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ARTICLE

Received 15 Jun 2015 | Accepted 1 Jul 2016 | Published 16 Aug 2016

DOI: 10.1038/ncomms12436 OPEN

miR-625-3p regulates oxaliplatin resistance by targeting MAP2K6-p38 signalling in human colorectal adenocarcinoma cells

Mads Heilskov Rasmussen1,*, Iben Lyskjr1,*, Rosa Rakownikow Jersie-Christensen2, Line Schmidt Tarpgaard3, Bjarke Primdal-Bengtson1, Morten Muhlig Nielsen1, Jakob Skou Pedersen1, Tine Plato Hansen4,w,Flemming Hansen5, Jesper Velgaard Olsen2, Per Pfeiffer3,6, Torben Falck rntoft1 & Claus Lindbjerg Andersen1

Oxaliplatin resistance in colorectal cancers (CRC) is a major medical problem, and predictive markers are urgently needed. Recently, miR-625-3p was reported as a promising predictive marker. Herein, we show that miR-625-3p functionally induces oxaliplatin resistance in CRC cells, and identify the signalling networks affected by miR-625-3p. We show that the p38 MAPK activator MAP2K6 is a direct target of miR-625-3p, and, accordingly, is downregulated in non-responder patients of oxaliplatin therapy. miR-625-3p-mediated resistance is reversed by anti-miR-625-3p treatment and ectopic expression of a miR-625-3p insensitive MAP2K6 variant. In addition, reduction of p38 signalling by using siRNAs, chemical inhibitors or expression of a dominant-negative MAP2K6 protein induces resistance to oxaliplatin. Transcriptome, proteome and phosphoproteome proles conrm inactivation of MAP2K6-p38 signalling as one likely mechanism of oxaliplatin resistance. Our study shows that miR-625-3p induces oxaliplatin resistance by abrogating MAP2K6-p38-regulated apoptosis and cell cycle control networks, and corroborates the predictive power of miR-625-3p.

1 Department of Molecular Medicine, Department of Clinical Medicine, Aarhus University Hospital, Palle Juul-Jensens Boulevard 99, DK-8200 Aarhus, Denmark. 2 Department of Proteomics, Faculty of Health and Medical Sciences, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Blegdamsvej 3b, DK-2200 Copenhagen, Denmark. 3 Institute of Clinical Research, University of Southern Denmark, Winslwparken 19, DK-5000 Odense, Denmark. 4 Department of Pathology, Institute of Clinical Research, Odense University Hospital, Winslwparken 15, DK-5000 Odense, Denmark. 5 Department of Oncology, Aarhus University Hospital, Nrrebrogade 44, DK-8200 Aarhus, Denmark. 6 Department of Oncology, Odense University Hospital, Sdr. Boulevard 29, DK-5000 Odense, Denmark. * These authors contributed equally to this work. w Present address: Institute for Clinical

Medicine, University of Copenhagen, Kettegrd All 30, DK-2650 Hvidovre, Denmark. Correspondence and requests for materials should be addressed to C.L.A. (email: mailto:[email protected]

Web End [email protected] ).

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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms12436

Colorectal cancer (CRC) is the third most commonly diagnosed malignant disease in the western world1. Approximately 25% of the patients present with a

disseminated, stage IV disease and in further 1015% of patients with initially localized disease, metastases will develop within 5 years. However, no predictive biomarker for standard chemotherapeutic treatment is available and as many as 50% of the patients do not obtain an objective response to rst-line treatment2. Thus, the identication of predictive biomarkers for response is of great importance.

MicroRNAs (miRNAs) are endogenous, small non-coding RNAs that play essential roles in the regulation of gene expression3, and which have been linked to chemotherapy resistance4. Recently, miR-625-3p was reported to be positively associated with lack of response to rst-line oxaliplatin (oxPt)-based treatment in two independent cohorts of patients with metastatic CRC (mCRC)5. While that study suggested high expression of miR-625-3p to be a novel predictive marker for oxPt-resistance in a subset of mCRC patients, a possible functional relationship between miR-625-3p and cellular drug sensitivity was not examined.

Here, we have constructed a transposon-based doxycycline (DOX) inducible vector to investigate the role of miR-625-3p in modulating oxPt sensitivity in CRC cells in vitro. Our results show that on exposure to oxPt ectopic expression of miR-625-3p increases cell viability by decreasing apoptosis. Furthermore, we have identied direct and indirect targets of miR-625-3p dysregulation in these cells and in mCRC patients treated with rst-line oxPt. We show that miR-625-3p directly targets and inhibits the mitogen activated protein kinase (MAPK) kinase MAP2K6 (also known as MKK6). As a consequence, we nd that miR-625-3p-induced resistance is associated with reduced MAP kinase signal transduction after genotoxic stress leading to a reduction of p38-mediated apoptosis and an increase in cell cycle progression signals.

ResultsEctopic expression of miR-625-3p promotes oxPt resistance. We constructed a Sleeping Beauty (SB) transposon vector (pSBInducer), which allows for stable expression of small interfering RNAs (siRNAs) and miRNAs in a DOX-inducible manner (Supplementary Fig. 1), and consequently, robust downregulation of targeted genes in mammalian cells (Supplementary Fig. 2).

We used pSBInducer to introduce miR-625-3p expression (or control shRNA designed not to target any human transcripts) in the microsatellite stable and microsatellite instable CRC cell lines SW620 and HCT116, respectively (Supplementary Fig. 1). Forty-eight hours of DOX induction raised the level of miR-625-3p approximately three-fold in HCT116.625 cells, which is comparable to the previously reported difference in miR-625-3p expression between responder and non-responder patients (Supplementary Fig. 3)5. In SW620.625 cells, DOX treatment induced miR-625-3p by more than 400 fold (Supplementary Fig. 3). Ectopic expression of miR-625-3p had no signicant effect on cell growth in SW620 cells, whereas in HCT116 cells, a slight (28%) increased viability was observed (Fig. 1a).

DOX-induced SW620.625, HCT116.625 and control cells were next treated with increasing concentrations of oxPt for 48 h and cell viability assessed. In both cell lines miR-625-3p induction increased oxPt resistance over a range of concentrations (Fig. 1b), which translated into an increase in the half maximum inhibitory concentration IC50 (causing 50% inhibition of viability) from1.6 mM in HCT116.ctrl to 28.8 mM in HCT116.625, and from1.3 mM in SW620.ctrl control cells to 6.1 mM in SW620.625 cells (Fig. 1c). There was no difference in IC50 between vector control

cells and their parental wild-type counterparts (Fig. 1c). This indicates that miR-625-3p functionally is associated with increased resistance to oxPt in CRC cells.

Increased miR-625-3p expression reduces oxPt-induced cell death. To determine whether inhibition of cell death was a contributing factor to the observed oxPt resistance in HCT116.625 and SW620.625 cells, we performed a lactate dehydrogenase activity (LDH) assay. Induction of miR-625-3p in HCT116.625 cells inhibited drug-induced cell death when exposed to oxPt (Fig. 2a). A small decrease in cell death was also observed for 2 and 8 mM oxPt in miR-625-3p overexpressing

SW620.625 cells although this was only borderline signicant (Fig. 2a).

To conrm that the oxPt resistance phenotype was a general consequence of miR-625-3p induction, we used a ow cytometry-based Annexin-V/propidium iodide (PI) cell death assay on three randomly selected, independent HCT116.625 single cell clones (these are biological replicates since Sleeping Beauty mediated transposition is near-random and individual low-passage cell clones harbour unique pSBInducer integrations6). In agreement

Untreated cells

1.5

1.0

Proliferation

0.5

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ctrl 625 ctrl 625

HCT116 SW620

b oxPt treated (48 h) cells

HCT116

IC50 (48 h oxPt)

ctrl 625

wt ctrl 625

1.0

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0.5

0.0

SW620

ctrl 625

1.0

0.5

0.0

0 HCT116

SW620

0 0.5 1 2 4 8 16 64

M oxPt

Figure 1 | Ectopic expression of miR-625-3p is associated with increased viability in oxPt medium. (a) Cell proliferation upon DOX induction of miR-625-3p in the CRC cell lines HCT116.625, SW620.625 and control cells expressing a scrambled shRNA was determined by an MTT assay after 72 h of growth. Displayed as means.e.m. (n 3). (b) Cell proliferation after

48 h of oxPt treatment was assessed by MTT in DOX-induced HCT116.625, SW620.625. Displayed relative to untreated cells as means.e.m. (n 3).

(c) IC50 values were calculated on the basis of experiments from b as well as from wild-type cells not subjected to pSBInducer transposition.

Displayed as mean IC50s.e.m. (n 3). *Pr0.05 (t-test); NS, not

signicant.

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms12436 ARTICLE

miR-625-3p reduces cell death

miR-625-3p reduces apoptosis in single cell clones ctrl / 0 M

ctrl / 64 M

ctrl

HCT116

625

LDH

ctrl

SW620

625

P = 0.08

P = 0.06

Propidium iodide

miR-625#1 / 0 M miR-625#1 / 64 M

0 0 2 8 64M oxPt

Anti-miR-625-3p restores oxPt sensitivity

Annexin V

P = 0.14

Single cell clone

OxPt(64 M) Q1 (%) Q2 (%) Q3 (%) Q4 (%)

Death rate (%)

LDH 64 M / 0 M

ctrl

5 8

4 71

1 4

9017 81

miR-625 #1

4 9

6 20

0 3

9069 23

miR-625 #2

4 13

3 25

0 4

9259 36

Anti-625:

+ +

HCT116. 625#1

HCT116. ctrl

miR-625 #3

5 17

3 33

0 3

9248 48

Figure 2 | miR-625-3p inhibits oxPt-induced cell death in CRC cell lines. (a) DOX-induced HCT116.625 and SW620.625 together with control cells were treated for 48 h with oxPt. Cell death was determined with the LDH assay as 100%*(LDHmedium/(LDHmedium LDH

lysate)). Displayed as means.e.m. (n 3). (b) DOX-induced HCT116.625 and HCT116.ctrl single colony-derived cells were treated with media containing 0 or 64 mM oxPt for 48 h. In the top

panel, representative results from HCT116.ctrl and HCT116.625#1 single cell clones are shown. Quadrants Q1, Q2 and Q3 contain early apoptotic, late apoptotic and necrotic cells, respectively, while quadrant Q4 contains living cells. The bottom panel reports the fraction of cells in each quadrant for three independent HCT116.625 single cell clones. The death rate was calculated as 100%*(1 [Q464 m

M/Q40 mM]). (c) HCT116.625#1 and HCT116.ctrl cells were induced with DOX and transfected with 20 nM anti-miR-625-3p oligo. Twenty-four hours after transfection, cells were cultivated in 0 or 64 mM oxPt for 48 h before cell death was assessed by LDH assay. Data are presented as mean increase in 64 mM oxPt-induced cell deaths.e.m. (n 5).

*Pr0.05 (t-test).

with the LDH assay, the Annexin-V/PI assay demonstrated that miR-625-3p indeed reduced oxPt-induced cell death (Fig. 2b). The percentage of apoptotic cells in non-treated cells was similar in control and miR-625-3p cell clones, while the death rate upon exposure to oxPt was reduced from 81% in control cells to below 50% in the HCT116.625 cell clones. The same experiment was also performed with a single cell-derived SW620 clone, which revealed a similar effect (reduction in death rate from 51% in SW620.ctrl to 33% in SW620.625 cells; Supplementary Table 1).

To investigate whether sensitivity towards oxPt could be restored by reducing miR-625-3p levels, the most oxPt-resistant HCT116.625 clone (clone #1) was transfected with an inhibitor of miR-625-3p (an anti-miR). The anti-miR signicantly increased oxPt sensitivity towards 64 mM oxPt as assessed by LDH assay compared with mock transfected HCT116.625#1 cells (Fig. 2c). Anti-miR treatment also increased the sensitivity of control cells toward oxPt, although the difference was only borderline signicant (P 0.140, t-test), presumably reecting an effect of

downregulating the endogenous miR-625-3p (Fig. 2c). Finally, decreased apoptosis in the HCT116.625 single cell clones upon exposure to oxPt was also supported by xCELLigence real-time proliferation assays (Supplementary Fig. 4).

In conclusion, our data demonstrate that ectopic expression of miR-625-3p promotes resistance towards oxPt in CRC cells, and that this resistance is caused, at least in part, by inhibition of oxPt-induced cell death.

miR-625-3p transcripts are associated with oxPt response. To identify genes associated with the oxPt-resistant phenotype, transcriptional proles of DOX-induced SW620.625 and SW620.ctrl cells were generated (Fig. 3a). We reasoned that a stronger impact on target mRNAs would be seen in SW620.625 cells as compared with HCT116.625 cells owing to the higher miR-625-3p levels in the former (Supplementary Fig. 3).

In total, 216 and 163 genes were up- and downregulated, respectively, in miR-625-3p expressing SW620.625 cells (absolute fold change 41.5; Supplementary Data 1). We noted upregulation of several genes encoding ATP-binding cassette (ABC) transporter proteins (for example, ABCA6, FC 17.4; and

ABCA9, FC 2.8, see Supplementary Data 1), however, the

particular ABC proteins previously implicated in multi-drug resistance (for example, MDR1/ABCB1 and MRP1/ABCC1) were not dysregulated. Since no obvious pathways or single genes

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Genes upregulated in SW620.625 cells

ES = 0.367 P = 0.036

Genes upregulated in non-responder (NR) patients

Genes upregulated in responder (R) patients

SW620.625 SW620.ctrl

Gene rankNR-R

Figure 3 | miR-625-3p regulates networks associated with therapy response. (a) Unsupervised clustering of the most variable probe sets across three SW620.625 and three SW620.ctrl samples after DOX induction. (b) Expression proles of primary tumours from rst-line oxPt-treated mCRC patients were generated and 20,693 genes ranked according to difference in median expression between non-responder (n 9) and responder (n 17) patients.

Genes upregulated in the SW620.625 cells (black vertical bars) were signicantly associated with the non-responder phenotype (enrichment score 0.367, P 0.036, KolmogorovSmirnov test).

with strong connection to drug resistance were identied, we addressed whether the dysregulated genes might be relevant in a clinical setting. To this end, we proled 26 microsatellite stable primary tumours from mCRC patients receiving oxPt-based therapy as rst-line treatment. Objective best response to treatment was then used as phenotype labels (Non-responder and Responder) in a gene set enrichment analysis7. Interestingly, we found enrichment for SW620.625 upregulated genes among the non-responding patients (Fig. 3b). These data indicate a clinical relevance for the oxPt-resistant phenotype induced by ectopic miR-625-3p overexpression.

The MAPK kinase MAP2K6 is a direct target of miR-625-3p. To identify miR-625-3p target genes, we searched the transcriptional prole for mRNAs with miR-625-3p target sequences that were downregulated in the SW620.625 cells. Overall, we found enrichment for mRNAs containing the miR-625-3p 7-mer target sequence (CTATAGT) in their 30UTR among down-regulated genes (Fig. 4a). To select putative target genes for experimental validation, we used the miRmap tool, which applies multiple predictors to generate a combined score of miRNAmRNA repression strength (from 0 to 100; ref. 8). We selected the eight most downregulated genes with a miR-625-3p target sequence and a miRmap score above 75: MAP2K6, RCN1, BCL11A, COMMD8, MXI1, NUP35, ST18 and IRAK2 (Supplementary Table 2), and conrmed downregulation of these genes by quantitative real-time PCR (Supplementary Fig. 5). Next, we screened for downregulation of these genes in a set of independently induced SW620.625 and HCT116.625 cell populations (Fig. 4b). Although all genes could be validated as being downregulated in SW620.625 cells compared with control cells, only MAP2K6 was validated in HCT116.625 cells.

We used an anti-AGO2 antibody to immunoprecipitate RNA-induced silencing complex (RISC)-associated RNA9, which revealed increased AGO2 association for MAP2K6, MXI1 and IRAK2 in SW620.625 cells (Fig. 4c). At the protein level, however, only MAP2K6 were robustly downregulated after miR-625-3p induction in SW620.625 cells (Fig. 4d and Supplementary Fig. 6). In HCT116.625 cells, we also observed reduced MAP2K6 compared with ctrl cells although the effect appeared less pronounced than in SW620.625 cells (Fig. 4d). Since MAP2K6 levels in HCT116 cells approached the detection limit of

western blotting (Supplementary Fig. 7), we estimated miR-625-3p-associated MAP2K6 reduction by mass spectrometry quantication, which showed a mean downregulation of3.6- and 1.7-fold in SW620.625 and HCT116.625, respectively (Fig. 4e).

The MAP2K6 30UTR contains a putative 8mer miR-625-3p seed site with a miRmap score of 85.49 (Fig. 4f). To experimentally conrm this, an B175 base fragment of the

MAP2K6 30UTR centred around this putative seed site was cloned into the 30UTR of a Renilla Luciferase reporter gene construct (30UTR WT Luc reporter). When transfected into HEK293T cells together with pre-miR-625-3p, Luc expression was reduced with 75% as compared with mock transfected cells (that is, Luc reporter with no MAP2K6 30UTR) (Fig. 4g). The reduction was specically related to miR-625-3p since co-transfection with a control pre-miR (Scr) had no effect on Luc (Fig. 4g). Furthermore, specic mutation of the miR-625-3p seed sequence (30UTR mut1 and mut2) completely abolished miR-625-3p-mediated reduction of Luc (Fig. 4g).

Altogether, the data strongly support that MAP2K6 is a direct and functional target of miR-625-3p.

MAP2K6MAPK14 signalling mediates oxPt response. MAP2K6 is a dual specicity protein kinase, which transduces cellular and environmental stress signalling to its substrates, the p38 MAP kinases (MAPK1114; ref. 10). In support of miR-625-3p regulating MAP2K6 signalling, we observed reduced phosphorylation of MAPK14Tyr180/Y182 upon miR-625-3p-induction (Fig. 5a). To appreciate the resulting change in MAPK14 activity, we quantied the MAPK14 substrates HSPB1Ser82 (ref. 11), 4EBP1Ser65 (ref. 12) and CDC25cSer216 (ref. 13) from multiple western blots; this showed 1.72.5- and1.86.6-fold reduction of substrate phosphorylation in HCT116.625 and SW620.625, respectively (Fig. 5a).

To mechanistically investigate the role of MAP2K6 in oxPt response in CRC cells, we stably expressed MAP2K6 lacking the miR-625-3p binding site in HCT116.625 cells. Western blotting conrmed high ectopic MAP2K6 levels after DOX induction compared with endogenous MAP2K6 (Fig. 5b), which is relatively lowly expressed in HCT116 cells compared with other CRC cells (Fig. 4d and Supplementary Fig. 7). First, we addressed the immediate changes in MAPK14 activity upon 30 min oxPt treatment. OxPt exposure in HCT116.ctrl.mock control cells led

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms12436 ARTICLE

c d

SW620

Target gene dysregulation

SW620

Cumulative

distribution

Log(625/ctrl)

1.00.80.60.40.20.00.0 1.0

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MXI1

IRAK2

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RCN1

BCL11A

COMMD8

MXI1

NUP35

ST18

IRAK2

Gene rank

Mock 3UTR wt

3UTR mut1 3UTR mut2

MAP2K6

miR-625-3p

0.25

Renillaluc / firefly luc

MAP2K6 proteome intensity ratiosHCT116

SW620

0.20

Rep:

5-...CCUUGGAAUCUAUAGUAUAGAAUG...-3

5-...CCUUGGAAUUGAUAUCAUAGAAUG...-3

5-...CCUUGGAAUUGUACUCAUAGAAUG...-3

#1 #2 #1 #2 #3

3UTR wt

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miR-625-3p

0.10

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MAP2K6 MAP2K3/MAP2K6

0.05

Pre-miR: None miR-625-3p Scr

Figure 4 | MAP2K6 is a direct and functional target of miR-625-3p. (a) Genes were ranked according to differential expression after miR-625-3p induction in SW620 cells (log2(625/ctrl) (bottom panel). The cumulative distribution of genes with a miR-625-3p seed sequence (red) was signicantly different from the distribution of genes without a seed sequence (black) (P 1.9*10 5, KolmogorovSmirnov test; top panel). Note that for clarity, the

log2(625/ctrl)-scale is truncated at 1 and 1. (b) The 625/ctrl-expression ratios of eight candidate target genes were determined by qRTPCR after

induction of miR-625-3p (or control) in SW620 cells and HCT116 CRC cells. Shown are mean expression ratios.e.m. (n 3). (c) qRTPCR quantication

of candidate target genes in RNA from AGO2-associated precipitates and normalized to GAPDH in input. Mean associations.e.m. (n 3) displayed

relative to control cells. (d) Representative western blots of MAP2K6 in SW620 and HCT116 cells after DOX-induction for 48 h. b-Actin (ACTB) was used as loading control. Quantication of MAP2K6 band intensities (normalized to ACTB) is indicated. (e) Quantication of MAP2K6 downregulation after induction of miR-625-3p as determined by mass-spec proteome analysis of two (SW620) or three (HCT116) independent DOX inductions. Displayed as log2 mean peptide intensity ratio. For SW620 data, 2045 kDa proteins were excised from a denaturing gel and subjected to unlabelled proteome quantication. For HCT116 data, we used isotope-labelled whole cell lysates described below (see Fig. 6a). Note that while one MAP2K6 specic peptide was quantied in the SW620 lysates, only peptides (n 2) shared between MAP2K3 and MAP2K6 were detected in HCT116 cells. (f) Structure of the

30UTR of MAP2K6 (ENSG00000108984, miR-625-3p binding site at 30UTR position 173180). The close-up depicts miR-625-3p annealed to the wild-type target sequence (underlined) as well as the two mutated sequences used in g. (g) Mean normalized Renilla Luc signals.e.m. (n 3) from HEK293T cells

24 h after transfection with psiCHECK-2 reporter containing MAP2K 30UTR, either of the mutated 30UTR sequences shown in f or mock. Experiments where a miR-625-3p or control (Scr) pre-miR were co-transfected together with psiCHECK-2 are indicated. *Po0.05 (t-test); NS, not signicant.

to increased MAPK14Tyr180/Y182 phosphorylation and a concurrent increase in MAPK14 activity (3.0-, 4.6- and2.7-fold increased phosphorylation of HSPB1Ser82, 4EBP1Ser65 and CDC25cSer216, respectively; Fig. 5b). However, when cells with increased miR-625-3p levels (HCT116.625.mock) were exposed to oxPt, we observed lack of MAPK14 activation and even a small reduction in MAPK14 substrate phosphorylation levels (Fig. 5b). In contrast, oxPt treatment of HCT116.625.map2k6 cells was associated with increased MAPK14 substrate phosphorylation (Fig. 5b), indicating that ectopic MAP2K6 was able to rescue oxPt-induced MAP2K6 signalling. Interestingly, the moderate induction of MAPK14 activity (1.4-, 1.4- and 1.7-fold increased HSPB1Ser82, 4EBP1Ser65 and CDC25cSer216 phosphorylation) shows that MAP2K6 overexpression is not associated with hyperactivation of MAPK14 signalling under these conditions. To directly address whether ectopic MAP2K6 in itself made HCT116 cells hypersentitive to oxPt, we induced ectopic MAP2K6 in HCT116.ctrl cells (HCT116.ctrl.map2k6) for 48 h before treating them with oxPt for 30 min (Fig. 5c). No hyperactivation was observed, in fact the induced increase in HSPB1Ser82, 4EBP1Ser65 and CDC25cSer216 phosphorylation(2.2-, 1.5- and 1.6-fold increased HSPB1Ser82, 4EBP1Ser65 and CDC25cSer216 phosphorylation) was less than in HCT116.ctrl.mock cells and comparable to HCT116.625.map2k6 cells (Fig. 5c). This suggests the presence of feedback mechanisms such as the dual-specicity protein phosphatases14 or that other signalling components become limiting15.

We next investigated how ectopic expression of the miR-625-3p insensitive MAP2K6 variant affected the ability of miR-625-3p to inhibit oxPt-induced cell death (Fig. 5d). As expected, after 48 h of oxPt treatment cell death was reduced in HCT116.625.mock compared to HCT116.ctrl.mock cells. The introduction of ectopic MAP2K6, however, resensitized HCT116.625.map2k6 cells to oxPt, reaching the same level of cell death as HCT116.ctrl.mock cells. In agreement with the changes in MAPK14 activity assessed after 30 min oxPt treatment (Fig. 5b,c), the control experiment with HCT116.ctrl.map2k6 cells conrmed that expression of MAP2K6 did not lead to oxPt hypersensitivity (Fig. 5d). Taken together, these ndings indicate that increased oxPt resistance mediated by miR-625-3p is conveyed through its target MAP2K6.

To further corroborate the importance of MAP2K6 for mediating the effect of miR-625-3p in CRC cells, we generated stable HCT116 cell lines expressing a dominant-negative version of MAP2K6 harbouring a K82A mutation, which abolishes kinase activity16. Western blotting showed the dominant-negative MAP2K6 to be expressed at many times higher level than the endogenous MAP2K6 (Fig. 5e). Dominant-negative MAP2K6 expressing cells showed a B40% reduction in 64 mM oxPt-induced cell death compared with control HCT116 cells (Fig. 5e), and hence, mimics the phenotype of miR-625-3p overexpressing cells.

We nally asked whether MAP2K6 might be correlated with miR-625-3p and chemotherapy response in patients? Indeed, although not reaching signicance, we found that MAP2K6 was

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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms12436

ctrl.mock 625.mock 625.map2k6

Quantification of

MAPK14 substrates

HCT116 SW620

OX (64 M, 30 min): +

3.00

Phosphorylation change

Log 2 (64 M / 0 M)

3.00 p-hspb1

p-4ebp1

p-cdc25c

Phosphorylation change

Log 2 (625 / ctrl)

Quantification of

MAPK14 substrates

SW620

p-hspb1

p-4ebp1

p-cdc25c

MAP2K6

pT180/Y182-MAPK14

pS82-HSPB1

pS65-4EBP1

TUB ACTB

pS216-CDC25c

pT180/Y182-MAPK14

2.00

pS82-HSPB1

pS65-4EBP1

pS216-CDC25c

TUB

ctrl 625 ctrl 625

ACTB 3.00

1.00

0.00

1550 15

Quantification of

MAPK14 substrates

3.00

0.00

1.00

2.00

HCT116

3.00 625.map2k6

ctrl.mock

625.mock

0.5

ctrl.mock 625.mock

ctrl.map2k6 625.map2k6

P = 0.11

P = 0.06

P = 0.09

ctrl.map2k6

OX (64 M, 30 min): +

0.4

3.00

Phosphorylation change

Log 2 (64 M / 0 M)

MAP2K6 pT180/Y182-MAPK14

pS82-HSPB1

pS65-4EBP1

ACTB

2.00

p-hspb1

p-4ebp1

p-cdc25c

0.3

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LDH

25 15 50

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TUB

2.00

0.1

ctrl.map2k6

0 0.5 1 2 8 16 64 M oxPt

MAP2K6 DN

1.25

1.00

CRC samples

LDH 64 M OX / 0 M OX

(relative to ctrl)

MAP2K6mRNA (log 2)

MAP2K6 mRNA (log 2)

MAP2K6

and response

0.75

P = 0.055

0.50

0.25

0.00

2 4 6 miR6253p (log2)

MAP2K6 ACTB

NR R

ctrl DN

Figure 5 | miR-625-3p regulates resistance to oxPt through MAP2K6 and MAPK14. (a) Western blotting using antibodies against the phosphorylated forms of MAPK14T180/Y182, HSPB1Ser82, 4EBP1Ser64 and CDC25cS216 in HCT116.625 and SW620.625 cells 48 h after DOX induction. b-Actin and tubulin was used as loading control (left). Quantication of HSPB1Ser82, 4EBP1Ser64 and CDC25cS216 western blot bands from three to ve western blots normalized to a-tubulin and b-actin and displayed as log2(625/ctrl)s.e.m. In one case the HSPB1Ser82 signal in SW620.625 was below detection level, and for this sample the median value for the two other replicates was used (right). (b) Changes in phosphorylation of activated pT180/Y182-MAPK14 and downstream substrates in HCT116.ctrl.mock, HCT116.625.mock and HCT116.625.map2k6 cells after 48 h of DOX induction followed by 30 min of 64 mM oxPt treatment (left). Quantication of HSPB1Ser82, 4EBP1Ser64 and CDC25cS216 substrate phosphorylation from three to ve western blots normalized to a-tubulin and b-actin and displayed as oxPt-induced phosphorylation change compared with untreated cells (log2(64 mM/0 mM)s.e.m. (right). (c) Same as b for the HCT116.ctrl.map2k6 cells. (d) Cells were DOX-induced for 48 h and treated with 064 mM oxPt for 48 h before cell death was determined (LDH assay). Bars represent the mean percentage of cell deaths.e.m. (n 3). Signicant difference between HCT116.625.mock and HCT116.625.map2k cells is

indicated (*Pr0.05, t-test). (e) Control HCT116 cells (ctrl) and cells expression a dominant-negative version of MAP2K6 (DN) were induced for 48 h and treated with 64 mM oxPt or left untreated for 48 h before the increase in cell death (64 mM/0 mM) was determined by LDH. Results are displayed relative to control cells (set to 1; means.e.m. from n 4 experiments; *Pr0.05; t-test). Western blot against MAP2K6 (f) Correlation between MAP2K6 mRNA

levels and mir-625-3p in clinical samples (P 0.212, Pearsons correlation). (g) MAP2K6 mRNA was downregulated in tumours from mCRC patients not

responding (NR, n 9) compared with responders (R, n 17) to rst-line oxPt-based therapy (t-test).

negatively correlated with miR-625-3p expression in 26 mCRC tumours (Pearsons r 0.22; Fig. 5f). In addition, we also

found MAP2K6 to be slightly downregulated in non-responder patients compared with responder patients (Fig. 5g). Altogether, these data suggest that the oxPt-resistant phenotype induced by miR-625-3p in CRC cells operates through the direct target MAP2K6.

miR-625-3p dysregulates MAPK signalling pathways. The results presented above indicates that the p38 MAPK subfamily

(MAPK1114) could be implicated as a mediator of platinum drug-induced stress signalling including apoptosis, a concept that has been exploited by others17,18. We therefore proled the phosphoproteome of both untreated and oxPt-treated HCT116.ctrl and HCT116.625 cells using stable isotope labelling by amino acid (SILAC)-based mass spectrometry of TiO2-enriched phosphopeptides (Fig. 6a). We detected 9,423 distinct phosphopeptides on 3,217 different proteins including 177 kinases and 50 phosphatases (Supplementary Fig. 8a). We found between one and three phosphosites per protein on 475% of all detected proteins (Supplementary Fig. 8b), and

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MAPK1

CSNK2A1

PRKACA

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Figure 6 | Decreased MAPK signalling is associated with miR-625-3p induction. (a) Phosphopeptide-enriched SILAC mass-spectrometry analysis was performed on HCT116.ctrl and HCT116.625 cells in two experimental setups done in parallel (EXP1 and EXP2) each involving three experimental conditions labelled with light (L), medium (M) or heavy (H) isotopes. Each experimental condition was done in triplicate. Four data sets were generated by calculating the mean log2 peptide intensity ratios from triplicate experiments for the following conditions: (i) HCT116.625/HCT116.ctrl (red),(ii) oxPt-treated HCT116.625/HCT116.ctrl (black), (iii) oxPt-treated HCT116.ctrl/HCT116.ctrl (blue) and (iv) oxPt-treated HCT116.625/oxPt-treated HCT116.ctrl (yellow). (b) Proteins with signicantly dysregulated phosphopeptides after miR-625-3p induction were subjected to GO term and KEGG pathway enrichment analysis. The adjusted P-values for the most associated terms are shown. P 0.05 is indicated with a stippled line. (c) Kinase

substrate enrichment analysis (KSEA) on log2(625/ctrl) ratios was calculated for substrate groups as the difference in the number of phosphopeptides with increased phosphorylation minus the number with decreased phosphorylation, and displayed as the fractional delta count (fcount), that is, the delta count divided with the sum of phosphopeptides in each substrate group (coloured bars indicate Pr0.05, hypergeometric test). (d) Mean log2(625/ctrl)

phosphorylation levels were calculated for the signicant substrate groups in c), and signicance of the difference to the population mean (stippled line) calculated with a z-test (Pr0.05 indicated with coloured boxes). Mean and median are shown with a squared box and a horizontal line, respectively, the interquartile range is marked by the lower and upper hinges, respectively, and the whiskers indicate 1.5 times the interquartile range.

detected predominantly serine phosphorylations (8,582 versus 816 and 25 threonine and tyrosine phosphopeptides, respectively; Supplementary Fig. 8c).

We rst looked at the overall effect on the phosphoproteome after 48 h of increased miR-625-3p levels. Proteins with altered phosphorylations were mostly associated with GO terms involved with increased GTPase activity in nucleus, cytoplasm and adherence junction components, and with the mTOR, ErbB, insulin signalling and MAPK pathways (Fig. 6b). To look for changes in the activities of individual kinases, we did kinase substrate enrichment analysis (KSEA)19 using a merged collection of specic kinase phosphorylation site mappings obtained from the Human Protein Reference Database20, PhosphoSitePlus21 and PhosphoELM22 (see Methods section). KSEA indicated decreased activity of MAPK8, MAPK14, MAPK1 and MTOR kinases, and increased activity of the CDK7, PRKACA and CSNK2A1 kinases, respectively, after miR-625-3p induction (Fig. 6c). In agreement, the mean log2 ratios of MAPK14,

MTOR and MAPK1 substrate groups were signicantly lower than the experimental mean, and for the PRKACA substrate group it was signicantly higher (Fig. 6d). Collectively, this indicates that miR-625-3p overexpression leads to decreased activity of MTOR, MAPK1 and the MAPK14 kinases. The latter in agreement with the notion that miR-625-3p targets MAP2K6.

To more specically investigate the role of MAPK14 in oxPt resistance, we rst inactivated MAPK14 signalling (indicated by reduced HSPB1Ser82 phosphorylation) in HCT116 and SW620

cells through siRNA mediated depletion of MAPK14 (Supplementary Fig. 9). MAPK14 depletion was associated with increased resistance to oxPt-induced cell death. In HCT116 cells the induced death was reduced to 50% of control cells, and in SW620 cells to 85% of controls (Fig. 7a).

Second, we used the small molecule inhibitor SB203580 to obstruct oxPt-induced MAPK14 activation in HCT116 cells as indicated by reduction of HSPB1Ser82 phosphorylation (Fig. 7b). SB203580 treatment reduced oxPt-induced cell death to less than 75% of control cells (Fig. 7c). A similar reduction in oxPt-induced cell death was also observed in SW620 cells (Fig. 7c). We also tested a second MAPK14 inhibitor, SB202190, which reduced the oxPt sensitivity in HCT116 cells, but not SW620 cells (Fig. 7c). Taken together, our observations in HCT116 and SW620 support that abrogation of MAPK14 signalling plays a role in oxPt resistance.

Speculating whether the observations could be generalized and extended to additional CRC cell lines we generated stable, inducible miR-625-3p expression in the HCC2998 CRC cell line (Fig. 7d). This line represents a tumour etiology distinct from HCT116 and SW620 by being microsatellite stable, expressing a truncated TP53 variant, and displaying a hypermutator phenotype as a consequence of a POLE missense mutation23. miR-625-3p levels in HCC2998.625 cells after DOX induction was increased 420-fold (Supplementary Fig. 10) and associated with decreased MAP2K6 levels as well as with decreased MAPK14 activity (Fig. 7d). Similar to HCT116 and SW620 cells, ectopic

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Colo205

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LoVo

Figure 7 | Inhibition of MAPK14 induces oxaliplatin resistance in CRC cells. (a) MAPK14 was specically depleted from HCT116 and SW620 cells by transfection of a pool of MAPK14 targeting siRNAs (siRMAPK14) 48 h before being treated with 64 mM oxPt for 48 h (or left unexposed; see Supplementary

Fig. 9 for knockdown efciencies). The impact on cell death (64 mM/0 mM) was determined by LDH and are displayed relative to cells transfected with a scrambled siRNA (siRscr, set to 1). Means.e.m. from at least n 4 experiments with * indicating a signicant reduction in oxPt-induced cells death

compared with siRscr transfected cells (Pr0.05, t-test). (b) A phospho-specic western blot versus the MAPK14/MAPKAPK2 substrate Ser82-HSPB1 was applied to show increased MAPK14 activity after oxPt treatment and the inhibitory effect of 10 mM SB203580 on this activity. (c) HCT116 and SW620 cells were treated for 1 h with MAPK11/14 inhibitors SB203580 (10 mM, blue) or SB202190 (5 mM, purple), then exposed to 64 mM oxPt (or left unexposed) for 48 h before the increase in cell death (64 mM/0 mM) was determined by LDH. Presented relative to cells not treated with inhibitor (DMSO treated;

means.e.m. from at least n 4 experiments with * indicating a signicant reduction in oxPt-induced death compared with DMSO-treated cells, Pr0.05,

t-test). (d) Stable, inducible expression of miR-625-3p was generated using pSBInducer transposition in HCC2998 CRC cells (left). Phospho-specic western blot for MAP2K6 and MAPK14 activity 48 h after DOX induction of HCC2998.ctrl and HCC2998.625 cells (right). (e) HCC2298.ctrl and HCC2998.625 cells DOX-induced for 48 h, then treated (or left untreated) with 64 mM oxPt for 48 h before the increase in cell death (64 mM/0 mM) was determined by LDH. Results are displayed relative to control cells (set to 1; means.e.m. from n 3 experiments; *Pr0.05, t-test). (f,g) HCC2998,

Colo205, DLD1, HT29 and LoVo CRC cells were treated for 1 h with MAPK11/14 inhibitors SB203580 (10 mM, blue) or SB202190 (5 mM, purple)then exposed to 64 mM oxPt (or left unexposed) for 48 h before the increase in cell death (64 mM/0 mM) was determined by LDH. Displayed relative to DMSO-treated cells (means.e.m. from n 34 experiments with * indicating a signicant reduction, Pr0.05, t-test). (h) A schematic model

showing how miR-625-3p mediated downregulation of MAP2K6 could modulate response to oxPt by abrogating MAPK14 stress-induced signalling. In the canonical model MAP2K6 in complex with MAP2K3 phosphorylates and activates MAPK14, which in turndirectly or indirectly via substrate kinases such as MAPKAPK2activates a diverse number of target proteins central to stress-induced transcription, translation, cell cycle control and apoptosis.

miR-625-3p expression reduced the 64 mM oxPt-induced cell death to B75% of control cells (Fig. 7e). Using the same conditions as above (Fig. 7c), chemical inhibition of MAPK14 signalling in HCC2998 cells by SB202190 also reduced oxPt induced cell death to B70%, while SB203580 had no effect (Fig. 7f).

To further generalize the involvement of MAPK14 signalling in oxPt response, the two MAPK14 inhibitors were applied to four additional CRC cell lines. In all four cell lines, MAPK14 inhibition reduced the sensitivity to oxPt (Fig. 5g). Taken together, these data show that inhibition of MAPK14 phenocopies the effect of miR-625-3p overexpression and supports the notion that the MAP2K6MAPK14 signalling network plays a central functional role in miR-625-3p-induced oxPt resistance (Fig. 7h).

The phosphoproteomic response to oxPt in CRC cells. To further characterize the role of miR-625-3p during oxPt treatment in CRC cells, we delineated phosphorylation changes associated with the immediate (30 min) response to oxPt in control CRC cells. Totally, we detected 205 phosphopeptides with

phosphoserines/threonines preceding a glutamine, which are potential substrates of ATM and ATR DNA damage response kinases (Fig. 8a)24. The pS/pTQ motif was enriched among peptides that had increased phosphorylation after oxPt treatment (Fig. 8b), indicating that the DNA damage response signalling was induced after 30 min of oxPt exposure. Although phosphorylation of pS/pTQ motifs increased upon oxPt treatment, the general trend was the opposite. Indeed, we found more than three times as many phosphopeptides with decreased phosphorylation (n 993) compared with phosphopeptides

with increased phosphorylation (n 313) after oxPt treatment

(Fig. 8c), suggesting global dephosphorylation in CRC cells immediately after oxPt exposure similar to what has been observed after cisplatin treatment25. Dysregulated phosphoproteins were associated with processes involved in chromatin remodelling, mitotic cell cycle, microtubule organisation and pathways such as mTOR, cell cycle, ErbB and MAPK signalling (Supplementary Fig. 11). KSEA analysis suggested increased activities of ribosomal protein S6 kinases beta-1 and alpha-1 (RPS6KB1 and RPS6KA1), and various protein kinases known to be implicated in genotoxic stress signalling (PRKACA, PRKCD and PRKD1)2629 as well as AKT1 (Fig. 8d). Reduced activities were found for cyclin-dependent

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ATM/ATR pS/pTQ motifs

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Kinases associated with 30 min oxPt treatment

OxPt induces global dephosphorylation

RPS6KB1

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RPS6KB1

RPS6KA1

PRKD1

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AKT1

PRKACA

CDK2

CDK1

PLK1

Figure 8 | The phosphoproteome response to oxPt in CRC cells. (a) A sequence logo was generated based on 205 detected phosphopeptides with potential ATM/ATR phosphorylation sites (pS/pTQ). (b) Fishers exact test on counts of dysregulated (log2(ctrl OX/ctrl)0.58 and false discovery

rate (FDR) r0.1) phosphopeptides revealed signicantly increased upregulation of pS/pTQ motifs after oxPt treatment. (c) The number ofaltered phosphopeptides after 30 min of 16 mM oxPt treatment were counted and grouped into peptides with decreased phosphorylation(log2(ctrl OX/ctrl)o0.58) (Loss) and increased phosphorylation (log2(ctrl OX/ctrl)40.58) (Gain). (d) KSEA was done on log2(ctrl OX/ctrl)

ratios (as described in Fig. 5). Only substrate groups with indication of altered activities after oxPt exposure are shown (*Pr0.05, hypergeometric test). (e) Mean log2 phosphorylation ratios for the nine substrate groups in d; (coloured boxes indicate Pr0.05, z-test). NS, not signicant.

kinase 1 and 2 (CDK1 and CDK2) and polo-like kinase 1 (PLK1; Fig. 8d), in agreement with all three being positively involved in cell cycle progression and inhibition of DNA damage response30,31. The importance of these kinases in the immediate cellular response to oxPt was also supported by increased mean log2 phosphorylation ratios for the RPS6KB1, RPS6KA1, PRKD1, AKT1 and PRKACA substrate groups, and by decreased ratios for the CDK1 and CDK2 substrate groups (Fig. 8e).

miR-625-3p blocks the normal cellular response to oxPt. We next investigated whether miR-625-3p expression affected the

predicted activities of the oxPt-regulated kinases identied by KSEA (see Fig. 8d,e). In the 625 OX/ctrl OX experiment, a

mean log2 ratio different from zero is expected for kinases whose activities after oxPt treatment are altered by increased miR-625-3p levels, while unaffected kinases will have a mean log2 ratio around zero. The mean phosphorylation ratios for the oxPt-induced PRKD1 and AKT1 substrate groups were decreased, while CDK1 and CDK2 substrates on average showed increased phosphorylation levels (Fig. 9a). Strikingly, the strongest change in mean log2 phosphorylation ratios were found for the MAPKAPK2 substrate group (MAPK14 substrate and binding partner) whose log2 ratio was decreased after oxPt

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R-pS pS/pTP pS/pTP-R/K

pS-D

..R.R..S.......

....RR.S.......

....R..S.......

....RS.S.......

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ACTB

CDK

FZR1

Active protein Unstable/inactive protein p Phosphorylation

Mitotic dissembly of nuclear lamina

ctrl 625

64 M OxPt

ACTB

ctrl 625

64 M OxPt

Figure 9 | Critical components of the cellular response to oxPt are blocked in cells with increased miR-625-3p levels. (a) Mean log2 ratios of substrates groups involved in oxPt response were calculated for the 625 OX/ctrl OX data. (b) Mean log2 for the MAPKAPK2 substrate group. (c) The most

signicantly altered substrate phosphorylation motifs identied for the ctrl OX/ctrl and 625 OX/ctrl OX experiments identied using KSEA

(displayed as fcounts, Pr0.05 indicated with "*"). Mean log2 ratios for substrates with these motifs were calculated. Coloured boxes in the boxplots of a, b and c indicate Pr0.05, z-test; and NS, not signicant. On the basis of similarity, the 16 individual motifs were grouped into the four motif groups indicated above. Note that the experimental mean log2 ratios for clarity have been omitted in b and c. (d) oxPt treatment in HCT116.ctrl cells led to dephosphorylation of Serine 130 (S130) of Cyclin-Dependent Kinase Inhibitor 1 (CDKN1A, also known as p21CIP1), which has been linked to increased stability of CDKN1A and inhibition of CDK/cyclin-mediated cell cycle progression65. In contrast, increased S130 phosphorylation was seen in cells with ectopic miR-625-3p expression. As indicated, this phosphorylation may itself be mediated by elevated CDK activity66. Increased CDK activity at the G1/S checkpoint or in early M phase was also indicated by S138/S151 phosphorylations on inactivated FZR1 (also known as CDH1) in miR-625-3p expressing cells, whereas unphosphorylated FZR1 in control cells suggested decreased CDK signalling at G0 or early G1 (ref. 67). In support of mitotic-induced nuclear lamina breakdown, increased phosphorylation was observed on multiple residues on LMNA in miR-625-3p cells; On the contrary, these became dephosphorylated after oxPt treatment in control cells indicating decreased cell cycle progression (also see Supplementary Fig. 14). (e) Western blotting against the CDK1 substrate phospho-LAMIN A/CS22 on lysates from oxPt-treated HCT116.ctrl and HCT116.625 cells. Quantication of bands representing Lamin A and C isoforms are indicated (normalized to b-actin signal). (f) Western blotting against the phosphorylated CDK motif p-TPXK on lysates from oxPt-treated HCT116.ctrl and HCT116.625 cells. Individual substrates are indicated with a dot with red and black indicating increase or decrease/no change in intensity, respectively, in HCT116.625 as compared with HCT116.ctrl cells.

treatment in HCT116.625 cells (Fig. 9b). The mean log2 ratios for all the ve substrate groups were in the opposite direction in the 625 OX/ctrl OX as compared with the OX ctrl/ctrl

experiment. In agreement with the miR-625-3p-induced oxPt resistance phenotype (Fig. 2a,b), this suggested that miR-625-3p blocks signalling cascades central in the normal response to DNA damage.

Further, we investigated whether miR-625-3p-mediated blockage of oxPt-induced signalling also was evident on a phosphorylation motif level. KSEA analysis and mean log2 phosphorylation ratios on motif groups (that is, phosphopeptides with a similar 15 amino acid-motif centred on the phosphorylated residue) suggested that oxPt treatment of control cells led to increased kinase activities directed towards serines that are preceded by one or two basic arginine residues (R-pS motifs), or followed by an acidic aspartate (pS-D motifs) (Fig. 9c). Dephosphorylation after oxPt treatment was seen on proline directed motifs with or without a single trailing basic residue

(pS/pTP-R/K and pS/pTP motifs; Fig. 9c), which are typically associated with the CDK, MAPK and GSK families32. In contrast, the oxPt response in the context of miR-625-3p led to increased pS/pTP-R/K-associated kinase activity, and generally, decreased R-pS-directed activity, while phosphorylations on pS/pTP motifs, in general, were similar in ctrl and 625 cells (Fig. 9c).

We used the network-based NetworKIN data set33 to identify kinases most likely associated with the differentially phosphorylated R-pS, pS-D and pS/pTP-R/K motifs (Supplementary Fig. 12). A signicant association was found between the oxPt-induced motifs (R-pS and pS-D) and multiple kinase families including AKT1 and AKT2 kinases, protein kinase A, Calcium/Calmodulin-Dependent Protein Kinase II kinases (CAMKII), as well as HIPK2 and PAK kinases. The miR-625-3p specic pS/pTP-R/K motif was most strongly associated with cyclin-dependent kinases (CDK1, CDK2 and CDK5), and to a lesser extent with MAP kinases and TTK kinase. As expected, many of these kinases are involved in DNA damage response

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(for example, AKT, CAMKII, HIPK2 and PAK) and cell cycle regulation (for example, CDK, MAPK and TKK). Furthermore, several of them overlap with the kinases identied in the substrate group analysis.

To identify individual phosphoproteins associated with the observed progressive cell cycle signalling, we rst dened regulatory classes based on those phosphopeptides with phosphorylation changes in opposite direction in the ctrl OX/ctrl

and 625 OX/ctrl OX experiments (Supplementary Fig. 13).

Among these, we identied several cell cycle-associated proteins, including CDKN1A, FZR1 and LAMIN A/C (Fig. 9d), with differential phosphorylation patterns that supported increased cell cycle progression in oxPt-treated HCT116.625 cells compared with control cells (Supplementary Fig. 14).

Phosphospecic western blotting against LAMIN A/CSer22 (Fig. 9e)a known CDK1 target at the onset of mitosis34 conrmed the observed increase in lamin phosphorylation (Supplementary Fig. 14), which is a marker of nuclear envelope disassembly during mitosis. Interestingly, increased LAMIN A/CSer22 phosphorylation in oxPt-treated HCT116.625 cells appeared to be a consequence of an increase in the LAMIN C over the LAMIN A isoform (Fig. 9e).

To conrm increased CDK activity after oxPt treatment in HCT116.625 cells, we did phosphospecic western blotting against the most differentially phosphorylated CDK motif pTPXK (Fig. 9c, a target for CDKs 1 and 2, among others). This revealed increased phosphorylation in oxPt-treated HCT116.625 cells at the majority of CDK substrates consistent with increased activity (Fig. 9f).

Finally, we found that phosphorylation of ATM/ATR pT/pSQ motifs in the oxPt-treated HCT116.625 cells was signicantly increased (Po0.05, Fishers exact test), indicating that alteration of cell cycle signalling in these cells was not related to lack of DNA damage sensing per se (Supplementary Fig. 15a,b). This suggests that miR-625-3p acts after, or independently of, the immediate ATM/ATR-mediated DNA damage response (Supplementary Fig. 15c).

Altogether, these analyses are in support of the hypothesis that miR-625-3p induces blockage of signalling pathways involved in normal oxPt response, which, among other things, culminates in increased cell cycle progression signals relative to control cells.

DiscussionPreviously, we reported that high expression of miR-625-3p in primary tumours of mCRC patients was associated with an odds ratio above 6 for a poor response to rst-line oxPt-based therapy5. In the present work, we have shown that miR-625-3p functionally leads to oxPt resistance by preventing the DNA damage response system to induce cell cycle arrest and apoptosis. Furthermore, we have identied MAP2K6 as a functional target for miR-625-3p, and as a mediator of miR-625-3p-induced oxPt resistance. To the best of our knowledge, MAP2K6 is the rst functionally documented target of miR-625-3p, and conversely, miR-625-3p is the rst described microRNA targeting MAP2K6. MAP2K6 (together with MAP2K3) catalyses dual phosphorylation of the TGY motif in the activation loop of the four p38 MAPK isoforms (MAPK1114; refs 3537), and as such conveys p38-mediated cellular stress signalling10. The presented results are consistent with a model were miR-625-3p through downregulation of MAP2K6 impairs p38-MAPK stress signalling (Fig. 7h and Supplementary Fig. 15c). It is important to emphasize, however, that our model only addresses miR-625-3p signalling through MAP2K6. It is likely that miR-625-3p additionally could mediate resistance by regulating other unknown target proteins.

On the basis of our results using chemical inhibitors and MAPK14 knockdown, and in agreement with other studies38,39,

we are inclined to believe that the MAPK14 isoform of p38 is a mediator of miR-625-3p-induced oxPt resistance. We are aware of the discrepancy in the effect on oxPt sensitity after chemical inhibition in two (SW620 and HCC2998) out of seven cell lines tested, which we attribute to the cell-specic off-targeting effects known to exist for SB203580 and SB202190 (refs 40,41). Our phosphoproteome data in exponentially growing unstressed CRC cells also revealed that MAPK14 was the kinase whose activity (on a substrate level) was mostly affected by miR-625-3p induction. Finally, oxPt treatment showed increased activity of the MAPKAPK2 kinase, which is a canonical MAPK14 substrate and binding partner responsible for nuclear translocation of MAPK14 after stress42. This suggests that MAPK14MAPKAPK2 activation plays a role during oxPt response in cancer cells. Such notion is further supported by our observation of reduced activity of MAPKAPK2 in oxPt-resistant HCT116.625 cells.

We observed resistance to oxPt after miR-625-3p induction in all three cell modelswith the strongest phenotype obtained in HCT116 cellsdespite different levels of induction (3 in

HCT116, 25 in HCC2998 and 4400 in SW620) and

different degrees of MAP2K6 reduction (0.8 in HCT116,

0.4 in HCC2998 and 0.2 in SW620). This indicates that the

resulting level of MAP2K6 proteinrather than changes in miR-625-3p and MAP2K6 per sedetermines response to oxPt. Alternative explanations include cell-specic wiring and dependencies of the MAP2K6MAPK14 signalling pathway15, and diversity in a stress mediator downstream of MAPK14. An interesting candidate is TP53, which is mutated in SW620 and HCC2998 cells but wild type in HCT116. These hypotheses will have to be addressed in future studies.

Induction of p38 signalling by platinum-based drugs has been ascribed a pro-apoptotic role in multiple types of cancer cells10,17,39,43,44. On the other hand, p38 may also induce survival signals after cytotoxic stress4547. In fact, MAP2K3/6-p38-MAPKAPK2/3 activation has recently emerged as a third signalling axis during DNA damage response, alongside ATM-CHEK2 and ATR-CHEK1 (refs 48,49). In this setting, p38 signalling functions as a cell cycle checkpoint by deactivating CDC25s, cyclinE and CDK1 to prevent premature mitotic entry48,50. Thus, the outcome from dysregulated p38 signalling in drug-treated cancer cells appears to be a function of several factors including the extent and nature of the cellular insult. In that respect, we note that increased sensitivity to the topoisomerase I inhibitor irinotecan (another drug used to treat CRC patients) has been shown to correlate with decreased p38 phosphorylation in CRC patients51. Following this, CRC patients with high mir-625-3p levels and reduced MAP2K6MAPK14 signalling, and therefore resistance to oxPt, may instead benet from irinotecan treatment as rst-line therapy.

The ndings reported suggest that the expression level of miR-625-3p, possibly in combination with the expression level and activity of MAP2K6 and MAPK14, has the potential to serve as a biomarker for predicting response to oxPt. Since up to 20% of mCRC patients show high miR-625-3p expression5, the number of patients that potentially could benet from quantication of the miR-625-3p biomarker is substantial. In addition, the observation that anti-miR-625-3p treatment makes cells with high miR-625-3p level responsive to oxPt, indicates that it may be possible to sensitize patients with high miR-625-3p expressing cancers to oxPt by miR-625-3p antagonist treatment before, or simultaneously with, oxPt treatment.

In conclusion, we have shown that overexpression of miR-625-3p in CRC cells can induce resistance to oxPt by directly targeting MAP2K6 and consequently inactivating genotoxic stress signalling conveyed by the MAP2K6MAPK14 pathway.

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Methods

Patients. Fresh frozen primary tumour biopsies originated from 26 patients who were treated with oxPt and 5-FU as rst-line therapy for mCRC in the Departments of Odense University Hospital and Aarhus University Hospital, Denmark, as described in ref. 52. Informed consent was obtained from allthe patients. The study was approved by the national ethics committees and governmental authorities in Denmark and was conducted in accordance with the Declaration of Helsinki. The patients were grouped according to objective therapy response into nine poor responders (best response being either Progressive disease or Stable disease) and 17 good responders (Partial response or Complete response).

Cell lines. HEK293 Flp pFRT/eGFP was a gift from Jacob Giehm Mikkelsen, Aarhus University, while CRC cells originated from the ATCC and NCI-60 repositories (kind gift from Nils Brnner, University of Copenhagen). The cell lines were authenticated by our in-house STR analysis (http://identicell.dk

Web End =http://identicell.dk), and were tested negative for mycoplasma using MycoSensor PCR Assay Kit (Stratagene). All the cell lines were grown in RPMI medium 1640 with L-glutamine (Life Technologies) supplemented with 10% heat-inactivated fetal calf serum (Life Technologies). The cells were propagated in 37 C at 90% air humidity and with 5% CO2. For oxPt treatment cells were rst induced for 48 or 72 h with 50 ng ml 1 doxycycline hyclate (Sigma-Aldrich), and then cultured in medium supplemented with the indicated concentrations of oxPt (Fresenius Kabi) together with doxycycline. Chemical inhibitors SB203580 (Invivogen) and SB202190 (Invivogen) were dissolved in dimethyl sulphoxide (DMSO) and kept in aliquots at 20 C

until use. The cells were pre-incubated 1 h with inhibitor (or DMSO) supplemented medium before exposure to oxPt (or medium) containing inhibitor (or DMSO).

Vectors. The pSBinducer vector was made by modication of the pINDUCER vector53. Using the pSBT-PGK-Puro plasmid as a template the SB right inverted repeat (SB-RIR) and the mouse phosphoglycerate kinase 1 polyadenylation segment (PGApA) cloning fragments were PCR-amplied with primer pairs MreI-SB-RIR and HindIII-XcaI-c (SB-RIR), and HindIII-PacI-PGKpA and MreI-c (PGKpA), respectively (the oligos are shown in Supplementary Data 3). The SB left inverted repeat (SB-LIR) cloning fragment was amplied from pT2_CMV-eGFP-SV40-neo using primers SgrDI-SB-LIR and NheI-c (SB-LIR). The PGKpA and SB-RIR fragments were digested with MreI (Fermentas) and ligated together with T4 ligase (New England Biolabs), before cloned into pUC18 using HindIII digestion. Lentiviral elements from the pINDUCER vector were removed by restriction digestion with SgrDI (Fermentas) and NheI (Fermentas) and the SB-LIR fragment inserted using the same restriction sites. The PGKpA.SB-RIR fragment was excised from pUC18.PGKpA.SB-RIR using PacI (Fermentas) and XcaI (Bst1107I, Fermentas) and introduced into pINDUCER.PGKpA.SB-LIR using the same restriction sites to generate the nal pSBInducer vector. A DNA oligo constructed to allow expression of a specic shRNA when inserted into the pSBInducer vector was amplied using the universal primers miR30PCRXhoI and miR30PCREcoRI and cloned into pSBInducer using XhoI and EcoRI restriction sites (Supplementary Data 3). To generate DOX-inducible SB-mediated stable expression of recombinant FLAG-MAP2K6 and FLAG-MAP2K6DN, we rst used the pINDUCER11 vector (in which the puromycin resistance gene is replaced by EGFP53) to make an EGFP expressing pSBInducer version according to the strategy outlined above. From this, we removed the tRFP-miR30-shRNA-miR30 element (see Supplementary Fig. 1) using AgeI and MluI restriction digestion and a multiple cloning site-N-FLAG sequence (MCS-NFLAG, PCR amplied) inserted using the same restriction sites. The MAP2K6 ORF was then PCR amplied from pcMKK6wt or pcMKK6(S87A)54,55 using primers BspEI-map2k6 and NotI-map2k6 and cloned using BspEI and NotI to generate the pSBInducer.map2k6. Mock vector (pSBInducer.mock) was made by removing MAP2K6 by restriction cloning.

In all the steps, plasmid DNA was puried with GeneElute Plasmid Miniprep Kit (Sigma-Aldrich). Correct insertion was conrmed by sequencing and with appropriate restriction digestions.

Generation of pSBInducer cells. To generate pSBInducer cells, approximately 10 mio. cells were transfected with 1,500 ng pSBInducer.shRNA DNA (siREGFP, miR-625-3p or scramble) and 1,500 ng pCMV-SB100XCO helper plasmid(or, as a negative control, 1,500 ng pUC19 DNA) using 15 ml Lipofectamine 2000 (Invitrogen) in 500 ml Opti-Mem I Medium (Gibco, Invitrogen-Life Technologies). After transfection, cells were incubated for 24 h before refreshing the media. The cells were treated with a puromycin concentration of 1 mg ml 1 (HCT116 and

HCC2998) or 2 mg ml 1 puromycin (SW620 and HEK293 Flp pFRT/eGFP) for5 days to eliminate control transfected cells. We used the tRFP uorescence marker to sort for cell populations expressing the shRNA after induction; these cells were frozen and used for subsequent experiments. All the experiments were conducted with low-passage (o10 passages after sorting) cell populations. Single cell clones were generated from single RFP-positive cells sorted directly into 96 wells from where they were propagated and frozen. We generated MAP2K6 (or Mock) expressing cells by transposing HCT116.625 (and HCT116.ctrl) cells with pSBInducer.map2k6 (and pSBInducer.mock) as described above, except that we used FACS to isolate EGFP/tRFP double positive cells. We used western blotting and

quantitative PCR with reverse transcription (qRTPCR) to conrm expression of FLAG-MAP2K6 protein and miR-625-3p, respectively. Sorting was performed at the FACS Core Facility, The Faculty of Health Sciences, Aarhus University, Denmark, on a FACSAria IIII (BD Biosciences).

Western blotting. Protein extraction and western blotting analysis were performed according to standard procedures. Antibodies were GFP (1:1,000, Abcam, ab1218), b-actin (1:25,000, Abcam, ab49900), tubulin (1:5,000, Abcam, ab7291), p38a/MAPK14 (1:500, Santa Cruz Biotechnologies, SC-81621), MAP2K6/MKK6 (1:5001:1,000, Cell Signaling, #8550), MXI1 (1:200, Santa Cruz Biotechnologies, SC-1042), IRAK2 (1:1,000, Cell Signaling, #4367), phospho-Thr180/Tyr182-p38a/MAPK14 (1:750, Cell Signaling, #9211), phospho-Ser82-HSPB1 (1:2,000, Cell Signaling, #2406), phospho-Ser216-CDC25c (1:750, Cell Signaling, #4901), phospho-Ser65-4EBP1 (1:7501:1,000, Cell Signaling, #9456), phospho-Ser22-Lamin A/C (1:1,000, Cell Signaling, #2026) and phospho-CDK Substrate[pTPXK] (1:1,000, Cell Signaling, #14371). Densitometrical quantication of MAP2K6 protein and MAPK14 phospho-substrates was done in ImageJ using b-actin and a-tubulin as loading controls (Supplementary Fig. 16).

RNA extraction, reverse transcription and qRTPCR. Total RNA from cell lines was puried using QIAzol Lysis Reagent (Qiagen) according to the manufacturers guidelines. RNA quality and integrity was ensured according to Agilent 2100 Bioanalyzer runs (RIN score 49.5 for all samples; Agilent Technologies). Small

RNA expression levels were quantied with qRTPCR according to the protocol of the Universal cDNA synthesis kit (Exiqon) using miRCURY LNA Universal RT microRNA PCR assays (Exiqon) and SYBR Green master mix (Exiqon) according to the manufacturers instructions. For mRNA detection, single-strand cDNA was synthesized using the Superscript Reverse Transcriptase Kit (Life Technologies) and qRTPCR was performed using SYBR Green PCR Master Mix (Applied Biosystems) as described in the protocol. Small RNA and mRNA expression was normalized with 5S and GAPDH, respectively. Samples with a mean Ct440 were assigned Undetermined. All qRTPCR measurements were done on a 7900 HT instrument (Applied Biosystems). MAPK14 mRNA was detected using TaqMan Assay Hs01051152_m1 (Cat# 4331182 Applied Biosystems) and normalizedto UBC.

Cell viability and death assays. Cell viability was measured using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay (Roche Applied Science). Cellular death (LDH release) was measured using the Cytotoxicity Detection Kit PLUS (LDH) (Roche Applied Science). Fluorescence signal was measured using a multi-well ELISA reader (Synergy HT-reader, BioTek).

Annexin VPI apoptosis assay. For the apoptosis assay, cells were DOX-induced and treated with 64 mM oxPt for 48 h. Adherent and non-adherent cells were collected, pooled and stained using the Annexin V-FITC Apoptosis Detection Kit (Sigma-Aldrich) according to the manufacturers protocol. Flow cytometry was performed at the FACS Core Facility, The Faculty of Health Sciences, Aarhus University, Denmark on a FACSAria IIII (BD Biosciences). FlowJo software version 8.8.3 (Tree Star Inc.) was used for data analysis. Initially, cells were gated with forward scatter-area (FSC-A) versus side scatter-area (SSC-A) followed by FSC-A versus forward scatter-height (FSC-H) to obtain cell singlets after which the percentage of cells in each quadrant of the uorescein isothiocyanate (FITC) versus PI plot were obtained. For clarity only n 8,000 cells were visualized although

typically at least 50,000 cells were counted.

Anti-miR and siRNA experiments. For anti-miR experiments, cells were DOX-induced for 24 h prior transfection with anti-miR (MH12612, mirVana miRNA inhibitor (miRBase ID: hsa-miR-625-3p) catalogue (Cat.) #4464084, Life Technologies) or control miR (Pre-miR miRNA Precursor MoleculesNegative Control #2 Cat. #AM17111, Life Technologies) for 24 h before incubation in the presence of 0 or 64 mM oxPt for additional 48 h. To knock down MAPK14, we used

SMARTpool, siGENOME MAPK14 siRNA (#M-003512-06-0005, Dharmacon Cat.). The cells were transfected with 20 nM siRNA 48 h prior LDH oxPt treatment. A scrambled siRNA (Cat. #4390843, Ambion) were transfected at 20 nM in parallel and used as control.

AGO2 pull-down. The SW620.625 and control cells were scraped off culture asks on ice in gentle lysis buffer (20 mM TRIS pH 7.5, 10 mM NaCl, 0.5% NP-40, 2 mM EDTA supplemented with RNase inhibitor RNaseOut (Life Technologies) and Complete Mini protease inhibitor cocktail (Roche Applied Science)), incubated for 5 min before being hypertonically lysed by increasing the NaCl concentration to 150 mM and incubated for additionally 5 min on ice. After 4 C centrifugation at 19,000g for 10 min, the supernatant was collected and subjected to pull-down (10% was used for input control) by incubation with monoclonal AGO2 antibody 11A9 (Sigma-Aldrich, Cat. #SAB4200085)-bound Protein G-coupled Magnetic Dynabeads (Life Technologies; 15 mg 11A9 per 25 ml beads) following the

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manufacturers recommendation. All washing steps were performed in ice cold washing buffer (50 mM TRIS pH 7.5, 150 mM NaCl and 0.05% NP-40), and total RNA from input and immunoprecipitate fractions puried with QIAzol (Qiagen). A parallel pull-down using monoclonal M2 anti-FLAG antibody (Sigma-Aldrich, Cat. #F1804) was performed as negative control.

miR-625-3p luciferase reporter assay. Wild-type and mutated versions of part of the MAP2K6 30UTR centred on the miR-625-3p target site were generated by primer extension PCR using oligos notI-map2k6.3UTR-rev and either of xhoI-map2k6.3UTR.wt-fwd, xhoI-map2k6.3UTR.mut1-fwd and xhoI-map2k6. 3UTR.mut2-fwd (see Supplementary Data 3), and cloned into the psiCHECK-2 plasmid after the Renilla luciferase (Rluc) reporter gene. psiCHECK-2 contains Firey luciferase, which enables accurate control of differences in transfection efciency between experiments and wells. psiCHECK-2 vector without MAP2K6 30UTR gave a robust signal and were used as mock control. 8000 HEK293T cells were transfected with Lipofectamin-2000 (Thermo Scientic) in 96-well plates using 50 ng mock, 30UTR wild-type or mutated vectors alone, or cotransfected together with 20 nM miR-625-3p pre-miR (Life Technology, #AM17100) or scramble control pre-miR (Negative Control2 Life Technology #AM17111). One day after transfection, the cells were lysed and Renilla and Firey substrates added using the Dual-Glo Luciferase Assay System (Promega #E2920) following the manufacturers recommendations, and luminescence read in a multi-well ELISA reader (Synergy HT-reader, Bio-tek). RLuc signals were normalized to Firey luminescence.

Microarray proling and RNA data processing. Expression proling on cell lines was performed on three biological replicates on total RNA (all with RIN 10)

isolated from SW620.625 and SW620.ctrl cells treated with 50 ng ml 1 DOX for 48 h using GeneChip Human Gene 2.0 ST arrays (Affymetrix) according to manufacturers recommendation. Expression proling on clinical samples was performed on RNA (median RIN 6.5) from pure cancer epithelium obtained

using laser microdissection as described52. RNA was amplied using the Ovation Pico WTA system (Nugen) and proled on GeneChip Human Gene 1.0 ST arrays (Affymetrix) according to manufacturers recommendation. All samples passed basic quality control measures as performed in Affymetrix Gene Expressing Console. The data were loaded into the GeneSpring v.12.5 software (Agilent Technologies), and probe intensities quantile normalized and summarized into probe set values using the IterPLIER16 algorithm. Probe sets were excluded if they had (i) a mean log2(expression) o5 in both SW620.625 and control cells;

(ii) an association with multiple gene symbols (that is, overlapping genes);(iii) no association with a gene symbol; (iv) an association with gene symbols mapping to several probes sets. Genes with a fold change 41.5 were considered dysregulated. Unsupervised hierarchical cluster analysis on SW620.625 and SW620.ctrl expression data were done in Cluster3 (ref. 56) using the most variable probe sets (that is, with a variance on the log2 expression values 40.25 across all the six samples) applying an absolute correlation similarity metric, and visualized using Treeview57. To identify putative miR-625-3p target genes, 30UTR sequences were obtained from TargetScan (6.2) for all human annotated transcripts, and the longest 30UTR sequence was chosen for isoforms with identical gene symbol. Genes were ranked by fold change in expression between miR-625-3p induction and scrambled control. Subsequently, 30UTR sequences were scored for the presence of the target motif complementary to the seed sequence of miR-625p-3p (that is, CTATAGT). The empirical distribution of the ranks for genes with and without the target motif was compared using a KolmogorovSmirnov test. The web-based miRmap tool (http://mirmap.ezlab.org/app/

Web End =http://mirmap.ezlab.org/app/) 8 was used with standard parameters using the options Species Human and miRNA hsa-miR-625-3p; Candidate

target genes with a miRmap score 475 were extracted.

SILAC labelling and phosphopeptide enrichment. The HCT116.625 and HCT116.ctrl cells were grown in SILAC RPMI 1640 medium (PAA, Cat. #E15-087) with 2 mM L-glutamine and 10% dialysed fetal bovine serum (Sigma, Cat. #F0392) supplemented with heavy isotopes Arg10-13C6,15N4 (1.14 mM) and Lys8-13C6,15N2 (0.22 mM; Cambridge Isotope Laboratories, Cat. #CNLM-539-H and #CNLM-291-H), medium isotopes Arg6-13C and Lys4-D4 (Cat. #CLM-2265-H and #DLM-2640-O) or light isotopes Arg0 and Lys0 (Sigma). After 46 cell passages, we ensured that the incorporation rate was 495%. We also ensured that the HCT116.625 cells retained oxPt resistance after DOX induction compared with the HCT116.ctrl cells. The cell triplicates were labelled, induced with DOX and exposed to 16 mM oxPt according to the protocol described in Fig. 6a and

Supplementary Fig. 10. Total protein lysates were harvested after several washes in ice-cold PBS using radioimmunoprecipitation assay buffer (50 mM TRIS pH 7.5, 150 mM NaCl, 50 mM EDTA, 0.1% sodium deoxycholate, 1% NP-40) supplemented with 1 mM Na-orthovanadate, 5 mM NaF, 5 mM b-glycerophosphate and complete Protease Inhibitor Cocktail (Roche). Lysates were pelleted at 4 C at 15,000g for 15 min and the supernatant transferred to ice-cold acetone. The precipitated proteins were resuspended in 6 M urea, 2 M thiourea and 10 mM HEPES pH 8.0 and concentration was estimated with Bradford assay. Two triple SILAC experiments were designed to cover all conditions (Supplementary Fig. 10). After mixing proteins 1:1:1, these were reduced in 1 mM dithiothreitol followed by

alkylation with 5 mM chloroacetamide, both steps for 45 min. The mixtures were pre-digested with LysC (Wako) in an enzyme/protein ratio of 1:100 (w/w) for 3 h followed by dilution with 50 mM ABC pH 8.0 to 2 M urea and further digested overnight with trypsin 1:100 (w/w). The digestion was quenched with triuoroacetic acid TFA to a nal concentration of 2% and the peptide mixture was washed and eluted from Sep-Pak (C18 Classic Cartridge, Waters). Elution was done with 2 ml 40% acetonitrile (ACN), 0.1% TFA followed by 4 ml 60% ACN,0.1% TFA. The sample volume was doubled by addition of 12% TFA in ACN and subsequently enriched with TiO2 beads (5 mm, GL Sciences Inc., Tokyo, Japan) as previously described58, and nally enriched for a second and third time.

MS/msproteome and phosphoproteome processing. The peptide mixture was separated on an in-house made 50 cm capillary column packed with 1.9 mm Reprosil-Pur C18 beads (Dr Maisch, Germany) using an EASY-nLC 1,000 system (Thermo Scientic). The column temperature was maintained at 50 C using a column oven (PRSO-V1, Sonation GmbH, Biberach, Germany) and the LC system was interfaced online with the Q Exactive mass spectrometer (Thermo Scientic). Formic acid 0.1% was used to buffer the pH in the two running buffers used. The total gradient was 250 min followed by a 15 min washout and re-equilibration. In detail, the ow rate started at 250 nl min 1 and 5% ACN with a linear increase to 25% ACN over 220 min followed by 30 min linear increase to 60% ACN. The washout followed with 60% ACN for 5 min followed by re-equilibration with a5 min linear gradient back down to 5% ACN, which were maintained for the last 5 min. For phosphopeptide-enriched samples, the Q Exactive was operated with a data-dependent method using Top10. Full scan resolutions were set to 70,000 at 200 m/z with a target value of 3 106 and a maximum ll time of 20 ms. Mass

range was set to 3001,750 m/z. Fragment scan resolution were set to 35,000 with target value 1 105 and maximum ll time 108 ms. Proteome data were acquired

with a Top12 method and fragment scan resolution 17,500 and 44 ms ll time. Isolation width was 2 m/z and normalized collision energy (NCE) 28 for phosphor-enriched samples and 2.2 m/z and 25 NCE for proteome samples. All raw LC-MS/MS data were analysed by MaxQuant v1.4.1.4 (ref. 59), and searched against the human Uniprot database (April 2012 release). Carbamidomethylation of cysteine was specied as xed modication for both groups. For the proteome data, variable modications considered were oxidation of methionine, protein amino (N)-terminal acetylation and pyro-glutamate formation from glutamine. The phosphoproteome data were additionally searched with phosphorylation as a variable modication of serine, threonine and tyrosine residues. The match between run option was enabled, and the minimum score for both modied and unmodied peptides were set to 25, we used false discovery rate limit of 1% on peptide level.

Proteome and phosphoproteome data analyses. From the two (HCT116.625 and HCT116.ctrl) triplicate proteome intensity data, we made log2(625/ctrl) ratios, and used only distinct proteins that were detected in all three ratios (n 2,410). All

proteins with an absolute log2(625/ctrl) 40.58 are listed in Supplementary Data 2. SW620 proteome data were generated by separating protein lysates on a denaturing Bis-Tris gel, excise proteins between 20 and 45 kDa, which were then subjected to in-gel digestion followed by nLC-MS60. In HCT116 cells, MAP2K6 were quantied using peptides GAYGVVEK and INPELNQK shared with the MAP2K3 paralogue (since no MAP2K6 specic peptides were detected), whereas we used the MAP2K6 specic peptide DVKPSNVLINALGQVK in SW620 cells. Phosphopeptide positions were mapped to the Homo sapiens canonical UniProtKB data set61. We used HGNC gene names obtained from UniProtKB, which together with the phosphorylation positions acted as unique identiers. Log2 ratios of normalized phosphopeptide intensities were generated for each triplicate ctrl OX/ctrl,

625/ctrl, 625 OX/ctrl and 625 OX/ctrl OX experiment, and the mean log2

ratio calculated for phosphopeptides detected in at least two of three replicates, while singly detected phospopeptides were discarded. We used a t-test to test the null hypothesis of no difference, that is, H0: log2 ratio 0, and estimated the local

false discovery rate to obtain the chance of individual log2 ratios being false positives62. The Enrichr tool63 was used to obtain enrichment scores (P-values) to KEGG pathways and GO-terms, using HGNC names as input. Only the top ve (lowest P values) for each category were reported. To detect changes in kinase activities, we applied the KSEA framework developed by Cassado et al.19 This is based on assigning individual phosphopeptides to one or more substrate groups according to the kinase(s) known or believed to catalyse the phosphorylation.

To obtain such information, we merged and manually curated three collections of kinase phosphorylation mappings obtained from the Human ProteinReference Database20, PhosphoSitePlus21 and PhosphoELM22. From this collection, we extracted those phosphorylated substrates detected in our experiments, and used this to make 25 substrate groups with at least 10 substrate members. The fractional delta-count (fcount) was then calculated as the number of substrates with increased (log2 ratio 40, NI) minus the number of substrates with decreased (log2 ratio o0, ND) phosphorylation divided by the total number of substrates in the group, that is: fcount (NI ND)/(NI ND). A BenjaminHochberg corrected P-value

from a hypergeometric test was calculated to indicate the statistical signicance of obtaining NI (and ND). In addition to the fcount measure, we also calculated the mean log2 ratio (x) for all substrate log2 ratios within individual substrate groups, and tested whether this diverged from the population (experimental) mean (m)

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using the z statistics (z (x m)/(s/N0.5), where s is the population standard

deviation and N the number of substrates in the group). The Z values were converted into two-sided P values via the standard normal distribution(P 2*F( abs(z))), which were corrected for multiple testing with the

BenjaminHochberg procedure. To nd altered phosphorylation motifs by KSEA for the ctrl OX/ctrl and 625 OX/ctrl OX data, we rst extracted 15 amino

acids-windows (7 1 7) around the central phosphorylated amino acid for all

serine and threonine phosphopeptides (detected in two out of three experimental triplicates). Proteins with phosphorylations within seven amino acids from theN or carboxy (C) terminus were discarded. Phosphopeptides were then subjected to the motif-x algorithm64 using the following parameters: occurrences 5,

signicance 0.000001 and background IPI Human Proteome. This resulted in

84 different motifs based on 7,850 phosphopeptides detected in the experiment. We further restricted KSEA to 51 motifs that were detected at least 50 times. To nd kinases whose activities were associated with individual substrates and motifs, we obtained the netwoKIN data set of kinase substrate mappings33, but restricted us to entries with a high condence (arbitrarily chosen as a networkin_score 43), corresponding to 36,972 out of the total 304,338 distinct kinase substrates in networKIN. To predict kinase activities associated with the differentially phosphorylated motifs (R-pS, pS-D and pS/pTP-R-K) we simplied the 15 amino acid motifs to 11 amino acids (5 1 5) motifs to directly use the 11 amino

acids-phosphorylation windows provided for substrates in the networKIN data. Note that this resulted in the yy.SPy.K. motif was removed from the analysis since it is minimally described by a 6 1 6 format. We counted the

enrichment score (ES) for each motif being associated with a given kinase as: ESm,k (nm,k/Nm)/(Nk/N), where nm,k is the number of times a kinase k is mapped to

a motif m, Nm is the total number of motifs m, Nk is the total number kinases k and N is the total number of kinase motif mappings. We used a BH-corrected P value from a two-sided Fishers exact test as signicance for the association, and for each motif selected the top-10 kinases with the smallest P value and with at least 100 observations for a motif (nm,kZ100). Due to overlap between motifs, this resulted in 39 distinct kinases.

Statistical analysis. If not mentioned otherwise, a two-sided Students t-test was performed to evaluate statistical signicance of differences in means, and the Fishers exact test used to test independence of count data. Experiments were performed at least three times and P values r0.05 were considered signicant (indicated with * in gures).

Data availability. Proteome data are available via ProteomeXchange with identier PXD002172. Clinical and cell line expression data can be obtained via GEO with identiers GSE83129 and GSE83131, respectively. The authors declare that all other data supporting the ndings of this study are available within the article and its Supplementary Information Files or from the corresponding author upon reasonable request.

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Acknowledgements

We thank Thomas Westbrook, Baylor College of Medicine, for providing us with INDUCER vectors. The plasmids encoding MAP2K6 and MAP2K6DN were kindly provided from Richard Vaillancourt, University of Arizona College of Pharmacy. HEK293 Flp pFRT/eGFP cells, pSBT-PGK-Puro and pT2_CMV-eGFP-SV40-neo plasmids were a generous gift from Jacob Giehm Mikkelsen, Aarhus University. All CRC cells were a kind gift from Nils Brnner, University of Copenhagen. We thank the FACS Core Facility, Aarhus University and the MOMA Microarray Core Center, Aarhus University Hospital, for helping with the analyses. Excellent technical assistance was provided by Hanne Steen, Maria Engtoft, Birgitte Trolle, Susanne Bruun and Louise Nielsen.

Author contributions

M.H.R. and I.L. designed and performed the experiments, as well as analysed most of the data. M.H.R., I.L., R.R.J.-C. and J.V.O. designed, performed and analysed the proteome part. L.S.T., T.P.H., F.H. and P.P. collected patient material and evaluated responsesto therapy. M.H.R., I.L. and B.P.-B. designed the vectors. M.H.R., M.M.N. and J.S.P. performed the bioinformatics. M.H.R., I.L., T.F.. and C.L.A. conceived the overall study design and wrote the paper.

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How to cite this article: Rasmussen, M. H. et al. miR-625-3p regulates oxaliplatin resistance by targeting MAP2K6-p38 signalling in human colorectal adenocarcinoma cells. Nat. Commun. 7:12436 doi: 10.1038/ncomms12436 (2016).

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Oxaliplatin resistance in colorectal cancers (CRC) is a major medical problem, and predictive markers are urgently needed. Recently, miR-625-3p was reported as a promising predictive marker. Herein, we show that miR-625-3p functionally induces oxaliplatin resistance in CRC cells, and identify the signalling networks affected by miR-625-3p. We show that the p38 MAPK activator MAP2K6 is a direct target of miR-625-3p, and, accordingly, is downregulated in non-responder patients of oxaliplatin therapy. miR-625-3p-mediated resistance is reversed by anti-miR-625-3p treatment and ectopic expression of a miR-625-3p insensitive MAP2K6 variant. In addition, reduction of p38 signalling by using siRNAs, chemical inhibitors or expression of a dominant-negative MAP2K6 protein induces resistance to oxaliplatin. Transcriptome, proteome and phosphoproteome profiles confirm inactivation of MAP2K6-p38 signalling as one likely mechanism of oxaliplatin resistance. Our study shows that miR-625-3p induces oxaliplatin resistance by abrogating MAP2K6-p38-regulated apoptosis and cell cycle control networks, and corroborates the predictive power of miR-625-3p.

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Title

miR-625-3p regulates oxaliplatin resistance by targeting MAP2K6-p38 signalling in human colorectal adenocarcinoma cells

Author

Rasmussen, Mads Heilskov; Lyskjær, Iben; Jersie-christensen, Rosa Rakownikow; Tarpgaard, Line Schmidt; Primdal-bengtson, Bjarke; Nielsen, Morten Muhlig; Pedersen, Jakob Skou; Hansen, Tine Plato; Hansen, Flemming; Olsen, Jesper Velgaard; Pfeiffer, Per; Ørntoft, Torben Falck; Andersen, Claus Lindbjerg

Pages

12436

Publication year

2016

Publication date

Aug 2016

Publisher

Nature Publishing Group

e-ISSN

20411723

Source type

Scholarly Journal

Language of publication

English

DOI

https://doi.org/10.1038/ncomms12436

ProQuest document ID

1811566359

miR-625-3p regulates oxaliplatin resistance by targeting MAP2K6-p38 signalling in human colorectal adenocarcinoma cells

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