Tumor and Stem Cell Biology An miRNA Expression Signature for the Human Colonic Stem Cell Niche Distinguishes Malignant from Normal Epithelia Vignesh Viswanathan 1,2,3 , Shirish Damle 4 , Tao Zhang 1,2,4 , Lynn Opdenaker 1,2 , Shirin Modarai 1,2 , Monica Accerbi 5 , Skye Schmidt 5 , Pamela Green 5 , Deni Galileo 2 , Juan Palazzo 4 , Jeremy Fields 6 , Sepehr Haghighat 1,2,4 , Isidore Rigoutsos 4 , Greg Gonye 4,7 , and Bruce M. Boman 1,2,4 Abstract Malignant transformation of tissue stem cells (SC) may be the root of most cancer. Accordingly, we identified miRNA expres- sion patterns in the normal human colonic SC niche to under- stand how cancer stem cells (CSC) may arise. In profiling miRNA expression in SC-enriched crypt subsections isolated from fresh, normal surgical specimens, we identified 16 miRNAs that were differentially expressed in the crypt bottom, creating an SC signature for normal colonic epithelia (NCE). A parallel analysis of colorectal cancer tissues showed differential expression of 83 miRNAs relative to NCE. Within the 16 miRNA signature for the normal SC niche, we found that miR-206, miR-007-3, and miR- 23b individually could distinguish colorectal cancer from NCE. Notably, miR-23b, which was increased in colorectal cancer, was predicted to target the SC-expressed G protein-coupled receptor LGR5. Cell biology investigations showed that miR-23b regu- lated CSC phenotypes globally at the level of proliferation, cell cycle, self-renewal, epithelial–mesenchymal transition, invasion, and resistance to the colorectal cancer chemotherapeutic agent 5- fluorouracil. In mechanistic experiments, we found that miR- 23b decreased LGR5 expression and increased ALDH þ CSCs. CSC analyses confirmed that levels of LGR5 and miR-23b are inversely correlated in ALDH þ CSCs and that distinct subpop- ulations of LGR5 þ and ALDH þ CSCs exist. Overall, our results define a critical function for miR-23b, which, by targeting LGR5, contributes to overpopulation of ALDH þ CSCs and colorectal cancer. Cancer Res; 77(14); 3778–90. Ó2017 AACR. Introduction Mounting evidence indicates that (i) stem cells (SC) are the cells of origin of cancer (1, 2), (ii) SC overpopulation drives tumor initiation and progression (3–5), and (iii) SCs are resistant to conventional anticancer therapies. We found that ALDH1 is a marker for normal and malignant human colonic SC and tracks SC overpopulation during colon tumorigenesis. Although this finding and others indicate that cancer SC (CSC) overpopulation drives tumor growth, it is incompletely understood which dysre- gulated mechanisms cause the SC overpopulation. Because evi- dence points to an important role for miRNAs in the pathogenesis of various diseases, we studied miRNAs as a possible mechanism in colorectal cancer. Aberrantly expressed miRNAs lead to widespread transcription- al dysregulation and cancer (6–10). In colorectal cancer, differ- ential miRNA expression has been related to stage and site of the disease (11). Mounting evidence also indicates a role for miRNAs in the maintenance of the CSC phenotype (12–16). We investigated dysregulated mechanisms in colonic SCs in colorectal cancer that are due to changes in miRNA expression. Our initial goal was to identify the set of miRNAs and their target genes that are specific to the normal colonic SC niche, and then identify the subset of these miRNAs that are aberrantly expressed in colorectal cancers compared with normal colonic epithelium (NCE). Our second goal was to see if some miRNAs are key to regulation of normal colonic SC populations, and when dysregulated contribute to SC overpopulation and colon tumorigenesis. Accordingly, we devised an innovative strategy for miRNA profiling of human colonic SCs. Because colonic SCs have unique functional properties, the crypt bottom, which contains most colonic SCs, should have a unique gene expression profile that should be discernable by microarray analysis. Therefore, in the current study, we: (i) isolated pure crypts from surrounding stromal elements, (ii) isolated crypt subsections (bottom 1/10 and top 9/10), and then (iii) used microarray-based miRNA expression profiling to compare the SC-enriched crypt with the crypt top. We hypothesized that specific miRNAs are selectively expressed in the normal crypt SC niche and that some or all of 1 Center for Translational Cancer Research, Helen F Graham Cancer Center and Research Institute, Newark, Delaware. 2 Department of Biological Sciences, University of Delaware, Newark, Delaware. 3 Department of Gastroenterology, Beth Israel Deaconess Medical Center, Boston, Massachusetts. 4 Thomas Jeffer- son University and Kimmel Cancer Center, Philadelphia, Pennsylvania. 5 Depart- ment of Plant and Soil Sciences, Delaware Biotechnology Institute, Newark, Delaware. 6 CA  TX Inc., Gladwyne, Pennsylvania. 7 Nanostring Technologies, Seattle, Washington. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Author: Bruce M. Boman, Center for Translational Cancer Research, Helen F Graham Cancer Center, Newark, DE 19716. Phone: 302-831- 2792; Fax: 302-623-4554; E-mail: BrBoman@ChristianaCare.org doi: 10.1158/0008-5472.CAN-16-2388 Ó2017 American Association for Cancer Research. Cancer Research Cancer Res; 77(14) July 15, 2017 3778 on April 24, 2020. © 2017 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst May 9, 2017; DOI: 10.1158/0008-5472.CAN-16-2388