Plexins Promote Hedgehog Signaling Through Their Cytoplasmic GAP Activity

Hedgehog signaling controls tissue patterning during embryonic and postnatal development and continues to play important roles throughout life. Characterizing the full complement of Hedgehog pathway components is essential to understanding its wide-ranging functions. Previous work has identified Neuropilins, established Semaphorin receptors, as positive regulators of Hedgehog signaling. Neuropilins require Plexin co-receptors to mediate Semaphorin signaling, but a role for Plexins in Hedgehog signaling has not yet been explored. Here, we provide evidence that multiple Plexins promote Hedgehog signaling in NIH/3T3 fibroblasts and that Plexin loss-of-function in these cells results in significantly reduced Hedgehog pathway activity. Catalytic activity of the Plexin GTPase activating protein (GAP) domain is required for Hedgehog signal promotion, and constitutive activation of the GAP domain further amplifies Hedgehog signaling. Additionally, we demonstrate that Plexins promote Hedgehog signaling at the level of GLI transcription factors and that this promotion requires intact primary cilia. Finally, we find that Plexin loss-of-function significantly reduces the response to Hedgehoga pathway activation in the mouse dentate gyrus. Together, these data identify Plexins as novel components of the Hedgehog pathway and provide insight into their mechanism of action.

Hedgehog-induced ciliary trafficking of kinesin-4 motor KIF7 requires intraflagellar transport but not KIF7’s microtubule binding

The kinesin-4 motor KIF7 is a conserved regulator of the Hedgehog signaling pathway. In vertebrates, Hedgehog signaling requires the primary cilium, and KIF7 and Gli transcription factors accumulate at the cilium tip in response to Hedgehog activation. Unlike conventional kinesins, KIF7 is an immotile kinesin and its mechanism of ciliary accumulation is unknown. We generated KIF7 variants with altered microtubule binding or motility. We demonstrate that microtubule binding of KIF7 is not required for the increase in KIF7 or Gli localization at the cilium tip in response to Hedgehog signaling. In addition, we show that the immotile behavior of KIF7 is required to prevent ciliary localization of Gli transcription factors in the absence of Hedgehog signaling. Using an engineered kinesin-2 motor that enables acute inhibition of intraflagellar transport (IFT), we demonstrate that kinesin-2 KIF3A/KIF3B/KAP mediates the translocation of KIF7 to the cilium tip in response to Hedgehog pathway activation. Together, these results suggest that KIF7’s role at the tip of the cilium is unrelated to its ability to bind to microtubules.

Mebendazole Mediates Proteasomal Degradation of GLI Transcription Factors in Acute Myeloid Leukemia

The prognosis of elderly AML patients is still poor due to chemotherapy resistance. The Hedgehog (HH) pathway is important for leukemic transformation because of aberrant activation of GLI transcription factors. MBZ is a well-tolerated anthelmintic that exhibits strong antitumor effects. Herein, we show that MBZ induced strong, dose-dependent anti-leukemic effects on AML cells, including the sensitization of AML cells to chemotherapy with cytarabine. MBZ strongly reduced intracellular protein levels of GLI1/GLI2 transcription factors. Consequently, MBZ reduced the GLI promoter activity as observed in luciferase-based reporter assays in AML cell lines. Further analysis revealed that MBZ mediates its anti-leukemic effects by promoting the proteasomal degradation of GLI transcription factors via inhibition of HSP70/90 chaperone activity. Extensive molecular dynamics simulations were performed on the MBZ-HSP90 complex, showing a stable binding interaction at the ATP binding site. Importantly, two patients with refractory AML were treated with MBZ in an off-label setting and MBZ effectively reduced the GLI signaling activity in a modified plasma inhibitory assay, resulting in a decrease in peripheral blood blast counts in one patient. Our data prove that MBZ is an effective GLI inhibitor that should be evaluated in combination to conventional chemotherapy in the clinical setting.

Cytoskeletal regulation of a transcription factor by DNA mimicry

A long-established strategy for transcription regulation is the tethering of transcription factors to cellular membranes. In contrast, the principal effectors of Hedgehog signaling, the Gli transcription factors, are regulated by microtubules in the primary cilium and the cytoplasm. How Gli is tethered to microtubules remains unclear. We uncover DNA mimicry by the ciliary kinesin Kif7 as a mechanism for the recruitment of Gli to microtubules, revealing a new mode of tethering a DNA-binding protein to the cytoskeleton. Gli increases the Kif7-microtubule affinity and consequently modulates the localization of both proteins to microtubules and the cilium tip. Thus, the kinesin-microtubule system is not a passive Gli tether but a regulatable platform tuned by the kinesin-transcription factor interaction. We re-tooled the unique DNA-mimicry-based Gli-Kif7 interaction for inhibiting the nuclear and cilium localization of Gli. This strategy can be potentially exploited for downregulating erroneously activated Gli in human cancers.

The exocyst complex and intracellular vesicles mediate soluble protein trafficking to the primary cilium

Efficient transport of proteins into the primary cilium is a crucial step for many signaling pathways. Dysfunction of this process can lead to the disruption of signaling cascades or cilium assembly, resulting in developmental disorders and cancer. Previous studies on ciliary trafficking were mostly focused on the membrane-embedded receptors. In contrast, how soluble proteins are delivered into the cilium is poorly understood. In our work, we identify the exocyst complex as a key player in the ciliary trafficking of soluble Gli transcription factors. Considering that the exocyst mediates intracellular vesicle transport, we demonstrate that soluble proteins, including Gli2/3 and Lkb1, can use the endosome recycling machinery for their delivery to the primary cilium. Finally, we identify GTPases: Rab14, Rab18, Rab23, and Arf4 involved in vesicle-mediated Gli protein ciliary trafficking. Our data pave the way for a better understanding of ciliary transport and uncover novel transport mechanisms inside the cell.

The Role of Smoothened-Dependent and -Independent Hedgehog Signaling Pathway in Tumorigenesis

The Hedgehog (Hh)-glioma-associated oncogene homolog (GLI) signaling pathway is highly conserved among mammals, with crucial roles in regulating embryonic development as well as in cancer initiation and progression. The GLI transcription factors (GLI1, GLI2, and GLI3) are effectors of the Hh pathway and are regulated via Smoothened (SMO)-dependent and SMO-independent mechanisms. The SMO-dependent route involves the common Hh-PTCH-SMO axis, and mutations or transcriptional and epigenetic dysregulation at these levels lead to the constitutive activation of GLI transcription factors. Conversely, the SMO-independent route involves the SMO bypass regulation of GLI transcription factors by external signaling pathways and their interacting proteins or by epigenetic and transcriptional regulation of GLI transcription factors expression. Both routes of GLI activation, when dysregulated, have been heavily implicated in tumorigenesis of many known cancers, making them important targets for cancer treatment. Hence, this review describes the various SMO-dependent and SMO-independent routes of GLI regulation in the tumorigenesis of multiple cancers in order to provide a holistic view of the paradigms of hedgehog signaling networks involving GLI regulation. An in-depth understanding of the complex interplay between GLI and various signaling elements could help inspire new therapeutic breakthroughs for the treatment of Hh-GLI-dependent cancers in the future. Lastly, we have presented an up-to-date summary of the latest findings concerning the use of Hh inhibitors in clinical developmental studies and discussed the challenges, perspectives, and possible directions regarding the use of SMO/GLI inhibitors in clinical settings.

Embryology of the upper limb

Recent advances in research technology have allowed a rapid progression in the understanding of upper limb embryogenesis. Limb development occurs between 4 and 8 weeks of gestation and progresses along three axes, as controlled by three centres: proximal–distal by the apical ectodermal ridge, anterior–posterior by the zone of polarizing activity, and dorsal–ventral by the dorsal ectoderm. In addition, research into molecular anatomy has revealed the central role of the sonic hedgehog protein and the complex interconnections linking all three axes of development. Despite these advances in understanding normal limb development, the field of pathembryogenesis remained limited. Recent discoveries about the roles of HOX and TBX genes, as well as bone morphogenetic proteins and GLI transcription factors, have allowed important insights into the causations of common clinical conditions. This expanding field of knowledge has facilitated the establishment of a new classification system based entirely on embryogenesis, which should catalyse closer surgeon/scientist collaborations.

SPOP and CUL3 Modulate the Sonic Hedgehog Signal Response Through Controlled Degradation of GLI Family Transcription Factors

The speckle-type POZ protein (SPOP) functions as a guardian of genome integrity and controls transcriptional regulation by functioning as a substrate adaptor for CUL3/RING-type E3 ubiquitin ligase complexes. SPOP-containing CUL3 complexes target a myriad of DNA-binding proteins involved in DNA repair and gene expression, and as such, are essential modulators of cellular homeostasis. GLI transcription factors are effectors of the Hedgehog (HH) pathway, a key driver of tissue morphogenesis and post-developmental homeostasis that is commonly corrupted in cancer. CUL3-SPOP activity regulates amplitude and duration of HH transcriptional responses by controlling stability of GLI family members. SPOP and GLI co-enrich in phase separated nuclear droplets that are thought to serve as hot spots for CUL3-mediated GLI ubiquitination and degradation. A similar framework exists in Drosophila, in which the Hedgehog-induced MATH (meprin and traf homology) and BTB (bric à brac, tramtrack, broad complex) domain containing protein (HIB) targets the GLI ortholog Cubitus interruptus (Ci) for Cul3-directed proteolysis. Despite this functional conservation, the molecular mechanisms by which HIB and SPOP contribute to Drosophila and vertebrate HH signaling differ. In this mini-review we highlight similarities between the two systems and discuss evolutionary divergence in GLI/Ci targeting that informs our understanding of how the GLI transcriptional code is controlled by SPOP and CUL3 in health and disease.

A PKA Inhibitor Motif within Smoothened Controls Hedgehog Signal Transduction

The Hedgehog (Hh) cascade is central to development, tissue homeostasis, and cancer. A pivotal step in Hh signal transduction is the activation of GLI transcription factors by the atypical G protein-coupled receptor (GPCR) Smoothened (SMO). How SMO activates GLI has remained unclear for decades. Here we show that SMO employs a decoy substrate sequence to physically block the active site of the PKA catalytic subunit (PKA-C) and extinguish its enzymatic activity. As a result, GLI is released from phosphorylation-induced inhibition. Using a combination of in vitro, cellular, and organismal models, we demonstrate that interfering with SMO / PKA pseudosubstrate interactions prevents Hh signal transduction. The mechanism we uncovered echoes one utilized by the Wnt cascade, revealing an unexpected similarity in how these two essential developmental and cancer pathways signal intracellularly. More broadly, our findings define a new mode of GPCR-PKA communication that may be harnessed by a range of membrane receptors and kinases.

Drug Delivery Systems for Hedgehog Inhibitors in the Treatment of SHH-Medulloblastoma

Medulloblastoma (MB) is a highly aggressive pediatric tumor of the cerebellum. Hyperactivation of the Hedgehog (HH) pathway is observed in about 30% of all MB diagnoses, thereby bringing out its pharmacological blockade as a promising therapeutic strategy for the clinical management of this malignancy. Two main classes of HH inhibitors have been developed: upstream antagonists of Smoothened (SMO) receptor and downstream inhibitors of GLI transcription factors. Unfortunately, the poor pharmacological properties of many of these molecules have limited their investigation in clinical trials for MB. In this minireview, we focus on the drug delivery systems engineered for SMO and GLI inhibitors as a valuable approach to improve their bioavailability and efficiency to cross the blood–brain barrier (BBB), one of the main challenges in the treatment of MB.