Pancreatic ductal adenocarcinomas (PDAC) are highly metastatic with poor prognosis, mainly due to delayed detection. We hypothesized that intercellular communication is critical for metastatic progression. Here, we show that PDAC- derived exosomes induce liver pre-metastatic niche formation in naïve mice and consequently increase liver metastatic burden. Uptake of PDAC-derived exosomes by Kupffer cells caused transforming growth factor β secretion and upregulation of fibronectin production by hepatic stellate cells. This fibrotic microenvironment enhanced recruitment of bone marrow-derived macrophages. We found that macrophage migration inhibitory factor (MIF) was highly expressed in PDAC-derived exosomes, and its blockade prevented liver pre-metastatic niche formation and metastasis. Compared to patients whose pancreatic tumors did not progress, MIF was markedly higher in exosomes from stage I PDAC patients who later developed liver metastasis. These findings suggest that exosomal MIF primes the liver for metastasis and may be a prognostic marker for the development of PDAC liver metastasis.

Summary Tumor-secreted extracellular vesicles (EVs) are critical mediators of intercellular communication between tumor cells and stromal cells in local and distant microenvironments. Accordingly, EVs play an essential role in both primary tumor growth and metastatic evolution. EVs orchestrate multiple systemic pathophysiological processes, such as coagulation, vascular leakiness, and reprogramming of stromal recipient cells to support pre-metastatic niche formation and subsequent metastasis. Clinically, EVs may be biomarkers and novel therapeutic targets for cancer progression, particularly for predicting and preventing future metastatic development.

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SignificanceIncreasing evidence suggests that extracellular vesicles (EVs) can transfer genetic material to recipient cells. However, the mechanism and role of this phenomenon are largely unknown. Here we have made a remarkable discovery: EVs can harbor the full mitochondrial genome. These extracellular vesicles can in turn transfer their mtDNA to cells with impaired metabolism, leading to restoration of metabolic activity. We determined that hormonal therapy induces oxidative phosphorylation-deficient breast cancer cells, which can be rescued via the transfer of mtDNA-laden extracellular vesicles. Horizontal transfer of mtDNA occurred in cancer stem-like cells and was associated with increased self-renewal potential of these cells, leading to resistance to hormonal therapy. We propose that mtDNA transfer occurs in human cancer via EVs.

Rapid deadenylation was impaired, indicating a crucial role for KSRP in this step of mRNA degradation. The two IL-8 ARE domains both contribute to interaction with KSRP, corresponding to the importance of both domains for rapid degradation. Exposure to the inflammatory cytokine IL-1 has been shown to stabilize IL-8 mRNA through p38 mitogen-activated protein (MAP) kinase and MK2. IL-1 treatment impaired the interaction of KSRP with the IL-8 ARE in a manner dependent on p38 MAP kinase but apparently independent of MK2. Instead, evidence that TTP, a target of MK2, can also destabilize the IL-8 ARE reporter mRNA is presented. In a comprehensive approach to identify mRNAs controlled by KSRP, two criteria were evaluated by microarray analysis of (i) association of mRNAs with KSRP in pulldown assays and (ii) increased amounts in KSRP knockdown cells. According to both criteria, a group of 100 mRNAs is controlled by KSRP, many of which are unstable and encode proteins involved in inflammation. These results indicate that KSRP functions as a limiting factor in inflammatory gene expression.Rapid degradation controls the levels of many mRNAs that are translated into transiently expressed proteins. These include cytokines, growth factors, proto-oncogene products, and other proteins participating in acute reactions. The short half-lives of their mRNAs depend on regulatory RNA sequences, the most widely distributed being AU-rich elements (AREs) located in their 3Ј untranslated regions (UTRs) (8,39). AREs have been divided into three classes, differing in the sequences and modes of degradation imposed by them (8). Class I AREs contain one to three scattered AUUUA motifs, and class II AREs contain multiple overlapping AUUUA motifs. Class III AREs are less well defined and lack an AUUUA motif. With the search pattern WWWU(AUUUA)UUUW, 4,000 human mRNAs have been reported to contain AREs and grouped into the ARED database (1), where the class II AREs are further subdivided into different groups, depending on the number of AUUUA motifs present in an ARE.Interleukin-8 (IL-8) is a member of the CXC chemokine family, released from different types of cells in response to direct cell stress, pathogens, or the proinflammatory cytokines tumor necrosis factor (TNF) and IL-1 (reference 25 and references therein). It attracts and activates leukocytes and also plays a role in angiogenesis. Studying its induction in response to IL-1, we previously observed that in addition to transcriptional activation of the IL-8 gene, its mRNA is stabilized (26,46). The latter response involves the activation of p38 mitogenactivated protein (MAP) kinase and its substrate kinase MK2. Stabilization of IL-8 mRNA can contribute to enhanced IL-8 expression, e.g., in viral infection (22). Our recent studies showed that the IL-8 mRNA contains an ARE which consists of two functionally distinct domains. They cooperate for maximal destabilization and interaction with cytoplasmic proteins in vitro (44).Control of mRNA degradation by AREs involves the function of protei...

We identified nicotinamide phosphoribosyltransferase (NAMPT), also known as pre-B cell colony enhancing factor (PBEF), as an essential enzyme mediating granulocyte colony-stimulating factor (G-CSF)-triggered granulopoiesis in healthy individuals and in individuals with severe congenital neutropenia. Intracellular NAMPT and NAD(+) amounts in myeloid cells, as well as plasma NAMPT and NAD(+) levels, were increased by G-CSF treatment of both healthy volunteers and individuals with congenital neutropenia. NAMPT administered both extracellularly and intracellularly induced granulocytic differentiation of CD34(+) hematopoietic progenitor cells and of the promyelocytic leukemia cell line HL-60. Treatment of healthy individuals with high doses of vitamin B3 (nicotinamide), a substrate of NAMPT, induced neutrophilic granulocyte differentiation. The molecular events triggered by NAMPT include NAD(+)-dependent sirtuin-1 activation, subsequent induction of CCAAT/enhancer binding protein-alpha and CCAAT/enhancer binding protein-beta, and, ultimately, upregulation of G-CSF synthesis and G-CSF receptor expression. G-CSF, in turn, further increases NAMPT levels. These results reveal a decisive role of the NAD(+) metabolic pathway in G-CSF-triggered myelopoiesis.

Pathways defining susceptibility of normal cells to oncogenic transformation may be valuable therapeutic targets. We characterized the cell of origin and its critical pathways in MN1-induced leukemias. Common myeloid (CMP) but not granulocyte-macrophage progenitors (GMP) could be transformed by MN1. Complementation studies of CMP-signature genes in GMPs demonstrated that MN1-leukemogenicity required the MEIS1/AbdB-like HOX-protein complex. ChIP-sequencing identified common target genes of MN1 and MEIS1 and demonstrated identical binding sites for a large proportion of their chromatin targets. Transcriptional repression of MEIS1 targets in established MN1 leukemias demonstrated antileukemic activity. As MN1 relies on but cannot activate expression of MEIS1/AbdB-like HOX proteins, transcriptional activity of these genes determines cellular susceptibility to MN1-induced transformation and may represent a promising therapeutic target.

Rett syndrome is a neurological, X chromosomal-linked disorder associated with mutations in the MECP2 gene. MeCP2 protein has been proposed to play a role in transcriptional regulation as well as in chromatin architecture. Since MeCP2 mutant cells exhibit surprisingly mild changes in gene expression, we have now explored the possibility that Rett mutations may affect the ability of MeCP2 to bind and organize chromatin. We found that all but one of the 21 missense MeCP2 mutants analyzed accumulated at heterochromatin and about half of them were significantly affected. Furthermore, two-thirds of all mutants showed a significantly decreased ability to cluster heterochromatin. Three mutants containing different proline substitutions (P101H, P101R and P152R) were severely affected only in heterochromatin clustering and located far away from the DNA interface in the MeCP2 methyl-binding domain structure. MeCP2 mutants affected in heterochromatin accumulation further exhibited the shortest residence time on heterochromatin, followed by intermediate binding kinetics for clustering impaired mutants. We propose that different interactions of MeCP2 with methyl cytosines, DNA and likely other heterochromatin proteins are required for MeCP2 function and their dysfunction lead to Rett syndrome.