Wnt signaling in adult intestinal stem cells and cancer - an insight from transgenic mice

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1 Wnt signaling in adult intestinal stem cells and cancer - an insight from transgenic mice Vladimir Korinek Department of Cell and Developmental Biology Institute of Molecular Genetics, v.v.i., Prague, CZ

2 Hierarchical model of adult tissue - - all tissues contain three basic cell compartments stem cells ( divisions/life) selfrenewal via asymetric cell division progenitors (Transit Amplifying cells; TA cells) differentiated cells Two fundamental questions of stem cell biology How to find stem cells? How to prove that you have them? Find unique marker(s) and perform functionality tests: transplantation to recipient = reconstitution of given tissue in vivo reconstitution of given tissue in vitro lineage tracing experiment track stem cell and its progeny in mouse

3 The gastrointestinal epithelia: the most rapidly renewing tissue of the human body

4 Stem cells reside in the crypts villi: non-dividing cells life span 5 days Small intestine Colon colon lumen: non-dividing cells life span 5 days crypts: dividing cells crypts: dividing cells stem cells stem cells

5 Ki67 staining in the small intestine villi: non-dividing cells The crypts contain both dividing and non-dividing cells crypts: dividing cells

6 Stem cell in the epithelium differentiates in four main cell types Crosnier et al., 2006; Cheng & Leblond, 1974

7 Assymetric division of stem cell: one stem cell and its progeny is shown in blue

8 Signaling pathways governing the architecture of the intestinal epithelium Krausova & Korinek, 2012

9 The Wnt signaling pathway (a simplified schema) The Wnt gene family mammals: 19 members (i.e. Wnts) all metazoans contain elements of Wnt signaling genes activated by Wnt signaling: c-myc, Cyclin D1, Twist, Axin2 Wnt signaling regulates various cellular processes during development and in adult tissues aberrant activation can lead to cancer For details visit the Wnt signaling homepage: adopted from Reya and Clevers, 2006

10 b-catenin, a protein with a dual function: adhesion and signaling b-catenin binds cadherins the protein is highly conserved during evolution Adherens junctions: strong mechanical attachments between adjacent cells anti-b-catenin staining P P P P cadherin protein stability transactivation domain

11 Nuclear b-catenin can be detected in the crypts of the large (and small) intestine van de Wetering et al., 2002

12 In the mouse the Wnt-signaling deficient gut displays dramatically reduced numbers of epithelial cells Existence of gene families complicates gene knockout studies (problems with redundancy) wild type 19 Wnt genes Tcf4 KO Korinek et al Lef/Tcf genes

13 The APC gene, the main tumor suppressor gene in the intestine polyp, a small clump of cells that forms on the lining of the intestine 1-10% of polyps progress to malignancy (carcinoma) the majority of colorectal cancer is sporadic (no familial history) 10-15% of colorectal cancers occur in dominantly inherited patterns (Lynch syndrome, FAP syndrome) Polyp observed during colonoscopy Resected carcinoma inactivation of APC is the rate-limiting step in tumor initiation APC mutations are found in >40% sporadic colorectal tumors Rizk & Barker, 2012

14 Aberrant Wnt signaling in colorectal tumors APC binds b-catenin and mediates b-catenin degradation APC-deficient colorectal tumors display constitutively active Wnt signaling Situation in tumor cell (no Wnt added but the APC gene is mutated) Tumor histology (b-catenin + hematoxyline stainings) anti-b-catenin staining in tumor; detail

15 : search for intestinal stem cells = search for markers of these cells Cheng and Leblond (1974): description of CBC cells = Crypt Base Columnar cells prediction that CBC cells are the intestinal stem cells van de Wetering et al. (2002): high throughput screen for Wnt signaling target genes in the intestinal cells Barker et al. (2007): Lgr5 is the marker of CBC stem cells Lgr5=leucine-rich-repeat-containing G-protein coupled receptor 5 Transmission Electron Microscopy (bottom of the crypt) Lgr5 mrna in situ hybridization in the small intestine Paneth cell CBC cells Porter et al Barker et al. 2007

16 Labeling of Lgr5-positive cells using knock-in technique Lgr5-EGFP-IRES-CreERT knock-in mouse Barker et al., 2007 Lgr5 + cells (green fluorescence) in the small intestine CBC stem cells (Lgr5) Paneth cells (lysosyme) Snipert et al. 2010

17 Growing organoid derived from one Lgr5 + cell

18 Epithelial organoids (or miniguts ) an in vitro system to study the gut epithelium freshly isolated crypt 2 days 7 days 9 days Organoids culturing conditions: EGF noggin (BMP signaling inhibitor) R-spondin (Wnt signaling agonist)

19 Organoid transplantation assay

20 The lineage tracing experiments: Cre recombinase is used to mark irreversibly one cell and its progeny Cre is type I topoisomerase of phage P1 Deletion of floxed sequences catalyzes recombination between loxp sites (34 bps) sequence of the loxp site is not present in mammalian genome Cre recombinase fused to the estrogen receptor (=CreERT) is used Reporter mouse Rosa26 gene is active in all cell types lacz encodes b-galactosidase enzyme (=reporter gene) genome of the reporter mouse contains floxed transcription blocker lacz is produced only when the blocker is recombined out by Cre CreERT-expressing mouse intercrossing

21 Lineage tracing using CreERT-expressing and ROSA26 reporter mouse Cre recombinase fused to the estrogen receptor (CreERT) can be regulated by tamoxifen Progeny of the breeding Situation in genomic DNA of cells expressing CreERT upon addition of tamoxifen + tamoxifen

22 The CreERT/lacZ induction in the adult Lgr5-EGFP-IRES-CreERT mice by tamoxifen Situation in genomic DNA of Lgr5 + cells + tamoxifen Days after tamoxifen injection Barker et al. 2007

23 Our aim: to find genes important for physiology of healthy and diseased gut tissue 2007: Chromatin Immunoprecipitation (ChIP) with DNA microarray analysis (ChIP-on-chips) of chromatin isolated from colorectal cancer cells displaying aberrant Wnt signaling APC-deficient cells: Colo320, DLD1, SW480 Cells harboring oncogenic mutations in b-catenin: LS174T Antibodies: anti-tcf; anti-b-catenin Promoter regions (bound by TCF4) of 18 genes were precipitated in all (four) tested cell lines Two genes Troy and Nkd1 were selected for further study Troy mrna in situ hybridization tumor (APC-Min mouse) crypts

24 Generation of Troy reporter mouse using BAC transgenesis Cre expression/activity = Troy expression + Tamoxifen Situation in genomic DNA of Troy + cells TROY-CreERT x Rosa26 reporter mouse Troy-CreERT2 transgene expression phenocopies the expression pattern of endogenous Troy

25 Long-term labeling of cells (originally) expressing Troy LacZ staining in the small intestine of Troy-CreERT x Rosa26 reporter mice (indicated days after tamoxifen injection) COLON 1 day 60 days 1 day 3 days 60 days Conclusions of the study Troy is Wnt signaling target gene Troy is a marker of CBC stem cells Troy interacts with Lgr5 Troy inhibits (negatively modulates) Wnt signaling The additional aim: Analysis of the Nkd1 gene

26 All details can be found in the recently published article:

27 The Crew Bobeš Fafílek Nikol Baloghová Kateřina Gajdůšková Adéla Hlavatá/Monika Horázná Dušan Hrčkulák Jitka Stančíková Michaela Krausová Vít Kříž Eva Šloncová Jiří Švec Lucie Tůmová Martina Vojtěchová Collaborators Zbynek Kozmik & his lab different mouse strains and ingredients Radek Sedlacek, Inken Beck (Transgenic Unit) transgenic mice Antonio Pombinho & Petr Bartunek HTS David Stanek & his lab microscopy Hynek Strnad & Jan Paces & Michal Kolar (Vlcek Lab) Chips and Bioinformatics

28 To produce conditional knockouts you need two mouse strains (1) Mouse expressing Cre in a specific tissue (the mouse is generated by transgenesis) (2) Mouse harboring floxed alleles of gene of interest (gene targeting in ES cells; floxed is mostly smaller but essential part of the gene) Conditional (= tissue-specific) deletion of gene of interest

29 Testing Cre-expressing mice (question: in which tissue is Cre produced?) Reporter mouse (Rosa26 gene/locus is active in all cell types) Cre-expressing mouse X Situation in cells expressing Cre Progeny of the reporter and Cre mouse + X-gal Example: Cre is driven by a promoter active in tracheal endoderm of the lung lacz = b-galactosidase

30 Inactivation of the floxed-apc gene in Lgr5(Troy)-positive CBC stem cells without Tamoxifen + Tamoxifen APC is deleted in CBC stem cells Non-functional APC gene/protein in Lgr5 + cells

31 Tumor formation in Lgr5-CreERT mouse harboring a conditional allele of the APC gene

32 b-catenin-positive tumors in the large and small intestine 14 days after tamoxifen injection (floxed-apc x Lgr5-CreERT2 mice) 0.5 mg of tamoxifen per animal 2 mg of tamoxifen per animal small intestine small intestine colon

33 The Yin and Yang of the Wnt pathway in the intestine Nuclear b-catenin can be detected in the crypts of the healthy large (and small) intestine polyp: aberrant Wnt signaling Adherens junctions van de Wetering et al., 2002 Reminder: nucleus of the cell stimulated with Wnt the crypt: physiological Wnt signaling

34 APC-Min mice a mouse model of the intestinal cancer produced using chemical mutagenesis of laboratory mice in the lab (Moser et al., 1990) APC-Min mice are APC +/- (Su et al., 1992) Min=Multiple intestinal neoplasia Reminder: wild-type APC (2842 AA) truncated APC protein (produced in APC-Min mice)