The long non-coding RNA MALAT1, also known as MALAT-1 or NEAT2, is a highly conserved nuclear ncRNA and a predictive marker for metastasis development in lung cancer. To uncover its functional importance, we developed a MALAT1 knockout model in human lung tumor cells by genomically integrating RNA destabilizing elements using Zinc Finger Nucleases. The achieved 1000-fold MALAT1 silencing provides a unique loss-of-function model. Proposed mechanisms of action include regulation of splicing or gene expression. In lung cancer, MALAT1 does not alter alternative splicing but actively regulates gene expression including a set of metastasis-associated genes. Consequently, MALAT1-deficient cells are impaired in migration and form fewer tumor nodules in a mouse xenograft. Antisense oligonucleotides blocking MALAT1 prevent metastasis formation after tumor implantation. Thus, targeting MALAT1 with antisense oligonucleotides provides a potential therapeutic approach to prevent lung cancer metastasis with MALAT1 serving as both, predictive marker and therapeutic target. Lastly, regulating gene expression, but not alternative splicing is the critical function of MALAT1 in lung cancer metastasis. In summary, ten years after the discovery of the lncRNA MALAT1 as a biomarker for lung cancer metastasis, our loss-of-function model unravels the active function of MALAT1 as a regulator of gene expression governing hallmarks of lung cancer metastasis.

In Alzheimer's disease (AD), memory impairment is the most prominent feature that afflicts patients and their families. Although reactive astrocytes have been observed around amyloid plaques since the disease was first described, their role in memory impairment has been poorly understood. Here, we show that reactive astrocytes aberrantly and abundantly produce the inhibitory gliotransmitter GABA by monoamine oxidase-B (Maob) and abnormally release GABA through the bestrophin 1 channel. In the dentate gyrus of mouse models of AD, the released GABA reduces spike probability of granule cells by acting on presynaptic GABA receptors. Suppressing GABA production or release from reactive astrocytes fully restores the impaired spike probability, synaptic plasticity, and learning and memory in the mice. In the postmortem brain of individuals with AD, astrocytic GABA and MAOB are significantly upregulated. We propose that selective inhibition of astrocytic GABA synthesis or release may serve as an effective therapeutic strategy for treating memory impairment in AD.

Genome-wide analyses have identified thousands of long noncoding RNAs (lncRNAs). Malat1 (metastasisassociated lung adenocarcinoma transcript 1) is among the most abundant lncRNAs whose expression is altered in numerous cancers. Here we report that genetic loss or systemic knockdown of Malat1 using antisense oligonucleotides (ASOs) in the MMTV (mouse mammary tumor virus)-PyMT mouse mammary carcinoma model results in slower tumor growth accompanied by significant differentiation into cystic tumors and a reduction in metastasis. Furthermore, Malat1 loss results in a reduction of branching morphogenesis in MMTV-PyMTand Her2/neu-amplified tumor organoids, increased cell adhesion, and loss of migration. At the molecular level, Malat1 knockdown results in alterations in gene expression and changes in splicing patterns of genes involved in differentiation and protumorigenic signaling pathways. Together, these data demonstrate for the first time a functional role of Malat1 in regulating critical processes in mammary cancer pathogenesis. Thus, Malat1 represents an exciting therapeutic target, and Malat1 ASOs represent a potential therapy for inhibiting breast cancer progression.

Next-generation sequencing technologies have greatly expanded our understanding of cancer genetics. Antisense technology is an attractive platform with the potential to translate these advances into improved cancer therapeutics, because antisense oligonucleotide (ASO) inhibitors can be designed on the basis of gene sequence information alone. Recent human clinical data have demonstrated the potent activity of systemically administered ASOs targeted to genes expressed in the liver. Here, we describe the preclinical activity and initial clinical evaluation of a class of ASOs containing constrained ethyl modifications for targeting the gene encoding the transcription factor STAT3, a notoriously difficult protein to inhibit therapeutically. Systemic delivery of the unformulated ASO, AZD9150, decreased STAT3 expression in a broad range of preclinical cancer models and showed antitumor activity in lymphoma and lung cancer models. AZD9150 preclinical activity translated into single-agent antitumor activity in patients with highly treatment-refractory lymphoma and non-small cell lung cancer in a phase I dose escalation study.

The DNA polymerase from Thermus aquaticus (Taq polymerase), famous for its use in the polymerase chain reaction, is homologous to Escherichia coli DNA polymerase I (pol I) Like pol I, Taq polymerase has a domain at its amino terminus (residues 1-290) that has 5' nuclease activity and a domain at its carboxy terminus that catalyses the polymerase reaction. Unlike pol I, the intervening domain in Taq polymerase has lost the editing 3'-5' exonuclease activity. Although the structure of the Klenow fragment of pol I has been known for ten years, that of the intact pol I has proved more elusive. The structure of Taq polymerase determined here at 2.4 A resolution shows that the structures of the polymerase domains of the thermostable enzyme and of the Klenow fragment are nearly identical, whereas the catalytically critical carboxylate residues that bind two metal ions are missing from the remnants of the 3'-5' exonuclease active site of Taq polymerase. The first view of the 5' nuclease domain, responsible for excising the Okazaki RNA in lagging-strand DNA replication, shows a cluster of conserved divalent metal-ion-binding carboxylates at the bottom of a cleft. The location of this 5'-nuclease active site some 70 A from the polymerase active site in this crystal form highlights the unanswered question of how this domain works in concert with the polymerase domain to produce a duplex DNA product that contains only a nick.

Considerable interest has emerged in the possibility of exploiting the apoptotic effects of TRAIL 1 for the treatment of cancer. TRAIL is a member of the tumor necrosis factor (TNF) family of cytokines that is capable of inducing apoptosis (1).The apoptosis-inducing receptors for TRAIL include Trail-R1 (DR4) and Trail-R2 (DR5), which are transmembrane type I receptors expressed on the surface of many types of cell. However, TRAIL also binds to non-apoptosis-inducing decoy receptors, which compete with death receptors for ligand and suppress apoptosis, including DcR1, DcR2, and osteoprotegerin (reviewed in Refs. 2 and 3). Empiric analysis of the effects of TRAIL on normal and malignant cells has provided compelling evidence that recombinant TRAIL protein preferentially induces apoptosis of cancer cells without harming most types of untransformed cells (reviewed in Ref. 2). When properly prepared and purified, recombinant trimeric TRAIL also lacks significant toxicity in primate species that possess receptors capable of binding human TRAIL (4, 5).Preclinical studies of recombinant TRAIL (extracellular domain) in mice have demonstrated impressive anti-tumor activity and synergy with cytotoxic anticancer drugs (6). However, not all tumors respond to TRAIL. This lack of response may be attributed either to unfavorable ratios of death and decoy receptors or because of intracellular resistance mechanisms (3, 7-10). With respect to intracellular resistance mechanisms, the FLIP protein has been identified as a blocker of apoptosis induced by TNF family death receptors (reviewed in Ref. 11). FLIP binds to and neutralizes adapter proteins and procaspases normally recruited to the cytosolic domains of apoptosis-inducing TRAIL receptors upon ligand stimulation, thus interrupting early steps in TRAIL signaling. Furthermore, overexpression of FLIP protein has been documented in cancers (12).PPAR␥ is a member of the steroid/retinoid superfamily of ligand-activated transcription factors. Agonistic ligands of PPAR␥ include modified fatty acids, cyclopentenone-containing prostaglandins, triterpenoids, and the thiazolidinediones, a class of insulin-sensitizing drugs used in the treatment of type II diabetes (reviewed in Ref. 13). Anti-tumor properties of PPAR␥ agonists have been reported. For example, thiazolidinediones have been shown to suppress the growth of human colon and breast cancer cell lines in vitro and in vivo in the mouse (14, 15), and a member of a new class of PPAR␥ agonists (tyrosine analogs) suppresses mammary carcinogenesis in a standard rat model (16). However, troglitazone increases incidence of colonic polyps in a mouse model in which one allele of adenomatous polyposis coli is inactive (17, 18), suggesting complex effects on neoplasia. Moreover, the concentrations of thiazolidinediones required for some apoptotic effects are beyond clinically attainable ranges (15).Here we explored the effects of PPAR␥ ligands on TRAILinduced apoptosis in epithelial cancers cell lines. Our findings demonstrate a new PPAR␥-inde...

The conversion of castasterone (CS) to brassinolide (BL), a Baeyer-Villiger oxidation, represents the final and rate-limiting step in the biosynthesis of BL in plants. Heterologously expressed Arabidopsis thaliana CYP85A2 in yeast mediated the conversion of CS to BL as well as the C-6 oxidation of brassinosteroids (BRs). This indicated that CYP85A2 is a bifunctional enzyme that possesses BR C-6 oxidase and BL synthase activity. CYP85A2 is thus a cytochrome P450 that mediates Baeyer-Villiger oxidation in plants. Biochemical, physiological, and molecular genetic analyses of Arabidopsis CYP85A2 loss-of-function and overexpression lines demonstrated that CS has to be a bioactive BR that controls the overall growth and development of Arabidopsis plants. Mutant studies also revealed that BL may not always be necessary for normal growth and development but that Arabidopsis plants acquire great benefit in terms of growth and development in the presence of BL

The concept of reprogramming of somatic cells has opened a new era in regenerative medicine. Transduction of defined factors has successfully achieved pluripotency. However, during the generation process of induced pluripotent stem (iPS) cells, genetic manipulation of certain factors may cause tumorigenicity, which limits further application. We report that that a single transfer of embryonic stem (ES) cell-derived proteins into primarily cultured adult mouse fibroblasts, rather than repeated transfer or prolonged exposure to materials, can achieve full reprogramming up to the pluripotent state without the forced expression of ectopic transgenes. During the process, gene expression and epigenetic status were converted from somatic to ES-equivalent status. We verified that protein-based reprogramming was neither by the contamination of protein do-