The transmembrane metalloprotease-disintegrin ADAM8 mediates cell adhesion and shedding of ligands, receptors and extracellular matrix components. Here, we report that ADAM8 is abundantly expressed in breast tumors and derived metastases compared to normal tissue, especially in triple-negative breast cancers (TNBCs). Furthermore, high ADAM8 levels predicted poor patient outcome. Consistently, ADAM8 promoted an aggressive phenotype of TNBC cells in culture. In a mouse orthotopic model, tumors derived from TNBC cells with ADAM8 knockdown failed to grow beyond a palpable size and displayed poor vascularization. Circulating tumor cells and brain metastases were also significantly reduced. Mechanistically, ADAM8 stimulated both angiogenesis through release of VEGF-A and transendothelial cell migration via β1-integrin activation. In vivo, treatment with an anti-ADAM8 antibody from the time of cell inoculation reduced primary tumor burden and metastases. Furthermore, antibody treatment of established tumors profoundly decreased metastases in a resection model. As a non-essential protein under physiological conditions, ADAM8 represents a promising novel target for treatment of TNBCs, which currently lack targeted therapies and frequently progress with fatal dissemination.Subject Category Cancer

Striated muscle is a highly specialized collection of tissues with contractile properties that vary according to functional needs. Although muscle fiber types are established postnatally, lifelong plasticity facilitates stimulus-dependent adaptation. Functional adaptation requires molecular adaptation, which is partially provided by miRNA-mediated post-transcriptional regulation. miR-206 is a muscle-specific miRNA enriched in slow muscles. We investigated whether miR-206 drives the slow muscle phenotype or is merely an outcome. We found that miR-206 expression increases in both physiological (including female sex and endurance exercise) and pathological conditions (muscular dystrophy and adrenergic agonism) that promote a slow phenotype. Consistent with that observation, the slow soleus muscle of male miR-206-knockout mice displays a faster phenotype than wild-type mice. Moreover, left ventricles of male miR-206 knockout mice have a faster myosin profile, accompanied by dilation and systolic dysfunction. Thus, miR-206 appears to be necessary to enforce a slow skeletal and cardiac muscle phenotype and to play a key role in muscle sexual dimorphisms.

Circulating tumor cell clusters (CTCCs) are significantly more likely to form metastases than single tumor cells. We demonstrate the potential of backscatter-based flow cytometry (BSFC) to detect unique light scattering signatures of CTCCs in the blood of mice orthotopically implanted with breast cancer cells and treated with an anti-ADAM8 or a control antibody. Based on scattering detected at 405, 488, and 633 nm from blood samples flowing through microfluidic devices, we identified 14 CTCCs with large scattering peak widths and intensities, whose presence correlated strongly with metastasis. These initial studies demonstrate the potential to detect CTCCs via label-free BSFC.

BackgroundMyogenesis is driven by specific changes in the transcriptome that occur during the different stages of muscle differentiation. In addition to controlled transcriptional transitions, several other post-transcriptional mechanisms direct muscle differentiation. Both alternative splicing and miRNA activity regulate gene expression and production of specialized protein isoforms. Importantly, disruption of either process often results in severe phenotypes as reported for several muscle diseases. Thus, broadening our understanding of the post-transcriptional pathways that operate in muscles will lay the foundation for future therapeutic interventions.MethodsWe employed bioinformatics analysis in concert with the well-established C2C12 cell system for predicting and validating novel miR-1 and miR-206 targets engaged in muscle differentiation. We used reporter gene assays to test direct miRNA targeting and studied C2C12 cells stably expressing one of the cDNA candidates fused to a heterologous, miRNA-resistant 3′ UTR. We monitored effects on differentiation by measuring fusion index, myotube area, and myogenic gene expression during time course differentiation experiments.ResultsGene ontology analysis revealed a strongly enriched set of putative miR-1 and miR-206 targets associated with RNA metabolism. Notably, the expression levels of several candidates decreased during C2C12 differentiation. We discovered that the splicing factor Srsf9 is a direct target of both miRNAs during myogenesis. Persistent Srsf9 expression during differentiation impaired myotube formation and blunted induction of the early pro-differentiation factor myogenin as well as the late differentiation marker sarcomeric myosin, Myh8.ConclusionsOur data uncover novel miR-1 and miR-206 cellular targets and establish a functional link between the splicing factor Srsf9 and myoblast differentiation. The finding that miRNA-mediated clearance of Srsf9 is a key myogenic event illustrates the coordinated and sophisticated interplay between the diverse components of the gene regulatory network.

11Striated muscle is a highly specialized collection of tissues with contractile properties varying according to 12 functional needs. Although muscle fiber types are established postnatally, lifelong plasticity facilitates stimulus-13 dependent adaptation. Functional adaptation requires molecular adaptation, partially provided by miRNA-14 mediated post-transcriptional regulation. miR-206 is a muscle-specific miRNA enriched in slow muscles. We 15 investigated whether miR-206 drives the slow muscle phenotype or is merely an outcome. We found that miR-16 206 expression increases in both physiologic (including female sex and endurance exercise) and pathologic 17 conditions that promote a slow phenotype. Consistent with that observation, the slow soleus muscle of male miR-18 206 knockout mice displays a faster phenotype than wild-type mice. Moreover, their left ventricles have a faster 19 myosin profile accompanied by male-specific dilation and systolic dysfunction. Thus, miR-206 appears necessary 20 to enforce a slow skeletal and cardiac muscle phenotype and to play a key role in muscle sexual dimorphisms. 21 22 2014). Notably, despite a general sex difference in muscle fiber types, it is not known whether biological sex 72 influences miR-206 expression. Thus, we sought to determine in both sexes whether the miR-206 slow muscle 73 enrichment is an outcome or a driver of the oxidative phenotype. 74 Materials and Methods 75Cloning and mutagenesis: The minimal promoter (minTATA) and MyoG enhancer firefly luciferase reporter gene 76 consructs were previously described (Cheung et al., 2007). We cloned the miR-206 enhancer (GRCm38/mm10 77 chr1: 20,678,678,259) in the same manner as described for MyoG. We generated E-box point mutations 78 (CANNTG à CANNTA) with the QuikChange II site-directed mutagenesis kit (Agilent, 200523, Santa Clara, 79 CA) per the manufacturer's instructions. We verified all clones by Sanger sequencing. MRF expression constructs 80 were a kind gift from Dr. Xuedong Liu (University of Colorado Boulder). Primer sequences are listed in 81 Supplementary Table 1. 82 Cell culture and transfection: We grew C2C12 myoblasts in Growth Medium (GM): high glucose DMEM 83 (Invitrogen, 11960069, Waltham, MA) supplemented with 20% fetal bovine serum, 2 mM L-glutamine, 100 84 U/mL penicillin and 100 μg/mL streptomycin, and 1 mM sodium pyruvate. We differentiated them to myotubes 85 by changing media to Differentiation Medium (DM): high glucose DMEM supplemented with 5% adult horse 86serum, 2 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin, and 1 mM sodium pyruvate. When 87 differentiating, we refreshed DM every day to prevent media acidification. We grew 10T ½ cells in GM but with 88 10% FBS. For luciferase assays, we plated cells in triplicate in 6-well dishes at a density of 50,000 cells/well 89 (C2C12) or 100,000 cells/well (10T ½) 24 hours before transfection. We repeated each experiment at least twice 90 with independent thaws of cells. We transfected with TransIT-LT1 (Mirus Bio, MIR 2305, Madiso...