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Human Cancer Biology

Colon Cancer Cells Escape 5FUChemotherapy-Induced CellDeath by Entering Stemness and Quiescence Associatedwith the c-Yes/YAP Axis

Yasmine Touil1,2, Wassila Igoudjil3, Matthieu Corvaisier3, Anne-Fr�ed�erique Dessein3, J�erome Vandomme1,Didier Mont�e8, Laurence Stechly3, Nicolas Skrypek3, Carole Langlois4, Georges Grard3, Guillaume Millet3,5,Emmanuelle Leteurtre3, Patrick Dumont7, St�ephanie Truant3,5, Francois-Ren�e Pruvot5, Mohamed Hebbar6,Fan Fan10, Lee M. Ellis10, Pierre Formstecher1, Isabelle Van Seuningen3, Christian Gespach9,Renata Polakowska1, and Guillemette Huet3

AbstractPurpose: Metastasis and drug resistance are the major limitations in the survival and management of

patients with cancer. This study aimed to identify the mechanisms underlying HT29 colon cancer cell

chemoresistance acquired after sequential exposure to 5-fluorouracil (5FU), a classical anticancer drug for

treatment of epithelial solid tumors. We examined its clinical relevance in a cohort of patients with colon

cancer with liver metastases after 5FU-based neoadjuvant chemotherapy and surgery.

Results: We show that a clonal 5F31 cell population, resistant to 1 mmol/L 5FU, express a typical cancer

stem cell–like phenotype and enter into a reversible quiescent G0 state upon reexposure to higher 5FU

concentrations. These quiescent cells overexpressed the tyrosine kinase c-Yes that became activated and

membrane-associated upon 5FU exposure. This enhanced signaling pathway induced the dissociation of the

Yes/YAP (Yes-associated protein) molecular complex and depleted nuclear YAP levels. Consistently, YES1

silencing decreased nuclear YAP accumulation and induced cellular quiescence in 5F31 cells cultured in 5FU-

freemedium. Importantly,YES1 andYAP transcript levelswerehigher in livermetastases of patientswith colon

cancer after 5FU-based neoadjuvant chemotherapy. Moreover, the YES1 and YAP transcript levels positively

correlated with colon cancer relapse and shorter patient survival (P < 0.05 and P < 0.025, respectively).

Conclusions: We identified c-Yes and YAP as potential molecular targets to eradicate quiescent cancer

cells and dormant micrometastases during 5FU chemotherapy and resistance and as predictive survival

markers for colon cancer. Clin Cancer Res; 1–10. �2013 AACR.

IntroductionTreatment of colon cancer involves the surgical resection

of the primary tumor. For patients with stage III disease, asurvival advantage is obtained with 5-fluorouracil (5FU)/

leucovorin-based adjuvant chemotherapy used in combi-nation with oxaliplatin or irinotecan (1–3). In the last 8years, cetuximab and panitumumab, twomonoclonal anti-bodies that target the epidermal growth factor receptor,were shown to be effective in combination with chemo-therapy or as single agents in patients with wild-type KRAStumors (4–6). In addition, antiangiogenic therapy targetingvascular endothelial growth factor (bevacizumab) confers abenefit when used in combination with chemotherapy (7).However, the development of drug resistance remains amajor limitation in the efficacy of the clinical response tochemotherapeutic and targeted therapy regimens.

A growing body of evidence suggests that the majority oftumors comprise a population of tumor-initiating or cancerstem cells (CSC) that are responsible for the developmentandmaintenance of tumors and resistance to cytotoxic drugs(8). In breast cancer, studies using clinical tumor samplessupport the hypothesis that the residual disease after neoad-juvant chemotherapy is enriched with CSCs. Cell suspen-sions derived from chemotherapy-treated patients showedan increase in mammosphere formation, self-renewal, andenrichment in CD44þCD24�/low stem-like cells (9). Inmurinemodels of colon cancer cell xenografts, the treatment

Authors' Affiliations: 1INSERM U837 Team 4 "Molecular and CellularTargeting of Cancers"; 2SIRIC ONCOLille; 3INSERM U837 Team 5 "Mucins,Epithelial Differentiation, and Carcinogenesis" Jean-Pierre Aubert ResearchCentre, Universit�e Lille and CHU, Univ Nord de France; 4Unit of Biostatistics;5Department of Digestive Surgery and Transplantation; 6Department ofMedical Oncology, CHU, Univ Nord de France; 7IBL UMR-8161 CNRS,Universit�e Lille Nord de France, Institut Pasteur, Lille; 8IRI USR 3078 CNRS,Villeneuve d'Ascq; 9INSERMU938,Molecular andClinical Oncology, HopitalSaint Antoine, Universit�e Pierre et Marie Curie 6, Paris, France; and 10TheUniversity of Texas MD Anderson Cancer Center, Houston, Texas

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

Y. Touil, W. Igoudjil, C. Gespach, R. Polakowska, and G. Huet contributedequally to this work.

Corresponding Author: Guillemette Huet, INSERM, Batiment Biserte, rueMichel Polonowski, Lille Cedex 59045, France. Phone: 0033-3-20-29-88-50; Fax: 0033-3-20-53-85-62; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-13-1854

�2013 American Association for Cancer Research.

ClinicalCancer

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of mice with chemotherapeutic agents enriched the tumorxenografts in ESAþCD44þ and ESAþCD44þCD166þ CSCs(10). In other studies, CSCs were isolated from various typesof tumors and analyzed for chemoresistance ex vivo. Inhuman colon cancer, CD133-positive CSCs were highlyresistant to 5FU and oxaliplatin (11).When the colon cancercell line HT29 was treated continuously with 5FU or oxa-liplatin, the emergent resistant subpopulations were highlyenriched in cells expressing stem cell markers, includingCD133 (16- to 30-fold) and CD44 (2-fold; ref. 12). Thesefindings suggest that drug-resistant subpopulations may beenriched in CSCs.

The intrinsic resistance of CSCs to chemotherapeuticagentsmay be explained by high expression of ATP-bindingcassette (ABC) multidrug transporters, antiapoptotic pro-teins, and by the resistance to DNA damage. Accumulatingevidence has indicated that CSC quiescence may alsoaccount for a possible mechanism of resistance because theactivity of several cytotoxic agents is dependent on cell-cycleprogression (13–16). Cellular quiescence is a basic mech-anism of clinical tumor dormancy, although angiogenicdormancy and escape from immune system control alsoplay important roles (17).

We previously reported that chronic treatment of theHT29 colon cancer cell line with chemotherapeutic agentsresulted in the emergence of drug-resistant HT29 subpopu-lations overexpressing the chemokine (C-X-C) motif recep-tor 4 (CXCR4). In addition, overexpression and autocrineactivation of CXCR4 played a role in the metastatic spread-ing to the lungs in immunodeficientmice (18, 19).Here, wereport the acquisition of a complexmechanismof chemore-sistance to 5FU involving selection for colonCSCs and their

quiescence linked to the activation of the c-Yes tyrosinekinase. We show that c-Yes controls the balance betweenquiescence and cycling of 5FU chemoresistant cells as wellas the cytoplasmic/nuclear ratio of the Yes-associated pro-tein (YAP) transcription coactivator. Finally, we discovereda direct relationship between c-Yes/YAP expression levelsand overall and disease-free survival of patients with colo-rectal livermetastases after 5FU-basedneoadjuvant therapy.

Materials and MethodsHuman colorectal carcinoma cell lines and patients’tissue samples

HT29 parental cell line, 5FU (10�6 and 10�5 mol/L)-resistant HT29 subpopulations (HT29FU) and 5F7 and5F31 clonal derivatives as well as 5FU-resistant (8 � 10�6

mol/L) RKO colon cancer cell line (RKO) cells (RKO-FU)and oxaliplatin-resistant (2� 10�6mol/L) RKO cells (RKO-oxaliplatin [OXA]) and HT29OXA cells subpopulationswere cultured as previously described (20–22). Liver metas-tases of colon adenocarcinoma were resected from 49patients and were processed and stored by the Tumor Celland Tissue Bank of the Regional Reference Cancer Center ofLille. After hepatic resection, fragments were taken frommacroscopic metastases, snap-frozen in liquid nitrogen,and stored at �80�C. The whole remaining tissue was fixedin 10% formalin and several other fragments were takenfrom the fixedmetastases and embedded in paraffin. Hema-toxilin, eosin, saffron, and Astra blue-stained sections wereexamined by an experienced pathologist to establish thehistological diagnosis. Fragments used in this study con-tained at least 60% malignant cells. Informed consent wasobtained from all patients. The curative microscopicallycomplete R0 liver resections in all patients were performedbetween February 2002 and May 2009. Neoadjuvant che-motherapy was administered for 28 patients before surgeryaccording to the recommendations of the French Thesaurusof Digestive Cancerology: Folfox in 17 patients, Folfiri in 1patient, Folfiri-bevacizumab in 10 patients. Informationabout sites of tumor recurrence or metastasis developmentafter curative hepatectomy and delay of recurrence werecollected for each patient.

YES1 silencingYES1 silencing was performed by retroviral infection of

5F31 cells using a pRETRO-Super vector as previouslydescribed (23). Short hairpin RNAs (shRNA) targetingYES1transcript sequences were also previously described (24).The selection of the YES1–silenced populations was per-formed with puromycin (1 mg/mL).

Statistical analysesAll data were expressed asmean (standard deviation) and

categorical variables by percentage (frequency). Overallsurvival (OS) and disease-free survival (DFS) were calcu-lated by the Kaplan–Meier method, and the differencesbetween groups were compared using the log-rank test. Toidentify predictive variables of OS or DFS, continuous

Translational RelevanceMetastasis, chemoresistance, and dormancy are the

major limitations in the management of patientswith cancer. Here, we show that 5-fluorouracil (5FU)chemoresistance in human colon cancer cells preselectstwo distinct drug-resistant clonal populations eachenriched in cells expressing specific stem cell markers anddifferential self-renewing and metastatic potentials. The5FU-induced quiescence is linked to activation of the c-Yes tyrosine kinase and nuclear depletion of the c-Yes–associated YAP oncogene. Consistently, YES1 silencingdecreasednuclearYAPaccumulationand inducedcellularquiescence in 5FU-free conditions. We further demon-strate the prognostic relevance of our data in colon cancerliver metastases treated by neoadjuvant chemotherapyregimens based on 5FU. High YES1 and YAP transcriptlevels measured in residual livermetastases after adjuvantchemotherapy are observed in patients at risk for coloncancer relapse and shorter survival. Therefore, these twomarkers identify candidates for adapted treatment target-ing the c-Yes/YAP axis in chemoresistant quiescent coloncarcinomas and liver metastases.

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variables were analyzed by a Cox proportional hazardsmodel and qualitative variables using the log-rank test.Expectationmaximization algorithmwas used to determinethe cutoff values of Yes and YAP. All analyses were per-formed using the SAS software version 9.2 (SAS InstituteInc.). A P value of <0.05 was considered significant.

Results5FU-resistant HT29 clones exhibited heterogeneity intheir chemoresistancePrevious studies have reported the emergence of stable

HT29 cell subpopulations resistant to 5FU thatwere capableto differentiate into goblet cell–like or enterocyte-like phe-notypes (20). Cloning of the HT29 subpopulation resistantto 10�6 mol/L 5FU (HT29FU) by limiting dilution gaverise to several HT29 cell clonal subpopulations (HT29 5Fclones; ref. 21). The analysis of IC50 values in HT29 5Fclones toward newly added 5FU revealed differential intrin-sic levels of chemoresistance to this drug. As shown in Fig. 1,the 5F31 clonewashighly 5FU resistant (IC50¼19.6�10�6

mol/L 5FU), whereas 5F7 clone was much less resistant(IC50 ¼ 1.9 � 10�6 mol/L) but still more resistant thanthe parental HT29 cell line (IC50 ¼ 1.1� 10�6 mol/L). Thehighly 5FU-resistant 5F31 cells were able to recover prolif-erative capacity after drug withdrawal.

Drug resistance is associated with a diverse expressionpattern of stem cell markers5FU-resistant clones highly expressed several colon CSC

surface markers as shown by flow cytometry using aquadruple labeling of CD24, CD44, CD133, and CXCR4(Supplementary Fig. S1A). In contrast, parental HT29 cellpopulation contained a low percentage of cells expressingstem cellmarkers, ranging from4% (CXCR4) to 32% (CD24;Supplementary Table S1). The two 5FU-resistant clones con-

tainedmore than 90% of cells expressing at least onemarker.The 5F31 cell population mainly contained CD24þ/CD44þ

cells (55%), CD24þ/CD44þ/CD133þ cells (14%), andCD24þ cells (14%). The 5F7 cell population was composedof CD24þ/CD44þ/CD133þ/CXCR4þ cells (50%) and ofCD24þ/CD44þ/CXCR4þ cells (31%). The 5FU-resistant(HT29FU) and oxaliplatin-resistant (HT29OXA) subpopu-lations (22) also contained an important proportion ofCD24, CD44, CD133, and CXCR4-positive cells (Supple-mentaryTable S1).Moreover, thepercentages ofCD133- andCXCR4-positive cells varied substantially in 5F7 and 5F31.This heterogeneity was further confirmed using the CSCmarker aldehyde dehydrogenase (ALDH)1A3, an isoformof ALDH (Supplementary Fig. S1B; 25). ALDH1A3 wasstrongly downregulated (74-fold) in 5F7 cells and converselyupregulated in 5F31 cells (3-fold), as compared with theparental HT29 cells. Total ALDH activity was also higher in5F31 versus 5F7 cells. Altogether, our data show that resistantclones were enriched in stem cell–like cancer cells that differin the expression pattern of stem cell markers.

Drug-resistant HT29 clones differed in theirproliferation and metastatic potentials

Stem cells are defined by their combined ability to self-renew and to differentiate. We investigated the self-renewalpotential of 5F7, 5F31, andHT29 cells and their 5FU-treatedcounterparts using anchorage-independent growth (Fig.2A). 5F31 cells formed well-delimitated regular spheres,whereas 5F7 and parental cells produced irregular sphereswith a strong tendency to form aggregates. The self-renewalpotential of sphereswas evaluated by an increase ratio in thenumber of spheres formed over three consecutive genera-tions (Fig. 2B). The ratio between third and first generationof spheres was significantly enhanced in 5FU-treated 5F7and 5F31 cells (1.9- and 5.8-fold, respectively) as comparedwith their untreated counterparts (1.3- and 2.5-fold, respec-tively). Also, the HT29FU and HT29OXA subpopulationswere enriched in the sphere-forming, self-renewing cellswhen compared with their parental cell line.

The 5F7 and 5F31 clones were then assessed for theirtumorigenic and metastatic potential using orthotopicxenografts. Both clones produced fairly well-differentiatedadenocarcinomas with cell layers and glands containingmucus in their lumen (Fig. 2C). The presence of lungmicrometastases was observed in both 5F7- and 5F31-xenografted mice; however, the number of lung metastaseswas higher in 5F7 xenografts in comparison with 5F31xenografts (9 vs. 2 per mouse). In addition, only 5F31 cellsgenerated metastases in lymph vessels around the liver,demonstrating that these two clones have similar tumori-genic but differential metastatic abilities.

Variations in drug resistance of HT29 clones areconnected to different phases of the cell cycle

Cellular responses to 5FU treatments using 1/2IC50 and2� IC50 5FU concentrations for 5F7 cells (1 and 4 mmol/L)and 5F31 cells (10 and 40 mmol/L) were examined by theflow cytometry cell-cycle analysis (Supplementary Table S2).

Figure 1. 5FU resistance of 5F7 and 5F31 cells. Survival curves of 5FU-resistant 5F7 and 5F31 clones after subsequent exposure to 5FUconcentrations for 5 days. The IC50 was defined as the concentration of5FU producing a 50% decrease in the number of cells compared withuntreated controls. Error bars, mean � SD for 6 replicates.

Cancer Cell Chemoresistance and Yes/YAP Axis

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Treatment ofHT29parental cells byhigh5FUconcentrationsinduced typical cell accumulation in the S-phase as previ-ously shown (26). Similarly, 5F7 cells accumulated at the S-phase in response to gradedhigh5FU concentrations, aswellas at the G2–M-phase. In contrast, subsequent exposure of5F31 cells to 5FU induced a selective and concentration-dependent accumulation of cells at the G0-phase of the cellcycle, from 2.9% in control cells to 12.3 and 50.4% in 5F31cells exposed to 10 and 40 mmol/L 5FU, respectively. Inaddition, the percentage of quiescent cells in the 5FU-treated5F31 cell line gradually increased over time, from 8.0% (1hour) to 14.7% (4hours), 32.5% (72 hours), and 50.4% (96hours). Because nondividing cells are not targeted by che-motherapeutic drugs targeting proliferating cells, our datasuggest that 5F31 cells evade 5FU treatment through entryin a quiescent G0 state. Importantly, a high percentage ofquiescencewas observed in other drug-resistant colon cancercell subpopulations, i.e., RKO-OXA (8.5%), -FU (9.8%)versus control RKO cells (3.1%), and HCT-116-OXA(11.1%), -FU (21.8%) versus control HCT-116 cells(4.7%). As these 5FU- and OXA-resistant cells are enrichedin stemcellmarkers (ourdata and ref. 12),we concluded thatthe entry into quiescence is a more general mechanismregulating colon cancer stem survival and chemoresistance.

5FU resistance of 5F7 and 5F31 HT-29 clones wasconnected to differential activation of Chk-2 and c-Yeskinases

Control- and subsequently 5FU-treated 5F7 and 5F31resistant cells were analyzed by phospho-kinase array and

Western blotting (Fig. 3A and B). Treatment of 5F7 cellswith 5FU primarily induced checkpoint kinase 2 (Chk-2)phosphorylation at the threonine 68 residue. As shownpreviously, Chk-2 is activated by the ataxia telangiectasiamutated (ATM)-mediated responses to DNA damage (27).Activation of this DNA damage–sensing pathway preventsprogression through the cell cycle and recruits the DNArepair machinery. Our result showing accumulation of 5F7cells in the S- and G2–M-phase upon 5FU treatment isconsistent with these observations. In contrast, the level ofphosphorylatedChk-2 remained unchanged in 5FU-treated5F31 cells (Fig. 3A and B). Instead, they had higher levels ofphosphorylated c-Yes. This finding was confirmed byWest-ern blotting of c-Yes in antiphosphotyrosine precipitates(Fig. 3C). The c-Yes phosphorylation level was increased by3.8-fold upon 5FU exposure. c-Yes phosphorylation wasfurther confirmed by ELISA assay (Fig. 3D). Control- and5FU-treated 5F31 cells showed higher c-Yes protein levelsthan the parental HT29 or 5F7 cells (4.5- and 4.9-fold,respectively, P < 0.01; Fig. 3B). Also, they expressed 7.7-foldhigher levels of YES1 transcripts, whereas 5F7 cells showedonly 3-fold higher than parental HT29 cells (Fig. 3E).

YES1 silencing in 5F31 cells induced quiescence andrestricted YAP nuclear accumulation

To investigate whether c-Yes is involved in the 5FU-induced cellular quiescence of 5F31, we silenced YES1 inthese cells using shRNA. Two silenced sh1 and sh2 5F31populations showing differential levels of YES1 silencingwere obtained, as compared with control transfected cells

Figure 2. 5FU-resistant 5F7 and 5F31 clones differ in their self-renewal and metastatic potentials. A, sphere formation in anchorage-independent cultureconditions, using parental HT29 cells and the drug-resistant clones. B, self-renewal capacity in spheres arising from untreated or treated cells. Thespheres obtained from cells cultured in the absence of 5FU (left) or presence of 5FU (4 days, 1/2IC50, right) were dissociated and subsequently reseededover three passages. The HT29FU and HT29OXA subpopulations were analyzed in comparison (right). Results are the ratio of the number of countedspheres between the third and first generations of spheres. Histograms are the means from at least two independent experiments. HT29 parental population(white), 5F7 (bright gray), 5F31 (dark gray), HT29FU (hatched), and HT29OXA (grid). C, tumorigenic and metastatic potential of 5F7 and 5F31 clones inimmunodeficient mice using orthotopic intracecal xenografts. Intracecal tumors and metastases were analyzed by conventional histology (hematoxylin,eosin, saffron and Astra blue, HESAB), magnification, �200. Intracecal xenografts seem as adenocarcinoma with cell rows and mucus-secreting glands.Both 5F7 and 5F31 tumors produce lung metastases but only 5F31 tumors generate extrahepatic lymph metastases.

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with the scrambled shRNA sequence (Scr; Fig. 4A). Consis-tent with recent data published on YES1–silenced HT29cells (24), YES1 silencing in 5F31 cells was associated withincreased b-catenin levels (Fig. 4A). Most interestingly,YES1 silencing levels were directly correlated with the emer-genceofG0quiescent 5F31 cells (Fig. 4B), as observed in sh2(50% silencing, 10% in G0 state) and sh1 cells (86%silencing, 29% cells in G0 state) as compared with Scr cells(4.6% in G0 state). Next, we compared the proliferationcapacity of 5FU-treated sh1 cells versus control Scr cells after5FU retreatment (40 mmol/L, 5 days) and subsequent drugwithdrawal. The population doubling time of silenced sh1cells was 57 hours, whereas the control Scr cells dividedevery 36 hours. These data demonstrate that depletion of c-Yes arrests 5F31 cells in the G0-phase of the cell cycle and

suggest that c-Yes is required for the reentry of 5F31 quies-cent cells into the proliferative state. As YAP is a growth-promoting transcriptional coactivator that promotes stemcell self-renewal and progenitors expansion (28), we ana-lyzed the consequences of YES1 silencing on its fate. Asshown in Fig. 4A, total YAP levels remained unchangedupon YES1 silencing, however, the YAP nuclear/cyto-plasmic ratio decreased in sh1 cells as compared withcontrol Scr cells (39%–61% vs. 48%–52%; Fig. 4C), imply-ing that c-Yes controls levels of nuclear YAP.

The nuclear accumulation of YAPwas restricted in 5FU-treated quiescent 5F31 cells

Because the majority of 5F31 cells submitted to high 5FUconcentration enter quiescence, we examined the effect of

Figure 3. 5FU-resistant 5F7 and5F31 clones selectively activatedifferent signaling pathwaysfollowing subsequent exposure to5FU. A, total cell lysates fromcontrol- and 5FU-treated 5F7 and5F31 cells (4 days) were analyzedusing phospho-kinase arrays. Chk-2 serine/threonine kinase, c-Yestyrosine kinase (arrows). Otherkinases explored in thesephospho-antibody arrays areinvolved in cancer cell proliferationand cell-cycle controls, survival,adhesion, and transcription (seethe phospho-array coordinates inMaterials and Methods). F17, F18,G5, G6: negative controls; A1, A2,A17, A18, G1, G2: positivecontrols. B, Western blots of thetotal cell lysates using specificantibodies against P-Chk-2 (T68),Chk-2, and c-Yes. Actin was usedas loading control. The data arerepresentative of two independentexperiments. C, analysis of c-Yesphosphorylation byimmunoprecipitation using theantiphosphotyrosine antibodyfollowed by Western blotting withthe anti-c-Yes antibody. D,analysis of c-Yes phosphorylationusing the phospho-c-Yes (Y426)ELISA assay. Error bars, mean �SD for three replicates. E, analysisof c-Yes mRNA levels in 5F7 and5F31 cells in comparison withHT29 cells.






5FU treatment upon the nuclear distribution of YAP. Asshown in Fig. 4D and E, the nuclear pool of YAP decreasedmarkedly upon 5FU exposure in 5F31 cells (27% vs. 55%),whereas total YAP level was not significantly altered. Ourdata support a possible link between nuclear YAP depletionand cellular quiescence. As stated above, c-Yes becomesphosphorylated in 5F31 cells reexposed to 5FU. A confocalmicroscopy analysis showed that 5FU exposure induced therecruitment of cytoplasmic c-Yes to the cell membrane (Fig.4F). This shift in the c-Yes subcellular distribution is con-sistent with its activation because the c-Yes kinase inhibitorSU6656 prevents c-Yes interaction with integral membraneproteins (29, 30). Thus, we hypothesized that 5FU treat-ment induces c-Yes phosphorylation and dissociates the c-Yes/YAP complex in 5F31 cells and that the released c-Yes istargeted to the plasma membrane. To test this hypothesis,YAP coimmunoprecipitation experiments were performedusing the anti-YAP antibody. Interestingly, c-Yes was foundin YAP immunoprecipitates prepared from control 5F31cells but not from 5FU-treated 5F31 cells, showing that thec-Yes/YAP complex was lost upon 5FU exposure (Fig. 4G).

YES1 and YAP expression levels were increased bychemotherapy in human colon livermetastases and arecorrelated with colon cancer relapse

To examine the clinical significance of our data, we ana-lyzed the level of YES1 and YAP transcripts in a cohort of49 patients with colon cancer who underwent surgical resec-tion of liver metastases. Twenty-eight patients received thechemotherapeutic regimens prior surgery. As shown in Fig.5A, quantification of YES1 and YAP transcript expressions by

quantitative real-time PCR showed enhanced YES1 (4.2-fold) and YAP (1.7-fold) transcript levels in patients treatedby chemotherapy (P ¼ 0.035 and 0.026, respectively;Fig. 5A). Consistently, increased c-Yes protein levels wereobserved in 5FU- and OXA-resistant HT29 and RKO coloncancer cells, respectively. Of note, YAP protein levels werestrongly elevated in OXA-resistant HT29 and RKO cells(Supplementary Fig. S2). This indicates that chemotherapymay preselect for quiescent colon CSCs harboring a dereg-ulationof the c-Yes/YAPaxis.Most importantly,YES1 expres-sion negatively correlated with the OS (threshold of 2.25,P ¼ 0.0231) and DFS (threshold of 1.35, P ¼ 0.0433;Fig. 5B). Interestingly, YAP expression also correlated withshorter OS (threshold of 2.62, P ¼ 0.025) and DFS (thresh-old of 2.75, P ¼ 0.0088). The neoadjuvant chemotherapytreatment is administered when liver metastases are ini-tially unresectable or marginally resectable (�5 bilateralnodules). Thus, the treated patients have generally a moresevere disease. As expected, in the studied population therewas no difference in OS and DFS between the groups oftreated and nontreated patients because of the advantagesprovided by neoadjuvant chemotherapy. In the nontreatedgroup, there was no correlation between YES1 and YAP tran-script levels and the clinical outcomes (DFS and OS).In the group of treated patients, YES1 overexpression wasnegatively correlated with OS, with statistical significance(P¼ 0.022) andwithnear significancewithDFS (P¼ 0.071).About YAP transcript, a trend was observed for both OS andDFS (P ¼ 0.092 and P ¼ 0.1, respectively). Thus, the sub-group of treated patients with liver metastases expressinghigh YES1 transcript levels is associated with poorer

Figure 4. YES1 silencing and 5FU inhibit YAP nuclear translocation. A, Western blot analysis of c-Yes, YAP, and b-catenin in YES1–silenced Sh1 and Sh2cells as compared with control Scr cells. B, the percentage of cells arrested at the quiescent G0 state in YES1–silenced Sh1 and Sh2 cells. Results areexpressed as the percentage of quiescent cells in G0 state. C, immunoblot of YAP and transcription factor Sp1 (nuclear marker) in nuclear and cytoplasmicfractions from YES1–silenced Sh1 cells and control Scr cells. D, immunoblots of YAP and b-catenin in control- and 5FU-treated (2� IC50, 4 days) 5F31 cells.E, impact of subsequent 5FU treatment (2� IC50, 4 days) on the nuclear distribution of YAP in 5F31 cells. Western blots are representative of atleast two independent experiments. F, subcellular localization of c-Yes by confocal microscopy analysis in control- and 5FU-treated 5F31 cells. G,coimmunoprecipitation (IP) of c-Yes with the anti-YAP antibody followed by immunoblotting using the c-Yes antibody and the YAP antibody in control- and5FU-treated 5F31 cells. Nonspecific c-Yes immunoprecipitation was monitored using a nonrelevant immunoglobulin G (IgG).

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outcomes. These clinical data are consistent with our ex-perimental study showing a direct correlation between c-Yesexpression and the quiescence of chemoresistant HT29colon cancer cells treated with 5FU. Altogether, these resultsshow that a poorer clinical outcome segregates with theincreased YES1 and YAP transcript levels and concerns pati-ents who received 5FU-based neoadjuvant chemotherapy.

DiscussionCurrently, treatment of synchronous or metachronous

colorectal liver metastases requires neoadjuvant chemo-therapy before hepatic surgery. Chemotherapeutic regi-mens consisting of 5FU and folinic acid combined withoxaliplatin (Folfox), irinotecan (Folfiri), or irinotecan andbevacizumab (Folfiri–bevacizumab) are scheduled every 15days during 3months (1–3). Drugs are withdrawn 1monthbefore surgery. Although temporarily efficient, this treat-ment rarely cures cancer and disease relapses from the drug-resistant cells. To investigate chemoresistance mechanismsoccurring upon sequential chemotherapy regimens, 5FU-resistant clones propagated in drug-free mediumwere reex-posed to 5FU. We show that 5FU-resistant cells could usedifferent strategies to survive rechallenge with 5FU. On theone hand, 5F7 cells that show a weak chemoresistantpotential respond to the drug by activating a typicalATM/Chk-2 pathway that prevents cell-cycle progressionuntil DNA repair is completed (27). On the other hand,5F31 cells although derived from the same parental che-moresistant subpopulation resist high 5FU concentrationsby entering a protective quiescent state, reversible upondrug withdrawal. The escape into quiescence may recapit-ulate clinical observations linked to tumor dormancy andcancer relapse following chemotherapy. This is an impor-tant observation that should be investigated further todetermine therapeutic regimens targeting also quiescentCSCs. Interestingly, both 5FU- and OXA-resistant cells

expressed colon CSC markers, implying that 5FU-basedchemotherapy fails to eradicate quiescent CSCs. The vari-ability in the expression pattern of several stem cell markersidentified in 5FU-resistant 5F7 and 5F31 cells suggests thatthe resistant clones emerge from different CSC subtypes.This notion reflects, again, clinical observations that atumor may contain heterogeneous populations of CSCs(31). Almost an exclusive expression of CSC markersCXCR4 by 5F7 cells and ALDH1A3 by 5F31 cells might bepredictive of the metastatic potential (32, 33). This isconsistent with our previous finding that CXCR4 overex-pression stimulates metastatic spreading of 5F7 subcutane-ous xenografts to the lungs (19). Using orthotopic murinexenografts, we found here that 5F31 cells expressing highALDH1A3 levels disseminated tomoremetastatic sites than5F7 cells overexpressing CXCR4, emphasizing a strong linkbetween the tumorigenic and metastatic potential and theCSC phenotype. We argue that onemechanism responsiblefor such a link may involve c-Yes/YAP signaling pathways.

The YES1 gene is amplified in 5FU-resistant cancer celllines due to its chromosomal localization close to the thy-midylate synthase gene, a 5FU target (19, 34, 35). 5F31 cells,characterized by the high chemoresistance potential and theability to enter quiescence upon 5FU exposure, displayed aparticularly elevated level of c-Yes. We have thereforeexplored the possible implicationof c-Yes andYAP indriving5FU resistance and quiescence of 5F31 cells. YAP stimulatesthe proliferation of mouse intestinal stem cells and main-tains their pluripotency (28, 36, 37). YAP is kept under aninactive state in differentiated cells through the Hippo path-way that phosphorylates YAP at the 127 serine residue andinduces its cytoplasmic sequestration and degradation (38).Here, we found that the entry of 5FU-resistant 5F31 cells inquiescenceupona5FU rechallenge induced the repressionofnuclearYAP levels, suggesting that this repressioncontributesto cancer cell dormancy. In agreement, we observed that the5FU-induced c-Yes phosphorylation and its translocation to

Figure 5. Correlations betweenYES1 and YAP expression andpatient with colon cancer survival.A, comparison of YES1 and YAPtranscript levels in colon livermetastases resected from patientswho have received or notneoadjuvant chemotherapy.B, Kaplan–Meier survival analysisof patients with colon canceraccording to the expressionof YES1 and YAP transcripts.






the plasmamembrane are associatedwith a loss of the c-Yes/YAP complex and depletion of nuclear YAP. In addition, wehave shown thatYES1 silencing induced the accumulationof5F31 cells at theG0-phaseof the cell cycle aswell as depletionof nuclear YAP levels. Consequently, the c-Yes–dependentregulation of YAP activity in 5FU-resistant 5F31 cells can beconsidered as a potentialmechanism involved in the controlof the quiescence/proliferation balance of these cells. Takentogether, our data support the notion that the c-Yes/YAPsignaling pathway contributes to 5FU resistance through thecombined acquisition of both cellular quiescence and stemcell–like phenotype. In support of this conclusion, recentdata indicate that the Yes/YAP-TEA domain2 (TEAD2) sig-naling cascade downstreamof the leukemia inhibiting factor(LIF) is necessary for the YAP nuclear translocation and self-renewal of mouse embryonic stem cells (39). YAP growth-promoting activity ismediated by the interaction between itsN-terminal domain and the TEAD family of DNA-bindingproteins (40).OncogenicYAP is overexpressed inmost colontumors and promotes the proliferation of colon cancer celllines (37, 41, 42). Consistently, YAP was recently incrimi-nated in the metastatic potential of breast cancer and mel-anoma cells through its TEAD interaction domain (43). Incancer-associated fibroblasts, YAP promotes matrix stiffen-ing, cancer cell invasion, and angiogenesis (44). YAP and itsparalog transcriptional co-activator with PDZ-bindingmotif(TAZ) are two downstream targets of the Hippo pathwayregulated by Lats 1/2 kinases. Upstream Hippo signals areinitiated by extracellular diffusible signals and receptor/non-receptor tyrosine kinases such as c-Yes and c-Abl (45).Accordingly, several survival effectors are known to actthroughG-protein receptors and tyrosine phosphorylations,including the src family kinases. Further studies areneeded todetermine the possible connections of the c-Yes/YAP axiswith the core components of the Hippo pathway. In ourstudy, it is also plausible that the oxidative stress responseinduced by 5FU is implicated in c-Yes activation (46).Finally, the c-Yes/cdc42 pathway was shown to repress thetranscription factornuclear factorof activatedT cellsmember1 (NFAT1) by inducing the formation of a cytoplasmiccomplex with casein kinase 1a (CK1a), and Lats-inducedYAP phosphorylation primes YAP for CK1-induced phos-phorylation (47, 48).

We have shown that 5FU-based neoadjuvant chemother-apy of patients with metastatic colon cancer increased levelsof both YES1 and YAP transcripts in liver metastases, thusproviding one explanation for the higher aggressiveness andmetastatic potential of relapsed drug-resistant tumors. Fur-thermore, we found that YES1 and YAP transcript levels werecorrelated with the reduced DFS and OS, supporting thepotential implication of the c-Yes/YAP signaling cascade intumor relapse. Our data demonstrate that 5FU chemother-apy in the HT29 colon cancer cell line preselects two distinct

drug-resistant clonal populations each enriched in cellsexpressing their specific set of stem cell markers and differentself-renewing potential, suggesting that in clinic, 5FU che-moresistant cancer cells may require clone-selective andadapted therapeutic strategies to be eradicated. Our datapoint to the c-Yes/YAP axis as signaling elements involvedin the acquisition and maintenance of cellular quiescencelinked to 5FU resistance in colon cancer. Taken togetherwithour clinical study, we demonstrated here that the c-Yes/YAPsignaling pathway should be considered as a potential ther-apeutic target to kill drug-resistant quiescent cancer cells.Alternatively, the identification of new compounds targetingCSCs in a quiescent state might prove to be beneficial forpatients with cancer with colon liver metastases and detect-able alterations of the c-Yes/YAP signaling axis.

Disclosure of Potential Conflicts of InterestL.M. Ellis is a consultant and advisory board member of Genentech/

Roche, Lilly/Imclone, and Amgen. No potential conflicts of interest weredisclosed by the other authors.

Authors' ContributionsConceptionanddesign:Y. Touil,W. Igoudjil, A.-F.Dessein, P. Formstecher,C. Gespach, R. Polakowska, G. HuetDevelopment of methodology: Y. Touil, W. Igoudjil, M. Corvaisier, A.-F.Dessein, J. Vandomme, D. Mont�e, L. Stechly, G. Grard, P. Dumont, F. Fan,L.M. Ellis, R. Polakowska, G. HuetAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): M. Corvaisier, A.-F. Dessein, J. Vandomme,D. Mont�e, N. Skrypek, G. Millet, E. Leteurtre, P. Dumont, S. Truant, F.-R.Pruvot, I. Van Seuningen, G. HuetAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): Y. Touil, M. Corvaisier, A.-F. Dessein,J. Vandomme, L. Stechly, N. Skrypek, C. Langlois, G. Millet, P. Dumont,S. Truant, M. Hebbar, C. Gespach, R. Polakowska, G. HuetWriting, review, and/or revision of the manuscript: W. Igoudjil, A.-F.Dessein, C. Langlois, M. Hebbar, P. Formstecher, I. Van Seuningen,C. Gespach, R. Polakowska, G. HuetAdministrative, technical, or material support (i.e., reporting ororganizing data, constructing databases): W. Igoudjil, A.-F. Dessein,P. Dumont, S. Truant, I. Van Seuningen, C. Gespach, R. Polakowska, G. HuetStudy supervision: F.-R. Pruvot, C. Gespach, R. Polakowska, G. Huet

AcknowledgmentsThe authors thank Nathalie Jouy (Flow Cytometry Core Facility IFR114/

IMPRT), M.H. Gevaert and R. Siminsky (Department of Histology, Facultyof Medicine, University of Lille 2), M. Samyn and V. Dumetz (Laboratoryof Immunohistochemistry, Centre deBiologie-Pathologie, CHRU-Lille), andLaurence Wicquart ("Tumoroth�eque du Centre Regional de R�ef�erence enCanc�erologie").

Grant SupportThis work was supported by Fondation pour la recherche sur le cancer

(ARC), Ligue contre le Cancer (Comit�e du Nord), INSERM, SIRIC ONCO-Lille Grant INCa-DGOS-inserm 6041 and Institut de Recherche sur le cancerde Lille (IRCL).

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 July 5, 2013; revised October 22, 2013; accepted November 8,2013; published OnlineFirst December 9, 2013.

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Published OnlineFirst December 9, 2013.Clin Cancer Res Yasmine Touil, Wassila Igoudjil, Matthieu Corvaisier, et al. with the c-Yes/YAP AxisDeath by Entering Stemness and Quiescence Associated Colon Cancer Cells Escape 5FU Chemotherapy-Induced Cell

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