| indicates complementary base pairing and?: indicates a weak binding interaction. DDAH1 is expressed in breast cancer cell lines and inversely correlates with expression of miR-193b We sought to quantify expression of DDAH1 and miR-193b in a selection of breast cancer cell lines. In DDAH1? MCF7 cells, inhibition of miR-193b elevated DDAH1 expression. Luciferase reporter assays demonstrated as a direct target of miR-193b. MDA-MB-231 cells organised into tube structures in an assay of VM, which was significantly inhibited by DDAH1 knockdown or miR-193b expression. Mechanistically, we found miR-193b regulates cell proliferation and migration of MDA-MB-231 cells, whilst DDAH1 knockdown inhibited cell migration. These studies represent the first evidence for DDAH1 expression, regulation and function in breast SR 59230A HCl cancer cells, and highlights SR 59230A HCl SR 59230A HCl that targeting DDAH1 expression and/or enzymatic activity may be a valid option in the treatment of aggressive breast cancers. Introduction Breast cancer is the most common cancer among women and accounts for a significant proportion of cancer-related death in western countries1. Currently there is no gold standard therapy for breast cancer due to its highly heterogeneous nature. Whilst the majority of breast cancers are positive for estrogen receptor (ER+), progesterone receptor (PR+) and/or human epidermal growth factor receptor 2 (HER2+), and can thus be treated with targeted endocrine therapy2, a small subset of breast cancers are negative for all three receptors. These tumours, termed triple SR 59230A HCl negative breast cancer (TNBC), are typically treated with a less-successful combinatorial approach of chemotherapy, radiation therapy and surgery. In addition, TNBC presents as a highly proliferative and aggressive disease with rapid growth and early metastases, resulting in significantly higher mortality rates and a reduced life expectancy when compared to other molecular subtypes3. Access to a blood supply plays a central role in both local tumour growth and distant metastasis of breast cancer4. Intra-tumoural vascular networks formed by angiogenesis, the sprouting and extension of pre-existing blood vessels, has previously been considered the only process responsible for tumour vascularisation and blood supply. However, despite the theoretical efficacy of anti-angiogenic treatments to target this process, the benefits obtained are often modest and have not proved beneficial in regards to long-term survival5,6. Recently, a new tumour vascular paradigm independent of endothelial cell-mediated angiogenesis has been described. Vasculogenic mimicry (VM) describes the formation of vessel-like networks directly by the tumour cells themselves7,8. In contrast to vessels lined by endothelial cells, channels formed by VM are lined by tumour cells yet can still fuse to a conventional vascular network to provide an adequate blood supply for tumour growth9. The presence of VM networks is predictive of PDGFRA poor survival and increased metastatic potential through entrance of tumour cells into the vasculature10,11, and VM inhibition is reported to abrogate tumour development12. The molecular mechanisms regulating VM, and whether these overlap with classical angiogenesis, are currently not well understood. However, it has SR 59230A HCl been suggested that an upregulation of angiogenesis-related genes may be involved13. Nitric oxide (NO) is an important cellular signalling molecule14. Synthesis of NO is mediated by the family of nitric oxide synthase (NOS) enzymes through conversion of arginine to L-citrulline. The methylated arginines asymmetric dimethylarginine (ADMA) and monomethyl arginine (L-NMMA) are competitive endogenous inhibitors of all isoforms of NOS15,16. Dimethylarginine dimethylaminohydrolase (DDAH) is the primary enzyme involved in the metabolism of ADMA and L-NMMA17. Whilst two isoforms of DDAH are observed in human (DDAH1 and DDAH2), current evidence suggests DDAH1 is the critical enzyme for ADMA and L-NMMA clearance18, 19 and is thus important for the tight regulation of NO production. NO has various functions in many processes including angiogenesis and cancer20,21. Specifically, endothelium-derived NO promotes angiogenesis through inhibition of apoptosis22 and enhancement of endothelial cell proliferation and migration23,24. In cancer the roles of NO are diverse, and are proposed to have dual pro- and anti-tumour effects depending on local concentration25. An increase in inducible NOS (iNOS) expression is documented in many solid tumours including those of the breast26C29. Furthermore, DDAH overexpression enhances angiogenesis in tumours with an accompanied increase in metastatic potential30,31. Inhibition of NO synthesis significantly suppresses angiogenesis with some beneficial effects in cancer32,33. These findings suggest a key role for DDAH1 in the modulation of angiogenesis of endothelial cells. A family of small non-coding RNAs (21C25 nt) called microRNAs (miRNA or miR) have recently emerged as major post-transcriptional regulators of gene expression34. The post-transcriptional regulatory function of miRNAs is mediated through target mRNA degradation and/or inhibition of protein translation, promoted through their binding to miRNA target sites typically located within the 3-untranslated region (3UTR) of target mRNAs. Each miRNA contains a unique seed sequence corresponding to nucleotides 2C7 from its 5 terminus which determines.