GLI2-mediated melanoma invasion and metastasis.
BACKGROUND
The transforming growth factor-beta (TGF-beta) 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-beta 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 mm(2), difference = 1.88 mm(2), 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-beta-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-beta vs shCtrl + TGF-beta, 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.


CONCLUSIONS
GLI2 was directly involved in driving melanoma invasion and metastasis in this preclinical study.
Alexaki
Vasileia-Ismini
VI
Curie Institute, INSERM U1021/CNRS UMR3347, University Center, Orsay Cedex, France.
Javelaud
Delphine
D
Van Kempen
Leon C L
LC
Mohammad
Khalid S
KS
Dennler
Sylviane
S
Luciani
Flavie
F
Hoek
Keith S
KS
Juàrez
Patricia
P
Goydos
James S
JS
Fournier
Pierrick J
PJ
Sibon
Claire
C
Bertolotto
Corine
C
Verrecchia
Franck
F
Saule
Simon
S
Delmas
Veronique
V
Ballotti
Robert
R
Larue
Lionel
L
Saiag
Philippe
P
Guise
Theresa A
TA
Mauviel
Alain
A
D000818:Animals; D015153:Blotting, Western; D001859:Bone Neoplasms / Q000378:metabolism* / Q000556:secondary*; D015820:Cadherins / Q000378:metabolism; D045744:Cell Line, Tumor; D003094:Collagen; D004338:Drug Combinations; D015972:Gene Expression Regulation, Neoplastic; D053823:Hedgehog Proteins / Q000378:metabolism; D006801:Humans; D016867:Immunocompromised Host; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000378:metabolism*; D007797:Laminin; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D008545:Melanoma / Q000378:metabolism* / Q000473:pathology* / Q000556:secondary; D051379:Mice; D009361:Neoplasm Invasiveness; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011509:Proteoglycans; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction; D012878:Skin Neoplasms / Q000378:metabolism* / Q000473:pathology*; D015854:Up-Regulation; D000075746:Zinc Finger Protein Gli2
D015820:Cadherins; D004338:Drug Combinations; C082593:GLI2 protein, human; D053823:Hedgehog Proteins; D051741:Kruppel-Like Transcription Factors; D007797:Laminin; D009687:Nuclear Proteins; D011509:Proteoglycans; D012333:RNA, Messenger; C509239:SHH protein, human; D000075746:Zinc Finger Protein Gli2; C059485:matrigel; D003094:Collagen; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9