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

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