Medical applications of nanotechnology typically focus on drug delivery and biosensors. Here, we combine nanotechnology and bioengineering to demonstrate that nanoparticles can be used to remotely regulate protein production in vivo. We decorated a modified temperature-sensitive channel, TRPV1, with antibody-coated iron oxide nanoparticles that are heated in a low-frequency magnetic field. When local temperature rises, TRPV1 gates calcium to stimulate synthesis and release of bioengineered insulin driven by a Ca2+-sensitive promoter. Studying tumor xenografts expressing the bioengineered insulin gene, we show that exposure to radio waves stimulates insulin release from the tumors and lowers blood glucose in mice. We further show that cells can be engineered to synthesize genetically encoded ferritin nanoparticles and inducibly release insulin. These approaches provide a platform for using nanotechnology to activate cells.

Metastatic progression of cancer is a complex and clinically daunting process. We previously identified a set of human microRNAs (miRNAs) that robustly suppress breast cancer metastasis to lung and bone and which display expression levels that predict human metastasis. Although these findings revealed miRNAs as suppressors of cell-autonomous metastatic phenotypes, the roles of non-coding RNAs in non-cell-autonomous cancer progression processes remain unknown. Here we reveal that endogenous miR-126, an miRNA silenced in a variety of common human cancers, non-cell-autonomously regulates endothelial cell recruitment to metastatic breast cancer cells, in vitro and in vivo. It suppresses metastatic endothelial recruitment, metastatic angiogenesis and metastatic colonization through coordinate targeting of IGFBP2, PITPNC1 and MERTK--novel pro-angiogenic genes and biomarkers of human metastasis. Insulin-like growth factor binding protein 2 (IGFBP2) secreted by metastatic cells recruits endothelia by modulating IGF1-mediated activation of the IGF type-I receptor on endothelial cells; whereas c-Mer tyrosine kinase (MERTK) receptor cleaved from metastatic cells promotes endothelial recruitment by competitively antagonizing the binding of its ligand GAS6 to endothelial MERTK receptors. Co-injection of endothelial cells with breast cancer cells non-cell-autonomously rescues their miR-126-induced metastatic defect, revealing a novel and important role for endothelial interactions in metastatic initiation. Through loss-of-function and epistasis experiments, we delineate an miRNA regulatory network's individual components as novel and cell-extrinsic regulators of endothelial recruitment, angiogenesis and metastatic colonization. We also identify the IGFBP2/IGF1/IGF1R and GAS6/MERTK signalling pathways as regulators of cancer-mediated endothelial recruitment. Our work further reveals endothelial recruitment and endothelial interactions in the tumour microenvironment to be critical features of metastatic breast cancer.

Highlights d Circulating levels of eNAMPT decline with age in both mice and humans d Increasing eNAMPT promotes NAD + , counteracts aging, and extends healthspan in mice d eNAMPT is contained exclusively in extracellular vesicles (EVs) in mice and humans d Supplementing eNAMPT in EVs improves physical activity

SUMMARY Nicotinamide phosphoribosyltransferase (NAMPT), the key NAD+ biosynthetic enzyme, has two different forms, intra- and extracellular (iNAMPT and eNAMPT), in mammals. However, the significance of eNAMPT secretion remains unclear. Here we demonstrate that deacetylation of iNAMPT by the mammalian NAD+-dependent deacetylase SIRT1 predisposes the protein to secretion in adipocytes. NAMPT mutants reveal that SIRT1 deacetylates lysine 53 and enhances eNAMPT activity and secretion. Adipose tissue-specific Nampt knockout and knockin (ANKO and ANKI) mice show reciprocal changes in circulating eNAMPT, affecting hypothalamic NAD+/SIRT1 signaling and physical activity accordingly. The defect in physical activity observed in ANKO mice is ameliorated by nicotinamide mononucleotide (NMN). Furthermore, administration of a NAMPT-neutralizing antibody decreases hypothalamic NAD+ production, and treating ex vivo hypothalamic explants with purified eNAMPT enhances NAD+, SIRT1 activity, and neural activation. Thus, our findings indicate a critical role of adipose tissue as a modulator for the regulation of NAD+ biosynthesis at a systemic level.

SUMMARY Photoreceptor death is the endpoint of many blinding diseases. Identifying unifying pathogenic mechanisms in these diseases may offer global approaches for facilitating photoreceptor survival. We found that rod or cone photoreceptor-specific deletion of nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme in the major NAD+ biosynthetic pathway beginning with nicotinamide, caused retinal degeneration. In both cases, we could rescue vision with nicotinamide mononucleotide (NMN). Significantly, retinal NAD+ deficiency was an early feature of multiple mouse models of retinal dysfunction, including light-induced degeneration, streptozotocin-induced diabetic retinopathy, and age-associated dysfunction. Mechanistically, NAD+ deficiency caused metabolic dysfunction and consequent photoreceptor death. We further demonstrate that the NAD+-dependent mitochondrial deacylases SIRT3/SIRT5 play important roles in retinal homeostasis and that NAD+ deficiency causes SIRT3 dysfunction. These findings demonstrate that NAD+ biosynthesis is essential for vision, provide a foundation for future work to further clarify the mechanisms involved, and identify a unifying therapeutic target for diverse blinding diseases.

Post-transcriptional regulators have emerged as robust effectors of metastasis and display deregulated expression through unknown mechanisms. Here, we reveal that the human microRNA-335 locus undergoes genetic deletion and epigenetic promoter hypermethylation in every metastatic derivative obtained from independent patients' malignant cell populations. Genetic deletion of miR-335 is a common event in human breast cancer, is enriched for in breast cancer metastases, and also correlates with ovarian cancer recurrence. We furthermore identify miR-335 as a robust inhibitor of tumor reinitiation. We thus implicate the miR-335 locus on 7q32.2 as the first selective metastasis suppressor and tumor initiation suppressor locus in human breast cancer.Supplemental material is available for this article.Received July 24, 2010; revised version accepted December 13, 2010.The progression of carcinoma from its inception to that of a macroscopic, invasive, disseminating, and distal organ colonizing metastasis is the predominant cause of death from solid neoplasms ( The mechanistic dissection of the metastatic cascade has benefitted much from both pioneering mouse xenograft models of human cancer cells (Fidler 2003) and transcriptomic profiling techniques (van de Vijver et al. 2002). In an unbiased search for regulators of metastatic progression, we previously discovered the first set of human small noncoding RNAs (microRNAs [miRNAs]) that suppress breast cancer metastasis to lung and bone (Tavazoie et al. 2008)-two primary sites of clinical metastasis. The expression levels of these metastasis suppressor miRNAs were significantly reduced in the primary breast neoplasms of patients who went on to develop metastases. Two of these miRNAs, miR-335 and miR-206, were found to suppress migration, invasion, and metastatic colonization without inhibiting overall tumor growth, while miR-126 was found to suppress metastasis in part through the inhibition of growth (Tavazoie et al. 2008). miR-335 was found to inhibit metastasis through the targeting of a set of newly identified metastasis genes, including the transcription factor SOX4 and the extracellular matrix protein Tenascin-C (TNC) (Tavazoie et al. 2008)-genes that have been shown recently to also mediate metastatic phenotypes in hepatocellular carcinoma (Liao et al. 2008) and pancreatic adenocarcinoma (Chen et al. 2009), respectively. Here we identify the upstream mechanisms of silencing that impact this robust metastasis suppressor miRNA. In addition, we uncover the etiologic basis for miR-335 expression silencing in primary breast neoplasms, despite its lack of primary tumor growth inhibition. Results and DiscussionTo determine the mechanisms responsible for silencing of the miR-335 metastasis suppressor miRNA, we first turned our attention to the MDA-MB-231 breast cancer population and its in vivo selection derived lung (LM2) and bone (BoM2) metastatic sublines. All of these metastatic derivatives were previously found to display silencing of the miR-335 metastasis suppressor by u...

Individual cells within a tumour can exhibit distinct genetic and molecular features. The impact of such diversification on metastatic potential is unknown. Here we identify clonal human breast cancer subpopulations that display different levels of morphological and molecular diversity. Highly variable subpopulations are more proficient at metastatic colonization and chemotherapeutic survival. Through single-cell RNA-sequencing, inter-cell transcript expression variability is identified as a defining feature of the highly variable subpopulations that leads to protein-level variation. Furthermore, we identify high variability in the spliceosomal machinery gene set. Engineered variable expression of the spliceosomal gene SNRNP40 promotes metastasis, attributable to cells with low expression. Clinically, low SNRNP40 expression is associated with metastatic relapse. Our findings reveal transcriptomic variability generation as a mechanism by which cancer subpopulations can diversify gene expression states, which may allow for enhanced fitness under changing environmental pressures encountered during cancer progression.

Post-transcriptional deregulation is a defining feature of metastatic cancer. While many microRNAs have been implicated as regulators of metastatic progression, less is known about the roles and mechanisms of RNA-binding proteins in this process. We identified muscleblind-like 1 (MBNL1), a gene implicated in myotonic dystrophy, as a robust suppressor of multiorgan breast cancer metastasis. MBNL1 binds the 3 ′ untranslated regions (UTRs) of DBNL (drebrin-like protein) and TACC1 (transforming acidic coiled-coil containing protein 1)-two genes that we implicate as metastasis suppressors. By enhancing the stability of these genes' transcripts, MBNL1 suppresses cell invasiveness. Consistent with these findings, elevated MBNL1 expression in human breast tumors is associated with reduced metastatic relapse likelihood. Our findings delineate a post-transcriptional network that governs breast cancer metastasis through RNA-binding protein-mediated transcript stabilization.