Sonic Hedgehog Mutations Identified in Holoprosencephaly Patients Can Act in a Dominant Negative Manner

Sonic Hedgehog (SHH) plays an important instructional role in vertebrate development, as exemplified by the numerous developmental disorders that occur when the SHH pathway is disrupted. Mutations in the SHH gene are the most common cause of sporadic and inherited Holoprosencephaly (HPE), a developmental disorder that is characterized by defective prosencephalon development. SHH HPE mutations provide a unique opportunity to better understand SHH biogenesis and signaling, and to decipher its role in the development of HPE. Here, we analyzed a panel of SHH HPE missense mutations that encode changes in the amino-terminal active domain of SHH. Our results show that SHH HPE mutations affect SHH biogenesis and signaling at multiple steps, which broadly results in low levels of protein expression, defective processing of SHH into its active form and protein with reduced activity. Additionally, we found that some inactive SHH proteins were able to modulate the activity of wt SHH in a dominant negative manner, both in vitro and in vivo . These findings show for the first time the susceptibility of SHH driven developmental processes to perturbations by low-activity forms of SHH. In conclusion, we demonstrate that SHH mutations found in HPE patients affect distinct steps of SHH biogenesis to attenuate SHH activity to different levels, and suggest that these variable levels of SHH activity might contribute to some of the phenotypic variation found in HPE patients

Hedgehog signaling regulates E-cadherin expression for the maintenance of the actin cytoskeleton and tight junctions

In the stomach, strictly regulated cell adherens junctions are crucial in determining epithelial cell differentiation. Sonic Hedgehog (Shh) regulates epithelial cell differentiation in the adult stomach. We sought to identify whether Shh plays a role in regulating adherens junction protein E-cadherin as a mechanism for epithelial cell differentiation. Mouse nontumorigenic gastric epithelial (IMGE-5) cells treated with Hedgehog signaling inhibitor cyclopamine and anti-Shh 5E1 antibody or transduced with short hairpin RNA against Skinny Hedgehog (IMGE-5Ski) were cultured. A mouse model expressing a parietal cell-specific deletion of Shh (HKCre/ShhKO) was used to identify further changes in adherens and tight junctions. Inhibition of Hedgehog signaling in IMGE-5 cells caused loss of E-cadherin expression accompanied by disruption of F-actin cortical expression and relocalization of zonula occludens-1 (ZO-1). Loss of E-cadherin was also associated with increased proliferation in IMGE-5Ski cells and increased expression of the mucous neck cell lineage marker MUC6. Compared with membrane-expressed E-cadherin and ZO-1 protein in controls, dissociation of E-cadherin/β-catenin and ZO-1/occludin protein complexes was observed in HKCre/ShhKO mice. In conclusion, we demonstrate that Hedgehog signaling regulates E-cadherin expression that is required for the maintenance of F-actin cortical expression and stability of tight junction protein ZO-1

Selective Inhibitor of Proteasome's Caspase-like Sites Sensitizes Cells to Specific Inhibition of Chymotrypsin-like Sites

SummaryProteasomes degrade most proteins in mammalian cells and are established targets of anticancer drugs. All eukaryotic proteasomes have three types of active sites: chymotrypsin-like, trypsin-like, and caspase-like. Chymotrypsin-like sites are the most important in protein degradation and are the primary target of most proteasome inhibitors. The biological roles of trypsin-like and caspase-like sites and their potential as cotargets of antineoplastic agents are not well defined. Here we describe the development of site-specific inhibitors and active-site probes of chymotrypsin-like and caspase-like sites. Using these compounds, we show that cytotoxicity of proteasome inhibitors does not correlate with inhibition of chymotrypsin-like sites and that coinhibition of either trypsin-like and/or caspase-like sites is needed to achieve maximal cytotoxicity. Thus, caspase-like and trypsin-like sites must be considered as cotargets of anticancer drugs

Hedgehog-Producing Cancer Cells Respond to and Require Autocrine Hedgehog Activity

A number of Smoothened (SMO) pathway antagonists are currently undergoing clinical trials as anti-cancer agents. These drugs are proposed to attenuate tumor growth solely through inhibition of Hedgehog (HH), which is produced in tumor cells but acts on tumor stromal cells. The pivotal argument underlying this model is that the growth inhibitory properties of SMO antagonists on HH-producing cancer cells are due to their off-target effects. Here we show that the tumorigenic properties of such lung cancer cells depend on their intrinsic level of HH activity. Notably, reducing HH signaling in these tumor cells decreases HH target gene expression. Taken together, these results question the dogma that autocrine HH signaling plays no role in HH-dependent cancers, and does so without using SMO antagonists

Hedgehog-Producing Cancer Cells Respond to and Require Autocrine Hedgehog Activity

A number of Smoothened (SMO) pathway antagonists are currently undergoing clinical trials as anti-cancer agents. These drugs are proposed to attenuate tumor growth solely through inhibition of Hedgehog (HH), which is produced in tumor cells but acts on tumor stromal cells. The pivotal argument underlying this model is that the growth inhibitory properties of SMO antagonists on HH-producing cancer cells are due to their off-target effects. Here we show that the tumorigenic properties of such lung cancer cells depend on their intrinsic level of HH activity. Notably, reducing HH signaling in these tumor cells decreases HH target gene expression. Taken together, these results question the dogma that autocrine HH signaling plays no role in HH-dependent cancers, and does so without using SMO antagonists

Hedgehog Signaling Regulates Bladder Cancer Growth and Tumorigenicity

The role of HEDGEHOG (HH) signaling in bladder cancer remains controversial. The gene encoding the HH receptor and negative regulator PATCHED1 (PTCH1) resides on a region of chromosome 9q, one copy of which is frequently lost in bladder cancer. Inconsistent with PTCH1 functioning as a classic tumor suppressor gene, loss-of-function mutations in the remaining copy of PTCH1 are not commonly found. Here, we provide direct evidence for a critical role of HH signaling in bladder carcinogenesis. We show that transformed human urothelial cells and many urothelial carcinoma (UC) cell lines exhibit constitutive HH signaling, which is required for their growth and tumorigenic properties. Surprisingly, rather than originating from loss of PTCH1, the constitutive HH activity observed in UC cell lines was HH ligand-dependent. Consistent with this finding, increased levels of HH and the HH target gene product GLI1 were found in resected human primary bladder tumors. Furthermore, based on the difference in intrinsic HH dependence of UC cell lines, a gene expression signature was identified that correlated with bladder cancer progression. Our findings therefore indicate that therapeutic targeting of the HH signaling pathway may be beneficial in the clinical management of bladder cancer

The Hedgehog processing pathway is required for NSCLC growth and survival

Considerable interest has been generated from the results of recent clinical trials using SMOOTHENED (SMO) antagonists to inhibit the growth of HEDGEHOG (HH) signaling dependent tumors. This interest is tempered by the discovery of SMO mutations mediating resistance, underscoring the rationale for developing therapeutic strategies that interrupt HH signaling at levels distinct from those inhibiting SMO function. Here, we demonstrate that HH dependent non-small cell lung carcinoma (NSCLC) growth is sensitive to blockade of the HH pathway upstream of SMO, at the level of HH ligand processing. Individually, the use of different lentivirally delivered shRNA constructs targeting two functionally distinct HH-processing proteins, SKINNY HEDGEHOG (SKN) or DISPATCHED-1 (DISP-1), in NSCLC cell lines produced similar decreases in cell proliferation and increased cell death. Further, providing either an exogenous source of processed HH or a SMO agonist reverses these effects. The attenuation of HH processing, by knocking down either of these gene products, also abrogated tumor growth in mouse xenografts. Finally, we extended these findings to primary clinical specimens, showing that SKN is frequently over-expressed in NSCLC and that higher DISP-1 expression is associated with an unfavorable clinical outcome. Our results show a critical role for HH processing in HH-dependent tumors, identifies two potential druggable targets in the HH pathway, and suggest that similar therapeutic strategies could be explored to treat patients harboring HH ligand dependent cancers

The Full-length Unprocessed Hedgehog Protein Is an Active Signaling Molecule*

The hedgehog (HH) family of ligands plays an important instructional role in metazoan development. HH proteins are initially produced as ∼45-kDa full-length proteins, which undergo an intramolecular cleavage to generate an amino-terminal product that subsequently becomes cholesterol-modified (HH-Np). It is well accepted that this cholesterol-modified amino-terminal cleavage product is responsible for all HH-dependent signaling events. Contrary to this model we show here that full-length forms of HH proteins are able to traffic to the plasma membrane and participate directly in cell-cell signaling, both in vitro and in vivo. We were also able to rescue a Drosophila eye-specific hh loss of function phenotype by expressing a full-length form of hh that cannot be processed into HH-Np. These results suggest that in some physiological contexts full-length HH proteins may participate directly in HH signaling and that this novel activity of full-length HH may be evolutionarily conserved