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A novel PTEN-dependent link to ubiquitination controls FLIPS stability and TRAIL sensitivity in glioblastoma

Amith Panner¹, Courtney A Crane, Changjiang Weng

¹Brain Tumor Research Center, Department of Neurological Surgery and University of California San Francisco Comprehensive Cancer Center, University of California San Francisco, San Francisco, California 94158-9001, USA.

Cancer Research

|October 8, 2009

Abstract:

Phosphatase and tensin homologue (PTEN) loss and activation of the Akt-mammalian target of rapamycin (mTOR) pathway increases mRNA translation, increases levels of the antiapoptotic protein FLIP(S), and confers resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in glioblastoma multiforme (GBM). In PTEN-deficient GBM cells, however, the FLIP(S) protein also exhibited a longer half-life than in PTEN mutant GBM cells, and this longer half-life correlated with decreased FLIP(S) polyubiquitination. FLIP(S) half-life in PTEN mutant GBM cells was reduced by exposure to an Akt inhibitor, but not to rapamycin, suggesting the existence of a previously undescribed, mTOR-independent linkage between PTEN and the ubiquitin-dependent control of protein stability. Total levels of the candidate FLIP(S) E3 ubiquitin ligase atrophin-interacting protein 4 (AIP4) were comparable in PTEN wild-type (WT) and PTEN mutant GBM cells, although in PTEN-deficient cells, AIP4 was maintained in a stable polyubiquitinated state that was less able to associate with FLIP(S) or with the FLIP(S)-containing death inducing signal complex. Small interfering RNA-mediated suppression of AIP4 levels in PTEN WT cells decreased FLIP(S) ubiquitination, prolonged FLIP(S) half-life, and increased TRAIL resistance. Similarly, the Akt activation that was previously shown to increase TRAIL resistance did not alter AIP4 levels, but increased AIP4 ubiquitination, increased FLIP(S) steady-state levels, and suppressed FLIP(S) ubiquitination. These results define the PTEN-Akt-AIP4 pathway as a key regulator of FLIP(S) ubiquitination, FLIP(S) stability, and TRAIL sensitivity and also define a novel link between PTEN and the ubiquitin-mediated control of protein stability.

Insights

Loss of PTEN in glioblastoma activates Akt, stabilizing the anti-apoptotic protein FLIP(S) via the E3 ligase AIP4. This PTEN-Akt-AIP4 pathway regulates FLIP(S) stability and TRAIL resistance in GBM.

Area of Science:

Oncology
Molecular Biology
Cellular Biology

Background:

Loss of Phosphatase and tensin homologue (PTEN) and activation of the Akt-mammalian target of rapamycin (mTOR) pathway promote glioblastoma multiforme (GBM) growth and resistance to apoptosis.
The anti-apoptotic protein FLIP(S) plays a crucial role in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis resistance in GBM.
PTEN deficiency in GBM cells leads to increased FLIP(S) half-life and decreased polyubiquitination, suggesting a link to protein stability.

Purpose of the Study:

To investigate the novel, mTOR-independent mechanism linking PTEN status to the ubiquitin-dependent control of FLIP(S) protein stability.
To elucidate the role of the E3 ubiquitin ligase atrophin-interacting protein 4 (AIP4) in regulating FLIP(S) stability and TRAIL sensitivity in PTEN-mutant GBM.
To define the PTEN-Akt-AIP4 pathway's contribution to TRAIL resistance in glioblastoma.

Main Methods:

Comparative analysis of FLIP(S) half-life and ubiquitination in PTEN wild-type (WT) and PTEN-mutant GBM cells.
Treatment of PTEN mutant GBM cells with Akt inhibitor and rapamycin to assess effects on FLIP(S) half-life.
Assessment of AIP4 levels, ubiquitination status, and association with FLIP(S) in different PTEN contexts.
Small interfering RNA (siRNA)-mediated suppression of AIP4 in PTEN WT cells.
Analysis of Akt activation effects on AIP4 ubiquitination and FLIP(S) levels and ubiquitination.

Main Results:

PTEN-deficient GBM cells exhibit a longer FLIP(S) half-life due to decreased FLIP(S) polyubiquitination, an effect partially reversed by Akt inhibition.
AIP4 levels are comparable, but AIP4 ubiquitination and association with FLIP(S) are altered in PTEN-deficient cells, with stable polyubiquitinated AIP4 showing reduced binding.
AIP4 suppression in PTEN WT cells mimics PTEN deficiency by increasing FLIP(S) half-life and TRAIL resistance.
Akt activation increases AIP4 ubiquitination, leading to suppressed FLIP(S) ubiquitination and increased FLIP(S) levels, thereby enhancing TRAIL resistance.

Conclusions:

The PTEN-Akt-AIP4 signaling axis is a critical regulator of FLIP(S) ubiquitination and stability.
This pathway significantly influences TRAIL-induced apoptosis sensitivity in glioblastoma multiforme.
A novel link between PTEN status and ubiquitin-mediated control of protein stability is established, impacting GBM pathogenesis.

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