Abstract 4076: Redox dependent regulation of cFLIP promoter activity and gene expression by PTEN.
Authors: Subramaniam Kothandharaman , Hirpara Jayshree L. , Clement Marie-Veronique , Pervaiz Shazib 
Published: August 2014

Reactive oxygen species (ROS) regulate cell fate signals by engaging distinct signaling networks such as cell proliferation, survival and apoptosis. While an overwhelming increase in ROS trigger cell/tissue damage and death, a slight pro-oxidant state on the other hand serves as a secondary messenger for cell survival and proliferation. Recent evidence also indicates crosstalk between cellular redox status and the PI3K/Akt axis. Consistent with this, in an attempt to understand the survival pathways induced by a slight increase in cellular pro-oxidant status, in particular superoxide anion, we investigated the effect of an altered redox status on the expression of the death inhibitory protein cFLIP. We show that an increase in superoxide resulted in an increase in cFLIP promoter activity as well as protein expression. Interestingly, overexpression of Akt1 or Akt 2 also resulted in cFLIP induction. Of note, the increase in cFLIP promoter activity measured by a luciferase expression system was significantly suppressed when PTEN was restored by over expression. On the other hand, co-expression of Akt1 and Akt2 abrogated PTEN mediated down regulation of cFLIP, further demonstrating the positive regulatory effect of Akt species on cFLIP transcription. More interestingly, the effect of Akt on cFLIP correlated with its ability to increase intracellular superoxide levels (measured by dihydroethidium (DHE) staining as well as Lucigenin-based chemiluminescence), which was in turn blocked upon overexpression of PTEN. Phosphorylation-inefficient mutant AKt construct (dominant negative Akt for S473 and T308 residues) lost 50% of its activity to upregulate cFLIP transcription, indicating that Akt phosphorylation/activation is required for ROS production and cFLIP transcriptional activation. Using functional mutants of PTEN (generously provided by Donald J. Tindall, Mayo Foundation, MN) we further show that the lipid phosphatase function of PTEN is required for its regulation of cFLIP expression. Furthermore, PTEN mediated down regulation of FLIP transcription was rescued by over expression of c-Jun and wild type CREB in a concentration dependent manner, which was corroborated by the observation that pCREB level was lower upon enforced PTEN expression. Finally, to provide a functional relevance of these findings, we demonstrate that PTEN restoration in PTEN-/- LnCaP cells resulted in enhanced sensitivity to TRAIL-induced apoptosis, which could be rescued by over expression of isoforms of cFLIP, viz., cFLIPs and cFLIPL. Taken together, our preliminary data suggest that loss of PTEN leads to up-regulation of cFLIP transcription via redox dependent mechanisms, thus contributing to survival and chemoresistance in prostate cancer cells.

Citation Format: Kothandharaman Subramaniam, Jayshree L. Hirpara, Marie-Veronique Clement, Shazib Pervaiz. Redox dependent regulation of cFLIP promoter activity and gene expression by PTEN. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4076. doi:10.1158/1538-7445.AM2013-4076