Abstract 4070: Mutant tristetraprolin enhances growth factor downregulation and inhibition of cell growth in malignant glioma

Abstract

Abstract Background: Tristetraprolin (TTP) negatively regulates short-lived mRNAs by binding to AU-rich elements in the 3′ untranslated region (3′UTR). A number of mRNA targets, including vascular endothelial growth factor (VEGF), and interleukin (IL)-8, play an important role in tumor progression. We have shown previously that TTP downregulates IL-8 and VEGF in malignant glioma (MG) cells via RNA destabilization. In primary glioblastoma tumors, TTP is heavily phosphorylated which may render the protein less active. Hypothesis: We postulate that the destabilizing effect of TTP on target growth factor mRNAs is held in check by hyperphosphorylation which leads to altered subcellular localization and protein stability. Methods: Wild-type TTP and two mutant forms in which select serines were exchanged with alanines were cloned with a Flag epitope into pTRE2 and pEGFP vectors. U251 Tet On MG cells were transfected with pTRE2–TTP plasmids to produce stable clones. TTP expression was induced using doxycycline. Cellular location was assessed by fluorescence microscopy and Western blotting. Protein stability was measured by assessment of TTP levels following cycloheximide treatment. VEGF and IL-8 mRNA and protein levels were measured by qRT-PCR and ELISA. For assessment of RNA half-lives, cells were treated with actinomycin D. Cell growth, proliferation and cell viability were assessed using biochemical assays, soft agar, and Trypan Blue method. Results: Both IL-8 and VEGF mRNA levels were significantly lowered in the mutant TTP clones compared to wild-type. Half-lives of these mRNAs were also shortened, and ELISA analysis showed a correspondingly greater decrease in protein expression. There was a marked impact on cellular phenotype with greater inhibition of proliferation and loss of cell viability. A Soft agar colony formation was decreased by five-fold in mutant clones versus wild-type and a greater than ten-fold decrease versus the parent cell line. With TNF-α, there was a significant shift of mutant TTP to the nuclear compartment compared to WT TTP. Treatment with cycloheximide revealed that the WT TTP was less stable compared to dephosphorylated mutant. Conclusion: Removal of key serine residues from TTP enhances its function as an RNA destabilizer of VEGF and IL-8 mRNs and as a negative regulator of tumor cell growth in MG. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4070.