ORIGINAL ARTICLE Attenuation of the beta-catenin/TCF4 complex in colorectal cancer cells induces several growth-suppressive microRNAs that target cancer promoting genes T Schepeler 1 , A Holm 1 , P Halvey 2 , I Nordentoft 1 , P Lamy 1 , EM Riising 3 , LL Christensen 1 , K Thorsen 1 , DC Liebler 2 , K Helin 3 , TF Ørntoft 1 and CL Andersen 1 1 Department of Molecular Medicine (MOMA), Aarhus University Hospital, Aarhus N, Denmark; 2 Department of Biochemistry, Jim Ayers Institute for Precancer Detection and Diagnosis, Vanderbilt University School of Medicine, Nashville, TN, USA and 3 Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark Aberrant activation of the Wnt signaling pathway is causally involved in the formation of most colorectal cancers (CRCs). Although detailed knowledge exists regarding Wnt-regulated protein-coding genes, much less is known about the possible involvement of non-coding RNAs. Here we used TaqMan Array MicroRNA Cards, capable of detecting 664 unique human microRNAs (miRNAs), to describe changes of the miRNA transcrip- tome following disruption of beta-catenin/TCF4 activity in DLD1 CRC cells. Most miRNAs appeared to respond independent of host gene regulation and proximal TCF4 chromatin occupancy as inferred from expression micro- array and ChIP–chip data. A module of miRNAs induced by abrogated Wnt signaling in vitro was downregulated in two independent series of human primary CRCs (n ¼ 76) relative to normal adjacent mucosa (n ¼ 34). Several of these miRNAs (miR-145, miR-126, miR-30e-3p and miR- 139-5p) markedly inhibited CRC cell growth in vitro when ectopically expressed. By using an integrative approach of proteomics and expression microarrays, we found numer- ous mRNAs and proteins to be affected by ectopic miR- 30e-3p levels. This included HELZ and PIK3C2A that were directly repressed by several miRNA binding sites as confirmed by luciferase reporter assays in combination with mutational analyses. Finally, small interfering RNA- mediated downregulation of PIK3C2A, but not HELZ, was sufficient on its own to restrict CRC cell growth. Collectively, our study demonstrates that multiple miR- NAs are upregulated as a consequence of forced attenua- tion of Wnt signaling in CRC cells, and some of these miRNAs inhibit cell growth with concomitant suppression of several growth-stimulatory cancer-related genes. Oncogene (2012) 31, 2750–2760; doi:10.1038/onc.2011.453; published online 3 October 2011 Keywords: microRNA; Wnt; cancer; miR-30e-3p; PIK3C2A; TCF4 Introduction Colorectal cancer (CRC) is a very common malignancy and responsible for a large fraction of cancer deaths (Weitz et al., 2005). Most CRCs are caused by aberrant Wnt signaling that fuel excessive cell growth through increased activity of growth-stimulating genes while keeping negative growth regulators (for example, FOXO3A, DAPK2, HATH1 and CDKN1A/p21) in check through direct and indirect mechanisms (van de Wetering et al., 2002; Tsuchiya et al., 2007; Dehner et al., 2008; Li et al., 2009). The widespread effects on gene expression imposed by the Wnt signaling pathway is initially executed by the bipartite transcription factor complex beta-catenin/TCF that re- cruits numerous auxiliary co-regulators to achieve activa- tion or inhibition of target gene promoters in an often context-dependent manner (van de Wetering et al., 2002; Theisen et al., 2007; Blauwkamp et al., 2008; Cselenyi and Lee, 2008; Vlad et al., 2008; Railo et al., 2009). Interestingly, recent studies have hinted at the tantalizing possibility that microRNAs (miRNAs), often functioning as negative posttranscriptional regulators (Filipowicz et al., 2008), are also embedded in the Wnt signaling network. Among the first connections between Wnt signaling and miRNAs, was the observation that impairment of Wnt signaling in Drosophila melanogaster fly larvae led to increased activity of the bantam miRNA (Brennecke et al., 2003). The regulatory mechanism was not further defined, but a subsequent study suggested that it depended on modulation of Notch activity (Herranz et al., 2008). In another developmental setting, ectopic stimulation of Wnt signaling in Xenopus laevis frog embryos led to decreased miR-15/-16 levels (Martello et al., 2007). Levels of miR-29 family members were reported to increase during Wnt-induced differentiation of mouse and human osteoblast bone cells in vitro (Kapinas et al., 2009, 2010), and miR-30e levels increased during differentiation of rat intestinal cells in a beta-catenin/TCF4-dependent manner (Liao and Lonnerdal, 2010). In addition, Wang et al. demonstrated that reconstitution of APC activity affects miRNA levels in the APC mutant CRC cell line HT29 (Wang et al., 2009). Collectively, existing evidence supports the rationale that the Wnt targetome may be even more intricate than previously anticipated. Given the paramount Received 10 March 2011; revised 13 August 2011; accepted 30 August 2011; published online 3 October 2011 Correspondence: Dr CL Andersen, Department of Molecular Medicine (MOMA), Aarhus University Hospital, Brendstrupgaardsvej 100, Skejby, DK-8200 Aarhus N, Denmark. E-mail: cla@ki.au.dk Oncogene (2012) 31, 2750–2760 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc