Binding of eef1a2 to the rna-dependent protein kinase pkr modulates its activity and promotes tumour cell survival

ABSTRACT BACKGROUND Through several not-fully-characterised moonlighting functions, translation elongation factor _eEF1A2_ is known to provide a fitness boost to cancer cells. Furthermore,

_eEF1A2_ has been demonstrated to confer neoplastic characteristics on preneoplastic, nontumourigenic precursor cells. We have previously shown that _eEF1A2_ is the target of plitidepsin, a

marine drug currently in development for cancer treatment. Herein, we characterised a new signalling pathway through which _eEF1A2_ promotes tumour cell survival. METHODS Previously unknown

binding partners of _eEF1A2_ were identified through co-immunoprecipitation, high-performance liquid chromatography-mass spectrometry and proximity ligation assay. Using plitidepsin to

release _eEF1A2_ from those protein complexes, their effects on cancer cell survival were analysed in vitro. RESULTS We uncovered that double-stranded RNA-activated protein kinase (PKR) is a

novel _eEF1A2_-interacting partner whose pro-apoptotic effect is hindered by the translation factor, most likely through sequestration and inhibition of its kinase activity. Targeting

_eEF1A2_ with plitidepsin releases PKR from the complex, facilitating its activation and triggering a mitogen-activated protein kinase signalling cascade together with a nuclear

factor-κB-dependent activation of the extrinsic apoptotic pathway, which lead to tumour cell death. CONCLUSIONS Through its binding to PKR, _eEF1A2_ provides a survival boost to cancer

cells, constituting an Achilles heel that can be exploited in anticancer therapy. SIMILAR CONTENT BEING VIEWED BY OTHERS BLOCKADE OF EIF5A HYPUSINATION LIMITS COLORECTAL CANCER GROWTH BY

INHIBITING MYC ELONGATION Article Open access 10 December 2020 THE PLASTICITY OF MRNA TRANSLATION DURING CANCER PROGRESSION AND THERAPY RESISTANCE Article 02 August 2021 EIF3A REGULATION OF

MTOR SIGNALING AND TRANSLATIONAL CONTROL VIA HUR IN CELLULAR RESPONSE TO DNA DAMAGE Article 12 March 2022 INTRODUCTION Human translation elongation factor 1α2, encoded by the _eEF1A2_ gene,

has been identified as a pro-oncogenic protein. Absent from the majority of body tissues, with the exceptions of brain, heart and skeletal muscle1 (tissues with very low cell death), it is

expressed in many cancer types,1,2,3 where it provides tumour cells with improved fitness and survival. Moreover, _eEF1A2_ has been demonstrated to confer neoplastic characteristics to

preneoplastic, nontumourigenic human ovarian precursor cells4 and to NIH-3T3 mouse fibroblasts.2 Although the canonical function of _eEF1A2_ is delivering aminoacyl-transfer RNAs to the

ribosome during translation, other “moonlighting” functions have been described for the protein.5 For example, sphingosine kinase 1 (SPHK1) activity is enhanced by direct interaction with

the guanosine diphosphate (GDP)-bound form of _eEF1A2_6,7 favouring tumour cell growth. Similarly, _eEF1A2_ enhances peroxiredoxin-1 (PRDX1) activity, providing cells with extraordinary

resistance to oxidative stress-induced cell death.8 We have recently reported that _eEF1A2_ is the molecular target for plitidepsin, a marine drug under development for the treatment of

cancer patients.9 We have shown that plitidepsin binds with high affinity (_K_D ~80 nM) and persistence (residence time ∼9 min) to _eEF1A2_.9 While a contribution to oncogenicity of

_eEF1A2_’s canonical function cannot be ruled out, it seems clear that some other functions of the protein are essential to convey its pro-tumourigenic effect.8,10,11,12 Here we investigate

the role of new “moonlighting functions” of _eEF1A2_ in the maintenance of tumour phenotype. Using plitidepsin to target _eEF1A2_, we demonstrate that the pro-oncogenic activities of

_eEF1A2_ are important for the fitness and survival of tumour cells. Most unexpectedly, a previously undescribed regulatory interaction between _eEF1A2_ and interferon-induced,

dsRNA-activated protein kinase (PKR, EIF2AK2) has been found to be disrupted by the binding of plitidepsin to the elongation factor. This new _eEF1A2_–PKR complex has been proven essential

for the survival of cancer cells. In addition, binding of plitidepsin to _eEF1A2_ inhibits its interaction with PRDX1 and blocks the activation of SPHK. Taken together, these results show

that the fitness boost that the moonlighting functions of _eEF1A2_ provide to cancer cells constitutes an Achilles heel that can be purposely exploited in anticancer therapy. MATERIALS AND

METHODS REAGENTS Plitidepsin (CAS No. 137219-37-5) was synthesised at PharmaMar (Madrid, Spain). Poly(I:C) was from InvivoGen (San Diego, CA, USA). C16, BAY 11-7082, PF-543, and anti-FAS

(CH11) antibodies (Abs) were from Merck-Millipore (Danvers, MA, USA). PRDX1-Myc plasmid, anti-myc-tag and anti-tGFP-tag magnetic beads and anti-tGFP monoclonal Abs were from Origene

(Rockville, MD, USA). hTNF-α (human tumour necrosis factor-α), anti-phospho-JNK (c-Jun N-terminal kinase), anti-phospho-p38, anti-myc-tag, anti-phospho-eIF2α, anti-eIF2α, anti-NF-κB p65,

anti-IκBα, anti-BCL2, anti-c-Myc, anti-cyclin-D1, anti-FAS, anti-Mcl1, anti-caspase-8, and anti-FLAG Abs were from Cell Signaling Technologies (Danvers, MA, USA). Anti-_eEF1A2_ Ab was from

GeneTex (Irvine, CA, USA). Anti-FAS (SM1/23) Ab was from Enzo Life Sciences (Farmingdale, NY, USA). Anti-XIAP Ab was from BD (San Jose, CA, USA). Step Human High-Yield IVT,

anti-poly(ADP-ribose) polymerase (PARP) Ab, Alexa-Fluor®-488 goat anti-rabbit IgG, pT7CFE1-NHis-GST-CHA vector, glutathione agarose and glutathione _S_-transferase (GST)-tagged human

rhinovirus-3C protease were from Thermo Scientific (Rockford, IL, USA). Anti-SPHK1, anti-SPHK2, anti-PKR and anti-phospho-PKR Abs were from Abcam (Cambridge, UK). Anti-_eEF1A2_ Ab for

proximity ligation assay (PLA) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Lipofectamine® 2000 and Opti-MEM™ were from Life Technologies (Carlsbad, CA, USA). Bright-Glo™

and ADP-Glo™ were from Promega (Madison, WI, USA). SPHK Activity Assay and Sphingosine-1-phosphate (S1P) Competitive ELISA Kits were from Echelon Biosciences (Salt Lake City, UT, USA). All

other reagents, including Duolink® PLA, were purchased from Sigma-Aldrich (St Louis, MO, USA). CELL CULTURE HeLa cervix adenocarcinoma (ATCC CCL-2) cells were purchased from ATCC (Manassas,

VA, USA). Cell lines were further authenticated through the AACR authentication service. Plitidepsin-resistant HeLa cells (HeLa-APL-R) and stably transfected HeLa APL-A2-tGFP and APL-A1-tGFP

cells were generated at PharmaMar.9,13 PKR-null (PKR−/−) and eIF2α S51A mutant mouse embryonic fibroblasts (MEFs) and their wild-type (wt) counterparts were a gift from César de Haro

(CBMSO, Madrid, Spain). Cell culture, proliferation assays, transfection and clone selection procedures were described elsewhere.8 IMMUNOPRECIPITATION Cells were lysed with lysis buffer (1%

Triton X-100, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA) and centrifuged at 13,000 × _g_ for 20 min. Supernatant protein was quantified and 150 to 500 µg of total protein was

immunoprecipitated overnight at 4 °C with appropriate Ab-coated beads. Beads were extensively washed, boiled for 10 min in Laemmli buffer, subjected to polyacrylamide gel electrophoresis,

electro-blotted onto polyvinylidene fluoride membranes and hybridised with the appropriate antibodies. Immunoreactive bands were analysed with a Bio-Rad ChemiDoc Touch Imager (Hercules, CA,

USA). Densitometry for the quantification of the immunoblotting bands was performed with the Bio-Rad Image Lab v.6 software (Hercules, CA, USA). Experiments were repeated at least three

times to ensure consistency. IMMUNOFLUORESCENCE Cells (25,000 per well) were cultured in 16-well slides and treated with vehicle, 450 nM plitidepsin or 25 ng/mL hTNF-α for the indicated

times. Then, cells were washed with phosphate-buffered saline (PBS), fixed for 10 min with 4% paraformaldehyde, washed with PBS and permeabilised for 10 min with PBS-0.2% Triton X-100 at

room temperature (RT). Slides were blocked with 5% bovine serum albumin (BSA) at RT for 1 h and incubated with anti-NF-κB Ab (1:100 in 5% BSA–0.2% Triton X-100) for 1 h at RT. Slides were

then washed with PBS–0.2% Triton X-100 and incubated for 1 h with Alexa Fluor® 488 goat anti-rabbit IgG diluted 1:200 in 5% BSA–0.2% Triton X-100. Slides were finally rinsed with PBS–0.2%

Triton X-100, mounted and observed through a Zeiss AxioVert 200M Apotome microscope (Oberkochen, Germany). Images were acquired using the AxioVision Release 4.8.2.SP2 software. PROXIMITY

LIGATION ASSAY For the assay, Duolink® PLA reagents and protocol were used following the instructions of the supplier. Sixteen-well slides were seeded with 40,000 HeLa wt cells and cultured

for 24 h before being treated either with vehicle (0.1% dimethyl sulfoxide) or 450 nM plitidepsin for 1 h. Slides were then stained with the appropriate antibodies following the instruction

of the manufacturer, mounted and observed through a Zeiss AxioVert 200M Apotome microscope (Oberkochen, Germany). Images were acquired using the AxioVision Release 4.8.2.SP2 software. PKR

ACTIVITY ASSAY _eEF1A2_ was purified from rabbit muscle as described in ref.9; rabbit and human _eEF1A2_ sequences are 100% identical. _eEF1A2_ from the muscle was mainly in GDP-bound

conformation. To determine GDP and guanosine triphosphate (GTP) occupation within _eEF1A2_ and to obtain the GTP-bound conformation by exchanging GDP with the non-hydrolyzable GTP analogue

GppNHp, we followed the procedures described by Smith and Rittinger.14 Human PKR (HGNC:9437) was cloned into pT7CFE1-NHis-GST-CHA vector. PKR was produced using the Step Human High-Yield IVT

Kit. The identity of the protein was confirmed by Western blot. Typically 1 mL of translation reaction using 40 µg of plasmid yielded 100 µg of recombinant protein. PKR activity was

monitored using myelin basic protein (MBP) as substrate, measuring the adenosine diphosphate (ADP) produced after 60 min. Reactions were performed in a final volume of 5 µL with 80 nM PKR,

10 µM ATP and 5 µM MBP in 25 mM Tris-HCl, pH 7.5, 20 mM MgCl2, 500 µM dithiothreitol and in the presence or absence of 1 µg/mL poly(I:C), 2 µM C16 and different concentrations of _eEF1A2_

either in GDP-bound or GTP-bound conformations with 1 µM GDP or GppNHp, respectively. Mixtures were incubated at RT for 60 min and ADP detected with the ADP-Glo™ Kinase Assay System

following the vendor’s instructions. TRANSACTIVATION LUCIFERASE ASSAY NF-κB transactivation was assayed using the Bright-Glo™ Luciferase Assay System following the manufacturer’s

instructions. MDA-MB-231 human breast cancer cells stably transfected with 3×NF-κB-tk-Luc plasmid (containing three NF-κB binding sites, a minimal promoter and a Renilla luciferase gene)

were exposed to 50 ng/mL TNFα (positive control) or 450 nM plitidepsin for the indicated times. IκB kinase (IKK) inhibitors were used as controls, either alone or combined with TNFα or

plitidepsin. Luminescence was measured in a Perkin-Elmer Victor3 reader (Waltham, MA, USA). A cell proliferation assay was simultaneously performed to control the cytotoxicity of the

compounds. SPHK1 ACTIVITY ASSAY SPHK1 activity was quantified with the Sphingosine Kinase Activity Assay Kit following the manufacturer’s instructions. S1P QUANTIFICATION S1P levels were

analysed with the S1P Competitive ELISA Kit following the manufacturer’s instructions. CERAMIDE QUANTIFICATION Total lipid extracts from cell homogenates (2 × 106 cells) were divided into

three aliquots, two of them for the analysis of levels of sphingoid bases and the third for lipid phosphorus quantification. The first aliquot was hydrolysed for 1 h at 100 °C with 1 M KOH

in methanol to deacylate ceramides and derivatives in total sphingoid bases. The second aliquot was used to determine the free sphingoid bases through mild alkaline hydrolysis at 37 °C (0.1 

M KOH in chloroform–methanol 2:1 (v/v)). Long-chain bases from both aliquots were derivatised with _o_-phthalaldehyde, separated and quantified by high-performance liquid chromatography

(HPLC) as described in ref.15 Results were expressed as picomoles of ceramide per nanomole of lipid phosphorus. RESULTS INTERACTION OF _EEF1A2_ WITH PKR AFFECTS ITS CATALYTIC ACTIVITY To

identify new _eEF1A2_-interacting proteins, we performed immunoprecipitation experiments followed by a mass spectrometry-based proteomics approach. HeLa cells stably transfected with

_eEF1A2_-tGFP were lysed and immunoprecipitated with an anti-tGFP Ab. As a control, wt HeLa cells were processed identically. After filtering for elongation factor isoforms, proteins

involved in translation, common interacting actors such as chaperonins, proteins known to bind non-specifically to the immunoprecipitation matrix16 and proteins considered artefacts inherent

to the experimental procedure (e.g. immunoglobulins), six proteins remained selected (Table 1). Our attention was drawn to PKR, a known regulator of cell survival in response to stress

whose role in cancer has been unveiled in recent years.17,18 Since the interaction between _eEF1A2_ and PKR had not been reported yet, we set out to confirm this result. Extracts from HeLa

cells expressing chimeric _eEF1A2_-tGFP or eEF1A1-tGFP constructs were immunoprecipitated with either anti-GFP or anti-PKR antibodies. PKR was detected in the anti-GFP-immunoprecipitated

material from _eEF1A2_-tGFP-expressing HeLa cells, but not in the anti-GFP-immunoprecipitated material from eEF1A1-tGFP-expressing HeLa cells (Fig. 1a). Similarly, _eEF1A2_-GFP, but not

eEF1A1-tGFP, was identified in the PKR-immunoprecipitated sample (Fig. 1a), demonstrating the specificity of the binding between PKR and _eEF1A2_. Immunoprecipitation of extracts from HeLa

cells ectopically overexpressing human _eEF1A2_-tGFP, as well as myc-tagged human PRDX1, SPHK1 or SPHK2, confirmed the previously described interaction between these three proteins and

_eEF1A2_ (Supplementary Figures 1, 2A). To understand whether the kinase function of PKR was affected by its interaction with _eEF1A2_, we measured the activity of the recombinant human

enzyme produced by an in vitro translation system. Figure 1b shows that the kinase was functionally active and its activity was boosted by the viral dsRNA mimetic poly(I:C) and abated by the

specific PKR inhibitor C16.19 Interestingly, the presence of rabbit _eEF1A2_ caused an inhibitory effect on the kinase activity of PKR, more pronounced when the PKR–_eEF1A2_ molar ratio was

1:2 (_p_ < 0.005). This inhibition was observed for both nucleotide-dependent conformations of _eEF1A2_, although the GTP-bound form was slightly more efficient. Taken together, these

results demonstrate that _eEF1A2_ directly interacts with PKR and impairs its catalytic activity. _EEF1A2_–PKR COMPLEX FORMATION INHIBITS THE PRO-APOPTOTIC ACTIVITY OF PKR The observation of

_eEF1A2_–PKR complexes prompted us to investigate their biological role in tumour cells. We first examined whether targeting of _eEF1A2_ by plitidepsin was able to modulate _eEF1A2_–PKR

complex formation in HeLa cells. Taking advantage of the PLA, we observed by fluorescent microscopy the formation of _eEF1A2_–PKR complexes within HeLa wt cells (Fig. 1c, upper panel). These

complexes were lost when cells were incubated for 1 h with 450 nM plitidepsin (Fig. 1c, second panel). Controls lacking each of the primary antibodies used for the assay are shown in the

third and fourth panels. Wt and _eEF1A2_-tGFP-transfected HeLa cells were treated for different times with 450 nM plitidepsin and cell lysates subjected to immunoprecipitation. Plitidepsin

treatment resulted in a rapid and sustained reduction of _eEF1A2_–PKR complexes, as deduced by the disappearance of the anti-PKR Ab immunoreactive band in the anti-tGFP-immunoprecipitated

material (Fig. 1d) or the anti-GFP Ab immunoreactive band in the anti-PKR-immunoprecipitated material (Fig. 1e). Moreover, plitidepsin also disrupted, in a time-dependent manner,

_eEF1A2_–PRDX1 complexes (Supplementary Fig. 1). Of note, the interaction between _eEF1A2_ and both SPHK1 and SPHK2 was not affected by the drug (Supplementary Figs. 2, 3). To gain further

insight into the biological effects of plitidepsin-induced _eEF1A2_–PKR complex disruption, we evaluated the sensitivity to the drug of PKR−/−, PKR−/− with re-expressed PKR and wt MEFs. As

observed in Fig. 2a (upper panel), sensitivity to plitidepsin was significantly reduced in PKR−/− MEFs and recovered to the levels shown by wt MEFs when PKR was ectopically re-expressed in

the null fibroblasts, hence suggesting a critical role for PKR in the antiproliferative effect of this drug after binding to _eEF1A2_. To notice, the effect of a control compound

(doxorubicin, DOX) was not affected by the presence of PKR (Fig. 2a, lower panel). Indeed, PKR appears essential for leading MEFs into apoptosis upon plitidepsin treatment as evidenced by

the absence of PARP processing in drug-treated PKR−/− cells (Fig. 2b). To confirm these effects in tumour cells, we explored whether plitidepsin induced the activation of PKR, as measured by

its self-phosphorylation on Thr-451, in HeLa cells. Figure 2c demonstrates that treatment of HeLa cells with 450 nM plitidepsin elicited a sustained increase in the levels of

autophosphorylated PKR, while the total kinase levels remained unaffected. However, such phosphorylation was significantly reduced when HeLa cells had been pretreated with the PKR inhibitor

C16 before plitidepsin exposure (Fig. 2d). Moreover, reduction in PKR self-phosphorylation caused by co-treatment with C16 was concomitant with a decrease in the ability to induce PARP

processing, hence supporting the essential role of PKR in plitidepsin-promoted apoptosis. To corroborate this conclusion, the antiproliferative effect of plitidepsin on HeLa cells was

evaluated in the presence and absence of C16. Figure 2e demonstrates that, indeed, C16 cotreatment protected HeLa cells from the lethal effect of plitidepsin, clearly evidencing the critical

role of PKR in the mechanism of action of the drug. Altogether, these results show that the presence of _eEF1A2_–PKR complexes is necessary to inhibit the pro-apoptotic functions of PKR in

tumour cells. INHIBITION OF _EEF1A2_ WITH PLITIDEPSIN INDUCES APOPTOSIS DEPENDENT ON PKR ACTIVATION As mentioned above, targeting of _eEF1A2_ with plitidepsin induces the activation of

proteins involved in pro-apoptotic signalling such as JNK and p38/mitogen-activated protein kinase (MAPK). Figure 3a, b show the rapid activation of these proteins in wt MEFs treated with

450 nM plitidepsin, the highest activation reached between 0.5 and 1 h post-treatment. However, PKR removal in these cells led to a significantly weaker activation of JNK. Likewise,

plitidepsin induced the rapid and sustained phosphorylation of eIF2α in wt MEFs but not in PKR−/− cells. The lack of eIF2α phosphorylation in these latter cells, even in the presence of the

PKR activator poly-I:C,20 confirmed the null expression of PKR in this cell line (Fig. 3a). Interestingly, although poly-I:C effectively promoted eIF2α phosphorylation in wt MEFs, it did not

promote per se JNK or p38 activation. These findings clearly point to a unique involvement of PKR in the plitidepsin-induced activation of these signalling kinases. Nonetheless, despite the

essential role of PKR in the apoptotic effect of plitidepsin, PKR-dependent phosphorylation of eIF2α seemed not to be critical for plitidepsin lethality. Indeed, MEFs expressing a

non-phosphorylatable eIF2α (eIF2αS51A) were as sensitive to plitidepsin as wt MEFs (Fig. 3c). In addition, treatment of these mutant MEFs (completely lacking eIF2α phosphorylation,

Supplementary Fig. 4) with plitidepsin induced the same pro-apoptotic signalling events observed in wt cells, namely rapid JNK activation and later PARP processing (Supplementary Fig. 4). We

have finally checked the activation pattern of PKR in plitidepsin-resistant tumour cells with reduced _eEF1A2_ protein levels.9 Treatment with 450 nM plitidepsin induced a clear,

time-dependent increase in phosphorylated PKR in HeLa cells (Fig. 3d). However, in _eEF1A2_-deficient cells (HeLa-APL-R), phospho-PKR levels did not change significantly after plitidepsin

treatment. These differences in PKR self-phosphorylation, mirrored in the levels of phosphorylated eIF2α, were parallel to differences in the signature of the drug in both HeLa strains, that

is, JNK activation and PARP processing, which were visible in wt HeLa cells but not in the resistant ones (Fig. 3d). This result confirmed the essential role of PKR in the apoptosis induced

after plitidepsin binding to _eEF1A2_. _EEF1A2_–PKR COMPLEXES REGULATE THE NF-ΚB SIGNALLING PATHWAY We then checked the effect of targeting _eEF1A2_ by plitidepsin on the NF-κB signalling

pathway, which is involved in the downstream pro-apoptotic effect of PKR.21 As a marker of NF-κB activation, we first investigated whether IκB was phosphorylated by IKK and, thus, degraded

by the proteasome following plitidepsin treatment. Indeed, wt MEFs treated with 450 nM plitidepsin showed a clear IκB degradation after 3 h, absent in PKR−/− MEFs (Fig. 4a). To ensure that

NF-κB activation also occurred in cancer cells in an _eEF1A2_-dependent fashion, wt and APL-R HeLa cells were treated with 450 nM plitidepsin for the indicated times and the levels of IκB

checked by Western blot. While in wt HeLa cells plitidepsin induced a clear degradation of IκB, in APL-R HeLa cells, lacking _eEF1A2_, no alteration of IκB levels was observed (Fig. 4b).

Then, we analysed by immunofluorescence the cellular localisation of NF-κB in wt or PKR−/− MEFs treated with plitidepsin 450 nM for 6 h or, as a positive activation control, with 50 ng/mL of

TNFα for 1 h (Fig. 4c). NF-κB shuttled to the nucleus in wt MEFs after treatment with either TNFα or plitidepsin, demonstrating the activation of the pathway. In contrast, in PKR−/− MEFs we

could not see any shuttling of NF-κB after TNFα or plitidepsin treatments, indicating that the activation observed in wt MEFs was dependent on PKR. Since NF-κB localised to the nuclei in

plitidepsin-treated wt MEFs, we checked whether the transcriptional activity of the protein was also activated. To that end, we took advantage of MDA-MB-231 cells stably transfected with an

NF-κB luciferase reporter plasmid. We treated the cells with either 450 nM plitidepsin for times ranging from 0.5 to 6 h, 50 ng/mL TNFα (an activator of NF-κB), 100 µM BAY11-7082, 20 µM

PS1145 (both IKK inhibitors) or combinations of them and quantified the luciferase activity under each condition. Even in the presence of 50 ng/mL TNFα, plitidepsin clearly inhibited the

production of luciferase indicating that transactivation from NF-κB was inhibited in the presence of the drug (Fig. 4d). Taken together, these findings demonstrate that NF-κB activation by

plitidepsin was dependent on the presence of _eEF1A2_ and its interaction with PKR. We then checked whether the expression of some NF-κB-responsive genes was affected by plitidepsin. HeLa

cells were treated with 450 nM plitidepsin for times ranging from 0.5 to 6 h and the cellular levels of FAS, Bcl2, Mcl1, XIAP, c-myc and cyclin D1 were analysed by Western blot. Plitidepsin

clearly inhibited the expression of Bcl2, Mcl1, XIAP, c-myc and cyclin D1, while FAS expression was activated (Fig. 4e). _EEF1A2_–PKR COMPLEXES REGULATE THE EXTRINSIC APOPTOTIC PATHWAY After

these results, we were interested in learning whether NF-κB activation was necessary for plitidepsin-induced apoptosis. HeLa cells were treated with 450 nM plitidepsin, 100 µM BAY11-7082 or

a combination of both for 6 h and PARP1 cleavage was analysed as apoptosis marker. Co-treatment with plitidepsin and the IKK inhibitor BAY11-7082 clearly reduced the pro-apoptotic effect of

the drug, showing that NF-κB activation was indeed necessary for plitidepsin-induced apoptosis (Fig. 5a). Fas signalling has been shown to be activated by NF-κB at the transcriptional

level22,23 and PKR-induced apoptosis depends on the activation of Fas by NF-κB.21,24 Noteworthy, plitidepsin has been shown to induce the extrinsic apoptotic pathway in Jurkat leukaemia

cells.25,26 Thus, we analysed whether plitidepsin was activating the extrinsic apoptotic pathway also in HeLa cells. Caspase-8 was, indeed, activated by auto-cleavage in plitidepsin-treated

HeLa cells, and co-treatment with the IKK inhibitor BAY11-7082 partially inhibited this activation (Fig. 5a). To check whether the NF-κB-dependent apoptotic activation induced by plitidepsin

was dependent on PKR, we treated HeLa cells with 450 nM plitidepsin, 100 µM C16 (PKR inhibitor) or a mixture of both for 6 h and checked by Western blot the levels of PKR phosphorylation,

caspase-8 activation and PARP1 cleavage. Inhibition of PKR with C16 blocked both caspase-8 activation and PARP1 cleavage clearly indicating that plitidepsin-induced apoptosis was dependent

on PKR (Fig. 5b). Finally, we explored whether the activation of the extrinsic apoptotic pathway by plitidepsin was due to Fas clustering (as previously demonstrated in Jurkat cells26) or

through other external stimuli (e.g. FasL). We treated HeLa cells for 6 h with 450 nM plitidepsin, 50 µM caspase-8 inhibitor Z-IETD, 0.5 µg/mL Fas-activating Ab CH11 or 1 µg/mL

Fas-inhibiting Ab SM1/23 (or combinations thereof, pre-incubating 1 h with the inhibitors Z-IETD or SM1/23 when present) and checked caspase-8 activation. Plitidepsin-induced caspase-8

activation was specifically inhibited by co-treatment with Z-IETD. Furthermore, co-treatment with SM1/23 did not reduce the pro-apoptotic effect of plitidepsin, indicating that the effect of

this drug was likely due, at least in part, to the induction of Fas clustering rather than pathway activation by FasL (Fig. 5c). DISCUSSION For a long time, human _eEF1A2_ has been known to

behave as a pro-oncogenic protein, its aberrant expression increasing cancer cell fitness through the inhibition of apoptosis,12 control of misfolded proteins degradation,11 heat-shock

response,27 reorganisation of actin cytoskeleton10 and regulation of oxidative stress.8 Several interaction partners have been described for _eEF1A2_ that are important to convey its

pro-oncogenic properties, including PRDX1,8 SPHK16,7 and others. We have recently demonstrated that _eEF1A2_ is the main molecular target of plitidepsin, a marine drug currently in

development for the treatment of multiple myeloma patients.9 Here we show that PKR is a novel _eEF1A2_-interacting partner, whose pro-apoptotic activity is regulated by the elongation factor

likely through the inhibition of its kinase activity. Targeting of _eEF1A2_ with plitidepsin releases PKR from the complex, facilitating its activation and, in doing so, it triggers the

MAPK-dependent and NF-κB-dependent activation of the extrinsic apoptotic pathway. Our data stress the important role of the “moonlighting” effects of _eEF1A2_ in the maintenance of cancer

phenotype. PKR is a dsRNA-dependent serine/threonine protein kinase better known for its role in the innate immune response against viral infections.28 PKR can also be activated, in a

dsRNA-independent manner, through the PKR-associated activator (PACT) in response to oxidative stress (H2O2)29 or the second messenger ceramide.30 Given that PKR functions as a tumour

suppressor controlling cell growth and proliferation,17,31,32 it can be considered a good target for cancer therapy, the rationale being selectively activating the kinase to trigger tumour

cell apoptosis.17 Herein, we show that _eEF1A2_ binds to PKR, most likely keeping it inactive since _eEF1A2_ reduces the phosphorylation of MBP catalysed by PKR. Remarkably, upon plitidepsin

binding to _eEF1A2_, PKR is released from this complex and becomes activated, probably through several cellular stimuli such as oxidative stress or increased ceramide levels. Indeed,

through its binding to _eEF1A2_, plitidepsin could actively generate both oxidative stress, by decreasing PRDX1 activity, and increased ceramide levels, inhibiting SPHK1 and displacing the

sphingolipid equilibrium towards ceramide synthesis. Once activated, PKR is a potent apoptotic inducer.17 Induction of apoptosis by PKR has been related to the phosphorylation of eIF2α,33

the activation of MAPKs (JNK and p38)34 or the overexpression of Fas (CD95/Apo-1), a TNFR family member,24 through transactivation by NF-κB. We now show that plitidepsin binding to _eEF1A2_

does, indeed, induce hyper-phosphorylation of eIF2α in a PKR-dependent fashion, although this phosphorylation seems to be irrelevant for the antiproliferative activity of the compound given

that non-phosphorylatable eIF2αS51A homozygous MEFs are as sensitive to the drug as wt MEFs. MAPKs p38 and JNK have been shown to be activated by plitidepsin, JNK being essential for the

apoptotic effect of this drug.35 Here we show that JNK activation by plitidepsin is, at least in part, dependent on the disruption of _eEF1A2_–PKR complexes, since PKR-deficient fibroblasts

have diminished JNK activation and apoptosis in response to the compound. Furthermore, in HeLa-APL-R cells lacking _eEF1A2_, plitidepsin was unable to induce PKR self-phosphorylation and,

therefore, JNK activation was also greatly diminished. Thus, signalling through JNK is important for the apoptogenic effect of plitidepsin-activated PKR. A third way to execute PKR-induced

apoptosis is the NF-κB signalling pathway. This may appear paradoxical since, through the transactivation of a plethora of anti-apoptotic genes, NF-κB is widely accepted as one of the main

pro-survival pathways in the cell.36 Nevertheless, there is also evidence on the pro-apoptotic effect of the NF-κB pathway, through the transactivation of pro-apoptotic genes such as _Fas_,

_FasL_, _TRAIL_, _p53_ or _Bcl-xS_ (reviewed in ref.37) We now report that plitidepsin, in a PKR-dependent and _eEF1A2_-dependent way, activates the NF-κB pathway, inducing the degradation

of IκB and the nuclear translocation of NF-κB. Furthermore, plitidepsin-induced apoptosis was partially dependent on NF-κB activation, since pretreatment of HeLa cells with the IKK inhibitor

BAY11-7082 clearly diminished cell death. We show that plitidepsin blocks TNFα-induced luciferase transactivation in MDA-MB-231 cells transfected with an NF-κB-responsive luciferase

reporter plasmid. Moreover, during plitidepsin treatment in HeLa cells, most NF-κB-transactivated genes, especially those involved in growth and survival such as c-myc,38 cyclin D1,39

Bcl2,40 Mcl141 or XIAP42 were down-regulated, with the exception of pro-apoptotic Fas (CD95)22,23,43 whose expression was boosted. Fas has been shown to be involved in the pro-apoptotic

effect of plitidepsin in human leukaemic cells.25,26 Here we demonstrate that caspase-8 is activated by plitidepsin in HeLa cells, concomitant to an induction of apoptosis that can be

suppressed with the IKK inhibitor BAY11-7082 or with the PKR inhibitor C16. Thus, though NF-κB is acknowledged for the transactivation of pro-survival genes, activation of the pathway by

plitidepsin is essential for the induction of apoptosis in cancer cells. Fas clustering, rather than binding to its ligand FasL, has been previously shown to activate the extrinsic apoptotic

pathway in Jurkat T cell leukaemia cells treated with plitidepsin.26 We now extend this discovery to other cell types, demonstrating that HeLa cells treated with plitidepsin activate the

extrinsic apoptotic pathway through Fas clustering, by NF-κB transactivation of the receptor, rather than through lipid raft modification. From a structural viewpoint, the best-characterised

binding partners for PKR are eIF2α44 and the vaccinia virus protein K3L, which mimics the 3D structure of eIF2α and its mode of interaction with PKR, thereby competitively blocking eIF2α

phosphorylation on Ser51.45 The GTP-binding pocket of eIF2 is contained within the γ-subunit and the guanine nucleotide exchange factor for eIF2 is eIF2B,46 which exchanges GDP for GTP on

the γ-subunit of eIF2 and is inhibited by stress-induced phosphorylation of eIF2α.47 This α-subunit of eIF2 consists of two domains,48 the C-terminal one being structurally very similar to

the C-terminal domain of eEF1Bα, that is, the α-subunit of the guanine nucleotide exchange factor for _eEF1A2_.49 Thus, structural and functional similarities exist between eIF2α–eIF2γ and

eEF1Bα–eEF1A pairs.48 Interestingly, the crystal structure of _eEF1A2_ from rabbit muscle in complex with GDP revealed a tightly bound homodimer in the asymmetric unit,50 whereas the complex

between eIF2α and the catalytic domain of PKR disclosed a binding mode that nicely accounts for the accessibility of the (missing) loop containing Ser51 into the catalytic cleft of PKR.44 A

non-classical pathway leading to the full catalytic activation of PKR is triggered by direct interaction with PACT/RAX, a cellular protein that can heterodimerise directly with PKR.51 It

seems feasible that dissociation of the _eEF1A2_ homodimer from PKR can release the intramolecular auto-inhibition effected by the dsRNA-binding domains and lead to activation of the kinase

domain and the observed eIF2α phosphorylation. In summary, our findings shed light on several moonlighting functions of _eEF1A2_ (proposed mechanism in Fig. 6), which support the

pro-oncogenic properties of this alternative elongation factor, and uncover PKR as a new protein binding partner whose regulation may be essential for the inhibition of apoptosis. It is

tempting to think of _eEF1A2_ as an essential cell death regulator, necessary for the survival of tissues with low rates of cell division after embryonic development, mostly in brain, heart

and muscle. This would be the reason why embryonic eEF1A1 expression in those tissues is switched off in the adult and replaced by _eEF1A2_: the A2 homologue would inhibit apoptosis in

tissues with low cell renewal, helping them to maintain their special homeostasis. In cancer cells, aberrant _eEF1A2_ expression would provide resistance to cell death, an essential

advantage that would help tumour cells to overcome their intrinsic weaknesses, allowing them to grow. Plitidepsin, through its binding to _eEF1A2_, would release the apoptotic brake imposed

by this elongation factor, exposing the frailty of the tumour cells, especially when compared with healthy tissues expressing _eEF1A2_, and leading them to a PKR-dependent apoptotic cell

death. The fitness boost that these _eEF1A2_ non-canonical functions provide to cancer cells would then be important for their growth and survival. All in all, _eEF1A2_ emerges as an

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by PACT or dsRNA. _Proc. Natl. Acad. Sci. USA_ 103, 10005–10010 (2006). Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS We thank Doctors César de Haro and Juan José

Berlanga for fruitful discussions and for providing essential reagents. We also thank Rafael Sánchez for his technical assistance. The proteomic analysis was performed in the Proteomics Unit

of Complutense University of Madrid that belongs to ProteoRed, PRB2-ISCIII, supported by grant PT13/0001. F.G. is the recipient of a Spanish MEC/MICINN (SAF2015-64629-C2-2-R) grant. AUTHOR

CONTRIBUTIONS Conceptualisation and design: A.L., M.J.M.-A., M.M.-D., F.G., J.M.D., J.F.M.-L. and C.M.G.; investigation: A.L., M.J.M.-A., M.M.-D. and J.F.M.-L.; writing original draft: A.L.,

F.G., J.M.D., J.F.M.-L. and C.M.G.; editing final version: A.L., F.G., J.M.D., J.F.M.-L and C.M.G.; approving the final version: A.L., M.J.M.-A, M.M-D, F.G., J.M.D, J.F.M.-L and C.M.G.

AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Departamento de Biología Celular y Farmacogenómica, Pharma Mar S.A., Colmenar Viejo, 28770, Madrid, Spain Alejandro Losada, María José

Muñoz-Alonso, Marta Martínez-Díez, Juan Manuel Domínguez, Juan Fernando Martínez-Leal & Carlos M. Galmarini * Departamento de Ciencias Biomédicas, Unidad Asociada al IQM-CSIC,

Universidad de Alcalá, Madrid, Spain Federico Gago Authors * Alejandro Losada View author publications You can also search for this author inPubMed Google Scholar * María José Muñoz-Alonso

View author publications You can also search for this author inPubMed Google Scholar * Marta Martínez-Díez View author publications You can also search for this author inPubMed Google

Scholar * Federico Gago View author publications You can also search for this author inPubMed Google Scholar * Juan Manuel Domínguez View author publications You can also search for this

author inPubMed Google Scholar * Juan Fernando Martínez-Leal View author publications You can also search for this author inPubMed Google Scholar * Carlos M. Galmarini View author

publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to Juan Fernando Martínez-Leal. ETHICS DECLARATIONS COMPETING INTERESTS A.L.,

M.J.M.-A., M.M.-D., J.M.D. J.F.M.-L and C.M.G. are employees and shareholders of Pharma Mar S.A.; F.G. has received a research grant from Pharma Mar S.A. AVAILABILITY OF DATA AND MATERIAL

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Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Losada, A., Muñoz-Alonso, M.J., Martínez-Díez, M. _et al._ Binding of _eEF1A2_ to the RNA-dependent protein kinase PKR

modulates its activity and promotes tumour cell survival. _Br J Cancer_ 119, 1410–1420 (2018). https://doi.org/10.1038/s41416-018-0336-y Download citation * Received: 12 April 2018 *

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