The Down syndrome-associated DYRK1A kinase has been reported as a stimulator of the developmentally important Hedgehog (Hh) pathway, but cells from Down syndrome patients paradoxically display reduced Hh signaling activity. Here, we find that DYRK1A stimulates GLI transcription factor activity through phosphorylation of general nuclear localization clusters. In contrast, in vivo and in vitro experiments reveal that DYRK1A kinase can also function as an inhibitor of endogenous Hh signaling by negatively regulating ABLIM proteins, the actin cytoskeleton and the transcriptional co-activator MKL1 (MAL). As a final effector of the DYRK1A–ABLIM-Actin-MKL1 sequence, we identify the MKL1-interactor Jumonji-domain demethylase 1A (JMJD1A) as a novel Hh pathway component stabilizing the GLI1 protein in a demethylase-independent manner. Furthermore, a Jumonji-specific small molecule antagonist represents a novel and powerful inhibitor of Hh signal transduction by inducing GLI1 protein degradation in vitro and in vivo.

The Hedgehog (HH) pathway has been identified as an important deregulated signal transduction pathway in pancreatic ductal adenocarcinoma (PDAC), a cancer type characterized by a highly metastatic phenotype. In PDAC, the canonical HH pathway activity is restricted to the stromal compartment while HH signaling in the tumor cells is reduced as a consequence of constitutive KRAS activation. Here we report that in the tumor compartment of PDAC the HH pathway effector transcription factor GLI1 regulates epithelial differentiation. RNAi-mediated knockdown of GLI1 abolished characteristics of epithelial differentiation, increased cell motility and synergized with TGFβ to induce an epithelial-to-mesenchymal transition (EMT). Notably, EMT conversion in PDAC cells occurred in the absence of induction of SNAIL or SLUG, two canonical inducers of EMT in many other settings. Further mechanistic analysis revealed that GLI1 directly regulated the transcription of E-cadherin, a key determinant of epithelial tissue organization. Collectively, our findings identify GLI1 as an important positive regulator of epithelial differentiation, and they offer an explanation for how decreased levels of GLI1 are likely to contribute to the highly metastatic phenotype of PDAC.

Uncontrolled Hedgehog (Hh) signaling is the cause of several malignancies, including the pediatric cancer medulloblastoma, a neuroectodermal tumor affecting the cerebellum. Despite the development of potent Hh pathway antagonists, medulloblastoma drug resistance is still an unresolved issue that requires the identification of novel drug targets. Following up on our observation that histone deacetylase 6 (HDAC6) expression was increased in Hh-driven medulloblastoma, we found that this enzyme is essential for full Hh pathway activation. Intriguingly, these stimulatory effects of HDAC6 are partly integrated downstream of primary cilia, a known HDAC6-regulated structure. In addition, HDAC6 is also required for the complete repression of basal Hh target gene expression. These contrasting effects are mediated by HDAC6 0 s impact on Gli2 mRNA and GLI3 protein expression. As a result of this complex interaction with Hh signaling, global transcriptome analysis revealed that HDAC6 regulates only a subset of Smoothened-and Gli-driven genes, including all well-established Hh targets such as Ptch1 or Gli1. Importantly, medulloblastoma cell survival was severely compromised by HDAC6 inhibition in vitro and pharmacologic HDAC6 blockade strongly reduced tumor growth in an in vivo allograft model. In summary, our data describe an important role for HDAC6 in regulating the mammalian Hh pathway and encourage further studies focusing on HDAC6 as a novel drug target in medulloblastoma.

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Nature Communications 6: Article number:8023 (2015); Published: 27 August 2015; Updated: 26 October 2015 In Fig. 2j of this Article, the image in the lower left panel was inadvertently duplicated from the upper left panel during the final stages of manuscript preparation. The correct version of the figure appears below.

In a recent publication (1), we showed that low levels of the GLI1 transcription factor sensitize pancreatic ductal adenocarcinoma (PDAC) cells to induce an epithelial-to-mesenchymal transition (EMT). Analysis of the mechanism identified Ecadherin (encoded by the CDH1 gene) as a direct target of GLI1 in PDAC cells. We showed direct binding of GLI1 to the CDH1 promoter and transcriptional activation of the CDH1 gene by GLI1 in pancreatic cancer cells using expression and reporter studies. Finally, we were able to show a positive correlation between GLI1 and E-cadherin expression in cultured PDAC cells and in patient samples.Our data were questioned in a letter to the editor from Inaguma and colleagues who recently published that GLI1 induces mucin MUC5AC expression in PDAC cells leading to Ecadherin protein destabilization (2). In their letter, the authors raise concerns about several points of our manuscript, and we would like to reply to the major points of criticism in this response letter.One of these points refers to the fact that we did not show changes in E-cadherin levels upon GLI1 overexpression in several PDAC cell lines, a somewhat astonishing criticism given the fact that the authors themselves never properly showed alterations in E-cadherin expression upon full-length GLI1 overexpression in their own manuscript (2). However, we assume that the transcriptional machinery that controls the GLI1-CDH1 axis might be saturated in these cells and therefore no change in CDH1 levels upon further increase of GLI1 by means of transfection can be seen. One exception was MiaPaca-2 cells, which upregulated CDH1 expression upon GLI1 transfection, despite the reported hypermethylation of the CDH1 promoter in these cells. In agreement with our data, others have also reported induction of CDH1 in MiaPaca-2 cells upon transfection with transcription factor expression plasmids, suggesting that the promoter methylation does not fully prevent transcriptional activity at this gene locus under certain circumstances (3). Also not discussed in the letter by Inaguma and colleagues is our finding that a transcriptionally inactive GLI1 lacking the DNA-binding domain is able to suppress E-cadherin expression and induce an EMT in Panc1 cells. We interpret these data as dominant-negative behavior of the mutant GLI1 on the wild-type form. Taken together, we concluded that in most of the cell lines analyzed, the effects of GLI1 on CDH1 are saturated, and only a reduction in GLI1 is translated into effects on CDH1. A slightly alternative possible scenario would be that GLI1 is required to maintain CDH1 expression once the promoter has been activated by a second factor. In such a case, the second factor would define the level of CDH1 expression and a reduction in the maintenance factor (GLI1) would decrease the CDH1 expression but increasing GLI1 levels would not further stimulate it.Another point of concern that Inaguma and colleagues pointed out was that our clinical samples were not microdissected and therefore the positive correlati...

<p>PDf file - 405K</p>

<p>PDf file - 405K</p>