mTOR controls FLIPS translation and TRAIL sensitivity in glioblastoma multiforme cells

Abstract

The tumor-selective, proapoptotic, death receptor ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a mediator of antitumor drug activity and in itself is a promising agent for the treatment of human malignancies. Like many tumors, however, glioblastoma multiforme (GBM), the most fatal form of glioma, exhibits a range of TRAIL sensitivity, and only a small percentage of GBM tumors undergo TRAIL-induced apoptosis. We here show that TRAIL resistance in GBM is a consequence of overexpression of the short isoform of the caspase-8 inhibitor, c-FLICE inhibitory protein (FLIP(S)), and that FLIP(S) expression is in turn translationally enhanced by activation of the Akt-mammalian target of rapamycin (mTOR)-p70 S6 kinase 1 (S6K1) pathway. Conversely, pharmacologic or genetic inhibition of mTOR, or the mTOR target S6K1, suppresses polyribosomal accumulation of FLIP(S) mRNA, FLIP(S) protein expression, and TRAIL resistance. In archived material from 12 human GBM tumors, PTEN status was a predictor of activation of the Akt-mTOR-S6K1 pathway and of FLIP(S) levels, while in xenografted human GBM, activation status of the PTEN-Akt-mTOR pathway distinguished the tumors inherently sensitive to TRAIL from those which could be sensitized by the mTOR inhibitor rapamycin. These results define the mTOR pathway as a key limiter of tumor elimination by TRAIL-mediated mechanisms, provide a means by which the TRAIL-sensitive subset of GBM can be identified, and provide rationale for the combined use of TRAIL with mTOR inhibitors in the treatment of human cancers.

FIG. 1.

Identification of TRAIL-sensitive and TRAIL-resistant GBM cell lines. Human GBM cells were incubated with TRAIL (0 to 1,000 ng/ml; 24 h), stained with propidium iodide, and analyzed by flow cytometry for the percentage of cells having a <2N DNA content (apoptotic cells). Data shown are the means ± standard errors derived from three independent experiments. *, P < 0.05.

FIG. 2.

TRAIL-resistant GBM cells fail to activate caspase-8 and caspase-3 in response to TRAIL. TRAIL-sensitive (A) or TRAIL-resistant (B) GBM cells were incubated with TRAIL (800 ng/ml; 0 to 7 h) and assayed for procaspase-8 and procaspase-3 cleavage by Western blotting. Data presented are representative of three independent experiments.

FIG. 3.

TRAIL sensitivity correlates with FLIP protein expression in GBM cells. TRAIL-resistant (U373/A172) or TRAIL-sensitive (U87/U251) cells were analyzed for FLIP_L and FLIP_S expression by Western blotting. Values listed are the means derived from three independent experiments. *, P < 0.05.

FIG. 4.

Overexpression of FLIP_S, but not FLIP_L, suppresses TRAIL-induced apoptosis in GBM cells. TRAIL-sensitive (U87/U251) GBM cells were sham infected (CTRL) or stably infected with blank (pFB-Neo), FLIP_S- or FLIP_L-encoding constructs. Following selection, cells were either analyzed for FLIP_S and FLIP_L expression by Western blot analysis (middle panels) or were exposed to TRAIL (800 ng/ml; 24 h), incubated with propidium iodide and a fluorescein isothiocyanate-conjugated annexin V antibody, and analyzed by flow cytometry for the percentage of propidium iodide (+) (y axis)/annexin V(+) (x axis) apoptotic cells (bottom panels). Data shown are representative of three independent experiments. Top panels are graphic representations of the data in the bottom panels and from similar studies in U251 cells (not shown). Values listed are the means ± standard errors. *, P < 0.05.

FIG. 5.

Translational regulation of FLIP_S expression. TRAIL-sensitive (U87/U251) or TRAIL-resistant (U373/A172) cells were lysed, spiked with Drosophila RPL3 RNA (to normalize for equal isolation/loading), and analyzed by RT-PCR (A, top) or Northern blotting (A, bottom) for levels of FLIP_S, FLIP_L, GAPDH, and RPL3 mRNA. Lysed cells were also subjected to sucrose density gradient (5 to 70%) centrifugation, with subsequent RNA from fractions containing unassembled ribosomal subunits (fractions 1 to 6) or assembled polyribosomes (fractions 7 to 12) (B), spiked with RPL3 RNA and analyzed for FLIP_S, FLIP_L, and RPL3 content by Northern blotting (C). (D) Graphic representation of the percentage of total FLIP_S or FLIP_L mRNA found in the combined monosomal or polysomal fractions. Data shown are representative of three independent experiments. Values listed are the means ± standard errors. *, P < 0.05.

FIG. 6.

The Akt-mTOR pathway plays a role in the translational regulation of FLIP_S expression and TRAIL sensitivity. (A) U87 cells retrovirally infected with a 4HT-inducible Akt-encoding construct were incubated with 4HT (0 or 10 nM; 24 h), lysed, and subjected to an Akt in vitro kinase assay using GSK-3α/β as the substrate and endogenous GSK-3α/β protein to normalize for equal protein loading. (B to E) U87 cells infected with a 4HT-inducible Akt-encoding construct were incubated with 4HT (0 or 10 nM) or 4HT plus rapamycin (100 nM) for 30 min, after which TRAIL was added (800 ng/ml; 24 h). Cells were then lysed, spiked with Drosophila RPL3 RNA, and either analyzed by Western blotting for FLIP_L and FLIP_S expression (B); analyzed by Northern blotting for levels of FLIP_S, GAPDH, and RPL3 mRNA (C); or separated into monosomal and polysomal fractions, after which levels of FLIP_S, FLIP_L, GAPDH, and RPL3 mRNA in each fraction were assessed by Northern blot analysis (D). (E) A graphic representation of data derived in panel D, with FLIP_S values normalized to GAPDH RNA levels and compared to the FLIP_S/GAPDH RNA ratio in control cells. (F) The percentage of cells having a <2N DNA content (apoptotic cells) following TRAIL exposure (800 ng/ml; 24 h). Data shown are representative of three independent experiments. Values listed are the means ± standard errors (where shown). *, P < 0.05.

FIG. 7.

Overexpression of FLIP_S, but not FLIP_L, sensitizes Akt-overexpressing U87 cells to TRAIL-induced apoptosis. U87 cells infected with a 4HT-inducible Akt-encoding construct were incubated with 4HT (0 or 10 nM) for 30 min, after which either a scramble siRNA or an siRNA targeting FLIP_S or FLIP_L was added for 96 h. Cells were then either analyzed by Western blotting for FLIP_L and FLIP_S expression (A) or exposed to TRAIL (800 ng/ml; 24 h), incubated with propidium iodide, and a fluorescein isothiocyanage-conjugated annexin V antibody and analyzed by flow cytometry for the percentage of propidium iodide (+)/annexin V (+) apoptotic cells (B). (C) A graphic representation of the data derived in panel B. Data shown are representative of three independent experiments. Values listed are the means ± standard errors (where shown). *, P < 0.05.

FIG. 8.

Rapamycin reduces FLIP_S protein levels and sensitizes GBM cells to TRAIL-induced cell death. TRAIL-resistant (U373 and A172) GBM cells were incubated with rapamycin (0 or 100 nM; 30 min), after which the cells were exposed to TRAIL (0 or 800 ng/ml; 24 h). Cells were then either analyzed by Western blotting for FLIP_L and FLIP_S expression in the absence of TRAIL (A) or lysed, spiked with Drosophila RPL3 RNA, and analyzed for the effects of rapamycin (in the absence of TRAIL) on total levels of FLIP_S, FLIP_L, GAPDH, and RPL3 mRNA by Northern blot analysis (B, bottom panel) and quantitative RT-PCR (B, top panel). Cellular lysates were also separated into monosomal and polysomal fractions and spiked with Drosophila RPL3 RNA, after which RNA was isolated and levels of FLIP_S, FLIP_L, GAPDH, and RPL3 mRNA in each ribosomal fraction of control or rapamycin-treated U373 and A172 cells was assessed by Northern blot analysis (C). (D) A graphic representation of the data derived in panel C, with values expressed as the percentage of GAPDH, FLIP_S, or FLIP_L mRNA detected in the pooled monosomal or polysomal fractions. (E) Cells were stained with propidium iodide and analyzed by flow cytometry for the percentage of cells having <2N DNA content (apoptotic cells) following TRAIL exposure (800 ng/ml; 24 h). Data shown are representative of three independent experiments. All values listed are the means ± standard errors (where shown). *, P < 0.05.

FIG. 9.

Rapamycin sensitizes GBM cells to TRAIL-induced apoptosis through an mTOR-dependent mechanism. TRAIL-resistant (U373 and A172) GBM cells were infected with a blank retroviral construct or a construct encoding either an AU1-tagged KD or RR mTOR. Following selection, cells were lysed and expression of the AU1-tagged proteins of interest were verified by Western blotting (bottom panels). Cells were then incubated with 100 nM rapamycin and 800 ng/ml TRAIL for 24 h, after which the cells were stained with propidium iodide and analyzed by flow cytometry for the percentage of cells having <2N DNA content (apoptotic cells). The data shown are the means ± standard errors derived from three independent experiments. *, P < 0.05 in comparison to control cells; **, P < 0.05 in comparison to cells expressing either KD or RR mTOR constructs.

FIG. 10.

Differences in the activation of the S6K1 arm of the mTOR signaling pathway in TRAIL-sensitive versus TRAIL-resistant GBM cells. TRAIL-sensitive (U87 and U251) or TRAIL-resistant (U373 and A172) cells were analyzed for expression of phosphorylated (activated) Akt, phospho-S6K1, phospho-S6, 4E-BP1, eIF4E, and FLIP_S by Western blotting. Values listed are the means derived from three independent experiments. *, P < 0.05.

FIG. 11.

Genetic suppression of the S6K1 pathway reduces expression of FLIP_S protein and sensitizes GBM cells to TRAIL-induced apoptosis. TRAIL-resistant (U373 and A172) GBM cells were sham transfected, transiently transfected with either siRNA targeting S6K1 or a nonspecific scrambled siRNA (96 h), or stably transfected with an empty vector control (pcDNA3-Neo) or a construct encoding the eIF4E inhibitor 4E-BP1. Cells were then analyzed by Western blotting for levels of phospho-S6K1, phospho-S6 protein, 4E-BP1, and FLIP_S (A and B) or lysed, separated into monosomal and polysomal fractions, and spiked with Drosophila RPL3 RNA, after which RNA was isolated and analyzed for FLIP_S mRNA levels by quantitative RT-PCR (C, top) and Northern blot analysis (C, bottom). (D) Cells were stained with propidium iodide and analyzed by flow cytometry for the percentage of cells having <2N DNA content (apoptotic cells) after exposure to TRAIL (800 ng/ml; 24 h). Data shown are representative of three independent experiments. All values listed are the means ± standard errors (where shown). In panel D, polysomal FLIP_S mRNA levels were normalized to GAPDH mRNA levels and then to FLIP/GAPDH mRNA ratios in control (CTRL) cells for each cell line to generate the mean ± standard error values presented. *, P < 0.05.

FIG. 12.

Overexpression of S6K1 enhances FLIP_S expression and confers resistance to TRAIL-induced apoptosis. TRAIL-sensitive (U87 and U251) cells were either sham transfected or stably transfected with an empty vector (pCAN-neo or pRK7-neo) or constructs encoding either wild-type S6K1, wild-type eIF4E, or both. Following selection, cells were analyzed by Western blotting for levels of phospho-S6K1, phospho-S6 protein, eIF4E, and FLIP_S (A and B), stained with propidium iodide, and analyzed by flow cytometry for the percentage of cells having <2N DNA content (apoptotic cells) 24 h after exposure to 800 ng/ml TRAIL (F); or lysed, separated into monosomal and polysomal fractions, and spiked with Drosophila RPL3 RNA, after which RNA was isolated and analyzed for FLIP_S mRNA levels by quantitative RT-PCR (E, top) and Northern blot analysis (E, bottom). TRAIL-sensitive U87 and U251 cells were also sham infected with empty control vectors (pBabe-puro or pMV7-neo) or a construct encoding wild-type S6K1, an inactive S6K1 (K100R), a rapamycin-resistant S6K1, a wild-type eIF4E, or an inactive eIF4E (W73A). Following selection, cells were analyzed for levels of S6K1 protein, phospho-S6 protein, eIF4E, and FLIP_S/FLIP_L expression by Western blot analysis (C and D). All values are the means ± standard errors (where shown) derived from three independent experiments. *, P < 0.05.

FIG. 13.

Analysis of the linkage between PTEN, Akt, pS6K, FLIP_S, and TRAIL sensitivity in primary human GBM and GBM cell lines. Cellular lysates from surgically resected primary GBM (A) or xenografted human GBM (B) of known PTEN status were assessed for levels of pAkt, pS6K1, FLIP_S, and FLIP_L by Western blot analysis. (C) Cells from GBM xenografts were incubated with 0 or 100 nM rapamycin and 0 or 800 ng/ml TRAIL for 24 h, after which the cells were stained with propidium iodide and analyzed by flow cytometry for the percentage of apoptotic cells. The data shown are the means ± standard errors derived from three independent experiments. Values listed are the means derived from three independent experiments.