PROJECT SUMMARY Post-transcriptional regulatory programs play a major role in shaping the aberrant gene expression landscape that is a hallmark of cancer progression. We have recently demonstrated that specific classes of non-coding RNAs, such as tRNAs (Goodarzi et al, Cell, 2016) and tRNA fragments (Goodarzi et al, Cell, 2015), play major roles in breast cancer metastasis as post-transcriptional regulators of gene expression. However, these regulatory RNAs are but a fraction of non-coding RNAs that are differentially expressed in highly metastatic cells. For example, antisense RNAs are a large but often ignored class of non-coding RNAs with poorly understood functions in the cell. We recently performed a systematic analysis of antisense RNAs expressed in a panel of poorly and highly metastatic breast cancer cells. We observed tens of antisense RNAs that are associated with metastatic capacity. Antisense RNAs can function as post-transcriptional regulators through their ability to form stable duplexes with their sense counterparts. However, the underlying molecular mechanisms and their role in gene expression regulation remains largely unknown. Here, we propose a detailed dissection of our top candidate antisense RNA, which targets the 3' UTR of the gene NQO1 and is hence named NQO1-AS, and its function in promoting breast cancer metastasis. We hypothesize that NQO1-AS forms a stable duplex with the 3' UTR of NQO1 which precludes the protein HNRNPC from binding. HNRNPC likely acts as a regulator of alternative poly(A) site selection in NQO1 mRNA, shifting the equilibrium towards an isoform with a truncated 3' UTR. We hypothesize that this truncated transcript isoform has a shorter life-span. Thus, by over-expressing NQO1-AS, highly metastatic cells post-transcriptionally increase the expression of NQO1. Enhanced NQO1 activity enables cancer cells to withstand the oxidative stress experienced during metastasis. In this study, we seek to perform a detailed dissection of this pathway, understand its role in metastasis, and test its clinical relevance in patients. Importantly, the enhanced NQO1 activity in highly metastatic cells can be exploited, using adjuvant therapies, to specifically target metastatic cells in patients with invasive breast cancer.