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 10

Inhibition of ERK does not account for effects of SMAP/B56-PP2A on 4E-BP1.

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Inhibition of ERK does not account for effects of SMAP/B56-PP2A on 4E-BP...
(A) Model showing upstream kinases that regulate 4E-BP1 phosphorylation. mTOR is a major 4E-BP1 kinase, and ERK has also been implicated in phosphorylation of specific sites on the protein. AKT and/or ERK are also upstream regulators of mTOR and TFEB/TFE3. (B and C) PDAC or CRC cells were treated with vehicle, 20 μM DT-061, or the indicated concentrations of SCH772984 for 1–4 hours before immunoblot analysis. pERK, ERK1/2 phosphorylated on Thr202/Thr204; p-RSK, phospho-RSK. The CRC samples analyzed in B are the same as in Figure 2G, which confirms effects on 4E-BP1. (D) RT-qPCR analysis of 4E-BP1 mRNA expression (expressed relative to 18S rRNA and normalized to control) in BxPC-3 cells treated as in C. (E) As in C except that cells were preincubated (30 minutes) with SCH772984 before addition of DT-061 for 4 hours. (F) As in D except that BxPC-3 cells were treated as in E. Immunoblot data are representative of 3 or more independent experiments, and RT-qPCR data are averages (± SEM) of 3 independent experiments. *P < 0.05 for increase induced by DT-061 (Holm-Bonferroni–adjusted 1-sided Student’s t test).