Secreted PGK1 and IGFBP2 contribute to the bystander effect of miR-10b gene editing in glioma.

MicroRNA-10b (miR-10b) is an essential glioma driver and one of the top candidates for targeted therapies for glioblastoma and other cancers. This unique miRNA controls glioma cell cycle and viability via an array of established conventional and unconventional mechanisms. Previously reported CRISPR-Cas9-mediated miR-10b gene editing of glioma cells in vitro and established orthotopic glioblastoma in mouse models demonstrated the efficacy of this approach and its promise for therapy development. However, therapeutic gene editing in patients' brain tumors may be hampered, among other factors, by the imperfect delivery and distribution of targeting vectors. Here, we demonstrate that miR-10b gene editing in glioma cells triggers a potent bystander effect that leads to the selective cell death of the unedited glioma cells without affecting the normal neuroglial cells. The effect is mediated by the secreted miR-10b targets phosphoglycerate kinase 1 (PGK1) and insulin-like growth factor binding protein 2 (IGFBP2) that block cell-cycle progression and induce glioma cell death. These findings further support the feasibility of therapeutic miR-10b editing without the need to target every cell of the tumor.

Identification of kinases activated by multiple pro-angiogenic growth factors.

Antiangiogenic therapy began as an effort to inhibit VEGF signaling, which was thought to be the sole factor driving tumor angiogenesis. It has become clear that there are more pro-angiogenic growth factors that can substitute for VEGF during tumor vascularization. This has led to the development of multi-kinase inhibitors which simultaneously target multiple growth factor receptors. These inhibitors perform better than monotherapies yet to date no multi-kinase inhibitor targets all receptors known to be involved in pro-angiogenic signaling and resistance inevitably occurs. Given the large number of pro-angiogenic growth factors identified, it may be impossible to simultaneously target all pro-angiogenic growth factor receptors. Here we search for kinase targets, some which may be intracellularly localized, that are critical in endothelial cell proliferation irrespective of the growth factor used. We develop a quantitative endothelial cell proliferation assay and combine it with "kinome regression" or KIR, a recently developed method capable of identifying kinases that influence a quantitative phenotype. We report the kinases implicated by KIR and provide orthogonal evidence of their importance in endothelial cell proliferation. Our approach may point to a new strategy to develop a more complete anti-angiogenic blockade.

YAP regulates cell size and growth dynamics via non-cell autonomous mediators.

The Hippo pathway regulates organ size, regeneration, and cell growth by controlling the stability of the transcription factor, YAP (Yorkie in Drosophila). When there is tissue damage, YAP is activated allowing the restoration of homeostatic tissue size. The exact signals by which YAP is activated are still not fully understood, but its activation is known to affect both cell size and cell number. Here we used cultured cells to examine the coordinated regulation of cell size and number under the control of YAP. Our experiments in isogenic HEK293 cells reveal that YAP can affect cell size and number by independent circuits. Some of these effects are cell autonomous, such as proliferation, while others are mediated by secreted signals. In particular CYR61, a known secreted YAP target, is a non-cell autonomous mediator of cell survival, while another unidentified secreted factor controls cell size.

Bayesian Confidence Intervals for Multiplexed Proteomics Integrate Ion-statistics with Peptide Quantification Concordance.

Multiplexed proteomics has emerged as a powerful tool to measure relative protein expression levels across multiple conditions. The relative protein abundances are inferred by comparing the signals generated by isobaric tags, which encode the samples' origins. Intuitively, the trust associated with a protein measurement depends on the similarity of ratios from the protein's peptides and the signal-strength of these measurements. However, typically the average peptide ratio is reported as the estimate of relative protein abundance, which is only the most likely ratio with a very naive model. Moreover, there is no sense on the confidence in these measurements. Here, we present a mathematically rigorous approach that integrates peptide signal strengths and peptide-measurement agreement into an estimation of the true protein ratio and the associated confidence (BACIQ). The main advantages of BACIQ are: (1) It removes the need to threshold reported peptide signal based on an arbitrary cut-off, thereby reporting more measurements from a given experiment; (2) Confidence can be assigned without replicates; (3) For repeated experiments BACIQ provides confidence intervals for the union, not the intersection, of quantified proteins; (4) For repeated experiments, BACIQ confidence intervals are more predictive than confidence intervals based on protein measurement agreement. To demonstrate the power of BACIQ we reanalyzed previously published data on subcellular protein movement on treatment with an Exportin-1 inhibiting drug. We detect ∼2× more highly significant movers, down to subcellular localization changes of ∼1%. Thus, our method drastically increases the value obtainable from quantitative proteomics experiments, helping researchers to interpret their data and prioritize resources. To make our approach easily accessible we distribute it via a Python/Stan package.

Transcriptomic insights into genetic diversity of protein-coding genes in X. laevis.

We characterize the genetic diversity of Xenopus laevis strains using RNA-seq data and allele-specific analysis. This data provides a catalogue of coding variation, which can be used for improving the genomic sequence, as well as for better sequence alignment, probe design, and proteomic analysis. In addition, we paint a broad picture of the genetic landscape of the species by functionally annotating different classes of mutations with a well-established prediction tool (PolyPhen-2). Further, we specifically compare the variation in the progeny of four crosses: inbred genomic (J)-strain, outbred albino (B)-strain, and two hybrid crosses of J and B strains. We identify a subset of mutations specific to the B strain, which allows us to investigate the selection pressures affecting duplicated genes in this allotetraploid. From these crosses we find the ratio of non-synonymous to synonymous mutations is lower in duplicated genes, which suggests that they are under greater purifying selection. Surprisingly, we also find that function-altering ("damaging") mutations constitute a greater fraction of the non-synonymous variants in this group, which suggests a role for subfunctionalization in coding variation affecting duplicated genes.

The Nuclear Proteome of a Vertebrate.

The composition of the nucleoplasm determines the behavior of key processes such as transcription, yet there is still no reliable and quantitative resource of nuclear proteins. Furthermore, it is still unclear how the distinct nuclear and cytoplasmic compositions are maintained. To describe the nuclear proteome quantitatively, we isolated the large nuclei of frog oocytes via microdissection and measured the nucleocytoplasmic partitioning of ∼9,000 proteins by mass spectrometry. Most proteins localize entirely to either nucleus or cytoplasm; only ∼17% partition equally. A protein's native size in a complex, but not polypeptide molecular weight, is predictive of localization: partitioned proteins exhibit native sizes larger than ∼100 kDa, whereas natively smaller proteins are equidistributed. To evaluate the role of nuclear export in maintaining localization, we inhibited Exportin 1. This resulted in the expected re-localization of proteins toward the nucleus, but only 3% of the proteome was affected. Thus, complex assembly and passive retention, rather than continuous active transport, is the dominant mechanism for the maintenance of nuclear and cytoplasmic proteomes.

Droplet barcoding for single-cell transcriptomics applied to embryonic stem cells.

It has long been the dream of biologists to map gene expression at the single-cell level. With such data one might track heterogeneous cell sub-populations, and infer regulatory relationships between genes and pathways. Recently, RNA sequencing has achieved single-cell resolution. What is limiting is an effective way to routinely isolate and process large numbers of individual cells for quantitative in-depth sequencing. We have developed a high-throughput droplet-microfluidic approach for barcoding the RNA from thousands of individual cells for subsequent analysis by next-generation sequencing. The method shows a surprisingly low noise profile and is readily adaptable to other sequencing-based assays. We analyzed mouse embryonic stem cells, revealing in detail the population structure and the heterogeneous onset of differentiation after leukemia inhibitory factor (LIF) withdrawal. The reproducibility of these high-throughput single-cell data allowed us to deconstruct cell populations and infer gene expression relationships. VIDEO ABSTRACT.

A noncanonical Frizzled2 pathway regulates epithelial-mesenchymal transition and metastasis.

Wnt signaling plays a critical role in embryonic development, and genetic aberrations in this network have been broadly implicated in colorectal cancer. We find that the Wnt receptor Frizzled2 (Fzd2) and its ligands Wnt5a/b are elevated in metastatic liver, lung, colon, and breast cancer cell lines and in high-grade tumors and that their expression correlates with markers of epithelial-mesenchymal transition (EMT). Pharmacologic and genetic perturbations reveal that Fzd2 drives EMT and cell migration through a previously unrecognized, noncanonical pathway that includes Fyn and Stat3. A gene signature regulated by this pathway predicts metastasis and overall survival in patients. We have developed an antibody to Fzd2 that reduces cell migration and invasion and inhibits tumor growth and metastasis in xenografts. We propose that targeting this pathway could provide benefit for patients with tumors expressing high levels of Fzd2 and Wnt5a/b.

Exploiting polypharmacology for drug target deconvolution.

Polypharmacology (action of drugs against multiple targets) represents a tempting avenue for new drug development; unfortunately, methods capable of exploiting the known polypharmacology of drugs for target deconvolution are lacking. Here, we present an ensemble approach using elastic net regularization combined with mRNA expression profiling and previously characterized data on a large set of kinase inhibitors to identify kinases that are important for epithelial and mesenchymal cell migration. By profiling a selected optimal set of 32 kinase inhibitors in a panel against six cell lines, we identified cell type-specific kinases that regulate cell migration. Our discovery of several informative kinases with a previously uncharacterized role in cell migration (such as Mst and Taok family of MAPK kinases in mesenchymal cells) may represent novel targets that warrant further investigation. Target deconvolution using our ensemble approach has the potential to aid in the rational design of more potent but less toxic drug combinations.

Vascular disrupting agent drug classes differ in effects on the cytoskeleton.

Vascular disrupting agents (VDAs), anti-cancer drugs that target established tumor blood vessels, fall into two main classes: microtubule targeting drugs, exemplified by combretastatin A4 (CA4), and flavonoids, exemplified by 5,6-dimethylxanthenone-4-acetic acid (DMXAA). Both classes increase permeability of tumor vasculature in mouse models, and DMXAA in particular can cause massive tumor necrosis. The molecular target of CA4 is clearly microtubules. The molecular target(s) of DMXAA remains unclear. It is thought to promote inflammatory signaling in leukocytes, and has been assumed to not target microtubules, though it is not clear from the literature how carefully this assumption has been tested. An earlier flavone analog, flavone acetic acid, was reported to promote mitotic arrest suggesting flavones might possess anti-microtubule activity, and endothelial cells are sensitive to even mild disruption of microtubules. We carefully investigated whether DMXAA directly affects the microtubule or actin cytoskeletons of endothelial cells by comparing effects of CA4 and DMXAA on human umbilical vein endothelial cells (HUVEC) using time-lapse imaging and assays for cytoskeleton integrity. CA4 caused retraction of the cell margin, mitotic arrest and microtubule depolymerization, while DMXAA, up to 500 µM, showed none of these effects. DMXAA also had no effect on pure tubulin nucleation and polymerization, unlike CA4. We conclude that DMXAA exhibits no direct anti-microtubule action and thus cleanly differs from CA4 in its mechanism of action at the molecular level.

Genome sequencing reveals insights into physiology and longevity of the naked mole rat.

The naked mole rat (Heterocephalus glaber) is a strictly subterranean, extraordinarily long-lived eusocial mammal. Although it is the size of a mouse, its maximum lifespan exceeds 30 years, making this animal the longest-living rodent. Naked mole rats show negligible senescence, no age-related increase in mortality, and high fecundity until death. In addition to delayed ageing, they are resistant to both spontaneous cancer and experimentally induced tumorigenesis. Naked mole rats pose a challenge to the theories that link ageing, cancer and redox homeostasis. Although characterized by significant oxidative stress, the naked mole rat proteome does not show age-related susceptibility to oxidative damage or increased ubiquitination. Naked mole rats naturally reside in large colonies with a single breeding female, the 'queen', who suppresses the sexual maturity of her subordinates. They also live in full darkness, at low oxygen and high carbon dioxide concentrations, and are unable to sustain thermogenesis nor feel certain types of pain. Here we report the sequencing and analysis of the naked mole rat genome, which reveals unique genome features and molecular adaptations consistent with cancer resistance, poikilothermy, hairlessness and insensitivity to low oxygen, and altered visual function, circadian rythms and taste sensing. This information provides insights into the naked mole rat's exceptional longevity and ability to live in hostile conditions, in the dark and at low oxygen. The extreme traits of the naked mole rat, together with the reported genome and transcriptome information, offer opportunities for understanding ageing and advancing other areas of biological and biomedical research.