Mechanisms of hedgehog signaling activation in cancer development

Hedgehog (HH) signaling has an important role in many physiological processes, and deregulation of this pathway can result in a wide range of malignancies. The aim of this thesis is to identify and evaluate the role of various posttranscriptional mechanisms, including alternative splicing, RNA editing and antisense RNAs, associated with different key components of HH signaling. In PAPER l, we studied the mechanism of action and biological significance of the carboxy-terminal truncated variant of SUFU, SUFU-C, in rhabdomyosarcoma. Our investigations revealed that SUFU-ΔC mRNA was generally expressed at lower to comparable levels than SUFU-FL mRNA but the protein level of SUFU-ΔC was very low compared with SUFUFL. SUFUΔC could repress GLI2 and GLI1ΔN, but not GLI1FL, transcriptional activity to the same extent as SUFUFL. Co-expression of GLI1-FL with SUFU-ΔC in Hek293 cells indicated that SUFU-ΔC but not SUFU-FL reduced the protein levels of GLI1FL. Confocal microscopy revealed a co-localization of GLI1FL with SUFU-ΔC in aggregate structures. Moreover, knockdown of endogenous SUFU-ΔC with shRNA constructs in RMS13 cells caused an increase in GLI1FL protein levels and up-regulation of Hedgehog signaling targets (PTCH1-1B and PTCH1-1C). In PAPER ll, we studied the prevalence and impact of GLI1 RNA editing in modulating its oncogenic properties. GLI1 mRNA is edited at nucleotide 2179, which results in adenosine (A) to inosine (I) substitution, leading to a change from Arg to Gly at position 701. This editing event is prevalent (around 50%) in a number of human normal tissues. However, in tumors biopsies and tumor cell lines, the extent of GLI1 editing is reduced. SiRNA mediated knockdown revealed both ADAR isoforms (ADAR1 and ADAR2) are needed for GLI1 RNA editing. Edited GLI1 has a higher capacity to activate most of the transcriptional targets and is less susceptible to inhibition by SUFU. Moreover, the edited GLI1 is less responsive to activation by the Dual-specificity Tyrosine Phosphorylation-regulated Kinase 1A (Dyrk1A) compared with the non-edited GLI1. Finally, we showed that GLI1 editing affects GLI1-dependent cellular growth. In PAPER lll, we unveiled the regulatory mechanisms employed by non-coding transcripts overlapping the GLI1 gene, GLI1AS, in normal development and carcinogenesis. GLI1AS is positioned head-to-head with the gene encoding GLI1. The expression of the 885-nucleotide, three-exon GLI1AS RNA was consistently lower but concordant with GLI1. SiRNA knockdown of GLI1AS up-regulated GLI1 and increased cellular proliferation. Overexpression of GLI1AS resulted in down-regulation of GLI1 and the GLI1 target genes PTCH1 and PTCH2, and decreased cellular proliferation. ChIP assays indicate a local alteration of chromatin structure via H3K27me3 and H3K4me3 remodeling. We also observed a reduction in RNA polymerase II recruitment at the GLI1 promoter region upon overexpression of GLI1AS, which is in-line with the chromatin-remodeling phenomena. Additionally, GLI1 knockdown reduced GLI1AS, while GLI1 overexpression increased GLI1AS, demonstrating a regulatory feedback loop on GLI1/GLI1AS expression. In PAPER lV, we analyzed GLI1 target genes, using single molecule RNAseq, employing two complementary approaches, overexpression of GLI1 and edited GLI1 combined with GLI1 depletion using siRNAs. Gene ontology (GO) analysis revealed that GLI1 and edited GLI1 are involved in developmental and metabolic processes, cellular proliferation, KEGG pathways in cancer, basal cell carcinomas and thyroid cancer. Moreover, these candidate target genes were further filtered via the FANTOM5 dataset resulting in 29 targets. Validation of the 20 targets, which have a Spearman correlation > 0.1 with the FANTOM dataset, by qPCR indicated that 15 targets are down-regulated in knockdown experiments with Rh36 rhabdomyosarcoma cells. Additionally, 4 targets (FOXS1, SOSTDC1, LOC100507346 and SOX18) are also up-regulated in overexpression experiments with Rh36 cells. Moreover, knockdown of FOXS1 in Rh36 cells resulted in down-regulation of GLI1, highlighting a FOXS1/GLI1 regulatory loop. Finally, GLI1 knockdown and Smoothened agonist SAG treatment in HH signaling responsive Daoy medulloblastoma cells modulate the expression of 9 out of the 15 targets, including SOSTDC1 and FOXS1

RNA editing of the GLI1 transcription factor modulates the output of Hedgehog signaling

The Hedgehog (HH) signaling pathway has important roles in tumorigenesis and in embryonal patterning. The Gliomaassociated 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. Copyright © 2013 Landes Bioscience

Novel Mechanism of Action on Hedgehog Signaling by a Suppressor of Fused Carboxy Terminal Variant

<div><p>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 <em>PTCH1</em> and <em>HHIP</em>. 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.</p> </div

RNA editing of the GLI1 transcription factor modulates the output of Hedgehog signaling.

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

Dose-dependent SUFU repression of GLI1 activity in constitutively active <i>Ptch1−/−</i> MEFs.

<p>Different amounts of Myc-tagged SUFU-FL or SUFU-ΔC expression constructs were co-transfected with 50 ng GLI1 expression constructs (Panel <b>A</b>, GLI1FL; panel <b>B</b>, GLI1ΔN), 12xGLIBS-luc and pRL-SV reporter plasmids, and the luciferase activity was measured. Error bars indicate the standard deviation. Note the increased capacity of SUFU-ΔC relative to SUFU-FL in inhibiting GLI1ΔN transcriptional activity.</p

Knock-down of SUFU-ΔC expression in RMS13 cells results in increased GLI1FL protein and up-regulation of HH targets.

<p><b>A</b>, Real-time RT-PCR analysis of the relative expression of SUFU-ΔC and SUFU-FL following transfection of the sh4, sh5 or shEGFP construct. <b>B</b>, Western blot analysis of soluble protein fractions with a GLI1 antibody following transfection of the sh4, sh5 or shEGFP construct. A quantification of the protein levels is shown to the right. <b>C</b>, Real-time RT-PCR analysis of the relative expression of HHIP, GLI1, PTCH1-1B and PTCH1-1C following transfection of the sh4, sh5 or shEGFP construct. In both the A and C panels the expression levels relative to the EGFP control, normalized to the mean expression of the housekeeping genes RPLPO and TBP, are shown. Error bars indicate the standard deviation. *, Statistical significant, p<0,05, compared with control (Student’s <i>t</i> test). Note that the more effective the knock-down of SUFU-ΔC, the higher the increase of GLI1FL protein and the expression of HH signaling target genes.</p

Dose-dependent SUFU repression of GLI activity in NIH3T3 cells. A

<p>. Western blot analysis of extracts from NIH3T3 cells, transfected with expression constructs for the SUFU variants, and detected with a SUFU antibody. Due to the partial co-migration with endogenous mouse Sufu, the SUFU-FL expression in the right lane was calculated by subtracting from the total signal the mouse Sufu signal. This was determined based on the Sufu expression in the left lane following normalization with the tubulin internal control. Note that the quantitation revealed that the level of SUFU-FL (right lane, upper protein band) is 188% the level of SUFU-ΔC (middle lane, lower protein band). <b>B–E</b>, Different amounts of Myc-tagged SUFU-FL or SUFU-ΔC expression constructs were co-transfected with 50 ng GLI expression constructs (Panel <b>B</b>, GLI1FL; panel <b>C</b>, GLI1ΔN; panel <b>D</b>, GLI2FL; panel <b>E</b>, GLI2ΔN), 12xGLIBS-luc and pRL-SV reporter plasmids, and the luciferase activity was measured. Error bars indicate the standard deviation.</p

Dose-dependent SUFU repression of GLI activity in <i>Sufu−/−</i> MEFs.

<p>Different amounts of SUFU-FL or SUFU-ΔC FLAG-tagged expression constructs were co-transfected with 8xGLIBS-luc and pRL-SV reporter plasmids, and the luciferase activity was measured. Error bars indicate the standard deviation. NS, non-specific; co-transfection with 8xmutatedGLIBS-luc.</p