transcription factor prox1 suppresses notch pathway ...molecular cell biology transcription factor...

Molecular Cell Biology

Transcription Factor PROX1 Suppresses NotchPathway Activation via the NucleosomeRemodeling and Deacetylase Complex inColorectal Cancer Stem–like CellsJenny H€ogstr€om1, Sarika Heino1, Pauliina Kallio1, Marianne L€ahde1,Veli-Matti Lepp€anen1, Diego Balboa2, Zolt�an Wiener1, and Kari Alitalo1

Abstract

The homeobox transcription factor PROX1 is induced byhigh Wnt/b-catenin activity in intestinal adenomas and colo-rectal cancer, where it promotes tumor progression. Here wereport that in LGR5þ colorectal cancer cells, PROX1 suppressesthe Notch pathway, which is essential for cell fate in intestinalstem cells. Pharmacologic inhibition of Notch in ex vivo 3Dorganoid cultures from transgenic mouse intestinal adenomamodels increased Prox1 expression and the number of PROX1-positive cells. Notch inhibition led to increased proliferationof the PROX1-positive colorectal cancer cells, but did not affecttheir ability to give rise to PROX1-negative secretory cells.Conversely, PROX1 deletion increased Notch target geneexpression and NOTCH1 promoter activity, indicating recip-

rocal regulation between PROX1 and the Notch pathway incolorectal cancer. PROX1 interacted with the nucleosomeremodeling and deacetylase (NuRD) complex to suppress theNotchpathway. Thus, our data suggests that PROX1andNotchsuppress each other and that PROX1-mediated suppression ofNotch mediates its stem cell function in colorectal cancer.

Significance: These findings address the role of thePROX1 homeobox factor as a downstream effector ofWnt/b-catenin singling in colorectal cancer stem cells andshow that PROX1 inhibits the Notch pathway and helps toenforce the stem cell phenotype and inhibit differentiation.Cancer Res; 78(20); 5820–32. �2018 AACR.

IntroductionActivation of the Wnt pathway is the initiating event and one

of the key determinants of further pathogenesis in the majorityof human colorectal cancers (1). Cellular levels of free b-cateninare normally strictly controlled through a multiprotein com-plex that comprises the APC protein. Activation of the Wntpathway prevents the proteasomal degradation of b-catenin,which translocates to the nucleus and binds to and activates theTcf/Lef1 transcription factors, leading to activation of targetgenes stimulating cell-cycle progression and tumorigenesis (2).The homeobox transcription factor PROX1 is directly regulatedby abnormally high levels of Wnt/b-catenin signaling activity incolorectal cancer (3). Altered levels of PROX1 have been dem-

onstrated in several cancers and PROX1 has been implicated inregulating cell fate in stem and progenitor cells (reviewed inref. 4). In the normal intestine, PROX1 is expressed in only fewsecretory cells (3, 5). PROX1 expression is induced in intestinaltumor progression, where it is associated with high-gradedysplasia and an invasive phenotype (3, 6). Importantly, asubpopulation of the PROX1-expressing adenoma and colo-rectal cancer cells displays stem cell features and deletion orsilencing of Prox1 reduces tumor size and the number of LGR5þ

stem cells (3, 5).Cross-talk between the Notch and Wnt signaling pathways has

an important function in adult intestinal homeostasis (7, 8).Genetic or chemical inhibition of the Notch pathway results inreduced number of LGR5þ intestinal stem cells and accumulationof differentiated secretory cells (9, 10). In intestinal adenomas,Notch activity is regulated downstream of Wnt signaling throughb-catenin–mediated transcriptional activation of the Notchligand Jagged1, which contributes to intestinal tumorigenesis inApcmin/þ mice (11). Consistently with these results, ectopic over-expression of the active NOTCH1 intracellular domain (NICD1)leads to an elevated number of adenomas in Apc-mutantmice (7, 12). However, despite increased numbers of tumorsinApcmin/þ;NICD1;villin-CreERT2mice, these tumorswerepredom-inantly low-grade adenomas, whereas control tumors fromApcmin/þ mice displayed features of high-grade adenomas, sug-gesting that active NOTCH1 is needed for tumor formation ratherthan tumor progression. Interestingly, aberrant NOTCH1 recruitsthe histone methyltransferase SET1 domain bifurcated 1(SETDB1) to suppress Wnt target genes, including PROX1 (12).Although the supportive role of Notch activity in stem cells is a

1Translational Cancer Biology Research Program, University of Helsinki, Helsinki,Finland. 2Molecular Neurology Program and Biomedicum Stem Cell Center,University of Helsinki, Helsinki, Finland.

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

Z. Wiener and K. Alitalo are the senior coauthors of this article.

Current address for Z. Wiener: Department of Genetics, Cell- and Immunobiol-ogy, Semmelweis University, Budapest H-1089, Hungary.

Corresponding Author: Kari Alitalo, Translational Cancer Biology ResearchProgram, Biomedicum Helsinki, University of Helsinki, PO Box 63 (Haartmanin-katu 8), Helsinki FI-00014, Finland. Phone: 3589-1912-5511; Fax: 3589-1912-5510;E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-18-0451

�2018 American Association for Cancer Research.

CancerResearch

Cancer Res; 78(20) October 15, 20185820

on January 18, 2021. © 2018 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst August 28, 2018; DOI: 10.1158/0008-5472.CAN-18-0451

http://crossmark.crossref.org/dialog/?doi=10.1158/0008-5472.CAN-18-0451&domain=pdf&date_stamp=2018-9-29

http://cancerres.aacrjournals.org/

widely accepted concept, these data raised the possibility thatNotch activity is not required in PROX1-expressing colorectalcancer stem-like cells. Here, we set out to study the relationshipbetween PROX1 and the Notch pathway in colorectal cancer.

Materials and MethodsIn vivo experiments

The National Animal Experiment Board at the Provincial StateOffice of Southern Finland approved all experiments performedwith mice (ESAVI/6306/04.10.07/2016 and ESAVI/7945/04.10.07/2017). All mice were maintained in the C57Bl/6Jbackground.

The Apcflox/flox (13), Prox1flox/flox (14) and Lgr5-EGFP-IRES-CreERT2 (15) mice were crossed to generate intestinal stem cell–specific deletion of the Apc and/or Prox1 gene. To delete Apc andinduce tumorigenesis, 2 mg tamoxifen (Sigma #T5648) dissolvedin 100 mL corn oil (Sigma #8001-30-7)was given by oral gavage atthe age of 8–10 weeks. To chemically inhibit the Notch pathway,mice were given intraperitoneal injections of the g-secretaseinhibitor dibenzazepine (Tocris #4489; 20 mmol/kg/day) dilutedin 100 mL 0.5% (Hydroxylpropyl)methylcellulose (Sigma#H7509)-0.1% Tween20 (Acros organics #BP337-500) or vehicleonfive consecutive days, starting at day 5or day 21 after tamoxifenadministration. The in vivo experiment was repeated twice. Foreach experiment, 6–10 mice were used.

To analyze the effect of Notch inhibition on Prox1 lineagetracing in intestinal adenomas, dibenzazepine (20 mmol/kg/day)was administered intraperitoneally on four consecutive days to16-week-old Apcmin/þ;Rosa26-LSL-TdTomato (Jackson Laboratories,Stock 007914); Prox1-CreERT2 (16) mice. Prox1 lineage tracingwas activated by a single dose of 2mg tamoxifen after the last doseof dibenzazepine and the mice were terminated after 24 or 72hours. The in vivo experiment was repeated three times. For eachexperiment, 10–12 mice were used.

To assess cell proliferation,mice were given one intraperitonealinjection of 1 mg/mL 5-ethynyl-20-deoxyuridine (EdU; ThermoFisher Scientific #A10044) dissolved in 100 mL 0.9% NaCl andterminated 4 hours later. For analysis of EdU incorporation, EdUþ

cells of 50–60 tumors from both groups were quantified andnormalized to the total number of tumor cells.

Intestinal organoid culturesThe Ethical Committee of Helsinki University Central Hospital

approved all the experiments involving patient samples. Weobtained written informed consent from the patients and thestudies were conducted in accordance with the Declaration ofHelsinki. Tissue biopsies from patients were processed accordingto previously published method (17). Patient I harbored aKRASG12D mutation, but neither of the patients had clinicallyrelevant BRAF or PI3KCA mutations (6). Intestinal crypts fromApcflox/flox;villin-CreERT2 (18), Apcflox/flox; Lgr5-EGFP-IRES-CreERT2,Apcmin/þ;Rosa26LSL-TdTomato;Prox1-CreERT2andApcmin/þ; Prox1flox/flox;villin-CreERT2 mice were isolated and cultured as describedpreviously (5, 19). To activate gene deletion, cultures were treatedwith 300 nmol/L 4-hydroxytamoxifen (4-OH-Tam) for 24 hours.Organoidswith endogenously activeb-catenin/TCFpathwaywerethen selected and cultured in growth factor–deficient medium.When indicated, organoids were cultured in the presence of 10mmol/L of DAPT (Tocris #2634) or dibenzazepine (Tocris #4489)forfivedays, starting onday3 for an early timeperiodor day12 for

a later time period after Apc deletion. For determination of thereplating efficiency, the organoids were extensively trypsinized toobtain clusters of 4–5 cells, which were counted and embeddedintoMatrigel (Corning #35623) in equal numbers. Thenumber oforganoids/well was counted under microscope 3–4 days afterreplating. To analyze changes in gene expression after Apc dele-tion, organoids were lysed at different time points after additionof 4-OH-Tam. Changes in gene expression were compared withnormal organoids (d0).

Cell cultureThe SW1222, SW620, and LS174T colorectal cancer cell lines

were cultured in DMEM (Lonza #BE12-707F) þ 10 % FBS (Bio-west, S181B-500), 2 mmol/L L-glutamine (Corning #25-005-Cl),100 U/mL penicillin and streptomycin (Lonza #DE-17-602E).HEK293FT cells (Thermo Fisher Scientific #R70007) were cul-tured in high-glucose DMEM (Gibco #11960-044) þ 10% FBS,2 mmol/L L-glutamine, and 100 U/mL penicillin and streptomy-cin. Cells weremaintained at 37�C in a humidified incubator with5% CO2 and passaged every 2–4 days. The SW620 cell line wasobtained from ATCC (CCL-227). The SW1222 and LS174T celllines were a kind gift from Dr. Meenhard Herlyn, Wistar Institute(Philadelphia, PA; 2010) and Dr. Olli Kallioniemi, Institute forMolecular Medicine Finland (Helsinki, Finland; 2015), respec-tively. The SW620 and LS174T cells were originally authenticatedby ATCC. All the cells were routinely authenticated by morpho-logic inspection and expression of specific markers, and tested forMycoplasma by DAPI staining. The HEK293FT cells were usedwithin approximately 20 passages after thawing original stocks.For experimental procedures, colorectal cancer cell lineswere usedbetween 5 and 15 passages after thawing. When indicated, cellswere cultured in the presence of 1 mmol/L of entinostat (LCLaboratories #E-3866) or vorinostat (LC Laboratories #V-8477). For analysis of "megacolony" formation (20), 1,000SW1222 cells were embedded in Matrigel and analyzed at day10. Spheroidswere imagedwith EVOSFL inverted epifluorescencemicroscope (Thermo Fisher Scientific) using 10� or 20� LD PhAir objectives.

Flow cytometryOrganoids were centrifuged at 1,500 rpm for 5minutes and the

Matrigel was removed. To obtain a single-cell suspension, orga-noids were incubated in Hank’s Balanced Salt Solution (HBSS;Gibco #14175-053) containing 1 mg/mL collagenase type 1(Worthington #LS004196), 1 mg/mL collagenase H (Roche#11074032001), 4 mg/mL dispase II (Sigma #04942078001),and 1,000 U/mL benzonase (ChemCruz #sc-202391) for 30minutes at 37�C with gentle shaking, followed by 10-minuteincubation with trypsin. After fixation with 2% PFA, the cells wereblocked with mouse Fc block (1 mg/106 cells, BD Biosciences#553141) andpermeabilizedwith 1%BSAþ 0.1%TritonX-100 inHBSS. Cells were then incubated with goat anti-hPROX1 (R&DSystems #AF2727) for 20 minutes, followed by 20-minute incu-bationwithAlexa647donkey anti-goat secondary antibody.HBSSwashes were performed in between each step. The samples weremeasuredwith aGuava EasyCyte instrument (Millipore). Theflowcytometric analysis was repeated twice, using biological replicates.

Single-cell RNA sequencing and data analysisApcflox/flox;villin-CreERT2 organoids were treated with vehicle or

dibenzazepine for 5 days, starting on day 3 after Apc deletion.

Prox1-NuRD Regulation of Notch in Colorectal Cancer

www.aacrjournals.org Cancer Res; 78(20) October 15, 2018 5821




Organoids were then dissociated to obtain a single-cell suspen-sion. Cells in 0.04%BSA-PBS were analyzed using the ChromiumSingle Cell 30RNA-sequencing system (10x Genomics) with theReagent Kit v2 according to the manufacturer's instructions.Briefly, the cells were loaded into Chromium Single Cell Chipv2 (10x Genomics) and gel beads in emulsion (GEM) generationwas performed aiming at 3,000 cell captures per sample. Subse-quent cDNA purification, amplification (12 cycles), and libraryconstruction (sample index PCR 14 cycles) was performed asinstructed. Sample libraries were sequenced on the IlluminaNovaSeq 6000 system using S1 flow cell (Illumina) with follow-ing read lengths: 26 bp (Read 1), 8 bp (i7 Index), 0 bp (i5 Index),and 91 bp (Read 2), resulting in 115,754 and 113,705mean readsper cell for the sample vehicle and dibenzazepine, respectively.The Cell Ranger v 2.1.1 mkfastq and count pipelines (10x Geno-mics)were used todemultiplex and convert Chromiumsingle-cell30 RNA-sequencing barcodes and read data to FASTQ files and togenerate aligned reads and gene-cell matrices. Reads were alignedtomouse reference genomemm10.We used the Seurat R packagefor quality control, filtering, and analysis of the data (21). Cellswere filtered on the basis of UMI counts and percentage ofmitochondrial genes. Cells with more than 5% of mitochondrialgenes were filtered out. The expression matrix was further filteredby removing genes with expression in less than 10 cells and cellswith less than 800 expressed genes. The minimum expressionthresholdwas set at 1. Thefinal dataset consistedof 968 cells in theVehicle sample and 1,240 cells in the dibenzazepine sample. Tobe able to compare the two samples, we performed canonicalcorrelation analysis (CCA) to identify shared correlation struc-tures and aligned the dimensions using dynamic time warping.After this, we performed clustering using tSNE and set the reso-lution at 0.5. The single-cell RNA-sequencing data can be accessedfrom the Gene Expression Omnibus under accession numberGSE118055.

PlasmidsThe shHDAC1 constructs (TRCN0000004817, TRCN-

0000004818), shHDAC2 (TRCN0000196590, TRCN0000-197086), shMTA1 (TRCN0000013361, TRCN0000013362), andshScramble (SH002) were acquired from the TRC library. TheNOTCH1 promoter-based luciferase reporter was described previ-ously (22). BirA-hPROX1 fusion protein was cloned into a FUWlentiviral vector. Myc-BirA (23) was amplified using primersGAAGCTTGGGCTGCAGGTCGACTCTAGAGCCACCATGGAACA-AAAACTCATCTCAG and AGGGCTGTGCTGTCATGGTCAGGCAT-AGATCCTGAGCCCTTCTCT. hPROX1was amplified using primersATGCCTGACCATGACAGCACAG and TTATCGATAAGCTTG-ATATCGAATTCGGCGCGCCCTACTCATGAAGCAGCTCTTG. Gel-purified fragments were then assembled to FUW-Xbal-Ascl-CIP.Transfection and transduction were performed as describedpreviously (5).

BirA-mediated proximity labelingSW1222 cells were transducedwith FUW-BirA-PROX1 or FUW-

BirA-NLS-Cherry lentivirus, plated at 1� 107 cells per sample andincubated with 50 mmol/L biotin (Sigma Aldrich #B4501). After24 hours, the cells were washed three times with PBS, stored at�80�C and later lysed on ice for 10 minutes in PLCLB buffer(50 mmol/L HEPES, 150 mmol/L NaCl, 10% glycerol, 1% TritonX-100, 1.5 mmol/L MgCl2, 1 mmol/L EGTA, 10 mmol/LNa4P2O7, 100 mmol/L NaF), supplemented with 1.0 mmol/L

PMSF and 10 mL/mL protease inhibitor cocktail (Sigma-Aldrich#P8340), 0.1% SDS, and 80 U/mL Benzonase (ChemCruz#sc-202391), followed by three cycles of sonication and centri-fugation twice at 16,000� g at 4�C to remove insoluble material.Cleared lysates were loaded into Bio-Spin chromatographycolumns (Bio-Rad Laboratories #732-6008) loaded with Strep-Tactin sepharose (400 mL 50% slurry; IBA Lifesciences #2-1201-010) and washed with ice-cold PLCLB lysis buffer and ice-coldPLCLB buffer without Triton X-100 and protease inhibitors.Bound proteins were eluted twice with 300 mL of freshly prepared0.5 mmol/L biotin in PLCLB buffer without Triton X-100and protease inhibitors. Liquid chromatography–tandem massspectrometry analysis was performed as described previously(24). Two biological replicates were used.

Luciferase assaySW1222 cells transduced with lentivirus were trypsinized and

100,000 cells were plated into wells of a 24-well plate. Cells weretransfected with 1 mg of NOTCH1 promoter–based luciferasereporter (N1PR-luc) using Fugene6 transfection agent accordingto manufacturer's instructions (Promega #E2692) and lysed 48hours later. Luciferase measurement was done using the dualfirefly luciferase assay kit and preformed according to manufac-turer's instruction (Promega #E1910). All experiments were donein quadruplicate and repeated 2–3 times. Luciferase value wasnormalized to protein concentration.

Gene editing with the CRISPR/Cas9 systemFour different guide RNAs (gRNAs) targeting PROX1 start codon

region were designed using http://crispr.mit.edu/ (gPROX1.1:GCTCAAGAATCCCGGGACCCTGG, gPROX1.2: ATCTTCAAAAG-CTCGTCAGCTGG, gPROX1.3: CCTGAGAGCAAAGCGCGCCCG-GG, gPROX1.4: CGGGTTGAGAATATAATTCGGGG). gRNA-PCRtranscriptional units were assembled as described previously (25)and tested by transfection to HEK293 cells together with WTSpCas9 expressing plasmid CAG-Cas9-T2A-EGFP-ires-puro(Addgene plasmid #78311). Deletion generation was assessed byPCR of the targeted region (PROX1_Fw: GTGCCATAAATCCCA-GAGCCTATG, PROX1_Rv: ACTTTCTCGGGGACTCACAGAC).gRNAs pairs (1þ3 and 1þ4) generating deletions most efficientlywere cloned into a modified version of LentiCRISPR v2 backbone(Addgene plasmid #52961). SW1222 cells were transduced withlentivirus containing the sgPROX1-1 (gRNApair 1þ3), sgPROX1-2(gRNApair 1þ4), or sgCTRL constructs and selected using 5 mg/mLpuromycin (Merck #540222-100). The cells were then used forfurther experiments. PROX1 deletion was confirmed by Westernblot analysis.

Microarray experiments and gene set enrichment analysisThe quality of RNA was determined with Bioanalyzer (Agilent

Technologies) and further analyzed on genome-wide IlluminaMouse WG-6 v2 Expression BeadChips (Illumina). Illumina'sGenomeStudio software was used for initial data analysis andquality control and the detailed data analysis was performed withthe Chipster software (www.chipster.csc.fi). The data were nor-malized with quantile normalization. When comparing theexpression profiles of TdTomatoþ and TdTomato� organoids,the datasets derived from hybridizations at different time pointswere normalized separately with quantile and gene average nor-malizations and the data were then unified. For pathway enrich-ment analysis, the Enrichr software (http://amp.pharm.mssm.

H€ogstr€om et al.

Cancer Res; 78(20) October 15, 2018 Cancer Research5822



http://crispr.mit.edu/

www.chipster.csc.fi

http://amp.pharm.mssm.edu/Enrichr/


edu/Enrichr/) was used. The gene expression dataset was trans-ferred to the Gene Set Enrichment Analysis software (http://www.broadinstitute.org/gsea; refs. 26, 27) and the analysis was carriedout with default parameters except that the ¹exclude smaller sets"was set to 25 and gene permutation was applied. We used amodified list of the kegg.v3.1.symbols gene set (http://www.broadinstitute.org/gsea/msigdb) with the addition intestinalstem cell-specific genes (28), genes activated during Apcmin/þ

tumorigenesis and suppressed byNOTCH1 activation, and genesupregulated in NOTCH1-activated tumors (12). FDR q < 0.1 wasregarded significant. Themicroarray data can be accessed from theGene Expression Omnibus under the accession numberGSE117981.

Statistical analysisData are presented as meanþ SEM unless otherwise indicated.

Statistical comparison of two groups was done by two-tailedunpaired t test using the GraphPad Prism 6.0 software.P < 0.05 was considered statistically significant and the signifi-cance is marked by �, P < 0.05; ��, P < 0.01; and ��, P < 0.005.

Other methods are detailed in the Supplementary Materialsand Methods.

ResultsNotch activity is essential for intestinal tumor developmentduring a transient phase after Apc deletion

NOTCH1 was previously reported to suppress PROX1 expres-sion in intestinal adenoma cells (12). To study this process inadenoma stem cells, we established organoids derived fromApcflox/flox;Lgr5-EGFP-IRES-CreERT2 (LApc) or Apcflox/flox;villin-CreERT2 (VApc) mice. For induction of tumorigenesis, 4-OH-Tamwas added to theorganoid cultures, leading toApcdeletiononly inthe LGR5þ intestinal stem cells (LApcD/D) or in all intestinalepithelial cells (VApcD/D). Tomodel the effect of Notch inhibitionafter Apc deletion, we inhibited Notch by adding the g-secretaseinhibitor (GSI) dibenzazepine or DAPT to the cultures three dayslater (Fig. 1A). Notch inhibition decreased the number of viableorganoids and RNAs encoding the Notch target genes hairy/enhancer of split (Hes1), Notch regulated ankyrin repeat protein(Nrarp) and Olfactomedin 4 (Olfm4), and the intestinal stem cellmarkers Leucine-rich repeat-containing G-protein–coupledreceptor 5 (Lgr5) and Achaete-Scute family BHLH transcriptionfactor 2 (Ascl2) in both Apc-deleted and control organoids(Supplementary Fig. S1A and S1B). Lgr5-EGFPþ intestinal stemcell numberwas alsodecreased, indicating thatNotch is critical forLGR5þ stem cell activity early after Apc deletion (Fig. 1B). Fur-thermore,Notch inhibition increased the expressionofProx1RNAand the number of PROX1þ cells (Fig. 1B; Supplementary Fig. S1Aand S1B). Although the overall number of Lgr5-EGFPþ stem cellsdecreased, only a minor decrease of the Lgr5-EGFPþ PROX1þ cellpopulation was observed (Fig. 1B). Furthermore, single-cellRNA analysis of dibenzazepine or vehicle-treated Apcflox/flox;villin-CreERT2 organoids showed that the Prox1-expressing clusterwas enriched for intestinal stem cell markers Lgr5 and Tnfrsf19(15, 29) after Notch inhibition (Fig. 1C). These data suggestedthat the PROX1þ stem-like cells had a selective growth advantagewhen Notch was inhibited.

Kinetic analysis of 4-OH-Tam–treated Apcflox/flox;villin-CreERT2

organoids revealed that increased expression of Prox1 and theWnttarget genes Axin2, Sox9, c-Myc correlated with decreased expres-

sion of Notch targets Olfm4 and Hes1 (Fig. 2A–D). However,Notch inhibition on day 12 after Apc deletion no longer affectedthe proportion of viable organoids, their colony formation effi-ciency, or the viability of patient-derived colorectal cancer orga-noids (Fig. 2E and F), suggesting that active Notch signaling wasno longer required tomaintain Apc-mutant intestinal epithelium.In agreement with this, the Lgr5-EGFPþ cells in the controlintestine ofApcflox/flox;Lgr5-EGFP-IRES-CreERT2micewere uniform-ly positive for NICD1 (Fig. 2G), whereas only 36% of the Lgr5-EGFPþ cells were positive for NICD1 21 days after Apc deletion(Fig. 2H). A majority of the Lgr5-EGFPþ stem cells were PROX1þ,and PROX1þ cells were negative for NICD1 (Fig. 2I and J),supporting the notion that PROX1þ cells have a selective growthadvantage after Apc deletion. Collectively, these results suggestthat the Notch pathway is important for intestinal stem cellactivity during a transient phase after Apc deletion, but is inacti-vated in later appearing PROX1-expressing stem-like cells.

Chemical Notch inhibition provides the PROX1þ adenomacells a growth advantage

To assess whether the PROX1þ adenoma cells retain their stemcell activity even when Notch activity is inhibited, we inducedtumorigenesis by tamoxifen treatment of Apcflox/flox; Lgr5-EGFP-IRES-CreERT2 mice and applied the g-secretase inhibitor dibenza-zepine 5 days later for 5 days. As reported previously, dibenza-zepine treatment converted intestinal epithelial cells intoMucin2þ (MUC2) goblet cells and reduced the number ofLgr5-EGFPþ stem cells in the normal intestine (7), leading todeath of themicewithin 8 days after the start of the dibenzazepinetreatment. Although the number of Lgr5-EGFPþ adenoma stemcells was decreased at day 10 after Apc deletion when the Notchpathway was inhibited, the majority of them were PROX1þ

(Fig. 3A and B). In contrast, Notch inhibition starting 21 daysafterApc deletion, when themajority of the Lgr5-EGFPþ cells werePROX1þ, did not significantly decrease the number of Lgr5-EGFPþ stem cells (Fig. 3C). Furthermore, the MUC2þ goblet cellsoccurred selectively in the PROX1- tumor cell population(Fig. 3D–F; Supplementary Fig. S2A). Dibenzazepine treatmentalso promoted increased proliferation of PROX1þ tumor cells anddecreased proliferation of PROX1� cells on days 10 and 26 afterApc deletion (Fig. 3G–I; Supplementary Fig. S2B), suggesting thatNotch inhibition provides the PROX1þ cells an additional growthadvantage.

To further analyze the PROX1þ stem-like cells, we inducedlineage tracing in Apcmin/þ;Rosa26-LSL-TdTomato;Prox1-CreERT2 miceand treated the mice with dibenzazepine or vehicle for 4 days.TdTomato fluorescence 3 days after the last dose of dibenzazepineindicated that some PROX1þ cells had differentiated to PROX1�/TdTomatoþ Paneth cells (lysozymeþ) and goblet cells (MUC2þ;Supplementary Fig. S2C and S2D). Furthermore, Notch inhibi-tion increased the number of proliferating PROX1þ tumor cellsand the number of proliferating TdTomato-labeled PROX1�

progeny in more advanced tumors of the Apcmin/þ mice (Fig.3J–L), suggesting that continued growth of the tumors originatesfrom the PROX1þ tumor cells.

Induction of NOTCH1 reduces tumor stem cell activityAs PROX1þ adenoma cells retained their stem cell activity after

Notch inhibition, we asked whether NOTCH1 overexpressionaffects the stem cell properties of PROX1þ cells in mousetumor organoid cultures and human colorectal cancer cells. We





http://amp.pharm.mssm.edu/Enrichr/

http://www.broadinstitute.org/gsea

http://www.broadinstitute.org/gsea

http://www.broadinstitute.org/gsea/msigdb

http://www.broadinstitute.org/gsea/msigdb


B

PR

OX

1

DBZ

20

40

60

80

PR

OX

1+ C

ells

(%)

DBZ

20

40

60

80

Lgr5

-EG

FP+

Cel

ls (%

)

C

Vehicle

Vehicle

Vehicle DBZ

Prox1

−20

0

20

Tnfrsf19

−20

0

20

tSN

E_2 Lgr5

−20

0

20

Lgr5-EGFP

tSNE_1

−1 0 1 2

Scaled expression

100 101 10 2 10 3 10 4 10 5100

101

102

103

104

105

100

101

102

103

104

105

100 101 102 103 104 105

Vehicle DBZ

*

*

A

d0

4-OH-Tam

d3

10 µmol/L GSI

d8

RT-qPCR

Flow cytomerty

Analysis ofviable

organoids

PROX1+

EGFP+

PROX1+/EGFP+

Crypt isolation

Apcflox/flox;Lgr5-EGFP-IRES-CreERT2

Dissociated cells

Single cellRNA-seq

Apcflox/flox;villin-CreERT2

Figure 1.

Notch activity is essential for themaintenance of intestinal stem cells during a transient phase afterApc deletion.A, Schematic illustration of the experimental design.B, Quantification of Lgr5-EGFPþ and PROX1þ cells based on flow cytometry of Lgr5-EGFP fluorescence and PROX1 immunostaining in LApcD/D d8 organoids.C,RNA expression analysis of Prox1, Lgr5, and Tnfrsf19-expressing single cells inApcflox/flox; villin-CreERT2 day 8 organoids. Feature heatmap and t-SNE plot was usedto visualize clustering of RNA-sequenced single cells. Organoids were treated for 5 days with the g-secretase inhibitor dibenzazepine (DBZ), starting onday three after the addition of 4-OH-Tam. Student unpaired t test, n ¼ 3þ3; � , P < 0.05.






transduced Apc-mutant organoids derived from Apcmin/þ;Rosa26-LSL-TdTomato; Prox1-CreERT2 mice with NICD1-EGFP or con-trol lentivirus and then subjected them to short-term geneticlabeling by applying 4-OH-Tam. Sixteen hours after the additionof 4-OH-Tam, only cells positive for PROX1 displayed redTdTomato fluorescence (Fig. 4A and B), confirming the specificityof the labeling. The NICD1-overexpressing cells formed lesscolonies, which had fewer TdTomatoþ cells than control lentivi-rus–transfected cells (Fig. 4C and D), reflecting Prox1 suppressionin these cells. NICD1 overexpression in the PROX1þ SW620 and

SW1222 human colorectal cancer cell lines that are enriched forstem-like cells (20, 30) indicated that the NICD1-overexpressingcells suppressed PROX1 RNA and protein (Fig. 4E–G), supportingprevious findings (12). Furthermore, when the control andNICD1-transducedSW1222cellswere grownas three-dimensionalcultures inMatrigel, theNICD1-overexpressing cellswereunable toform "megacolonies" (Fig. 4H), indicating that they lack stem cellproperties (31). These results suggest that NOTCH1 activationsuppresses PROX1 and thus stem cell properties of PROX1þ

colorectal cancer cells.

Lgr5

-EG

FP N

ICD

1 D

AP

I

LApc∆/∆ d21LApc

LApc∆/∆ d21

36±11%

G H

Hes

1 R

NA

2

4

6

A

10

20

30

RN

A

Axin2Sox9c-Myc

B

0.6

0.4

0.2

0.8

1.0

1.2

Via

ble

orga

noid

s

Num

ber o

f new

col

onie

s

C

20

40

60

80

100 VehicleDBZ

Via

ble

orga

noid

s (%

)

D E F

0.6

0.4

0.2

0.8

1.0

1.2 VehicleDAPT

Lgr5

-EG

FPN

ICD

1D

AP

I

Lgr5

-EG

FP N

ICD

1 D

AP

ILg

r5-E

GFP

NIC

D1

PR

OX

1

LApc∆/∆ d21I J

Pro

x1 R

NA

50

100

150

0 2 5 8 11 14

Olfm

4 R

NA

5

10

15

20

0 2 5 8 11 14Day 0 2 5 8 11 14

0 2 5 8 11 14

Day Pt. I Pt. II

Figure 2.

Prox1 induction correlates withdecreased Notch target geneexpression after Apc deletion. A–D,Kinetic analysis of RNA encoding Prox1(A), the Wnt target genes Axin2, Sox9,and c-Myc (B), and Notch-targetsOlfm4(C), and Hes1 (D) after Apc deletion inthe Apcflox/flox; villin-CreERT2 organoids.E, The ratio of viable organoids, andnumber of new colonies afterreplating of DAPT-treated Apcflox/flox;Lgr5-EGFP-IRES-CreERT2 (LApcD/D)organoids. The treatment was started 12days after Apc deletion for fivedays. F, The percentage of viableorganoids in dibenzazepine (DBZ)-treated patient-derived colorectalcancer organoids. G and H,Immunofluorescence staining of Lgr5-EGFP and NICD1 in Apcflox/flox; Lgr5-EGFP-IRES-CreERT2 intestine before (G)and 21 days (H) after Apc deletion. Notethat whereas all EGFPþ cells are NICD1þ

in the control crypt, only some Lgr5-EGFPþ cells are NICD1þ in theadenomas. I and J, Lgr5-EGFP, NICD1,and PROX1 immunofluorescence inApcflox/flox; Lgr5-EGFP-IRES-CreERT2

intestines 21 days after tamoxifenadministration. Note that PROX1þ Lgr5-EGFPþ cells are negative for NICD1.Arrowheads, NICD1þ cells.Student unpaired t test, n ¼ 3þ3.Scale bars, 20 mm.






PROX1 regulates the Notch pathway in colorectal cancerAs increased Prox1 expression correlated with decreased Olfm4

and Hes1 expression, we next assessed whether PROX1 regulatesthe Notch pathway. We first analyzed the gene expression signa-

ture of FACS-isolated TdTomatoþ/PROX1þ cells by microarrayanalysis. As expected (5), we found that Prox1-expressing cellsdisplayed an enrichment of the intestinal stem cell gene signature(Supplementary Fig. S3A; ref. 28), which had similarity to gene

F

A VehicleDBZ

5

10

15

MU

C2+

Cel

ls d

10 (

%)

C

EdU

+ C

ells

d26

(%)

UdE

otamoTdT

1XO

RP

Apcmin/+

D

10

20

30

40

20

40

60

80

100

Rat

io o

f TdT

om+ /

PRO

X1−

10

20

30

40*

*

*EdU

+ C

ells

(%)

E

B

G H

** **** **

**

**

**

MU

C2

PR

OX

1 D

AP

I

UdE

1X

OR

PI

PA

D

2.5

5.0

7.5

10

MU

C2+

Cel

ls d

26 (

%)

*

*10

20

30

40E

dU+

Cel

ls d

10 (%

)I

K LJ

LApc∆/∆ d10Vehicle DBZLg

r5-E

GFP

PR

OX

1 D

AP

I

20

40

60

Lgr5

-EG

FP+

Cel

ls d

10 (%

)

PROX1+Total

20

40

60

80

100

***

*

Lgr5

-EG

FP+

Cel

ls d

26 (%

)

PROX1−

PROX1+Total

PROX1−

PROX1+Total

PROX1−

PROX1+Total

PROX1−

PROX1+Total

PROX1−

PROX1+

Total

PROX1−

PROX1+Total

PROX1−

EdU+

EdU−

Figure 3.

Chemical Notch inhibition provides the PROX1þ adenoma cells a growth advantage. A–C, Lgr5-EGFP and PROX1 immunostaining (A), and quantification of PROX1þ

and PROX1� Lgr5-EGFPþ cells (B and C) in Apc-deleted Apcflox/flox; Lgr5-EGFP-IRES-CreERT2 intestines treated with the g-secretase inhibitor dibenzazepine(DBZ). Arrowheads, Lgr5-EGFPþ cells.D–F,Goblet cell (MUC2) and PROX1 immunostaining (D), and quantification of PROX1þ and PROX1�MUC2þ tumor cells (E andF) in the same intestine. Arrowheads, MUC2þ cells. G–I, EdU and PROX1 immunostaining (G), and quantification of PROX1þ and PROX1� EdUþ tumor cells(H and I) in the same intestine. Arrowheads, PROX1þEdUþ cells. Dibenzazepine treatment was started on day 5 (A, B, D, E, G, and H) or day 21 (C, F, and I) afterthe addition of tamoxifen. J–L, EdU, TdTomato, and PROX1 immunostaining (J), and quantification (K) of PROX1 negative EdUþ and EdU� cells derivedfrom the PROX1þ cells (TdTomatoþ) and total percentage of EdUþ cells (L) in tumors ofApcmin/þ;Rosa26LSL-tdTomato; Prox1-CreERT2mice treatedwith dibenzazepine.Analysis was performed three days after the last injection of dibenzazepine. Arrowheads, TdTomatoþ/EdUþ/PROX1� cells. Mice were given four (J–L)or five (A–I) intraperitoneal injections of dibenzazepine (20 mmol/kg/day). EdU (1 mg/kg) was administered 4 hours prior to termination. Student unpairedt test, n ¼ 10–14; � , P < 0.05; �� , P < 0.01. Scale bars, 20 mm.






sets upregulated by Apc mutation and suppressed by NICD1overexpression in intestinal adenomas (Supplementary Fig.S3B; ref. 12). Accordingly, the Prox1 cell transcriptome showeda negative enrichment of genes induced byNICD1overexpression(Supplementary Fig. S3C), indicating that Notch signaling issuppressed in the Prox1-expressing cells.

We next induced Prox1 and Apc deletion by tamoxifen treat-ment of Apcflox/flox;Prox1flox/flox;Lgr5-EGFP-IRES-CreERT2 (LApcPD/D)mice to analyze PROX1 regulation of the Notch pathway inintestinal adenoma stem cells. Interestingly, Prox1 deletionincreased the ratio of OLFM4þ cells in the Lgr5-EGFPþ cellpopulation, suggesting Notch activation in these cells (Fig. 5A

A

CTRL-GFP NICD1-GFP

D

E NICD1-GFP CTRL-GFP

GFP

DA

PI

PR

OX

1 D

AP

I

0.2

0.4

0.6

0.8

GFP

+ C

olon

ies

***

1.0

NICD1-GFP CTRL-GFP SW1222 SW620

0.2

0.4

0.6

0.8

1

1.2

PR

OX

1 R

NA

SW12

22

SW62

0

** *

SW12

22

SW62

0

20

40

60

80

100

GFP

+ /P

RO

X1−

Cel

ls (%

)

CTRL-GFPNICD1-GFP

B C

0.2

0.4

0.6

0.8

1.0

TdTo

m+ /

GFP

+ C

olon

ies

***

CTRL-GFPNICD1-GFP

** **

PROX1

tdTo

mat

o

PR

OX

1 td

Tom

ato

F G H

Figure 4.

Induction of NOTCH1 reduces tumor stem cell activity. A and B, Immunofluorescence image of an organoid (A) and flow cytometry (B) of PROX1 staining andTdTomato signals in single cells derived from Apcmin/þ; Rosa26LSL-tdTomato; Prox1-CreERT2 organoids 16 hours after addition of 4-OH-Tam. C, Comparison ofthe relative numbers of single-cell–derived GFP control versus NICD1-GFP colonies among Apcmin/þ; Rosa26LSL-tdTomato; Prox1-CreERT2 organoids. D, Comparison ofthe relative number of GFP control and NICD1-GFP colonies among the tdTomatoþ colonies. E–G, NICD1-GFP and PROX1 immunofluorescence staining (E),PROX1 RNA (F), and the ratio of GFPþ/PROX1� cells (G) in SW1222 and SW620 cells four days after lentiviral transduction of GFP control or NICD1-GFP. Note thatthe NICD1þ cells are PROX1� (arrowheads). H, Images of colonies formed by SW1222 cells transduced with NICD1-GFP and GFP control lentiviruses. Notethat NICD1-transduced cells fail to form colonieswithmultiple lumens. Student unpaired t testwas used. � ,P <0.05; �� ,P <0.01; �� ,P <0.005. Scale bars, 20mm(E) or100 mm (H).






and B). Also, Prox1 deletion in organoids derived from Apcmin/þ;Prox1flox/flox;villin-CreERT2 mice resulted in increased expression ofthe Notch targets Olfm4 and Hey2 (Fig. 5C). Furthermore, Prox1deletion decreased the expression of Pleiotrophin (Ptn), Pyruvatedehydrogenase kinase 4 (Pdk4), and Distal-less homebox 3(Dlx3), genes that are suppressed by NICD1 overexpression, andincreased the expression of Tripartite motif-containing protein 31(Trim31) and Carbonic anhydrase 13 (Car13), transcripts thatare induced by NICD1 in intestinal adenomas (SupplementaryFig. S3D; ref. 12).

To explore PROX1 regulation of NOTCH in human colorectalcancer, we employed CRISPR/Cas9 to delete PROX1 exon 2(Supplementary Fig. S3E and S3F). SW1222 cells were lentivirallytransduced with Cas9 plus two different PROX1 gRNAs andselected with puromycin. PROX1 deletion was first confirmed byWestern blotting (Supplementary Fig. S3G), and the cells werethen transfected with a NOTCH1 promoter–driven luciferasereporter. When analyzed 48 hours later, the PROX1-deleted cellsshowed a 2-fold higher luciferase signal than the control cells(Fig. 5D). Furthermore, PROX1 deletion increased expression ofthe Notch targetsOLFM4 andHEY2 (Fig. 5E). PROX1 overexpres-

sion in the NOTCH1-active LS174T cells (8) also decreased theexpression ofOLFM4 andHEY2 (Fig. 5F). These data indicate thatPROX1 suppresses the Notch pathway in mouse adenomas andhuman colorectal cancer cells.

PROX1 recruits the NuRD complex to suppress the Notchpathway

To investigate the mechanism of how PROX1 suppresses theNotch pathway, we performed BirA-mediated proximity labelingto identify PROX1-interacting proteins in SW1222 cells (Supple-mentary Fig. S4A and S4B). Remarkably, we found that PROX1interacts with several proteins that have chromatin remodelingfunction. In particular, several components of the NuRD core-pressor complex were among the top 10 hits (SupplementaryTable S1). By coimmunoprecipitation, we determined that at leastHDAC1 and MTA1, key components of the complex, coprecipi-tated with PROX1 from lysates of SW1222 and SW620 cells(Fig. 6A). The prior report that the NuRD complex suppressesNotch pathway in Schwann cells (32), raised the possibility thatPROX1 suppresses theNotchpathway in colorectal cancer cells viathe NuRD complex. To assess this, we treated SW1222 cells with

A

C

***

**

**

sgCTRLsgPROX1-1sgPROX1-2

D E

*****

0.5

1

1.5

2

2.5

3

N1P

R L

ucife

rase

OLFM4

HEY2

0.5

1

1.5

2

2.5R

NA

**

***

**

F

LApc

∆/∆

LApc

P∆/∆

Lgr5-EGFP OLFM4 PROX1 Merge

Rat

io o

f Lgr

5-E

GFP

+ ce

lls

PROX1+OLFM4−PROX1−OLFM4+PROX1+OLFM4+PROX1−OLFM4−

LApc

P∆/∆

LApc

∆/∆

***20

40

60

80

100

***

***

B

Prox1

Olfm4

Hey2

1

2

3

RN

A

Prox1∆/∆Ctrl4

RN

A

CTRLPROX1

***

PROX1

OLFM4

0.5

1.0

1.5

2.030

40

50

HEY2

****

Figure 5.

Prox1 suppresses the Notch1 signaling pathway. A and B, Representative image of Lgr5-GFP fluorescence and OLFM4 and PROX1 immunostaining (A), andquantification of the ratio of PROX1þOLFM4�, PROX1�OLFM4þ, PROX1þOLFM4þ, and PROX1�OLFM4� Lgr5�EGFPþ cells (B) in adenomas derived fromApcflox/flox;Lgr5-EGFP-IRES-CreERT2 (LApcD/D) and Apcflox/flox; Prox1flox/flox; Lgr5-EGFP-IRES-CreERT2 (LApcPD/D) mice 21 days after addition of Tam. C, RNAexpression of Notch target genes in Apcmin/þ (Ctrl) and Apcmin/þ; Prox1flox/flox; villin-CreERT2 (Prox1D/D) organoids three days after addition of 4-OH-Tam. D andE, NOTCH1 promoter–driven luciferase assay (D) and RNA expression of Notch target genes (E) in SW1222 cells transduced with sgCTRL or sgPROX1lentiviruses. F, RNA expression of PROX1 and NOTCH1 target genes in LS174T cells transduced with CTRL- or PROX1-overexpressing lentivirus. Data arepresented as fold change, mean þ SD (C–F), n ¼ 4þ4; Student unpaired t test, � , P < 0.05; �� , P < 0.01; �� , P < 0.005. Scale bars, 20 mm.






CA

1

2

35

10

15

OLF

M4

shSCR

shHDAC2-1

shHDAC2-2

2

4

6

8

10

shSCR

shHDAC1

shHDAC2

shMTA

1

5

10

15

N1P

R L

ucife

rase

**

shMTA

1

-3.63

to -3

.54

-1.58

to -1

.47

-0.97

to -0

.74

0.01

0.02

0.03

0.04

0.05

% o

f Inp

ut

IgGPROX1

0.005

0.010

0.015

0.020 IgGHDAC1

-3.63

to -3

.54

-1.58

to -1

.47

-0.97

to -0

.74

B D***

***

***

**

**

* **

*

PR

OX

1

E

**

**

2

4

6

8

10

12

shSCR

shMTA1-1

shMTA1-2

*

F G H

0.05

0.10

0.15 IgGMTA1

-3.63

to -3

.54

-1.58

to -1

.47

-0.97

to -0

.74

** ***

I

PROX1 NOTCH1

-1.5 and -0.9 kb

OFF

NuRD complex

Wnt pathwayβ-catenin/TCF4

J

IgG InputHDAC1

IgG InputMTA1

PR

OX

1SW1222

SW1222

SW620

SW620

***

***

shSCR

shHDAC1-1

shHDAC1-2

**

PROX1+

CTRL

PROX1

shHDAC1

shHDAC2

1

2

3

OLF

M4

PROX1

CHD3/4

GATAD2a/bMTA1-3

RBBP4/7

HDAC2

MBD

2/3

HDAC1

PROX1

Figure 6.

Implication of the PROX1-NuRD corepressor complex in suppression of the NOTCH pathway. A, Coimmunoprecipitation of HDAC1 and MTA1 with PROX1 fromlysates of SW1222 and SW620 cells. B–E, NOTCH1 promoter luciferase assay (B) and expression of OLFM4 RNA in SW1222 cells transduced with shSCR,shHDAC1 (C), shHDAC2 (D), or shMTA1 (E) lentivirus. F, Expression of OLFM4 RNA in LS174T cells expressing CTRL or PROX1 lentivirus and transfected with shSCR,shHDAC1, shHDAC2, or shMTA1. G–I, Chromatin immunoprecipitation of PROX1, HDAC1, and MTA1 in SW620 cells, followed by RT-qPCR of three regionsof theNOTCH1 promoter. J, Schematic presentation of the PROX1-NuRD complex–mediated suppression of the NOTCH1. Data are presented as fold change, meanþSD (B–F) or percentage of input (G–I), n ¼ 4þ4 (B–F), 2þ2 (G–I); Student unpaired t test, � , P < 0.05; �� , P < 0.01; �� , P < 0.005.






two HDAC inhibitors for 72 hours to inhibit HDAC1 andHDAC2. This increased the expression of the Notch target genesOLFM4 andHEY2 (Supplementary Fig. S4C). Lentiviral silencingof HDAC1, HDAC2, or MTA1 increased the NOTCH1 promoter-luciferase reporter expression and the expression of OLFM4(Fig. 6B–E; Supplementary Fig. S4D–S4F), indicating that theNuRD complex suppresses Notch signaling in colorectal cancer.Furthermore, HDAC1, HDAC2, or MTA1 silencing in PROX1-overexpressing LS174T cells blocked the PROX1-mediated sup-pression of OLFM4 (Fig. 6F), suggesting that PROX1 acts via theNuRD complex to regulate the Notch signaling pathway incolorectal cancer. Chromatin immunoprecipitation using anti-bodies against PROX1, HDAC1, and MTA1, followed by qPCR ofthree regions of the NOTCH1 promoter, revealed two potentialPROX1-NuRD-binding sites at �1.5 kb and �0.8 kb, whereasonly MTA1 bound weakly to the NOTCH1 promoter at �3.5 kb(Fig. 6G–I; Supplementary Fig. S4G). These data indicate thatPROX1 suppresses the Notch pathway by recruiting the NuRDcomplex to the NOTCH1 promoter (Fig. 6J).

DiscussionIn this study, we provide evidence that the PROX1 transcription

factor regulates colorectal cancer stem-like cells via a bidirectionalinteraction with the Notch pathway. We show that Notch inhi-bition in Apcwild-type or mutant organoids decreases expressionof intestinal stem cell markers while increasing PROX1þ cells.Chemical Notch inhibition provided PROX1þ cells a furthergrowth advantage in the emerging tumors. Interestingly, Prox1deletion increased expression of Notch target genes andNOTCH1promoter activity. Mechanistic analysis of this finding indicatedthat PROX1 recruits theNuRD complex to theNOTCH1promoterto suppress the Notch pathway in colorectal cancer.

Intestinal crypt cells are plastic and compensatory mechanismshave been reported to produce fast-cycling crypt base columnarstem cells following injury (33–35). Similarly, colorectal tumorsare composed of a flexible hierarchy of stem-like cells, progeni-tors, and more differentiated cells (reviewed in ref. 36). In thenormal intestine, PROX1 is expressed in enteroendocrine cellsand some Paneth cells (3, 5). Furthermore, the PROX1þ enter-oendocrine cells can function as injury-inducible stem cells (35).In colorectal cancer, PROX1 is directly regulated by the b-catenin/TCF4 pathway. Notch inhibition at an early phase after Apcdeletion resulted in increased production of PROX1þ adenomacells, but Notch inhibition had only a minor effect later duringtumor development due to Notch downregulation in PROX1þ

cells. Analysis of single-cell RNA sequencingof control andNotch-inhibited organoids showed that Prox1þ cell clusters express someintestinal stem cell markers, suggesting that the PROX1þ cellsform a subpopulation of intestinal stem-like cells. Furthermore,the LGR5þPROX1� cells were decreased by Notch inhibition,indicating that a subpopulation of the stem-like cells is Notchdependent. In line with this, Schmidt and colleagues demonstrat-ed that high NOTCH activity marks a distinct colon cancersubpopulation with low levels of WNT and MAPK activity(37). Furthermore, NOTCH1 was shown to be coexpressed withthe intestinal stemcellmarker BMI1,whereas the LGR5þ stem-likecells expressed Wnt markers (38). Considering that PROX1 is aWnt target, these studies suggest that PROX1 marks a high Wntand Notch negative stem-like cell population.

NOTCH1 is known to suppress PROX1expression in lymphaticendothelial cells (39), thyroid cancer cells (40), myoblasts (41),in the developing Drosophila intestine (42), and in mouse intes-tinal adenomas (12). Although the functionof theNotchpathwayin theWT intestinal stem cells iswell established (8), how it affectscolorectal cancer progression is unclear. NOTCH1 has beenshown to be essential in the initiation of colorectal cancer (7,11, 12).However, deletion of theNotch effectorRbpjdidnot affecttumorigenesis in Apc-mutant intestine (43). Furthermore, aber-rant NOTCH1 expression decreases during tumor progressionand metastasis, suggesting that the Notch pathway functionsmainly in the early phase of colorectal cancer progression (44).On the other hand, PROX1 was shown to be important incolorectal cancer progression and metastatic outgrowth of Wnthigh colon cancer cells (3, 45), rather than for adenoma initiation.In our models, PROX1 and the Notch pathway suppressed eachother, indicating that high PROX1 expression and active Notchsignaling are mutually exclusive. PROX1 has previously beenshown to suppress the Notch pathway to allow differentiationof myoblasts (41) and neurons (46). We show evidence thatPROX1 suppresses Notch pathway in colorectal cancer via recruit-ing the NuRD complex to the NOTCH1 promoter. The NuRDcomplex consists of seven subunits that function together inchromatin remodeling and histone deacetylation (reviewedin ref. 47). Previous studies have shown that the NuRD complexmaintains the silencing of tumor suppressor genes in colorectalcancer (48). Furthermore, deficiencyofMbd2, a component of thecomplex, reduces tumor burden in Apcmin/þ mice and attenuatesWnt signaling (49, 50). These studies indicate that the NuRDcomplex performs an important function in colorectal cancer.Importantly, aberrant NOTCH1 expression suppressed the stemcell activity of the PROX1-positive stem-like cells. These observa-tions raise the possibility that PROX1 suppresses the Notchpathway to promote malignant growth.

In conclusion, we demonstrate that PROX1 interacts with theNuRD complex to suppress Notch signaling in colorectal cancerstem-like cells. Furthermore, we show that ectopic expression ofactive NOTCH1 suppresses PROX1 and the stem cell activityof PROX1-positive cells, suggesting a reciprocal interaction ofPROX1 and Notch pathways in regulation of colorectal cancerstem-like cells. On the basis of our results, we propose a modelwhere the LGR5þ stem cells require active Notch signaling earlyafter Apc deletion. However, some of these cells start to expressPROX1, which then suppresses the Notch pathway. ThesePROX1þ LGR5þ cells proliferate and support tumor growthindependently of the Notch pathway. Our studies thus give newinsight into the complex regulation of stem-like cells and identifyPROX1 as amarker of a Notch-independent stem cell population.

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

Authors' ContributionsConception and design: J. H€ogstr€om, Z. Wiener, K. AlitaloDevelopment of methodology: J. H€ogstr€om, S. Heino, D. BalboaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J. H€ogstr€om, S. Heino, P. Kallio, M. L€ahde,V.-M. Lepp€anen, Z. WienerAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J. H€ogstr€om, S. Heino, K. AlitaloWriting, review, and/or revision of the manuscript: J. H€ogstr€om, Z. Wiener,K. Alitalo






Administrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): J. H€ogstr€om, K. AlitaloStudy supervision: K. Alitalo

AcknowledgmentsThis work was funded by the Academy of Finland (grants nos. 292816 and

273817, Centre of Excellence Program 2014–2019: Translational Cancer Biol-ogy grant no. 307366), Cancer Foundation in Finland, Sigrid Juselius Foun-dation, Hospital District of Helsinki and Uusimaa Research Grants, HelsinkiInstitute of Life Sciences (HiLIFE), Biocenter Finland (all to K. Alitalo), SwedishCultural Foundation in Finland, Magnus Ehrnrooth Foundation, BiomedicumHelsinki Foundation, IdaMontini Foundation, K Albin Johansson Foundation,Orion Research Foundation, Medicinska underst€odsf€oreningen Liv&H€alsa andMaud KuistilaMemorial Foundation (all to J. H€ogstr€om), J�anos Bolyai Grant bythe Hungarian Academy of Sciences (to Z. Wiener). We thank Dr MarkkuVarjosalo and the proteomics unit at the Institute of Biotechnology, Universityof Helsinki, for performing mass spectromerty, Dr. Seppo Kaijalainen forcloning the BirA-PROX1 construct, Dr. Timo Otonkoski from the MolecularNeurology Program and Biomedicum Stem Cell Center, University of Helsinki

for supporting the cloning of the sgCTRL and sgPROX1 constructs, Dr. Guil-lermoOliver from the FeinbergCardiovascular Research Institute,NorthwesternUniversity, Chicago for the Prox1flox/flox and Prox1-CreERT2 mice; Dr. TohruKiyono from the National Cancer Center, Japan for the NOTCH1 promoter-based luciferase reporter, Dr. Riikka Kivel€a for discussions, Drs. Pekka Katajisto,Saara Ollila, and Cecilia Sahlgren for reading and commenting of the manu-script, and David He, Kirsi Mattinen, Tanja Laakkonen, and Tapio Tainola fortechnical assistance. We also thank the Single Cell Analytics core facility at theInstitute forMolecularMedicine Finland, the Biomedicum functional genomicsunit and Biomedicum imaging unit, University of Helsinki for providingmaterials and services.

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 February 12, 2018; revised June 25, 2018; accepted August 20, 2018;published first August 28, 2018.

References1. Fearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis.

Cell 1990;61:759–67.2. Gregorieff A, Clevers H. Wnt signaling in the intestinal epithelium: from

endoderm to cancer. Genes Dev 2005;19:877–90.3. Petrova TV, Nyk€anen A, Norrm�en C, Ivanov KI, Andersson LC, Haglund C,

et al. Transcription factor PROX1 induces colon cancer progression bypromoting the transition from benign to highly dysplastic phenotype.Cancer Cell 2008;13:407–19.

4. Elsir T, Smits A, Lindstr€om MS, Nist�er M. Transcription factor PROX1: Itsrole in development and cancer. Cancer Metastasis Rev 2012;31:793–805.

5. Wiener Z, H€ogstr€om J, Hyv€onen V, Band AM, Kallio P, Holopainen T, et al.Prox1 promotes expansion of the colorectal cancer stem cell population tofuel tumor growth and ischemia resistance. Cell Rep 2014;8:1943–56.

6. Elyada E, Pribluda A, Goldstein RE,Morgenstern Y, Brachya G, CojocaruG,et al. CKIalpha ablation highlights a critical role for p53 in invasivenesscontrol. Nature 2011;470:409–13.

7. Fre S, Pallavi SK, Huyghe M, Lae M, Janssen KP, Robine S, et al. Notch andwnt signals cooperatively control cell proliferation and tumorigenesis inthe intestine. Proc Natl Acad Sci U S A 2009;106:6309–14.

8. Tian H, Biehs B, Chiu C, Siebel CW, Wu Y, Costa M, et al. Opposingactivities of notch and wnt signaling regulate intestinal stem cells and guthomeostasis. Cell Rep 2015;11:33–42.

9. Pellegrinet L, Rodilla V, Liu Z, Chen S, Koch U, Espinosa L, et al. Dll1-anddll4-mediated notch signaling are required for homeostasis of intestinalstem cells. Gastroenterology 2011;140:1230,1240.

10. van Es JH, van Gijn ME, Riccio O, van den Born M, Vooijs M, Begthel H,et al. Notch/g-secretase inhibition turns proliferative cells in intestinalcrypts and adenomas into goblet cells. Nature 2005;435:959–63.

11. Rodilla V, Villanueva A, Obrador-Hevia A, Robert-Moreno A, Fernandez-Majada V, Grilli A, et al. Jagged1 is the pathological link between wnt andnotch pathways in colorectal cancer. Proc Natl Acad Sci U S A 2009;106:6315–20.

12. Kim HA, Koo BK, Cho JH, Kim YY, Seong J, Chang HJ, et al. Notch1counteracts WNT/beta-catenin signaling through chromatin modificationin colorectal cancer. J Clin Invest 2012;122:3248–59.

13. Shibata H, Toyama K, Shioya H, Ito M, Hirota M, Hasegawa S, et al. Rapidcolorectal adenoma formation initiated by conditional targeting of the apcgene. Science 1997;278:120–3.

14. Harvey NL, Srinivasan RS, Dillard ME, Johnson NC, Witte MH, Boyd K,et al. Lymphatic vascular defects promoted by Prox1 haploinsufficiencycause adult-onset obesity. Nat Genet 2005;37:1072–81.

15. Barker N, Van Es JH, Kuipers J, Kujala P, Van Den Born M, Cozijnsen M,et al. Identificationof stemcells in small intestine and colonbymarker geneLgr5. Nature 2007;449:1003–7.

16. Srinivasan RS, Dillard ME, Lagutin OV, Lin FJ, Tsai S, Tsai MJ, et al. Lineagetracing demonstrates the venous origin of the mammalian lymphaticvasculature. Genes Dev 2007;21:2422–32.

17. Sato T, Stange DE, Ferrante M, Vries RG, Van Es JH, Van den Brink S, et al.Long-term expansion of epithelial organoids from human colon, adeno-ma, adenocarcinoma, and barrett's epithelium. Gastroenterology 2011;141:1762–72.

18. El Marjou F, Janssen K, HungJunn Chang B, Li M, Hindie V, Chan L, et al.Tissuespecific and inducible Cremediated recombination in the gut epi-thelium. Genesis 2004;39:186–93.

19. Sato T, Vries RG, Snippert HJ, Van De Wetering M, Barker N, Stange DE,et al. Single Lgr5 stem cells build crypt villus structures in vitro without amesenchymal niche. Nature 2009;459:262–5.

20. Ashley N, Yeung T, Bodmer WF. Stem cell differentiation and lumenformation in colorectal cancer cell lines and primary tumours. Cancer Res2013;73:5798–809.

21. Butler A, Hoffman P, Smibert P, Papalexi E, Satija R. Integrating single-celltranscriptomic data across different conditions, technologies, and species.Nat Biotechnol 2018;36:411.

22. Yugawa T, Handa K, Narisawa-Saito M, Ohno S, Fujita M, Kiyono T.Regulation of Notch1 gene expression by p53 in epithelial cells. Mol CellBiol 2007;27:3732–42.

23. Roux KJ, Him DI, Raifa M, Burke B. A promiscuous biotin ligase fusionprotein identifies proximal and interacting proteins in mammalian cells.J Cell Biol 2012;196:801–10.

24. Yadav L, Tamane F, G€o€os H, van Drogen A, Katainen R, Aebersold R, et al.Systematic analysis of human protein phosphatase interactions anddynamics. Cell Syst 2017;4:430–44.

25. Balboa D, Weltner J, Eurola S, Trokovic R, Wartiovaara K, Otonkoski T.Conditionally stabilized dCas9 activator for controlling gene expression inhuman cell reprogramming and differentiation. Stem Cell Rep 2015;5:448–59.

26. Mootha VK, Lindgren CM, Eriksson K, Subramanian A, Sihag S, Lehar J,et al. PGC-1a-responsive genes involved in oxidative phosphorylationare coordinately downregulated in human diabetes. Nat Genet 2003;34:267–73.

27. SubramanianA, TamayoP,Mootha VK,Mukherjee S, Ebert BL,GilletteMA,et al. Gene set enrichment analysis: a knowledge-based approach forinterpreting genome-wide expression profiles. Proc Natl Acad Sci U S A2005;102:15545–50.

28. van der Flier LG, vanGijnME, Hatzis P, Kujala P, Haegebarth A, Stange DE,et al. Transcription factor achaete scute-like 2 controls intestinal stem cellfate. Cell 2009;136:903–12.

29. Fafilek B, KrausovaM, VojtechovaM, Pospichalova V, Tumova L, SloncovaE, et al. Troy, a tumor necrosis factor receptor familymember, interacts withlgr5 to inhibit wnt signaling in intestinal stem cells. Gastroenterology2013;144:381–91.

30. Wang C, Xie J, Guo J, Manning HC, Gore JC, Guo N. Evaluation of CD33and CD133 as cancer stem cell markers for colorectal cancer. Oncol Rep2012;28:1301–8.






31. Yeung TM, Gandhi SC, Wilding JL, Muschel R, Bodmer WF. Cancer stemcells from colorectal cancer-derived cell lines. Proc Natl Acad Sci U S A2010;107:3722–7.

32. Wu LM, Wang J, Conidi A, Zhao C, Wang H, Ford Z, et al. Zeb2 recruitsHDAC-NuRD to inhibit Notch and controls Schwann cell differentiationand remyelination. Nat Neurosci 2016;19:1060–72.

33. Tian H, Biehs B, Warming S, Leong KG, Rangell L, Klein OD, et al. A reservestem cell population in small intestine renders Lgr5-positive cells dispens-able. Nature 2011;478:255.

34. Tetteh PW, Basak O, Farin HF, Wiebrands K, Kretzschmar K, BegthelH, et al. Replacement of lost Lgr5-positive stem cells throughplasticity of their enterocyte-lineage daughters. Cell Stem Cell 2016;18:203–13.

35. Yan KS, Gevart O, Zheng GXY, Anchang B, Probert CS, Larkin KA, et al.Intestinal enteroendocrine lineage cells possess homeostatic and injury-inducible stem cell activity. Cell Stem Cell 2017;21:78–90.

36. Stange DE, Clevers H. Concise review: the yin and yang ofintestinal (cancer) stem cells and their progenitors. Stem Cells2013;31:2287–95.

37. Schmidt EM, Lamprecht S, Blaj C, Schaaf C, Krebs S, BlumH, et al. Targetingtumor cell plasticity by combined inhibition of NOTCH and MAPKsignaling in colon cancer. J Exp Med 2018;215:1693–708.

38. Srinivasan T, Walters J, Bu P, Than EB, Tung KL, Chen KY, et al. NOTCHsignaling regulates asymmetric cell fate of fast- and slow-cycling coloncancer-initiating cells. Cancer Res 2016;76:3411–21.

39. Kang J, Yoo J, Lee S, Tang W, Aguilar B, Ramu S, et al. An exquisite cross-control mechanism among endothelial cell fate regulators directs theplasticity and heterogeneity of lymphatic endothelial cells. Blood 2010;116:140–150.

40. Choi D, Ramu S, Park E, Jung E, Yang S, JungW, et al. Aberrant activation ofNotch signaling inhibits PROX1 activity to enhance the malignant behav-ior of thyroid cancer cells. Cancer Res 2016;76:582–93.

41. Salmela I, Nguyen YH, Petrova TV, Koistinen HA, Wiener Zet al. TheTranscription factor Prox1 is essential for satellite cell differentiation andmuscle fiber-type regulation. Nat Commun 2016;7:13124.

42. Guo Z, Ohlstein B. Bidirectional Notch signaling regulates Drosophilaintestinal stem cell multipotency. Science 2015;20:350.

43. PeignonG,Durand A, CacheuxW, Ayrault O, Terris B, Laurent-Puig P, et al.Complex interplay between beta-catenin signalling and notch effectors inintestinal tumorigenesis. Gut 2011;60:166–76.

44. Veenendaal LM,KranenburgO, SmakmanN, KlompA, Borel Rinkes IH, vanDiest PJ. Differential notch andTGFb signaling in primary colorectal tumorsand their corresponding metastases. Analyt Cell Pathol 2008;30:1–11.

45. Ragusa S, Cheng J, Ivanov KI, Zangger N, Ceteci F, Bernier-Latmani J, et al.PROX1 promotes metabolic adaptation and fuels outgrowth of wnt highmetastatic colon cancer cells. Cell Rep 2014;8:1957–73.

46. Kaltezioti V, Kouroupi G, Oikonomaki M, Mantouvalou E, StergiopoulosA, Charonis A, et al. Prox1 regulates the Notch1-mediated inhibition ofneurogenesis. PLoS Biol 2010;21:8 e1000565.

47. Lai AY, Wade PA. NuRD: A multi-faceted chromatin remodeling complexin regulating cancer biology. Nat Rev Cancer 2011;11:588–59.

48. Cai Y, Geutjes EJ, de Lint K, Roepman P, Bruurs L, Yu LR, et al. The NuRDcomplex cooperates with DNTMs to maintain silencing of key colorectaltumor suppressor genes. Oncogene 2014;33:2157–68.

49. SansomOJ, Berger J, Bishop SM, Hendrich B, Bird A, Clarke AR. Deficiencyof Mbd2 suppresses intestinal tumorigenesis. Nat Genet 2003;34:145–7.

50. Phesse TJ, Parry L, Reed KR, Ewan KB,Dale TC, SansomOJ, et al. Deficiencyof Mbd2 attenuates Wnt signaling. Mol Cell Biol 2008;28:6094–103.






2018;78:5820-5832. Published OnlineFirst August 28, 2018.Cancer Res Jenny Högström, Sarika Heino, Pauliina Kallio, et al.

like Cells−olorectal Cancer Stemvia the Nucleosome Remodeling and Deacetylase Complex in CTranscription Factor PROX1 Suppresses Notch Pathway Activation

Updated version

10.1158/0008-5472.CAN-18-0451doi:

Access the most recent version of this article at:

Material

Supplementary

http://cancerres.aacrjournals.org/content/suppl/2018/08/28/0008-5472.CAN-18-0451.DC1

Access the most recent supplemental material at:

Cited articles

http://cancerres.aacrjournals.org/content/78/20/5820.full#ref-list-1

This article cites 50 articles, 16 of which you can access for free at:

Citing articles

http://cancerres.aacrjournals.org/content/78/20/5820.full#related-urls

This article has been cited by 1 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/78/20/5820To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/lookup/doi/10.1158/0008-5472.CAN-18-0451

http://cancerres.aacrjournals.org/content/suppl/2018/08/28/0008-5472.CAN-18-0451.DC1

http://cancerres.aacrjournals.org/content/78/20/5820.full#ref-list-1

http://cancerres.aacrjournals.org/content/78/20/5820.full#related-urls

http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://cancerres.aacrjournals.org/content/78/20/5820


transcription factor prox1 suppresses notch pathway ...molecular cell biology transcription factor...

Documents