N-methylated adenine (mA) is the most frequent posttranscriptional modification in eukaryotic mRNA. Turnover of RNA generates N-methylated AMP (N-mAMP), which has an unclear metabolic fate. We show that and human cells require an N-mAMP deaminase (ADAL, renamed MAPDA) to catabolize N-mAMP to inosine monophosphate in vivo by hydrolytically removing the aminomethyl group. A phylogenetic, structural, and biochemical analysis revealed that many fungi partially or fully lack MAPDA, which coincides with a minor role of NA-RNA methylation in these organisms. MAPDA likely protects RNA from mA misincorporation. This is required because eukaryotic RNA polymerase can use N-mATP as a substrate. Upon abrogation of , root growth is slightly reduced, and the N-methyladenosine, N-mAMP, and N-mATP concentrations are increased in Arabidopsis. Although this will potentially lead to mA misincorporation into RNA, we show that the frequency is too low to be reliably detected in vivo. Since N-mAMP was severalfold more abundant than N-mATP in mutants, we speculate that additional molecular filters suppress the generation of N-mATP. Enzyme kinetic data indicate that adenylate kinases represent such filters being highly selective for AMP versus N-mAMP phosphorylation. We conclude that a multilayer molecular protection system is in place preventing N-mAMP accumulation and salvage.

Paracrine activation of cells contained in the tumor microenvironment promotes tumor progression and metastasis. In breast cancer, malignant cells recruit and educate macrophages into a M2 tumor-promoting phenotype that supports the metastatic spread of cancer cells. Here, we show that miR-149 functions as a metastasissuppressing microRNA in breast cancer cells by limiting colonystimulating factor-1 (CSF1)-dependent recruitment and M2 polarization of macrophages. In lymph node-positive, triple-negative breast cancer (TNBC) tissues, low miR-149 expression correlated with macrophage infiltration and reduced patient survival. By directly targeting CSF1, miR-149 expression in TNBC cell lines (MDA-MB-231 and BT-549) inhibited the recruitment of human monocytic THP-1 cells and primary human macrophages. Furthermore, in macrophages cocultured with MDA-MB-231 cells expressing miR-149, epidermal growth factor (EGF) and amphiregulin expression levels were strongly reduced, resulting in reduced EGF receptor activation in the cancer cells. In vivo, lung metastases developing from orthotopic MDA-MB-231 tumors were reduced by 75% by miR-149 expression, and this was associated with impaired M2 macrophage infiltration of the primary tumors. These data suggest that miR-149 downregulation functionally contributes to breast tumor progression by recruiting macrophages to the tumor and facilitating CSF1 and EGF receptor cross-talk between cancer cells and macrophages.

The PI3K-Akt pathway is one of the most commonly dysregulated cancer-associated signaling pathways. Here we report an oncogenic function for the miR-181 family in luminal breast cancer cells that involves Akt hyperactivation. We show that miR-181a and miR-181d posttranscriptionally suppress the expression of PHLPP2 and INPP4B phosphatases, resulting in elevated growth factor-induced Akt phosphorylation. Ectopic expression of miR-181a and miR-181d promoted S-phase entry and cell proliferation, which was reversed by pharmacological Akt inhibition. Importantly, the expression of miR-181 family members and PHLPP2/INPP2B are inversely correlated in primary human estrogen receptor-positive breast cancers, supporting the clinical relevance of our findings.

Matrix metalloproteinases (MMPs) degrade several ECM components and are crucial modulators of cell invasion and tissue organization. Although much has been reported about their function in remodeling ECM in health and disease, their trafficking across the Golgi apparatus remains poorly understood. Here we report that the cis-Golgi protein nucleobindin-1 (NUCB1) is critical for MMP2 and MT1-MMP trafficking along the Golgi apparatus. This process is Ca2+-dependent and is required for invasive MDA-MB-231 cell migration as well as for gelatin degradation in primary human macrophages. Our findings emphasize the importance of NUCB1 as an essential component of MMP transport and its overall impact on ECM remodeling.

<p>Characterization of MDA-MB-231 cells stably expressing miR-149</p>