Maintenance of adenomatous polyposis coli (APC)-mutant colorectal cancer is dependent on Wnt/β-catenin signaling Alix Scholer-Dahirel1, Michael R. Schlabach, Alice Loo, Linda Bagdasarian, Ronald Meyer, Ribo Guo, Steve Woolfenden, Kristine K. Yu, Judit Markovits, Karen Killary, Dmitry Sonkin, Yung-Mae Yao, Markus Warmuth, William R. Sellers, Robert Schlegel, Frank Stegmeier, Rebecca E. Mosher, and Margaret E. McLaughlin2 Novartis Institutes for Biomedical Research, Cambridge, MA 02139 Edited by Bert Vogelstein, Johns Hopkins University, Baltimore, MD, and approved August 23, 2011 (received for review March 18, 2011) Persistent expression of certain oncogenes is required for tumor progenitors. Consequently, colorectal cancer cells undergo dif- maintenance. This phenotype is referred to as oncogene addiction ferentiation (7, 14, 15). By imposing a proliferative crypt pro- and has been clinically validated by anticancer therapies that genitor phenotype, aberrant Wnt pathway activation may allow specifically inhibit oncoproteins such as BCR-ABL, c-Kit, HER2, benign tumors (polyps) to persist for many years, providing an PDGFR, and EGFR. Identifying additional genes that are required opportunity for the acquisition of further mutations (e.g., in for tumor maintenance may lead to new targets for anticancer KRAS, SMAD-4, and P53 genes) required for the development of drugs. Although the role of aberrant Wnt pathway activation in malignant colorectal tumors (16). the initiation of colorectal cancer has been clearly established, Although the role of Wnt pathway activation in the initiation it remains unclear whether sustained Wnt pathway activation of colon tumorigenesis has been well established, it is unclear is required for colorectal tumor maintenance. To address this whether tumors that have acquired additional mutations in question, we used inducible β-catenin shRNAs to temporally con- oncogenes or tumor suppressor genes remain dependent on Wnt trol Wnt pathway activation in vivo. Here, we show that active pathway activation. Although β-catenin siRNA inhibits engraft- β Wnt/ -catenin signaling is required for maintenance of colorectal ment of colorectal cancer cells (17), a recent study reports that APC tumor xenografts harboring mutations. Reduced tumor inhibition of Wnt signaling in established colorectal xenografts β growth upon -catenin inhibition was due to cell cycle arrest and (mutant for the β-catenin gene) by inducible β-catenin shRNA β differentiation. Upon reactivation of the Wnt/ -catenin pathway had no significant effect on tumor growth (18). Human co- colorectal cancer cells resumed proliferation and reacquired a crypt lorectal tumors with mutations in β-catenin are usually less ag- progenitor phenotype. In human colonic adenocarcinomas, high gressive and smaller than those with APC mutations (19), levels of nuclear β-catenin correlated with crypt progenitor but suggesting that APC and β-catenin mutations are not functionally not differentiation markers, suggesting that the Wnt/β-catenin equivalent. Consistently, in addition to its function in the β-cat- pathway may also control colorectal tumor cell fate during the enin degradation complex, APC can also directly contribute to maintenance phase of tumors in patients. These results support the regulation of mitosis and apoptosis (20). Such β-catenin– efforts to treat human colorectal cancer by pharmacological inhi- fl bition of the Wnt/β-catenin pathway. independent APC functions may in uence the degree of depen- dency on Wnt pathway activation for colorectal tumor mainte- nance. Given the large preponderance of APC mutations in utational activation of the Wnt pathway occurs in the vast human colorectal cancer, it is crucial to determine whether Mmajority of colorectal cancers through truncating muta- sustained Wnt pathway activation is required for maintenance of APC AXIN2 tions in adenomatous polyposis coli ( )or or muta- APC-mutant colorectal tumors. β-catenin – tions in GSK3-target residues in (1 5). In normal In this study, we provide evidence that Wnt/β-catenin pathway intestinal cells, APC associates with axin, glycogen synthase ki- activation is required for maintenance of colorectal tumors β β β nase-3 (GSK-3 ), and casein kinase 1 (CK1) to form a -catenin harboring APC mutations. We show that β-catenin inhibition β destruction complex. This complex phosphorylates -catenin, in vivo strongly inhibited the growth of established APC-mutant resulting in its ubiquitylation and subsequent degradation by the colorectal tumor xenografts. Upon β-catenin inhibition, co- APC proteasome (6). In contrast, in cells harboring mutations in , lorectal cancer cells underwent cell cycle arrest and rapidly AXIN2 β-catenin β ,or , -catenin accumulates and upon its trans- started to differentiate into polarized intestinal epithelial cells, location to the nucleus, interacts with the T-cell factor/lymphoid producing mucins and displaying microvilli. Such differentiated MEDICAL SCIENCES enhancer factor (TCF/LEF) family of transcription factors to cancer cells, when relieved from β-catenin inhibition, could de- fi activate speci c Wnt target genes (7, 8). differentiate and regain their proliferative potential in vitro. In Considerable evidence from human genetic studies and mouse genetic models suggests that mutational activation of the Wnt pathway initiates the process of colon tumorigenesis. Inherited Author contributions: A.S.-D., M.R.S., A.L., Y.-M.Y., M.W., W.R.S., R.S., F.S., R.E.M., and APC mutations cause familial adenomatous polyposis, and ac- M.E.M. designed research; A.S.-D., M.R.S., A.L., L.B., R.M., R.G., S.W., K.K.Y., J.M., K.K., quired APC mutations represent the earliest genetic alteration so D.S., and M.E.M. performed research; A.S.-D., M.R.S., and D.S. contributed new reagents/ analytic tools; A.S.-D., M.R.S., A.L., L.B., R.M., R.G., J.M., D.S., and M.E.M. analyzed data; far detected in the genesis of sporadic colorectal cancer (9). Rare and A.S.-D., M.R.S., A.L., M.W., W.R.S., R.S., F.S., R.E.M., and M.E.M. wrote the paper. mutations in AXIN2 or β-catenin can also be present in small The authors declare no conflict of interest. neoplastic lesions (5, 10). In experimental mouse models, loss of This article is a PNAS Direct Submission. APC (11, 12) or expression of constitutively active β-catenin (13) fi 1Present address: Institut National de la Santé et de la Recherche Médicale, U830, Genetic is suf cient to drive polyp formation. and Biology of Cancers, Institut Curie, F-75248 Paris Cedex 05, France. Inhibition of the Wnt pathway in colorectal cancer cells 2To whom correspondence should be addressed. E-mail: margaret.mclaughlin@novartis. in vitro by overexpression of dominant-negative TCF4 or in- com. β-catenin ducible siRNA results in rapid cell cycle arrest and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. blocks a genetic program that is physiologically active in crypt 1073/pnas.1104182108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1104182108 PNAS | October 11, 2011 | vol. 108 | no. 41 | 17135–17140 Downloaded by guest on September 30, 2021 addition, we report that in human colonic adenocarcinomas high -catenin AXIN2 c-MYC A 1.5 1.5 1.5 levels of nuclear β-catenin correlated with crypt progenitor but *** *** *** *** *** *** not differentiation markers, suggesting that the Wnt/β-catenin 1.0 1.0 1.0 pathway may also control colorectal tumor cell fate during the 0.5 0.5 0.5 0.0 0.0 0.0 maintenance phase of tumors in patients. Dox: - + - + - + - + - + - + - + - + - + NTC NTC NTC sh35 sh36 Results sh35 sh36 sh35 sh36 Sustained Wnt/β-Catenin Pathway Activation Is Required for -catenin Maintenance of Colorectal Tumors Harboring APC Mutations. To B C Vehicle Dox Total analyze the role of sustained Wnt pathway activation in main- 200 ** *** tenance of colorectal tumors, we used doxycycline-inducible 150 β-catenin shRNAs to temporally control Wnt pathway activation NTC 100 in vivo. We infected LS411N and SW403 colorectal cancer cells 50 (both APC mutant) with a robust inducible single-lentiviral Mean Intensity 0 vector pLKO-Tet-On (21), containing either control nontarget- β-catenin– 150 Nuclear ing (NTC) shRNA or two distinct targeting shRNA sh35 ns ** *** sequences (shRNA-35 or shRNA-36). Polyclonal stable lines 100 were obtained after puromycin selection and inoculated s.c. in 50 fi Nu/Nu mice. To con rm that the doxycycline treatment led to Mean Intensity 0 inhibition of the Wnt/β-catenin pathway, we treated mice bearing sh36 Dox: - + - + - + 3 established tumors (100–300 mm ) with doxycycline for 3 d and NTC sh35 sh36 analyzed various molecular components of the Wnt/β-catenin pathway. Upon doxycycline treatment, β-catenin expression was D Vehicle Dox 1500 NTC sh35 sh36 dramatically reduced at the level of both mRNA (knockdown 1000 efficiency >95%, Fig. 1A) and protein in the β-catenin shRNA 500 tumors but not in the NTC shRNA tumors (Fig. 1 B and C and LS411N A B β-catenin volume Tumor 0 Fig. S1 and ). As expected, silencing of caused 10 20 30 10 20 30 10 20 30 a concomitant reduction of β-catenin target genes AXIN2 and c- Days post implant MYC at the mRNA and protein levels (Fig. 1A and Fig. S1 E–H). β We noted that -catenin inhibition had a more dramatic effect E 1500 NTC sh35 sh36 on c-MYC expression in SW403 versus LS411N cells (up to 99% 1000 c-MYC and 50% reduction of nuclear staining intensity, re- 500 spectively). Specificity of the β-catenin shRNAs was confirmed SW403 Tumor volume Tumor 0 in vitro: Decreased cell viability was noted only in LS411N and 12 20 28 12 20 28 12 20 28 SW403 colorectal cancer cell lines, not in RKO colorectal cancer Days post implant cells that are wild type for β-catenin and APC (Fig.