TRIB2 confers resistance to anti-cancer therapy

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Received 23 Nov 2015 | Accepted 23 Jan 2017 | Published 9 Mar 2017

Richard Hill1,2,3, Patricia A. Madureira2, Bibiana Ferreira2, Ins Baptista2, Susana Machado2, Laura Colao2, 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 identication 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 signicantly 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 Shefeld, Shefeld 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: mailto:[email protected]

Web End [email protected] ).

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DOI: 10.1038/ncomms14687 OPEN

TRIB2 confers resistance to anti-cancer therapy by activating the serine/threonine protein kinase AKT

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14687

The emergence of drug-resistant tumour cells is a major obstacle for both conventional chemotherapeutics as well as novel targeted therapeutics1. As FOXO

proteins play a key role in the action of several anticancer drugs, proteins that are capable of suppressing FOXO activity are strong candidates to confer drug resistance27. Our previous large scale genetic screen identied the kinase-like protein Tribbles 2 (TRIB2) as a FOXO suppressor protein8, while TRIB2 has also been implicated in the development and progression of melanoma and leukaemia811.

In our study presented here, we describe a new regulatory mechanism underlying drug resistance in different cancer entities and suggests that TRIB2 functions as a critical regulatory component of the PI3K signalling network, activating AKT in cancer cells. In addition, our data support the use of TRIB2 as a biomarker for both prognosis and personalized cancer therapy, as well as identifying this protein as a molecular target for combination cancer treatment.

ResultsTRIB2-conferred resistance to anti-cancer therapeutics. To test our hypothesis that the TRIB2 protein confers resistance to anti-cancer drugs, we rst generated and then examined the sensitivity of several stable, isogenic TRIB2 in vitro models (Supplementary Table 1; Supplementary Fig. 1) after treatment with the dual PI3K/mTOR kinase inhibitor BEZ235. High-TRIB2 expression signicantly increased cell resistance to BEZ235 treatment characterized by a signicantly lower sub-G1 cell population and a reduction of caspase-3 cleavage (Fig. 1a,b). To characterize the prole of kinases that were affected by TRIB2 status, we tested a range of inhibitors that display distinct kinase selectivity (summarized in Fig. 1c). TRIB2 protein expression signicantly inuenced isogenic cell line sensitivity to BAY236 (BAY 80-6946) and BAY1082439 treatment (Fig. 1d). This response was independent of the cell cycle (Supplementary Fig. 2ac), indicating that TRIB2 reduces cell death induced by PI3K inhibitors and thus is capable of conferring resistance to these drugs. Strikingly, isogenic cell line sensitivity to inhibition of the central effector protein AKT (ref.12) and to the mTORC1/2 inhibitor TORIN1 was independent of TRIB2 status (Fig. 1e). We investigated this further and exposed our isogenic cell lines to rapamycin, a specic mTORC1 inhibitor. Noticeably, TRIB2 status correlated with resistance to this compound (Fig. 1f). Taken together our data raises the intriguing possibility that TRIB2 acts upstream of these proteins affecting AKT and mTORC2.

TRIB2 protein level correlates with AKT activation. To investigate this further, we performed western blot analyses before and after clinically representative drug treatment with each compound using our isogenic cell lines. We note a prominent TRIB2-dependent disparity in the levels of pSer473-AKT1 and total AKT using our in vitro models. In melanoma and osteosarcoma cells with high-TRIB2 protein expression, we identied signicantly higher levels of total AKT and pSer473-AKT and the opposite was observed in cancer cells with TRIB2 depletion (Fig. 2a). Consistent with our previous data, this was not observed for pThr308-AKT1, where there were readily detectable levels of this AKT isoform independent of TRIB2 status in each in vitro model. These data support the intriguing possibility that TRIB2 might promote the further activation of AKT via Ser473 phosphorylation in a PI3K and mTORC1 independent manner. In this way, drugs targeting these proteins within the PI3K pathway are not effective in tumours where TRIB2 expression is up-regulated. To investigate this possibility,

we analysed signalling components within the PI3K/AKT network and found that TRIB2 over expression increased the activation of pSer473-AKT before and in the presence of PI3K and mTOR inhibitors (Fig. 2b). Importantly, while TRIB2 protein over expression signicantly increased pSer473-AKT1 levels, isogenic cell line exposure to our inhibitor compounds reduced the level of pSer473-AKT, indicating that the upstream components of this network remain functional within these in vitro models. To support this, we transiently transfected wildtype AKT, a phospho-mimetic AKT (AKTSer473D), wildtype FOXO3a or a phosphor-mutant FOXO3a (FOXO3a-AAA) into our isogenic cell lines (Supplementary Fig. 3a,b). We note that the over-expression of either AKTSer473D or FOXO3a-AAA negated the TRIB2-dependent resistance to BEZ235 treatment. In contrast, over-expression of either WT AKT or FOXO3a did not. For this we conclude that TRIB2-mediated resistance was AKT-dependent via FOXO3a. Interestingly there was no signicant difference in protein expression for PDK1, pSer241-PDK1, RAPTOR, pSer792-RAPTOR, p70S6K and only a slightly increased level of pThr389-p70S6K correlated with TRIB2 expression (Supplementary Fig. 4) Taken together we conclude that TRIB2-conferred resistance to PI3K and mTORC1 inhibitors is independent of PDK1 and p70S6K.

TRIB2 and AKT interact and form a protein-protein complex. FOXOs have been shown to be the major transcriptional effectors following PI3K inhibition13 and consistent with this nding, we observe signicantly elevated pSer235-FOXO3a protein levels and a concomitant suppression of FOXO-dependent gene expression in cells with high-TRIB2 protein expression (Fig. 2c,d). Our data raise the hypothesis that TRIB2-dependent FOXO suppression is indirect and rather, could be mediated via p473-AKT1 activation. To address this hypothesis we investigated the mTORC2/AKT/FOXO3a cascade as mTORC2 is responsible for the phosphorylation of ser473 within AKT1 (ref. 14). We questioned if there was physical interaction between TRIB2, AKT1 and pThr1135-RICTOR using co-immunoprecipitation and protein fragment complementation assays1517. The results showed a strong interaction between TRIB2 and AKT1 but could not observe TRIB2/RICTOR binding (Fig. 2e,f). Strikingly, the TRIB2/AKT1 complex (including the intensity of the interaction) was as strong as the known AKT1/JIP1 interaction18. Developing these ndings further, we addressed the functional importance of FOXO and RICTOR in TRIB2-dependent drug resistance. Four different shRNA constructs were used to stably silence FOXO3a expression (Supplementary Fig. 5). The depletion of FOXO3a expression signicantly increased in vitro resistance to PI3K inhibitors regardless of TRIB2 status (Fig. 3a). Furthermore, FOXO3a depletion abolished the TRIB2-dependent difference, we previously observed for FOXO-regulated gene transcription in our isogenic cell lines (Fig. 3b). In addition to phosphorylating and directing FOXO3a for proteasome mediated degradation, AKT1 can also suppress apoptosis by activating the E3 ubiquitin ligase mouse double minute 2 homologue (MDM2), thus inhibiting p53-mediated apoptosis6,19. To analyse the role of TRIB2 within this arm of the AKT network, we broadened our study to include rst line chemotherapeutics known to act through p53 that are independent of FOXO (Fig. 3c, Supplementary Fig. 6). Remarkably, TRIB2 over expression signicantly increased cell resistance to dacarbazine (DTIC), gemcitabine and 5-uorouracil (5-FU) induced cell death independent of FOXO. Accordingly, high-TRIB2 expression was associated with notably elevated pSer166-MDM2 protein levels (Fig. 3d). Therefore, we questioned whether p53 regulation was disrupted by increased TRIB2 protein levels. We found that

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a b

BEZ235

0.040 0.035

0.005 0.034

Relative cleaved caspase3

Relative TRIB2

SubG 1cells (%)

U2OS

0 48

12 24 0 48

12 24

hr. post

TRIB2

Actin

(empty-GFP)

(TRIB2-GFP)

U2OS empty

U2OS TRIB2

Protein expression (relative density/ actin)

BEZ235

0.0033 **

Pro-caspase 3

Caspase 3

0.1698 ns

0.0715 ns

0.0238 *

0.0046 **

0.5531 ns

0.6090 ns

0.0027 **

0.3281 ns

TRIB2

Actin

0 NT 12 24 48

0 NT 12 24 48 NT 12 24 48

U2OS GFP

U2OS TRIB2

293T GFP

293T TRIB2

G361 sc. shRNA

G361 TRIB2 shRNA

SK-Mel28 sc. shRNA

SK-Mel28 TRIB2 shRNA

hr. post BEZ235 hr. post BEZ235

hr. post

G361

0 48

12 24 0 48

12 24

G361 sc. shRNA

G361 shTRIB2

(sc. shRNA) (TRIB2 sh)

Protein expression

(relative density/ actin)

0.0023 **

0.5220 ns

0.0647 ns

Pro-caspase 3

Caspase 3

0.0002 ***

0.0004 ***

0.0029 **

0.9699 ns

0.8854 ns

0.3224 ns

0 NT 12 24 48

0 NT 12 24 48 NT 12 24 48

hr. post BEZ235 hr. post BEZ235

c d

Receptor tyrosine

BAY236

BAY439

50 0.002

0.003

0.001

SubG 1cells (%)

0.005

0.009

0.002

[PI3K IC

[PI3K IC50

[PI3K IC

[PI3K IC [mTOR IC

Empty

TRIB2

sc.shRNA

TRIB2 sh

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TRIB2 sh

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BAY931

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AKTVIII

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TORIN1

Rapamycin

SubG 1cells (%)

40 0.033

0.241

0.332

40 0.418

0.290

0.097

40 0.0014 **

0.405

0.053

30 0.005 0.002

0.001

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TRIB2

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Scramble

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TRIB2

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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 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 means.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% SDSPAGE).

(right) Densitometry for cleaved caspase3 and TRIB2 under each condition. Data normalized to actin and analysed in ImageJ. Data indicates relative mean intensitys.e.m and analysed by two-way ANOVA. (c) Schematic showing the target specicity for each compound, the inhibitory concentration50% (IC50) and commercial name. (d) FACS analysis of TRIB2 isogenic cell lines 72 h post exposure to various pre-clinical PI3K inhibitors (n 6).

(e) Isogenic TRIB2 cell line FACS analysis 72 h post-AKT, mTOR1/2 or mTOR1 inhibition (n 6). P values are indicated for each comparison by two-way

analysis of variance (ANOVA) and data represent the means.d.

high-TRIB2 protein expression correlated with a signicantly lower p53 protein level in the absence of DNA damage or cellular stress consistent with increased MDM2 activity (Fig. 3d). Concomitant to this reduced p53 protein level, we observed the diminished expression of p53 target genes including p21, MDM2, Bax and PUMA (Fig. 3e). Altogether these data highlight the involvement of TRIB2 within the PI3K signalling network,

affecting cell line resistance to various anti-cancer agents by activating AKT1, inhibiting FOXO and p53.

It has been previously reported that TRIB2 associated with and inhibited the transcription factor CCAAT/enhancer-binding protein alpha (C/EBPa)11. Gene expression analysis in our isogenic cell lines revealed that high-TRIB2 expression leads to reduced number of C/EBPa transcripts and the downregulation of

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C/EBPa target genes (Supplementary Table 5), which might contribute to the response to drug treatment.

The COP1 domain of TRIB2 is required for AKT activation. To identify directly the TRIB2 functional domains that effect AKT activation, FOXO3a suppression and confer resistance to PI3K inhibitors, we employed a variety of mutated TRIB2

proteins (Supplementary Fig. 7a)20. The DC, kinase domain (KD), 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 resistance to each of the investigated therapeutics indicating that the COP1 region could be driving cell line resistance. In DC,

KD, DCOP1 and N-terminal transfected cells, we observe little to no change in the level of pSer473-AKT1. In contrast, in untreated

a b

U2OS

Osteosarcoma

Breast

Melanoma

Renal

GFP TRIB2

293T (TRIB2-GFP)

MCF-7

U2OS (empty-GFP)

MDA-489

293T (Empty-GFP)

SK-Mel28(sc. shRNA)

SK-Mel28(TRIB2 sh.)

G361(TRIB2 shRNA)

G361 (sc.shRNA)

BEZ235

GFP TRIB2

BAY236

GFP TRIB2

BAY439

GFP TRIB2

Rapa

Rapa.

M14

UACC62

A375

TRIB2 Total-AKT pSer473-AKT

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pThr308-AKT

TRIB2

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pSer473-AKT pThr308-AKT

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BAY439

U2OS

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p27expression

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pThr308-AKT

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0 1 6 12

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pSer473-AKT

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Total FOXO3a

BIM

FasLG

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IP -TRIB2

IgG NT IgG NT IgG NT

U2OS TRIB2

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SK-MEL 28

U2OS-TRIB2

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TRIB2 Total-AKT

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BAY236

BAY439

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AKT1

JIP1

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Akt1 V1 trb2 V2

Akt2 V1 trb2 V2

Akt1 V1 JIP1 V2

Akt2 V1 JIP1 V2

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cells transfected with full length, DN or the C-terminal TRIB2 constructs, the levels of pSer473-AKT1, pSer253-FOXO3a and pSer166-MDM2 increased signicantly (Fig. 3h). Alongside, these phosphorylation events we observed a signicant reduction of FOXO3a-dependent gene and protein expression (Supplementary Fig. 7b,c). Taken together, these data support that the TRIB2 COP1 domain promotes AKT activation repressing FOXO3a and p53 activity, thereby fuelling cellular resistance to PI3K inhibitor regimes.

TRIB2 protein confers in vivo resistance to BEZ235 treatment. To address if TRIB2 over expression ablated tumour regression following the in vivo administration of PI3K inhibitors, we established isogenic 293T subcutaneous tumours in the anks of NOD/Scid mice. Tumours formed equivalently in these mice irrespective of TRIB2 status. The daily oral administration of BEZ235 resulted in the regression of 293T-GFP xenograft tumours and a statistically signicant increase in survival. In contrast, 293T-TRIB2 tumours were highly resistant to treatment, correlating with our in vitro studies indicating that TRIB2 over expression signicantly reduced the effectiveness of BEZ235 treatment in vivo (Fig. 4a,b). To further support our in vitro analysis, we also collected (where possible) tumours from each mouse and evaluated the protein expression proles (Fig. 4c). We note that while there was variable overall protein expression within the various treatment groups, that 293T-TRIB2 BEZ235 treated tumours had signicantly higher total and pSer473 AKT compared to 293T-GFP BEZ235 treated tumours. In contrast 293T-TRIB2 BEZ235 treated tumours show attenuated FasLG protein expression.

TRIB2 status corresponds to clinical outcome. We next questioned if these ndings are representative of the clinical situation. We collected tumour tissue samples from patients with melanoma, pancreatic and colon cancer prior to standard anti-cancer therapies (DTIC, gemcitabine or paclitaxel). Compared to normal tissue samples, TRIB2 transcription and TRIB2 protein expression was signicantly increased in metastatic melanoma, primary colon and primary pancreatic cancer tissue samples (Fig. 4df; Supplementary Fig. 8). We did not detect a signicant increase in either TRIB1 or TRIB3 expression in any of the samples analysed (Supplementary Fig. 9). Concomitant with TRIB2 status, we found that pSer473-AKT1 and pSer253-FOXO3a protein levels were signicantly higher, while the transcript and protein levels of the FOXO-dependent genes BIM, FasLG and TRAIL were signicantly lower in ex vivo melanoma samples compared to normal control tissue samples. Importantly, when we classied our samples based on the clinical

outcome of the respective patients, we noted that TRIB2 expression correlated with a signicantly worse prognosis (Fig. 4d). We next investigated whether our transcription data correlated with protein levels. First, TRIB2 protein expression was distinctly increased in melanoma tissue samples compared to normal skin and second, the highest TRIB2 protein expression was observed in melanoma tissues samples from patients with a poor clinical outcome (Fig. 4e; Supplementary Fig. 10). We next 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 signicantly elevated total AKT, pSer473-AKT1, pSer253-FOXO3a and pSer166-MDM2 and no signicant difference regarding pThr308-AKT1. Our study also demonstrated that pSer473-AKT1 and pSer253-FOXO3a levels correlated with melanoma patient prognosis.Further examination of AKT in our clinical samples revealed that TRIB2 and pSer473-AKT1 co-localize in melanoma cells from patients with progressive disease (Fig. 4f). Finally, we examined the clinical signicance of our ex vivo data within large patient cohorts. Melanoma, colon and pancreatic tumour tissue samples were analysed based on TRIB2 expression (low Z1.5, high Z2.5 versus normal tissue expression) and KaplanMeier survival curves plotted. For melanoma and colon cancer patients, high-TRIB2 expression correlated with a signicantly worse clinical outcome (Fig. 4g,h; Supplementary Fig. 11). For pancreatic cancer, there was only a strong trend for high-TRIB2 expression, primarily due to the extremely poor overall prognosis exhibited in pancreatic cancer patients. We further analysed the GSE65904 data set21 addressing several clinical parameters, which when combined with TRIB2 expression were used to determine if TRIB2 expression was dependent or independent of these prognostic parameters. Using a multivariate cox regression model we note that TRIB2 transcription is a prognostic factor, independent of disease stage, tissue involvement, age and gender (Supplementary Tables 6 and 7). Furthermore, survival data from the GSE65904 dataset revealed a highly signicant TRIB2-dependent distant metastasis-free survival time or disease-specic survival time response (Fig. 4g,h). Interestingly, this correlation to patient prognosis was only observed for TRIB2 and not for TRIB1 or TRIB3 (Supplementary Fig. 12af).

DiscussionOverall, our data reveal novel and important mechanisms of TRIB2-dependent intrinsic resistance to standard chemo therapies and PI3K inhibitors. A patient tumour population with a high-TRIB2 protein level prior to treatment would be predicted to respond poorly to these treatments by promoting 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% SDSPAGE. sc indicates scramble shRNA within the indicated cell lines. (b) Immunoblot proles for matched isogenic

TRIB2 cell lines for the AKT signalling network. High-TRIB2 expression correlated with signicantly increased post-translational modication(s) of downstream AKT targets. (c) Temporal inhibition of PI3K-dependent signalling following exposure to PI3K inhibitors. Total protein lysate of50200 mg was loaded per lane and separated by 612% SDSPAGE. (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 thes.d. ns indicates that the comparison was not statistically signicant. (e) (left) Co-immunoprecipitation of TRIB2 or total AKT1 from indicated cell lines, (500 mg total protein lysate immunoprecipitated per lane) and separated by 812% SDSPAGE. (right) Total protein levels in each indicated cell line for co-IP targets (100 mg total protein loaded per lane and separated by 812% SDSPAGE). (f) Representative yellow uorescent 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 usedas 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 eld. (g) (top) Percentage of YFP positive cells determined by FACS. (bottom) Mean uorescent intensity of YFP positive cells. One-way ANOVA with Dunnetts multiple comparison test, n 4 (*Pr0.05, **Pr0.01, ***Pr0.001). All analysis was conducted

compared to zipV1trib3V2 (lane 1).

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a b c d

BEZ235

p27

Dazcarbine Gemcitabine

MDM2

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Sub G 1cells (%)

Gene expression

(vs/GAPDH)

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293T GFP

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G361 sc. shRNA

G361 TRIB2 shRNA

SKMel28 sc. shRNA

SKMel28 TRIB2 shRNA

U2OS GFP

U2OS TRIB2

293T GFP

293T TRIB2

G361 sc. shRNA

G361 TRIB2 shRNA

SKMel28 sc. shRNA

SKMel28 TRIB2 shRNA

293T (TRIB2-GFP)

293T (Empty-GFP)

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U2OS (empty-GFP)

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U2OS TRIB2 FOXO3a shRNA

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U2OS TRIB2 shRNA

U2OS GFP FOXO3a shRNA

U2OS TRIB2 FOXO3a shRNA

293T GFP FOXO3a shRNA

293T TRIB2 FOXO3a shRNA

293T GFP sc. shRNA

293T TRIB2 shRNA

293T GFP FOXO3a shRNA

293T TRIB2 FOXO3a shRNA

U2OS

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FOXO3a shRNA

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Figure 3 | TRIB2-mediated resistance to chemotherapeutics is via AKT activation, suppressing FOXO3a and p53. (a) Matched isogenic TRIB2 cell line FACS analysis following the knockdown of FOXO3a and subsequent exposure to BEZ235, BAY236 (copanlisib) or BAY439 (BAY1082439) (n 6). P values

are indicated for each comparison by two-way ANOVA and data represent the means.d. (b) Quantitative real time PCR (qRT-PCR) analysis of FOXO3a-dependent gene expression after FOXO3a knockdown and isogenic cell line treatment for 24 h with PI3K inhibitors (n 6), (*Pr0.05, **Pr0.01 and were

analysed by two-way ANOVA). Data represent the means.d. (c) Matched isogenic TRIB2 cell line FACS analysis after 72 h exposure to various chemotherapeutics (n 6). P values are indicated for each comparison and data represent the means.d. (d) I Representative immunoblot analysis

showing 50 mg (MDM2), 100 mg (MDM2-Ser166), 50 mg (p53) total protein lysate per lane separated by 610% SDSPAGE. (e) p53-dependent gene expression was evaluated following TRIB2 isogenic cell line treatment with each indicated chemotherapeutic agent for 24 h. P values are shown for comparison by 2-way ANOVA (*Pr0.05, **P r0.001, ***P r0.0001) and data shown indicates means.d. (f) Representative immunoblot analysis showing TRIB2 protein expression (100 mg total protein loaded per lane) following exposure to each indicated PI3K inhibitor. (g) FACS analysis of 293Tcells after transfection of each TRIB2 construct and subsequent exposure to BEZ235, BAY236 (BAY 80-6946) or BAY439 (BAY1082439) (n 6) for 72 h. P

values are indicated for each comparison by two-way ANOVA and data represent the means.d. P values are shown for each comparison where no signicant difference was noted. Data was analysed by 2-way ANOVA *Pr0.05, **Pr0.001. (h) Representative immunoblot analysis showing 50 mg (GFP), 100 mg (AKT-Ser473), 100 mg (FOXO3a-Ser253), 100 mg (MDM2-Ser166) protein expression 48 h post-transfection of the indicated GFP tagged

TRIB2 plasmid constructs.

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AKT activation (and subsequent inactivation of FOXO and p53) may also be highly relevant for other therapeutics that target the upstream PI3K/AKT network such as trastuzumab approved for the treatment of breast and gastric cancers and cetuximab for the treatment of colon cancer22. Thus, patients whose tumours display high-TRIB2 expression may not benet from

specic PI3K inhibitor therapeutics, rendering TRIB2 as a suitable biomarker predicting treatment outcome and selecting patients for individualized therapy. Furthermore, TRIB2 inhibitors might synergize with current therapies targeting the PI3K/AKT pathway hereby improving treatment outcome and patient survival.

a b c

293T GFP Vehicle

293T GFP BEZ235

293T TRIB2 BEZ235

Tumour

100

Survival (percentage)

5,000

Tumour volume (mm3 )

293T-TRIB2 BEZ235

1 2 3 4 5 1 2 3 1 2 3 4 5

293T-GFP BEZ235

4,000

293T-GFP Vehicle

3,000

0.0339 *0.0083 **

0.6374 ns

2,000

Total AKT

Ser473 AKT

Ser253 FOXO3a Total p53

FasLG Cleaved Caspase3

actin

1,000

Total FOXO3a

0 5 10 15 20 25

1 3 5 7 9 11 13

Time post treatment (days)

Normal

Metastatic melanoma

*** *** ***

12.0

8.0

2.0

1.0

0.0

< 0.0001 ***

15.0

Normal

Complete response

Gene expression (fold change/GAPDH)

< 0.0001

*** 0.0003 ***

< 0.0001 ***

< 0.0001

*** 0.1010 NS

10.0

< 0.0001 ***

Stable disease

Progressive disease

5.0

3.0

** ***

* *

** **

2.0

1.0

***

** *

0.0

TRIB2 BIM p27 p16 p21 FasLG TRAIL AKT MDM2

e f

Normal tissue Progressive disease.

Stable

Complete disease

response

Progressive disease.

TRIB2

Total AKT

p-Ser473 AKT

MDM2

Total p53

BIM

FasLG

actin

Merged

TRIB2 + pSer473

AKT TRIB2

pSer473 AKT

p-Thr308 AKT

63x

100x

Total FOXO3a

pSer253-FOXO3a

pSer166-MDM2

Melanoma TRIB2 GSE65904

Low TRIB2 expression High TRIB2 expression

Receptor tyrosine

1.0

0.8

0.6

0.4

0.2

0.0

0 2,000 4,000 6,000

Distant metastasis-free survival (days)

High expression

Low expression

Cumulative survival

P = <0.001

P = 0.005

Cumulative survival

1.0

0.8

0.6

0.4

0.2

0.0

High expression Low expression

0 2,000 4,000 6,000

Disease specific survival (days)

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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14687

Western blot analysis. For the preparation of whole cell lysate, cells were harvested and lysed using RIPA buffer (50 mM TrisHCl pH 7.4, 1% NP-40,0.5% Na-deoxychlorate, 150 mM NaCl, 1 mM EDTA, 2 mM NaF, 2 mM NaVO4 and 1 protease inhibitor cocktail (PIC) (Sigma). For SDSpolyacrylamide gel

electrophoresis (SDSPAGE), protein samples were boiled for 510 min in protein sample buffer (50 mM Tris pH 6.8, 1% SDS, 10% glycerol, 0.01% Bromophenol Blue, b mercaptoethanol (50 ml per 950 ml sample buffer)). Following electrophoresis, proteins were transferred onto nitrocellulose membrane (BioRad).

The membrane was blocked for 1 h at room temperature or overnight at4 C 5% BSA 0.1% tween20 blocking buffer. Primary antibodies were addedto the membrane (Supplementary Table 2) overnight at 4 C or for 2 h at room temperature. Secondary antibody was added (Santa Cruz Biotechnology) at typically 1:5,000 dilution for 1 h at room temperature. Visualization of signalwas achieved using a ChemiDocXRS Imaging System (BioRad). Full original

membranes are shown in Supplementary Figs 1325.

Co-immunoprecipitation. Co-immunoprecipitations (Co-IP) were performed as described in (ref. 18). Cells were lysed in cold lysis buffer (50 mM Tris-Cl at pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.25% sodium deoxycholate, protease inhibitor mixture). Cell extracts (500 mg) were incubated with the rst antibodies (Supplementary Table 2) or control normal IgG on a rotator overnight at 4 C, followed by addition of protein G magnetic beads (Invitrogen) for 2 h at 4 C.Beads were then washed four times using the lysis buffer. The immune complexes were subjected to SDSPAGE followed by immunoblotting with the secondary antibody.

Quantitative Real time PCR (qRT-PCR). Total RNA was extracted by using TRI-reagent (Sigma, PT). cDNA was generated using NZY First-Strand cDNA Synthesis Kit (NZYTech, PT). Real Time PCR was performed on a BioRad CFX96 quantitative real time PCR machine using the SYBR Green JumpStart master mix (Sigma, PT). The primer sequences (NZYTech, PT) for measuring p16, p27, FasLG, Bim, TRAIL, p21, Bax, PUMA, MDM2, TRIB1, TRIB2, TRIB3 and GAPDH are shown in Supplementary Table 3. Data analysis was carried out using the2 DDCT method24.

Flow cytometric cell cycle analysis. Cells were grown to 70% conuence. Cells were mock treated/exposed to each compound for the time points indicated.Samples were collected, washed (PBS) and xed (70% ethanol). Ethanol was removed and samples were resuspended in PBS. Propidium iodide (2.5 mg ml) was added to each sample. Samples were run on a Fluorescence Activated Cell Scanner (FACS) and the percentage populations (sub-G1, G1, S and G2 phases) determined.

50,000 total events were scored per study Data was analysed using Innicyt (Cytognos).

In vivo studies. In vivo efcacy studies were performed using 293T-GFP and 293T-TRIB2 cells injected subcutaneously in the ank of female NOD/SCID mice.Animals were treated with either vehicle alone (20% N-Methyl-2-pyrrolidone (NMP) 80% polyethylene glycol (PEG) 300) or BEZ235 (30 mg kg 1 by oral gavage. Treatment was administered daily for the indicated length of time.

Tumours were measured manually by calliper daily. All in vivo modellingwas carried out according to guidelines, regulations set out in Portuguese law (Portaria 1005/02 and Portaria 1131/97), which transcribes the European Guideline 86/609/EC and approved by the CBMR Biotrio ethics board.

Figure 4 | TRIB2-conferred resistance to PI3K inhibitors in vivo and ex vivo and correlates with poor clinical prognosis. (a) KaplanMeier analysis of isogenic TRIB2 cell lines grow subcutaneously in NOD/Scid mice treated with vehicle (n 7), of BEZ235 (n 7). The presence of TRIB2 signicantly

reduced survival (log rank P 0.033) when treated daily with BEZ235. (b) TRIB2 overexpressing 293T tumours (n 7) show little to no response to daily

administration of BEZ235 compared to isogenic lines with endogenous (low, n 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 20) versus normal control tissue (n 10). Data were analysed by two-way ANOVA) and values

represent the means.d. (right panel), Quantitative real time PCR (qRT-PCR) analysis of gene expression in ex vivo metastatic melanoma samples (n 20)

versus normal control tissue (n 10) classied by clinical response to rst line chemotherapy (complete response n 5, stable disease n 5 or progressive

disease n 10). Data were analysed by two-way ANOVA) *Pr0.05, **Pr0.01, ***Pr0.001) and values represent the means.d. (e) Representative

immunoblot analysis of the AKT signalling cascade from ex vivo metastatic melanoma samples compared to normal tissue samples. (f) Representative immunouorecent images of metastatic melanoma dual stained for TRIB2 and pSer473-AKT1 demonstrating co-staining and interaction. (g) KaplanMeier analysis from melanoma patients in the GSE65904 dataset classied based on low (Z1.5 fold) or high (Z2.5 fold) TRIB2 expression. Log-rank tests reveal that TRIB2 expression is highly signicant for patient prognosis with median survival for low (4,450 days) and high (394 days) expression respectively.

(h) KaplanMeier 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.

Online Content, Any additional Methods, Extended Data display items and Source Data are available in the online version of the paper; references unique to these sections appear only in the online paper.

Methods

Tissue samples. Surgically excised tumour tissue samples from colon cancer primaries, pancreatic cancer primaries and melanoma metastases were obtained from patients prior to rst-line systemic therapy. Core tumour samples of colon carcinoma were obtained from patients with colon cancer at the time of their surgery. For each colon cancer patient, a section of matched normal colontissue was extracted adjacent to the tumour site. Pancreatic tumour tissue(and matched normal tissue) was obtained during surgery (employing the Whipple 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 split into smaller sections for RNA extraction using TRI-Reagent (Sigma). The clinical data of the corresponding patients were extracted from the patient les.

Cell lines and reagents. The human cell lines SK-Mel28, A375, N14, G361, UACC62, M14 (melanoma), HEK293T (renal cell carcinoma), U2OS [p53 / ],

Soas2 [p53 / ] (osteosarcoma), MCF-7 and MDA-MB231 (breast cancer) were maintained in DMEM supplemented with 10% FBS (Sigma, PT) and antibiotics (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 were used for our immunoblots and signal visualization was achieved using a ChemidocXRS system (BioRad, PT). Dacarbazine (Sigma, PT), gemcitabine

hydrochloride (Eli Lilly #VL7502), AKT inhibitor VIII (Calbiochem, US), BEZ235 (Novartis, US), BAY236, (BAY 80-6946) BAY439 (BAY1082439), BAY1001931(a gift from Bayer AG, Germany), rapamycin (SIGMA, PT), actinomycin D (Sigma, PT), and cyclohexamide (Sigma, PT) and MG132 (Sigma, PT) were used at concentrations described in the text.

Constructs and shRNA. A 3062 bp fragment encoding the entire human Trib2 (hTRIB2) cDNA was sub-cloned into pEGFP-N1 or pIREPuro2 plasmids,(one co-expressing GFP, the other containing a V5 tag.). Full length human TRIB2 (hTRIB2, 1-343 aa), dN (63-343aa hTRIB2, dC (1-304 aa) hTRIB2, KD (63-304 aa) hTRIB2, NT (DCT, 1250 aa) hTRIB2, CT (DNT, 270-343 aa) hTRIB2 and DCOP1 hTRIB2 were kindly provided by W.S. Pear. Each was cloned into pMigR1-myc plasmid as described in ref. 20. Full length pAKT1-S473D and pCMV6HA-mAKT1 were kindly provided by A. Newton. pECE-Foxo3a-(A)3 and pCLne-wt-Foxo3a were kindly provided by M. Greenberg. All complementary DNAs (cDNAs) were 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 shRNA-917 or hTRIB2-918 were sub-cloned into pRetroSuper. Each construct was transfected into indicated cell lines for selection and screening. Full sequences are provided in our Supplementary Information. For our protein fragment complementation assays, cells were co-transfected with the Venus plasmid constructs23 for AKT1 (1032-2471aa), AKT2 (1032-2477aa) or TRIB2 (1012-2042aa). YFP signal was quantied using ow cytometry (FacsCalibur, Becton Dickinson). The images of the interactions were captured by a Leica DMI 4000B inverted uorescence microscope.

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Fixing and staining samples for immunouorescence. To x samples/cells, slides/coverslips were xed and permeabilised for 15 min in dry methanol at

20 C and rehydrated in PBS. Samples/cells were blocked (donkey serum 1:30) for 1 h at 37 C in a humied chamber incubated with primary antibody(either Anti-pSer473AKT1 or TRIB2) for 1 h at 37 C in a humied chamber followed by PBS washing and incubation with a uorescent secondary antibody (Molecular Probes A21207 or A11055) for 1 h at 37 C in a humied chamber. Antibody solutions were made in PBS (Sigma). Following labelling procedures, samples/cells were mounted on glass slides in Clear Mount mounting solution (Invitrogen).

Statistical analysis. Statistical signicance was assessed by two-way analysis of variance (ANOVA) or the two-tailed Students t-test. Statistical signicance was dened as PZ0.05. Results are expressed as the means.d. or s. e. of the mean (s.e.m.) and are described in each gure legend when applied.

Data availability. The authors declare that the data supporting the ndings of this study are available within the paper and its Supplementary Information les.

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Acknowledgements

This work was supported by a grant from Bayer A.G. (Grants4Target 2012-08-0765) and by Fundaao para a Cincia e a Tecnologia (FCT) Research Center Grant UID/BIM/04773/2013 Centre for Biomedical Research 1334. R. Hill is the recipientof a FCT 2012 research grant (SFRH/BPD/84634/2012). The research performed by P.A. Madureira is a recipient of an FCT Investigator Program contract (ref: IF/00614/ 2014) from the Foundation for Science and Technology of Portugal. B.I. Ferreira is the recipient of a FCT 2014 research grant SFRH/BPD/100434/2014. S. Machado is the recipient of a ProRegem grant PD/BD/114258/2016. We thank S. Tenbaum andP. Castelo-Branco for helpful discussions and critical reading of this paper. We acknowledge the expert technical assistance of D. Cebrin and A. Mozes. We are indebted to W. Pear for providing plasmids. A. Adrienn and E.K. Toth were supported by the Fondation Leducq transatlantic network of excellence programme.

Author contributions

R.H., P.A.M., B.F., I.B., M.S., N.L., A.A. and E.K.T. conducted and analysed in vitro cell based experiments. R.H., P.A.M. and M.S. conducted and analysed in vivo experiments. S.M. and A.D. analysed high-throughput screen data. N.L. provided chemical compounds and S.U. clinical samples. M.I. and A.G. analysed all large patient cohort data sets. R.H., E.K.T. and W.L. directed the research. W.L. secured funding. All authors contributed to the writing of the manuscript and approved the nal version.

Additional information

Supplementary Information accompanies this paper at http://www.nature.com/naturecommunications

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Competing nancial interests: All authors declare that there are no competing nancial interests except N.L. who is an employee of Bayer AG.

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Abstract

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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.

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Title

TRIB2 confers resistance to anti-cancer therapy by activating the serine/threonine protein kinase AKT

Author

Hill, Richard; Madureira, Patricia A; Ferreira, Bibiana; Baptista, Inês; Machado, Susana; Colaço, Laura; Dos Santos, Marta; Liu, Ningshu; Dopazo, Ana; Ugurel, Selma; Adrienn, Angyal; Kiss-toth, Endre; Isbilen, Murat; Gure, Ali O; Link, Wolfgang

Pages

14687

Publication year

2017

Publication date

Mar 2017

Publisher

Nature Publishing Group

e-ISSN

20411723

Source type

Scholarly Journal

Language of publication

English

DOI

https://doi.org/10.1038/ncomms14687

ProQuest document ID

1875377682

TRIB2 confers resistance to anti-cancer therapy by activating the serine/threonine protein kinase AKT

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