Published OnlineFirst July 27, 2010; DOI: 10.1158/0008-5472.CAN-10-1616 Published OnlineFirst on July 27, 2010 as 10.1158/0008-5472.CAN-10-1616 Tumor and Stem Cell Biology Cancer Research Dvl2 Promotes Intestinal Length and Neoplasia in the ApcMin Mouse Model for Colorectal Cancer Ciara Metcalfe1, Ashraf E.K. Ibrahim1, Michael Graeb1, Marc de la Roche1, Thomas Schwarz-Romond1, Marc Fiedler1, Douglas J. Winton2, Anthony Corfield3, and Mariann Bienz1 Abstract APC mutations cause activation of Wnt/β-catenin signaling, which invariably leads to colorectal cancer. Similarly, overexpressed Dvl proteins are potent activators of β-catenin signaling. Screening a large tissue microarray of different staged colorectal tumors by immunohistochemistry, we found that Dvl2 has a strong tendency to be overexpressed in colorectal adenomas and carcinomas, in parallel to nuclear β-catenin and Axin2 (a universal transcriptional target of Wnt/β-catenin signaling). Furthermore, deletion of Dvl2 reduced Min the intestinal tumor numbers in a dose-dependent way in the Apc model for colorectal cancer. Interestingly, the small intestines of Dvl2 mutants are shortened, reflecting in part a reduction of their crypt diameter and cell size. Consistent with this, mammalian target of rapamycin (mTOR) signaling is highly active in normal intestinal crypts in which Wnt/β-catenin signaling is active, and activated mTOR signaling (as revealed by Min staining for phosphorylated 4E-BP1) serves as a diagnostic marker of Apc mutant adenomas. Inhibition Min of mTOR signaling in Apc mutant mice by RAD001 (everolimus) reduces their intestinal tumor load, sim- ilarly to Dvl2 deletion. mTOR signaling is also consistently active in human hyperplastic polyps and has a significant tendency for being active in adenomas and carcinomas. Our results implicate Dvl2 and mTOR in the progression of colorectal neoplasia and highlight their potential as therapeutic targets in colorectal cancer. Cancer Res; 70(16); OF1–10. ©2010 AACR. Introduction it binds to TCF factors to operate a transcriptional switch. Apc mutations in mice also initiate intestinal tumorigenesis Most colorectal cancers are initiated by hyperactivation of (5), and the transcriptional program activated by APC loss the Wnt/β-catenin pathway in the intestinal epithelium, typ- resembles that of the normal intestinal crypts, which com- ically by loss-of-function mutations of the APC tumor sup- prise the intestinal stem cell compartment (2). One of the pressor (1, 2). APC is a negative regulator of β-catenin; it key APC effector genes in normal crypts and tumorigenesis binds to Axin to promote the phosphorylation of β-catenin is c-myc (6). by glycogen synthase kinase 3β, thus earmarking it for pro- Loss of APC function mimics β-catenin activation by Wnt teasomal degradation (3). APC truncations such as those typ- signals in normal cells, which critically depends on Dishev- ically found in colorectal cancer lack their Axin-binding elled (Dvl; ref. 7): upon Wnt stimulation, Dvl binds to and domain and, therefore, retain only partial function (4); thus, recruits Axin to the plasma membrane by virtue of its poly- β-catenin accumulates in the cytoplasm and nucleus where merizing activity (8, 9), thus assembling signalosomes that also contain Frizzled receptor and LRP6 coreceptor and pro- moting the phosphorylation of the LRP6 cytoplasmic tail (10, Authors' Affiliations: 1Medical Research Council Laboratory of 11). The latter acts as a direct inhibitor of glycogen synthase Molecular Biology and 2Cancer Research UK Cambridge Research β β Institute, Li Ka Shing Centre, Cambridge, United Kingdom; and kinase 3 (12, 13), which allows unphosphorylated -catenin 3Clinical Sciences at South Bristol, Bristol Royal Infirmary, Marlborough to accumulate and trigger a transcriptional switch, much Street, Bristol, United Kingdom like APC loss. Notably, if Dvl is expressed at high levels, it Note: Supplementary data for this article are available at Cancer Research potently activates β-catenin, independently of Wnt stimula- Online (http://cancerres.aacrjournals.org/). tion; it recruits Axin into cytoplasmic signalosomes (8, 9) Current address for C. Metcalfe: Genentech, South San Francisco, CA and inhibits glycogen synthase kinase 3β through LRP6 94080. phosphorylation (14). Current address for T. Schwarz-Romond: EMBO, Meyerhofstr. 1, 69117 In binding to Axin, Dvl blocks the activity of the Axin-APC Heidelberg, Germany. complex in downregulating β-catenin; if overexpressed, Dvl Corresponding Author: Mariann Bienz, Medical Research Council Lab- oratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, United could thus synergize with a partially functional Axin-APC Kingdom. Phone: 44-1223-402055; Fax: 44-1223-412-142; E-mail: complex and further promote Wnt/β-catenin pathway activ- [email protected]. ity. This is the case in Drosophila, in which dishevelled is es- doi: 10.1158/0008-5472.CAN-10-1616 sential for Wnt pathway activity in hypomorphic APC mutant ©2010 American Association for Cancer Research. embryos (15). The same could be true in colorectal cancer www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2010 American Association for Cancer Research. Published OnlineFirst July 27, 2010; DOI: 10.1158/0008-5472.CAN-10-1616 Metcalfe et al. cells, which carry hypomorphic APC mutations (4), which de- Small interfering RNA knockdown, Western blot, and lete the Axin-binding domain from APC, and thus allow only real-time quantitative PCR analysis indirect association of the two proteins through β-catenin Small interfering RNA–mediated depletion of Dvl2, TOP- (16); indeed, these cancer cells could be particularly sensitive FLASH luciferase reporter assays, Western blot, and real-time to Dvl expression levels, and their β-catenin hyperactivation quantitative PCR analyses (Supplementary Figs. S1 and S3) might reflect both their Dvl signaling activity and their APC were done as described (for details of cell lines used, see Sup- loss. If so, Dvl and its signaling partners (which include sev- plementary Information; refs. 8, 24). To detect endogenous eral kinases; ref. 7) could have potential as targets for thera- Dvl2 (Supplementary Fig. S1C; Fig. 1B), a rabbit antiserum peutic intervention. was generated against human Dvl2 (amino acids 78–250), To examine the possible role of Dvl in colorectal cancer, was affinity purified, and was characterized in overexpres- we screened a large tissue microarray (TMA) of colorectal sion and small interfering RNA–mediated depletion experi- tumor samples and found that Dvl2 has a strong tendency ments (Graeb, data not shown) as described (8, 25). to become overexpressed during the tumor progression. Fur- thermore, we show that lowering the dose of Dvl2 reduces Colorectal tumor samples Min the numbers of intestinal tumors in the Apc mouse model, Tissue samples for Western blots (Supplementary Fig. S1C) indicating a tumor-promoting role of Dvl2 in the intestine. were collected from patients undergoing elective surgery for We also discovered that Dvl2−/− mice have shortened intes- colorectal resections in accordance with standard procedures tines, and we present evidence that this reflects partly fewer (26); ethical approval for this collection was granted by the intestinal crypts, and partly reduced crypt diameters, sug- United Bristol Hospital Trust Research and Development Eth- gesting that Dvl2 may promote crypt cell growth. Consistent ical Committee (SU/2005/2114 and 05/Q2006/164). For anal- with this observation, we show that crypts exhibit high levels ysis by immunohistochemistry (Supplementary Fig. S2; Figs. 1 of phosphorylated 4E-BP1 (p4E-BP1), a key read-out of acti- and 6), two TMAs were constructed from multiple (2–4) rep- vated mammalian target of rapamycin (mTOR) signaling that licate tissue cores from 64 patients undergoing colectomy re- promotes cell growth (17), consistent with earlier results (18). sections for colorectal cancer at Addenbrookes Hospital, Indeed, we find high p4E-BP1 levels to be a diagnostic mark- Cambridge, England4. Ethical approval was obtained from Min er for nascent polyps and larger intestinal tumors of Apc the Cambridgeshire Local Research Ethics Committee (04/ mutant mice, and we confirm that inhibition of this pathway Q0108/125 and 06/Q0108/307). Samples were selected on by the rapamycin-like inhibitor RAD001 reduces the tumor the basis of availability of paraffin blocks with adequate cel- numbers in this model (18). Importantly, we find that mTOR lularity. H&E-stained slides of all cases were reviewed, signaling is highly active in human hyperplastic polyps, and marked, and used to guide the sampling from morphological- also within a subset of adenomas and colorectal carcinomas, ly representative regions of the tissue blocks. Five-micrometer indicating the therapeutic potential of mTOR inhibitors in sections were obtained from paraffin-embedded blocks and colorectal cancer. were deparaffinized and rehydrated with xylene and alcohol. Antigen retrieval was performed with EDTA buffer (pH 9.0) at Materials and Methods 100°C for 20 mintues. The antibodies used were as follows: affinity-purified α-Dvl2 (1:50), α-β-catenin (1:200; BD Trans- Mouse models duction Laboratories), α-Axin2 (1:500; Abcam), and α-pS6 Animal care and procedures were done in accordance with (1:100; Cell Signaling Technology). A commercially available the standards set by the United Kingdom Home Office. α-Dvl2 antiserum was also tested on some samples