MEK and TAK1 Regulate Apoptosis in Colon Cancer Cells with ...MEK pathway–dependent cancers due to...

Cell Death and Survival

MEK and TAK1 Regulate Apoptosis in ColonCancer Cells with KRAS-Dependent Activationof Proinflammatory SignalingKelsey L. McNew,William J.Whipple, Anita K. Mehta, Trevor J. Grant,Leah Ray, Connor Kenny, and Anurag Singh

Abstract

MEK inhibitors have limited efficacy in treating RAS–RAF–MEK pathway–dependent cancers due to feedback pathwaycompensation and dose-limiting toxicities. Combining MEKinhibitors with other targeted agents may enhance efficacy.Here, codependencies of MEK, TAK1, and KRAS in colon cancerwere investigated. Combined inhibition of MEK and TAK1potentiates apoptosis in KRAS-dependent cells. Pharmacologicstudies and cell-cycle analyses on a large panel of colon cancercell lines demonstrate that MEK/TAK1 inhibition induces celldeath, as assessed by sub-G1 accumulation, in a distinct subsetof cell lines. Furthermore, TAK1 inhibition causes G2–M cell-cycle blockade and polyploidy in many of the cell lines. MEKplus TAK1 inhibition causes reduced G2–M/polyploid cellnumbers and additive cytotoxic effects in KRAS/TAK1-depen-dent cell lines as well as a subset of BRAF-mutant cells. Mech-anistically, sensitivity to MEK/TAK1 inhibition can be conferredby KRAS and BMP receptor activation, which promote expres-sion of NF-kB-dependent proinflammatory cytokines, driving

tumor cell survival and proliferation. MEK/TAK1 inhibitioncauses reduced mTOR, Wnt, and NF-kB signaling in TAK1/MEK-dependent cell lines concomitant with apoptosis. A Wnt/NF-kB transcriptional signature was derived that stratifies pri-mary tumors into three major subtypes: Wnt-high/NF-kB-low,Wnt-low/NF-kB-high and Wnt-high/NF-kB-high, designatedW, N, and WN, respectively. These subtypes have distinctcharacteristics, including enrichment for BRAF mutations withserrated carcinoma histology in the N subtype. Both N and WNsubtypes bear molecular hallmarks of MEK and TAK1 depen-dency seen in cell lines. Therefore, N and WN subtype signa-tures could be utilized to identify tumors that are most sensitiveto anti-MEK/TAK1 therapeutics.

Implications: This study describes a potential therapeutic strategyfor a subset of colon cancers that are dependent on oncogenicKRAS signaling pathways, which are currently difficult to blockwith selective agents. Mol Cancer Res; 14(12); 1204–16. �2016 AACR.

IntroductionColon cancers are molecularly and histologically heteroge-

neous with multiple oncogenic driver mutations promotingtumorigenesis via deregulated MAPK, Wnt, BMP, and NF-kBsignaling pathway activation. KRAS and BRAF mutations occurfrequently and drive MEK–ERK mitogenic pathway activation.KRAS mutations cooperate with inactivating APC and SMAD4mutations to hyperactivate deregulated canonical Wnt and TGFb/BMP receptor signaling, respectively, causing accelerated andaggressive tumorigenesis (1–4). KRAS,Wnt, and TGFb/BMP path-ways are subject to extensive crosstalk through complex, context-

dependentmechanisms leading tomolecular andhistologic intra-and intertumor heterogeneity. This complexity is illustrated byglobal gene expression profiling andmolecular subtype classifica-tions (5–8). KRAS-mutant tumors do not classify into a distinctsubtype anddisplay highly diversemolecular signatures. Recently,molecular diversity has been documented in KRAS-mutant lungcancers, where cooccurring mutations in STK11/LKB1 and TP53generate distinct molecular subtypes with selective pharmacolog-ic vulnerabilities (9, 10). Identifying subtype-selective vulnerabil-ities in RAS/RAF pathway–dependent cancers may yield moreefficacious therapeutics.

Using a transcriptional signature associated with KRAS depen-dency in colon cancer cell lines, we identified the TGFb-activatedkinase (TAK1) as a critical cell survival mediator in KRAS-depen-dent cells (11). We blocked TAK1 kinase activity with an anti-inflammatory agent, 5Z-7-oxozeaenol (5Z-7-oxo), which inducesapoptosis in KRAS-dependent cells. In this study, we determinedthat 5Z-7-oxo has off-target MEK kinase inhibitory activity. Thisprompted our interest in evaluating the cytotoxic effects ofcombining MEK and TAK1 inhibition with single agents. KRAS-mutant colon cancer cell lines exhibit a spectrum of MEK depen-dencies, whereas BRAF-mutant cell lines are significantly moreMEK dependent (12). Furthermore, MEK inhibitor sensitivitiescan be correlated with distinct transcriptional signatures (13). Wehypothesized that combining MEK and TAK1 inhibitors wouldinduce additive cytotoxic effects in a KRAS-dependent subtype of

Department of Pharmacologyand Experimental Therapeutics, Centerfor Cancer Research, Boston University School of Medicine, BostonMassachusetts.

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

Current address for K.L. McNew: Vanderbilt School of Medicine, Nashville,Tennessee.

Corresponding Author:Anurag Singh, Boston University School of Medicine, 72E. Concord St., Room K712B, Boston, MA 02118. Phone: 617-638-4175; Fax: 617-638-4176; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-16-0173

�2016 American Association for Cancer Research.

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colon cancer cell lines. Indeed, previous studies have describedeffective combination approaches with MEK kinase inhibitors totreat KRAS-driven cancers (14–16).

TAK1 mediates innate immunity and proinflammatory signal-ing via regulation of NF-kB and AP-1 (Jun/Fos) dependent tran-scriptional programs (17). Autocrine or paracrine proinflamma-tory signaling drives KRAS-dependent tumor cell survival (18–23). However, the underlying mechanisms and implications ofKRAS-dependent proinflammatory signaling for treatment ofRAS/RAF pathway–dependent tumors has yet to be fully deter-mined. In this study, we analyzed MEK/TAK1 dependencies in acomprehensive panel of colon cancer cell lines that displayvarying molecular and phenotypic characteristics. The overarch-ing goal was to identify definitive molecular correlates of MEK/TAK1 codependencies. Given the role of TAK1 in proinflamma-tory signaling, we investigated the role of the KRAS–TAK1 axis inregulating inflammatory cytokine expression levels and subse-quent effects onMEK/TAK1dependencies. Finally,wedeterminedwhether molecular hallmarks of MEK/TAK1 dependencies corre-late with molecular subtype classifications of primary tumorsfrom patients with colon cancer.

Materials and MethodsOligonucleotide microarray analyses

Robust multiarray averaged (RMA) normalized primary tumordata frompatients with colon cancer were used for gene expressionanalyses of the canonical Wnt/NF-kB signatures and are availablethrough the NCBI GEO database (Affymetrix Human Exon Array -GSE39582; ref. 7). All genome-scale datasets were processed andanalyzed using R and Bioconductor software packages. A set ofgenes whose expression correlated significantly with the canonicalWnt target gene AXIN2 was first identified. Within this list, RNF43was the most correlated gene with AXIN2. RNF43-correlated geneswere then isolated using Pearson correlations with 0.5 and�0.5 ascut-off thresholds. This yielded a set of 184 positively and nega-tively correlated genes. Expression data for this 184-gene set acrossthe 566 primary tumors was extracted, followed by hierarchicalclustering using Euclidean distance as a similarity metric. Heat-mapswere generated using the pheatmappackage in R. TheCancerGenome Atlas (TCGA) data for colorectal cancer primary tumorcohorts (colorectal adenocarcinoma;COAD)wereobtainedvia theBroad Institute's Firebrowse web portal. For Ingenuity PathwayAnalysis (IPA; Fig. 6E), differentially expressed genes in Wnt-high/NF-kB-low, Wnt-low/NF-kB-high, and Wnt-high/NF-kB-high (W,N, andWN, respectively) subtypeswere identifiedby linearmodel-ing using the limma package in R.

High-content imaging analyses of cell-cycle phaseCells were imaged using the Celigo fluorescence imaging plate

reader (Nexelcom Inc.). Image analysis software was used to countindividual nuclei and correspondingfluorescence intensity for eachnucleus.Whenplottedasahistogram,distinctpeakswereobserved,which indicated cells at various stages of the cell cycle. Formost celllines, a prominent histogram peak was seen corresponding to cellsin the G1 phase, with a second smaller peak of double the intensityof the G1 peak, corresponding to cells in the G2–M phase. Anysignals to the left of the G1 peak are considered "sub-G1" andindicative of apoptotic or necrotic cells with less that 2N DNAcontent. Signals to the right of the G2–M peak are consideredpolyploid and indicate cells that have greater that 4N content.

Thus, relative fractions of cells in each cell-cycle phase were deter-mined in control or inhibitor-treated cells. Tab-delimited object-level data were compiled and processed using scripts written in the"R" software package. Scripts are available upon request.

Cell lines, reagents, and plasmid constructsHuman colon cancer–derived cell lines were used throughout

this study as described previously (11). 5Z-7-oxozeanol (5Z-7-oxo), AZD6244, and AZ-TAK1 were purchased from Sigma, Sell-eckChem Inc., and Toronto Research Chemicals Inc., respectively.The following lentiviral-based expression plasmids were used inthis study: pWPI (gift from Didier Trono, Ecole PolytechniqueFederale de Lausanne, Switzerland, Addgene plasmid #12254),pLEX307 (gift from David Root, BROAD Institute, MIT/Harvard,Cambirdge, MA, USA, Addgene plasmid #41392), and pLenti6-CMV (Invitrogen).

Western blottingCell lysates were prepared in Laemmli buffer. SDS-PAGE and

transfer to polyvinylidene difluoride membranes were performedfor antibody incubations. Enhanced chemiluminescence (ECL)was used to detect proteins with West-Pico or West-Dura reagents(Pierce-Thermo). ECL imaging was performed on a Syngene G-BoxXT4 systemwith theGeneSys imaging software. The followingantibodies were used forWestern blotting: PARP, Axin2, p-AMPK,t-AMPK, caspase-8, pS6, S6, pERK, ERK, pS6K, S6K, pAKT, AKT,V5, pTAK1, TAK1, pSAPK/JNK (Cell Signaling Technology), pan-RAS (BD Biosciences), ERK, and GAPDH (Santa Cruz Biotech-nology). All antibodies were diluted in 2% BSA/TBS-T solution.Secondary anti-mouse or rabbit HRP–conjugated antibodieswerepurchased from Cell Signaling Technology.

Lentiviral shRNA experimentsAll shRNA constructs were in the pLKO.1 backbone from the

Broad Institute's RNAi Consortium (TRC). Lentiviral particleswere generated using a three-plasmid system, as described previ-ously (24, 25). Target cells were spin-infected with recombinantlentiviruses in the presence of 8 mg/mL polybrene at 1,200� g for1 hour. Stable selection of cell lines was performed by treatingcells with 1 mg/mL puromycin for 1 week.

Cellular proliferation and viability assaysCell lines were obtained from ATCC and were validated by the

ATCC by testing for cell line–specific short tandem repeats (STR).Cell viability assays were performed in a 96-well plate formatusing AlamarBlue reagent. Cells were treated with compounds for72 hours or subjected to shRNA-mediated gene depletions for 96hours followed by incubation with 50 mg/mL AlamarBlue for 2hours at 37�C/5%CO2. AlamarBlue fluorescence was quantitatedusing a Fluostar Optima plate reader (BMG Labtech) at lEx/lEm590 nm. For clonogenic assays, 2 � 104 cells were plated in thewells of 12-well plates and treated with indicated inhibitors oragents. Clones were grown for twoweeks withmedia changes andaddition of indicated drug concentrations every 3 days. Cloneswere quantified by measuring pixel density of images using theImageJ software package.

Luciferase reporter assaysTOP-FLASH assays were performed using a stable TCF4-binding

element containing reporter (gift from Roel Nusse, Stanford Uni-versity, Stanford, CA, USA, Addgene plasmid #24308). NF-kB

KRAS, MEK, and TAK1 Codependencies in Colon Cancer

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assays were performed using a lentiviral expression construct offirefly luciferase under the control ofkBbinding elements, pHAGE-NF-kB-LUC(gift fromAndrewWilson,BostonUniversity SchoolofMedicine, Boston, MA). Cells stably expressing the reporter con-structs were plated in 96-well plates at a density of 5� 103 cells perwell in100-mL total volume. Reporter assayswereperformed in livecells using 100mg/mLD-luciferin followed by a "glow-based" assayfor luminescence with 5-second read-time per well. Measurementswere obtained using a Fluostar Optima plate reader.

Cytokine and antibody arraysHuman Inflammation Antibody Array C3 (RayBiotech Inc.)

was used to detect cytokines and factors in conditioned mediafrom cells under indicated conditions. Cells were plated in 150-mmdishes in 20mL of serum-free RPMI. Twenty-four hours later,media were concentrated 20-fold using 3 kDa Centrifugal FilterUnits (Millipore). Binding and visualizationwas performed as perthemanufacturer's recommended guidelines and imagedwith theSyngene G-Box XT4 system/GeneSys software. Human CytokineArray Panel A (R&D Systems Inc.) was used in Supplementary Fig.S4 according to the manufacturer's guidelines.

qPCR-based gene expression analysesCytokines and BMP/Wnt target mRNA levels were measured

using TaqMan assays. RNA was extracted from cells using theRNeasy Kit (Qiagen) and reverse transcribed with the HighCapacity cDNA Synthesis Kit (Applied Biosystems). DilutedcDNA was used in TaqMan qPCR assays with standard recom-mended/validated probe-based assays for each gene and relativequantitation determinations were performed in a StepOne Plussystem (Applied Biosystems).

ResultsTAK1 and MEK dependency in KRAS-dependent colon cancercell lines

A subset of KRAS-dependent colon cancer cell lines aresensitive to the naturally occurring anti-inflammatory agent5Z-7-oxo, reported to be a selective irreversible TAK1 kinaseinhibitor (11, 26). We subsequently noted that 5Z-7-oxo inhi-bits MEK and a number of other kinases (MRC, InternationalCenter for Kinase Profiling; http://www.kinase-screen.mrc.ac.uk/kinase-inhibitors). To confirm the effects on MEK, we trea-ted SK-CO-1, KRAS-dependent, colon cancer cells with 5Z-7-oxo over a time course and observed almost complete loss ofERK phosphorylation (pERK) after 1 hour of treatment, indi-cating strong MEK inhibition (Supplementary Fig. S1A). How-ever, pERK levels steadily rose after 2 hours reaching approx-imately 25%maximal levels at 7 hours. This reactivation of ERKhas been documented in previous studies of MEK inhibition inKRAS-mutant cancer cell lines (27). As PI3K–mTOR signaling isa key survival pathway downstream of oncogenic KRAS, weassessed 5Z-7-oxo effects on phosphorylation and activation ofdownstream PI3K/mTOR pathway components, ribosomalprotein S6, S6 kinase, and AKT (pS6, pS6K, and pAKT). Incontrast to pERK levels, pS6K/pS6 levels steadily decreasedfollowing 5Z-7-oxo treatment, yet remained low at longer timepoints, up to 7 hours. An increase in pAKT levels was seenfollowing drug treatment, which occurs in many cancer celllines upon MEK inhibition due to feedback mitogenic pathwayactivation (28).

Subsequently, we used the more selective, allosteric MEKinhibitor selumetinib/AZD6244(MEKi) in a similar time courseexperiment. Like 5Z-7-oxo, MEKi caused rapid reduction in pERKlevels, which increased steadily but remained at lower levels ascompared with 5Z-7-oxo treatment at the same dose (Supple-mentary Fig. S1B).MEKi also caused reduced pS6 levels albeit to alesser extent than 5Z-7-oxo.We then tested the effects of 5Z-7-oxoand MEKi on apoptosis induction. Although both inhibitorspromoted loss of ERK phosphorylation, 5Z-7-oxo induced higherlevels of cleaved PARP than MEKi at a concentration of 5 mmol/L,suggesting that the apoptotic effects of 5Z-7-oxo are not solely duetoMEK inhibition (Supplementary Fig. S1C).We confirmedTAK1dependency in SK-CO-1 cells using siRNA oligonucleotide trans-fection to deplete TAK1 (Supplementary Fig. S1D). Finally, wecould rescue the apoptotic effects of TAK1 depletion using RNAi-resistant (RR) mutants of two TAK1 splice variants A and D whenreconstituted together into KRAS-dependent cells (Supplementa-ry Fig. S1E). TAK1-A encodes a 75-kDa isoform and TAK1-Dencodes a shorter 50-kDa isoform that differ in their C-terminiand have nonoverlapping functions (29). TAK1-A-RR reconstitu-tion partially rescued TAK1 shRNA–induced effects on caspase-8cleavage as well as p-AMPK levels (Supplementary Fig. S1F).Interestingly, TAK1 overexpression led to increased pERK levels,indicating that TAK1 can promote MEK–ERK pathway activation.

As 5Z-7-oxo nonselectively inhibits MEK as well as TAK1, weidentifieda selectiveTAK1kinase inhibitor.AZ-TAK1inhibits TAK1kinase activity in cells at submicromolar doses (30). AZ-TAK1treatment of KRAS-dependent Gp5D cells led to increases in PARPcleavage, indicating apoptotic cell death (Fig. 1A). This was asso-ciated with dose-dependent decreases in levels of the Wnt targetgene Axin2 and decreased ribosomal S6 phosphorylation.Of note,AZ-TAK1 treatment alone had no significant effect on pERK levels,indicating that AZ-TAK1 does not inhibit MEK and that MEKinhibition is not necessary for apoptosis induction. Next, wecompared the effects of AZ-TAK1 treatment in KRAS-dependent(KRAS mutant) SK-CO-1 cells versus KRAS-independent (KRASwild-type) HT-29 cells (Fig. 1B). TAK1 inhibition caused a dose-dependent increase in PARP cleavage in SK-CO-1 cells but had noeffect on PARP in HT-29 cells. TAK1 regulates proinflammatorysignaling, which is commonly associated with extrinsic deathreceptor–mediated apoptosis, for example, through TNFa andTRAIL. Therefore, we assessed caspase-8 cleavage, which occursupon death receptor activation. TAK1 inhibition led to dose-dependent increases in cleaved caspase-8 levels in SK-CO-1 cells,but had no effect on caspase-8 inHT-29 cells. The apoptotic effectsof TAK1 inhibition were associated with reduced levels of pS6. Wecompared the effects of TAK1 inhibition in two KRAS-mutant celllines with contrasting KRAS dependencies, SW620 (KRAS-depen-dent) versus SW837 (KRAS-independent; Fig. 1C). AZ-TAK1caused dose-dependent increases in PARP and caspase-8 cleavagein SW620 cells, which did not occur in SW837 cells. KRAS-inde-pendent SW837 cells had high basal levels of cleaved caspase-8,which didnot further increase uponAZ-TAK1 treatment. PARPandcaspase-8 cleavage were associated with decreased pS6 levels inSW620 cells following TAK1 inhibition. As with the other cell linestested, TAK1 inhibition did not cause reduced pERK levels in eitherSW620 or SW837 cells.

As the apoptotic effects of 5Z-7-oxo associate with concomitantTAK1 and MEK inhibition, we assessed the cytotoxic effects ofcombining individual TAK1 and MEK inhibitors in colon cancercell lines.We compared clonogenic growth of twoKRASwild-type

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(KRAS-independent) and two KRAS-mutant (KRAS-dependent)colon cancer cell lines, all harboring APC mutations, followinginhibition of TAK1 and MEK alone or in combination (Fig. 1D;Supplementary Fig. S1G). The KRAS wild-type cell line C2BBe1showed increased growth following TAK1 inhibition. Clonogenicgrowth of C2BBe1 cells increased slightly more with combinedTAK1/MEK inhibition. HT-29 cells, which are BRAF-mutant,showed slightly reduced growth following MEK inhibition butdid not respond additionally to combined MEK and TAK1 inhi-bition. TAK1 inhibition alone caused 50% reduced growth in theKRAS-dependent Gp5D cell line. Upon combined TAK1/MEKinhibition,we observed over 90% reduction of clonogenic growthin Gp5D cells, indicating additive effects of the combinationtreatment. SK-CO-1 cells were highly sensitive to TAK1 inhibitionalone. Combining TAK1 andMEK inhibition had little additionaleffects compared with TAK1 inhibition alone. In contrast, the

reduced cell viability induced by combined TAK1/MEK inhibitioninGp5D cells corresponded tomarked induction of apoptotic celldeath as assessed by PARP and caspase-8 cleavage (Fig. 1E).Furthermore, Axin2 and pS6 levels were additively decreased withthe combination treatment. Taken together, we conclude thatcombined MEK and TAK1 inhibition induces additive cytotoxiceffects in mutant KRAS–dependent cells.

MEK/TAK1 inhibition causes altered cell-cycle progression incolon cancer cell lines

To determine the spectrum of MEK/TAK1 dependencies incolon cancer cell lines, we performed pharmacologic studies ofAZ-TAK1 andAZD6244/MEKi in a comprehensive panel of 30 celllines with varying molecular and histologic characteristics. TheMDCK (canine kidney derived) cell line was included as a "nor-mal" control. To generate a comprehensive phenotypic dataset,

Figure 1.

KRAS-dependent colon cancer cell lines are TAK1- andMEK-dependent.A,Dose-dependent effects of the reversible selective ATP-competitive TAK1 kinase inhibitorAZ-TAK1 in KRAS-dependent Gp5D cells. Cells were treated for 24 hours at the indicated doses or with DMSO vehicle control (0). Cleaved PARP levelsindicate activation of apoptosis. Phosphorylated ribosomal S6 (pS6) and pERK levels are indicated. Axin2 levels indicate activation of canonical Wnt signaling. TotalERK and S6 levels serve as gel loading controls. B, Effects of TAK1 inhibition on indicated protein expression levels in SK-CO-1 (KRAS-mutant/mut) versusHT-29 (KRAS-wild type/WT) cells. GAPDH serves as an additional gel loading control. C, Western blot analyses of AZ-TAK1 treatment of KRAS-mutant cell linesSW620 (KRAS-dependent, DEP) and SW837 (KRAS-independent, IND).D,Clonogenic growth effects of AZ-TAK1 (1mmol/L) andAZD6244 (MEKi, 0.2mmol/L) eitheralone or in combination over a 7-day period in KRAS wild-type (WT) versus mutant (MUT) cell lines. Data are represented as the mean of three replicates� SEM (� , �� , �� level of significance as assessed by Student t test derived P values). E, Combined effects of AZ-TAK1 and MEKi on apoptosis induction as well ascanonical Wnt (Axin2) and RAS (S6, ERK) signaling pathways.






weperformed high-content imaging analyses on inhibitor-treatedcells (72-hour treatment) using Hoechst 33526 nuclear staining,followed by fluorescence imaging and nuclear intensity quanti-fication (see Materials and Methods). First, we generated proba-bility density plots for each cell line treated with increasing dosesof AZ-TAK1 plus MEKi at two different doses. In subsets of celllines, TAK1 inhibition induces polyploidy as assessed by theemergence of distinct peaks in the density plots correspondingto cells with 8N, 16N, and 32N DNA content. These effects weremost clearly evident in the RKO cell line, which is TAK1-depen-dent and BRAF-mutant (31). A low dose of 0.25 mmol/L AZ-TAK1

caused a marked increase in cells with 8N and 16NDNA content.At 0.5 mmol/L AZ-TAK1, cells with 32N DNA content emerged(Fig. 2A). MEKi alone caused an increase in cells with 8N DNAcontent. Strikingly, combiningMEKiwith AZ-TAK1 negated someof these polyploidy-inducing effects of AZ-TAK1,with the32Ncellsubpopulation completely abolished (Fig. 2A). Similar resultswere seen with a number of KRAS-mutant cell lines includingDLD-1 and HCT116 (Supplementary Fig. S2A and S2B; ref. 11).

Cell counts for each phase of the cell cycle were determined bygating the peaks from the density plots shown in Fig. 2A usingimage analysis software. Thus, relative fractions of cells in each

Figure 2.

TAK1 andMEK inhibition cause altered cell-cycle progression in subsets of colon cancer cell lines.A,Representative rawDNA intensity density plots of Hoechst33526-stained RKO cells treated with indicated doses of AZ-TAK1 (left) or AZ-TAK1 plus MEKi (right). Peaks are labeled by relative DNA content, 2N equivalentto diploid DNA content and 4N or greater indicative of tetraploid cells. Cells were treated for 72 hours with inhibitors. Data are cumulated from 4 wells of a 96-wellplate for each condition. B, Representative stacked bar plots of Gp5D KRAS-dependent cells indicating relative proportions of cell subpopulations in eachof the indicated cell-cycle phases (G1–S, G2–M, polyploid/>4Nor sub-G1) following treatmentwith increasingdoses ofAZ-TAK1 combinedwith either DMSOcontrol or0.2/1 mmol/L of MEKi. Data are derived from cell-cycle plots as shown in A following gating at each phase (e.g., 2N peaks represent G1–S cells, 4N peaksrepresent G2–M cells). C, Heatmap representation of data shown in B for a 30 cell line cohort with mixed molecular and histologic characteristics. Cell lines areclustered by similarity using Euclidean distance. The heatmap data are segregated by rows into each cell-cycle phase and treatment condition. The nontransformedMDCK cell line is highlighted in green. All data are means of four independent replicates per condition.

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phase of the cell cycle were quantitated. Using this approach, welooked at the effects of AZ-TAK1 and MEKi on Gp5D cells. AZ-TAK1 caused amarked increase in the sub-G1 population ofGp5Dcells indicating induction of cell death. AZ-TAK1 treatment alsocaused increased numbers of polyploid Gp5D cells (Fig. 2B).When MEKi was combined with AZ-TAK1, the number of poly-ploid cells decreased. Total cell counts were additively decreasedin many cell lines when MEKi and AZ-TAK1 were combined(Supplementary Fig. S2C). After compiling cell-cycle phase datafor the 30 cell line cohort, we performed hierarchical clusteringanalysis of the data to identify coclustered cell lines exhibitingsimilar responses to AZ-TAK1/MEKi treatment (Fig. 2C). Of note,sensitivity to AZ-TAK1 did not significantly relate to underlyingoncogenic KRAS or BRAF mutations. We identified a cluster of 3cell lines that were very sensitive to AZ-TAK1 treatment, asassessed bymarked increases in sub-G1 cells upon drug treatment.These were CL11, MDST8, and SW48. Of the 30 cell lines tested,17 underwent significant polyploidization following AZ-TAK1treatment. A number of cell lines did not show appreciableresponsiveness to AZ-TAK1 treatment, including CL40, HT55,and LS180 cells. As a number of KRAS WT cells were TAK1dependent, we determined whether these cell lines were alsoKRAS-dependent. Indeed, shRNA-mediated KRAS depletion inKRASWT/TAK1-dependent cell lines RKOandMDST8 induced anapoptotic response as assessed by PARP and caspase-8 cleavage(Supplementary Fig. S2D). In contrast, HT29 cells, which areinsensitive to TAK1 inhibition, did not undergo apoptosis fol-lowing KRAS depletion. In summary, these data indicate thatsubsets of colon cancer cell lines undergo cell death or mitoticdefects following TAK1 and MEK inhibition.

KRAS and BMP receptor activation sensitize to MEK/TAK1inhibition

BMP receptor–mediated TAK1 activation occurs in many KRAS-dependent colon cancer cells (11). We can confer TAK1 depen-dency by introducing mutant KRAS or constitutively active BMPreceptor (BMPR1A-CA) in KRAS-WT HT-29 cells (SupplementaryFig. S3A and S3B; ref. 11). When coexpressed in HT-29 cells, KRASand BMPR1A-CA caused a 10-fold leftward shift in the dose–response curve for 5Z-7-oxo (Fig. 3A). To understand the under-lying mechanisms for this synthetic lethality, we generated stablecell line variants of HT-29, which express 4-hydroxytamoxifen (4-HT)–inducible ER-KRAS (iKRAS) alone or coexpressed withBMPR1A-CA. Addition of 4-HT to these cells caused rapid iKRASactivation. We either chronically induced iKRAS for two weeks orpulse-activated iKRAS for 24 hours and passaged the cells for twoweeks following deactivation of iKRAS. Cells with chronic iKRASactivation were sensitized to 5Z-7-oxo treatment, leading to cas-pase-8 and PARP cleavage (Fig. 3B). In cells with activated iKRAS/BMPR1A-CA, Axin2, pTAK1, pERK, and pS6 levels were elevated.5Z-7-oxo treatment reduced Axin2 levels and levels of phosphor-ylated TAK1, ERK, and S6. We noted that pulse-activated iKRAS/BMPR1A-CA–expressing cells maintained sensitivity to 5Z-7-oxodespite no increase in baseline pERK activation, indicating apossible metastable epigenetic switch induced by transient iKRASactivation. 5Z-7-oxo treatment did not cause appreciable changesin pERK levels in control/vehicle–treatedHT-29 cells, but did causereduced pERK in iKRAS-activated cells. The reasons for thesevariable effects on ERK are unclear, but could relate to para-crine/autocrine activation of MAPK signaling by cytokines orgrowth factors that are induced following treatmentwith5Z-7-oxo.

We noted that overexpression of oncogenic forms of fourclosely related RAS isoforms (H, N, K-4A, and K-4B) causesinduction of caspase-8 cleavage (Supplementary Fig. S3C). Thissuggests that oncogenic RAS proteins trigger death receptor sig-naling, likely through the induction of proinflammatory cyto-kines such as TNFa. Mutant HRAS and KRAS-4A caused thestrongest induction of caspase-8 cleavage. Furthermore, KRAS-4A induced stronger TAK1 dependency in HT-29 cells comparedwith the KRAS-4B isoform (Supplementary Fig. S3D). Therefore,for subsequent experiments, we generated stable cell line variantsof HT-29–expressing mutant KRAS-4A and/or BMPR1A-CA.KRAS/BMPR1A activation caused reduced expression of the neg-ative BMP signaling regulator BAMBI (Fig. 3C). In contrast, Wnttarget gene expressionwas elevated inKRAS/BMPR1A-CA–expres-sing cells, includingAXIN2 aswell as PROX1 andMSX2, which areassociated with TAK1 dependency in colon cancer cell lines (11).

KRAS-4A/BMPR1A-CA coexpression sensitized HT-29 cells toMEKi and AZ-TAK1 (Fig. 3D). KRAS/BMPR1A-CA coexpressingcells displayed stronger PARP/caspase-8 cleavage following TAK1/MEK inhibition comparedwith vector control cells.Of note,MEKicaused reduced pS6 but increased pAKT (S473) levels in KRAS/BMPR1A-CA–expressing cells indicating reduced mTORC1 andincreased mTORC2 activities (32). AZ-TAK1 caused reduced pS6levels in control, but not in KRAS/BMPR1A-CA cells. Conversely,AZ-TAK1 reduced pAKT levels only in cells with mutant KRAS.BothpS6 andpAKT levelswere abrogatedwith theMEKi/AZ-TAK1combination. As expected, pERK levels were significantly dimin-ished by MEKi, but did not change significantly with AZ-TAK1.Thus, oncogenic KRAS and BMPR1A-CA cooperate to "rewire" thesignaling network in HT-29 cells such that TAK1 promotesmTORC2 activity, whereas MEK predominately activatesmTORC1.We conclude that the induction of cell death followingMEK/TAK1 inhibition in cells expressing oncogenic KRAS andBMPR1A-CA is associated with combined effects of dualmTORC1/mTORC2 blockade by parallel mechanisms.

The effects of KRAS/BMPR1A on TAK1 dependency were val-idated with TET-inducible shTAK1 introduced into the KRAS/BMPR1A-CA–expressing HT-29 cells. TAK1 expression was ablat-ed with doxycycline (Fig. 3E). Transient expression of mutantKRAS-4A inHT-29 cells followed by TAK1depletion, led to higherlevels of cleaved caspase-8 compared with KRAS-4B expressionand TAK1 depletion (Supplementary Fig. S3D). In stable KRAS-4A/BMPR1A-CA HT-29–variant cells, TAK1 depletion and MEKiled to increased caspase-8 andPARP cleavage, similar to the effectsobserved with combined TAK1/MEK inhibition. In vector controlHT-29 cells, pS6 levels were additively reduced following com-bined TAK1depletion andMEKi,whichwasnot seen in theKRAS/BMPR1A-CA–expressing cells. Finally, we observed reduced pAKTlevels with combined MEKi and TAK1 depletion in the KRAS/BMPR1A-CA cells, but not in vector control cells, similar to theeffects we saw with TAK1/MEK pharmacologic inhibition. Thus,apoptotic effects of combined TAK1 depletion and MEKi areassociated with mTORC1 regulation of pS6 levels and mTORC2effects on AKT phosphorylation.

KRAS and BMP signaling interactions modulateproinflammatory cytokine expression

Given the role of TAK1 in proinflammatory signaling, wehypothesized that inflammatory cytokine signaling would beinvolved in KRAS-dependent tumor cell survival. To that end, weperformed a series of experiments to characterize the expression






Figure 3.

KRAS and BMP receptor coactivation sensitizes cells to TAK1 and MEK inhibition. A, Dose–response curves for 5Z-7-oxo in control or mutant KRAS(G12V) andBMPR1A-CA (Q233D constitutively activated mutant) expressing HT-29 cells. B, HT-29 cells stably expressing inducible ER-KRAS(G12V) (iKRAS) alone(control/iKRAS) or coexpressed with BMPR1A-CA and treated with EtOH control (left 2 lanes), treated chronically with 4-HT for two weeks (middle 2 lanes) or pulsetreated for 24 hours and passaged in the absence of 4-HT for twoweeks (right 2 lanes). Cells were subsequently treatedwith DMSO vehicle control or 2 mmol/L 5Z-7-oxo for 24 hours. PARP and caspase-8 cleavage indicate effects on apoptosis. TAK1 autophosphorylation levels (pTAK1) indicate activation of TAK1. Phospho-ERK,-S6, and -AKT levels indicate activation of canonical RAS signaling. Total levels of proteins serve as gel loading controls. V5 levels indicate expression ofV5 epitope–tagged BMPR1A-CA protein. C, Relative expression of Wnt pathway target genes by TaqMan-based qRT-PCR in Luciferase (Luc) control–expressingversusKRAS/BMPR1A-CA–expressing cells. Data areplotted ona log2 scale as themeanof three replicates�SEM.D,Effects of TAK1/MEK inhibition inHT-29–derivedstable cell lines expressing either vector control or mutant V5-KRAS-4A/BMPR1A-CA. Expression levels of apoptotic markers and phosphorylated/activatedRAS/MAPK pathway components JNK, ERK, S6, and AKT are shown. E, HT-29–derived stable cell lines expressing a tet-R–controlled shTAK1 constructas well as control vector or activated KRAS-4A(G12V) plus BMPR1A-CA. TAK1 depletion is induced upon doxycycline (DOX) treatment (0.1 ng/mL). Effects onexpression of apoptotic markers and canonical RAS pathway components ERK, S6, and AKT are indicated. Total protein or GAPDH levels serve as gel loadingcontrols. Data are representative of two or three independent experiments.

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levels of proinflammatory secreted factors in KRAS/BMPR1A-CA–expressing HT-29 cells. Secreted factors in conditioned mediafrom control or KRAS/BMPR1A-CA HT-29–stable lines weredetected using an antibody array–based proteomics approach(Fig. 4A and B). GM-CSF was the most upregulated factor inKRAS/BMPR1A-CA–expressing cells, with over a 32-fold induc-tion. Other strongly induced factors included sTNFRII, a solubledecoy receptor for TNFa, as well as IL10, RANTES/CCL5, andPDGF-B. We performed a similar experiment with an alternativeantibody array (R&D Systems Inc.) and obtained similar results,observing strong induction of GM-CSF, RANTES, IP-10/CXCL10,and TNFa by mutant KRAS (Supplementary Fig. S4A and S4B).

Next, we quantitatively assessedmRNA expression levels of keycytokines by qRT-PCR in the KRAS/BMPR1A-CA HT-29–stablelines. We noted strong mRNA induction of IL8, CSF2 (encodingGM-CSF), CCL2, and TNF (encoding TNFa), in KRAS/BMPR1A-

CA–expressing cells (Fig. 4C; Supplementary Fig. S4C). CCL5,which encodes RANTES, was elevated when either KRAS orBMPR1A was expressed alone, but slightly decreased in theKRAS/BMPR1A-CA coexpressing cells. The discrepancy withresults from the antibody array could be explained by feedbackcontrol of CCL5 transcription in cells with high protein expres-sion. Although elevated in single mutant KRAS–expressing cells,we noted reduced expression of CXCL9, IL1RN, and CXCL10 inKRAS/BMPR1A-CA double mutant cells. IL1RN encodes the IL1receptor antagonist (IL1RA), which functions as an anti-inflam-matory decoy receptor (33). To test whether IL1RA or CXCL9 hadantiproliferative effects on KRAS-dependent cell lines, weexpressed recombinant IL1RA and CXCL9 in 293T cells. IL1RNand CXCL9 mRNA expression in 293T cells was verified by qRT-PCR (Supplementary Fig. S4F). IL1RA and CXCL9 293T condi-tionedmediawas applied to SK-CO-1 andGp5Dcells, resulting in

Figure 4.

KRAS and BMP receptor activation cooperate to induce proinflammatory cytokine expression, which contributes to tumor cell survival.A,Antibody array analysis ofinflammatory cytokines in conditioned media from indicated stable cell line derivatives of HT-29–expressing KRAS-4A(G12V), BMPR1A-CA, or combinationsof each. Most prominently induced cytokines are highlighted in colored boxes. B, Quantitation of relative fold induction of cytokines in indicated HT-29–derivedstable cell lines compared with vector control cells. Data are the mean of duplicates � SEM and are representative of two independent experiments. C,Transcriptional induction of cytokine expression inHT-29–stable lines depicted inA andB as assessedbyqRT-PCR.Data are themean of triplicates�SEM.CXCL9 andIL1RN, which encodes the IL1RA are highlighted in red. D, Clonogenic growth effects of treating SK-CO-1 and Gp5D cells with exogenous recombinantIL1RA and CXCL9. E, Quantitation of clonogenic cell densities as depicted in D. Data in D and E are representative of three independent experiments.






50% or more reduction in clonogenic growth (Fig. 4D and E).CXCL9 had a stronger growth-inhibitory effect in Gp5D com-pared with SK-CO-1 cells.

KRAS, BMP, TAK1, andMEK regulate canonical Wnt and NF-kBsignaling

AsKRAS, BMP, andWnt signaling are constitutively activated inKRAS-dependent cells, we investigated the regulation of cytokineexpression by endogenous KRAS, BMP, and canonical Wnt path-ways using shRNAs against KRAS, BMP7 (which encodes a ligandfor the BMP receptor), and CTNNB1/b-catenin. Depletion ofKRAS and BMP7 had similar effects on reducing levels of CSF2,CXCL10, TNF, IL1A, and IL8 in concordance with the gain-of-function experiments utilizing mutant KRAS and BMPR1A-CA(Fig. 5A). In contrast, Wnt pathway blockade by CTNNB1 deple-tion (Fig. 5A) or dominant-negative TCF4 (Supplementary Fig.S4E) caused a reduction in cytokine mRNA expression levels,

suggesting that canonical Wnt signaling negatively regulates tran-scriptional regulation of cytokine expression. CXCL9 levels didnot change significantly following KRAS andBMP7depletion, butwere upregulated following CTNNB1 depletion. CTNNB1 deple-tion caused reduced expression of canonical Wnt target genes, asexpected (Supplementary Fig. S4D).

AsNF-kB signaling is critical for proinflammatory signaling andis activated downstream of TAK1, we determined the effects ofTAK1 and MEK inhibition on NF-kB signaling, as assessed by aluminescence reporter assay using a kB-binding element-fireflyluciferase construct. In all cell lines tested (RKO, MDST8, HT29,and SK-CO-1), regardless of the sensitivity to MEK and TAK1inhibition, we observed dose-dependent decreases in NF-kBtranscriptional activity following AZ-TAK1 treatment (Fig. 5B).In the more sensitive cell lines (RKO, MDST8, and SK-CO-1), wenoted that NF-kB activity was more strongly reduced at the 24-hour time point compared with 9 hours, in contrast to the

Figure 5.

MEK and TAK1 inhibition suppresses canonical NF-kB and Wnt signaling. A, Cytokine expression levels in SK-CO-1 cells following depletion of indicatedgenes by lentiviral shRNA–mediated delivery. Data are the mean of triplicates � SEM. B, NF-kB luciferase reporter assays showing relative reporter activity in cellstreated with increasing doses of AZ-TAK1 at two time points, 9 and 24 hours. Data are the mean of four independent replicates � SEM. C, Effects of combiningAZ-TAK1 and MEKi on NF-kB luciferase reporter activity, at 9- and 24-hour time points. Data are presented as mean values of three replicates � SEM,relative to vehicle control–treated cells at 9 hours. D, TOP-FLASH luciferase reporter assays showing reporter activity following either AZ-TAK1 or MEKi treatment.Data are presented as mean values of four replicates � SEM.

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AZ-TAK1–insensitive HT-29 cells. Conversely, MEKi caused aslight increase in NF-kB reporter activity in SK-CO-1 cells, whichcould be inhibited with combined AZ-TAK1 treatment (Fig. 5C).We also compared the effects of MEKi to AZ-TAK1 on canonicalWnt signaling as measured by TOP-FLASH reporter assays (Fig.5D). Both MEKi and AZ-TAK1 caused reduced TOP-FLASH activ-ity in 3 of the 4 cell lines tested (MDST8, SK-CO-1, andHT-29) buthad little effect in the RKO cell line. Of note, RKO cells harbor a

G659 frameshift–inactivating mutation in RNF43, whereas theother 3 cell lines have APC mutations.

Molecular subtype classifications of primary tumors bycanonical Wnt/NF-kB signatures

To determine the physiological relevance of Wnt and NF-kBpathway activation and crosstalk in colon cancer cell lines, weanalyzed gene expression microarray data from a 566 primary

Figure 6.

Canonical Wnt/NF-kB transcriptional signatures define three major molecular subtypes of primary colon tumors.A, Subtype classifications of primary colon tumorsfrom the Marisa and colleagues dataset (7). The flow chart depicts derivation of a canonical Wnt signature using Pearson correlation coefficients to identifygenes correlated with RNF43 expression. The heatmap represents gene expression in three major subtypes revealed by hierarchical clustering of the 184 RNF43-correlated gene set. Genes and samples are clustered by Euclidean distance. Samples are color coded according to KRAS/BRAF mutations, the original sixsubtype classification byMarisa and colleagues,Wnt/NF-kB subtypes (W¼Wnt-high;N¼NFkB-high;WN¼WntþNF-kB-high) andmismatch repair (MMR) status(d, deficient; p, proficient). B, Kaplan–Meier curves showing relapse-free survival of patients with tumors classified into N/W/WN subtypes. C, Boxplotsdepicting expression of selected canonical Wnt targets (AXIN2 and RNF43) and canonical NFkB targets (IL1A and IL1RN) in the threemajor subtypes. Boxplots showminimum/maximum range, mean and interquartile range, as well as outliers. D, Statistical enrichment for KRAS and BRAF mutations in the three majorsubtypes depicted in B as determined by pairwise Fisher exact tests (� , P¼ 0.047; �� , P¼ 0.001; �� , P < 0.0001). E, Upstream activator analysis using the IPA toolshowing predicted upstream activating molecules for differentially expressed genes in each of the three major subtypes as determined by activation Z-score.






tumor cohort (7). We derived a canonical Wnt signature byidentifying genes whose expression correlated with theWnt targetgene RNF43 (see Materials and Methods; Fig. 6A). Clustering ofthe 566 tumor cohort using the 184 RNF43 gene set yielded 3major molecular subtypes. This was confirmed by k-means clus-tering of the data and analysis of within group sum of squares,showing a bend in the plot at 3 clusters (Supplementary Fig. S5A).Upon examination, the three subtypes were either comprised ofWnt target genes (Wnt-high/W), NF-kB target genes (NFkB high/N), or a subtype with intermediate expression of both Wnt andNF-kB target genes (WN; Fig. 6A). We next analyzed relapse-freesurvival data for this patient cohort and determined that thepatients with tumors of theWN subtype had the worst prognosis,compared with W subtype tumors (Fig. 6B). To confirm therelationship of the subtypes to Wnt and NF-kB signaling, wedetermined that the expression of Wnt targets AXIN2 and RNF43were high in the W and WN subtypes but low in the N subtype(Fig. 6C). Conversely, expression of NF-kB targets were highest inN subtype compared with W and WN subtypes. Finally, weperformed a similar subtype classification using an RNA-seq–based RNF43 signature derived from the Cancer Genome Atlas(TCGA) colorectal adenocarcinoma (COAD) cohort andobserved three major subtypes analogous to the W, N, and WNsubtypes (Supplementary Fig. S4B and S4C).

We hypothesize that the WN subtype has hallmarks of KRAS-dependent cell lines as Wnt and NF-kB pathways are concomi-tantly activated in these cells. Indeed, we noted significant enrich-ment or overrepresentation of oncogenic KRAS mutations in theWN subtype (Fig. 6D). Conversely, KRASmutations were under-represented in the W and N subtypes, whereas BRAF mutationswere over-represented in the N subtype. Of note, the N subtypewas significantly enriched with mismatch repair pathway–defec-tive (dMMR) tumors (Fig. 6A). Using the IPA tool, we analyzeddifferentially expressed genes in each subtype for upstream path-way activation. In the W subtype, anti-inflammatory mediatorsIL10RA, LIF, and IL1RNwere predicted to be activated (Fig. 6E). Incontrast, the N subtype had predicted activation of proinflamma-tory mediators TNFa and IFNa. The WN subtype genes wererelated to activation of FOS, a component of the AP-1 complex, aswell as the NF-kB1/p105/p50 and Rel-A/p65 subunits of thecanonical NF-kB transcriptional complex. Taken together, molec-ular subtype classification of primary colon tumors by virtue of acanonical Wnt/NF-kB signature reveals reciprocal regulation ofproinflammatory and anti-inflammatory pathways, with a dualpathway–activated WN subtype bearing molecular hallmarks ofKRAS dependency.

DiscussionIn this study, we comprehensively profiled TAK1 and MEK

dependencies in a large panel of colon cancer cell lines. First, weshowed that the potent cytotoxic effects of the putative TAK1inhibitor, 5Z-7-oxo, in KRAS-dependent cancer cell lines can beattributed to combined inhibition of MEK and TAK1. A subset ofKRAS-mutant colon cancer cell lines is sensitive to the MEKinhibitor selumetinib/AZD6244 (MEKi; ref. 34), prompting con-cern that 5Z-7-oxo apoptosis-inducing effects may be due toMEKinhibition alone. To refute this contention, we show that thealternative TAK1 kinase inhibitor, AZ-TAK1, causes reduced cellviability and induces apoptosis in KRAS-dependent cells with noeffects on MEK activity and subsequent ERK phosphorylation

(see Fig. 1). We also compared the effects of MEKi to 5Z-7-oxo onapoptosis induction in SK-CO-1 cells (Supplementary Fig. S1C).Both compounds caused reduced ERK phosphorylation. WhencomparedwithMEKi, 5Z-7-oxo induced stronger apoptotic effectsassociated with increased suppression of canonical Wnt and NF-kB signaling. Therefore, although 5Z-7-oxo and AZD6224/MEKiboth inhibit MEK, the signaling and phenotypic effects of the twocompounds are distinct.

We showed that more selective TAK1 inhibition with AZ-TAK1induces apoptosis as well as mitotic defects in a significantnumber of colon cancer cell lines. When combined with TAK1inhibition, MEK inhibition caused additive cytotoxic and apo-ptotic effects in some KRAS-dependent as well BRAF-mutantcolon cancer cells. Recent studies indicate that BRAF-mutantcolon cancer cells are codependent on the EGFR pathway, whichcan activate wild-type KRAS (35, 36). This supports our datashowing that cell lines, such as RKO and MDST8, are partiallyKRAS-dependent, which could occur via constitutive EGFR path-way activation of wild-type KRAS. Importantly, we showed thatcombining individual TAK1 and MEK inhibitors had additivecytotoxic effects in subsets of colon cancer cell lines revealing apotential therapeutic strategy for KRAS/BRAF-dependent coloncancers.

We have uncoveredmolecular hallmarks andmechanisms thatgovern TAK1 and MEK dependencies in colon cancer cell lines.KRAS, TAK1, and MEK codependencies correlate with oncogenicKRAS and BMP receptor activation, which promote autocrine/paracrine maintenance of tumor cell survival by inducing expres-sion of proinflammatory cytokines (see Fig. 4). We hypothesizethat a feed-forward loop potentiates KRAS-TAK1 antiapoptoticsignaling, creating an oncogene "addicted" state. KRAS, via MEKand TAK1, activates NF-kB signaling, which likely plays a majorrole in maintaining tumor cell survival, in part via autocrine/paracrine cytokine signaling. Indeed, GM-CSF and RANTES haverecently been implicated as autocrine/paracrine factors that driveKRAS-mediated tumorigenesis in models of pancreatic and lungcancer, respectively (19, 21).

In this study, we uncover a complex relationship betweenTAK1, NF-kB, and Wnt that is subject to positive and negativefeedback loops. Importantly, Wnt pathway blockade via deple-tion of CTNNB1/b-catenin results in strong induction of NF-kB–mediated cytokine expression. This indicates an inhibitory func-tion for Wnt/b-catenin in proinflammatory signaling. Indeed,canonical Wnt and NF-kB signaling are reciprocally regulated incolon cancer cells (37). However, Wnt and NF-kB transcriptionalprograms are concomitantly activated in KRAS-dependent cells.NF-kB is known to induce death receptor ligands, such as TNFa,which can trigger extrinsic apoptotic cell death. We hypothesizethat concomitant activation of canonical Wnt signaling in KRAS-dependent cells serves to limit the deleterious effects of the NF-kBproinflammatory cytokine network, leading to increased cellularproliferation. Furthermore, under conditions of nutrient andgenotoxic stress, TAK1 plays a central role in coordinating proin-flammatory signaling. Therefore, perturbation of TAK1 leads to ashift in the balance between the prosurvival and prodeath signals,leading to apoptosis. Identifying the critical prosurvival andprodeath cytokines that regulate KRAS-dependent tumorigenesisis a key goal in future studies.

A crucial proinflammatory factor that regulates survival is IL1,which likely plays a pivotal role in maintaining KRAS-dependentcolon cancer cell survival, in concordance with previous studies of

McNew et al.





IL1-dependent antiapoptotic signaling and promotion of KRAS-driven tumorigenesis (18, 38). A role for IL1 in tumorigenesis hasbeen demonstrated through the identification of gain-of-functionpolymorphisms in the IL1B gene in gastric cancer (39). Expressionlevels of IL1RA and CXCL9 are downregulated in cells withactivated KRAS/BMPR1A-CA suggesting a negative role inKRAS-dependent signaling. Indeed, exposure of KRAS-dependentcells to IL1RA and CXCL9 causes reduced cell viability. CXCL9 isan ELR-negative (Glu/Leu/Arg) cytokine and angiostatic,highlighting an antitumorigenic role for CXCL9 (40). Cell non-autonomous roles for KRAS-mediated cytokine expression arelikely to affect the behavior of tumor stromal components in themicroenvironment. Thus, reduced expression of CXCL9 andincreased expression of VEGF could lead to increased angiogen-esis. Mutant KRAS expression promotes GM-CSF production,which is likely to affect the behavior of macrophages that mod-ulate evasion of immune surveillance. The modulation of thetumor microenvironment by the KRAS–TAK1–MEK axis will bean interesting avenue for future studies.

To validate our in vitro findings, we performed clusteringanalyses of primary tumor gene expression datasets employinga transcriptional signature of genes positively and negativelycorrelated with RNF43 expression. RNF43 is a Wnt target genethat regulates the Wnt pathway in a negative feedback loop (31).TheRNF43 signature can robustly classify tumors into threemajorsubtypes, which can be characterized by activation of canonicalWnt and NF-kB signaling, designated W, N, and WN. As NF-kB ishyperactivated in cell lines that are TAK1/MEK dependent, wehypothesize that tumors classified as WN or N subtypes will bemost sensitive to anti-TAK1/MEK agents. TheWN subtype ismostclosely related to the C3 subtype reported by Marisa and collea-gues (7). They identified six major subtypes that segregate basedon molecular and histologic characteristics, including subtypesfrom serrated versus conventional carcinomas. The N subtype isenriched with CpG Island Methylator Phenotype positive(CIMPþ) and mismatch repair–deficient (dMMR) tumors. Inter-estingly, a recent study identified three major subtypes in coloncancer, one of which is a serrated carcinoma–derived poor prog-nosis subtype (8). This could be analogous to the WN subtypeidentified in this study. This suggests some clinical utility for theW/N/WN signature as a method to stratify patients based onpredicted responsiveness to anti-TAK1 agents.

In conclusion, we describe context-dependent control ofproinflammatory signaling by oncogenic KRAS, mediated by

downstream effector kinases TAK1 and MEK. Our findings willbe relevant for new approaches to "synthetic lethality" loss-of-function screens to identify candidate therapeutic targets formutant KRAS-dependent cancers. Previous mutant KRAS syn-thetic lethal screens yielded inconclusive results, perhaps due tothe contextual complexity of KRAS-dependent signaling net-works (41, 42). Among hits from these screens were the innateimmunity kinase TBK-1 and the GATA2 transcription factor,which regulate hematopoiesis and inflammation (43). Theseprevious hits combined with our findings converge on proin-flammatory signaling networks as critical mediators of KRAS-dependent tumorigenesis. We propose that KRAS-dependentproinflammatory signaling networks will provide new oppor-tunities for therapeutic intervention.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: A. SinghDevelopment of methodology: K.L. McNew, W.J. Whipple, A.K. Mehta,A. SinghAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K.L. McNew, T.J. Grant, L. Ray, C. Kenny, A. SinghAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): K.L. McNew, W.J. Whipple, A. SinghWriting, review, and/or revision of the manuscript: K.L. McNew, A.K. Mehta,A. SinghAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): W.J. Whipple, A. SinghStudy supervision: A. Singh

AcknowledgmentsWewould like to thank Dr. Hui Feng, Dr. Neil Ganem, and Dr. Rachel Flynn

for advice and reagents, Dr. Tom Gilmore, Dr. Gerald Denis, and Dr. NeilGanem for critical evaluation of themanuscript andDr. AndrewWilson andDr.Darrel Cotton for the NF-kB reporter plasmid.

Grant SupportThis work was supported by NIH/NCI grants R00 CA149169(to A. Singh)

and NIHT32 GM008541(to T.J. Grant and L. Ray).The costs of publication of this articlewere defrayed inpart by the payment of

page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

ReceivedMay 13, 2016; revised August 23, 2016; accepted September 1, 2016;published OnlineFirst September 21, 2016.

References1. Haigis KM, Kendall KR, Wang Y, Cheung A, Haigis MC, Glickman JN, et al.

Differential effects of oncogenic K-Ras and N-Ras on proliferation, differ-entiation and tumor progression in the colon. Nat Genet 2008;40:600–8.

2. SansomOJ,Meniel V, Wilkins JA, Cole AM,Oien KA,Marsh V, et al. Loss ofApc allows phenotypic manifestation of the transforming properties of anendogenous K-ras oncogene in vivo. Proc Natl Acad Sci U S A 2006;103:14122–7.

3. Hung KE, MaricevichMA, Richard LG, ChenWY, RichardsonMP, Kunin A,et al. Development of a mouse model for sporadic and metastatic colontumors and its use in assessing drug treatment. Proc Natl Acad Sci U S A2010;107:1565–70.

4. Fearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell1990;61:759–67.

5. Sadanandam A, Lyssiotis CA, Homicsko K, Collisson EA, Gibb WJ, Wulls-chleger S, et al. A colorectal cancer classification system that associatescellular phenotype and responses to therapy. Nat Med 2013;19:619–25.

6. Budinska E, Popovici V, Tejpar S, D'Ario G, Lapique N, Sikora KO, et al.Gene expression patterns unveil a new level of molecular heterogeneity incolorectal cancer. J Pathol 2013;231:63–76.

7. Marisa L, de Reynies A, Duval A, Selves J, Gaub MP, Vescovo L, et al. Geneexpression classification of colon cancer into molecular subtypes: charac-terization, validation, and prognostic value. PLoSMed 2013;10:e1001453.

8. De Sousa EMF,WangX, JansenM, Fessler E, TrinhA, de Rooij LP, et al. Poor-prognosis colon cancer is defined by a molecularly distinct subtype anddevelops from serrated precursor lesions. Nat Med 2013;19:614–8.

9. Kim HS, Mendiratta S, Kim J, Pecot CV, Larsen JE, Zubovych I, et al.Systematic identification of molecular subtype-selective vulnerabilities innon-small-cell lung cancer. Cell 2013;155:552–66.

10. Skoulidis F, Byers LA, Diao L, Papadimitrakopoulou VA, Tong P, Izzo J,et al. Co-occurring genomic alterations define major subsets of KRAS-Mutant lung adenocarcinoma with distinct biology, immune profiles, andtherapeutic vulnerabilities. Cancer Discov 2015;5:860–77.






11. Singh A, Sweeney MF, Yu M, Burger A, Greninger P, Benes C, et al. TAK1inhibition promotes apoptosis in KRAS-dependent colon cancers. Cell2012;148:639–50.

12. Janakiraman M, Vakiani E, Zeng Z, Pratilas CA, Taylor BS, Chitale D, et al.Genomic and biological characterization of exon 4 KRAS mutations inhuman cancer. Cancer Res 2010;70:5901–11.

13. Dry JR, Pavey S, Pratilas CA, Harbron C, Runswick S, Hodgson D, et al.Transcriptional pathway signatures predict MEK addiction and response toselumetinib (AZD6244). Cancer Res 2010;70:2264–73.

14. Knight ZA, Lin H, Shokat KM. Targeting the cancer kinome throughpolypharmacology. Nat Rev Cancer 2010;10:130–7.

15. Corcoran RB, Cheng KA, Hata AN, Faber AC, Ebi H, Coffee EM, et al.Synthetic lethal interaction of combined BCL-XL and MEK inhibitionpromotes tumor regressions in KRAS mutant cancer models. Cancer Cell2013;23:121–8.

16. Roper J, SinnamonMJ, Coffee EM, Belmont P, Keung L, Georgeon-RichardL, et al. Combination PI3K/MEK inhibition promotes tumor apoptosis andregression in PIK3CA wild-type, KRAS mutant colorectal cancer. CancerLett 2014;347:204–11.

17. Sakurai H.Targeting of TAK1 in inflammatory disorders and cancer. TrendsPharmacol Sci 2012;33:522–30.

18. Ling J, Kang Y, Zhao R, Xia Q, Lee DF, Chang Z, et al. KrasG12D-inducedIKK2/beta/NF-kappaB activation by IL-1alpha and p62 feedforward loopsis required for development of pancreatic ductal adenocarcinoma. CancerCell 2012;21:105–20.

19. Pylayeva-Gupta Y, LeeKE,HajduCH,MillerG, Bar-SagiD.Oncogenic Kras-induced GM-CSF production promotes the development of pancreaticneoplasia. Cancer Cell 2012;21:836–47.

20. Ancrile B, Lim KH, Counter CM. Oncogenic Ras-induced secretion of IL6 isrequired for tumorigenesis. Genes Dev 2007;21:1714–9.

21. Zhu Z, Aref AR, Cohoon TJ, Barbie TU, Imamura Y, Yang S, et al. Inhibitionof KRAS-driven tumorigenicity by interruption of an autocrine cytokinecircuit. Cancer Discov 2014;4:452–65.

22. Mayo MW, Wang CY, Cogswell PC, Rogers-Graham KS, Lowe SW, DerCJ, et al. Requirement of NF-kappaB activation to suppress p53-independent apoptosis induced by oncogenic Ras. Science 1997;278:1812–5.

23. Gapuzan ME, Schmah O, Pollock AD, Hoffmann A, Gilmore TD. Immor-talized fibroblasts from NF-kappaB RelA knockout mice show phenotypicheterogeneity and maintain increased sensitivity to tumor necrosis factoralpha after transformation by v-Ras. Oncogene 2005;24:6574–83.

24. Moffat J, Grueneberg DA, Yang X, Kim SY, Kloepfer AM, Hinkle G, et al. Alentiviral RNAi library for human and mouse genes applied to an arrayedviral high-content screen. Cell 2006;124:1283–98.

25. Naldini L, Blomer U, Gallay P, Ory D, Mulligan R, Gage FH, et al. In vivogene delivery and stable transduction of nondividing cells by a lentiviralvector. Science 1996;272:263–7.

26. Ninomiya-Tsuji J, Kajino T, Ono K, Ohtomo T, Matsumoto M, Shiina M,et al. A resorcylic acid lactone, 5Z-7-oxozeaenol, prevents inflammation byinhibiting the catalytic activity of TAK1 MAPK kinase kinase. J Biol Chem2003;278:18485–90.

27. Lito P, Saborowski A, Yue J, SolomonM, JosephE,Gadal S, et al.Disruptionof CRAF-mediated MEK activation is required for effective MEK inhibitionin KRAS mutant tumors. Cancer Cell 2014;25:697–710.

28. Turke AB, Song Y, Costa C, Cook R, Arteaga CL, Asara JM, et al. MEKinhibition leads to PI3K/AKT activation by relieving a negative feedback onERBB receptors. Cancer Res 2012;72:3228–37.

29. Dempsey CE, Sakurai H, Sugita T, Guesdon F. Alternative splicing and genestructure of the transforming growth factor beta-activated kinase 1. Bio-chim Biophys Acta 2000;1517:46–52.

30. Buglio D, Palakurthi S, Byth K, Vega F, Toader D, Saeh J, et al. Essentialrole of TAK1 in regulating mantle cell lymphoma survival. Blood 2012;20:347–55.

31. Giannakis M, Hodis E, JasmineMu X, Yamauchi M, Rosenbluh J, CibulskisK, et al. RNF43 is frequentlymutated in colorectal and endometrial cancers.Nat Genet 2014;46:1264–6.

32. Zoncu R, Efeyan A, Sabatini DM.mTOR: from growth signal integration tocancer, diabetes and ageing. Nat Rev Mol Cell Biol 2011;12:21–35.

33. Cohen S, Hurd E, Cush J, Schiff M, Weinblatt ME, Moreland LW, et al.Treatment of rheumatoid arthritis with anakinra, a recombinant humaninterleukin-1 receptor antagonist, in combination with methotrexate:results of a twenty-four-week, multicenter, randomized, double-blind,placebo-controlled trial. Arthritis Rheum 2002;46:614–24.

34. Tentler JJ, Nallapareddy S, Tan AC, Spreafico A, Pitts TM, Morelli MP, et al.Identification of predictive markers of response to the MEK1/2 inhibitorselumetinib (AZD6244) in K-ras-mutated colorectal cancer. Mol CancerTher 2010;9:3351–62.

35. Corcoran RB, Ebi H, Turke AB, Coffee EM, Nishino M, Cogdill AP, et al.EGFR-mediated re-activation of MAPK signaling contributes to insensitiv-ity of BRAFmutant colorectal cancers to RAF inhibition with vemurafenib.Cancer Discov 2012;2:227–35.

36. Prahallad A, SunC,Huang S, Di Nicolantonio F, Salazar R, ZecchinD, et al.Unresponsiveness of colon cancer to BRAF(V600E) inhibition throughfeedback activation of EGFR. Nature 2012;483:100–3.

37. Deng J, Miller SA, Wang HY, Xia W, Wen Y, Zhou BP, et al. beta-catenininteracts with and inhibits NF-kappa B in human colon and breast cancer.Cancer Cell 2002;2:323–34.

38. Takeuchi O, Akira S. Pattern recognition receptors and inflammation. Cell2010;140:805–20.

39. El-Omar EM, Carrington M, ChowWH, McColl KE, Bream JH, Young HA,et al. Interleukin-1 polymorphisms associated with increased risk of gastriccancer. Nature 2000;404:398–402.

40. Coussens LM, Werb Z. Inflammation and cancer. Nature 2002;420:860–7.41. Barbie DA, Tamayo P, Boehm JS, Kim SY, Moody SE, Dunn IF, et al.

Systematic RNA interference reveals that oncogenic KRAS-driven cancersrequire TBK1. Nature 2009;462:108–12.

42. Luo J, EmanueleMJ, Li D, CreightonCJ, SchlabachMR,Westbrook TF, et al.A genome-wide RNAi screen identifies multiple synthetic lethal interac-tions with the Ras oncogene. Cell 2009;137:835–48.

43. Kumar MS, Hancock DC, Molina-Arcas M, Steckel M, East P, DiefenbacherM, et al. The GATA2 transcriptional network is requisite for RAS oncogene-driven non-small cell lung cancer. Cell 2012;149:642–55.


McNew et al.




2016;14:1204-1216. Published OnlineFirst September 21, 2016.Mol Cancer Res Kelsey L. McNew, William J. Whipple, Anita K. Mehta, et al. KRAS-Dependent Activation of Proinflammatory SignalingMEK and TAK1 Regulate Apoptosis in Colon Cancer Cells with

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