P14ARF inhibits human glioblastoma–induced angiogenesis by upregulating the expression of TIMP3
Abdessamad Zerrouqi, … , Daniel J. Brat, Erwin G. Van Meir
Abdessamad Zerrouqi, … , Daniel J. Brat, Erwin G. Van Meir
Published March 1, 2012
Citation Information: J Clin Invest. 2012;122(4):1283-1295. https://doi.org/10.1172/JCI38596.
View: Text | PDF
Research Article Oncology

P14ARF inhibits human glioblastoma–induced angiogenesis by upregulating the expression of TIMP3

Abstract

Malignant gliomas are the most common and the most lethal primary brain tumors in adults. Among malignant gliomas, 60%–80% show loss of P14ARF tumor suppressor activity due to somatic alterations of the INK4A/ARF genetic locus. The tumor suppressor activity of P14ARF is in part a result of its ability to prevent the degradation of P53 by binding to and sequestering HDM2. However, the subsequent finding of P14ARF loss in conjunction with TP53 gene loss in some tumors suggests the protein may have other P53-independent tumor suppressor functions. Here, we report what we believe to be a novel tumor suppressor function for P14ARF as an inhibitor of tumor-induced angiogenesis. We found that P14ARF mediates antiangiogenic effects by upregulating expression of tissue inhibitor of metalloproteinase–3 (TIMP3) in a P53-independent fashion. Mechanistically, this regulation occurred at the gene transcription level and was controlled by HDM2-SP1 interplay, where P14ARF relieved a dominant negative interaction of HDM2 with SP1. P14ARF-induced expression of TIMP3 inhibited endothelial cell migration and vessel formation in response to angiogenic stimuli produced by cancer cells. The discovery of this angiogenesis regulatory pathway may provide new insights into P53-independent P14ARF tumor-suppressive mechanisms that have implications for the development of novel therapies directed at tumors and other diseases characterized by vascular pathology.

Authors

Abdessamad Zerrouqi, Beata Pyrzynska, Maria Febbraio, Daniel J. Brat, Erwin G. Van Meir

×

Figure 5

The upregulation of TIMP3 gene expression by P14ARF is SP1 dependent.

Options: View larger image (or click on image) Download as PowerPoint
The upregulation of TIMP3 gene expression by P14ARF is SP1 dependent. ...
(A) The silencing of SP1 (siSP1) abrogates upregulation of TIMP3 mRNA (Northern) and protein (Western) expression by P14ARF in A5 cells. The noncontiguous lane of the composite blot separated by a thin white line was run on the same gel and exposed similarly. (B) Western blot showing the pharmacological inhibition of SP1 activity with mithramycin (Mith; 70 and 100 nM, 36 hours) prevents TIMP3 induction by P14ARF. (C) Luciferase reporter assay showing SP1 is required for TIMP3 promoter activation by P14ARF. A5 cells were transfected with a TIMP3 promoter–luciferase reporter and 24 hours later treated with siRNAs (siCtrl or siSP1). Twenty-four hours later, P14ARF expression was induced with dox with or without mithramycin (70 and 100 nM) for 36 hours. Cell lysates were then analyzed for luciferase activity. The values of firefly luciferase normalized to protein content are presented as the mean ± SD of triplicates; *P < 0.05, paired Student’s t test; n = 2. (D) ChIP assay demonstrating P14ARF-dependent binding of SP1 to the TIMP3 promoter. Chromatin fragments prepared from A5 cells with or without P14ARF expression were immunoprecipitated with rabbit anti–human SP1 (PEP2) or rabbit IgGs. Fw and Rev primers framing the SP1 binding sites in the TIMP3 promoter were used for PCR. The binding of rabbit IgG to the TIMP3 promoter served as negative control. Note: Dox treatment did not alter the basal level of SP1 binding in parental LN229-L16 cells, excluding nonspecific effects of dox. Three independent repeats (n = 3) gave similar results.