Stat1 stimulates cap-independent mRNA translation to PNAS PLUS inhibit cell proliferation and promote survival in response to antitumor drugs Shuo Wanga, Christos Patsisa,b, and Antonis E. Koromilasa,c,1 aLady Davis Institute for Medical Research, McGill University, Sir Mortimer B. Davis-Jewish General Hospital, Montreal, Quebec H3T 1E2, Canada; bDivision of Experimental Medicine, Department of Medicine, Faculty of Medicine, McGill University, Montreal, Quebec H3A 1A3, Canada; and cDepartment of Oncology, Faculty of Medicine, McGill University, Montreal, Quebec H2W 1S6, Canada Edited by Nahum Sonenberg, McGill University, and accepted by the Editorial Board March 20, 2015 (received for review October 29, 2014) The signal transducer and activator of transcription 1 (Stat1) promoter of mouse leukemogenesis caused by the activation functions as a tumor suppressor via immune regulatory and cell- of either Abelson murine leukemia viral oncogene homolog v-Abl autonomous pathways. Herein, we report a previously unidentified or translocation-Ets-leukemia locus (TEL) and Janus kinase 2 fu- cell-autonomous Stat1 function, which is its ability to exhibit both sion protein through immune regulatory mechanisms independent antiproliferative and prosurvival properties by facilitating trans- of IFN-γ (15). Although Stat1 is not mutated in human cancers, lation of mRNAs encoding for the cyclin-dependent kinase inhibitor posttranslational modifications by phosphorylation, acetylation, and p27Kip1 and antiapoptotic proteins X-linked inhibitor of apoptosis SUMOylation have been thought to control its function (16, 17). and B-cell lymphoma xl. Translation of the select mRNAs requires Exposure of cells to various extracellular stimuli, such as the transcriptional function of Stat1, resulting in the up-regulation hormones, mitogens, and growth factors, leads to the activation of the p110γ subunit of phosphoinositide 3-kinase (PI3K) class IB of phosphoinositide 3-kinase (PI3K) and the recruitment of the and increased expression of the translational repressor translation serine-threonine kinase Akt/PKB to the plasma membrane, initiation factor 4E (eIF4E)-binding protein 1 (4EBP1). Increased which results in its activation by phosphorylation at threonine PI3Kγ signaling promotes the degradation of the eIF4A inhibitor (T) 308 and serine (S) 473 (18). Phosphorylation of Akt at T308 BIOCHEMISTRY programmed cell death protein 4, which favors the cap-independent is mediated by the PI3K-dependent kinase 1 (PDK1), whereas translation of the select mRNAs under conditions of general Akt S473 phosphorylation is induced by the mammalian target of inhibition of protein synthesis by up-regulated eIF4E-binding pro- rapamycin complex 2 (mTORC2) kinase (19). Akt mediates the tein 1. As such, Stat1 inhibits cell proliferation but also renders cells activation of several effector proteins including mTORC1, which increasingly resistant to antiproliferative effects of pharmacological is essential for cell growth (20). Regulation of protein synthesis is inhibitors of PI3K and/or mammalian target of rapamycin. Stat1 also a well-characterized function of mTORC1, which is exerted protects Ras-transformed cells from the genotoxic effects of doxoru- through its ability to mediate, directly or indirectly, the phos- bicin in culture and immune-deficient mice. Our findings demonstrate phorylation of ribosomal proteins and translation initiation fac- an important role of mRNA translation in the cell-autonomous Stat1 tors (eIFs) (21). Tumor cells display increased mRNA trans- functions, with implications in tumor growth and treatment with lation, which is mainly exerted at the initiation level through the chemotherapeutic drugs. coordinated action of several eIFs, facilitating mRNA recruit- ment to the ribosomes and its positioning at the initiation codon Stat1 | phosphoinositide 3-kinase | programmed cell death protein 4 | eIF4E-binding protein 1 | mRNA translation (22). The cap-binding protein eIF4E together with eIF4A and eIF4G form the eIF4F complex, which is essential for translation tat1 is essential for innate immunity by protecting the host Significance Sfrom infections with viruses and other pathogens (1). Stat1 mediates the transcriptional induction of IFN-inducible genes by acting downstream of type I (α/β) and II (γ) IFN receptors, Stat1 functions as a tumor suppressor by inhibiting cell pro- resulting in the synthesis of proteins with antimicrobial and im- liferation and mediating antitumor immune responses. Thus, mune regulatory properties (1). Both types of IFNs induce Stat1 Stat1 is thought to be a suitable target for the implementation phosphorylation at tyrosine (Y) 701, which is mediated by re- of effective antitumor therapies. However, recent findings ceptor-associated Janus kinases (Jaks) and is required for have shown that Stat1 can mediate resistance to antitumor homodimerization as well as heterodimerization with other Stat drugs through mechanisms that are not well understood. family members (1). Stat1 Y701 phosphorylation is crucial for Herein, we demonstrate the ability of Stat1 to induce phos- DNA binding and transcriptional function, whereas serine (S) phoinositide 3-kinase γ (PI3Kγ) signaling and inhibit general 727 phosphorylation promotes gene transactivation in response protein synthesis, which results in the translation of select to IFNs (1). Nevertheless, unphosphorylated Stat1 also possesses mRNAs encoding for proteins that inhibit cell proliferation or transcriptional functions in cells infected with viruses or exposed render cells increasingly resistant to antitumor drugs. Our work to DNA damage (1–3). may result in the design of therapies that disarm the pro- Genetic inactivation of mouse Stat1 has demonstrated that it survival function of Stat1 in tumors without compromising its functions as a tumor suppressor via two different but not mu- ability to mount an effective antitumor immune response. tually exclusive mechanisms (4, 5). One mechanism depends on Author contributions: S.W. and A.E.K. designed research; S.W. and C.P. performed re- the induction of antitumor immune responses (6) and the other search; S.W., C.P., and A.E.K. analyzed data; and S.W. and A.E.K. wrote the paper. on the suppression of oncogenic signaling in a cell-autonomous The authors declare no conflict of interest. (tumor cell-specific) manner (7–11). The antitumor properties of This article is a PNAS Direct Submission. N.S. is a guest editor invited by the Editorial Stat1 have been best documented in breast cancers in which Board. Stat1 assumes both immune regulatory and cell-autonomous 1To whom correspondence should be addressed. Email: [email protected]. α functions to suppress either ErbB2/HER2 or estrogen receptor This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (ERα)-mediated tumorigenesis (4, 10–14). Stat1 can also act as a 1073/pnas.1420671112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1420671112 PNAS Early Edition | 1of7 Downloaded by guest on September 28, 2021 of the majority of capped mRNAs; eIF4F is negatively regulated by the eIF4E-binding proteins 1 and 2 (4E-BPs), which interact with eIF4E to prevent eIF4F formation (23). Activation of the PI3K pathway leads to mTORC1-mediated phosphorylation of 4E-BPs and their dissociation from eIF4E, resulting in an active eIF4F complex that stimulates cap-dependent translation (23). eIF4F activity is enhanced in tumor cells due to increased eIF4E expression and/or phosphorylation at S209 (24). Cells respond to IFNs by increasing PI3K signaling, leading to increased phosphorylation of 4EBPs and stimulation of cap- dependent translation of mRNAs transcriptionally induced by Jak-Stat activation (25–27). Herein, we demonstrate that Stat1 acts independent of IFNs to induce PI3K signaling by facilitating the expression of the p110γ catalytic subunit of PI3K class IB at the transcriptional level. We also show that the transcriptional properties of Stat1 are required to increase 4EBP1 expression, resulting in the inhibition of general protein synthesis and cell proliferation. Both pathways are mediated by unphosphorylated Stat1 and contribute to increased translation of select mRNAs encoding for the cyclin-dependent kinase (cdk) inhibitor p27Kip1, X-linked inhibitor of apoptosis (XIAP), and B-cell lymphoma xl (Bcl-xl) as a means to inhibit proliferation or promote survival in response to chemotherapeutic drugs. Results Stat1 Stimulates PI3K Signaling in Response to Mitogenic Treatment. Much is known about Stat1’s role in cytokine signaling (1), but its function in mitogenic responses is less understood. We observed that serum stimulation of primary mouse embryonic fibroblasts Fig. 1. Stat1 stimulates PI3K signaling and p110γ expression. (A and B) (MEFs) resulted in the induction of Akt phosphorylation at T308 Primary MEFs (A) were serum deprived for 18 h followed by 10% (vol/vol) + + − − and S473, which was higher in Stat1 / than Stat1 / MEFs (Fig. calf serum stimulation for the indicated time points. Control or Ha-Ras G12V- 1A). Serum stimulation induced the phosphorylation of the 70 kDa transformed cells were maintained in complete medium (B). Cell extracts (50 μg of protein) were subjected to immunoblot analyses for the indicated isoform of the ribosomal S6 kinase 1 (S6K1) at T398, which γ +/+ proteins. (C and D) Detection of PI3KC mRNA expression in immortalized is mediated by mTORC1, at higher levels in primary Stat1 (C) or Ha-Ras G12V-transformed cells