Prevention and Epidemiology NOTCH Signaling Is Required for Formation and Self-Renewal of Tumor-Initiating Cells and for Repression of Secretory Cell Differentiation in Colon Cancer Shaheen S. Sikandar 1 , Kira T. Pate 2 , Scott Anderson 1 , Diana Dizon 1 , Robert A. Edwards 3 , Marian L. Waterman 2 , and Steven M. Lipkin 4 Abstract NOTCH signaling is critical for specifying the intestinal epithelial cell lineage and for initiating colorectal adenomas and colorectal cancers (CRC). Based on evidence that NOTCH is important for the maintenance and self-renewal of cancer-initiating cells in other malignancies, we studied the role of NOTCH signaling in colon cancer–initiating cells (CCIC). Tumors formed by CCICs maintain many properties of the primary CRCs from which they were derived, such as glandular organization, cell polarity, gap junctions, and expression of char- acteristic CRC molecular markers. Furthermore, CCICs have the property of self-renewal. In this study, we show that NOTCH signaling is 10- to 30-fold higher in CCIC compared with widely used colon cancer cell lines. Using small-molecule inhibition and short hairpin RNA knockdown, we show that NOTCH prevents CCIC apoptosis through repression of cell cycle kinase inhibitor p27 and transcription factor ATOH1. NOTCH is also critical to intrinsic maintenance of CCIC self-renewal and the repression of secretory cell lineage differentia- tion genes such as MUC2. Our findings describe a novel human cell system to study NOTCH signaling in CRC tumor initiation and suggest that inhibition of NOTCH signaling may improve CRC chemoprevention and che- motherapy. Cancer Res; 70(4); 1469–78. ©2010 AACR. Introduction Colorectal cancer (CRC) is the second leading cause of U.S. cancer death (1). For metastatic CRC, the 5-year sur- vival rate is ∼10% (1). A mechanistic understanding of CRC initiation, recurrence, and metastasis is therefore an important goal. Several studies have shown that WNT and NOTCH pathways help maintain intestinal homeostasis, reg- ulate cell fate decisions, and play important roles in CRC tumorigenesis and progression (2–8). The majority of CRC tumors have increased WNT signaling (9). In normal intes- tinal homeostasis, WNT signaling stimulates intestinal stem and progenitor cell proliferation but paradoxically also causes terminal differentiation into Paneth cells. One might therefore expect that CRC cells would attempt to differen- tiate terminally into Paneth cells. However, other signaling pathways active in CRC prevent terminal differentiation and maintain self-renewal capacity. A candidate is the NOTCH signaling pathway. The role of NOTCH signaling in CRC is less well charac- terized than WNT. NOTCH signaling is triggered through the binding of a ligand on the membrane of one cell (Delta/Delta-like/Jagged/Serrate) to a receptor (NOTCH1/ NOTCH2/NOTCH3/NOTCH4) on the membrane of the con- tacting cell. This causes proteolytic cleavage of NOTCH receptors to release the cytoplasmic tail of NOTCH (NICD; ref. 10). NICD translocates to the nucleus and associates with CSL transcription factors (CBF1/RBPJκ/Suppressor of Hairless/Lag-1) and coactivator Mastermind to turn on transcription of target genes (11). The best-characterized targets of NOTCH are hairy/enhancer of split (HES) family of transcription factors, particularly HES1 in the intestine (12, 13). In normal mouse intestine, inhibition of NOTCH signaling results in exit from the proliferative compartment and differentiation into postmitotic goblet cells (7). Similar results are seen in knockouts of other critical NOTCH signal transduction components, including Hes1, Rbpjκ, Notch 1 and 2 receptors, and Pofut1 knockouts for normal intestine (6–8, 12, 14). Apc mutant intestinal adenoma cells, which have elevated WNT signaling, also respond to NOTCH signaling inhibition by terminal differentiation into goblet cells, accompanied by cell cycle arrest and/or apoptosis (7, 15). Therefore, suppression of NOTCH signaling is a powerful mechanism for directing both normal intestinal enterocyte progenitors and Apc mutant intestinal cancer cells to differentiate down a secretory lineage. NOTCH signaling plays an important role in intestinal tumor initiation but not progression in mice (15). Transgenic expression of NICD in the intestine leads to expansion of enterocyte progenitor cells (6) and increases the number of Authors' Affiliations: Departments of 1 Biological Chemistry, 2 Microbiology and Molecular Genetics, and 3 Pathology, School of Medicine, University of California, Irvine, California and 4 Department of Medicine, Weill Cornell College of Medicine, New York, New York Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Author: Steven M. Lipkin, Department of Medicine, Weill Cornell College of Medicine, New York, NY 10021. Phone: 212-774-7160; Fax: 212-774-7167; E-mail: stl2012@med.cornell.edu. doi: 10.1158/0008-5472.CAN-09-2557 ©2010 American Association for Cancer Research. Cancer Research www.aacrjournals.org 1469 Downloaded from http://aacrjournals.org/cancerres/article-pdf/70/4/1469/2645230/1469.pdf by guest on 20 June 2022