GLI2-mediated melanoma invasion and metastasis

Vasileia Ismini Alexaki, Delphine Javelaud, Leon C L Van Kempen, Khalid Mohammad, Sylviane Dennler, Flavie Luciani, Keith S. Hoek, Patricia Jurez, James S. Goydos, Pierrick J. Fournier, Claire Sibon, Corine Bertolotto, Franck Verrecchia, Simon Saule, Veronique Delmas, Robert Ballotti, Lionel Larue, Philippe Saiag, Theresa Guise, Alain Mauviel

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Abstract

Background The transforming growth factor-β (TGF-β) pathway, which has both tumor suppressor and pro-oncogenic activities, is often constitutively active in melanoma and is a marker of poor prognosis. Recently, we identified GLI2, a mediator of the hedgehog pathway, as a transcriptional target of TGF-β signaling. Methods We used real-time reverse transcription-polymerase chain reaction (RT-PCR) and western blotting to determine GLI2 expression in human melanoma cell lines and subsequently classified them as GLI2high or as GLI2low according to their relative GLI2 mRNA and protein expression levels. GLI2 expression was reduced in a GLI2high cell line with lentiviral expression of short hairpin RNA targeting GLI2. We assessed the role of GLI2 in melanoma cell invasiveness in Matrigel assays. We measured secretion of matrix metalloproteinase (MMP)-2 and MMP-9 by gelatin zymography and expression of E-cadherin by western blotting and RT-PCR. The role of GLI2 in development of bone metastases was determined following intracardiac injection of melanoma cells in immunocompromised mice (n = 5-13). Human melanoma samples (n = 79) at various stages of disease progression were analyzed for GLI2 and E-cadherin expression by immunohistochemistry, in situ hybridization, or RT-PCR. All statistical tests were two-sided. Results Among melanoma cell lines, increased GLI2 expression was associated with loss of E-cadherin expression and with increased capacity to invade Matrigel and to form bone metastases in mice (mean osteolytic tumor area: GLI2high vs GLI2low, 2.81 vs 0.93 mm2, difference = 1.88 mm2, 95% confidence interval [CI] = 1.16 to 2.60, P <. 001). Reduction of GLI2 expression in melanoma cells that had expressed high levels of GLI2 substantially inhibited both basal and TGF-β-induced cell migration, invasion (mean number of Matrigel invading cells: shGLI2 vs shCtrl (control), 52.6 vs 100, difference = 47.4, 95% CI = 37.0 to 57.8, P =. 024; for shGLI2 + TGF-β vs shCtrl + TGF-β, 31.0 vs 161.9, difference =-130.9, 95% CI =-96.2 to-165.5, P =. 002), and MMP secretion in vitro and the development of experimental bone metastases in mice. Within human melanoma lesions, GLI2 expression was heterogeneous, associated with tumor regions in which E-cadherin was lost and increased in the most aggressive tumors.ConclusionGLI2 was directly involved in driving melanoma invasion and metastasis in this preclinical study.

Original languageEnglish
Pages (from-to)1148-1159
Number of pages12
JournalJournal of the National Cancer Institute
Volume102
Issue number15
DOIs
StatePublished - Aug 4 2010

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Melanoma
Neoplasm Metastasis
Transforming Growth Factors
Cadherins
Reverse Transcription
Bone Development
Confidence Intervals
Cell Line
Polymerase Chain Reaction
Neoplasms
Western Blotting
Hedgehogs
Matrix Metalloproteinase 2
Matrix Metalloproteinase 9
Gelatin
Matrix Metalloproteinases
Small Interfering RNA
Cell Movement
In Situ Hybridization
Disease Progression

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

Alexaki, V. I., Javelaud, D., Van Kempen, L. C. L., Mohammad, K., Dennler, S., Luciani, F., ... Mauviel, A. (2010). GLI2-mediated melanoma invasion and metastasis. Journal of the National Cancer Institute, 102(15), 1148-1159. https://doi.org/10.1093/jnci/djq257

GLI2-mediated melanoma invasion and metastasis. / Alexaki, Vasileia Ismini; Javelaud, Delphine; Van Kempen, Leon C L; Mohammad, Khalid; Dennler, Sylviane; Luciani, Flavie; Hoek, Keith S.; Jurez, Patricia; Goydos, James S.; Fournier, Pierrick J.; Sibon, Claire; Bertolotto, Corine; Verrecchia, Franck; Saule, Simon; Delmas, Veronique; Ballotti, Robert; Larue, Lionel; Saiag, Philippe; Guise, Theresa; Mauviel, Alain.

In: Journal of the National Cancer Institute, Vol. 102, No. 15, 04.08.2010, p. 1148-1159.

Research output: Contribution to journalArticle

Alexaki, VI, Javelaud, D, Van Kempen, LCL, Mohammad, K, Dennler, S, Luciani, F, Hoek, KS, Jurez, P, Goydos, JS, Fournier, PJ, Sibon, C, Bertolotto, C, Verrecchia, F, Saule, S, Delmas, V, Ballotti, R, Larue, L, Saiag, P, Guise, T & Mauviel, A 2010, 'GLI2-mediated melanoma invasion and metastasis', Journal of the National Cancer Institute, vol. 102, no. 15, pp. 1148-1159. https://doi.org/10.1093/jnci/djq257
Alexaki VI, Javelaud D, Van Kempen LCL, Mohammad K, Dennler S, Luciani F et al. GLI2-mediated melanoma invasion and metastasis. Journal of the National Cancer Institute. 2010 Aug 4;102(15):1148-1159. https://doi.org/10.1093/jnci/djq257
Alexaki, Vasileia Ismini ; Javelaud, Delphine ; Van Kempen, Leon C L ; Mohammad, Khalid ; Dennler, Sylviane ; Luciani, Flavie ; Hoek, Keith S. ; Jurez, Patricia ; Goydos, James S. ; Fournier, Pierrick J. ; Sibon, Claire ; Bertolotto, Corine ; Verrecchia, Franck ; Saule, Simon ; Delmas, Veronique ; Ballotti, Robert ; Larue, Lionel ; Saiag, Philippe ; Guise, Theresa ; Mauviel, Alain. / GLI2-mediated melanoma invasion and metastasis. In: Journal of the National Cancer Institute. 2010 ; Vol. 102, No. 15. pp. 1148-1159.
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abstract = "Background The transforming growth factor-β (TGF-β) pathway, which has both tumor suppressor and pro-oncogenic activities, is often constitutively active in melanoma and is a marker of poor prognosis. Recently, we identified GLI2, a mediator of the hedgehog pathway, as a transcriptional target of TGF-β signaling. Methods We used real-time reverse transcription-polymerase chain reaction (RT-PCR) and western blotting to determine GLI2 expression in human melanoma cell lines and subsequently classified them as GLI2high or as GLI2low according to their relative GLI2 mRNA and protein expression levels. GLI2 expression was reduced in a GLI2high cell line with lentiviral expression of short hairpin RNA targeting GLI2. We assessed the role of GLI2 in melanoma cell invasiveness in Matrigel assays. We measured secretion of matrix metalloproteinase (MMP)-2 and MMP-9 by gelatin zymography and expression of E-cadherin by western blotting and RT-PCR. The role of GLI2 in development of bone metastases was determined following intracardiac injection of melanoma cells in immunocompromised mice (n = 5-13). Human melanoma samples (n = 79) at various stages of disease progression were analyzed for GLI2 and E-cadherin expression by immunohistochemistry, in situ hybridization, or RT-PCR. All statistical tests were two-sided. Results Among melanoma cell lines, increased GLI2 expression was associated with loss of E-cadherin expression and with increased capacity to invade Matrigel and to form bone metastases in mice (mean osteolytic tumor area: GLI2high vs GLI2low, 2.81 vs 0.93 mm2, difference = 1.88 mm2, 95{\%} confidence interval [CI] = 1.16 to 2.60, P <. 001). Reduction of GLI2 expression in melanoma cells that had expressed high levels of GLI2 substantially inhibited both basal and TGF-β-induced cell migration, invasion (mean number of Matrigel invading cells: shGLI2 vs shCtrl (control), 52.6 vs 100, difference = 47.4, 95{\%} CI = 37.0 to 57.8, P =. 024; for shGLI2 + TGF-β vs shCtrl + TGF-β, 31.0 vs 161.9, difference =-130.9, 95{\%} CI =-96.2 to-165.5, P =. 002), and MMP secretion in vitro and the development of experimental bone metastases in mice. Within human melanoma lesions, GLI2 expression was heterogeneous, associated with tumor regions in which E-cadherin was lost and increased in the most aggressive tumors.ConclusionGLI2 was directly involved in driving melanoma invasion and metastasis in this preclinical study.",
author = "Alexaki, {Vasileia Ismini} and Delphine Javelaud and {Van Kempen}, {Leon C L} and Khalid Mohammad and Sylviane Dennler and Flavie Luciani and Hoek, {Keith S.} and Patricia Jurez and Goydos, {James S.} and Fournier, {Pierrick J.} and Claire Sibon and Corine Bertolotto and Franck Verrecchia and Simon Saule and Veronique Delmas and Robert Ballotti and Lionel Larue and Philippe Saiag and Theresa Guise and Alain Mauviel",
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TY - JOUR

T1 - GLI2-mediated melanoma invasion and metastasis

AU - Alexaki, Vasileia Ismini

AU - Javelaud, Delphine

AU - Van Kempen, Leon C L

AU - Mohammad, Khalid

AU - Dennler, Sylviane

AU - Luciani, Flavie

AU - Hoek, Keith S.

AU - Jurez, Patricia

AU - Goydos, James S.

AU - Fournier, Pierrick J.

AU - Sibon, Claire

AU - Bertolotto, Corine

AU - Verrecchia, Franck

AU - Saule, Simon

AU - Delmas, Veronique

AU - Ballotti, Robert

AU - Larue, Lionel

AU - Saiag, Philippe

AU - Guise, Theresa

AU - Mauviel, Alain

PY - 2010/8/4

Y1 - 2010/8/4

N2 - Background The transforming growth factor-β (TGF-β) pathway, which has both tumor suppressor and pro-oncogenic activities, is often constitutively active in melanoma and is a marker of poor prognosis. Recently, we identified GLI2, a mediator of the hedgehog pathway, as a transcriptional target of TGF-β signaling. Methods We used real-time reverse transcription-polymerase chain reaction (RT-PCR) and western blotting to determine GLI2 expression in human melanoma cell lines and subsequently classified them as GLI2high or as GLI2low according to their relative GLI2 mRNA and protein expression levels. GLI2 expression was reduced in a GLI2high cell line with lentiviral expression of short hairpin RNA targeting GLI2. We assessed the role of GLI2 in melanoma cell invasiveness in Matrigel assays. We measured secretion of matrix metalloproteinase (MMP)-2 and MMP-9 by gelatin zymography and expression of E-cadherin by western blotting and RT-PCR. The role of GLI2 in development of bone metastases was determined following intracardiac injection of melanoma cells in immunocompromised mice (n = 5-13). Human melanoma samples (n = 79) at various stages of disease progression were analyzed for GLI2 and E-cadherin expression by immunohistochemistry, in situ hybridization, or RT-PCR. All statistical tests were two-sided. Results Among melanoma cell lines, increased GLI2 expression was associated with loss of E-cadherin expression and with increased capacity to invade Matrigel and to form bone metastases in mice (mean osteolytic tumor area: GLI2high vs GLI2low, 2.81 vs 0.93 mm2, difference = 1.88 mm2, 95% confidence interval [CI] = 1.16 to 2.60, P <. 001). Reduction of GLI2 expression in melanoma cells that had expressed high levels of GLI2 substantially inhibited both basal and TGF-β-induced cell migration, invasion (mean number of Matrigel invading cells: shGLI2 vs shCtrl (control), 52.6 vs 100, difference = 47.4, 95% CI = 37.0 to 57.8, P =. 024; for shGLI2 + TGF-β vs shCtrl + TGF-β, 31.0 vs 161.9, difference =-130.9, 95% CI =-96.2 to-165.5, P =. 002), and MMP secretion in vitro and the development of experimental bone metastases in mice. Within human melanoma lesions, GLI2 expression was heterogeneous, associated with tumor regions in which E-cadherin was lost and increased in the most aggressive tumors.ConclusionGLI2 was directly involved in driving melanoma invasion and metastasis in this preclinical study.

AB - Background The transforming growth factor-β (TGF-β) pathway, which has both tumor suppressor and pro-oncogenic activities, is often constitutively active in melanoma and is a marker of poor prognosis. Recently, we identified GLI2, a mediator of the hedgehog pathway, as a transcriptional target of TGF-β signaling. Methods We used real-time reverse transcription-polymerase chain reaction (RT-PCR) and western blotting to determine GLI2 expression in human melanoma cell lines and subsequently classified them as GLI2high or as GLI2low according to their relative GLI2 mRNA and protein expression levels. GLI2 expression was reduced in a GLI2high cell line with lentiviral expression of short hairpin RNA targeting GLI2. We assessed the role of GLI2 in melanoma cell invasiveness in Matrigel assays. We measured secretion of matrix metalloproteinase (MMP)-2 and MMP-9 by gelatin zymography and expression of E-cadherin by western blotting and RT-PCR. The role of GLI2 in development of bone metastases was determined following intracardiac injection of melanoma cells in immunocompromised mice (n = 5-13). Human melanoma samples (n = 79) at various stages of disease progression were analyzed for GLI2 and E-cadherin expression by immunohistochemistry, in situ hybridization, or RT-PCR. All statistical tests were two-sided. Results Among melanoma cell lines, increased GLI2 expression was associated with loss of E-cadherin expression and with increased capacity to invade Matrigel and to form bone metastases in mice (mean osteolytic tumor area: GLI2high vs GLI2low, 2.81 vs 0.93 mm2, difference = 1.88 mm2, 95% confidence interval [CI] = 1.16 to 2.60, P <. 001). Reduction of GLI2 expression in melanoma cells that had expressed high levels of GLI2 substantially inhibited both basal and TGF-β-induced cell migration, invasion (mean number of Matrigel invading cells: shGLI2 vs shCtrl (control), 52.6 vs 100, difference = 47.4, 95% CI = 37.0 to 57.8, P =. 024; for shGLI2 + TGF-β vs shCtrl + TGF-β, 31.0 vs 161.9, difference =-130.9, 95% CI =-96.2 to-165.5, P =. 002), and MMP secretion in vitro and the development of experimental bone metastases in mice. Within human melanoma lesions, GLI2 expression was heterogeneous, associated with tumor regions in which E-cadherin was lost and increased in the most aggressive tumors.ConclusionGLI2 was directly involved in driving melanoma invasion and metastasis in this preclinical study.

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