GLI1 modulation of the output of hedgehog signaling in human cancer
Author: Diao, Yumei
Date: 2016-12-09
Location: Red Seminar Room, 6th floor, Novum, Huddinge
Time: 09.30
Department: Inst för biovetenskaper och näringslära / Dept of Biosciences and Nutrition
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Thesis (6.526Mb)
Abstract
The Hedgehog (HH) signaling pathway has critical roles in embryonic development and tumorigenesis. Aberrant activation of HH signaling has been known to be involved in several types of malignant tumors. GLI1 is a transcription factor that acts not only as the terminal effector but also represents a pathway target gene, amplifying the HH signal. GLI1 expression/activity is modulated at different levels of gene regulation. The aim of the thesis is to explore the functional alterations in GLI1 transcriptional activity and further investigate the consequences on the output of HH signaling.
Paper I. We discovered an antisense transcript, which is a long non-coding RNA located head-to-head with the gene encoding GLI1, and termed it GLI1 antisense (GLI1AS). The expression of GLI1AS in cancer cells was concordant with GLI1 levels. GLI1AS knockdown upregulated GLI1, increased cellular proliferation and tumor growth in a xenograft model system. GLI1AS overexpression decreased the levels of GLI1, its target genes and cellular proliferation. Additionally, we demonstrated that GLI1 knockdown reduced GLI1AS, while GLI1 overexpression increased GLI1AS, highlighting a regulatory feedback loop on GLI1/GLI1AS expression. GLI1AS acts by modifying the chromatin landscape and reduces the recruitment of RNA polymerase II at the GLI1/GLI1AS locus.
Paper II. We examined the possible interplay of S6K1 and GLI1 signaling in neuroblastoma. mTOR/S6K1 signaling is known to have a role in the development of this disease and recent evidence also implicates the HH pathway. In this paper, it is shown that knockdown of either S6K1 or GLI1 reduces the cellular proliferation of neuroblastoma cells. However, there is little effect of S6K1 on the expression of GLI1 mRNA and protein and on the capacity of GLI1 to activate target genes. No detectable phosphorylation of GLI1 is observed prior or following S6K1 knockdown. Moreover, no additional growth inhibitory effects were detected when PI3K/mTOR and GLI signaling inhibitors were combined. Our results demonstrate that the impact of S6K1 kinase on neuroblastoma cells is not mediated through modulation of GLI1 expression/activity.
Paper III. We investigated HH signaling activation in breast cancer and the contribution of GLI1 to tamoxifen resistance. Our results showed that GLI1 expression was higher in tamoxifen resistant compared to sensitive cells. Tamoxifen resistant cells had stronger ERα transcriptional activity relative to sensitive cells, even though the ERα expression was similar in both cell types. Knockdown of GLI1 attenuated cell proliferation and reduced ERα transcriptional activity in both resistant and sensitive cells, irrespective of estrogen stimulation. Moreover, a positive correlation between GLI1 and ERα expression was identified in breast cancer samples. High GLI1 expression predicted worse distant metastasis-free survival in breast cancer patients. These data suggest that the HH pathway may be a new candidate for therapeutic targeting and prognosis in ERα-positive breast cancer.
Paper IV. We explored the common and differential target genes of GLI1 and edited GLI1, using single molecule RNA sequencing (RNA-seq), via overexpression and siRNA-mediated depletion approaches in rhabdomyosarcoma Rh36 cells. 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. 37 genes were differentially upregulated by edited GLI1, while 29 common target genes, including FOXS1, SOSTDC1 and SOX18, were identified by RNA-seq combined with correlation analysis to the FANTOM5 dataset. SOSTDC1 and FOXS1 expression was also modulated in HH signaling responsive medulloblastoma Daoy cells by GLI1 knockdown and Smoothened agonist (SAG) treatment. Reciprocally, GLI1 was downregulated in both Rh36 and Daoy cells following FOXS1 knockdown, highlighting a FOXS1/GLI1 regulatory loop.
Paper I. We discovered an antisense transcript, which is a long non-coding RNA located head-to-head with the gene encoding GLI1, and termed it GLI1 antisense (GLI1AS). The expression of GLI1AS in cancer cells was concordant with GLI1 levels. GLI1AS knockdown upregulated GLI1, increased cellular proliferation and tumor growth in a xenograft model system. GLI1AS overexpression decreased the levels of GLI1, its target genes and cellular proliferation. Additionally, we demonstrated that GLI1 knockdown reduced GLI1AS, while GLI1 overexpression increased GLI1AS, highlighting a regulatory feedback loop on GLI1/GLI1AS expression. GLI1AS acts by modifying the chromatin landscape and reduces the recruitment of RNA polymerase II at the GLI1/GLI1AS locus.
Paper II. We examined the possible interplay of S6K1 and GLI1 signaling in neuroblastoma. mTOR/S6K1 signaling is known to have a role in the development of this disease and recent evidence also implicates the HH pathway. In this paper, it is shown that knockdown of either S6K1 or GLI1 reduces the cellular proliferation of neuroblastoma cells. However, there is little effect of S6K1 on the expression of GLI1 mRNA and protein and on the capacity of GLI1 to activate target genes. No detectable phosphorylation of GLI1 is observed prior or following S6K1 knockdown. Moreover, no additional growth inhibitory effects were detected when PI3K/mTOR and GLI signaling inhibitors were combined. Our results demonstrate that the impact of S6K1 kinase on neuroblastoma cells is not mediated through modulation of GLI1 expression/activity.
Paper III. We investigated HH signaling activation in breast cancer and the contribution of GLI1 to tamoxifen resistance. Our results showed that GLI1 expression was higher in tamoxifen resistant compared to sensitive cells. Tamoxifen resistant cells had stronger ERα transcriptional activity relative to sensitive cells, even though the ERα expression was similar in both cell types. Knockdown of GLI1 attenuated cell proliferation and reduced ERα transcriptional activity in both resistant and sensitive cells, irrespective of estrogen stimulation. Moreover, a positive correlation between GLI1 and ERα expression was identified in breast cancer samples. High GLI1 expression predicted worse distant metastasis-free survival in breast cancer patients. These data suggest that the HH pathway may be a new candidate for therapeutic targeting and prognosis in ERα-positive breast cancer.
Paper IV. We explored the common and differential target genes of GLI1 and edited GLI1, using single molecule RNA sequencing (RNA-seq), via overexpression and siRNA-mediated depletion approaches in rhabdomyosarcoma Rh36 cells. 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. 37 genes were differentially upregulated by edited GLI1, while 29 common target genes, including FOXS1, SOSTDC1 and SOX18, were identified by RNA-seq combined with correlation analysis to the FANTOM5 dataset. SOSTDC1 and FOXS1 expression was also modulated in HH signaling responsive medulloblastoma Daoy cells by GLI1 knockdown and Smoothened agonist (SAG) treatment. Reciprocally, GLI1 was downregulated in both Rh36 and Daoy cells following FOXS1 knockdown, highlighting a FOXS1/GLI1 regulatory loop.
List of papers:
I. Villegas VE, Rahman MF, Fernandez-Barrena MG, Diao Y, Liapi E, Sonkoly E, Ståhle M, Pivarcsi A, Annaratone L, Sapino A, Ramírez Clavijo S, Bürglin TR, Shimokawa T, Ramachandran S, Kapranov P, Fernandez-Zapico ME, Zaphiropoulos PG. Identification of novel non-coding RNA-based negative feedback regulating the expression of the oncogenic transcription factor GLI1. Mol Oncol. 2014 Jul;8(5):912-26.
Fulltext (DOI)
Pubmed
View record in Web of Science®
II. Diao Y, Rahman MF, Villegas VE, Wickström M, Johnsen JI, Zaphiropoulos PG. The impact of S6K1 kinase on neuroblastoma cell proliferation is independent of GLI1 signaling. BMC Cancer. 2014 Aug 18;14:600.
Fulltext (DOI)
Pubmed
View record in Web of Science®
III. Diao Y, Azatyan A, Rahman MF, Zhao C, Zhu J, Dahlman-Wright K, Zaphiropoulos PG. Blockade of the Hedgehog pathway downregulates estrogen receptor alpha signaling in breast cancer cells. Oncotarget. 2016.
Fulltext (DOI)
Pubmed
IV. Rahman MF, Diao Y, Vyatkin Y, Azatyan A, St.Laurent G, Kapranov P, Zaphiropoulos PG. Global analysis of GLI1 and RNA-edited GLI1 target genes. [Manuscript]
I. Villegas VE, Rahman MF, Fernandez-Barrena MG, Diao Y, Liapi E, Sonkoly E, Ståhle M, Pivarcsi A, Annaratone L, Sapino A, Ramírez Clavijo S, Bürglin TR, Shimokawa T, Ramachandran S, Kapranov P, Fernandez-Zapico ME, Zaphiropoulos PG. Identification of novel non-coding RNA-based negative feedback regulating the expression of the oncogenic transcription factor GLI1. Mol Oncol. 2014 Jul;8(5):912-26.
Fulltext (DOI)
Pubmed
View record in Web of Science®
II. Diao Y, Rahman MF, Villegas VE, Wickström M, Johnsen JI, Zaphiropoulos PG. The impact of S6K1 kinase on neuroblastoma cell proliferation is independent of GLI1 signaling. BMC Cancer. 2014 Aug 18;14:600.
Fulltext (DOI)
Pubmed
View record in Web of Science®
III. Diao Y, Azatyan A, Rahman MF, Zhao C, Zhu J, Dahlman-Wright K, Zaphiropoulos PG. Blockade of the Hedgehog pathway downregulates estrogen receptor alpha signaling in breast cancer cells. Oncotarget. 2016.
Fulltext (DOI)
Pubmed
IV. Rahman MF, Diao Y, Vyatkin Y, Azatyan A, St.Laurent G, Kapranov P, Zaphiropoulos PG. Global analysis of GLI1 and RNA-edited GLI1 target genes. [Manuscript]
Institution: Karolinska Institutet
Supervisor: Zaphiropoulos, Peter
Issue date: 2016-11-11
Rights:
Publication year: 2016
ISBN: 978-91-7676-467-1
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