Abstract MicroRNAs (miRNAs) are small non coding RNAs that regulate gene expression at post-transcriptional level through translational inhibition and/or degradation of mRNA target genes. Recent evidence points to a widespread role for miRNAs in the initiation and progression of tumorigenesis in a plethora of tissues including the mammary gland. Aberrant miRNA expression profiles have been described in breast cancer specimens compared to normal tissues, discriminating breast tumors with different clinico-biological phenotypes. In our previous publications, we have demonstrated that miR-222/221 cluster is highly expressed in aggressive breast cancer and high levels of miR-222/221 may confer a proliferation advantage to cancer cells and resistance to therapeutic agents by repressing several genes such as ERα;, CDKN1B, BIM, FOXO3, CAV1, PTEN. To demonstrate the in vivo role of miR-222/221 cluster in breast cancer initiation and progression, we have genetically deleted miR-222/221 gene in the MMTV-PyVT mouse model that develops multifocal mammary adenocarcinoma and lung metastatic lesions in 80% of the population. The tumor latency and multiplicity were assessed and none of these parameters were significantly affected by miR-222/221 ablation. Interestingly, lack of miR-222/221 impairs the development of lung metastasis with a reduction of the metastatic load of the lung. Moreover, metastases arising from miR-222/221 deficient cells present a greater cytoarchitectural similarity and a reduced rate of mitosis and apoptosis. We also show that loss of miR-222/221 modulates the transcriptome and miRNA expression profiles of mouse breast tumors and cancer cells, stimulating a stronger estrogen receptor alpha transcriptional network and a suppression of the cancer stem cells population. Citation Format: Gianpiero Di Leva, Claudia Piovan, Douglas Cheung, Daniel Briskin, Arpan Kumar, Gerard Nuovo, Matteo Fassan, Vincenzo Coppola, Michela Garofalo, Carlo Croce. miR-222/221 in aggressive breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A085.