Mechanisms of hedgehog signaling activation in cancer development

Abstract

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.