Modulators of the hedgehog signaling pathway

Since its discovery by C. Nüsslein-Volhard and E. F. Wieschaus, hedgehog (hh) signaling has come a long way. Today it is regarded as a key regulator in embryogenesis where it governs processes like cell proliferation, differentiation, and tissue patterning. Furthermore, in adults it is involved in the maintenance of stem cells, and in tissue repair and regeneration. But hh signaling has a second—much darker—face: it plays an important role in several types of human cancers where it promotes growth and enables proliferation of tumor stem cells. The etiology of medulloblastoma and basal cell carcinoma is tightly linked to aberrant hh activity, but also cancers of the prostate, the pancreas, the colon, the breasts, rhabdomyosarcoma, and leukemia, are dependent on irregular hh activity. Recent clinical studies have shown that hh signaling can be the basis of an important new class of therapeutic agents with far-reaching implications in oncology. Thus, modulation of hh signaling by means of small molecules has emerged as a valuable tool in combating these hh-dependent cancers. Cyclopamine, a unique natural product with a fascinating history, was the first identified inhibitor of hh signaling and its story is closely linked to the progress in the whole field. In this review we will trace the story of cyclopamine, give an overview on the biological modes of hh signaling both in untransformed and malignant cells, and finally present potent modulators of the hh pathway—many of them already in clinical studies. For more than 30 years now the knowledge on hh signaling has grown steadily—an end to this development is far from being conceivable.     

Rab23, a negative regulator of hedgehog signaling, localizes to the plasma membrane and the endocytic pathway

The regulation of hedgehog signaling by vesicular trafficking was exemplified by the finding that Rab23, a Rab-GTPase vesicular transport protein, is mutated in open brain mice. In this study, the localization of Rab23 was analyzed by light and immunoelectron microscopy after expression of wild-type (Rab23-GFP), constitutively active Rab23 (Rab23Q68L-GFP), and inactive Rab23 (Rab23S23N-GFP) in a range of mammalian cell types. Rab23-GFP and Rab23Q68L-GFP were predominantly localized to the plasma membrane but were also associated with intracellular vesicular structures, whereas Rab23S23N-GFP was predominantly cytosolic. Vesicular Rab23-GFP colocalized with Rab5Q79L and internalized transferrin-biotin, but not with a marker of the late endosome or the Golgi complex. To investigate Rab23 with respect to members of the hedgehog signaling pathway, Rab23-GFP was coexpressed with either patched or smoothened. Patched colocalized with intracellular Rab23-GFP but smoothened did not. Analysis of patched distribution by light and immunoelectron microscopy revealed it is primarily localized to endosomal elements, including transferrin receptor-positive early endosomes and putative endosome carrier vesicles and, to a lesser extent, with LBPA-positive late endosomes, but was excluded from the plasma membrane. Neither patched or smoothened distribution was altered in the presence of wild-type nor mutant Rab23-GFP, suggesting that despite the endosomal colocalization of Rab23 and patched, it is likely that Rab23 acts more distally in regulating hedgehog signaling.     

Hedgehog信号通路在肿瘤中作用的研究进展

癌的发生是一个多因素多步骤的过程,同时伴随着一系列致癌因素所导致的基因突变,以及某些信号通路的异常激活。hedgehog（HH）信号通路参与了正常的胚胎发育过程以及组织的创伤与修复,特别是干细胞的自我更新,但它的异常激活却在多种人类恶性肿瘤中有发生。本文集中介绍HH在癌的发生、增殖、浸润及转移中所起的重要作用,通过对其致癌机制的综述,阐明它将可能成为一个有效治疗靶点,从而为肿瘤的防治提供了更多机遇。     

Genetic analysis of the two zebrafish patched homologues identifies novel roles for the hedgehog signaling pathway

Background  

Aberrant activation of the Hedgehog (Hh) signaling pathway in different organisms has shown the importance of this family of morphogens during development. Genetic screens in zebrafish have assigned specific roles for Hh in proliferation, differentiation and patterning, but mainly as a result of a loss of its activity. We attempted to fully activate the Hh pathway by removing both receptors for the Hh proteins, called Patched1 and 2, which are functioning as negative regulators in this pathway.     

Comprehensive methylome analysis of ovarian tumors reveals hedgehog signaling pathway regulators as prognostic DNA methylation biomarkers

Women with advanced stage ovarian cancer (OC) have a five-year survival rate of less than 25%. OC progression is associated with accumulation of epigenetic alterations and aberrant DNA methylation in gene promoters acts as an inactivating “hit” during OC initiation and progression. Abnormal DNA methylation in OC has been used to predict disease outcome and therapy response. To globally examine DNA methylation in OC, we used next-generation sequencing technology, MethylCap-sequencing, to screen 75 malignant and 26 normal or benign ovarian tissues. Differential DNA methylation regions (DMRs) were identified, and the Kaplan–Meier method and Cox proportional hazard model were used to correlate methylation with clinical endpoints. Functional role of specific genes identified by MethylCap-sequencing was examined in in vitro assays. We identified 577 DMRs that distinguished (p < 0.001) malignant from non-malignant ovarian tissues; of these, 63 DMRs correlated (p < 0.001) with poor progression free survival (PFS). Concordant hypermethylation and corresponding gene silencing of sonic hedgehog pathway members ZIC1 and ZIC4 in OC tumors was confirmed in a panel of OC cell lines, and ZIC1 and ZIC4 repression correlated with increased proliferation, migration and invasion. ZIC1 promoter hypermethylation correlated (p < 0.01) with poor PFS. In summary, we identified functional DNA methylation biomarkers significantly associated with clinical outcome in OC and suggest our comprehensive methylome analysis has significant translational potential for guiding the design of future clinical investigations targeting the OC epigenome. Methylation of ZIC1, a putative tumor suppressor, may be a novel determinant of OC outcome.     

Mouse dispatched mutants fail to distribute hedgehog proteins and are defective in hedgehog signaling

Hedgehog (Hh) signaling plays a major role in multiple aspects of embryonic development, which involves both short- and long-range signaling from localized Hh sources. One unusual aspect of Hh signaling is the autoproteolytic processing of Hh followed by lipid modification. As a consequence, the N-terminal fragment of Hh becomes membrane anchored on the cell surface of Hh-producing cells. A key issue in Hh signaling is to understand the molecular mechanisms by which lipid-modified Hh protein is transported from its sites of synthesis and subsequently moves through the morphogenetic field. The dispatched gene, which encodes a putative multipass membrane protein, was initially identified in Drosophila and is required in Hh-producing cells, where it facilitates the transport of cholesterol-modified Hh. We report the identification of the mouse dispatched (Disp) gene and a phenotypic analysis of Disp mutant mice. Disp-null mice phenocopy mice deficient in the smoothened gene, an essential component for Hh reception, suggesting that Disp is essential for Hh signaling. This conclusion was further supported by a detailed molecular analysis of Disp knockout mice, which exhibit defects characteristic of loss of Hh signaling. We also provide evidence that Disp is not required for Hh protein synthesis or processing, but rather for the movement of Hh protein from its sites of synthesis in mice. Taken together, our results reveal a conserved mechanism of Hh protein movement in Hh-producing cells that is essential for proper Hh signaling.     

Multiple positive and negative regulators of signaling by the EGF-receptor

Signaling via the epidermal growth factor (EGF)-receptor family is subject to regulation and modulation by multiple ligands, effectors and negative regulators, as well as regulation by heterodimerization between family members and crosstalk between heterologous signaling pathways. Besides serving as a paradigm for receptor tyrosine kinases in general, this family is crucial for development and is often mutated or amplified in human tumors.     

The ESC-E(Z) complex participates in the hedgehog signaling pathway

Polycomb group (PcG) genes are required for stable inheritance of epigenetic states throughout development, a phenomenon termed cellular memory. In Drosophila and mice, the product of the E(z) gene, one of the PcG genes, constitutes the ESC-E(Z) complex and specifically methylates histone H3. It has been argued that this methylation sets the stage for appropriate repression of certain genes. Here, we report the isolation of a well-conserved homolog of E(z), olezh2, in medaka. Hypomorphic knock-down of olezh2 resulted in a cyclopia phenotype and markedly perturbed hedgehog signaling, consistent with our previous report on oleed, a medaka esc. We also found cyclopia in embryos treated with trichostatin A, an inhibitor of histone deacetylase, which is a transient component of the ESC-E(Z) complex. The level of tri-methylation at lysine 27 of histone H3 was substantially decreased in both olezh2 and oleed knock-down embryos, and in embryos with hedgehog signaling perturbed by forskolin. We conclude that the ESC-E(Z) complex per se participates in hedgehog signaling.     

Ski and SnoN: negative regulators of TGF-beta signaling

Ski and SnoN are unique proto-oncoproteins in that they can induce both oncogenic transformation and terminal muscle differentiation when expressed at high levels. Recent studies using in vitro and in vivo approaches have begun to unravel the complex roles of Ski and SnoN in tumorigenesis and embryonic development. The identification of Ski and SnoN as important negative regulators of signal transduction by the transforming growth factor-beta superfamily of cytokines provides a valuable molecular basis for the complex functions of Ski and SnoN.     

Type-A Arabidopsis response regulators are partially redundant negative regulators of cytokinin signaling

Type-A Arabidopsis (Arabidopsis thaliana) response regulators (ARRs) are a family of 10 genes that are rapidly induced by cytokinin and are highly similar to bacterial two-component response regulators. We have isolated T-DNA insertions in six of the type-A ARRs and constructed multiple insertional mutants, including the arr3,4,5,6,8,9 hextuple mutant. Single arr mutants were indistinguishable from the wild type in various cytokinin assays; double and higher order arr mutants showed progressively increasing sensitivity to cytokinin, indicating functional overlap among type-A ARRs and that these genes act as negative regulators of cytokinin responses. The induction of cytokinin primary response genes was amplified in arr mutants, indicating that the primary response to cytokinin is affected. Spatial patterns of ARR gene expression were consistent with partially redundant function of these genes in cytokinin signaling. The arr mutants show altered red light sensitivity, suggesting a general involvement of type-A ARRs in light signal transduction. Further, morphological phenotypes of some arr mutants suggest complex regulatory interactions and gene-specific functions among family members.     

RNAi suppression of RPN12a decreases the expression of type-A ARRs, negative regulators of cytokinin signaling pathway, in Arabidopsis

The 26S proteasome is a 2-MDa complex with a central role in protein turn over. The 26S proteasome is comprised of one 20S core particle and two 19S regulatory particles (RPs). The RPN12a protein, a non-ATPase subunit of the 19S RP, was previously shown to be involved in cytokinin signaling in Arabidopsis. To further investigate cellular roles of RPN12a, RNAi transgenic plants of RPN12a were constructed. As expected, the 35S:RNAi-RPN12a plants showed cytokinin signaling defective phenotypes, including abnormal formation of leaves and inflorescences. Furthermore, RNAi knock-down transgenic plants exhibited additional unique phenotypes, including concave and heart-shape cotyledons, triple cotyledons, irregular and clustered guard cells, and defects in phyllotaxy, all of which are typical for defective cytokinin signaling. We next examined the mRNA level of cytokinin signaling components, including type-A ARRs, type-B ARRs, and CRFs. The expression of type-A ARRs, encoding negative regulators of cytokinin signaling, was markedly reduced in 35S:RNAi-RPN12a transgenic plants relative to that in wild type plants, while type-B ARRs and CRFs were unaffected. Our results also indicate that in vivo stability of the ARR5 protein, a negative regulator of cytokinin signaling, is mediated by the 26S proteasome complex. These results suggest that RPN12a participates in feedback inhibitory mechanism of cytokinin signaling through modulation of the abundance of ARR5 protein in Arabidopsis.     

Protein tyrosine phosphatases as negative regulators of mitogenic signaling

The regulation of tyrosine phosphorylation represents a key mechanism governing cell proliferation. In fibroblasts, inputs from both growth factor and extracellular matrix receptors are required for cell division. Triggering such receptors induces a wave of tyrosine phosphorylation on key signaling molecules, culminating in the activation of cyclin-dependent kinases and cell cycle progression. In general, protein tyrosine kinases stimulate, while protein tyrosine phosphatases inhibit, such cell proliferation pathways. The role of protein tyrosine kinases in mitogenesis has been extensively studied, but the identity and targets of the protein tyrosine phosphatases that regulate cell growth are not well described. In this review, I will survey recent advances in the identification and regulation of protein tyrosine phosphatases that downregulate cell proliferation. J. Cell. Physiol. 180:173–181, 1999. © 1999 Wiley-Liss, Inc.     

Type IV collagens and Dpp positive and negative regulators of signaling

Decapentaplegic (Dpp) is an essential morphogen in the TGF-β/BMP superfamily which patterns fields of cells during multiple stages of Drosophila development, including the ovary and embryo. We have found that type IV collagens bind to Dpp and play essential roles in the regulation of its signaling during these two developmental stages. This article primarily focuses on type IV collagens and embryonic Dpp signaling to discuss aspects of the type IV collagen mutant phenotype in the context of additional data from the field. In addition, the restriction of Dpp signaling in the the ovary by type IV collagens is described, as the differences between the embryonic and ovarian Dpp sources result in distinct effects of collagen IV proteins in the two systems.     

The nodal pathway acts upstream of hedgehog signaling to specify ventral telencephalic identity

The Nodal and Hedgehog signaling pathways influence dorsoventral patterning at all axial levels of the CNS, but it remains largely unclear how these pathways interact to mediate patterning. Here we show that, in zebrafish, Nodal signaling is required for induction of the homeobox genes nk2.1a in the ventral diencephalon and nk2.1b in the ventral telencephalon. Hedgehog signaling is also required for telencephalic nk2.1b expression but may not be essential to establish diencephalic nk2.1a expression. Furthermore, Shh does not restore ventral diencephalic development in embryos lacking Nodal activity. In contrast, Shh does restore telencephalic nk2.1b expression in the absence of Nodal activity, suggesting that Hedgehog signaling acts downstream of Nodal activity to pattern the ventral telencephalon. Thus, the Nodal pathway regulates ventral forebrain patterning through both Hedgehog signaling-dependent and -independent mechanisms.