Inhibition of SAPK2/p38 Enhances Sensitivity to mTORC1 Inhibition by Blocking IRES-Mediated Translation Initiation in Glioblastoma

Abstract

A variety of mechanisms confer hypersensitivity of tumor cells to the macrolide rapamycin, the prototypic mTORC1 inhibitor. Several studies have demonstrated that the status of the AKT kinase plays a critical role in determining hypersensitivity. Cancer cells in which AKT activity is elevated are exquisitely sensitive to mTORC1 inhibitors while cells in which the kinase is quiescent are relatively resistant. Our previous work has demonstrated that a transcript specific protein synthesis salvage pathway is operative in cells with quiescent AKT levels, maintaining the translation of crucial mRNAs involved in cell cycle progression in the face of global eIF-4E-mediated translation inhibition. The activation of this salvage pathway is dependent on SAPK2/p38-mediated activation of IRES-dependent initiation of the cyclin D1 and c-MYC mRNAs resulting in the maintenance of their protein expression levels. Here we demonstrate that both genetic and pharmacological inhibition of SAPK2/p38 in glioblastoma multiforme (GBM) cells significantly reduces rapamycin induced IRES-mediated translation initiation of cyclin D1 and c-MYC resulting in increased G1 arrest in vitro and inhibition of tumor growth in xenografts. Moreover, we observed that the AKT-dependent signaling alterations seen in vitro are also displayed in engrafted tumors cells and were able to demonstrate that combined inhibitor treatments markedly reduced the mRNA translational state of cyclin D1 and c-MYC transcripts in tumors isolated from mice. These data support the combined use of SAPK2/p38 and mTORC1 inhibitors to achieve a synergistic anti-tumor therapeutic response, particularly in rapamycin resistant quiescent AKT-containing cells.

IRES-dependent translation of egr2 is induced under inflammatory conditions

“…Thus, besides IL-1b, additional factors within the CM, which remain to be elucidated, contribute to enhanced egr2 translation. Furthermore, our data corroborate previous reports showing that p38-MAPK activity is important for IRES-dependent translation of cyclin D1 and c-myc (Shi et al 2005;Cloninger et al 2011).…”

Mechanistic Target of Rapamycin (mTOR) Inhibition Synergizes with Reduced Internal Ribosome Entry Site (IRES)-mediated Translation of Cyclin D1 and c-MYC mRNAs to Treat Glioblastoma

“…Protein and RNA Analyses-Western blotting and quantitative RT-PCR analyses were performed as described previously (20). Briefly, for Western blotting, cells or tissues were lysed in radioimmune precipitation assay (lysis) buffer containing protease inhibitor mixture and phosSTOP phosphatase inhibitor mixture (Roche), and extracts were resolved by SDS-PAGE.…”

“…RNA interference targeting hnRNP A1 was performed as described previously (18,23) using siRNAs with the target sequence 5Ј-gguucuauuuggaauuuau-3Ј obtained from Ambion (Applied Biosystems). Polysome analyses were performed as before (20). Briefly, cells were lysed in buffer containing 100 g/ml cycloheximide at 4°C.…”

“…Our previous studies have demonstrated that both allosteric and direct kinase inhibitors of mTOR can activate a transcript-specific protein synthesis salvage pathway, maintaining the translation of crucial mRNAs involved in cell cycle progression, resulting in resistance to mTOR therapies (17)(18)(19). The activation of this intrinsic pathway is dependent on SAPK2/p38-mediated activation of IRESdependent initiation of cyclin D1 and c-MYC mRNAs in GBM (20). Recently, we have described the therapeutic potential of targeting IRES-dependent c-MYC translation via the identification of a small molecule inhibitor that blocked c-MYC IRESmediated translation initiation (21).…”

Phosphorylation of the Hippo Pathway Component AMOTL2 by the mTORC2 Kinase Promotes YAP Signaling, Resulting in Enhanced Glioblastoma Growth and Invasiveness

“…Protein and mRNA Analyses, Immunoprecipitations, and in Vitro Kinase Assays-Western blot and quantitative RT-PCR analyses were performed as described previously (19). Briefly, for Western blotting, cells or tissues were lysed in RIPA (lysis) buffer containing protease inhibitor mixture and phosSTOP phosphatase inhibitor mixture (Roche Applied Science), and extracts were resolved by SDS-PAGE.…”