Cancer Letters

Cancer Letters

Volume 343, Issue 1, 1 February 2014, Pages 51-61
Cancer Letters

The GLI1 splice variant TGLI1 promotes glioblastoma angiogenesis and growth

https://doi.org/10.1016/j.canlet.2013.09.014Get rights and content

Highlights

  • TGLI1 transcripts and encoded protein were highly expressed in approximately half of the GBM specimens we have analyzed.

  • TGLI1-expressing GBM xenografts grow larger and present with greater tumor angiogenesis than GLI1-expressing GBM tumors.

  • The heparanase-1 (HPA1) gene is a novel transcriptional target of TGLI1.

  • TGLI1–associated upregulation of HPA1 and VEGF-A contributes to the excessive vascularity of GBM.

  • The study helps define TGLI1 as a novel mediator of growth and angiogenesis of GBM.

Abstract

We investigated truncated glioma-associated oncogene homolog 1 (TGLI1) that behaves as gain-of-function GLI1 and promotes tumor cell migration and invasion. Herein, we report that TGLI1 had a higher propensity than GLI1 to enhance glioblastoma angiogenesis and growth, both in vivo and in vitro. TGLI1 has gained the ability to enhance expression of pro-angiogenic heparanase. In patient glioblastomas, TGLI1 levels are correlated with heparanase expression. Together, we report that TGLI1 is a novel mediator of glioblastoma angiogenesis and that heparanase is a novel transcriptional target of TGLI1, shedding new light on the molecular pathways that support tumor angiogenesis and aggressive growth.

Introduction

The Hedgehog pathway is important for embryonic development [1], [2] and tumorigenesis [3], [4], [5]. It is also one of the most dysregulated pathways in human cancers and is considered to be an important target of anti-cancer therapy [6], [7]. The Hedgehog pathway is highly complex and can be activated following the binding of Sonic hedgehog (Shh) to its receptor patched, a repressor of a 7-transmembrane receptor smoothened, SMO. Shh’s binding to PTCH derepresses SMO which, in turn, activates nuclear import of GLI1. Nuclear GLI1 binds to the GLI1-binding element in GLI1-regulated genes, leading to their activation [8], [9].

The GLI family of zinc finger transcription factors constitutes the terminal effectors of the Shh pathway [10], [11]. Although the human GLI1 gene was initially identified as an amplified gene in glioblastoma (GBM) cells [12], GLI1 gene amplification was later found to be rare in GBM and some other cancer types [13], [14], [15]. Somatic mutations in the GLI1 gene have never been reported in any cell or tumor type. Very recently, the human GLI1 gene transcript was found to be alternatively spliced to yield two shorter isoforms, namely, GLI1ΔN identified in 2008 [16], and TGLI1 identified in our laboratory in 2009 [17]. GLI1ΔN contains a large deletion of 128 codons, lacks two functional domains and behaves as a weak GLI1 transcription factor [16]. The TGLI1 variant is uniquely distinct from the other two previously reported GLI1 variants in that it contains a small in-frame deletion of 123 bases (41 codons) spanning the entire exon 3 along with part of exon 4 of GLI1 and preserves functional domains of GLI1 and the ability to undergo nuclear import and to activate GLI1-targeted genes [17].

Additional evidence indicates that TGLI1 is highly expressed in GBM and breast cancer, but not detected in normal brain and mammary tissues [17], [18]. TGLI1 has gained the ability to regulate genes that are not targeted by GLI1, including CD24 [17]. More recently, our group further showed that TGLI1 transcriptionally activates expression of vascular endothelial growth factor-A (VEGF-A) and VEGFR2 genes in breast cancer and medulloblastoma cells [18], [19]. Using GBM and breast cancer models, we have shown that TGLI1, but not GLI1, enhances the propensity of cancer cells to migrate and invade [17], [18]. However, TGLI1 functionality relative to GLI1 remains poorly understood. To address this knowledge gap, we undertook the current study aiming to gain new insights into the relevance and role of TGLI1 in GBM, the most common brain cancer in adults and one of the deadliest and most therapeutically intractable human malignancies [20]. Of note, our results fill critical knowledge gaps in the Hedgehog-GLI1 pathway and in GBM vascularization by defining TGLI1 as a novel mediator of neoangiogenesis in cancers and potentially other diseases.

Section snippets

Patient GBM tumors, directly passaged xenografts and cell lines

Patient GBM Tumors were provided by Dr. Matthias Gromeier at Duke University, as well as, purchased from US Biomax (Rockville, MD). Patient GBM-derived xenografts and D54MG GBM cells were provided by the Preston Robert Tisch Brain Tumor Center at Duke University. Direct xenografts were derived patient GBM tumors that were excised during 2008–2010 and maintained in athymic mice as subcutaneous flank xenografts. The direct xenografts recapitulate features of patient tumors since patient GBMs lose

TGLI1 protein is highly expressed in patient GBM samples, patient GBM-derived direct xenografts and has a higher propensity than GLI1 to enhance in vivo growth and vascularity of GBM xenografts

In our 2009 study [17], we reported TGLI1 transcript was expressed in the majority of GBM cell lines, but not in normal brain or other normal tissues. Here, we further showed that TGLI1 protein is readily detected in patient GBMs and patient GBM-derived xenografts (Fig. 1A). Previous reports have shown that direct xenografts recapitulate features of the patient’s original tumor [21]. As shown in Fig. 1A, TGLI1 (146 kD) and GLI1 (150 kD) proteins were separated by prolonged SDS-PAGE followed by WB

Discussion

Kinzler and Vogelstein first reported the identification of the human GLI1 gene in 1987 [12] and characterized it as a member of the Kruppel family of zinc finger proteins in 1988 [10]. In the 1988 milestone study, the authors detected a shorter transcript in a human embryonal carcinoma cell line using northern blotting with a GLI1 probe [10]. Following an examination of the GLI1 genomic sequence in the cells, the authors concluded that no somatic abnormality was present in the GLI1 gene and

Conflict of Interest

The authors declared no conflict of interest.

Acknowledgements

This study was supported by the NIH grant K01-CA118423, and W81XWH-11-1-0600 from the U.S. Department of Defense, the Pediatric Brain Tumor Foundation, the Beez Foundation and the Intramural Division of Surgical Sciences Dani P. Bolognesi, Ph.D. Award and Clarence Gardner, Ph.D. Award (to H.-W. L). We also thank Dr. Matthias Gromeier at Duke University for providing protein lysates of primary GBM specimens.

References (52)

  • S. Kanda et al.

    Sonic hedgehog induces capillary morphogenesis by endothelial cells through phosphoinositide 3-kinase

    The Journal of Biological Chemistry

    (2003)
  • A. Merchant et al.

    Gli1 regulates the proliferation and differentiation of HSCs and myeloid progenitors

    Blood

    (2010)
  • Z. Ji et al.

    Oncogenic KRAS activates hedgehog signaling pathway in pancreatic cancer cells

    The Journal of Biological Chemistry

    (2007)
  • Q. Fan et al.

    Requirement of TGFbeta signaling for SMO-mediated carcinogenesis

    The Journal of Biological Chemistry

    (2010)
  • T. Sheng et al.

    Regulation of Gli1 localization by the cAMP/protein kinase A signaling axis through a site near the nuclear localization signal

    The Journal of Biological Chemistry

    (2006)
  • Q. Cai et al.

    Protein kinase Cdelta negatively regulates hedgehog signaling by inhibition of Gli1 activity

    The Journal of Biological Chemistry

    (2009)
  • N. Dahmane et al.

    The Sonic Hedgehog-Gli pathway regulates dorsal brain growth and tumorigenesis

    Development (Cambridge, England)

    (2001)
  • G. Rao et al.

    Sonic hedgehog and insulin-like growth factor signaling synergize to induce medulloblastoma formation from nestin-expressing neural progenitors in mice

    Oncogene

    (2004)
  • G. Feldmann et al.

    An orally bioavailable small-molecule inhibitor of Hedgehog signaling inhibits tumor initiation and metastasis in pancreatic cancer

    Molecular Cancer Therapeutics

    (2008)
  • R. Carpenter, H.W. Lo, Hedgehog pathway and GLI1 Isoforms in human cancer, Discovery Medicine 13,...
  • K.W. Kinzler et al.

    The GLI gene encodes a nuclear protein which binds specific sequences in the human genome

    Molecular and Cellular Biology

    (1990)
  • J.W. Yoon et al.

    Defining a role for Sonic hedgehog pathway activation in desmoplastic medulloblastoma by identifying GLI1 target genes

    International Journal of Cancer

    (2009)
  • K.W. Kinzler et al.

    The GLI gene is a member of the Kruppel family of zinc finger proteins

    Nature

    (1988)
  • H. Zhu et al.

    The human glioma-associated oncogene homolog 1 (GLI1) family of transcription factors in gene regulation and diseases

    Current Genomics

    (2010)
  • K.W. Kinzler et al.

    Identification of an amplified, highly expressed gene in a human glioma

    Science

    (1987)
  • A. Forus et al.

    Mapping of amplification units in the q13–14 region of chromosome 12 in human sarcomas: some amplica do not include MDM2

    Cell Growth & Differentiation

    (1993)
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    These authors are contributed equally to this work.

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