GLI1 is a key downstream transcription effector of the Hedgehog (Hh) signaling pathway that is involved in promoting cell growth, differentiation and tissue patterning in embryonic development. GLI1 over-activation and its nuclear localization has also been linked to the increased aggressiveness of a number of cancers. It has previously been demonstrated that DYRK1A (dual-specificity tyrosine-regulated kinase 1A) can phosphorylate GLI1 and promote GLI1 nuclear localization and its transcriptional activity. Utilizing recombinant human GLI1 and DYRK1A proteins and phospho-peptide mass spectrometry, we demonstrated that GLI1 is phosphorylated by DYRK1A at Ser408, a phospho-site that falls within the putative nuclear localization sequence (NLS) of GLI1 suggesting a possible mechanistic role in modulating its translocation. Further, we showed that the Ser408 site on GLI1 was not phosphorylated in the presence of the selective DYRK1A inhibitor harmine. The data described herein provide the first identification of a DYRK1A-mediated site of phosphorylation on GLI1 within its NLS and may serve as a valuable mechanism for further understanding Hh signaling modulation.
Activation of the Hedgehog (Hh) pathway effector GLI1 is linked to tumorigenesis and invasiveness in a number of cancers, with targeting of GLI1 by small molecule antagonists shown to be effective. We profiled a collection of GLI antagonists possessing distinct mechanisms of action for efficacy in phenotypic models of inflammatory and non-inflammatory breast cancer (IBC and non-IBC) that we showed expressed varying levels of Hh pathway mediators. Compounds GANT61, HPI-1, and JK184 decreased cell proliferation, inhibited GLI1 mRNA expression and decreased the number of colonies formed in TN-IBC (SUM149) and TNBC (MDA-MB-231 and SUM159) cell lines. In addition, GANT61 and JK184 significantly down-regulated GLI1 targets that regulate cell cycle (cyclin D and E) and apoptosis (Bcl2). GANT61 reduced SUM149 spheroid growth and emboli formation, and in orthotopic SUM149 tumor models significantly decreased tumor growth. We successfully utilized phenotypic profiling to identify a subset of GLI1 antagonists that were prioritized for testing in in vivo models. Our results indicated that GLI1 activation in TN-IBC as in TNBC, plays a vital role in promoting cell proliferation, motility, tumor growth, and formation of tumor emboli.
The data presented in this article support the accompanying research article “Identification of a DYRK1A-mediated phosphorylation site within the nuclear localization sequence of the hedgehog transcription factor GLI1” (Ehe et al., 2017) [1]. Although it has been demonstrated that DYRK1A (dual-specificity tyrosine-regulated kinase 1A) can phosphorylate the hedgehog pathway transcription factor GLI1 (GLIoma-associated oncogene homolog 1) and promote its nuclear localization, the DYRK1A-mediated sites of phosphorylation on GLI1 involved were not fully known. This article details the mass spectrometry methods and resulting dataset for the peptides identified from GLI1 when incubated with DYRK1A under varying conditions. The data include details of sequence coverage and all phospho-peptides identified.
Introduction: The transcription factor, glioma-associated oncogene homolog 1 (GLI1) is a downstream marker for hedgehog (Hh) pathway activation. Recently, Hh-GLI has emerged as a central pathway target in several human cancers (breast, leukemia, lung etc.) with elevated GLI1 in breast cancer having been linked to poor survival outcomes. A number of GLI targeted inhibitors have been shown to inhibit Hh/GLI signaling with some having efficacy in tumor models. We have previously demonstrated that siRNA downregulation of GLI1 expression in inflammatory breast cancer reduced proliferation and migration[1]. Further, we previously demonstrated the feasibility of employing a high throughput (HT) approach to profile drugs in dose response in cell line panels [2]. In this study, we have taken a comprehensive approach to profile a panel of GLI antagonists (including GANTS, HPIs, ATO and JK184) in a range of breast cancer cell lines with varying GLI1 expression levels to identify those with effects on growth. Experiment procedure: to assess cell proliferation, we employed an automated quantitative high throughput (qHT) approach to profile compounds on three phenotypic subtypes of breast cancer cell line models: triple negative breast cancer (TNBC), inflammatory breast cancer, and Claudin low breast cancer cell lines. High-content imaging of nuclear count utilizing Hoechst staining was also used as an alternative measure of proliferation. Quantitative PCR and Western blotting was used to assess the effects of the inhibitors on the GLI1 expression. Results: A subset of GLI antagonists were identified as decreasing cell growth and viability in all the cell lines. Conclusion: Several agents showed efficacy in in vitro BC cancer models demonstrating that GLI inhibitors may be a valid therapeutic approach for targeting GLI-dependent BC cancers. [1] Z. Thomas, W. Gibson, J. Sexton, K. Aird, S. Ingram, A. Aldrich, H. Lyerly, G. Devi, K. Williams, Targeting GLI1 expression in human inflammatory breast cancer cells enhances apoptosis and attenuates migration. British Journal of Cancer 104 (2011) 1575-1586. [2] K.P. Williams, J.L. Allensworth, S.M. Ingram, G.R. Smith, A.J. Aldrich, J. Z Sexton, G. R Devi, Quantitative high-throughput efficacy profiling of approved oncology drugs in inflammatory breast cancer models of acquired drug resistance and re-sensitization. Cancer Lett. 337 (2013) 77-89. Funded in part by DOD/CDMRP IDEA W81XWH-13-1-0141 award (KPW); NIH U54CA156735 (KPW); Duke Cancer Institute - Cancer and Environment Initiative P3917733 sub-award (GRD); W81XWH-13-1-041 subcontract (GRD) and National Cancer Institute training grant T32CA009111 (SJS). Citation Format: Helen Oladapo, Jodie M. Fleming, Kezia Addo, Mike Tarpley, Ben Ehe, Shalonda Ingram, Scott Sauer, Gayathri Devi, Kevin P. Williams. Identification of GLI1 antagonists for breast cancer therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 680. doi:10.1158/1538-7445.AM2015-680
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