The naevoid basal cell carcinoma syndrome (NBCCS) is caused by mutations in the hedgehog receptor PTCH gene. It is characterized by developmental defects and a predisposition to the development of certain tumours, such as basal cell carcinoma, medulloblastoma and meningioma, and potentially fetal rhabdomyomas and embryonal rhabdomyosarcomas. This study aimed to analyse PTCH status in an NBCCS patient with fetal rhabdomyoma and to investigate whether deregulation of hedgehog signalling, as shown by altered expression of hedgehog pathway components and/or genetic imbalances, is a general finding in sporadic rhabdomyomas and rhabdomyosarcomas. The NBCCS patient had a novel PTCH germ-line mutation, 1370insT, and developed a fetal rhabdomyoma that harboured a 30 bp in-frame deletion in the second allele resulting in homozygous inactivation of PTCH. Sporadic rhabdomyomas and rhabdomyosarcomas showed overexpression of PTCH (43/43) and GLI1 (41/43) mRNA, as determined by in situ hybridization, indicating ongoing active hedgehog signalling. Immunohistochemical staining revealed a subgroup of fetal rhabdomyomas and embryonal rhabdomyosarcomas (12/34) lacking PTCH immunoreactivity. Four of nine informative fetal rhabdomyomas and embryonal rhabdomyosarcomas showed loss of heterozygosity (LOH) in the PTCH region with two of these (one fetal rhabdomyoma and one embryonal rhabdomyosarcoma) also showing LOH in the SUFU region. These findings suggest that haploinsufficiency for the two tumour suppressor genes PTCH and SUFU, which are both active in the same signalling pathway, may be important for tumour development. Based on our results we propose that the pathogenesis of rhabdomyoblastic tumours, particularly fetal rhabdomyomas and embryonal rhabdomyosarcomas, involves deregulation of the hedgehog signalling pathway.

Synergism between the RAS and Hedgehog (HH) pathways has been suggested for carcinogenesis in the pancreas, lung and colon. We investigated the molecular cross-talk between RAS and HH signaling and found that, although mutant RAS induces or enhances SHH expression and favors paracrine HH signaling, it antagonizes autocrine HH signal transduction. Activated RAS can be found in primary cilia, the central organelle of HH signal transduction, but functions in a cilium-independent manner and interferes with Gli2 function and Gli3 processing. In addition, the cell-autonomous negative regulation of HH signal transduction involves the RAS effector molecule dual specificity tyrosine phosphorylated and regulated kinase 1B (DYRK1B). In line with a redirection of autocrine toward paracrine HH signaling by a KRAS-DYRK1B network, we find high levels of GLI1 expression restricted to the stromal compartment and not to SHH-expressing tumor cells in human pancreatic adenocarcinoma.

Hedgehog (HH) signaling is one of the key pathways with major significance for embryogenesis, tumorigenesis, and stem cell maintenance. Glioma-associated oncogene 1 (GLI1) is a transcription factor that acts as the terminal signaling effector but also represents a pathway target gene. Here we report the identification and functional properties of novel GLI1 splice variants generated by skipping exons 2 and 3 and encoding an N-terminal truncated GLI1 protein (GLI1⌬N). Analysis of the GLI1⌬N mRNAs in adult human tissues revealed comparable expression levels to the full-length GLI1 (GLI1FL), whereas in tumor cell lines a generally lower and more variable expression pattern was observed. Furthermore, GLI1⌬N is up-regulated by HH signaling to the same extent as GLI1FL but has a weaker capacity to activate transcription. However, in specific cellular contexts GLI1⌬N may be more potent than GLI1FL in activating endogenous gene expression. Moreover, the dual-specificity tyrosine phosphorylation-regulated kinase 1 (Dyrk1) potentiates the transcriptional activity of GLI1FL but not GLI1⌬N. Interestingly, GLI1FL, in contrast to GLI1⌬N, is localized solely at the nucleus, in line with its increased transcriptional capacity. The negative regulator of the pathway, Suppressor of Fused (SUFU), elicits a cytoplasmic retention of the GLI1 isoforms, which is more pronounced for GLI1FL, as this contains an N-terminal SUFU binding domain. Collectively, our findings reveal that the activation mechanism of the terminal transducer of the pathway, GLI1, is mediated not only by GLI1FL but also by the GLI1⌬N variant.The post-transcriptional process of alternative splicing is considered to be a pervasive phenomenon in eukaryotic gene expression that increases the diversity of mRNAs and proteins. Genome-wide analysis indicates that at least 75% of human multiexon genes have alternative splice variants (1, 2). Additionally, variations in the splicing pattern of gene products have been related to pathological states including cancer. It is now believed that a minimum of 15% of the point mutations responsible for human genetic diseases are in fact interfering with splicing regulatory events (3, 4). Alternative splice variants have the potential of being used as diagnostic markers and/or therapeutic targets (5).The Hedgehog (HH) 4 signaling pathway was first reported as a major pathway involved in pattern formation during development of Drosophila and embryonic developmental processes in vertebrates. Additionally, abnormal activation of the pathway has been linked to several cancers including basal cell carcinoma, medulloblastoma, rhabdomyosarcoma, lung, prostate, and pancreatic tumors (6 -9). Using Drosophila as the model organism significant findings on the mechanism of this pathway have been revealed. Active signal transduction is generally associated with binding of HH ligands to the Patched (PTCH) receptor. This releases the inhibitory effects of PTCH on the signaling molecule Smoothened (SMO), thus initiating a series of molecular events t...

The Hedgehog (HH) signaling pathway has important roles in tumorigenesis and in embryonal patterning. The Glioma-associated oncogene 1 (GLI1) is a key molecule in HH signaling, acting as a transcriptional effector and, moreover, is considered to be a potential therapeutic target for several types of cancer. To extend our previous focus on the implications of alternative splicing for HH signal transduction, we now report on an additional post-transcriptional mechanism with an impact on GLI1 activity, namely RNA editing. The GLI1 mRNA is highly edited at nucleotide 2179 by adenosine deamination in normal cerebellum, but the extent of this modification is reduced in cell lines from the cerebellar tumor medulloblastoma. Additionally, basal cell carcinoma tumor samples exhibit decreased GLI1 editing compared with normal skin. Interestingly, knocking down of either ADAR1 or ADAR2 reduces RNA editing of GLI1. This adenosine to inosine substitution leads to a change from Arginine to Glycine at position 701 that influences not only GLI1 transcriptional activity, but also GLI1-dependent cellular proliferation. Specifically, the edited GLI1, GLI1-701G, has a higher capacity to activate most of the transcriptional targets tested and is less susceptible to inhibition by the negative regulator of HH signaling suppressor of fused. However, the Dyrk1a kinase, implicated in cellular proliferation, is more effective in increasing the transcriptional activity of the non-edited GLI1. Finally, introduction of GLI1-701G into medulloblastoma cells confers a smaller increase in cellular growth relative to GLI1. In conclusion, our findings indicate that RNA editing of GLI1 is a regulatory mechanism that modulates the output of the HH signaling pathway.

Rhabdomyosarcoma (RMS) is the most frequent soft-tissue sarcoma in children. Embryonal rhabdomyosarcoma (E-RMS) represents the most common RMS subtype, but the molecular events driving this tumor are still largely unknown. The hedgehog (HH) pathway, a major signal transduction cascade, is linked with many cancers, including RMS. As we previously have detected loss of heterozygosity of PTCH1 in E-RMS, we now examined 8 E-RMS tumor samples and 5 E-RMS cell lines for the presence of PTCH1 mutations, but none was detected. However, in the E-RMS cell lines, a variable pattern of up-regulated expression of certain HH signaling target genes, including HHIP, PTCH1, SFRP1, and GLI1, was observed. Moreover, treatment with the small molecule HH signaling inhibitors cyclopamine and GANT61 inhibited cell proliferation in all E-RMS cell lines analyzed. Interestingly, GANT61 was more effective, and this was accompanied by increased apoptosis, while cyclopamine promoted necrotic events. Specific knockdown of SMO had no effect on the proliferation of E-RMS cells, indicating the presence of an SMO-independent HH signaling pathway in the E-RMS cell lines. Furthermore, in an in vivo xenograft model, tumor growth was significantly reduced by GANT61 treatment of E-RMS cells. Additionally, siRNA experiments provided evidence that inhibition of GLI1 or GLI3 but not GLI2 was sufficient to reduce proliferation of these cell lines. As GANT61 is known to block GLI1/GLI2 transcriptional activity, the inhibition of E-RMS growth by GANT61 is likely to be mediated through GLI1. In conclusion, our findings implicate that GLI1 could constitute an effective therapeutic target in pediatric E-RMS.

The Suppressor of Fused (SUFU) protein plays an essential role in the Hedgehog (HH) signaling pathway, by regulation of the GLI transcription factors. Two major isoforms of human SUFU are known, a full-length (SUFU-FL) and a carboxy-terminal truncated (SUFU- ΔC) variant. Even though SUFU- ΔC is expressed at an equivalent level as SUFU-FL in certain tissues, the function of SUFU-ΔC and its impact on HH signal transduction is still unclear. In two cell lines from rhabdomyosarcoma, a tumor type associated with deregulated HH signaling, SUFU-ΔC mRNA was expressed at comparable levels as SUFU-FL mRNA, but at the protein level only low amounts of SUFU-ΔC were detectable. Heterologous expression provided support to the notion that the SUFU-ΔC protein is less stable compared to SUFU-FL. Despite this, biochemical analysis revealed that SUFU-ΔC could repress GLI2 and GLI1ΔN, but not GLI1FL, transcriptional activity to the same extent as SUFU-FL. Moreover, under conditions of activated HH signaling SUFU-ΔC was more effective than SUFU-FL in inhibiting GLI1ΔN. Importantly, co-expression with GLI1FL indicated that SUFU-ΔC but not SUFU-FL reduced the protein levels of GLI1FL. Additionally, confocal microscopy revealed a co-localization of GLI1FL with SUFU-ΔC but not SUFU-FL in aggregate structures. Moreover, specific siRNA mediated knock-down of SUFU-ΔC resulted in up-regulation of the protein levels of GLI1FL and the HH signaling target genes PTCH1 and HHIP. Our results are therefore suggesting the presence of novel regulatory controls in the HH signaling pathway, which are elicited by the distinct mechanism of action of the two alternative spliced SUFU proteins.