TRIB2 confers resistance to anti-cancer therapy by ......5Genomics Unit, Centro Nacional de...

ARTICLE

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

TRIB2 confers resistance to anti-cancer therapy byactivating the serine/threonine protein kinase AKTRichard Hill1,2,3, Patricia A. Madureira2, Bibiana Ferreira2, Ines Baptista2, Susana Machado2, Laura Colaco2,

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.

DOI: 10.1038/ncomms14687 OPEN

1 Department of Biomedical Sciences and Medicine (DCBM), University of Algarve, Campus de Gambelas, Faro 8005-139, Portugal. 2 Centre for BiomedicalResearch (CBMR), University of Algarve, Campus de Gambelas, Faro 8005-139, Portugal. 3 Brain Tumour Research Centre, Institute of Biomedical andBiomolecular 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 HospitalEssen, Essen 45147, Germany. 7 Department of Cardiovascular Science, University of Sheffield, Sheffield S10 2RX, UK. 8 Department of Molecular Biology andGenetics, 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

mailto:[email protected]

http://www.nature.com/naturecommunications

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

proteins play a key role in the action of several anticancerdrugs, proteins that are capable of suppressing FOXO activityare strong candidates to confer drug resistance2–7. Our previouslarge scale genetic screen identified the kinase-like proteinTribbles 2 (TRIB2) as a FOXO suppressor protein8, whileTRIB2 has also been implicated in the development andprogression of melanoma and leukaemia8–11.

In our study presented here, we describe a new regulatorymechanism underlying drug resistance in different cancer entitiesand suggests that TRIB2 functions as a critical regulatorycomponent of the PI3K signalling network, activating AKT incancer cells. In addition, our data support the use of TRIB2as a biomarker for both prognosis and personalized cancertherapy, as well as identifying this protein as a molecular targetfor combination cancer treatment.

ResultsTRIB2-conferred resistance to anti-cancer therapeutics. To testour hypothesis that the TRIB2 protein confers resistance toanti-cancer drugs, we first generated and then examined thesensitivity of several stable, isogenic TRIB2 in vitro models(Supplementary Table 1; Supplementary Fig. 1) after treatmentwith the dual PI3K/mTOR kinase inhibitor BEZ235. High-TRIB2expression significantly increased cell resistance to BEZ235treatment characterized by a significantly lower sub-G1

cell population and a reduction of caspase-3 cleavage (Fig. 1a,b).To characterize the profile of kinases that were affected byTRIB2 status, we tested a range of inhibitors that displaydistinct kinase selectivity (summarized in Fig. 1c). TRIB2protein expression significantly influenced isogenic cell linesensitivity to BAY236 (BAY 80-6946) and BAY1082439 treatment(Fig. 1d). This response was independent of the cell cycle(Supplementary Fig. 2a–c), indicating that TRIB2 reduces celldeath induced by PI3K inhibitors and thus is capable of con-ferring resistance to these drugs. Strikingly, isogenic cell linesensitivity to inhibition of the central effector protein AKT (ref.12) and to the mTORC1/2 inhibitor TORIN1 was independent ofTRIB2 status (Fig. 1e). We investigated this further and exposedour isogenic cell lines to rapamycin, a specific mTORC1 inhibitor.Noticeably, TRIB2 status correlated with resistance to this com-pound (Fig. 1f). Taken together our data raises the intriguingpossibility that TRIB2 acts upstream of these proteins affectingAKT and mTORC2.

TRIB2 protein level correlates with AKT activation. To inves-tigate this further, we performed western blot analyses beforeand after clinically representative drug treatment with eachcompound using our isogenic cell lines. We note a prominentTRIB2-dependent disparity in the levels of pSer473-AKT1and total AKT using our in vitro models. In melanomaand osteosarcoma cells with high-TRIB2 protein expression,we identified significantly higher levels of total AKT and pSer473-AKT and the opposite was observed in cancer cells with TRIB2depletion (Fig. 2a). Consistent with our previous data, this wasnot observed for pThr308-AKT1, where there were readilydetectable levels of this AKT isoform independent of TRIB2status in each in vitro model. These data support the intriguingpossibility that TRIB2 might promote the further activation ofAKT via Ser473 phosphorylation in a PI3K and mTORC1 inde-pendent manner. In this way, drugs targeting these proteinswithin the PI3K pathway are not effective in tumours whereTRIB2 expression is up-regulated. To investigate this possibility,

we analysed signalling components within the PI3K/AKT net-work and found that TRIB2 over expression increased the acti-vation of pSer473-AKT before and in the presence of PI3K andmTOR inhibitors (Fig. 2b). Importantly, while TRIB2 proteinover expression significantly increased pSer473-AKT1 levels,isogenic cell line exposure to our inhibitor compounds reducedthe level of pSer473-AKT, indicating that the upstream compo-nents of this network remain functional within these in vitromodels. To support this, we transiently transfected wildtypeAKT, a phospho-mimetic AKT (AKTSer473D), wildtypeFOXO3a or a phosphor-mutant FOXO3a (FOXO3a-AAA) intoour isogenic cell lines (Supplementary Fig. 3a,b). We note thatthe over-expression of either AKTSer473D or FOXO3a-AAAnegated the TRIB2-dependent resistance to BEZ235 treatment.In contrast, over-expression of either WT AKT or FOXO3a didnot. For this we conclude that TRIB2-mediated resistancewas AKT-dependent via FOXO3a. Interestingly there was nosignificant difference in protein expression for PDK1, pSer241-PDK1, RAPTOR, pSer792-RAPTOR, p70S6K and only a slightlyincreased level of pThr389-p70S6K correlated with TRIB2expression (Supplementary Fig. 4) Taken together we concludethat TRIB2-conferred resistance to PI3K and mTORC1 inhibitorsis independent of PDK1 and p70S6K.

TRIB2 and AKT interact and form a protein-protein complex.FOXOs have been shown to be the major transcriptional effectorsfollowing PI3K inhibition13 and consistent with this finding, weobserve significantly elevated pSer235-FOXO3a protein levels anda concomitant suppression of FOXO-dependent gene expressionin cells with high-TRIB2 protein expression (Fig. 2c,d). Ourdata raise the hypothesis that TRIB2-dependent FOXOsuppression is indirect and rather, could be mediated via p473-AKT1 activation. To address this hypothesis we investigated themTORC2/AKT/FOXO3a cascade as mTORC2 is responsible forthe phosphorylation of ser473 within AKT1 (ref. 14). Wequestioned if there was physical interaction between TRIB2,AKT1 and pThr1135-RICTOR using co-immunoprecipitationand protein fragment complementation assays15–17. The resultsshowed a strong interaction between TRIB2 and AKT1 but couldnot observe TRIB2/RICTOR binding (Fig. 2e,f). Strikingly, theTRIB2/AKT1 complex (including the intensity of the interaction)was as strong as the known AKT1/JIP1 interaction18. Developingthese findings further, we addressed the functional importance ofFOXO and RICTOR in TRIB2-dependent drug resistance. Fourdifferent shRNA constructs were used to stably silence FOXO3aexpression (Supplementary Fig. 5). The depletion of FOXO3aexpression significantly increased in vitro resistance to PI3Kinhibitors regardless of TRIB2 status (Fig. 3a). Furthermore,FOXO3a depletion abolished the TRIB2-dependent difference, wepreviously observed for FOXO-regulated gene transcription inour isogenic cell lines (Fig. 3b). In addition to phosphorylatingand directing FOXO3a for proteasome mediated degradation,AKT1 can also suppress apoptosis by activating the E3 ubiquitinligase mouse double minute 2 homologue (MDM2), thusinhibiting p53-mediated apoptosis6,19. To analyse the role ofTRIB2 within this ‘arm’ of the AKT network, we broadened ourstudy to include first line chemotherapeutics known to actthrough p53 that are independent of FOXO (Fig. 3c,Supplementary Fig. 6). Remarkably, TRIB2 over expressionsignificantly increased cell resistance to dacarbazine (DTIC),gemcitabine and 5-fluorouracil (5-FU) induced cell deathindependent of FOXO. Accordingly, high-TRIB2 expressionwas associated with notably elevated pSer166-MDM2 proteinlevels (Fig. 3d). Therefore, we questioned whether p53 regulationwas disrupted by increased TRIB2 protein levels. We found that

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14687

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


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

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

It has been previously reported that TRIB2 associated with andinhibited the transcription factor CCAAT/enhancer-bindingprotein alpha (C/EBPa)11. Gene expression analysis in ourisogenic cell lines revealed that high-TRIB2 expression leads toreduced number of C/EBPa transcripts and the downregulation of

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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 (n¼ 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, (50mg 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 (n¼6).

(e) Isogenic TRIB2 cell line FACS analysis 72 h post-AKT, mTOR1/2 or mTOR1 inhibition (n¼ 6). P values are indicated for each comparison by two-way

analysis of variance (ANOVA) and data represent the mean±s.d.

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14687 ARTICLE



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

The COP1 domain of TRIB2 is required for AKT activation.To identify directly the TRIB2 functional domains that effectAKT activation, FOXO3a suppression and confer resistance toPI3K 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 conferresistance to any of the PI3K inhibitors tested (Fig. 3g). However,the full length, DN and the C-terminal constructs did conferresistance to each of the investigated therapeutics indicatingthat the COP1 region could be driving cell line resistance. In DC,KD, DCOP1 and N-terminal transfected cells, we observe little tono change in the level of pSer473-AKT1. In contrast, in untreated

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cells transfected with full length, DN or the C-terminal TRIB2constructs, the levels of pSer473-AKT1, pSer253-FOXO3a andpSer166-MDM2 increased significantly (Fig. 3h). Alongside, thesephosphorylation events we observed a significant reduction ofFOXO3a-dependent gene and protein expression (SupplementaryFig. 7b,c). Taken together, these data support that the TRIB2COP1 domain promotes AKT activation repressing FOXO3a andp53 activity, thereby fuelling cellular resistance to PI3K inhibitorregimes.

TRIB2 protein confers in vivo resistance to BEZ235 treatment.To address if TRIB2 over expression ablated tumour regressionfollowing the in vivo administration of PI3K inhibitors, weestablished isogenic 293T subcutaneous tumours in the flanks ofNOD/Scid mice. Tumours formed equivalently in these miceirrespective of TRIB2 status. The daily oral administration ofBEZ235 resulted in the regression of 293T-GFP xenografttumours and a statistically significant increase in survival. Incontrast, 293T-TRIB2 tumours were highly resistant to treatment,correlating with our in vitro studies indicating that TRIB2 overexpression significantly reduced the effectiveness of BEZ235treatment in vivo (Fig. 4a,b). To further support our in vitroanalysis, we also collected (where possible) tumours from eachmouse and evaluated the protein expression profiles (Fig. 4c). Wenote that while there was variable overall protein expressionwithin the various treatment groups, that 293T-TRIB2 BEZ235treated tumours had significantly higher total and pSer473 AKTcompared to 293T-GFP BEZ235 treated tumours. In contrast293T-TRIB2 BEZ235 treated tumours show attenuated FasLGprotein expression.

TRIB2 status corresponds to clinical outcome. We nextquestioned if these findings are representative of the clinicalsituation. We collected tumour tissue samples from patientswith melanoma, pancreatic and colon cancer prior to standardanti-cancer therapies (DTIC, gemcitabine or paclitaxel).Compared to normal tissue samples, TRIB2 transcriptionand TRIB2 protein expression was significantly increased inmetastatic melanoma, primary colon and primary pancreaticcancer tissue samples (Fig. 4d–f; Supplementary Fig. 8). We didnot detect a significant increase in either TRIB1 or TRIB3expression in any of the samples analysed (Supplementary Fig. 9).Concomitant with TRIB2 status, we found that pSer473-AKT1and pSer253-FOXO3a protein levels were significantly higher,while the transcript and protein levels of the FOXO-dependentgenes BIM, FasLG and TRAIL were significantly lower in ex vivomelanoma samples compared to normal control tissue samples.Importantly, when we classified our samples based on the clinical

outcome of the respective patients, we noted that TRIB2expression correlated with a significantly worse prognosis(Fig. 4d). We next investigated whether our transcription datacorrelated with protein levels. First, TRIB2 protein expression wasdistinctly increased in melanoma tissue samples compared tonormal skin and second, the highest TRIB2 protein expressionwas observed in melanoma tissues samples from patients witha poor clinical outcome (Fig. 4e; Supplementary Fig. 10). We nextquestioned whether there was also a de-regulated AKT signallingwithin our clinical samples. Consistent with both our in vitroand in vivo data, we observed significantly elevated totalAKT, pSer473-AKT1, pSer253-FOXO3a and pSer166-MDM2and no significant difference regarding pThr308-AKT1. Ourstudy also demonstrated that pSer473-AKT1 and pSer253-FOXO3a levels correlated with melanoma patient prognosis.Further examination of AKT in our clinical samples revealed thatTRIB2 and pSer473-AKT1 co-localize in melanoma cells frompatients with progressive disease (Fig. 4f). Finally, we examinedthe clinical significance of our ex vivo data within large patientcohorts. Melanoma, colon and pancreatic tumour tissue sampleswere analysed based on TRIB2 expression (low Z1.5, high Z2.5versus normal tissue expression) and Kaplan–Meier survivalcurves plotted. For melanoma and colon cancer patients, high-TRIB2 expression correlated with a significantly worse clinicaloutcome (Fig. 4g,h; Supplementary Fig. 11). For pancreaticcancer, there was only a strong trend for high-TRIB2 expression,primarily due to the extremely poor overall prognosis exhibited inpancreatic cancer patients. We further analysed the GSE65904data set21 addressing several clinical parameters, which whencombined with TRIB2 expression were used to determineif TRIB2 expression was dependent or independent of theseprognostic parameters. Using a multivariate cox regression modelwe note that TRIB2 transcription is a prognostic factor,independent of disease stage, tissue involvement, age andgender (Supplementary Tables 6 and 7). Furthermore, survivaldata from the GSE65904 dataset revealed a highly significantTRIB2-dependent distant metastasis-free survival time or disease-specific survival time response (Fig. 4g,h). Interestingly, thiscorrelation to patient prognosis was only observed for TRIB2 andnot for TRIB1 or TRIB3 (Supplementary Fig. 12a–f).

DiscussionOverall, our data reveal novel and important mechanismsof TRIB2-dependent intrinsic resistance to standard chemotherapies and PI3K inhibitors. A patient tumour populationwith a high-TRIB2 protein level prior to treatment would bepredicted to respond poorly to these treatments by promotingPI3K/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 100mg (TRIB2), 50mg (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

50–200mg 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

AKT protein/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, n¼4 (*Pr0.05, **Pr0.01, ***Pr0.001). All analysis was conducted

compared to zipV1trib3V2 (lane 1).




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U2OS (Empty)

BE

Z23

5N

T

BA

Y23

6B

AY

439

U2OS (TRIB2)

BE

Z23

5N

T

BA

Y23

6B

AY

439

TRIB2

β actin

Gen

e ex

pres

sion

(vs/

GA

PD

H)

0

2

4

6

ns

ns

****

*

*

p27

NT

NT

BE

Z23

5B

AY

236

BA

Y43

9

U2OSGFP

FOXO3ashRNAsc.

FOXO3ashRNAsc.

U2OSTRIB2

NT

NT

BE

Z23

5B

AY

236

BA

Y43

9

1

2

3

4

ns

ns

ns

ns

ns

ns

BIM

ns

ns

ns

ns

ns

ns

1

2

3

4FasLG

Gen

e ex

pres

sion

(vs/

GA

PD

H)

Gen

e ex

pres

sion

(vs/

GA

PD

H)

293T

moc

k29

3T T

RIB

2F

LC

OP

1ΔN ΔC K

DC

term

N te

rm

0

20

40

60

80

100

NS **

0.8296*

*

0.4943

0.7783

0.6837

***

0.6443*

0.54430.6130

*

0.7855

****

0.5696*

0.8028

0.9118*

0.8286

NT BEZ BAY236 BAY439

Sub

G1

cells

(%

)

293T

moc

k29

3T T

RIB

2F

LC

OP

1ΔN ΔC K

DC

term

N te

rm

293T

moc

k29

3T T

RIB

2F

LC

OP

1ΔN ΔC K

DC

term

N te

rm

293T

moc

k29

3T T

RIB

2F

LC

OP

1ΔN ΔC K

DC

term

N te

rm

pSer473-AKT

GFP

pSer253-FOXO3a

β actin

NT

pSer166-MDM2

293T

moc

k29

3T T

RIB

2F

L

CO

P1

ΔN ΔC KD

C te

rmN

term

Sub

G1

cells

(%

)

0

20

40

60

80 0.018 0.003 0.019 0.018

Dazcarbine

0.031 0.011 0.027 0.029

Gemcitabine

0

20

40

60

80

U2O

S G

FP

U2O

S T

RIB

229

3T G

FP

293T

TR

IB2

G36

1 sc

. shR

NA

G36

1 T

RIB

2 sh

RN

AS

KM

el28

sc.

shR

NA

SK

Mel

28 T

RIB

2 sh

RN

A

U2O

S G

FP

U2O

S T

RIB

229

3T G

FP

293T

TR

IB2

G36

1 sc

. shR

NA

G36

1 T

RIB

2 sh

RN

AS

KM

el28

sc.

shR

NA

SK

Mel

28 T

RIB

2 sh

RN

A

0.81610.5192

0.73350.5431

Dazcarbine

0

20

40

60

80

0

20

40

60

80 0.4221

0.7579

0.5101

0.8291

Gemcitabine

Sub

G1

cells

(%

)

U2O

S G

FP

sc.

shR

NA

U2O

S T

RIB

2 sh

RN

A

293T

GF

P s

c. s

hRN

A

293T

TR

IB2

shR

NA

U2O

S G

FP

FO

XO

3a s

hRN

A

U2O

S T

RIB

2 F

OX

O3a

shR

NA

293T

GF

P F

OX

O3a

shR

NA

293T

TR

IB2

FO

XO

3a s

hRN

A

U2O

S G

FP

sc.

shR

NA

U2O

S T

RIB

2 sh

RN

A

293T

GF

P s

c. s

hRN

A

293T

TR

IB2

shR

NA

U2O

S G

FP

FO

XO

3a s

hRN

A

U2O

S T

RIB

2 F

OX

O3a

shR

NA

293T

GF

P F

OX

O3a

shR

NA

293T

TR

IB2

FO

XO

3a s

hRN

A

Sub

G1

cells

(%

)S

ub G

1 ce

lls (

%)

Sub

G1

cells

(%

)

0.021

0

20

40

60

80BEZ235

0.00130.0016

0.0123

0

20

40

60

80BAY236

0.0002

0.006

0.0003

0.008

sc. s

hRN

AF

OX

O3a

shR

NA

Ric

tor

shR

NA

293T

TR

IB2

293T

empt

y

U2O

ST

RIB

2

U2O

Sem

pty

sc. s

hRN

AF

OX

O3a

shR

NA

Ric

tor

shR

NA

sc. s

hRN

AF

OX

O3a

shR

NA

Ric

tor

shR

NA

sc. s

hRN

AF

OX

O3a

shR

NA

Ric

tor

shR

NA

0

20

40

60

80BAY439

0.0005

0.016

0.0024

0.004

0.0012

0.026

0.0017

0.014

0.0003

0.021

0.002

0.006

0.0014

0.073

0.004

0.006

a b c d

e f

g h

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) (n¼6). P values

are indicated for each comparison by two-way ANOVA and data represent the mean±s.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 (n¼ 6), (*Pr0.05, **Pr0.01 and were

analysed by two-way ANOVA). Data represent the mean±s.d. (c) Matched isogenic TRIB2 cell line FACS analysis after 72 h exposure to various

chemotherapeutics (n¼6). P values are indicated for each comparison and data represent the mean±s.d. (d) I Representative immunoblot analysis

showing 50mg (MDM2), 100mg (MDM2-Ser166), 50mg (p53) total protein lysate per lane separated by 6–10% SDS–PAGE. (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 mean±s.d. (f) Representative immunoblot analysis

showing TRIB2 protein expression (100mg total protein loaded per lane) following exposure to each indicated PI3K inhibitor. (g) FACS analysis of 293T cells

after transfection of each TRIB2 construct and subsequent exposure to BEZ235, BAY236 (BAY 80-6946) or BAY439 (BAY1082439) (n¼6) for 72 h. P

values are indicated for each comparison by two-way ANOVA and data represent the mean±s.d. P values are shown for each comparison where no

significant difference was noted. Data was analysed by 2-way ANOVA *Pr0.05, **Pr0.001. (h) Representative immunoblot analysis showing 50mg

(GFP), 100mg (AKT-Ser473), 100mg (FOXO3a-Ser253), 100mg (MDM2-Ser166) protein expression 48 h post-transfection of the indicated GFP tagged

TRIB2 plasmid constructs.




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

specific PI3K inhibitor therapeutics, rendering TRIB2 as asuitable biomarker predicting treatment outcome and selectingpatients for individualized therapy. Furthermore, TRIB2inhibitors might synergize with current therapies targeting thePI3K/AKT pathway hereby improving treatment outcome andpatient survival.

Merged

TRIB2 +pSer473

AKT TRIB2pSer473

AKT

63x

100x

Progressive disease.Normal tissue Progressive disease.

Stable disease

Complete response

TRIB2

Total FOXO3a

pSer253-FOXO3a

Total AKT

p-Ser473 AKT

MDM2

Total p53

BIM

FasLG

β actin

pSer166-MDM2

p-Thr308 AKT

Normal

Metastatic melanoma

TRIB2 BIM p27 p16 p21 FasLG TRAIL AKT MDM2

12.0

8.0

2.0

1.0

0.0

< 0.0001***

< 0.0001***

< 0.0001*** 0.0003

***

< 0.0001***

< 0.0001***

< 0.0001*** 0.1010

NS

< 0.0001***

Gen

e ex

pres

sion

(fol

d ch

ange

/GA

PD

H)

0.0

1.0

2.0

3.05.0

10.0

15.0

Stable disease

Progressive disease

Normal

Complete response

TRIB2 BIM p27 p16 p21 FasLG TRAIL AKT MDM2

***

***

***

***

*** *

**** ***

*

* * * ** **

Gen

e ex

pres

sion

(fol

d ch

ange

/GA

PD

H)

Time post treatment (days)

Tum

our

volu

me

(mm

3 )

1 3 5 7 9 11 130

1,000

2,000

3,000

4,000

5,000

0.0062 **0.0299 *

0.6459 NS

Time post treatment (days)

0 5 10 15 20 250

20

40

60

80

100

Sur

viva

l (p

erce

ntag

e)

0.0339 *0.0083 **

0.6374 ns

293T GFP Vehicle

293T GFP BEZ235

293T TRIB2 BEZ235

Ser473 AKT

Ser253 FOXO3aTotal p53

FasLGCleaved Caspase3

β actin

Total AKT

Tumour

293T-GFPVehicle

293T-TRIB2BEZ235

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

293T-GFPBEZ235

Total FOXO3a

1.0

0.8

0.6

0.4

0.2

0.0

0 2,000 4,000 6,000

Distant metastasis-freesurvival (days)

Cum

ulat

ive

surv

ival

Melanoma TRIB2GSE65904

Low TRIB2 expression High TRIB2 expression

Receptor tyrosine kinases

PI3K

p27 BIM FasLG

p21 Bax PUMA

p53

Survival.Tumour growth.

Treatment failure.

PI3K inhibitor treatment(for example BEZ235)

AKTp473

MDM2p166 FOXO3a p253

mTORRICTOR

Receptor tyrosine kinases

PI3K

FOXO3a

p27 BIM FasLG

p21 Bax PUMA

p53

Apoptosis.Tumour regression.

clinical response.

MDM2

AKT

FOXO3a

PI3K inhibitor treatment(for example BEZ235)

mTORRICTOR

TRIB2COP1

High expressionLow expression

1.0

0.8

0.6

0.4

0.2

0.0

Cum

ulat

ive

surv

ival

0 2,000 4,000 6,000

Disease specificsurvival (days)

High expression

Low expression

P = <0.001

P = 0.005

a b c

d

e f

g

h

i




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

MethodsTissue samples. Surgically excised tumour tissue samples from colon cancerprimaries, pancreatic cancer primaries and melanoma metastases were obtainedfrom patients prior to first-line systemic therapy. Core tumour samples of coloncarcinoma were obtained from patients with colon cancer at the time of theirsurgery. 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 Whipplesurgical methodology). Melanoma tumour samples were obtained from metastaticlesions from patients with advanced melanoma in AJCC stage IV. Informedconsent was obtained for the use of these samples. The samples were freshly frozenand cryo-preserved until processing. Before analysis, the frozen tissue samples weresplit into smaller sections for RNA extraction using TRI-Reagent (Sigma). Theclinical data of the corresponding patients were extracted from the patient files.

Cell lines and reagents. The human cell lines SK-Mel28, A375, N14, G361,UACC62, M14 (melanoma), HEK293T (renal cell carcinoma), U2OS [p53þ /þ ],Soas2 [p53� /� ] (osteosarcoma), MCF-7 and MDA-MB231 (breast cancer) weremaintained in DMEM supplemented with 10% FBS (Sigma, PT) and antibiotics(Gibco, US). Cell lines were mycoplasma tested monthly within the CBMR byPCR (LookOut Mycoplasma PCR Detection Kit, SIGMA, PT). All cell lines areindicated in Supplementary Table 1. Antibodies shown in Supplementary Table 2were used for our immunoblots and signal visualization was achieved using aChemidocXRSþ system (BioRad, PT). Dacarbazine (Sigma, PT), gemcitabinehydrochloride (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 usedat 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, 1–250 aa) hTRIB2, CT (DNT, 270-343 aa) hTRIB2 and DCOP1hTRIB2 were kindly provided by W.S. Pear. Each was cloned into pMigR1-mycplasmid as described in ref. 20. Full length pAKT1-S473D and pCMV6HA-mAKT1were kindly provided by A. Newton. pECE-Foxo3a-(A)3 and pCLne-wt-Foxo3awere kindly provided by M. Greenberg. All complementary DNAs (cDNAs) weresequenced in their entirety to verify there were no mutations in any of ourconstructs. FOXO3a shRNA constructs originated from the NKI library, FOXO3a-826, FOXO3a-827 and FOXO3a-828 were cloned into pRetroSuper. hTRIB2shRNA-917 or hTRIB2-918 were sub-cloned into pRetroSuper. Each construct wastransfected into indicated cell lines for selection and screening. Full sequences areprovided in our Supplementary Information. For our protein fragment com-plementation assays, cells were co-transfected with the Venus plasmid constructs23

for AKT1 (1032-2471aa), AKT2 (1032-2477aa) or TRIB2 (1012-2042aa). YFPsignal was quantified using flow cytometry (FacsCalibur, Becton Dickinson). Theimages of the interactions were captured by a Leica DMI 4000B invertedfluorescence microscope.

Western blot analysis. For the preparation of whole cell lysate, cells wereharvested and lysed using RIPA buffer (50 mM Tris–HCl 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 SDS–polyacrylamide gelelectrophoresis (SDS–PAGE), protein samples were boiled for 5–10 min in proteinsample buffer (50 mM Tris pH 6.8, 1% SDS, 10% glycerol, 0.01% BromophenolBlue, b mercaptoethanol (50 ml per 950 ml sample buffer)). Following electro-phoresis, 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 roomtemperature. Secondary antibody was added (Santa Cruz Biotechnology) attypically 1:5,000 dilution for 1 h at room temperature. Visualization of signalwas achieved using a ChemiDocXRSþ Imaging System (BioRad). Full originalmembranes are shown in Supplementary Figs 13–25.

Co-immunoprecipitation. Co-immunoprecipitations (Co-IP) were performed asdescribed 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, proteaseinhibitor 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,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 complexeswere subjected to SDS–PAGE followed by immunoblotting with the secondaryantibody.

Quantitative Real time PCR (qRT-PCR). Total RNA was extracted by usingTRI-reagent (Sigma, PT). cDNA was generated using NZY First-Strand cDNASynthesis Kit (NZYTech, PT). Real Time PCR was performed on a BioRad CFX96quantitative real time PCR machine using the SYBR Green JumpStart mastermix (Sigma, PT). The primer sequences (NZYTech, PT) for measuring p16, p27,FasLG, Bim, TRAIL, p21, Bax, PUMA, MDM2, TRIB1, TRIB2, TRIB3 and GAPDHare shown in Supplementary Table 3. Data analysis was carried out using the2�DDCT method24.

Flow cytometric cell cycle analysis. Cells were grown to 70% confluence. Cellswere mock treated/exposed to each compound for the time points indicated.Samples were collected, washed (PBS) and fixed (70% ethanol). Ethanol wasremoved and samples were resuspended in PBS. Propidium iodide (2.5 mg ml) wasadded 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 Infinicyt(Cytognos).

In vivo studies. In vivo efficacy studies were performed using 293T-GFP and293T-TRIB2 cells injected subcutaneously in the flank 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 oralgavage. 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 Guideline86/609/EC and approved by the CBMR Bioterio 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 (n¼ 7), of BEZ235 (n¼ 7). The presence of TRIB2 significantly

reduced survival (log rank P¼0.033) when treated daily with BEZ235. (b) TRIB2 overexpressing 293T tumours (n¼ 7) show little to no response to daily

administration of BEZ235 compared to isogenic lines with endogenous (low, n¼ 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 (n¼ 20) versus normal control tissue (n¼ 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 (n¼ 20)

versus normal control tissue (n¼ 10) classified by clinical response to first line chemotherapy (complete response n¼ 5, stable disease n¼ 5 or progressive

disease n¼ 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.




Fixing and staining samples for immunofluorescence. To fix samples/cells,slides/coverslips were fixed 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 humified chamber incubated with primary antibody(either Anti-pSer473AKT1 or TRIB2) for 1 h at 37 �C in a humified chamberfollowed by PBS washing and incubation with a fluorescent secondary antibody(Molecular Probes A21207 or A11055) for 1 h at 37 �C in a humified 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 significance was assessed by two-way analysis ofvariance (ANOVA) or the two-tailed Students t-test. Statistical significance wasdefined as PZ0.05. Results are expressed as the mean±s.d. or s. e. of the mean(s.e.m.) and are described in each figure legend when applied.

Data availability. The authors declare that the data supporting the findings of thisstudy are available within the paper and its Supplementary Information files.

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AcknowledgementsThis work was supported by a grant from Bayer A.G. (Grants4Target 2012-08-0765)and by Fundacao para a Ciencia e a Tecnologia (FCT) Research Center GrantUID/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 byP.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 therecipient of a FCT 2014 research grant SFRH/BPD/100434/2014. S. Machado is therecipient of a ProRegem grant PD/BD/114258/2016. We thank S. Tenbaum andP. Castelo-Branco for helpful discussions and critical reading of this paper. Weacknowledge the expert technical assistance of D. Cebrian and A. Mozes. We areindebted to W. Pear for providing plasmids. A. Adrienn and E.K. Toth were supportedby the Fondation Leducq transatlantic network of excellence programme.

Author contributionsR.H., P.A.M., B.F., I.B., M.S., N.L., A.A. and E.K.T. conducted and analysed in vitro cellbased 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 chemicalcompounds and S.U. clinical samples. M.I. and A.G. analysed all large patient cohort datasets. R.H., E.K.T. and W.L. directed the research. W.L. secured funding. All authorscontributed to the writing of the manuscript and approved the final version.

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

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

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How to cite this article: Hill, R. et al. TRIB2 confers resistance to anti-cancer therapyby activating the serine/threonine protein kinase AKT. Nat. Commun. 8, 14687doi: 10.1038/ncomms14687 (2017).

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