Erbin Suppresses KSR1-Mediated RAS/RAF Signaling and … · Erbin Suppresses KSR1-Mediated RAS/RAF Signaling and Tumorigenesis in Colorectal Cancer Payton D. Stevens 1 ,Yang-An Wen

Molecular Cell Biology

Erbin Suppresses KSR1-Mediated RAS/RAFSignaling and Tumorigenesis in Colorectal CancerPayton D. Stevens1, Yang-An Wen2, Xiaopeng Xiong2, Yekaterina Y. Zaytseva3,Austin T. Li4, Chi Wang2, Ashley T. Stevens1, Trevor N. Farmer2, Tong Gan2,5,Heidi L.Weiss2, Masaki Inagaki6, Sylvie Marchetto7,8,9,10, Jean-Paul Borg7,8,9,10,and Tianyan Gao1,2

Abstract

Erbin belongs to the LAP (leucine-rich repeat and PDZdomain) family of scaffolding proteins that plays importantroles in orchestrating cell signaling. Here, we show thatErbin functions as a tumor suppressor in colorectal cancer.Analysis of Erbin expression in colorectal cancer patientspecimens revealed that Erbin was downregulated at bothmRNA and protein levels in tumor tissues. Knockdown ofErbin disrupted epithelial cell polarity and increased cellproliferation in 3D culture. In addition, silencing Erbinresulted in increased amplitude and duration of signalingthrough Akt and RAS/RAF pathways. Erbin loss inducedepithelial–mesenchymal transition, which coincided witha significant increase in cell migration and invasion. Erbininteracted with kinase suppressor of Ras 1 (KSR1) anddisplaced it from the RAF/MEK/ERK complex to preventsignal propagation. Furthermore, genetic deletion of Erbin

in Apc knockout mice promoted tumorigenesis and signif-icantly reduced survival. Tumor organoids derived fromErbin/Apc double knockout mice displayed increased tumorinitiation potential and activation of Wnt signaling. Resultsfrom gene set enrichment analysis revealed that Erbinexpression associated positively with the E-cadherinadherens junction pathway and negatively with Wnt signal-ing in human colorectal cancer. Taken together, our studyidentifies Erbin as a negative regulator of tumor initiationand progression by suppressing Akt and RAS/RAF signalingin vivo.

Significance: These findings establish the scaffold proteinErbin as a negative regulator of EMT and tumorigenesis incolorectal cancer through direct suppression of Akt and RAS/RAF signaling. Cancer Res; 78(17); 4839–52. �2018 AACR.

IntroductionErbin is a member of the leucine-rich repeat (LRR) and PDZ

domain (LAP) protein superfamily. It contains multiple protein–protein interaction modules including 16 LRRs followed by asingle PDZ domain at the C-terminus. Erbin is known to localizeprimarily to the adherens junctions and plays a role in maintain-ing the structural integrity of the junction in epithelial cells (1–3).The initial discovery of Erbin identifies Erbin as an ERBB2/Her2receptor–interacting protein that facilitates the localization of the

receptor to the basolateral membrane of epithelial cells (4). Inaddition, it has been shown that Erbin attenuates RAF activationby disrupting Shoc2-mediated RAS/RAF interaction (5, 6). More-over, downregulation of Erbin results in resistance to anoikis incervical cancer cells via activation of JAK/STAT signaling (7).However, the role of Erbin in regulating cell polarity and colo-rectal cancer tumorigenesis and progression remains elusive.

It has been well documented that epithelial cells, includingthose in the gastrointestinal tract, become polarized during thedifferentiation process (8). The polarization process, character-ized by the formation of specialized junctions between neigh-boring cells, also results in the segregation of two plasma mem-branedomains: the apical surface, facing the externalmediumandthe basolateral surface, connected to adjacent cells and extracel-lular matrix (9). The apical and basolateral membranes aresegregated by two highly organized junctions, tight junctions,and adherens junctions; and the many proteins that composethese junctions are assembled at the site of cell–cell contacts(10–12). Previous studies have demonstrated that loss of cellpolarity, through the disruption of these junctions, is associatedwith increased tumorigencity and accompanied by epithelial–mesenchymal transition (EMT; refs. 13–15). Therefore, the properestablishment of epithelial polarity allows cells to sense and torespond to signals that arise from the microenvironment in aspatiotemporally controlled manner.

By facilitating the assembly of tightly controlled signalingcomplexes through protein–protein interactions, scaffold pro-teins are known to play important roles in regulating spatiotem-poral responses in cell signaling (16). One of the best known

1Department of Molecular and Cellular Biochemistry, University of Kentucky,Lexington, Kentucky. 2Markey Cancer Center, University of Kentucky, Lexington,Kentucky. 3Department of Toxicology and Cancer Biology, University of Ken-tucky, Lexington, Kentucky. 4Paul Laurence Dunbar High School, Lexington,Kentucky. 5Department of Surgery, University of Kentucky, Lexington, Ken-tucky. 6Department of Physiology, Mie University Graduate School of Medicine,Tsu, Mie, Japan. 7Centre de Recherche en Canc�erologie de Marseille (CRCM),'Cell Polarity, Cell Signalling, and Cancer', Equipe Labellis�ee Ligue Contre leCancer, Inserm U1068, Marseille, France. 8CNRS, UMR7258, Marseille, France.9Institut Paoli-Calmettes, Marseille, France. 10Aix-Marseille University, UM 105,Marseille, France.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Tianyan Gao, University of Kentucky, 741 S Limestone,Lexington, KY 40536-0509. Phone: 859-323-3454; Fax: 859-257-6030; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-17-3629

�2018 American Association for Cancer Research.

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examples is the signal propagation in the RAS/RAF pathway,where the step-by-step activation process from RAS to ERK isfacilitated by scaffolding proteins, such as KSR and Shoc2(17, 18). KSR1, a kinase-like protein that lacks enzymatic activity,has been shown to promote cell proliferation and oncogenicpotential by enhancing signaling activation through the RAS/RAFpathway (19). KSR1 binds constitutively toMEK in the cytoplasmin unstimulated cells and translocates to the plasma membraneupon RAS activation (20, 21). At the plasma membrane, KSR1facilitates signaling propagation by organizing the formation ofRAF/MEK/ERK complex (21, 22). Although the molecular mech-anism by which scaffold proteins positively regulate RAS/RAFsignaling has been extensively studied, it remains largelyunknown whether scaffolding proteins are involved in signalingtermination.

Here, we report the identification of Erbin as a novel tumorsuppressor in colon cancer. We show that the mRNA and proteinexpression of Erbin is markedly decreased in colorectal cancerpatient specimens. Erbin negatively regulates RAS/RAF signalingby sequestering KSR1 and preventing the formation of KSR1/RAF1 complex. Functionally, knockdown of Erbin results in anincrease in cell motility by inducing EMT in colon cancer cells,and deletion of Erbin gene significantly decreases the lifespan ofApc-mutant mice and accelerates the tumor progression.

Materials and MethodsMice

All animal procedures were performed by following protocolsapproved by theUniversity of Kentucky Institutional Animal Careand Use Committee (IACUC). Erbin knockout (KO) mice onC57BL6 background as previously described (23) were main-tained by random intercrossing to sustain a heterogeneous mixedgenetic background. To produce animals used in the experiments,Erbinþ/� mice were bred with Apcf/f and Villin-cre (Vil-cre) mice(both were obtained from The Jackson Laboratory) on a C57BL6background. These mice were then intercrossed to produce threecohorts of animals, including Apcf/þ/Erbinþ/þ/Vil-cre (Apc/WT),Apcf/þ/Erbinþ/�/Vil-cre (Apc/Het), and Apcf/þ/Erbin�/�/Vil-cre(Apc/KO). To monitor survival, these cohorts of mice werefollowed for up to 6 months.

Cells and reagentsCaco2, HT29, and SW480 cells obtained from ATCC were

cultured in DMEM supplemented with 10% FBS (Sigma-Aldrich)and 1% penicillin/streptomycin. LIM2405 cells obtained fromLudwig Cancer Research Institute (New York, NY)were cultured inRPMI1640 supplemented with 10% FBS, 2 mmol/L L-Glutamine,25 mmol/L HEPES, and 1% penicillin/streptomycin. The muta-tion status of common colorectal cancer–associated genes isspecified in Supplementary Table S1 (24). Human colon cancercell lines were authenticated using short tandem repeat DNAprofiling and tested negative for Mycoplasma using PCR in March2016 (Genetica). Stable Erbin and KSR1 knockdown cells weregenerated by lentivirus-based shRNAs as described previously(25–27). The shRNA targeting sequences for human Erbin areas follows: 50-GCAGCCAAGTACAACCGTTAA-30 (A1) and 50-CGGGCTCAAGTTGCATTTGAA-30 (A2); and for mouse Erbinare: 50-GTTGGATTCAAATCAGATAAA-30 (B1) and 50-CAGTCAC-TACTCTAGATTATT-30 (B2). The shRNA targeting sequence forKSR1 is 50-CAACAAGGAGTGGAATGATTT-30. To generate wild-

type (WT) and Erbin KO mouse embryonic fibroblasts (MEF)cells, Erbin heterozygous mice were used for breeding, andindividual embryos at day 13 of gestation were isolated andgenotyped. MEF cells were produced from WT and Erbin-nullembryos by following standard protocols (28). The primary MEFcells were immortalized using retrovirus-mediated knockdownof p53 using pBabe-puro-shp53 (Addgene).

Transwell migration and invasion assayTranswell migration and invasion assays were performed by

following previously described procedures (25). Briefly, cellsgrown to 50% confluency were serum starved overnight. Formigration assays, 5 � 104 cells were seeded into Transwells in0.1% BSA and allowed to migrate for 4.5 hours at 37�C towardserum-free DMEM containing collagen (15 mg/mL) and EGF(10 ng/mL). For the invasion assay, cells were seeded into theupper chambers of Transwells that were precoated with Matrigel(BD Biosciences). The cells were allowed to migrate toward 5%FBS in the lower chamber for 24 hours. At the end of theincubation period, cells migrated to the bottom of Transwellswere fixed and stained with crystal violet. The numbers of cellswere counted using an invertedmicroscope at�20magnification.

Time-lapse live cell imaging and analysisControl and Erbin knockdown cells were serum starved for

4hours andplated as single cells onto collagen (15mg/mL)-coatedglass bottom culture dishes (MatTek). Cells were stimulatedwith EGF (10 ng/mL), and the trajectory of moving cells wascaptured using a Nikon BioStation IM equipped with a CO2

incubation chamber. Time-lapse phase images were taken every10 minutes for 6 hours with a 20� objective (25, 29). Therecorded movement of the cells was analyzed using NikonElements AR software.

Three-dimensional cell culture and immunofluorescencestaining

The polarization process of Caco2 cells was examined byfollowing procedures described previously (30). Briefly, single-cell suspension of Caco2 cells was cultured in 3D matrix consist-ing of 50% growth factor–reduced Matrigel (BD Biosciences) and50%Collagen I (Thermo Fisher Scientific). The cells were allowedto grow into acinar structures in the 3D matrix for 10 days. Thephase-contrast images of the acini were captured using a NikonEclipse Ti-E inverted microscope. For immunofluorescence stain-ing, the acini were fixed in 4% paraformaldehyde in PBS andpermeabilized with 0.5% Triton X-100 in PBS. Actin was stainedusing Alexa 488–conjugated phalloidin, while the nuclei of thecells were stained with DAPI-containing mounting medium. Todetect proliferating cells, the cells grown in3Dculturewere treatedwith 5-ethynyl-20-deoxyuridine (EdU) for 1 hour prior to fixation.TheEdU-positive cellswere stainedusingClick-iT EdUAlexa Fluor488 Imaging Kit (Thermo Fisher Scientific). Images were takenusing an Olympus FlowView FV1000 confocal laser-scanningmicroscope (Olympus).

Western blot analysisColon cancer cells or mouse tumor organoids were harvested

and detergent-solubilized cell lysates were obtained as describedpreviously (25–27, 31). Equal amounts of cell lysates wereresolved by SDS-PAGE and subjected to Western blot analysis.The Erbin antibody has been reported previously (3). The

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phospho-AKT (p-AKT for Ser473), pan-Akt, phospho-RAF1(p-RAF1 for Ser338), total RAF1, phospho-MEK1/2 (p-MEK forSer217/221), totalMEK1/2, phospho-ERK1/2 (p-ERK for Thr202/Tyr204), total ERK1/2, and E-cadherin antibodies were from CellSignaling Technology. The vimentin and N-cadherin antibodieswere from BD Biosciences. The b-actin and g-tubulin antibodieswere from Sigma-Aldrich.

Histologic analysis and IHC stainingMice were euthanized at indicated time points when showing

signs of intestinal neoplasia, such as hunched stature and rectalbleeding. Intestine segments were opened longitudinally ontofilter paper and made into "Swiss-roll" preparations as describedpreviously (25). For histologic analysis, H&E sections were pre-pared from fixed and paraffin-embedded Swiss-roll specimens byfollowing standard techniques. The colorectal cancer tissuemicro-array was created by the Biospecimen Procurement and Transla-tional Pathology Shared Resource Facility of the Markey CancerCenter (Lexington, KY), which contains 45 pairs of tumor andadjacent normal control tissues collected from patients withcolorectal cancer who had undergone surgery resections at theMarkey Cancer Center. For IHC staining, tissue sections weredeparaffinized, rehydrated, and treated with hydrogen peroxide.Antigen retrieval was performed using Dako Target RetrievalSolution (Dako Cytomation), and IHC staining was performedas described previously (26). The stained sections were visualizedusing a Nikon Eclipse 80i upright microscope.

Isolation and culture of mouse tumor organoidsIntestinal tumors were isolated from three cohorts of

Apc/Erbin compound mutant mice, including Apc/WT, Apc/Het,and Apc/KO, cultured in 3D Matrigel as described previously(32, 33). Dissociated tumor cells were embedded in 33% Matrigelin 3D growth medium [Advanced DMEM/F12 supplementedwith 1�GlutaMAX, 1�N-2, 1� B-27, 1mmol/LN-acetylcysteine,EGF (50 ng/mL) and 1% penicillin/streptomycin]. Tumor orga-noids were allowed to grow for 5 days and collected for proteinor RNA analysis. For colony formation assays, single-cell suspen-sions of 1,000 cells derived from tumor organoids were seededinto 3D Matrigel as described above. The number of coloniesformed after 5 days were counted using an inverted microscope.

Tumor organoids derived from Apcf/þ/KrasLSL-G12D/Villin-Cremice were generated and described previously (33). To silenceErbin expression, tumor organoids were dissociated into smallcell clusters using TrypLE (Thermo Fisher Scientific) and incubat-ed with sh-Erbin lentivirus in suspension for 6 hours in a 37�Cincubator. Cells were subsequently embedded in 33%Matrigel in3D growth medium (Advanced DMEM/F12 supplemented with1 � GlutaMAX, 1 � N-2, 1 � B-27, 1 mmol/L N-acetyl-L-cysteineand 1% penicillin/streptomycin), and puromycin was added2days later to select for stable knockdown cells. For colony forma-tion assays, tumor organoids were dissociated and single-cellsuspensions were seeded into 3DMatrigel. The number of tumororganoids formed after 6 days were counted and analyzed usingan inverted microscope.

Real-time PCRTotal RNA was isolated from mouse tumor organoids using

the RNeasy Mini Kit (Qiagen). Equal amounts of RNA wereused as templates for the synthesis of cDNA using RT2 HT FirstStrand Kit (Qiagen). Real-time PCR was performed using

mouse Lgr5-, Axin2- Cd44-, Ccnd1-, Ki67-, Alpi-, Fabp2-, andMuc2-specific probes using StepOne Real-Time PCR system(Applied Biosysems). All values were normalized to the levelof b-actin. The overall expression of b-actin mRNA remainedunchanged in different treatment groups as determined by theCt (threshold cycle) values.

Bioinformatics and statistical analysisIn experiments to assess gene or protein expression, rate of cell

migration, size and number of cell grown in 3D, and EdUincorporation were summarized using bar graphs, and pairwisecomparisons between different conditions were carried outusing two-sample t tests. To determine the relative expression ofErbin gene in human patients with colorectal cancer, microarrayand patient clinical data from two colorectal cancer studies weredownloaded from the Oncomine database. The Cancer GenomeAtlas (TCGA) dataset contains 192 adenocarcinoma and 22normal samples,withinwhich, 13werematchedpairs. Expressionof Erbin in tumor versus normal samples was compared usinglinear mixed models. Skrzypczak and colleagues' dataset con-tains 81 tumors and 24 normal samples. Expression of Erbinin tumor versus normal samples was compared on the basis of2-sample t test. All statistical analyses were performed using R(version 3.4.1).

For gene set enrichment analysis (GSEA), RNA sequencing(RNA-seq) data were obtained from the TCGA colorectal cancerstudy. Correlations between expressions of ERBIN and the othergenes were quantified by Spearman correlation coefficient. Thegenes were then ordered from highest to lowest based on thecorrelation coefficient. This ranked listwas inputted into theGSEADesktop Application (34) to identify pathways that are associatedwith ERBIN expression.

ResultsErbin is downregulated in colorectal cancer patient tumorsamples

To determine whether Erbin mRNA expression (gene symbol:ERBIN; previously known as ERBB2IP) is altered in patients withcolorectal cancer, we performed bioinformatic analysis of twomicroarray datasets of human colorectal cancer samples. Themicroarray and patient clinical data of the two studies (35, 36)were downloaded from the Oncomine database. The mRNAexpression of Erbin was significantly reduced in tumor sampleswhen compared with normal controls (Fig. 1A and B). Interest-ingly, additional analysis of Erbin expression in stage I–IV ofpatients with colorectal cancer revealed that Erbin was down-regulated upon tumor initiation when compared with normalcontrols, and no further loss of Erbin mRNA was observed astumors progressed through stage II–IV, thus suggesting that loss ofErbin expression is an early event in tumorigenesis (Supplemen-tary Fig. S1). In addition, the expression of Erbin protein wasdetected along the epithelial cell–cell junction in normal humancolon tissues by IHC staining,whereas the expression of Erbinwasmarkedly reduced andmislocalized to cytoplasm in tumor tissues(Fig. 1C). The basolateral distribution of Erbin in normal tissueswas consistent with those observed previously (3, 37). Quanti-tative results obtained from IHC staining of a colorectal cancertissue microarray revealed that basolateral membrane localiza-tion of Erbin was lost in all colorectal cancer tumor tissuesexamined (Fig. 1D; Supplementary Fig. S1). Furthermore, we

Erbin Functions as a Tumor Suppressor in Colorectal Cancer

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Figure 1.

Erbin is downregulated and mislocalized in colorectal cancer patient samples. A and B, Microarray and patient clinical data from two colorectal cancerstudies were downloaded from the Oncomine database (35, 36). Data from Skrzypczak and colleagues' study (A; ref. 35) contained 24 normal and 36adenocarcinoma samples; data from TCGA (B) contained 22 normal and 192 adenocarcinoma samples. Two sample t tests or linear mixed models were usedto compare ERBIN gene expression between adenocarcinoma and normal samples (P < 0.001). C, The expression of Erbin protein was detected in a tissuemicroarray using IHC staining. The tissue microarray contained 45 pairs of matched normal and tumor tissues. The basolateral localization of Erbin was detected innormal colonic epithelial cells as indicated by red arrows. Scale bar, 50 mm. D, Bar graph depicts the percentage of basolateral localization of Erbin in both normaland tumor tissues (n ¼ 45). E, Matched normal and tumor tissues from 7 patients with colorectal cancer were analyzed for Erbin expression using Westernblot analysis. F, The expression of Erbin protein was quantified by normalizing to tubulin or actin. The relative Erbin levels in tumor tissues were comparedwith that of normal tissues within the same patient. Data, mean � SEM (#, P < 0.0001).

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analyzed Erbin protein expression in matched normal and tumortissues obtained from 7 patients with colorectal cancer usingWestern blot analysis. Consistent with IHC staining results, Erbinprotein levels were significantly decreased in tumor tissues com-pared with normal controls (Fig. 1E and F; SupplementaryFig. S1). Collectively, our data obtained in patient samples pro-vided the first evidence, suggesting that Erbin may function astumor suppressor in colorectal cancer.

Knockdown of Erbin increases both Akt and RAS/RAF signalingPrevious studies have implicated that Erbin negatively regulates

ERK signaling (5, 38). To determine the function of Erbin incolorectal cancer, we silenced Erbin expression using two shRNAlentiviral targeting constructs (A1 and A2) in SW480, LIM2405,and Caco2 colon cancer cell lines. Consistently, knockdown ofErbin resulted in an increase in phosphorylation and activation ofboth the Akt and MEK/ERK pathways in all three cell lines(Fig. 2A). Because both Erbin shRNA targeting constructs hadsimilar effects on silencing Erbin expression and enhancing acti-vation of Akt and MEK/ERK phosphorylation, the subsequentexperiments were mostly performed using sh-Erbin-A2 shRNAand key findings were confirmed with sh-Erbin-A1 shRNA. Tofurther examine the effect of Erbin loss on the temporal activationof signaling, stable control and Erbin knockdown SW480 cellswere starved for 16 hours and subsequently stimulated with EGFfor the indicated time (Fig. 2B). Knockdown of Erbin increasedboth the amplitude and the duration of signaling through the Aktand RAF/MEK/ERK pathways (Fig. 2C). Similar results wereobtained in EGF-treated LIM2045 cells (Supplementary Fig. S2).

Erbin is required to maintain cell polarityWe next investigated the functional effects of Erbin down-

regulation using a 3D cell culture system. Control and ErbinknockdownCaco2 and SW480 cells were seeded into 3DMatrigeland allowed to grow for 10 days to form tumor spheroids. Asdescribed in previous studies (30, 39, 40), control Caco2 cellswere able to form acini-like spherical structures with a singlehollow lumen, which consisted of a layer of polarized epithelialcells as indicated by the apical localization of F-actin (Fig. 3A). Inmarked contrast, Erbin depletion altered the acinar structure byinducing the formation of multiple lumens (Fig. 3A). Moreover,although not fully polarized, control SW480 cells were able toform tumor spheroids with partially hollowed lumens; however,cell clusters formed by Erbin knockdown SW480 cells lackedlumen structure and exhibited no apical or basolateral differen-tiation (Fig. 3A). Together, these results suggested that Erbin playsan important role in maintaining epithelial polarity.

Furthermore, we found that knockdown of Erbin markedlyincreased the size of the spheroids in both Caco2 and SW480 cells(Fig. 3B). To determine whether decreased Erbin expression alterscell proliferation in 3D culture, tumor spheroids formed bycontrol and Erbin knockdown SW480 cells were labeled withEdU to mark proliferating cells (Fig. 3C). Quantitative resultsshowed that loss of Erbin expression increased cell proliferation(Fig. 3D). To determine themolecularmechanismbywhich Erbinloss induces polarity defects and promotes cell proliferation in 3Dculture, control and Erbin knockdown SW480 spheroids werecollected and analyzed byWestern blot analysis. Interestingly, themorphologic changes observed in sh-Erbin cells were accompa-nied by the downregulation of E-cadherin (an epithelial cellmarker) and upregulation of vimentin and N-cadherin (markers

of fibroblasts/mesenchymal cells), suggesting that Erbin knock-down cells had undergone EMT (Fig. 3E). Notably, increases inboth Akt and ERK activation were maintained in Erbin knock-down spheroids grown in 3D (Fig. 3E). Collectively, these datasuggested that downregulation of Erbin disrupts epithelial polar-ity and induces EMT.

Knockdown of Erbin promotes cell migration and invasion incolon cancer

As we have observed EMT-like phenotypes in Erbin knock-down colon cancer cells grown in 3D, we next determined therole of Erbin on regulating cell motility. The induction of EMTas indicated by downregulation of E-cadherin and upregulationof vimentin was confirmed in SW480 cells grown in regular 2Dculture (Fig. 4A). To monitor single-cell motility, time-lapseimages of control and Erbin knockdown SW480 and LIM2405cells were captured and the distances traveled of individual cellswere analyzed using Nikon BioStation. Results showed thatErbin knockdown cells were considerably more motile andaverage distances traveled by Erbin knockdown cells weresignificantly increased compared with the control cells(Fig. 4B and C), suggesting that Erbin loss increases cell motilityat the single-cell level.

In addition, control Erbin knockdown SW480 and LIM2405cells were subjected to Transwell migration and invasion assays.We found that both Erbin knockdown SW480 and LIM2405 cellsmigrated significantly faster than the control cells (Fig. 4D).Similarly, knockdown of Erbin inHT29 cells increased cell migra-tion as determined using Transwell assays (SupplementaryFig. S2), and silencing Erbin using two different targeting shRNAshad comparable effects on promoting migration in SW480 cells(Supplementary Fig. S2). Furthermore, knockdown of Erbin sig-nificantly increased the ability of SW480 cells to invade throughMatrigel (Fig. 4E), thus confirming that loss of Erbin promotes cellmigration and invasion. Because Erbin downregulation activatesboth Akt and ERK signaling, we further determined the functionalcontribution of these two pathways in regulating the cell motilitydownstream of Erbin. Interestingly, the effect of Erbin loss onpromoting cellmigrationwas completely blocked by treating cellswithMEK inhibitor (PD98059), whereas Akt inhibitor (MK2206)had no effect (Fig. 4F). The effect of MEK and Akt inhibitors onsuppressing ERK and Akt activation was confirmed usingWesternblot analysis (Fig. 4G). Collectively, these results suggested thatincreased cell migration observed in Erbin knockdown cells islikely a result of MEK/ERK signaling activation.

Erbin inhibits RAF/MEK/ERK signaling by disrupting theRAF1–KSR1 interaction

In our effort to further define themolecular mechanism under-lying Erbin-mediated inhibition of MEF/ERK signaling, we iden-tified KSR1 as an interacting protein of Erbin. KSR1 is known tofacilitate the formation of RAF/MEK/ERK complex upon RASactivation (20). We performed coimmunoprecipitation experi-ments to determine whether Erbin expression affects the interac-tion between KSR1 and RAF1. 293T cells transfected with Flag-RAF1 in the presence or absence of CFP-KSR1 andMyc-Erbinwereimmunoprecipitated with the anti-Flag antibody. The overex-pressed and endogenous KSR1 were found to interact with RAF1(Fig. 5A). However, coexpression of Erbin largely reduced theamount of KSR1interacting with RAF1 (Fig. 5A and B). In addi-tion, the interaction between endogenous Erbin and KSR1 was






readily detected in LIM2405 cells (Fig. 5C). Importantly, knock-down of Erbin resulted in an increase in formation of KSR1-RAF1complex in both Caco2 and LIM2405 cells (Fig. 5D). Further-more, we found that Erbin loss induced increase in ERK activationwas abolished in cells where KSR1was also silenced (Fig. 5E), thus

confirming the functional interplay between Erbin and KSR1 inregulating RAF/MEK/ERK signaling. Collectively, these resultssuggest that Erbin functions to prevent KSR1 from forming asignaling complex with RAF1 and inhibit signaling activationdownstream of RAF.

Figure 2.

Knockdown of Erbin in colorectalcancer cells increases Akt andRAS/RAF signaling. A, Cell lysatesprepared from stable control(sh-Control) and Erbin knockdown(sh-Erbin) cells, including Caco2,LIM2405, and SW480, were analyzedfor the expression of Erbin and thephosphorylation status of Akt, MEK,and ERK using Western blot analysis.Two different shRNA targetingsequences (A1 and A2) were used tosilence Erbin in each cell line. Therelative phosphorylation of Akt, MEK,and ERK was quantified bynormalizing ECL signals generated bythe phospho-specific antibodies tothat of total proteins. B, The stablecontrol and Erbin knockdownSW480cellswere stimulatedwith EGF(10 ng/mL) for the indicated times andactivation of signaling molecules wasanalyzed using Western blot analysis.C, The relative levels of p-Akt, p-MEK,and p-ERK in sh-Control and sh-ErbinSW480 cell lines were quantified bynormalizing ECL signals generated bythe phospho-specific antibodies tothat of total proteins and plotted overthe EGF stimulation time course.

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Figure 3.

Knockdown of Erbin disrupts epithelial cell polarity. A, Stable sh-Control and sh-Erbin Caco2 and SW480 cells were cultured for 10 days in 3D Matrigel.Immunofluorescence images were taken from cells stained with Alexa488-phalloidin (green) and DAPI (blue). Phase-contrast images were obtained using a NikonTE2000 invertedmicroscopewith 10� objective; scale bar, 50 mm; and confocal images of stained cells were obtained using anOlympus FlowView FV1000 confocallaser-scanning microscope; scale bar, 10 mm. B, The diameter of 15 to 35 randomly chosen spheroids of sh-Control and sh-Erbin Caco2 and SW480 cells weremeasured. Data, mean � SEM (� , P < 0.05). C, Stable sh-Control and sh-Erbin SW480 cells were labeled with EdU to mark proliferating cells. D, The percentageof EdU-positive cells was quantified and expressed graphically. Data, mean � SEM (� , P < 0.05). D, Stable sh-Control and sh-Erbin SW480 cells werecultured for 10 days in 3D Matrigel, and spheroids were collected and analyzed by Western blot analysis for the expression of EMT markers.






Figure 4.

Knockdown of Erbin increases cell motility in colorectal cancer cells. A, Stable sh-Control and sh-Erbin SW480 cells grown in 2D culture were analyzed forthe expression of Erbin, E-cadherin, and vimentin. B, Migration paths of stable sh-Control and sh-Erbin SW480 and LIM2405 cells were monitored using theNikon BioStation for 6 hours. The trajectories of 12 randomly chosen cells for each cell line are shown in the graphs. C, The averaged path lengths werequantified for sh-Control and sh-Erbin LIM2405 and SW480 cells. Data, mean � SEM (� , P < 0.05). D, Stable sh-Control and sh-Erbin SW480 and LIM2405 cellswere subjected to Transwell migration assays using collagen and EGF (10 ng/mL) as chemoattractants. The number of migrated cells per field of view was counted.Data, mean � SEM (n ¼ 12; #, P < 0.0001). E, Stable sh-Control and sh-Erbin SW480 cells were subjected to Transwell invasion assays using 5% FBS as thechemoattractant. The number of invaded cells per field of view was counted. Data, mean � SEM (#, P < 0.0001). F, Stable sh-Control and sh-Erbin SW480cells were treated overnight with MEK inhibitor PD98059 (10 mmol/L) or Akt inhibitor MK2206 (1 mmol/L), and subsequently subjected to Transwell migrationassays using collagen and EGF (10 ng/mL) as chemoattractants. The number of migrated cells per field of view was counted. Data, mean � SD (#, P < 0.001;�, P < 0.01). G, Cell lysates were prepared from sh-Control and sh-Erbin SW480 cells as treated in F and analyzed for the phosphorylation of Akt and ERKusing Western blot analysis.

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Genetic deletion of Erbin promotes tumorigenesis inApc-mutant mouse model

To determine the effect of Erbin loss on tumorigenesis ofcolorectal cancer in vivo, we crossed Erbin-null mice with theApcf/f/Vil-cremousemodel (41) to investigate the susceptibility ofmice deficient in Erbin to Apc-driven intestinal adenomas. ErbinKO mouse models have been used in previous studies to inves-tigate inflammatory responses, cardiac hypertrophy, and Her2/neu–mediated tumorigenesis in breast cancer (42–44). The ErbinKOmice did not develop spontaneous tumors. We compared theproliferation and differentiation of normal intestinal epithelialcells in WT and Erbin KO mice and found no difference in thenumber of proliferating cells and differentiated cells of differentcell linages (Supplementary Fig. S3). Consistently, the expressionof genes associatedwith normal intestinal stem and differentiatedcells remained unchanged in intestinal organoids derived fromErbin heterozygous and homozygous KO mice compared withWT mice (Supplementary Fig. S4). However, consistent with re-sults obtained in colorectal cancer cells, we found that the ampli-

tude and duration of EGF-stimulated Akt and Raf/Mek/Erksignaling were largely increased in Erbin KO MEF cells (Supple-mentary Fig. S4). Taken together, these results suggest that Erbinloss–induced activation of Akt and Erk signaling alone is notsufficient to initiate tumorigenesis in colon cancer.

To specifically assess the role of Erbin in tumor initiation andprogression in intestinal epithelium,we cross Erbin KOmice ontothe Apc-mutant background to generate the following threecohorts of mice: Apcf/þ/Vil-Cre/Erbinþ/þ, Apcf/þ/Vil-Cre/Erbinþ/�, and Apcf/þ/Vil-Cre/Erbin�/� (Apc/WT, Apc/Het, andApc/KO, respectively). The survival studies showed that knockoutof a single allele of Erbin was sufficient to markedly accelerate thetumorigenesis process, resulting in a significantly shorter lifespanwhen compared with Apc/WT mice. Knockout of both alleles ofErbin further accelerated this process and significantly decreasedsurvival (Fig. 6A). Histopathologic analysis revealed that adeno-mas were detected in both intestine and colon regions in all threecohorts of mice. However, when Apc/WT mice were sacrificed attime points that corresponded to average lifespan of Apc/KOmice

Figure 5.

Erbin disrupts the interactionbetween KSR1 and RAF1. A, 293T cellstransfected with Flag-RAF1 andCFP-KSR1 in the presence or absenceof Myc-Erbin coexpression werelysed and immunoprecipitated withFlag-affinity beads. The presence ofKSR1 and Erbin in theimmunoprecipitates was detected byWestern blot analysis. Arrow,endogenous KSR1coimmunoprecipitated by Flag-RAF1.B, The relative amount of KSR1coimmunoprecipitated with RAF1 inthe presence or absence of Myc-Erbincoexpression was quantified andexpressed graphically (n ¼ 3;� , P < 0.05). C, Erbin interacts withendogenous KSR1. Endogenous Erbinwas immunoprecipitated fromLIM2405 cell lysates, and the presenceof KSR1 in the immunoprecipitateswasdetected by Western blot analysis. D,Endogenous RAF1 wasimmunoprecipitated from sh-Controland sh-Erbin Caco2 and LIM2405 cells.The levels of KSR1coimmunoprecipitated with RAF1were detected by Western blotanalysis.E, The expression of KSR1 andErbin was silenced individually or incombination in SW480 cells usingcorresponding shRNA lentivirus. Celllysates were analyzed for thephosphorylation status of ERK. Therelative levels of ERK phosphorylationin different cells were quantified bynormalizing ECL signals generated byp-ERK to that of total ERK.






Figure 6.

Erbin deletion decreases survival and promotes the activation of Akt and Ras/Raf signaling in Apc-mutant mice. A, Erbin loss significantly reduces thelifespan of Apc-mutant mice. Kaplan–Meier curve shows the survival distribution of three cohorts of mice: Apcf/þ/Vil-Cre/Erbinþ/þ, Apcf/þ/Vil-Cre/Erbinþ/�, andApcf/þ/Vil-Cre/Erbin�/� (Apc/WT, Apc/Het, and Apc/KO, respectively). Numbers of mice in the three cohorts are: Apc/WT (n¼ 14), Apc/Het (n¼ 16), and Apc/KO(n ¼ 8). Statistical significance (determined by log-rank test) is given for comparison between Apc/WT and Apc/Het (P < 0.0001) and Apc/WT and Apc/KO(P < 0.0001). B, Histology analysis of intestine adenomas in age-matched Apc/WT and Apc/KO mice (at 2 months). Scale bar, 200 mm. C, The phosphorylationof Akt and Erk was analyzed in the intestinal tumors from Apc/WT, Apc/Het, and Apc/KO mice using IHC staining. The tissues were collected when the micewere at the terminal stage of tumor development. The age of the mice was 4.5 months for Apc/WT and Apc/Het and 2 months for Apc/KO mice, respectively.Scale bars, 50 mm. D, Tumor organoids were prepared from tumor tissues of Apc/WT and Apc/KOmice and grown in 3D Matrigel. Scale bar, 200 mm. E, Cell lysateswere prepared from tumor organoids as shown in D and analyzed for the phosphorylation status of Akt and ERK by Western blot analysis. The relative levelsof Akt and ErK phosphorylation were quantified by normalizing ECL signals generated by p-Akt and p-ErK to that of total Akt and ERK, respectively.


Stevens et al.




(2 months), the difference in tumor burdens was particular clearin that more than half of Apc/KO mice reached the maximumtumor burden at 2 months when no tumors were detected inApc/WTmice (Fig. 6B). The increased tumor burdenwas observedin Apc/Het mice sacrificed at 4.5 months (the average lifespan ofthis cohort) when compared with Apc/WT mice of same age,although to a less extent (Supplementary Fig. S5). At the terminalstage of tumor development (i.e., the mice were sacrificed dueto tumor burden), the total numbers and size of tumors in allthree cohorts were not significantly different. Thus, deletion ofeach Erbin allele significantly shortened the time needed to reachmaximal tumor burden in a gene dosage-dependent manner.

To determine the effect of Erbin loss on signaling activationin vivo, intestinal tumor tissues from Apc/WT, Apc/Het, andApc/KO mice were analyzed for the phosphorylation of Akt andErk using IHC staining. Because the tumorigenesis time coursefor Apc/KO mice was drastically accelerated, in which no tumorswere observed in Apc/WT mice at 2 months when majority ofApc/KO mice died (Fig. 6A and B), we collected tumor tissuesfrom different cohorts ofmice when they reachedmaximal tumorburden. Consistently, the levels of Akt and Erk phosphorylationwere markedly increased in Apc/Het and Apc/KO tumors com-pared with Apc/WT (Fig. 6C). In addition, tumor cells isolatedfrom Apc/WT and Apc/KOmice were allowed to grow into tumororganoids in 3D Matrigel. These organoids comprised of adeno-ma cells form cystic structure without budding (Fig. 6D).To determine whether Erbin inhibits signaling in mouse adeno-mas, protein lysates prepared from tumor organoids weresubjected to Western blot analysis. Both Akt and Erk phos-phorylation were markedly increased in Erbin KO organoids(Fig. 6E). Together, these data showed that Erbin loss promotesthe activation of Akt and Erk signaling and tumorigenesis inmouse models of colorectal cancer.

Loss of Erbin increases the tumor initiation potential of Apctumor organoids

Wenext analyzed the tumor initiation capacity ofmouse tumororganoids using the colony formation assay. Apc/WT andApc/KOorganoids were dissociated into single cells and reseeded intoMatrigel. The number of tumor organoids formed was countedafter 5 days in culture. Knockout of Erbin resulted in a 2-foldincrease in organoid formation (Fig. 7A), suggesting increasedtumor initiation capacity. In addition, we determined the profileof gene expression using quantitative RT-PCR analysis in tumororganoids. Apc/WT and Apc/KO organoids grown in 3D culturefor 5 days were collected and analyzed for the expression of Wnttarget genes that are known to associate with cancer stem cells(e.g., Lgr5, Axin2, and Cd44), intestinal cell differentiation (e.g.,Alpi, Fabp2, and Muc2), and cell proliferation (e.g., Ccnd1 andKi67). Tumor organoids derived from Apc/KO mice expressedhigher levels of genes associated with Wnt signaling and cellproliferation, which coincidedwith decreased expression of genesassociated with differentiated intestinal epithelial cells (Fig. 7B).Similarly, the expression of genes associated with Wnt signalingand cell proliferation were significantly increased and intestinaldifferentiation decreased in tumor organoids derived fromApc/Het mice (Supplementary Fig. S5), confirming that haplo-deficiency of Erbin is sufficient to promote tumorigenesis.

Intriguingly, Erbin loss didnot result inEMT-like phenotypes asthe expression of E-cadherin remained unchanged in Apc/KOtumor tissues and organoids compared with Apc/WT (data not

shown). Because human colon cancer cells used in this studycontain additional oncogenic alterations (such as KRAS orBRAF mutation), we performed additional experiments usingApc/KrasG12D double mutant mouse tumor organoids to deter-mine the effect of Erbin downregulation. Indeed, silencing Erbinusing two different lentiviral shRNAs in Apc/KrasG12D tumororganoids resulted in a decrease in E-cadherin expression andan increase in the phosphorylation of Akt and Erk (Fig. 7C). Inaddition, consistent with results obtained in Apc/KO organoids,the ability of Erbin knockdown Apc/KrasG12D cells to form tumororganoids in 3D was increased (Fig. 7D), suggesting thatdecreased expression of Erbin promotes tumor initiation poten-tial in Apc/KrasG12D tumors as well.

Furthermore, we determined whether Erbin expression is asso-ciated with cancer-related biological pathways by analyzing geneexpression data from TCGA colorectal cancer RNA-seq dataset.Results from the GSEA showed Erbin expression is positivelyassociated with the E-cadherin adherens junction pathway andnegatively associated with Wnt signaling (Fig. 7E). These datasupport our findings that loss of Erbin promotes the disruption ofepithelial polarity by reducing E-cadherin expression andenhances tumor initiation potential by increasing signalingthrough the Wnt pathway. Taken together, our results showedthat genetic deletion of Erbin potentiates tumor formation andprogression in vivo.

DiscussionLoss of epithelial polarity is a hallmark of advanced malignant

tumors. Emerging evidence supports the notion that disruption ofpolarity promotes themalignant transformationof epithelial cells(14, 45, 46). In this study, combining in vitro and in vivo analyses,we identify Erbin as a tumor suppressor in colorectal cancer. ThemRNA expression of Erbin is significantly downregulated inpatients with colorectal cancer. Knockdown of Erbin in coloncancer cells results in disruption of epithelial polarity, increasedcellmotility, and cell proliferation.Mechanistically, Erbin inhibitsthe activation RAF/MEK/ERK signaling by sequestering KSR1from forming a complex with RAF1. Finally, our in vivo studiesreveal that Erbin loss accelerates tumorprogression inApc-mutantmouse models.

Previous studies have suggested that Erbin inhibits the activa-tion of ERK by disrupting Shoc2-mediated RAS/RAF interaction(5, 6). However, Shoc2 is primarily localized to the endosomecompartment of the cell (47). It remains an open question howErbin, a basolateral membrane localized protein, interferes withShoc2-dependent activation of RAS/RAF signaling at the endo-some. In our study, we show that Erbin decreases RAF/MEK/ERKsignaling through directly competing with KSR1. KSR1 is knownto translocate to the plasma membrane upon RAS activation(20, 21). Results from our study and others demonstrate thatErbin is localized at the basolateral membrane. Being in closeproximity with receptor tyrosine kinases (such as EGFR) and thesite of RAS activation, the presence of Erbin may block the accessof KSR1 to RAS-bound RAF and reduces KSR1–RAF interaction. Itis interesting that Erbin downregulation promotes further acti-vation of ERK signaling cascade in colorectal cancer cells thatcontain KRAS or BRAF mutations. Thus, by providing a spatialcontrol of how signaling complexes are assembled, Erbin mayserve as a negative scaffold to restrict signaling output of onco-genic pathways mediated by WT or mutant KRAS and BRAF.






Figure 7.

Loss of Erbin enhances tumor initiation properties in tumor organoids.A, Tumor organoids derived fromApc/WT andApc/KOmicewere dissociated into single cells,and 1,000 cells were reseeded in 3D Matrigel. The number of cells that were able to successfully form colonies were quantified. Data, mean � SEM (� , P < 0.05).B, Tumor organoids derived from Apc/WT and Apc/KO mice grown in 3D Matrigel for 72 hours were collected and analyzed for the expression of Wnt targetgenes (Lgr5, Axin2, and Cd44), cell proliferation (Ccnd1 and Ki67), and intestinal differentiation (Alpi, Fabp2, and Muc2) using quantitative RT-PCR. Twodifferent mice of each genotype were used to prepare tumor organoids. RT-PCR analyses were performed separately with tumor organoids from individualanimals and results are combined and summarized in the bar graphs. Data, mean� SEM (#, P < 0.0001; � , P < 0.05). C, Tumor organoids derived from Apc/KrasG12D

double-mutant mice were infected with control and sh-Erbin lentivirus (B1 and B2) and selected to obtain stable Erbin knockdown tumor cells. Protein lysatesprepared from control and Erbin knockdown tumor organoids grown in 3D and analyzed for the expression of Erbin and E-cadherin and the phosphorylation of Aktand Erk. The relative levels of Akt and Erk phosphorylation were quantified by normalizing ECL signals generated by p-Akt and p-Erk to that of total Akt and Erk,respectively. D, Control and Erbin knockdown Apc/KrasG12D tumor organoids were dissociated into single cells and 1,000 cells were reseeded in 3D Matrigel.The number of cells that were able to successfully form colonies were quantified. Data, mean � SEM (�, P < 0.01). E, The GSEA was performed using the TCGAcolorectal cancer RNA-seqdataset to identify genes that havepositive or negative correlationswith Erbin expression. Enrichment plots showedsignificant correlationof the E-cadherin pathway (NES ¼ 2.32; FDR < 0.0001) and Wnt signaling (NES ¼ �2.65; FDR < 0.0001) with Erbin expression in patients with colorectal cancer.

Stevens et al.





Although the increased cell motility is mainly associated withactivation of MEK/ERK pathway in Erbin knockdown cells, theactivation of both Akt and MEK/ERK signaling likely contributesto increased tumorigenesis in Erbin KO mice. It has been shownrecently that oncogenic KRAS promotes Wnt signaling throughERK-mediated phosphorylation of LRP6 (48). However, treatingApc/KO tumor organoids with MEK or Akt inhibitor was unableto downregulateWnt target gene expression in our study (data notshown). It is possible that loss of Erbin expression may alter theorganization of epithelial junctions that allows the dissociation ofb-catenin from the cell membrane. Future studies are required todetermine the mechanism by which Erbin loss induces activationof Wnt signaling.

The role of Erbin in cancer has been controversial. Although anumber of studies have shown that Erbin negatively regulates cellproliferation and survival in different types of cancer cells (7, 49),other studies indicate that Erbin loss increases tumorigenesis(44, 50). Results from our study have provided several lines ofevidence supporting the tumor suppressor function of Erbin incolorectal cancer: (i) analysis of human colorectal cancer geneexpression datasets with large sample sizes indicates that ErbinmRNA expression is significantly downregulated in patients withcolorectal cancer; (ii) Erbin protein expression is decreased incolorectal cancer patient specimens by Western blot and IHCanalyses; (iii) knockout of Erbin in Apc-mutant mice promotestumor progression and reduces survival; and (iv) tumororganoidsderived from Erbin KO mice have increased tumor initiationpotential. Our findings are also corroborated by the bioinformat-ics analysis, in which Erbin expression is found to be down-regulated at early stage of colorectal cancer and associated withincreased E-cadherin junctions and decreased Wnt signaling.Additional gene sets that related to receptor tyrosine kinasesignaling, cell-cycle control, and protein translation are identifiedto be associated with Erbin expression in patients with colorectalcancer in our GSEA study (Supplementary Table S2), suggestingthat Erbin loss may have a broad impact on altering cell functionsin cancer. Moreover, a number of missense, nonsense, and frame-shift mutations are identified throughout the entire coding regionof Erbin based on an analysis of colorectal cancer datasets avail-able at cBioPortal (Supplementary Fig. S6; ref. 51). The mutationrate of Erbin is between 2% and 4% among different studies.However, future studies are needed to determine the mechanismby which Erbin is downregulated in colorectal cancer and howErbin loss induces polarity defect and EMT.

In summary, our study has uncovered a pivotal role of Erbin inmaintaining epithelial cell polarity and suppressing EMT incolorectal cancer. By developing novel in vivomouse models andtumor organoid systems, we demonstrate that Erbin exerts itstumor suppressor function by negatively regulating both Akt andRAF/MEK/ERK signaling and Erbin loss promotes tumor initia-tion and progression. The functional interplay between Erbin–KSR1 highlights the importance of scaffolding proteins in pro-viding the spatiotemporal control of cell signals. Future studies onErbin-dependent inhibition of tumor progression will help toexplore the potential of using Erbin as a diagnostic marker fordeveloping personalized treatment strategies in colorectal cancer.

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

Authors' ContributionsConception and design: P.D. Stevens, T. GaoDevelopment of methodology: P.D. Stevens, X. Xiong, T. GaoAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): P.D. Stevens, Y.-A. Wen, X. Xiong, Y.Y. Zaytseva,A.T. Stevens, T.N. Farmer, T. Gan, S. Marchetto, J.-P. Borg, T. GaoAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): P.D. Stevens, A.T. Li, C. Wang, A.T. Stevens,T.N. Farmer, H.L. Weiss, T. GaoWriting, review, and/or revision of the manuscript: P.D. Stevens, J.-P. Borg,T. GaoAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): X. Xiong, T. GanStudy supervision: M. Inagaki, T. Gao

AcknowledgmentsWe thank Dr. Emilia Galperin (University of Kentucky) for providing the

expression construct of CFP-KSR1. This work was supported by R01CA133429(T. Gao) and F31 CA196219 (P.D. Stevens). The studies were conducted withsupport provided by the Biospecimen Procurement and Translational Pathol-ogy and Biostatistics and Bioinformatics Shared Resource Facilities of theUniversity of Kentucky Markey Cancer Center (P30CA177558). J.P, Borg'slaboratory is funded by La Ligue Nationale Contre le Cancer (Label LigueJ.P. Borg), Fondation A�MIDEX, and SIRIC (INCa-DGOS-Inserm 6038).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received November 21, 2017; revised June 1, 2018; accepted July 2, 2018;published first July 6, 2018.

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Erbin Suppresses KSR1-Mediated RAS/RAF Signaling and … · Erbin Suppresses KSR1-Mediated RAS/RAF Signaling and Tumorigenesis in Colorectal Cancer Payton D. Stevens 1 ,Yang-An Wen