miR-9, a MYC/MYCN-activated microRNA, regulates E-cadherin and cancer metastasis Li Ma1,2, Jennifer Young1,7, Harsha Prabhala3,7, Elizabeth Pan1, Pieter Mestdagh4, Daniel Muth5, Julie Teruya-Feldstein6, Ferenc Reinhardt1, Tamer T. Onder1,2, Scott Valastyan1,2, Frank Westermann5, Frank Speleman4, Jo Vandesompele4, and Robert A. Weinberg1,2,8 1 Whitehead Institute for Biomedical Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA 2 MIT Ludwig Center for Molecular Oncology, Cambridge, MA 02142, USA 3 Medical Scientist Training Program, University of Virginia, Charlottesville, VA 22908, USA 4 Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium 5 Department of Tumor Genetics, German Cancer Center, Im Neuenheimer Feld 280, Heidelberg, Germany 6 Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA Abstract MicroRNAs (miRNAs) are increasingly implicated in regulating the malignant progression of cancer. Here we show that miR-9, the level of which is upregulated in breast cancer cells, directly targets CDH1, the E-cadherin-encoding mRNA, leading to increased cell motility and invasiveness. miR-9- mediated E-cadherin downregulation results in the activation of ��-catenin signaling, which contributes to upregulated expression of the gene encoding vascular endothelial growth factor (VEGF) this leads, in turn, to increased tumor angiogenesis. Overexpression of miR-9 in otherwise- non-metastatic breast tumor cells enables these cells to form pulmonary micrometastases in mice. Conversely, inhibiting miR-9 using a ���miRNA sponge��� in highly malignant cells inhibits metastasis formation. Expression of miR-9 is activated by MYC and MYCN, both of which directly bind to the mir-9-3 locus. Significantly, in human cancers, miR-9 levels correlate with MYCN amplification, tumor grade, and metastatic status. These findings uncover a regulatory and signaling pathway involving a metastasis-promoting miRNA that is predicted to directly target expression of the key metastasis-suppressing protein E-cadherin. Metastases are responsible for 90% of cancer-related mortality. These secondary growths arise as products of a multi-step process that begins when caner cells within primary tumors Users may view, print, copy, download and text and data- mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms 8Correspondence should be addressed to R.A.W. (weinberg@wi.mit.edu). 7These authors contributed equally to this work. AUTHOR CONTRIBUTIONS L.M. conceived the project. R.A.W. supervised research. L.M. and H.P. designed experiments. L.M., J.Y., H.P., E. P., J.T-F., and F.R. performed most of the experiments and analyzed data. P.M., D.M., F.W., F.P., and J.V. contributed MYCN and ChIP-on-chip data. T.T.O. contributed some of the constructs and shared unpublished observations. S.V. modified the miRNA sponge design for stable expression. L.M. and R.A.W. wrote the manuscript. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. NIH Public Access Author Manuscript Nat Cell Biol. Author manuscript available in PMC 2010 September 1. Published in final edited form as: Nat Cell Biol. 2010 March 12(3): 247���256. doi:10.1038/ncb2024. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

break away from neighboring cells and invade through the basement membrane1. This initial step of local invasion may frequently be triggered by contextual signals that carcinoma cells receive from the nearby stroma, causing them to undergo an epithelial-mesenchymal transition (EMT), a multi-faceted transdifferentiation program that enables tumor cells to acquire malignancy-associated phenotypes2. Subsequently, metastasizing cells can enter the circulation, doing so either directly or via lymphatics. Size constraints in the microvasculature cause many of these cells to be arrested at distant tissue sites, where they may extravasate and enter the foreign tissue parenchyma. At this point, they may remain dormant or, with low efficiency, proliferate from occult micrometastases to form angiogenic, clinically detectable metastases. The absence of EMT-inducing signals in the microenvironment of distant tissues may cause such disseminated cells to revert to an epithelial phenotype via a mesenchymal- epithelial transition (MET). Much research has been focused on identifying the critical regulators of the metastatic process these regulatory molecules include both proteins and microRNAs (miRNAs)3,4. MiRNAs are small non-coding RNA molecules that suppress gene expression by interacting with the 3��� untranslated regions (UTRs) of target mRNAs. These interactions may result in either inhibition of translation of the targeted mRNAs or their degradation5. In an initial real- time RT-PCR-based screen for differentially expressed miRNAs, we identified three miRNAs that are most significantly upregulated in human breast cancer cell lines ��� miR-155, miR-9, and miR-10b6. The subsequent functional studies of miR-10b validated its candidacy as a mechanistically important miRNA in cancer progression, as demonstrated by experiments showing that overexpression of miR-10b in otherwise-non-metastatic breast tumors initiated tumor invasion and distant metastasis in xenograft models6. Subsequently, several other miRNAs, including miR-373, miR-520c, miR-335, miR-206, miR-126, miR-21, and miR-31, have also been identified as either promoters or suppressors of metastasis7���11. In addition, the miR-200 family, whose role in regulating metastasis remains unclear, has emerged as a silencer of ZEB1 and ZEB2, two established EMT-inducing and metastasis-promoting transcription factors12,13, thereby representing yet another set of regulators of the EMT program. A second miRNA that stood out in our initial screen is miR-96, a miRNA that is selectively expressed in neural tissues under normal conditions14 and regulates their development15. Expression of this miRNA is higher in brain tumors than in tumors of other histological types, further demonstrating a tissue-specific expression pattern16. In the context of clinical breast cancer, miR-9 has been found to be upregulated in primary tumors relative to its expression in normal mammary tissues17. Interestingly, miR-9 was recently shown to be upregulated by 1,000-fold in c-myc-induced mouse mammary tumors18. In a preliminary survey, we used several computational algorithms, including the two most widely tested programs, TargetScan19 and PicTar20, to search for miRNAs that target evolutionarily conserved sequences present in the CDH1 mRNA this survey revealed that miR-9 was the only known miRNA that was predicted to target the CDH1 mRNA (Fig. 1a). CDH1 encodes the epithelial cell adhesion molecule E-cadherin, a trans-membrane glycoprotein that forms the core of the adherens junctions between adjacent epithelial cells21. The cytoplasmic tail of E-cadherin associates with a number of intracellular proteins that link E-cadherin to the actin cytoskeleton21. Given its well-established function in maintaining adherens junctions, E-cadherin inactivation presumably promotes metastasis by enabling the first step of the metastatic cascade ��� the dissociation of carcinoma cells from one another. In addition, its loss liberates �����catenin molecules that may move into the nucleus and activate pro-metastatic genes22. The significance of E-cadherin inactivation for metastasis has been demonstrated in a variety of in vitro and in vivo models22���27. Recently, we have found that E-cadherin loss in certain cell types can also trigger an EMT and a wide range of transcriptional and signaling changes that contribute to metastatic dissemination27. Thus, Ma et al. Page 2 Nat Cell Biol. Author manuscript available in PMC 2010 September 1. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

miR-9���s potential role as a suppressor of E-cadherin expression made this miRNA a strong candidate for promoting the acquisition of malignant phenotypes by carcinoma cells. RESULTS Effect of miR-9 on the expression of E-cadherin To determine whether miR-9 can indeed downregulate E-cadherin expression, we stably expressed miR-9 and, as a control, miR-10b, in two epithelial cell lines (Supplementary Information, Fig. S1). Ectopic expression of miR-9, but not miR-10b, led to an EMT-like conversion in HMLE non-transformed, immortalized human mammary epithelial cells28: these cells became scattered and assumed a spindle-like or star-like morphology (Fig. 1b) and displayed a 70% reduction in E-cadherin and a 5-fold increase in the mesenchymal marker vimentin (Fig. 1c). In contrast, in SUM149 human breast carcinoma cells29, miR-9 downregulated E-cadherin expression by ~50% but failed to induce vimentin, other mesenchymal markers, and a fibroblastic cell morphology (Fig. 1c, and data not shown). These differences in response were intrinsic to the two cell lines rather than a consequence of different degrees of E-cadherin suppression, because knockdown of E-cadherin by 90% using small- interfering RNA (siRNA) in both cell lines caused an EMT in HMLE cells27 but not in SUM149 cells (T.T.O. and R.A.W., unpublished observations). Hence, while miR-9 succeeds in suppressing E-cadherin expression in two epithelial cell lines, it only induces an EMT in one of them. To determine whether miR-9 directly targets the CDH1 mRNA, we performed reporter assays and found that miR-9 reduced the activity of a luciferase reporter that was fused to the wild- type 3��� UTR of the CDH1 mRNA but not to a mutant 3���UTR (Fig. 1d) the latter carried altered nucleotides that were introduced in the miR-9 ���seed-pairing���19 recognition site (Fig. 1a). Hence, the observed downregulation of E-cadherin by miR-9 depends directly on a single cognate recognition site in the CDH1 3���UTR. Inactivation of E-cadherin has been shown to promote cell migration and invasion in the presence or absence of an EMT27. Consistent with this, we found that ectopic expression of miR-9 led to a 3- to 5-fold increase in the motility and invasiveness of both HMLE and SUM149 cells in vitro (Fig. 1e, f). In order to determine whether these effects depend specifically on E- cadherin suppression, we employed an expression construct that encodes the entire E-cadherin coding sequence but lacks the 3��� UTR, yielding an mRNA that is resistant to miRNA-mediated suppression. Ectopic expression of E-cadherin with this construct reduced migration and invasion in the miR-9-overexpressing cells, but not in the control cells, which have a low basal level of miR-9 (Fig. 1e, f). This suggests that following miR-9 overexpression, a resulting reduction in E-cadherin is required in order for cells to exhibit increased motility and invasiveness. However, we cannot exclude the possibility that miR-9-mediated suppression of other targets is also required for the observed effects of this miRNA on cell phenotypes. Regulation of ��-catenin signaling and VEGF expression by miR-9 Having validated E-cadherin as a miR-9 target, we next sought to determine whether miR-9- mediated E-cadherin suppression would affect intracellular signaling. Binding of Wnt ligands to their receptors results in the stabilization of ��-catenin, allowing it to enter the cell nucleus, interact with the TCF/LEF family of transcription factors, and promote the transcription of genes 30. Independent of this, E-cadherin binds and sequesters a large pool of ��-catenin at the cytoplasmic membrane, thereby preventing its nuclear translocation and its function as a component of the TCF/LEF transcription factor complex22,31,32. Our previous work demonstrated that knockdown of E-cadherin in the experimentally transformed human mammary epithelial cells caused relocalization of ��-catenin from adherens junctions to the Ma et al. Page 3 Nat Cell Biol. Author manuscript available in PMC 2010 September 1. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript