins. Two gain-of-function mutations in human Smo that result in alterations in the seventh transmembrane domain and in the intracellular cytoplasmic C-terminal domain of the receptor that do not impair their interactions with Ptc have been identified in BCC sporadic tumors.138 The two individual mutations in Smo (but not wild-type Smo) can cooperate with adenovirus E1A to transform rat embryonic fibroblast cells in culture. Transgenic mice overexpressing the mutant Smo develop skin abnormalities similar to BCCs, supporting the role of Smo as a protooncogene.138 These findings suggest that pharmacological inhibition of Smo or its downstream effectors could provide effective treatment for BCCs and possibly for other cancers.

Cyclopamine, a teratogen found in the Veratrum californicum plant that blocks the synthesis of cholesterol, is an inhibitor of Hh/Smo signaling by acting on Smo and has recently been shown to lead to a rapid regression of skin tumors, while no adverse effects were noted on normal skin.140 The effects of oncogenic mutations of Smo and Ptc can be reversed by cyclopamine treatment141 and the modulation of the activities of Hh pathways has been suggested to offer improvements in the treatment of cancer.142 Cyclopamine and other small molecule inhibitors of Smo have been shown to be effective in cancer treatment.143145 Cyclopamine treatment has been shown to be effective in the inhibition of brain tumorigenesis146 and medulloblastoma growth147; the treatment of skin140 and colorectal tumors through the induction of apoptosis145; the prevention of ultraviolet B-induced BCC formation148; the inhibition of the invasiveness of cancer cells149; the growth of Hh pathway-activated breast carcinoma cells150; and the proliferation of metastatic prostate cancer cell lines.151 Cyclopamine also inhibits growth of small cell lung cancers and a wide range of foregut-derived malignancies both in vitro and in vivo.152 Modulation of the activity of the Hh pathway thus appears a promising avenue for the treatment of cancer. In addition, cyclopamide has recently been shown to be effective in a proof-of-principle study related to rapid clearing of skin lesions in psoriasis patients.153

Small non-peptidylic agonists and antagonists for Smo have recently been iden-tified.154 The Smo agonist designated Ag-1.4 mediates the activation of p44/p42 MAP kinase phosphorylation, which is abrogated in interferon (IFN)-a SHh-respon-sive cells,155 possibly explaining the effectiveness of IFN-based therapy in BCC treatment.

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