Abstract MicroRNAs (miRNAs) are noncoding single-stranded RNA molecules of ∼22 nucleotides in length. They have a critical role in regulating gene expression by targeting messenger RNAs (mRNAs) in a sequence-specific manner. Active mature miRNAs are produced from primary miRNA transcripts (pri-miRNAs) through sequential cleavages by the microprocessor complex, which includes DROSHA, DGCR8, and DICER proteins. Several miRNAs are specifically up-regulated in various types of tumors, and a wide range of studies have demonstrated how their down-regulation could potentially affect tumorigenesis, metastasis formation and drug resistance. However, most of these studies have indicated the possibility of modulating miRNA expression at the transcriptional level, while only a few focused on post-transcriptional control. Nucleolin (NCL) is a highly conserved multifunctional protein involved in ribosomal RNA (rRNA) biogenesis and in the stabilization of different mRNAs, in the nucleus and in the cytoplasm of normal and cancer cells. NCL was also found on the surface of different types of cancer cells, but not on their normal counterparts, shuttling between the inner and the outer part of the cell membrane. These observations suggested that NCL might be considered a cancer cell specific receptor, able to mediate tumor-selective uptake of specific ligands such as RNA and DNA G-rich aptamers. Here we show that NCL binds the terminal loop and promotes the maturation of a specific set of miRNAs, including miR-21, miR-103, miR-221 and miR-222, whose over-expression is causally associated with greater aggressiveness and resistance to anti-neoplastic therapies of several kind of tumors, such as breast cancer. Accordingly, a direct correlation between the expression levels of NCL and NCL-dependent miRNAs in human breast cancer samples was also observed. Conversely, NCL impairment down-regulated NCL-dependent miRNAs and up-regulates their target in vitro, affecting cancer cell proliferation, migration and anti-neoplastic drug resistance. Finally, we used AS1411, the first NCL-targeting G-rich aptamer that has reached phase II clinical trials for cancer therapy, to inhibit NCL activity in orthotopc xenograft models of breast cancer. Our in vivo data demonstrate that AS1411 down-regulates NCL-dependent miRNAs, hindering breast cancer metastasis. These findings provide insights into the molecular function of NCL in miRNA biology, as well as one of the first realistic strategies for miRNA regulation in cancer therapy. Citation Format: Dario Palmieri, Luciana De Luca, Jessica Consiglio, Alberto Rocci, Tiffany Talabere, Claudia Piovan, Alessandro Lagana, Luciano Cascione, Jingwen Guan, Pierluigi Gasparini, Veronica Balatti, Vincenzo Coppola, Craig Hofmeister, Guido Marcucci, John Byrd, Stefano Volinia, Charles Shapiro, Michael Freitas, Carlo Croce, Flavia Pichiorri. In vivo NCL-targeting affects breast cancer aggressiveness through miRNA regulation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1122. doi:10.1158/1538-7445.AM2013-1122