Embryonic stem (ES) cells represent a suitable model to analyze cell differentiation processes in vitro. Here, we report that pluripotent ES cells of the line BLC 6 differentiate in vitro into neuronal cells possessing the complex electrophysiological and immunocytochemical properties of postmitotic nerve cells. In the course of differentiation BLC 6-derived neurons differentially express voltage-dependent (K+, Na+, Ca2+) and receptor-operated (GABAA, glycine, AMPA, NMDA receptors) ionic channels. They generate fast Na(+)-driven action potentials and are functionally coupled by inhibitory (GABAergic) and excitatory (glutamatergic) synapses as revealed by measurements of postsynaptic currents. Moreover, BLC 6-derived neurons express neuron-specific cytoskeletal, cell adhesion and synaptic vesicle proteins and exhibit a Ca(2+)-dependent GABA secretion. Thus, the ES cell model enables the investigation of cell lineage determination and signaling mechanisms in the developing nervous system from a pluripotential stem cell to a differentiated postmitotic neuron. The in vitro differentiation of neurons from ES cells may be an excellent approach to study by targeted gene disruption a variety of neuronal functions.

Aerobic glycolysis (the Warburg effect) facilitates tumor growth, and drugs targeting aerobic glycolysis are being developed. However, how the Warburg effect is directly regulated is largely unknown. Here we show that transcription factor SIX1 directly increases the expression of many glycolytic genes, promoting the Warburg effect and tumor growth in vitro and in vivo. SIX1 regulates glycolysis through HBO1 and AIB1 histone acetyltransferases. Cancer-related SIX1 mutation increases its ability to promote aerobic glycolysis and tumor growth. SIX1 glycolytic function is directly repressed by microRNA-548a-3p, which is downregulated, inversely correlates with SIX1, and is a good predictor of prognosis in breast cancer patients. Thus, the microRNA-548a-3p/SIX1 axis strongly links aerobic glycolysis to carcinogenesis and may become a promising cancer therapeutic target.

Dysregulation of the epidermal growth factor receptor (EGFR) promotes cancer cell growth, invasion and metastasis. However, its relevant downstream effectors are still limited. Here, we show that EGFR promotes breast tumor growth and metastasis by downregulating the tumor suppressor micoRNA-338-3p (miR-338-3p) and activating the EYA2 (EYA transcriptional coactivator and phosphatase 2) oncoprotein. EGFR represses miR-338-3p expression largely through HIF1α transcription factor. miR-338-3p inhibits EYA2 expression by binding to the 3′-untranslated region of EYA2. EGFR increases EYA2 expression via HIF1α repression of miR-338-3p. Through the miR-338-3p/EYA2 pathway, EGFR increases breast cancer cell growth, epithelial-to-mesenchymal transition, migration, invasion and lung metastasis in vitro and in a allograft tumor mouse model in vivo. In breast cancer patients, miR-338-3p expression negatively correlates with the expression of EGFR and EYA2, EGFR status positively associates with EYA2 expression, and miR-338-3p and EYA2 predict breast cancer lung metastasis when expressed in primary breast cancers. These data suggest that the miR-338-3p/ EYA2 axis contributes to EGFR-mediated tumor growth and lung metastasis and that miR-338-3p activation or EYA2 inhibition or combination therapy targeting EGFR/miR-338-3p/EYA2 axis may be a promising way to treat patients with metastatic cancer. Cell Death and Disease (2017) 8, e2928; doi:10.1038/cddis.2017.325; published online 13 July 2017The epidermal growth factor receptor (EGFR) is a member of the ErbB (avian erythroblastosis oncogene B) family of receptors and activates multiple signaling pathways, including mitogen-activated protein kinase (MAPK)/extracellular signalregulated kinases (ERK) and phosphoinositide-3-kinase (PI3K)/V-AKT murine thymoma viral oncogene homolog (AKT) pathways. 1-3 EGFR activation regulates many biological processes, such as cell proliferation, invasion, metastasis and apoptosis. [4][5][6] EGFR is overexpressed in various human cancers, including lung cancer, breast cancer, colon cancer and glioblastoma, and is associated with tumor malignancy and poor prognosis. 7-10 Thus, EGFR and its downstream signaling effectors have become targets for cancer therapy. 11 Approximately 90% of deaths associated with cancer are due to distant metastases. 12 Many cancers can metastasize anywhere in body but primarily metastasizes to some organs or tissues. For instance, lungs and bones are frequent sites of breast cancer metastasis. Although EGFR dysregulation enhances cancer metastasis, the relevant downstream effectors are largely unknown.MicroRNAs (miRNAs) are small noncoding RNA molecules (about 22 nucleotides in length), which function in RNA silencing and post-transcriptional regulation of gene expression. miRNAs participate in many biological processes, such as cell proliferation, invasion, metastasis, and apoptosis. 13 Recently, EGFR has been shown to promote prostate cancer bone metastasis by decreasing the expression of miR-1, a tumor suppressor, and inc...

Integrin cell surface receptors play an important role for cell adhesion, migration, and differentiation during embryonic development by mediating cell-cell and cell-matrix interactions. Less is known about the function of integrins during commitment and lineage determination of early embryogenesis. Homozygous inactivation of the beta1 integrin gene results in embryonal death in mice around the time of implantation. In vitro, differentiation of embryonic stem (ES) cells which lack beta1 integrin (beta1-/-) into the cardiogenic lineage is delayed and results in a disordered cellular specification (Fässler et al., J. Cell Sci. 109, 2989-2999, 1996). To analyze beta1 integrin function during myogenesis and neurogenesis we studied differentiation of beta1-/- ES cells via embryoid bodies into skeletal muscle and neuronal cells in vitro. beta1-/- cells showed delayed and reduced myogenic differentiation compared to wildtype and heterozygous (beta1+/-) ES cells. RT-PCR analysis demonstrated delayed expression of skeletal muscle-specific genes in the absence of beta1 integrin. Immunofluorescence studies with antibodies against the sarcomeric proteins myosin heavy chain, titin, nebulin, and slow C-protein showed that myotubes formed, but their number was reduced and the assembly of sarcomeric structures was retarded. In contrast, neuronal cells differentiating from beta1-/- ES cells appeared earlier than wildtype and heterozygous (beta1+/-) ES cells. This was shown by the accelerated expression of neuron-specific genes and an increased number of neuronal cells in beta1-/- embryoid bodies. However, neuronal outgrowth was retarded in the absence of beta1 integrin. No functional difference between wildtype and beta1-/- cells was found with respect to secretion of gamma-aminobutyric acid, the main neurotransmitter of ES cell-derived neuronal cells. The lineage-specific effects of loss of beta1 integrin function, that is the inhibition of mesodermal and acceleration of neuroectodermal differentiation, were supported by differential expression of genes encoding lineage-specific transcription factors (Brachyury, Pax-6, Mash1) and signaling molecules (BMP-4 and Wnt-1). Because of the reduced and delayed expression of the BMP-4 encoding gene in beta1-/- cells, we analyzed in wildtype and beta1-/- cells the regulatory role of exogenously added BMP-4 on the expression of the mesodermal and neuronal marker genes, Brachyury and wnt-1, respectively. The data suggest that BMP-4 plays a regulatory role during differentiation of wildtype and beta1-/- cells by modifying mesodermal and neuronal pathways. The reduced expression of BMP-4 in beta1-/- cells may account for the accelerated neuronal differentiation in beta1-/- ES cells.

Tumour radiotherapy resistance involves the cell cycle pathway. CDC25 phosphatases are key cell cycle regulators. However, how CDC25 activity is precisely controlled remains largely unknown. Here, we show that LIM domain-containing proteins, such as FHL1, increase inhibitory CDC25 phosphorylation by forming a complex with CHK2 and CDC25, and sequester CDC25 in the cytoplasm by forming another complex with 14-3-3 and CDC25, resulting in increased radioresistance in cancer cells. FHL1 expression, induced by ionizing irradiation in a SP1- and MLL1-dependent manner, positively correlates with radioresistance in cancer patients. We identify a cell-penetrating 11 amino-acid motif within LIM domains (eLIM) that is sufficient for binding CHK2 and CDC25, reducing the CHK2–CDC25 and CDC25–14-3-3 interaction and enhancing CDC25 activity and cancer radiosensitivity accompanied by mitotic catastrophe and apoptosis. Our results provide novel insight into molecular mechanisms underlying CDC25 activity regulation. LIM protein inhibition or use of eLIM may be new strategies for improving tumour radiosensitivity.

Two ribosome-inactivating proteins, trichosanthin and alpha-momorcharin, have been studied in the forms of complexes with ATP or formycin, by an X-ray-crystallographic method at 1.6-2.0 A (0.16-0.20 nm) resolution. The native alpha-momorcharin had been studied at 2.2 A resolution. Structures of trichosanthin were determined by a multiple isomorphous replacement method. Structures of alpha-momorcharin were determined by a molecular replacement method using refined trichosanthin as the searching model. Small ligands in all these complexes have been recognized and built on the difference in electron density. All these structures have been refined to achieve good results, both in terms of crystallography and of ideal geometry. These two proteins show considerable similarity in their three-dimensional folding and to that of related proteins. On the basis of these structures, detailed geometries of the active centres of these two proteins are described and are compared with those of related proteins. In all complexes the interactions between ligand atoms and protein atoms, including hydrophobic forces, aromatic stacking interactions and hydrogen bonds, are found to be specific towards the adenine base. The relationship between the sequence conservation of ribosome-inactivating proteins and their active-centre geometry was analysed. A depurinating mechanism of ribosome-inactivating proteins is proposed on the basis of these results. The N-7 atom of the substrate base group is proposed to be protonated by an acidic residue in the active centre.

Many physiologically based pharmacokinetic (PBPK) models for environmental chemicals, drugs, and nanomaterials have been developed to aid risk and safety assessments using acslX. However, acslX has been rendered sunset since November 2015. Alternative modeling tools and tutorials are needed for future PBPK applications. This forum article aimed to: (1) demonstrate the performance of 4 PBPK modeling software packages (acslX, Berkeley Madonna, MATLAB, and R language) tested using 2 existing models (oxytetracycline and gold nanoparticles); (2) provide a tutorial of PBPK model code conversion from acslX to Berkeley Madonna, MATLAB, and R language; (3) discuss the advantages and disadvantages of each software package in the implementation of PBPK models in toxicology, and (4) share our perspective about future direction in this field. Simulation results of plasma/tissue concentrations/amounts of oxytetracycline and gold from different models were compared visually and statistically with linear regression analyses. Simulation results from the original models were correlated well with results from the recoded models, with time-concentration/amount curves nearly superimposable and determination coefficients of 0.86-1.00. Step-by-step explanations of the recoding of the models in different software programs are provided in the Supplementary Data. In summary, this article presents a tutorial of PBPK model code conversion for a small molecule and a nanoparticle among 4 software packages, and a performance comparison of these software packages in PBPK model implementation. This tutorial helps beginners learn PBPK modeling, provides suggestions for selecting a suitable tool for future projects, and may lead to the transition from acslX to alternative modeling tools.

BCL-2 is a novel direct target of miR-30a-5p. miR-30a-5p enhances NSCLC paclitaxel sensitivity in vitro and in vivo. miR-30a-5p sensitizes NSCLC cells to paclitaxel by inducing apoptosis through BCL-2 inhibition. miR-30a-5p negatively correlates with BCL-2 and predicts a favorable clinical outcome in NSCLC patients.