Discovery of a novel function for R-spondin1 as a suppressor of intestinal adenomas

Colorectal cancer (CRC) is the third leading diagnosed malignancy and the fourth most common cause of cancer-related mortality globally. CRC burden has been expected to increase by 60% within the next decade. Up to every third of the patients die from the disease despite the improvements in the CRC therapy. However, knowledge gained in understanding the molecular subtypes of the CRC has improved the ability to target CRC treatment strategies and to predict the treatment outcomes. Aberrant activation of the Wnt//B-catenin pathway drives intestinal tumorigenesis, often as a result of loss-of-function mutation of the tumor suppressor adenomatous polyposis coli (APC) gene. APC inactivation leads to disrupted proteasomal degradation of cytoplasmic B-catenin, which accumulates in the cytoplasm. This leads to dislocation of B-catenin into the nucleus, where it binds to transcription factors of the TCF/LEF family and initiates the transcription of target genes related to augmented cell cycle and tumorigenesis, such as Prospero homeobox protein 1 (PROX1), which has been shown to induce dysplasia and an invasive phenotype in CRC. R-spondins (RSPO1-3) are ligands that bind to leucine-rich G-protein-coupled receptors (LGR4-6) that promote Wnt signaling. Most of the RSPOs enhance the growth of intestinal stem cells (ISCs) in healthy mice. Furthermore, RSPO gene fusions were recently identified in human (h)CRC. Thus, inhibition of RSPOs was considered to suppress intestinal tumorigenesis. However, a recent report suggested that RSPO2 expression level negatively correlates with intestinal tumor cell differentiation, size, and metastasis. RSPO2 expression was also downregulated via promoter hypermethylation in hCRC, suggesting that RSPO2 would have a tumor-suppressive role in hCRC. One study suggested that LGR5 could enhance transforming growth factor B (TGFB)/SMAD signaling pathway upon RSPO1 stimulation in two tested hCRC cell lines. In general, the role of TGFB/SMAD signaling in CRC is exceptionally complex, as it can turn from tumor suppressive to pro-metastatic throughout the progression of adenomas to carcinomas. Thus, the role of RSPO in intestinal tumorigenesis remains controversial. I was interested in analyzing if exogenous Wnt-signals, especially RSPO1, could affect PROX1 expression induced by B-catenin activation in the early phase of intestinal tumorigenesis. For this analysis, we generated adeno-associated virus vectors (AAV) to induce systemic RSPO1 expression in mice. I then used Apc-deficient ApcMin/+ mice to investigate the effect of exogenous RSPO1 on intestinal adenomas, and surprisingly found that instead of enhancing intestinal tumorigenesis, RSPO1 functions as a suppressor of Wnt/-catenin signaling in Apc mutant adenoma cells and concomitantly activates the TGFB/SMAD signaling pathway, leading to apoptosis and growth inhibition of the adenoma cells. Thus, RSPO1 treatment leads to regression of intestinal adenomas in the ApcMin/+ mice, resulting in significantly increased survival of the mice. My thesis provides a novel insight into the regulation of intestinal stem cells in intestinal adenomas. Our data reveal a dual role of RSPO1 in modulating Wnt signaling, proliferation, and apoptosis in the intestinal stem cells. Furthermore, RSPO1 regulates the competition equilibrium between healthy intestinal stem cells and Apc-mutated tumor stem cells. These findings revise and expand our knowledge on the signaling pathways that regulate tumor progression and they could provide possibilities for the development of novel cancer treatment strategies.Kolorektaalisyöpä (CRC) on läntisen maailman kolmanneksi yleisin syöpä aiheuttaen neljänneksi eniten syöpäkuolemia. Tapausten odotetaan lisääntyvän peräti 60 % seuraavan vuosikymmenen aikana. Kehittyvistä hoitomuodoista huolimatta lähes kolmannes potilaista kuolee CRC:hen. Lisääntynyt tietämys CRC:n molekyylitason mekanismeista on mahdollistanut kohdennettujen hoitojen kehittämisen ja hoitotulosten tarkemman ennustamisen. Esimerkiksi kasvurajoitegeeni APC:n inaktivoiva mutaatio käynnistää poikkeavan solunsisäisen Wnt/B-catenin-signaloinnin ja solujen jakautumisen, mikä johtaa CRC:n kehityksen käynnistymiseen. LGR-reseptoreihin sitoutuvat R-spondiinit (RSPO1-3) ovat Wnt-signalointia vahvistavia ligandeja. R-spondiinien tiedetään myös kiihdyttävän terveiden hiirten suoliston kantasolujen kasvua. Lisäksi hiljattain löydettiin RSPO-geenifuusioita ihmisen CRC-soluissa, ja näin ollen RSPO-proteiinien eston on ajateltu estävän syövän kehitystä. Viimeaikaiset tutkimukset kuitenkin esittävät, että RSPO2-pitoisuus korreloisi negatiivisesti kasvaimen solujen erilaistumisasteen ja leviämistaipumuksen kanssa, ja näin ollen RSPO2 toimisi kasvun estäjänä CRC:ssä. Kahdella solulinjalla tehdyn tutkimuksen perusteella RSPO1 aktivoi TGFB/SMAD-signalointia ihmisen CRC-soluissa ja siten jarruttaa syövän kasvua. Toisaalta TGFB/SMAD-signaloinnin merkitys CRC-soluissa on monimutkainen, sillä syövän edetessä sen tiedetään muuttuvan kasvua hillitsevästä leviämistä edistäväksi. Tämän työn tarkoituksena oli analysoida solunulkoisten Wnt-ligandien merkitystä suolistokasvaimen kehityksen alkuvaiheissa. Kehitimme adenoviruksen kaltaisen vektorin, jonka avulla RSPO1-eritys käynnistyi hiiren maksasoluissa. Vektoria annettiin suolistokasvaimista kärsiville hiirille, ja sen sijaan, että kasvainten määrä ja kasvu olisivat kiihtyneet, RSPO1 yllättäen estikin Wnt-signalointia samalla aktivoiden TGFB/SMAD-signaalireittiä. Yhdessä nämä mekanismit johtivat kasvainsolujen apoptoosiin sekä kasvainten määrän ja koon pienenemiseen, pidentäen kasvaimista kärsivien hiirten elinikää merkittävästi. Väitöskirjatutkimukseni esittelee RSPO1:n kaksoisroolin Wnt-signaloinnin säätelijänä; terveessä suolessa se edistää kantasolujen kasvua ja jakautumista, kun taas kasvaimissa se estää solujen proliferaatiota ja kiihdyttää sisäsyntyistä solukuolemaa. RSPO1 siis säätelee terveen kudoksen ja kasvaimen solujen välistä kilpailuasetelmaa. Edellä mainitut havainnot laajentavat tietämystämme suolistosyövän ja terveen suolen solujen molekyylitason monimutkaisista mekanismeista ja eri signalointireittien välisistä interaktioista, ja siten tarjoavat mahdollisuuksia kehittää uusia hoitostrategioita eri syöpäsairauksiin

Transcription factor PROX1 suppresses Notch pathway activation via the nucleosome remodeling and deacetylase complex in colorectal cancer stem-like cells

The homeobox transcription factor PROX1 is induced by high Wnt/ß-catenin activity in intestinal adenomas and colorectal cancer (CRC), where it promotes tumor progression. Here we report that in LGR5+ CRC cells, PROX1 suppresses the Notch pathway, which is essential for cell fate in intestinal stem cells. Pharmacological inhibition of Notch in ex vivo 3D organoid cultures from transgenic mouse intestinal adenoma models increased Prox1 expression and the number of PROX1-positive cells. Notch inhibition led to increased proliferation of the PROX1-positive CRC cells but did not affect their ability to give rise to PROX1-negative secretory cells. Conversely, PROX1 deletion increased Notch target gene expression and NOTCH1 promoter activity, indicating reciprocal regulation between PROX1 and the Notch pathway in CRC. PROX1 interacted with the nucleosome remodeling and deacetylase (NuRD) complex to suppress the Notch pathway. Thus, our data suggests that PROX1 and Notch suppress each other and that PROX1-mediated suppression of Notch mediates its stem cell function in CRC

Expression of R-Spondin 1 in Apc(Min/+) Mice Suppresses Growth of Intestinal Adenomas by Altering Wnt and Transforming Growth Factor Beta Signaling

BACKGROUND & AIMS: Mutations in the APC gene and other genes in the Wnt signaling pathway contribute to development of colorectal carcinomas. R-spondins (RSPOs) are secreted proteins that amplify Wnt signaling in intestinal stem cells. Alterations in RSPO genes have been identified in human colorectal tumors. We studied the effects of RSPO1 overexpression in ApcMin/thorn mutant mice. METHODS: An adeno associated viral vector encoding RSPO1-Fc fusion protein, or control vector, was injected into ApcMin/thornmice. Their intestinal crypts were isolated and cultured as organoids. which were incubated with or without RSPO1-Fc and an inhibitor of transforming growth factor beta receptor (TGFBR). Livers were collected from mice and analyzed by immunohistochemistry. Organoids and adenomas were analyzed by quantitative reverse-transcription PCR, single cell RNA sequencing, and immunohistochemistry. RESULTS: Intestines from Apcthorn/thorn mice injected with the vector encoding RSPO1-Fc had significantly deeper crypts, longer villi, with increased EdU labeling, indicating increased proliferation of epithelial cells, in comparison to mice given control vector. AAV-RSPO1-Fctransduced ApcMin/thorn mice also developed fewer and smaller intestinal tumors and had significantly longer survival times. Adenomas of ApcMin/thorn mice injected with the RSPO1-Fc vector showed a rapid increase in apoptosis and in the expression of Wnt target genes, followed by reduced expression of messenger RNAs and proteins regulated by the Wnt pathway, reduced cell proliferation, and less crypt branching than adenomas of mice given the control vector. Addition of RSPO1 reduced the number of adenoma organoids derived from ApcMin/thorn mice and suppressed expression of Wnt target genes but increased phosphorylation of SMAD2 and transcription of genes regulated by SMAD. Inhibition of TGFBR signaling in organoids stimulated with RSPO1-Fc restored organoid formation and expression of genes regulated by Wnt. The TGFBR inhibitor restored apoptosis in adenomas from ApcMin/thorn mice expressing RSPO1Fc back to the same level as in the adenomas from mice given the control vector. CONCLUSIONS: Expression of RSPO1 in ApcMin/thorn mice increases apoptosis and reduces proliferation and Wnt signaling in adenoma cells, resulting in development of fewer and smaller intestinal tumors and longer mouse survival. Addition of RSPO1 to organoids derived from adenomas inhibits their growth and promotes proliferation of intestinal stem cells that retain the APC protein; these effects are reversed by TGFB inhibitor. Strategies to increase the expression of RSPO1 might be developed for the treatment of intestinal adenomas.Peer reviewe

Nainen työelämässä : työn ja perheen yhteensovittaminen

Tämän opinnäytetyön tarkoituksena oli tutkia naisen asemaa työelämässä, naisten koulutusrakennetta ja urakehitystä sekä työn ja perheen yhteensovittamista. Lisäksi selvitettiin naisyrittäjyyttä ja äitijohtajuutta sekä tasa-arvoa työelämässä ja perheen sisällä. Suomalaiset naiset ovat tehneet suurimmaksi osaksi maataloustyötä ja kotityötä sekä työtä perheyrityksen hyväksi. Naisten työskentelyn mahdollisuudet paranivat suomalaisen yhteiskunnan teollistuessa. Naisten palkkatyön rakenteeseen vaikutti suuresti naisten osallistuminen palveluammattityöhön 1900-luvulla. Naisille ja miehille maksettiin palkkaa eri perustein. Eri sukupuolten välinen palkkaluokkien käyttö kiellettiin vuonna 1962. Naisten palkkatyön lisääntyessä ja monipuolistuessa työmarkkinat eriytyivät nais- ja miesvaltaisiin aloihin. Naisten työssäkäynti on tänä päivänä kansainvälisesti tarkasteltuna korkealla, samoin naisten työllisyysaste. Työmarkkinat ovat voimakkaasti eriytyneet sukupuolen mukaan. Suomalaiset naiset saivat äänioikeuden vuonna 1906 ensimmäisinä naisina maailmassa. Suomessa noudatetaan lakia naisten ja miesten välisestä tasa-arvosta. Suomalaisilla työmarkkinoilla parhaiten koulutettu ikäryhmä on 25-34-vuotiaat. Koulutustaso on Suomessa korkea. Naisten osuus johto- ja esimiestehtävissä on kasvanut, vaikka usein naisten urakehitys pysähtyy tietylle tasolle. Naisyrittäjyydellä on pitkät perinteet ja se on ollut kautta historian tärkeä osa suomalaista yhteiskuntaa. Naisten yrityksien osuus on noin 33 % suomalaisista yrityksistä. Suomessa työn ja perhe-elämän yhteensovittamisen keskeinen ongelma on molempien vanhempien työssäkäynti ja heidän pääsääntöisesti tekemä kokopäivätyö. Työn ja perhe-elämän yhteensovittamisen ongelmallisuus on laaja alue, jota yhdistää työelämä, sosiaali- ja tasa-arvopolitiikka sekä talouselämä. Aihe on myös noussut viimeisen vuosikymmenen kuluessa tärkeäksi työelämän kehittämisalueeksi, vaikkei kyse ole uudesta yhteiskunnallisesta ongelmasta. Myös perhevapaajärjestelmä on osa suomalaista työelämää. Isyyden tutkiminen omana käsitteenään aloitettiin 1960-luvulla. Lapsi tarvitsee molemmilta vanhemmiltaan hoitoa ja huolenpitoa sekä heillä molemmilla on yhtäläiset oikeudet tutustua lapseensa. Vanhempainvapaat ovat olleet naisten ja miesten jaettavissa melkein kahden kymmenen vuoden ajan, mutta harva isä käyttää tätä oikeuttaan. Perhemyönteinen yrityskulttuuri vähentää muun muassa työntekijöiden työn ja perheen yhdistämisen vaikeutta ja työtyytymättömyyttä. Kun nainen toimii yrityksen johtajana, se vaatii myös hänen perhe-elämältäänkin enemmän.The purpose of this dissertation was to examine women’s position in working life, women’s educational structure career development and also how women consolidated work and family. In addition, this study clarified woman entrepreneurship and leadership and also equality in working life and inside the family. Finnish women have mostly done farm work and housework and worked for a family business. The opportunities of women to work improved when the Finnish society industrialized. The structure of women’s paid work was affected largely when women entered service trade work in the 1900`s. Men and women were paid on different grounds. The use of different wage brackets was denied in 1962. When women’s paid work procreated and diversified, the working life diverged on female- and male-dominated trades. Women are working a lot nowadays in Finland and their rate of employment high if we look at it internationally. The working life has heavily diverged on the basis of gender. The Finnish women got suffrage in 1906 and they were the first women in the world who get it as women. In Finland, we conform to a law of equality between men and women. The best educated age group is 25-34 years old in the Finnish labour market. Education level is high in Finland. The proportion of women in management and superior tasks has grown while often women career development stops at a certain level. Enterprise of women has long traditions and it has been throughout history a very important part of Finnish society. Women’s companies’ share of all companies in Finland is about 33 per cent. In Finland a salient problem in consolidation of work and family is that both parents have all-day jobs. The problematic nature in consolidation of work and family is an extensive area that connects working life, social and equality policy and economic life. This subject has arisen during the last decade as a important development area in working life even though this was not the case of some new social problem. A family off system is part of Finnish working life. Examination of fatherhood as its own concept was started in the 1960`s. A child needs tending and caring from both parents and parents have identical rights to get to know theirs children. Parents-offs have been distributed to a man and a woman for almost twenty years, but not many father uses this right. Family favorable enterprise culture reduces difficulties of consolidation of work and family and negative attitudes in workplace. When the woman is an executive of the company that also demands more from her family life

Blocking Angiopoietin-2 Promotes Vascular Damage and Growth Inhibition in Mouse Tumors Treated with Small Doses of Radiation

Abnormal vasculature in tumors leads to poor tissue perfusion and cytostatic drug delivery. Although drugs inducing vascular normalization, for example, angiopoietin-2 (Ang2)-blocking antibodies, have shown promising results in preclinical tumor models, clinical studies have so far shown only little efficacy. Because Ang2 is known to play a protective role in stressed endothelial cells, we tested here whether Ang2 blocking could enhance radiation-induced tumor vascular damage. Tumor-bearing mice were treated with anti-Ang2 antibodies every 3 or 4 days starting 3 days before 3 x 2 Gy or 4 x 0.5 Gy whole-body or tumor-focused radiation. Combination treatment with anti-Ang2 and radiation improved tumor growth inhibition and extended the survival of mice with melanoma or colorectal tumors. Single-cell RNA-sequencing revealed that Ang2 blocking rescued radiation-induced decreases inT cells and cells of the monocyte/macrophage lineage. In addition, anti-Ang2 enhanced radiation-induced apoptosis in cultured endothelial cells. In vivo, combination treatment decreased tumor vasculature and increased tumor necrosis in comparison with tumors treated with monotherapies. These results suggest that a combination of Ang2-blocking antibodies with radiation increases tumor growth inhibition and extends the survival of tumor-bearing mice. Significance: These findings offer a preclinical rationale for further testing of the use of radiation in combination with Ang2-blocking antibodies to improve the overall outcome of cancer treatment.Peer reviewe

Angiopoietin-2 blocking antibodies improve tumor growth inhibition and survival in mice treated with low doses of radiation

Radiation induced tumor cell death is strongly dependent on oxygen. As abnormal tumor vasculature promotes tumor hypoxia, drugs that induce vascular normalization, such as the anti-vascular endothelial growth factor (VEGF) antibody, have been tested as radiation sensitizers in preclinical and clinical settings. The insufficient benefit obtained with anti-VEGF therapy prompted us to test if antibodies blocking the endothelial growth factor angiopoietin-2 (Ang2) could improve the effect of radiation in mouse tumor allografts and human tumor xenografts. Mouse or human tumor cells were injected subcutaneously in isogenic immunocompetent or immunodeficient (NSG) mice, respectively, and tumors were allowed to form. The mice were injected with anti-Ang2 or control antibodies every three or four days starting three days before 3x2 Gy or 4x0.5 Gy whole-body radiation, followed by analysis of tumor growth, histology, vasculature, hypoxia and necrosis. Combination treatment with anti-Ang2 and radiation improved tumor growth inhibition and extended the survival of mice with melanoma or colorectal carcinoma allografts. A similar anti-Ang2 plus radiation response was also obtained in immunodeficient mice implanted with a human colorectal carcinoma xenograft, indicating that the adaptive immune response was not essential for the effect. Histological and immunohistochemical analysis of the tumors showed that the combination treatment decreased tumor vasculature, and increased tumor hypoxia and tumor necrosis in comparison with control tumors and tumors treated with the monotherapies. Our results suggest that a combination of Ang2 blocking antibodies and radiation increases tumor growth inhibition and extends the survival of tumor-bearing mice. Significance: These findings offer a preclinical rationale for further testing of the use of Ang2 blocking antibodies in combination with radiation to improve the overall outcome of cancer treatment

Lef1 restricts ectopic crypt formation and tumor cell growth in intestinal adenomas

Somatic mutations in APC or CTNNB1 genes lead to aberrant Wnt signaling and colorectal cancer (CRC) initiation and progression via-catenin-T cell factor/lymphoid enhancer binding factor TCF/LEF transcription factors. We found that Lef1 was expressed exclusively in Apc-mutant, Wnt ligand-independent tumors, but not in ligand-dependent, serrated tumors. To analyze Lef1 function in tumor development, we conditionally deleted Lef1 in intestinal stem cells of Apc(fl/fl) mice or broadly from the entire intestinal epithelium of Apc(fl/fl) or Apc(Min/+) mice. Loss of Lef1 markedly increased tumor initiation and tumor cell proliferation, reduced the expression of several Wnt antagonists, and increased Myc proto-oncogene expression and formation of ectopic crypts in Apc-mutant adenomas. Our results uncover a previously unknown negative feedback mechanism in CRC, in which ectopic Lef1 expression suppresses intestinal tumorigenesis by restricting adenoma cell dedifferentiation to a crypt-progenitor phenotype and by reducing the formation of cancer stem cell niches.Peer reviewe

Expression of R-spondin1 in ApcMin/+ Mice Reduces Growth of Intestinal Adenomas by Altering Wnt and TGFB Signaling

Suolistosyövät ovat läntisen maailman kolmanneksi yleisimpiä syöpiä. Alati kehittyvistä hoitomuodoista huolimatta edelleenkin joka kolmas suolistosyövistä leviää muihin elimiin, ja merkittävä osa potilaista menehtyy. Yleisesti tiedetään, että syöpäsolut vaativat jakautuakseen useita erilaisia mutaatioita, joiden ansiosta ne pääsevät karkaamaan elimistön kontrollista. Suolistosyövissä tyypillinen mutaatio on solunsisäistä Wnt-signalointireittiä säätelevässä APC-geenissä. APC:n periytyvä mutaatio aiheuttaa Familial Adenomatous Polyposis (FAP)-nimisen sairauden, jossa potilaan suolistoon kehittyy jopa satoja adenoomia. Nämä etenevät hoitamattomana syöväksi lähes 100% todennäköisyydellä. Tämän tutkielman tarkoituksena oli selvittää tarkemmin suolistosyövän kantasolujen ja niitä säätelevien, Wnt-signalointia vahvistavien kasvutekijöiden, R-spondiinien (RSPO), toimintaa, erityisesti kasvainten kehityksen alkuvaiheissa. Tätä tarkoitusta varten tuotettiin AAV-vektori, jonka avulla voitiin käynnistää systeeminen R-spondiinituotanto suolistosyövän Apc-mutantissa hiirimallissa. R-spondiinien tiedetään edistävän suoliston terveiden kantasolujen toimintaa, ja niiden on ajateltu olevan merkittävässä roolissa myös suolistosyöpien kehityksessä. AAV-RSPO1-vektori aikaansaikin villityypin hiirissä odotetusti suolen kantasolujen toiminnan kiihtymisen. Kuitenkin hiiren suolistokasvaimissa vaikutus oli päinvastainen; RSPO1-käsittely hidasti ApcMin-hiirten adenoomien kasvua, kiihdytti apoptoosia spesifisti adenoomasoluissa sekä pidensi kasvaimista kärsivien hiirten elinikää verrokkeihin nähden merkittävästi. Mekanistisissa tutkimuksissa kävi ilmi, että AAV-RSPO1-käsittely aktivoi TGFB-signalointireitin, jonka tiedetään aktivoivan solunsisäistä apoptoosikoneistoa yksinomaan kasvainsoluissa, ja siten rajoittavan niiden kasvua. Tämän seurauksena suolen terveet solut saavuttivat kilpailuedun kasvainsoluihin nähden, ja syrjäyttivät kasvainsoluja, jolloin adenoomista päästiin eroon lähes kokonaan. Näiden löydösten perusteella voisikin olla mahdollista kehittää uusia hoitomuotoja suolistosyöpiin, erityisesti FAP:tä sairastaville potilaille

NOTUM from Apc-mutant cells biases clonal competition to initiate cancer

Funding Information: Acknowledgements We thank the Core Services and Advanced Technologies at the Cancer Research UK Beatson Institute (C596/A17196 and A31287), and particularly the Biological Services Unit, Histology Service and Molecular Technologies; members of the Sansom and Katajisto laboratories for discussions of the data and manuscript; and BRC Oxford for supplying patient material. O.J.S. and his laboratory members were supported by Cancer Research UK (A28223, A21139, A12481 and A17196). D.J.F. and M.C.H. were supported by the UK Medical Research Council (MR/R017247/1 and MR/J50032X/1, respectively). SpecifiCancer CRUK Grand Challenge (C7932/A29055) is funded by Cancer Research UK and the Mark Foundation for Cancer Research. P.K. and his laboratory members were supported by the Academy of Finland Centre of Excellence MetaStem (266869, 304591 and 320185), the ERC Starting Grant 677809, the Swedish Research Council 2018-03078, the Cancerfonden 190634, the Jane and Aatos Erkko Foundation and the Cancer Foundation Finland. N.P. was supported by the Finnish Cultural Foundation, the Biomedicum Helsinki Foundation, the Orion Research Foundation sr and The Paulo Foundation. P.V.F. was supported by Alzheimer’s Research UK and The Francis Crick Institute. The ARUK UCL Drug Discovery Institute receives its core funding from Alzheimer’s Research UK (520909). The Francis Crick Institute receives its core funding from Cancer Research UK (FC001002), the UK Medical Research Council (FC001002) and the Wellcome Trust (FC001002). Publisher Copyright: © 2021, The Author(s), under exclusive licence to Springer Nature Limited.The tumour suppressor APC is the most commonly mutated gene in colorectal cancer. Loss of Apc in intestinal stem cells drives the formation of adenomas in mice via increased WNT signalling(1), but reduced secretion of WNT ligands increases the ability of Apc-mutant intestinal stem cells to colonize a crypt (known as fixation)(2). Here we investigated how Apc-mutant cells gain a clonal advantage over wild-type counterparts to achieve fixation. We found that Apc-mutant cells are enriched for transcripts that encode several secreted WNT antagonists, with Notum being the most highly expressed. Conditioned medium from Apc-mutant cells suppressed the growth of wild-type organoids in a NOTUM-dependent manner. Furthermore, NOTUM-secreting Apc-mutant clones actively inhibited the proliferation of surrounding wild-type crypt cells and drove their differentiation, thereby outcompeting crypt cells from the niche. Genetic or pharmacological inhibition of NOTUM abrogated the ability of Apc-mutant cells to expand and form intestinal adenomas. We identify NOTUM as a key mediator during the early stages of mutation fixation that can be targeted to restore wild-type cell competitiveness and provide preventative strategies for people at a high risk of developing colorectal cancer.Peer reviewe