Inhibition of ATF5 Sensitizes Glioblastoma Cells to the Cytotoxic Effects of TNF-Related Apoptosis Inducing-Ligand (TRAIL) in a CHOP-DR5 Dependent Manner
Markus D Siegelin, James M Angelastro, Lloyd A Greene. Columbia University College of Physicians and Surgeons, New York, NY; University of California, Davis, CA
Background: Glioblastoma, the most common primary brain tumor, is a devastating disease with a dismal prognosis. Recent research has shown that glioblastomas reveal high-expression of the Activating transcription factor 5 (ATF5), whereas non-neoplastic astrocytes and mature neuronal cells are devoid of ATF5 expression. Therefore, ATF5 represents a viable treatment target for glioblastoma. Here, we provide a novel mechanism of how interference with the ATF5 pathway induces a cellular stress response in glioblastoma cells and in turn can be utilized for a combinatorial treatment involving TRAIL.
Design: Glioblastoma cells (U87 and LN229) were transfected with a dominant negative form of ATF5 or the respective control plasmid. Subsequently, cells were treated with suboptimal concentrations of TRAIL. Cell death was analyzed by counting of apoptotic nuclei, using fluorescence microscopy. Western blots were performed to determine caspase cleavage (activation) and protein expression of CHOP and DR5.
Results: Glioblastoma cells treated with suboptimal dosages of TRAIL or a dominant-negative form of ATF5 (d/n-ATF5) reveal only minimal apoptosis induction after 24 hours of treatment. However, combining d/n-ATF5 with TRAIL causes nuclear fragmentation and cleavage (activation) of caspases in glioblastoma cell lines, indicative of rapid apoptosis. This combinatorial treatment approach appears independent of TP53 mutation status since both mutated and wild-type p53 cell lines responded to the combinatorial treatment regimen. Mechanistically, inhibition of ATF5 elicits an up-regulation of the stress response transcription factor, CHOP, coupled with an increase of DR5 protein levels. To clarify the role of CHOP in this pathway, CHOP protein levels were suppressed by a specific siRNA in glioma cells. Compared to glioma cells transfected with a non-targeting siRNA, cells with suppressed CHOP levels partially reversed the apoptotic effects of the combination treatment, consisting of TRAIL and d/n-ATF5. Mechanistically, siRNA mediated suppression of CHOP attenuated d/n-ATF5 driven DR5 up-regulation, suggesting that inhibition of ATF5 results in an enhancement of the extrinsic apoptotic cascade by elevating TRAIL receptors (DR5 levels).
Conclusions: Targeting the transcription factor ATF5 can be utilized to overcome TRAIL-resistance in glioblastoma cells.
Wednesday, March 6, 2013 1:00 PM
Poster Session VI # 239, Wednesday Afternoon