Tumors are phenotypically heterogeneous and include subpopulations of cancer cells with stemlike properties. The natural product salinomycin, a K+-selective ionophore, was recently found to exert selectivity against such cancer stem cells. This selective effect is thought to be due to inhibition of the Wnt signaling pathway, but the mechanistic basis remains unclear. Here, we develop a functionally competent fluorescent conjugate of salinomycin to investigate the molecular mechanism of this compound. By subcellular imaging, we demonstrate a rapid cellular uptake of the conjugate and accumulation in the endoplasmic reticulum (ER). This localization is connected to induction of Ca2+ release from the ER into the cytosol. Depletion of Ca2+ from the ER induces the unfolded protein response as shown by global mRNA analysis and Western blot analysis of proteins in the pathway. In particular, salinomycin-induced ER Ca2+ depletion up-regulates C/EBP homologous protein (CHOP), which inhibits Wnt signaling by down-regulating β-catenin. The increased cytosolic Ca2+ also activates protein kinase C, which has been shown to inhibit Wnt signaling. These results reveal that salinomycin acts in the ER membrane of breast cancer cells to cause enhanced Ca2+ release into the cytosol, presumably by mediating a counter-flux of K+ ions. The clarified mechanistic picture highlights the importance of ion fluxes in the ER as an entry to inducing phenotypic effects and should facilitate rational development of cancer treatments.

Targeting mRNA to eukaryotic cells is an emerging technology for basic research and provides broad applications in cancer immunotherapy, vaccine development, protein replacement, and in vivo genome editing. Although a plethora of nanoparticles for efficient mRNA delivery exists, in vivo mRNA targeting to specific organs, tissue compartments, and cells remains a major challenge. For this reason, methods for reporting the in vivo targeting specificity of different mRNA nanoparticle formats will be crucial. Here, we describe a straightforward method for monitoring the in vivo targeting efficiency of mRNA-loaded nanoparticles in mice. To achieve accurate mRNA delivery readouts, we loaded lipoplex nanoparticles with Cre-recombinase-encoding mRNA and injected these into commonly used Cre reporter mouse strains. Our results show that this approach provides readouts that accurately report the targeting efficacy of mRNA into organs, tissue structures, and single cells as a function of the used mRNA delivery system. The method described here establishes a versatile basis for determining in vivo mRNA targeting profiles and can be systematically applied for testing and improving mRNA packaging formats.

The t(8;21) chromosomal translocation activates aberrant expression of the AML1-ETO (AE) fusion protein and is commonly associated with core binding factor acute myeloid leukaemia (CBF AML). Combining a conditional mouse model that closely resembles the slow evolution and the mosaic AE expression pattern of human t(8;21) CBF AML with global transcriptome sequencing, we find that disease progression was characterized by two principal pathogenic mechanisms. Initially, AE expression modified the lineage potential of haematopoietic stem cells (HSCs), resulting in the selective expansion of the myeloid compartment at the expense of normal erythro- and lymphopoiesis. This lineage skewing was followed by a second substantial rewiring of transcriptional networks occurring in the trajectory to manifest leukaemia. We also find that both HSC and lineage-restricted granulocyte macrophage progenitors (GMPs) acquired leukaemic stem cell (LSC) potential being capable of initiating and maintaining the disease. Finally, our data demonstrate that long-term expression of AE induces an indolent myeloproliferative disease (MPD)-like myeloid leukaemia phenotype with complete penetrance and that acute inactivation of AE function is a potential novel therapeutic option.

BRCA1-associated protein 1 (BAP1) is a nuclear deubiquitinating enzyme that is associated with multiprotein complexes that regulate key cellular pathways, including cell cycle, cellular differentiation, cell death, and the DNA damage response. In this study, we found that the reduced expression of BAP1 pro6motes the survival of neuroblastoma cells, and restoring the levels of BAP1 in these cells facilitated a delay in S and G2/M phase of the cell cycle, as well as cell apoptosis. The mechanism that BAP1 induces cell death is mediated via an interaction with 14-3-3 protein. The association between BAP1 and 14-3-3 protein releases the apoptotic inducer protein Bax from 14-3-3 and promotes cell death through the intrinsic apoptosis pathway. Xenograft studies confirmed that the expression of BAP1 reduces tumor growth and progression in vivo by lowering the levels of pro-survival factors such as Bcl-2, which in turn diminish the survival potential of the tumor cells. Patient data analyses confirmed the finding that the high-BAP1 mRNA expression correlates with a better clinical outcome. In summary, our study uncovers a new mechanism for BAP1 in the regulation of cell apoptosis in neuroblastoma cells.

Colorectal cancer is one of the most commonly diagnosed cancers and the third most common cause of cancer-related death. Metastasis is the leading reason for the resultant mortality of these patients. Accordingly, development and characterization of novel anti-cancer drugs limiting colorectal tumor cell dissemination and metastasis are needed. In this study, we found that the small molecule Reversine reduces the migration potential of human colon carcinoma cells in vitro. A coupled kinase assay with bio-informatics approach identified the c-Jun N-terminal kinase (JNK) cascade as the main pathway inhibited by Reversine. Knockdown experiments and pharmacological inhibition identified JNK1 but not JNK2, as a downstream effector target in cancer cell migration. Xenograft experiments confirm the effect of JNK inhibition in the metastatic potential of colon cancer cells. These results highlight the impact of individual JNK isoforms in cancer cell metastasis and propose Reversine as a novel anti-cancer molecule for treatment of colon cancer patients.

Neuroblastoma is the most common paediatric cancer type. Patients diagnosed with high-risk neuroblastoma have poor prognosis and occasionally tumours relapse. As a result, novel treatment strategies are needed for relapse and refractory neuroblastoma patients. Here, we found that high expression of Mps1 kinase (mitotic kinase Monopolar Spindle 1) was associated with relapsefree neuroblastoma patient outcomes and poor overall survival. Silencing and inhibition of Mps1 in neuroblastoma or PDX-derived cells promoted cell apoptosis via the caspase-dependent mitochondrial apoptotic pathway. The mechanism of cell death upon Mps1 inhibition was dependent on the polyploidization/aneuploidization of the cells before undergoing mitotic catastrophe. Furthermore, tumour growth retardation was confirmed in a xenograft mouse model after Mps1inhibitor treatment. Altogether, these results suggest that Mps1 expression and inhibition can be considered as a novel prognostic marker as well as a therapeutic strategy for the treatment of highrisk neuroblastoma patients. Neuroblastoma is the most common extra-cranial solid tumour of childhood 1,2 causing approximately 10% of all childhood cancer-related deaths 3,4. Neuroblastoma is a very heterogeneous tumour compared to other cancers. It can regress spontaneously, however, in certain cases, can undergo treatment resistance 5,6. The International Neuroblastoma Risk Group (INRG) classifies neuroblastoma tumours into very low-risk, low-risk, intermediaterisk, and high-risk 7,8. Identification of the high-risk group is important for the development of personalised medicine for neuroblastoma patients with poor prognosis. Good prognosis has been observed in children with low-or intermediate-risk after chemotherapy in combination with surgical intervention. High-risk neuroblastoma represent 40% of all diagnosed cases, and these patients have poor prognosis harbouring tumour relapse and chemoresistance, and only 50% will attain long-term survival 9,10. MYCN gene amplification has been observed in less than half of the high-risk tumours 11. In non-MYCN high-risk neuroblastoma, point mutations in TP53 and Anaplastic Lymphoma Kinase (ALK) gene mutation have been observed 12 in less than 10% of neuroblastomas 13. Recently, targeting cell cycle and in particular mitosis has been proposed as an alternative therapeutic strategy for cancer treatment 14. Spindle Assembly Checkpoint or SAC generally monitor proper mitosis by controlling the correct attachment of the chromosomes to the microtubule spindle apparatus via their kinetochores 15. Once the chromosomes are fully arranged on the metaphase plate, the SAC is turned off, and chromosome segregation as well as cell division can be engaged 16. The Mps1 kinase (Monopolar spindle1) is an important regulator of the SAC and it phosphorylates target proteins principally on tyrosines, serines, and threonines 17. The most important function of Mps1 is to ensure proper biorientation of sister chromatids on the mitotic spindle at kinetochores. In early mitosi...

BackgroundConditional gene activation is an efficient strategy for studying gene function in genetically modified animals. Among the presently available gene switches, the tetracycline-regulated system has attracted considerable interest because of its unique potential for reversible and adjustable gene regulation.ResultsTo investigate whether the ubiquitously expressed Gt(ROSA)26Sor locus enables uniform DOX-controlled gene expression, we inserted the improved tetracycline-regulated transcription activator iM2 together with an iM2 dependent GFP gene into the Gt(ROSA)26Sor locus, using gene targeting to generate ROSA26-iM2-GFP (R26t1Δ) mice. Despite the presence of ROSA26 promoter driven iM2, R26t1Δ mice showed very sparse DOX-activated expression of different iM2-responsive reporter genes in the brain, mosaic expression in peripheral tissues and more prominent expression in erythroid, myeloid and lymphoid lineages, in hematopoietic stem and progenitor cells and in olfactory neurons.ConclusionsThe finding that gene regulation by the DOX-activated transcriptional factor iM2 in the Gt(ROSA)26Sor locus has its limitations is of importance for future experimental strategies involving transgene activation from the endogenous ROSA26 promoter. Furthermore, our ROSA26-iM2 knock-in mouse model (R26t1Δ) represents a useful tool for implementing gene function in vivo especially under circumstances requiring the side-by-side comparison of gene manipulated and wild type cells. Since the ROSA26-iM2 mouse allows mosaic gene activation in peripheral tissues and haematopoietic cells, this model will be very useful for uncovering previously unknown or unsuspected phenotypes.

CD8 DC-targeting trifunctional nanocarriers bear significant potential for antitumor immunotherapy.