TRIB2 Confers Resistance to Anti-Cancer Therapy by Activating the Serine/Threonine Protein Kinase AKT

ARTICLE

Received 23 Nov 2015 | Accepted 23 Jan 2017 | Published 9 Mar 2017 DOI: 10.1038/ncomms14687 OPEN TRIB2 confers resistance to anti-cancer therapy by activating the serine/threonine protein kinase AKT

Richard Hill1,2,3, Patricia A. Madureira2, Bibiana Ferreira2, Ineˆs Baptista2, Susana Machado2, Laura Colac¸o2, Marta dos Santos2, Ningshu Liu4, Ana Dopazo5, Selma Ugurel6, Angyal Adrienn7, Endre Kiss-Toth7, Murat Isbilen8, Ali O. Gure8 & Wolfgang Link1,2,9

Intrinsic and acquired resistance to chemotherapy is the fundamental reason for treatment failure for many cancer patients. The identiﬁcation of molecular mechanisms involved in drug resistance or sensitization is imperative. Here we report that tribbles homologue 2 (TRIB2) ablates forkhead box O activation and disrupts the p53/MDM2 regulatory axis, conferring resistance to various chemotherapeutics. TRIB2 suppression is exerted via direct interaction with AKT a key signalling protein in cell proliferation, survival and metabolism pathways. Ectopic or intrinsic high expression of TRIB2 induces drug resistance by promoting phospho- AKT (at Ser473) via its COP1 domain. TRIB2 expression is signiﬁcantly increased in tumour tissues from patients correlating with an increased phosphorylation of AKT, FOXO3a, MDM2 and an impaired therapeutic response. This culminates in an extremely poor clinical outcome. Our study reveals a novel regulatory mechanism underlying drug resistance and suggests that TRIB2 functions as a regulatory component of the PI3K network, activating AKT in cancer cells.

1 Department of Biomedical Sciences and Medicine (DCBM), University of Algarve, Campus de Gambelas, Faro 8005-139, Portugal. 2 Centre for Biomedical Research (CBMR), University of Algarve, Campus de Gambelas, Faro 8005-139, Portugal. 3 Brain Tumour Research Centre, Institute of Biomedical and Biomolecular Sciences, University of Portsmouth, PO1 2DT Portsmouth, UK. 4 Bayer AG, Drug Discovery Oncology Research, Berlin D-13342, Germany. 5 Genomics Unit, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid 28029, Spain. 6 Department of Dermatology, University Hospital Essen, Essen 45147, Germany. 7 Department of Cardiovascular Science, University of Shefﬁeld, Shefﬁeld S10 2RX, UK. 8 Department of Molecular Biology and Genetics, Bilkent University, Ankara 06533, Turkey. 9 Algarve Biomedical Center (ABC), University of Algarve, Campus de Gambelas, Faro 8005-139, Portugal. Correspondence and requests for materials should be addressed to W.L. (email: [email protected]).

NATURE COMMUNICATIONS | 8:14687 | DOI: 10.1038/ncomms14687 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14687

he emergence of drug-resistant tumour cells is a major we analysed signalling components within the PI3K/AKT net- obstacle for both conventional chemotherapeutics work and found that TRIB2 over expression increased the acti- Tas well as novel targeted therapeutics1. As FOXO vation of pSer473-AKT before and in the presence of PI3K and proteins play a key role in the action of several anticancer mTOR inhibitors (Fig. 2b). Importantly, while TRIB2 protein drugs, proteins that are capable of suppressing FOXO activity over expression significantly increased pSer473-AKT1 levels, are strong candidates to confer drug resistance2–7. Our previous isogenic cell line exposure to our inhibitor compounds reduced large scale genetic screen identified the kinase-like protein the level of pSer473-AKT, indicating that the upstream compo- Tribbles 2 (TRIB2) as a FOXO suppressor protein8, while nents of this network remain functional within these in vitro TRIB2 has also been implicated in the development and models. To support this, we transiently transfected wildtype progression of melanoma and leukaemia8–11. AKT, a phospho-mimetic AKT (AKTSer473D), wildtype In our study presented here, we describe a new regulatory FOXO3a or a phosphor-mutant FOXO3a (FOXO3a-AAA) into mechanism underlying drug resistance in different cancer entities our isogenic cell lines (Supplementary Fig. 3a,b). We note that and suggests that TRIB2 functions as a critical regulatory the over-expression of either AKTSer473D or FOXO3a-AAA component of the PI3K signalling network, activating AKT in negated the TRIB2-dependent resistance to BEZ235 treatment. cancer cells. In addition, our data support the use of TRIB2 In contrast, over-expression of either WT AKT or FOXO3a did as a biomarker for both prognosis and personalized cancer not. For this we conclude that TRIB2-mediated resistance therapy, as well as identifying this protein as a molecular target was AKT-dependent via FOXO3a. Interestingly there was no for combination cancer treatment. significant difference in protein expression for PDK1, pSer241- PDK1, RAPTOR, pSer792-RAPTOR, p70S6K and only a slightly Results increased level of pThr389-p70S6K correlated with TRIB2 TRIB2-conferred resistance to anti-cancer therapeutics. To test expression (Supplementary Fig. 4) Taken together we conclude that TRIB2-conferred resistance to PI3K and mTORC1 inhibitors our hypothesis that the TRIB2 protein confers resistance to anti-cancer drugs, we first generated and then examined the is independent of PDK1 and p70S6K. sensitivity of several stable, isogenic TRIB2 in vitro models (Supplementary Table 1; Supplementary Fig. 1) after treatment TRIB2 and AKT interact and form a protein-protein complex. with the dual PI3K/mTOR kinase inhibitor BEZ235. High-TRIB2 expression significantly increased cell resistance to BEZ235 FOXOs have been shown to be the major transcriptional effectors following PI3K inhibition13 and consistent with this finding, we treatment characterized by a significantly lower sub-G1 cell population and a reduction of caspase-3 cleavage (Fig. 1a,b). observe significantly elevated pSer235-FOXO3a protein levels and a concomitant suppression of FOXO-dependent gene expression To characterize the profile of kinases that were affected by TRIB2 status, we tested a range of inhibitors that display in cells with high-TRIB2 protein expression (Fig. 2c,d). Our distinct kinase selectivity (summarized in Fig. 1c). TRIB2 data raise the hypothesis that TRIB2-dependent FOXO protein expression significantly influenced isogenic cell line suppression is indirect and rather, could be mediated via p473- sensitivity to BAY236 (BAY 80-6946) and BAY1082439 treatment AKT1 activation. To address this hypothesis we investigated the (Fig. 1d). This response was independent of the cell cycle mTORC2/AKT/FOXO3a cascade as mTORC2 is responsible for (Supplementary Fig. 2a–c), indicating that TRIB2 reduces cell the phosphorylation of ser473 within AKT1 (ref. 14). We questioned if there was physical interaction between TRIB2, death induced by PI3K inhibitors and thus is capable of conferring resistance to these drugs. Strikingly, isogenic cell line AKT1 and pThr1135-RICTOR using co-immunoprecipitation and protein fragment complementation assays15–17. The results sensitivity to inhibition of the central effector protein AKT (ref. 12) and to the mTORC1/2 inhibitor TORIN1 was independent of showed a strong interaction between TRIB2 and AKT1 but could not observe TRIB2/RICTOR binding (Fig. 2e,f). Strikingly, the TRIB2 status (Fig. 1e). We investigated this further and exposed our isogenic cell lines to rapamycin, a specific mTORC1 inhibitor. TRIB2/AKT1 complex (including the intensity of the interaction) was as strong as the known AKT1/JIP1 interaction18. Developing Noticeably, TRIB2 status correlated with resistance to this compound (Fig. 1f). Taken together our data raises the intriguing these findings further, we addressed the functional importance of FOXO and RICTOR in TRIB2-dependent drug resistance. Four possibility that TRIB2 acts upstream of these proteins affecting AKT and mTORC2. different shRNA constructs were used to stably silence FOXO3a expression (Supplementary Fig. 5). The depletion of FOXO3a expression significantly increased in vitro resistance to PI3K TRIB2 protein level correlates with AKT activation. To inves- inhibitors regardless of TRIB2 status (Fig. 3a). Furthermore, tigate this further, we performed western blot analyses before FOXO3a depletion abolished the TRIB2-dependent difference, we and after clinically representative drug treatment with each previously observed for FOXO-regulated gene transcription in compound using our isogenic cell lines. We note a prominent our isogenic cell lines (Fig. 3b). In addition to phosphorylating TRIB2-dependent disparity in the levels of pSer473-AKT1 and directing FOXO3a for proteasome mediated degradation, and total AKT using our in vitro models. In melanoma AKT1 can also suppress apoptosis by activating the E3 ubiquitin and osteosarcoma cells with high-TRIB2 protein expression, ligase mouse double minute 2 homologue (MDM2), thus we identified significantly higher levels of total AKT and pSer473- inhibiting p53-mediated apoptosis6,19. To analyse the role of AKT and the opposite was observed in cancer cells with TRIB2 TRIB2 within this ‘arm’ of the AKT network, we broadened our depletion (Fig. 2a). Consistent with our previous data, this was study to include first line chemotherapeutics known to act not observed for pThr308-AKT1, where there were readily through p53 that are independent of FOXO (Fig. 3c, detectable levels of this AKT isoform independent of TRIB2 Supplementary Fig. 6). Remarkably, TRIB2 over expression status in each in vitro model. These data support the intriguing significantly increased cell resistance to dacarbazine (DTIC), possibility that TRIB2 might promote the further activation of gemcitabine and 5-fluorouracil (5-FU) induced cell death AKT via Ser473 phosphorylation in a PI3K and mTORC1 inde- independent of FOXO. Accordingly, high-TRIB2 expression pendent manner. In this way, drugs targeting these proteins was associated with notably elevated pSer166-MDM2 protein within the PI3K pathway are not effective in tumours where levels (Fig. 3d). Therefore, we questioned whether p53 regulation TRIB2 expression is up-regulated. To investigate this possibility, was disrupted by increased TRIB2 protein levels. We found that

2 NATURE COMMUNICATIONS | 8:14687 | DOI: 10.1038/ncomms14687 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14687 ARTICLE

ab BEZ235 U2OS Relative cleaved caspase3 Relative TRIB2

U2OS empty 80 0.0400.005 0.035 0.034 hr. post (empty-GFP) (TRIB2-GFP) 0.0238 U2OS TRIB2 0.0033 BEZ235 04812 24 012 24 48 * 60 0.0715 ** 3 0.1698 8 Pro-caspase 3 actin) ns ns 0.0046 0.5531 40 β ** 0.0027ns cells (%) 0.6090

1 Caspase 3 2 ** 4 0.3281 ns ns 20 TRIB2 1 SubG

0 Actin 0 0 Protein expression NT 12 24 48 NT 12 24 48 NT 12 24 48 (relative density/ G361 hr. post BEZ235 hr. post BEZ235 293T GFP

U2OS GFP G361 sc. shRNA 293T TRIB2 hr. post (sc. shRNA) (TRIB2 sh) 0.0029 U2OS TRIB2 ** BEZ235 04812 24 012 24 48 G361 shTRIB2 0.5220

G361 sc. shRNA 0.0647 10 10 0.0023 ns actin) 0.0004 ns 0.0002 ** Pro-caspase 3 β 0.9699 G361 TRIB2 shRNA *** SK-Mel28 sc. shRNA *** ns 0.3224 Caspase 3 0.8854 SK-Mel28 TRIB2 shRNA 5 5 ns ns TRIB2 0 0 Actin Protein expression NT 12 24 48 NT 12 24 48 NT 12 24 48 (relative density/ hr. post BEZ235 hr. post BEZ235

cd Receptor tyrosine BAY236 BAY439 kinases 50 0.009 0.002 0.001 50 40 0.003 40 0.005 30 α β 0.002 PI3K / PTEN cells (%) 30 1 20 α [PI3K IC50 4.9 nM] 20 BAY439 10

β SubG [PI3K IC50 15.0 nM] BAY931[IC 27 nM] 10 50 0 α 0 [PI3K IC50 4.0 nM] [PI3Kβ IC 75 nM] Empty 50 BEZ235 TRIB2 mTOR AKT [mTOR IC 6.0 nM] Empty TRIB2 50 TRIB2 sh TRIB2 sh sc.shRNA RICTOR RAPTOR sc.shRNA TRIB2 sh TRIB2 sh α sc.shRNA sc.shRNA [PI3K IC50 0.5 nM] β BAY236 [PI3K IC50 3.7 nM] U2OS G361 SK-MEL28 Rapamycin AKT inh.VIII U2OS G361 SK-MEL28 [IC5010 nM] TORIN1 [IC50 0.05 nM] [IC50 58 nM]

e BAY931 AKTVIII TORIN1 Rapamycin

50 0.097 50 50 50 0.001 40 40 40 40 0.033 0.0014 0.405 0.418 0.332 0.005 30 30 30 ** 0.053 30 0.002

cells (%) 0.290 0.241 1 20 20 20 20 10 10 10 10 SubG 0 0 0 0 Empty TRIB2 Empty TRIB2 Empty TRIB2 Empty TRIB2 TRIB2 Empty TRIB2 sh TRIB2 sh TRIB2 sh TRIB2 sh Scramble Scramble Scramble Scramble TRIB2 sh TRIB2 sh TRIB2 sh Scramble sc.shRNA sc.shRNA

U2OS G361 SK-MEL28 U2OS G361 SK-MEL28 U2OS 293T SK-MEL28 U2OS G361 SK-MEL28 Figure 1 | Overexpression of TRIB2 confers in vitro resistance to PI3K inhibitors. (a) Matched isogenic TRIB2 cell line FACS analysis following exposure to BEZ235 (n ¼ 6), sc.shRNA indicates scramble shRNA while TRIB2 shRNA indicates stable shRNA knockdown for TRIB2 in each cell lines. P values are indicated for each comparison by two-way analysis of variance (ANOVA) and data represent the mean±s.d. (b) (left) Higher pro-caspase3 and reduced cleavage of caspase3 in TRIB2 overexpressing cells after treatment with BEZ235, (50 mg total protein loaded per lane and separated by 10% SDS–PAGE). (right) Densitometry for cleaved caspase3 and TRIB2 under each condition. Data normalized to actin and analysed in ImageJ. Data indicates relative mean intensity±s.e.m and analysed by two-way ANOVA. (c) Schematic showing the target speciﬁcity for each compound, the inhibitory concentration

50% (IC50) and commercial name. (d) FACS analysis of TRIB2 isogenic cell lines 72 h post exposure to various pre-clinical PI3K inhibitors (n ¼ 6). (e) Isogenic TRIB2 cell line FACS analysis 72 h post-AKT, mTOR1/2 or mTOR1 inhibition (n ¼ 6). P values are indicated for each comparison by two-way analysis of variance (ANOVA) and data represent the mean±s.d.

high-TRIB2 protein expression correlated with a signiﬁcantly affecting cell line resistance to various anti-cancer agents by lower p53 protein level in the absence of DNA damage or cellular activating AKT1, inhibiting FOXO and p53. stress consistent with increased MDM2 activity (Fig. 3d). It has been previously reported that TRIB2 associated with and Concomitant to this reduced p53 protein level, we observed the inhibited the transcription factor CCAAT/enhancer-binding diminished expression of p53 target genes including p21, MDM2, protein alpha (C/EBPa)11. Gene expression analysis in our Bax and PUMA (Fig. 3e). Altogether these data highlight the isogenic cell lines revealed that high-TRIB2 expression leads to involvement of TRIB2 within the PI3K signalling network, reduced number of C/EBPa transcripts and the downregulation of

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C/EBPa target genes (Supplementary Table 5), which might proteins (Supplementary Fig. 7a)20. The DC, kinase domain (KD), contribute to the response to drug treatment. DCOP1 and the N-terminal TRIB2 constructs did not confer resistance to any of the PI3K inhibitors tested (Fig. 3g). However, the full length, DN and the C-terminal constructs did confer The COP1 domain of TRIB2 is required for AKT activation. resistance to each of the investigated therapeutics indicating To identify directly the TRIB2 functional domains that effect that the COP1 region could be driving cell line resistance. In DC, AKT activation, FOXO3a suppression and confer resistance to KD, DCOP1 and N-terminal transfected cells, we observe little to PI3K inhibitors, we employed a variety of mutated TRIB2 no change in the level of pSer473-AKT1. In contrast, in untreated

ab Osteosarcoma Melanoma U2OS

Renal Breast GFP TRIB2 GFP TRIB2 GFP TRIB2 GFP TRIB2 NT BEZ235 NT BEZ235 NT BAY236 NT BAY236 NT BAY439 NT BAY439 NT Rapa NT Rapa. TRIB2 Total-AKT pSer473-AKT

293T (TRIB2-GFP) 293T (Empty-GFP) U2OS (empty-GFP) MCF-7 MDA-489 SK-Mel28(TRIB2 sh.) SK-Mel28(sc. shRNA) UACC62 M14 A375 G361(TRIB2 shRNA) G361 (sc.shRNA) pThr308-AKT TRIB2 β-actin AKT pSer473-AKT pThr308-AKT β actin d c U2OS U2OS U2OS U2OS U2OS U2OS *** GFP TRIB2 GFP TRIB2 GFP TRIB2 ) 6 *** BEZ235 BEZ235 BAY236 BAY236 BAY439 BAY439 5 *** ns

GAPDH 4 ns 01612 01612 01612 01612 01612 01612 3 * Total AKT expression 2 pSer473-AKT 1 p27 0

pThr308-AKT (fold change/

) *** Total FOXO3a 4 *** pSer253-FOXO3a *** 3 ns FasLG GAPDH ns 2 BIM ns expression p27 1 β-actin BIM 0 (fold change/

e IP α-TRIB2 Total protein ) *** 4 *** ns U2OS 293T SK-MEL *** 3 ** TRIB2 TRIB2 28 GAPDH 2 ns expression IgG NT IgG NT IgG NT U2OS-TRIB2293T-TRIB2SK-MEL28 1 TRIB2 TRIB2

FasLG 0 Total-AKT Total-AKT (fold change/ NT NT BEZ235 BEZ235 fg BAY236 BAY439 BAY236 BAY439 BF GFP 15 U2OS U2OS *** ** ** 10 *** *** GFP TRIB2 Zip 5

V1-V2 cells (%)

YFP positive 0 AKT1 300 ** TRB2 *** *** 200 *** *** AKT1 100 JIP1 0 YFP +ve cells Signal mean of Akt1 V1 trb2 V2 Akt2 V1 trb2 V2 Akt1 V1 JIP1 V2 Akt2 V1 JIP1 V2 zip V1 V2 (+ve) zip V1 trb3 V2 (–ve)

4 NATURE COMMUNICATIONS | 8:14687 | DOI: 10.1038/ncomms14687 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14687 ARTICLE cells transfected with full length, DN or the C-terminal TRIB2 outcome of the respective patients, we noted that TRIB2 constructs, the levels of pSer473-AKT1, pSer253-FOXO3a and expression correlated with a significantly worse prognosis pSer166-MDM2 increased significantly (Fig. 3h). Alongside, these (Fig. 4d). We next investigated whether our transcription data phosphorylation events we observed a significant reduction of correlated with protein levels. First, TRIB2 protein expression was FOXO3a-dependent gene and protein expression (Supplementary distinctly increased in melanoma tissue samples compared to Fig. 7b,c). Taken together, these data support that the TRIB2 normal skin and second, the highest TRIB2 protein expression COP1 domain promotes AKT activation repressing FOXO3a and was observed in melanoma tissues samples from patients with p53 activity, thereby fuelling cellular resistance to PI3K inhibitor a poor clinical outcome (Fig. 4e; Supplementary Fig. 10). We next regimes. questioned whether there was also a de-regulated AKT signalling within our clinical samples. Consistent with both our in vitro and in vivo data, we observed significantly elevated total TRIB2 protein confers in vivo resistance to BEZ235 treatment. AKT, pSer473-AKT1, pSer253-FOXO3a and pSer166-MDM2 To address if TRIB2 over expression ablated tumour regression and no significant difference regarding pThr308-AKT1. Our following the in vivo administration of PI3K inhibitors, we study also demonstrated that pSer473-AKT1 and pSer253- established isogenic 293T subcutaneous tumours in the flanks of FOXO3a levels correlated with melanoma patient prognosis. NOD/Scid mice. Tumours formed equivalently in these mice Further examination of AKT in our clinical samples revealed that irrespective of TRIB2 status. The daily oral administration of TRIB2 and pSer473-AKT1 co-localize in melanoma cells from BEZ235 resulted in the regression of 293T-GFP xenograft patients with progressive disease (Fig. 4f). Finally, we examined tumours and a statistically significant increase in survival. In the clinical significance of our ex vivo data within large patient contrast, 293T-TRIB2 tumours were highly resistant to treatment, cohorts. Melanoma, colon and pancreatic tumour tissue samples correlating with our in vitro studies indicating that TRIB2 over were analysed based on TRIB2 expression (low Z1.5, high Z2.5 expression significantly reduced the effectiveness of BEZ235 versus normal tissue expression) and Kaplan–Meier survival treatment in vivo (Fig. 4a,b). To further support our in vitro curves plotted. For melanoma and colon cancer patients, high- analysis, we also collected (where possible) tumours from each TRIB2 expression correlated with a significantly worse clinical mouse and evaluated the protein expression profiles (Fig. 4c). We outcome (Fig. 4g,h; Supplementary Fig. 11). For pancreatic note that while there was variable overall protein expression cancer, there was only a strong trend for high-TRIB2 expression, within the various treatment groups, that 293T-TRIB2 BEZ235 primarily due to the extremely poor overall prognosis exhibited in treated tumours had significantly higher total and pSer473 AKT pancreatic cancer patients. We further analysed the GSE65904 compared to 293T-GFP BEZ235 treated tumours. In contrast data set21 addressing several clinical parameters, which when 293T-TRIB2 BEZ235 treated tumours show attenuated FasLG combined with TRIB2 expression were used to determine protein expression. if TRIB2 expression was dependent or independent of these prognostic parameters. Using a multivariate cox regression model TRIB2 status corresponds to clinical outcome. We next we note that TRIB2 transcription is a prognostic factor, questioned if these findings are representative of the clinical independent of disease stage, tissue involvement, age and situation. We collected tumour tissue samples from patients gender (Supplementary Tables 6 and 7). Furthermore, survival with melanoma, pancreatic and colon cancer prior to standard data from the GSE65904 dataset revealed a highly significant anti-cancer therapies (DTIC, gemcitabine or paclitaxel). TRIB2-dependent distant metastasis-free survival time or disease- Compared to normal tissue samples, TRIB2 transcription specific survival time response (Fig. 4g,h). Interestingly, this and TRIB2 protein expression was significantly increased in correlation to patient prognosis was only observed for TRIB2 and metastatic melanoma, primary colon and primary pancreatic not for TRIB1 or TRIB3 (Supplementary Fig. 12a–f). cancer tissue samples (Fig. 4d–f; Supplementary Fig. 8). We did not detect a significant increase in either TRIB1 or TRIB3 expression in any of the samples analysed (Supplementary Fig. 9). Discussion Concomitant with TRIB2 status, we found that pSer473-AKT1 Overall, our data reveal novel and important mechanisms and pSer253-FOXO3a protein levels were significantly higher, of TRIB2-dependent intrinsic resistance to standard chemo while the transcript and protein levels of the FOXO-dependent therapies and PI3K inhibitors. A patient tumour population genes BIM, FasLG and TRAIL were significantly lower in ex vivo with a high-TRIB2 protein level prior to treatment would be melanoma samples compared to normal control tissue samples. predicted to respond poorly to these treatments by promoting Importantly, when we classified our samples based on the clinical PI3K/PDK1-independent AKT activation (Fig. 4i). This

Figure 2 | TRIB2 protein expression correlates with AKT activation status and response to PI3K inhibition. (a) TRIB2 protein levels correlate with AKT-Ser473 phosphorylation in a broad range of model in vitro models. Representative images showing 100 mg (TRIB2), 50 mg (AKT-Ser473) total protein loaded per lane separated by 10% SDS–PAGE. sc indicates scramble shRNA within the indicated cell lines. (b) Immunoblot profiles for matched isogenic TRIB2 cell lines for the AKT signalling network. High-TRIB2 expression correlated with significantly increased post-translational modification(s)of downstream AKT targets. (c) Temporal inhibition of PI3K-dependent signalling following exposure to PI3K inhibitors. Total protein lysate of 50–200 mg was loaded per lane and separated by 6–12% SDS–PAGE. (d) FOXO3a-dependent gene expression was evaluated following 12 h treatment with PI3K inhibitors. NT indicates no treatment. *Po0.05 **Po0.01, ***Po0.005 by two-way ANOVA. Data represent the±s.d. ns indicates that the comparison was not statistically significant. (e) (left) Co-immunoprecipitation of TRIB2 or total AKT1 from indicated cell lines, (500 mg total protein lysate immunoprecipitated per lane) and separated by 8–12% SDS–PAGE. (right) Total protein levels in each indicated cell line for co-IP targets (100 mg total protein loaded per lane and separated by 8–12% SDS–PAGE). (f) Representative yellow fluorescent protein (YFP) complementation microscopy analysis assay for the assessment of the interaction between AKT1/2 and TRIB2. The leucine zipper Venus1 and Venus2 (ZIPV1-V2) constructs were used as a positive control, while the combination of TRIB3 V2 and V1 are our negative control. AKT1/2 JIP1 constructs were an additional control of a known AKTprotein/protein complex. BF indicates bright field. (g) (top) Percentage of YFP positive cells determined by FACS. (bottom) Mean fluorescent intensity of YFP positive cells. One-way ANOVA with Dunnett’s multiple comparison test, n ¼ 4(*Pr0.05, **Pr0.01, ***Pr0.001). All analysis was conducted compared to zipV1trib3V2 (lane 1).

NATURE COMMUNICATIONS | 8:14687 | DOI: 10.1038/ncomms14687 | www.nature.com/naturecommunications 5 RB lsi constructs. plasmid TRIB2 100 (GFP), (100 expression protein TRIB2 showing inﬁatdfeec a oe.Dt a nlsdb -a NV * ANOVA mean 2-way ( the (BAY1082439) by represent BAY439 analysed data or was and 80-6946) Data ANOVA (BAY BAY236 two-way noted. BEZ235, was by difference to comparison signiﬁcant exposure each subsequent for and indicated construct are values TRIB2 each of transfection after xrsinwseautdfloigTI2ioei elln ramn ihec niae hmteaetcaetfr2 h. 24 for agent chemotherapeutic indicated each with treatment (* ANOVA line 2-way cell by isogenic comparison TRIB2 following evaluated was expression hwn 50 showing hmteaetc ( chemotherapeutics nlsdb w-a NV) aarpeettemean the represent Data ANOVA). two-way by analysed 6 ( inhibitors PI3K with h 24 for treatment line cell isogenic mean and the knockdown represent ( FOXO3a (BAY1082439) data BAY439 after and or expression ANOVA (copanlisib) gene two-way BAY236 dependent by BEZ235, p53. comparison to and each exposure FOXO3a for subsequent suppressing indicated and activation, FOXO3a are AKT of via knockdown is the chemotherapeutics following analysis to FACS resistance TRIB2-mediated | 3 Figure ARTICLE gh e d abc SubG1 cells (%) Sub G cells (%) Sub G cells (%) Sub G cells (%) Gene expression 1 1 1 100 20 40 60 80 20 40 60 80 20 40 60 80 20 40 60 80 0 0 m 0

0 (fold-achange/ m ATSr7) 100 (AKT-Ser473), g MM) 100 (MDM2), g 0.0002 0.0005 GAPDH) 0.0013 U2OS sc. shRNA 0.0014 0.0003 0.0012 293T mock 0 1 2 3 4 5 FOXO3a shRNA 293T TRIB2 empty Rictor shRNA 0.016 0.006 0.021

FL 0.0804 BAY236 U2OS-empty NT BAY439 sc. shRNA BEZ235 NS U2OS NT 0.026 0.073 COP1 NS n FOXO3a shRNA 0.021 ΔN Rictor shRNA ¼ TRIB2 0.0024 0.0003 ΔC 0.0016 6). E TCGem DTIC BEZ KD 0.0021 293T sc. shRNA 0.004 0.002 0.0017 C term ** FOXO3a shRNA empty Rictor shRNA N term p21 P 0.0123 0.004 sc. shRNA 0.008 ausaeidctdfrec oprsnaddt ersn h mean the represent data and comparison each for indicated are values m 0.8296 0.0092 293T mock * 293T 0.006 0.014 FOXO3a shRNA 0.006 * MM-e16,50 (MDM2-Ser166), g 293T TRIB2 ** 0.4943 TRIB2 Rictor shRNA * 0.7783

P FL BEZ 0.6837 m r COP1 U2OS- TRIB2 * FX3-e23,100 (FOXO3a-Ser253), g ΔN 0.0114 .5 ** 0.05,

ΔC *

m KD Gene expression Gene expression Gene expression

oa rti oddprln)floigepsr oec niae IKihbtr ( inhibitor. PI3K indicated each to exposure following lane) per loaded protein total g C term

0 1 2 3 4 (vs/GAPDH) (vs/GAPDH) N term (vs/GAPDH) 1 2 3 4 1 2 3 4 0 2 4 6 0.1417

293T mock sc. 0.6443 ** P TBZDI Gem DTIC BEZ NT NS 293T TRIB2 * 0.5443 U2OS BAY236 r 0.6130 *

GFP NT FL FOXO3a shRNA ** ns 0.7855 ns ns NT ns

COP1 * .0,*** 0.001, ns ns * * ΔN 0.1223 BEZ235 NS ΔC BAY236 FasLG BIM p27 KD BAX BAY439 sc.

m C term

0.0093 NT p3 oa rti yaeprln eaae y61%SSPG.( SDS–PAGE. 6–10% by separated lane per lysate protein total (p53) g TRIB2 N term U2OS FOXO3a ** shRNA ns AUECOMMUNICATIONS NATURE ns NT ** ± ns ns ns ns ns ns

293T mock 0.5696 ** BEZ235 P ** ..( s.d.

293T TRIB2 0.8028 BAY236 BAY439 r * 0.0090 0.9118 m FL BAY439 ** 0.8286 MM-e16 rti xrsin4 ottaseto fteidctdGPtagged GFP indicated the of post-transfection h 48 expression protein (MDM2-Ser166) g COP1 .01 n aasonidctsmean indicates shown data and 0.0001) ΔN * c Δ ace sgncTI2cl ieFC nlssatr7 xouet various to exposure h 72 after analysis FACS line cell TRIB2 isogenic Matched )

C 0 1 2 3 KD SubG1 cells (%)

C term 0.2039

TBZDI Gem DTIC BEZ NT SubG cells (%)

NS 1 N term 20 40 60 80 20 40 60 80 0 0 P r 0.5192 .1 .0 0.019 0.003 0.018 0.0513 pSer253-FOXO3a U2OS GFP sc. shRNA U2OS GFP NS .5 ** 0.05, PUMA pSer166-MDM2 U2OS GFP FOXO3a shRNA U2OS TRIB2 Dazcarbine 0.7335 Dazcarbine pSer473-AKT U2OS TRIB2 shRNA 293T GFP 0.0254 U2OS TRIB2 FOXO3a shRNA

* 293T TRIB2 :48 O:1.08nom167|www.nature.com/naturecommunications | 10.1038/ncomms14687 DOI: | 8:14687 | 0.8161 β ±

P G361 sc. shRNA actin

r 293T GFP sc. shRNA GFP 0.0138

..( s.d. G361 TRIB2 shRNA 293T GFP FOXO3a shRNA 0.018 * 0.5431 .0.( 0.001. 293T TRIB2 shRNA SKMel28 sc. shRNA SKMel28 TRIB2 shRNA AUECMUIAIN O:10.1038/ncomms14687 DOI: | COMMUNICATIONS NATURE b 0 1 293T mock 2 293T TRIB2 FOXO3a shRNA uniaiera iePR(R-C)aayi fFOXO3a- of analysis (qRT-PCR) PCR time real Quantitative ) ±

293T TRIB2 20 40 60 80 0.1242 0 20 40 60 80 TBZDI Gem DTIC BEZ NT NS s.d.

FL 0 h

Δ NT

ersnaieimnbo nlsssoig50 showing analysis immunoblot Representative ) N 0.4221 ΔC U2OS GFP sc. shRNA U2OS GFP 0.027 0.011 0.031 P COP1 0.0373 Gemcitabine U2OS GFP FOXO3a shRNA Gemcitabine U2OS TRIB2 * ausaesonfrec oprsnweeno where comparison each for shown are values 0.7579 ± C term MDM2 N term U2OS TRIB2 shRNA 293T GFP ..( s.d. ± KD U2OS TRIB2 FOXO3a shRNA 0.0057 293T TRIB2 ..( s.d. ** 0.5101 G361 sc. shRNA 293T GFP sc. shRNA d G361 TRIB2 shRNA ersnaieimnbo analysis immunoblot Representative I ) 293T GFP FOXO3a shRNA 0.8291 0.029 0.0216 f 293T TRIB2 shRNA SKMel28 sc. shRNA ersnaieimnbo analysis immunoblot Representative ) * 293T TRIB2 FOXO3a shRNA SKMel28 TRIB2 shRNA n ¼ ( a pSer166 β TRIB2 ) (* 6), ace sgncTI2cl line cell TRIB2 isogenic Matched ) actin MDM2 MDM2 β actin f p53 g P ASaayi f293Tcells of analysis FACS ) r P (TRIB2)

NT U2OS ausaesonfor shown are values e .5 ** 0.05, 293T (TRIB2-GFP)

5-eedn gene p53-dependent ) BEZ235 BAY236 293T (Empty-GFP) BAY439 U2OS (TRIB2-GFP) U2OS (empty-GFP) P n (Empty)

NT U2OS r ¼ n BEZ235 .1adwere and 0.01 SK-Mel28(TRIB2 shRNA) ¼ )fr7 h. 72 for 6) BAY236 SK-Mel28(sc. shRNA) 6). BAY439 G361(TRIB2 shRNA)

P G361 (sc. shRNA) values m g P NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14687 ARTICLE

AKT activation (and subsequent inactivation of FOXO and p53) speciﬁc PI3K inhibitor therapeutics, rendering TRIB2 as a may also be highly relevant for other therapeutics that target suitable biomarker predicting treatment outcome and selecting the upstream PI3K/AKT network such as trastuzumab approved patients for individualized therapy. Furthermore, TRIB2 for the treatment of breast and gastric cancers and cetuximab inhibitors might synergize with current therapies targeting the for the treatment of colon cancer22. Thus, patients whose PI3K/AKT pathway hereby improving treatment outcome and tumours display high-TRIB2 expression may not beneﬁt from patient survival.

ab c 293T GFP Vehicle Tumour

100 ) 5,000 3 293T GFP BEZ235 293T-GFP 293T-GFP 293T-TRIB2 80 4,000 293T TRIB2 BEZ235 Vehicle BEZ235 BEZ235 60 0.0339 * 3,000 0.6459 NS 0.0083 ** 1 2345 123 12345 40 2,000 0.0299 * Total AKT 0.0062 ** Ser473 AKT 20 1,000 0.6374 ns Total FOXO3a

Survival (percentage) Ser253 FOXO3a 0 Tumour volume (mm 0 Total p53 0 5 10 15 20 25 135791113 FasLG Time post treatment (days) Time post treatment (days) Cleaved Caspase3 β actin

d < 0.0001 Normal *** Normal 12.0 Metastatic melanoma 15.0 *** Complete response ) ) < 0.0001 *** 10.0 Stable disease < 0.0001 *** < 0.0001 < 0.0001 *** *** *** Progressive disease

GAPDH *** 0.0003 0.1010 GAPDH 8.0 < 0.0001 *** < 0.0001 NS 5.0

3.0 ** ** *** ** *** ** * *

2.0 ** ** * * *

2.0 * Gene expression 1.0 Gene expression *

(fold change/ 1.0 (fold change/ *** 0.0 0.0 TRIB2 BIM p27 p16 p21 FasLG TRAIL AKT MDM2 TRIB2 BIM p27 p16 p21 FasLG TRAIL AKT MDM2 efComplete Stable Normal tissue response disease Progressive disease. Progressive disease.

TRIB2 TRIB2 + Total AKT pSer473 pSer473 Merged AKT TRIB2 AKT p-Ser473 AKT p-Thr308 AKT Total FOXO3a 63x pSer253-FOXO3a MDM2 pSer166-MDM2 Total p53 100x BIM FasLG β actin

g Melanoma TRIB2 i Low TRIB2 expression High TRIB2 expression GSE65904 Receptor tyrosine Receptor tyrosine kinases kinases 1.0 High expression 0.8 Low expression P = <0.001 PI3K PI3K 0.6 PI3K inhibitor treatment PI3K inhibitor treatment (for example BEZ235) (for example BEZ235) 0.4 mTOR mTOR RICTOR Apoptosis. RICTOR Survival.

0.2 Tumour regression. p473 Tumour growth. COP1 Cumulative survival AKT clinical response. AKT Treatment failure. 0.0 TRIB2 0 2,000 4,000 6,000 Distant metastasis-free MDM2 FOXO3a p166 MDM2 FOXO3a p253 h survival (days)

p27 BIM FasLG 1.0 High expression p27 BIM FasLG 0.8 Low expression FOXO3a P = 0.005 0.6 p21 Bax PUMA p21 Bax PUMA 0.4 p53 p53 0.2 Cumulative survival 0.0 0 2,000 4,000 6,000 Disease specific survival (days)

NATURE COMMUNICATIONS | 8:14687 | DOI: 10.1038/ncomms14687 | www.nature.com/naturecommunications 7 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14687

Online Content, Any additional Methods, Extended Data Western blot analysis. For the preparation of whole cell lysate, cells were display items and Source Data are available in the online version harvested and lysed using RIPA buffer (50 mM Tris–HCl pH 7.4, 1% NP-40, of the paper; references unique to these sections appear only in 0.5% Na-deoxychlorate, 150 mM NaCl, 1 mM EDTA, 2 mM NaF, 2 mM NaVO4 and 1 Â protease inhibitor cocktail (PIC) (Sigma). For SDS–polyacrylamide gel the online paper. electrophoresis (SDS–PAGE), protein samples were boiled for 5–10 min in protein sample buffer (50 mM Tris pH 6.8, 1% SDS, 10% glycerol, 0.01% Bromophenol Methods Blue, b mercaptoethanol (50 ml per 950 ml sample buffer)). Following electrophoresis, proteins were transferred onto nitrocellulose membrane (BioRad). Tissue samples. Surgically excised tumour tissue samples from colon cancer The membrane was blocked for 1 h at room temperature or overnight at primaries, pancreatic cancer primaries and melanoma metastases were obtained 4 °C 5% BSA 0.1% tween20 blocking buffer. Primary antibodies were added from patients prior to first-line systemic therapy. Core tumour samples of colon to the membrane (Supplementary Table 2) overnight at 4 °C or for 2 h at room carcinoma were obtained from patients with colon cancer at the time of their temperature. Secondary antibody was added (Santa Cruz Biotechnology) at surgery. For each colon cancer patient, a section of matched normal colon typically 1:5,000 dilution for 1 h at room temperature. Visualization of signal tissue was extracted adjacent to the tumour site. Pancreatic tumour tissue was achieved using a ChemiDocXRS þ Imaging System (BioRad). Full original (and matched normal tissue) was obtained during surgery (employing the Whipple membranes are shown in Supplementary Figs 13–25. surgical methodology). Melanoma tumour samples were obtained from metastatic lesions from patients with advanced melanoma in AJCC stage IV. Informed consent was obtained for the use of these samples. The samples were freshly frozen and cryo-preserved until processing. Before analysis, the frozen tissue samples were Co-immunoprecipitation. Co-immunoprecipitations (Co-IP) were performed as split into smaller sections for RNA extraction using TRI-Reagent (Sigma). The described in (ref. 18). Cells were lysed in cold lysis buffer (50 mM Tris-Cl at pH 7.4, clinical data of the corresponding patients were extracted from the patient files. 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.25% sodium deoxycholate, protease inhibitor mixture). Cell extracts (500 mg) were incubated with the first antibodies (Supplementary Table 2) or control normal IgG on a rotator overnight at 4 °C, Cell lines and reagents. The human cell lines SK-Mel28, A375, N14, G361, followed by addition of protein G magnetic beads (Invitrogen) for 2 h at 4 °C. UACC62, M14 (melanoma), HEK293T (renal cell carcinoma), U2OS [p53 þ / þ ], Beads were then washed four times using the lysis buffer. The immune complexes Soas2 [p53 À / À ] (osteosarcoma), MCF-7 and MDA-MB231 (breast cancer) were were subjected to SDS–PAGE followed by immunoblotting with the secondary maintained in DMEM supplemented with 10% FBS (Sigma, PT) and antibiotics antibody. (Gibco, US). Cell lines were mycoplasma tested monthly within the CBMR by PCR (LookOut Mycoplasma PCR Detection Kit, SIGMA, PT). All cell lines are indicated in Supplementary Table 1. Antibodies shown in Supplementary Table 2 Quantitative Real time PCR (qRT-PCR). Total RNA was extracted by using were used for our immunoblots and signal visualization was achieved using a TRI-reagent (Sigma, PT). cDNA was generated using NZY First-Strand cDNA ChemidocXRS þ system (BioRad, PT). Dacarbazine (Sigma, PT), gemcitabine Synthesis Kit (NZYTech, PT). Real Time PCR was performed on a BioRad CFX96 hydrochloride (Eli Lilly #VL7502), AKT inhibitor VIII (Calbiochem, US), BEZ235 quantitative real time PCR machine using the SYBR Green JumpStart master (Novartis, US), BAY236, (BAY 80-6946) BAY439 (BAY1082439), BAY1001931 mix (Sigma, PT). The primer sequences (NZYTech, PT) for measuring p16, p27, (a gift from Bayer AG, Germany), rapamycin (SIGMA, PT), actinomycin D FasLG, Bim, TRAIL, p21, Bax, PUMA, MDM2, TRIB1, TRIB2, TRIB3 and GAPDH (Sigma, PT), and cyclohexamide (Sigma, PT) and MG132 (Sigma, PT) were used are shown in Supplementary Table 3. Data analysis was carried out using the at concentrations described in the text. 2 À DDCT method24.

Constructs and shRNA. A 3062 bp fragment encoding the entire human Trib2 (hTRIB2) cDNA was sub-cloned into pEGFP-N1 or pIREPuro2 plasmids, Flow cytometric cell cycle analysis. Cells were grown to 70% confluence. Cells (one co-expressing GFP, the other containing a V5 tag.). Full length human TRIB2 were mock treated/exposed to each compound for the time points indicated. (hTRIB2, 1-343 aa), dN (63-343aa hTRIB2, dC (1-304 aa) hTRIB2, KD (63-304 aa) Samples were collected, washed (PBS) and fixed (70% ethanol). Ethanol was hTRIB2, NT (DCT, 1–250 aa) hTRIB2, CT (DNT, 270-343 aa) hTRIB2 and DCOP1 removed and samples were resuspended in PBS. Propidium iodide (2.5 mg ml) was hTRIB2 were kindly provided by W.S. Pear. Each was cloned into pMigR1-myc added to each sample. Samples were run on a Fluorescence Activated Cell Scanner plasmid as described in ref. 20. Full length pAKT1-S473D and pCMV6HA-mAKT1 (FACS) and the percentage populations (sub-G1,G1, S and G2 phases) determined. were kindly provided by A. Newton. pECE-Foxo3a-(A)3 and pCLne-wt-Foxo3a 50,000 total events were scored per study Data was analysed using Infinicyt were kindly provided by M. Greenberg. All complementary DNAs (cDNAs) were (Cytognos). sequenced in their entirety to verify there were no mutations in any of our constructs. FOXO3a shRNA constructs originated from the NKI library, FOXO3a- 826, FOXO3a-827 and FOXO3a-828 were cloned into pRetroSuper. hTRIB2 In vivo studies. In vivo efficacy studies were performed using 293T-GFP and shRNA-917 or hTRIB2-918 were sub-cloned into pRetroSuper. Each construct was 293T-TRIB2 cells injected subcutaneously in the flank of female NOD/SCID mice. transfected into indicated cell lines for selection and screening. Full sequences are Animals were treated with either vehicle alone (20% N-Methyl-2-pyrrolidone provided in our Supplementary Information. For our protein fragment com- (NMP) 80% polyethylene glycol (PEG) 300) or BEZ235 (30 mg kg À 1 by oral plementation assays, cells were co-transfected with the Venus plasmid constructs23 gavage. Treatment was administered daily for the indicated length of time. for AKT1 (1032-2471aa), AKT2 (1032-2477aa) or TRIB2 (1012-2042aa). YFP Tumours were measured manually by calliper daily. All in vivo modelling signal was quantified using flow cytometry (FacsCalibur, Becton Dickinson). The was carried out according to guidelines, regulations set out in Portuguese law images of the interactions were captured by a Leica DMI 4000B inverted (Portaria 1005/02 and Portaria 1131/97), which transcribes the European Guideline fluorescence microscope. 86/609/EC and approved by the CBMR Biote´rio ethics board.

Figure 4 | TRIB2-conferred resistance to PI3K inhibitors in vivo and ex vivo and correlates with poor clinical prognosis. (a) Kaplan–Meier analysis of isogenic TRIB2 cell lines grow subcutaneously in NOD/Scid mice treated with vehicle (n ¼ 7), of BEZ235 (n ¼ 7). The presence of TRIB2 significantly reduced survival (log rank P ¼ 0.033) when treated daily with BEZ235. (b) TRIB2 overexpressing 293T tumours (n ¼ 7) show little to no response to daily administration of BEZ235 compared to isogenic lines with endogenous (low, n ¼ 7) TRIB2 expression. (c) Representative immunoblot analysis of key proteins of interest from in vivo treated tumours. Only three tumours were present from the 293T-empty-BEZ235 treatment group. Red line (on 293-TRIB2 treated immunoblots highlight bands from the same membrane that were spliced due to lane gaps left on the original full immunoblot. Original full membranes are provided in Supplementary Information; Supplementary Fig. 19). (d) (left panel) Quantitative real time PCR (qRT-PCR) analysis of gene expression in ex vivo metastatic melanoma samples (n ¼ 20) versus normal control tissue (n ¼ 10). Data were analysed by two-way ANOVA) and values represent the mean±s.d. (right panel), Quantitative real time PCR (qRT-PCR) analysis of gene expression in ex vivo metastatic melanoma samples (n ¼ 20) versus normal control tissue (n ¼ 10) classified by clinical response to first line chemotherapy (complete response n ¼ 5, stable disease n ¼ 5 or progressive disease n ¼ 10). Data were analysed by two-way ANOVA) *Pr0.05, **Pr0.01, ***Pr0.001) and values represent the mean±s.d. (e) Representative immunoblot analysis of the AKT signalling cascade from ex vivo metastatic melanoma samples compared to normal tissue samples. (f) Representative immunofluorecent images of metastatic melanoma dual stained for TRIB2 and pSer473-AKT1 demonstrating co-staining and interaction. (g) Kaplan–Meier analysis from melanoma patients in the GSE65904 dataset classified based on low (Z1.5 fold) or high (Z2.5 fold) TRIB2 expression. Log-rank tests reveal that TRIB2 expression is highly significant for patient prognosis with median survival for low (4,450 days) and high (394 days) expression respectively. (h) Kaplan–Meier analysis for metastasis-free survival based on low (Z1.5 fold) or high (Z2.5 fold) TRIB2 expression (i) Proposed model of TRIB2- mediated drug resistance.

8 NATURE COMMUNICATIONS | 8:14687 | DOI: 10.1038/ncomms14687 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14687 ARTICLE

Fixing and staining samples for immunofluorescence. To fix samples/cells, 19. Mayo, L. D. & Donner, D. B. A phosphatidylinositol 3-kinase/Akt pathway slides/coverslips were fixed and permeabilised for 15 min in dry methanol at promotes translocation of Mdm2 from the cytoplasm to the nucleus. Proc. Natl À 20 °C and rehydrated in PBS. Samples/cells were blocked (donkey serum 1:30) Acad. Sci. USA 98, 11598–11603 (2001). for 1 h at 37 °C in a humified chamber incubated with primary antibody 20. Keeshan, K. et al. Transformation by Tribbles homolog 2 (Trib2) requires (either Anti-pSer473AKT1 or TRIB2) for 1 h at 37 °C in a humified chamber both the Trib2 kinase domain and COP1 binding. Blood 116, 4948–4957 followed by PBS washing and incubation with a fluorescent secondary antibody (2010). (Molecular Probes A21207 or A11055) for 1 h at 37 °C in a humified chamber. 21. Cirenajwis, H. et al. Molecular stratification of metastatic melanoma using gene Antibody solutions were made in PBS (Sigma). Following labelling procedures, expression profiling: Prediction of survival outcome and benefit from molecular samples/cells were mounted on glass slides in Clear Mount mounting solution targeted therapy. Oncotarget 6, 12297–12309 (2015). (Invitrogen). 22. Porta, C., Paglino, C. & Mosca, A. Targeting PI3K/Akt/mTOR Signaling in Cancer. Front Oncol. 4, 64 (2014). Statistical analysis. Statistical significance was assessed by two-way analysis of 23. Nagai, T. et al. A variant of yellow fluorescent protein with fast and variance (ANOVA) or the two-tailed Students t-test. Statistical significance was efficient maturation for cell-biological applications. Nat. Biotechnol. 20, 87–90 defined as PZ0.05. Results are expressed as the mean±s.d. or s. e. of the mean (2002). (s.e.m.) and are described in each figure legend when applied. 24. Schmittgen, T. D. & Livak, K. J. Analyzing real-time PCR data by the comparative C(T) method. Nat. Protoc. 3, 1101–1108 (2008). Data availability. The authors declare that the data supporting the findings of this study are available within the paper and its Supplementary Information files. Acknowledgements This work was supported by a grant from Bayer A.G. (Grants4Target 2012-08-0765) References and by Fundaçaõ para a Cieˆncia e a Tecnologia (FCT) Research Center Grant 1. Butler, E. B., Zhao, Y., Munoz-Pinedo, C., Lu, J. & Tan, M. Stalling the engine UID/BIM/04773/2013 Centre for Biomedical Research 1334. R. Hill is the recipient of resistance: targeting cancer metabolism to overcome therapeutic resistance. of a FCT 2012 research grant (SFRH/BPD/84634/2012). The research performed by Cancer Res. 73, 2709–2717 (2013). P.A. Madureira is a recipient of an FCT Investigator Program contract (ref: IF/00614/ 2. Berns, K. et al. 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Dissociation of Akt1 from its negative regulator JIP1 is To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ mediated through the ASK1-MEK-JNK signal transduction pathway during metabolic oxidative stress: a negative feedback loop. J. Cell Biol. 170, 61–72 (2005). r The Author(s) 2017

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