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.
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.
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 (GLI1DeltaN). Analysis of the GLI1DeltaN 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, GLI1DeltaN 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 GLI1DeltaN 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 GLI1DeltaN. Interestingly, GLI1FL, in contrast to GLI1DeltaN, 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 GLI1DeltaN variant.
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 common soft-tissue childhood cancer. Deregulation of the Hedgehog (HH) signaling pathway, which is essential for proper embryonic development, has been implicated as a driving force for this cancer. In paper I, we have shown that sporadic human RMS have an overactive HH signaling pathway, and exhibit loss of heterozygosity of the two tumor suppressor genes, PTCH1 and/or SUFU, indicating a role for the promotion of rhabdomyoblastic tumor development. Moreover, we also identified a novel PTCH1 germ-line mutation in a patient suffering from the Nevoid basal cell carcinoma syndrome and also demonstrated that fetal rhabdomyoma (RM), a benign rhabdomyoblastic tumor, is a true component of this disorder. We analysed 12 RM/RMS tumors and 5 E-RMS cell lines for the presence of mutations in PTCH1, but none were detected. To evaluate the functional importance of the deregulated HH pathway in specifically in embryonal RMS (E-RMS), we analysed the E-RMS cell lines for their dependence on HH activity (Paper II). All cell lines expressed HH signaling components and displayed upregulated HH target gene expression. Inhibition of HH signaling activity by the use of two small molecule antagonists, cyclopamine and GANT61, led to reduced proliferation of the cell lines. The effect of GANT61 was specific as HH target gene expression was reduced, whereas cyclopamine gave off-target effects. GANT61 induced apoptosis, and significantly reduced tumor growth in an in vivo model. Knockdown of the GLI transcription factors, the ultimate effectors of HH signaling, revealed that GLI1 and GLI3 were important for cell proliferation, whereas GLI2 was dispensable. As GANT61 inhibits GLI1/GLI2 transcriptional activity, the inhibition of E-RMS growth is likely to be mediated through GLI1. The HH pathway is a very complex and highly regulated pathway, and the complexity is further increased by the presence of several isoforms of HH pathway components. In paper III, we identified and analysed a novel GLI1 splice variant, which is generated by skipping exons 2 and 3 and encodes an N-terminal truncated GLI1protein (GLI1ΔN). The expression of this variant is downregulated in tumor tissues compared to normal samples. GLI1ΔN was upregulated by HH signaling to the same extent as full-length GLI1, but generally had a weaker capacity to activate transcription. Another negative regulator of HH signaling, SUFU, has also several isoforms. In paper IV, we have analysed a C-terminal truncated variant, SUFU-ΔC, for its impact on HH signal transduction. SUFU-ΔC mRNA was expressed at similar levels as SUFU-FL, but on the protein level only very low amounts of SUFU-ΔC could be detected in ERMS cell lines. Although SUFU-ΔC was shown to be less stable than SUFU-FL, it possesses an equal ability to repress GLI2 and GLI1ΔN, but not GLI1FL transcriptional activity. Co-transfection of SUFU-ΔC and SUFU-FL resulted in increased protein expression levels relative to individual transfections, implying a protein stabilizing capacity of the SUFU variants. In conclusion, we have shown a major role for the HH signaling pathway in the establishment and maintenance of RMS tumors. The analyses of the GLI1 and SUFU splice variants reveal increased complexity, and suggest novel regulatory mechanisms in the HH signaling pathway, in RMS but also in other HH pathway-related tumors. LIST OF PUBLICATIONS This thesis is based on the following publications, which are referred to in the text by their roman numerals: I. Tostar U, Malm CJ, Meis-Kindblom JM, Kindblom LG, Toftgard R and Unden AB. Deregulation of the hedgehog signaling pathway: a possible role for the PTCH and SUFU genes in human rhabdomyoma and rhabdomyosarcoma development. J Pathol. 2006, 208; 17-25 II. Tostar U, Toftgard R, Zaphiropoulos PG and Shimokawa T. Reduction of human embryonal rhabdomyosarcoma tumor growth by inhibition of the hedgehog signaling pathway. Genes & Cancer, 2010, in press. III. Shimokawa T, Tostar U*, Lauth M*, Palaniswamy R*, Kasper M, Toftgard R and Zaphiopoulos PG. Novel human glioma-associated oncogene1 (GLI1) splice variants reveal distinct mechanisms in the terminal transduction of the Hedgehog signal. J. Biol. Chem. 2008, 283, 14345-54 IV. Tostar U, Finta C, Zaphiropoulos PG, Shimokawa T. A suppressor of fused carboxy terminal variant and its impact on hedgehog signal transduction. (2010) Manuscript. * These authors contributed equally Related publication: Lauth M, Bergstrom A, Shimokawa S, Tostar U, Jin Q, Fendrich F, Guerra C, Barbacid M, Toftgard R. DYRK1B-dependent autocrine-to-paracrine shift of Hedgehog signaling by mutant RAS. Nat Struct Mol Biol. 2010 Jun; 17(6):718-25
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