Small-molecule modulators of B56-PP2A restore 4E-BP function to suppress eIF4E-dependent translation in cancer cells
Michelle A. Lum, Kayla A. Jonas, Shreya Parmar, Adrian R. Black, Caitlin M. O’Connor, Stephanie Dobersch, Naomi Yamamoto, Tess M. Robertson, Aidan Schutter, Miranda Giambi, Rita A. Avelar, Analisa DiFeo, Nicholas T. Woods, Sita Kugel, Goutham Narla, Jennifer D. Black
Michelle A. Lum, Kayla A. Jonas, Shreya Parmar, Adrian R. Black, Caitlin M. O’Connor, Stephanie Dobersch, Naomi Yamamoto, Tess M. Robertson, Aidan Schutter, Miranda Giambi, Rita A. Avelar, Analisa DiFeo, Nicholas T. Woods, Sita Kugel, Goutham Narla, Jennifer D. Black
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Research Article Cell biology Oncology

Small-molecule modulators of B56-PP2A restore 4E-BP function to suppress eIF4E-dependent translation in cancer cells

Abstract

Dysregulated eIF4E-dependent translation is a central driver of tumorigenesis and therapy resistance. eIF4E-binding proteins (4E-BP1/2/3) are major negative regulators of eIF4E-dependent translation that are inactivated in tumors through inhibitory phosphorylation or downregulation. Previous studies have linked PP2A phosphatase(s) to activation of 4E-BP1. Here, we leveraged biased small-molecule activators of PP2A (SMAPs) to explore the role of B56-PP2A(s) in 4E-BP regulation and the potential of B56-PP2A activation for restoring translational control in tumors. SMAP treatment promoted PP2A-dependent hypophosphorylation of 4E-BP1/2, supporting a role for B56-PP2As (e.g., B56α-PP2A) as 4E-BP phosphatases. Unexpectedly, SMAPs induced transcriptional upregulation of 4E-BP1 through a B56-PP2A→TFE3/TFEB→ATF4 axis. Cap-binding and coimmunoprecipitation assays showed that B56-PP2A(s) activation blocks assembly of the eIF4F translation initiation complex, and cap-dependent translation assays confirmed the translation-inhibitory effects of SMAPs. Thus, B56-PP2A(s) orchestrate a translation-repressive program involving transcriptional induction and activation of 4E-BP1. Notably, SMAPs promoted 4E-BP1–dependent apoptosis in tumor cells and potentiated 4E-BP1 function in the presence of ERK or mTOR inhibitors, agents that rely on inhibition of eIF4E-dependent translation for antitumor activity. These findings, combined with the ability of SMAPs to regulate 4E-BP1 in vivo, highlight the potential of PP2A activators for cancer therapy and overcoming therapy resistance.

Authors

Michelle A. Lum, Kayla A. Jonas, Shreya Parmar, Adrian R. Black, Caitlin M. O’Connor, Stephanie Dobersch, Naomi Yamamoto, Tess M. Robertson, Aidan Schutter, Miranda Giambi, Rita A. Avelar, Analisa DiFeo, Nicholas T. Woods, Sita Kugel, Goutham Narla, Jennifer D. Black

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Figure 7

SMAP/B56-PP2A induces 4E-BP1 upregulation at the level of transcription.

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SMAP/B56-PP2A induces 4E-BP1 upregulation at the level of transcription....
(A) Cells were treated with 20 μM DT-061 as indicated and analyzed by immunoblotting. LC: actin (SW-620, HCT-15, Capan-1, MiaPaCa-2) or GAPDH (FET). Dashed lines indicate rearrangement of lanes from a single blot for clarity. (B) HCT-116 cells were treated with DT-061 or vehicle for 1 hour before addition of CHX for the indicated times and analysis by immunoblotting. (C) As in A except that 4E-BP1 mRNA expression was determined by RT-qPCR, normalized to 18S rRNA, and expressed as relative to control. (D) Cells were pretreated with 1 μg/mL ActD for 1 hour as indicated, followed by addition of vehicle or DT-061 for 4 hours before analysis by immunoblotting. (E) Cells were treated with vehicle or DT-061 for 6 hours, with 5-ethynyl uridine (EU) added during the last hour. EU-labeled RNA was isolated, and 4E-BP1 mRNA was quantified by RT-qPCR, normalized to 18S rRNA, and expressed relative to control. Data in A, B, and D are representative of at least 3 independent experiments. Data in C and E are the average (± SEM) of at least 3 independent experiments. *P < 0.05, **P < 0.02 for increase over control (1-sided Student’s t test, Holm-Bonferroni adjusted for C).