Metabolic Glycan Labeling of Cancer Cells Using Variably Acetylated Monosaccharides.

Methylcyclopropene (Cyoc)-tagged tetra-acetylated monosaccharides, and in particular mannosamine derivatives, are promising tools for medical imaging of cancer using metabolic oligosaccharide engineering and the extremely fast inverse electron-demand Diels-Alder bioorthogonal reaction. However, the in vivo potential of these monosaccharide derivatives has yet to be fully explored due to their low aqueous solubility. To address this issue, we sought to vary the extent of acetylation of Cyoc-tagged monosaccharides and probe its effect on the extent of glycan labeling in various cancer cell lines. We demonstrate that, in the case of AcxManNCyoc, tri- and diacetylated derivatives generated significantly enhanced cell labeling compared to the tetra-acetylated monosaccharide. In contrast, for the more readily soluble azide-tagged sugars, a decrease in acetylation led to decreased glycan labeling. Ac3ManNCyoc gave better labeling than the azido-tagged Ac4ManNAz and has significant potential for in vitro and in vivo imaging of glycosylated cancer biomarkers.

Intestinal label-retaining cells are secretory precursors expressing Lgr5.

The rapid cell turnover of the intestinal epithelium is achieved from small numbers of stem cells located in the base of glandular crypts. These stem cells have been variously described as rapidly cycling or quiescent. A functional arrangement of stem cells that reconciles both of these behaviours has so far been difficult to obtain. Alternative explanations for quiescent cells have been that they act as a parallel or reserve population that replace rapidly cycling stem cells periodically or after injury; their exact nature remains unknown. Here we show mouse intestinal quiescent cells to be precursors that are committed to mature into differentiated secretory cells of the Paneth and enteroendocrine lineage. However, crucially we find that after intestinal injury they are capable of extensive proliferation and can give rise to clones comprising the main epithelial cell types. Thus, quiescent cells can be recalled to the stem-cell state. These findings establish quiescent cells as an effective clonogenic reserve and provide a motivation for investigating their role in pathologies such as colorectal cancers and intestinal inflammation.

Nuclear ARRB1 induces pseudohypoxia and cellular metabolism reprogramming in prostate cancer.

Tumour cells sustain their high proliferation rate through metabolic reprogramming, whereby cellular metabolism shifts from oxidative phosphorylation to aerobic glycolysis, even under normal oxygen levels. Hypoxia-inducible factor 1A (HIF1A) is a major regulator of this process, but its activation under normoxic conditions, termed pseudohypoxia, is not well documented. Here, using an integrative approach combining the first genome-wide mapping of chromatin binding for an endocytic adaptor, ARRB1, both in vitro and in vivo with gene expression profiling, we demonstrate that nuclear ARRB1 contributes to this metabolic shift in prostate cancer cells via regulation of HIF1A transcriptional activity under normoxic conditions through regulation of succinate dehydrogenase A (SDHA) and fumarate hydratase (FH) expression. ARRB1-induced pseudohypoxia may facilitate adaptation of cancer cells to growth in the harsh conditions that are frequently encountered within solid tumours. Our study is the first example of an endocytic adaptor protein regulating metabolic pathways. It implicates ARRB1 as a potential tumour promoter in prostate cancer and highlights the importance of metabolic alterations in prostate cancer.

Hyaluronan impairs vascular function and drug delivery in a mouse model of pancreatic cancer.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDA) is characterised by stromal desmoplasia and vascular dysfunction, which critically impair drug delivery. This study examines the role of an abundant extracellular matrix component, the megadalton glycosaminoglycan hyaluronan (HA), as a novel therapeutic target in PDA. METHODS: Using a genetically engineered mouse model of PDA, the authors enzymatically depleted HA by a clinically formulated PEGylated human recombinant PH20 hyaluronidase (PEGPH20) and examined tumour perfusion, vascular permeability and drug delivery. The preclinical utility of PEGPH20 in combination with gemcitabine was assessed by short-term and survival studies. RESULTS: PEGPH20 rapidly and sustainably depleted HA, inducing the re-expansion of PDA blood vessels and increasing the intratumoral delivery of two chemotherapeutic agents, doxorubicin and gemcitabine. Moreover, PEGPH20 triggered fenestrations and interendothelial junctional gaps in PDA tumour endothelia and promoted a tumour-specific increase in macromolecular permeability. Finally, combination therapy with PEGPH20 and gemcitabine led to inhibition of PDA tumour growth and prolonged survival over gemcitabine monotherapy, suggesting immediate clinical utility. CONCLUSIONS: The authors demonstrate that HA impedes the intratumoral vasculature in PDA and propose that its enzymatic depletion be explored as a means to improve drug delivery and response in patients with pancreatic cancer.

G-quadruplex DNA as a molecular target for induced synthetic lethality in cancer cells.

Synthetic lethality is a genetic concept in which cell death is induced by the combination of mutations in two sensitive genes, while mutation of either gene alone is not sufficient to affect cell survival. Synthetic lethality can also be achieved "chemically" by combination of drug-like molecules targeting distinct but cooperative pathways. Previously, we reported that the small molecule pyridostatin (PDS) stabilizes G-quadruplexes (G4s) in cells and elicits a DNA damage response by causing the formation of DNA double strand breaks (DSB). Cell death mediated by ligand-induced G4 stabilization can be potentiated in cells deficient in DNA damage repair genes. Here, we demonstrate that PDS acts synergistically both with NU7441, an inhibitor of the DNA-PK kinase crucial for nonhomologous end joining repair of DNA DSBs, and BRCA2-deficient cells that are genetically impaired in homologous recombination-mediated DSB repair. G4 targeting ligands have potential as cancer therapeutic agents, acting synergistically with inhibition or mutation of the DNA damage repair machinery.

Metabolic glycan imaging by isonitrile-tetrazine click chemistry.

Seeing the sugar coating: N-Acetyl-glucosamine and mannosamine derivatives tagged with an isonitrile group are metabolically incorporated into cell-surface glycans and can be detected with a fluorescent tetrazine. This bioorthogonal isonitrile-tetrazine ligation is also orthogonal to the commonly used azide-cyclooctyne ligation, and so will allow simultaneous detection of the incorporation of two different sugars.

Dual-sugar imaging using isonitrile and azido-based click chemistries.

We report the first account of metabolically labelling N-acetylglucosamine, in conjunction with either N-acetylgalactosamine or N-acetylmannosamine using a combination of isonitrile- and azide-based chemistries. With the appropriately labelled fluorescent probe molecules, that react with either the azido or isonitrile groups, the method enabled co-visualisation of cancer cell glycoproteins.

Imaging sialylated tumor cell glycans in vivo.

Cell surface glycans are involved in numerous physiological processes that involve cell-cell interactions and migration, including lymphocyte trafficking and cancer metastasis. We have used a bioorthogonal metabolic labeling strategy to detect cell surface glycans and demonstrate, for the first time, fluorescence and radionuclide imaging of sialylated glycans in a murine tumor model in vivo. Peracetylated azido-labeled N-acetyl-mannosamine, injected intraperitoneally, was used as the metabolic precursor for the biosynthesis of 5-azidoneuraminic, or azidosialic acid. Azidosialic acid-labeled cell surface glycans were then reacted, by Staudinger ligation, with a biotinylated phosphine injected intraperitoneally, and the biotin was detected by subsequent intravenous injection of a fluorescent or radiolabeled avidin derivative. At 24 h after administration of NeutrAvidin, labeled with either a far-red fluorophore or (111)In, there was a significant azido-labeled N-acetyl-mannosamine-dependent increase in tumor-to-tissue contrast, which was detected using optical imaging or single-photon-emission computed tomography (SPECT), respectively. The technique has the potential to translate to the clinic, where, given the prognostic relevance of altered sialic acid expression in cancer, it could be used to monitor disease progression.

Characterization of a heat resistant beta-glucosidase as a new reporter in cells and mice.

BACKGROUND: Reporter genes are widely used in biology and only a limited number are available. We present a new reporter gene for the localization of mammalian cells and transgenic tissues based on detection of the bglA (SYNbglA) gene of Caldocellum saccharolyticum that encodes a thermophilic beta-glucosidase. RESULTS: SYNbglA was generated by introducing codon substitutions to remove CpG motifs as these are associated with gene silencing in mammalian cells. SYNbglA expression can be localized in situ or detected quantitatively in colorimetric assays and can be co-localized with E. coli beta-galactosidase. Further, we have generated a Cre-reporter mouse in which SYNbglA is expressed following recombination to demonstrate the general utility of SYNbglA for in vivo analyses. SYNbglA can be detected in tissue wholemounts and in frozen and wax embedded sections. CONCLUSIONS: SYNbglA will have general applicability to developmental and molecular studies in vitro and in vivo.

Multiplexed Detection and Analysis of Low-Abundance Long Noncoding RNA Using RNAscope™ in Cultured Cells.

Detecting low-abundance long noncoding RNAs (lncRNAs) is extremely difficult due to their expression levels. Deeper sequencing with extensive protocols is required to detect these RNAs and high-throughput screens to examine the regulation of these RNAs are challenging. This protocol provides a multiplexed and robust method of detecting low-abundance RNAs, with improved signal-to-noise ratio using RNAscope-based RNA-FISH which utilizes a series of amplification steps. We have validated this protocol for investigating the regulation of low-abundance lncRNAs, which would be ideal for in vitro screening in 96-well plates.

Tissue Inhibitor of Metalloproteinase-3 (TIMP-3) induces FAS dependent apoptosis in human vascular smooth muscle cells.

Over expression of Tissue Inhibitor of Metalloproteinases-3 (TIMP-3) in vascular smooth muscle cells (VSMCs) induces apoptosis and reduces neointima formation occurring after saphenous vein interposition grafting or coronary stenting. In studies to address the mechanism of TIMP-3-driven apoptosis in human VSMCs we find that TIMP-3 increased activation of caspase-8 and apoptosis was inhibited by expression of Cytokine response modifier A (CrmA) and dominant negative FAS-Associated protein with Death Domain (FADD). TIMP-3 induced apoptosis did not cause mitochondrial depolarisation, increase activation of caspase-9 and was not inhibited by over-expression of B-cell Lymphoma 2 (Bcl2), indicating a mitochondrial independent/type-I death receptor pathway. TIMP-3 increased levels of the First Apoptosis Signal receptor (FAS) and depletion of FAS with shRNA showed TIMP-3-induced apoptosis was FAS dependent. TIMP-3 induced formation of the Death-Inducing Signalling Complex (DISC), as detected by immunoprecipitation and by immunofluorescence. Cellular-FADD-like IL-1 converting enzyme-Like Inhibitory Protein (c-FLIP) localised with FAS at the cell periphery in the absence of TIMP-3 and this localisation was lost on TIMP-3 expression with c-FLIP adopting a perinuclear localisation. Although TIMP-3 inhibited FAS shedding, this did not increase total surface levels of FAS but instead increased FAS levels within localised regions at the cell surface. A Disintegrin And Metalloproteinase 17 (ADAM17) is inhibited by TIMP-3 and depletion of ADAM17 with shRNA significantly decreased FAS shedding. However ADAM17 depletion did not induce apoptosis or replicate the effects of TIMP-3 by increasing localised clustering of cell surface FAS. ADAM17-depleted cells could activate caspase-3 when expressing levels of TIMP-3 that were otherwise sub-apoptotic, suggesting a partial role for ADAM17 mediated ectodomain shedding in TIMP-3 mediated apoptosis. We conclude that TIMP-3 induced apoptosis in VSMCs is highly dependent on FAS and is associated with changes in FAS and c-FLIP localisation, but is not solely dependent on shedding of the FAS ectodomain.

Synthetic lethality between androgen receptor signalling and the PARP pathway in prostate cancer.

Emerging data demonstrate homologous recombination (HR) defects in castration-resistant prostate cancers, rendering these tumours sensitive to PARP inhibition. Here we demonstrate a direct requirement for the androgen receptor (AR) to maintain HR gene expression and HR activity in prostate cancer. We show that PARP-mediated repair pathways are upregulated in prostate cancer following androgen-deprivation therapy (ADT). Furthermore, upregulation of PARP activity is essential for the survival of prostate cancer cells and we demonstrate a synthetic lethality between ADT and PARP inhibition in vivo. Our data suggest that ADT can functionally impair HR prior to the development of castration resistance and that, this potentially could be exploited therapeutically using PARP inhibitors in combination with androgen-deprivation therapy upfront in advanced or high-risk prostate cancer.Tumours with homologous recombination (HR) defects become sensitive to PARPi. Here, the authors show that androgen receptor (AR) regulates HR and AR inhibition activates the PARP pathway in vivo, thus inhibition of both AR and PARP is required for effective treatment of high risk prostate cancer.

Choline Kinase Alpha as an Androgen Receptor Chaperone and Prostate Cancer Therapeutic Target.

BACKGROUND: The androgen receptor (AR) is a major drug target in prostate cancer (PCa). We profiled the AR-regulated kinome to identify clinically relevant and druggable effectors of AR signaling. METHODS: Using genome-wide approaches, we interrogated all AR regulated kinases. Among these, choline kinase alpha (CHKA) expression was evaluated in benign (n = 195), prostatic intraepithelial neoplasia (PIN) (n = 153) and prostate cancer (PCa) lesions (n = 359). We interrogated how CHKA regulates AR signaling using biochemical assays and investigated androgen regulation of CHKA expression in men with PCa, both untreated (n = 20) and treated with an androgen biosynthesis inhibitor degarelix (n = 27). We studied the effect of CHKA inhibition on the PCa transcriptome using RNA sequencing and tested the effect of CHKA inhibition on cell growth, clonogenic survival and invasion. Tumor xenografts (n = 6 per group) were generated in mice using genetically engineered prostate cancer cells with inducible CHKA knockdown. Data were analyzed with χ(2) tests, Cox regression analysis, and Kaplan-Meier methods. All statistical tests were two-sided. RESULTS: CHKA expression was shown to be androgen regulated in cell lines, xenografts, and human tissue (log fold change from 6.75 to 6.59, P = .002) and was positively associated with tumor stage. CHKA binds directly to the ligand-binding domain (LBD) of AR, enhancing its stability. As such, CHKA is the first kinase identified as an AR chaperone. Inhibition of CHKA repressed the AR transcriptional program including pathways enriched for regulation of protein folding, decreased AR protein levels, and inhibited the growth of PCa cell lines, human PCa explants, and tumor xenografts. CONCLUSIONS: CHKA can act as an AR chaperone, providing, to our knowledge, the first evidence for kinases as molecular chaperones, making CHKA both a marker of tumor progression and a potential therapeutic target for PCa.

Continuous clonal labeling reveals small numbers of functional stem cells in intestinal crypts and adenomas.

Lineage-tracing approaches, widely used to characterize stem cell populations, rely on the specificity and stability of individual markers for accurate results. We present a method in which genetic labeling in the intestinal epithelium is acquired as a mutation-induced clonal mark during DNA replication. By determining the rate of mutation in vivo and combining this data with the known neutral-drift dynamics that describe intestinal stem cell replacement, we quantify the number of functional stem cells in crypts and adenomas. Contrary to previous reports, we find that significantly lower numbers of "working" stem cells are present in the intestinal epithelium (five to seven per crypt) and in adenomas (nine per gland), and that those stem cells are also replaced at a significantly lower rate. These findings suggest that the bulk of tumor stem cell divisions serve only to replace stem cell loss, with rare clonal victors driving gland repopulation and tumor growth.

Development and evaluation of new cyclooctynes for cell surface glycan imaging in cancer cells.

Two reagents have been synthesized for selective labeling of cell surface azidoglycans, an unusually stable version of a dibenzocyclooctyne (TMDIBO) and a third-generation difluorinated cyclooctyne (DIFO3). Both syntheses are efficient with minimal purification, and the dibenzocyclooctyne is stable under basic and acidic conditions. Flow cytometric measurements with azidosugar labeled cancer cells, in which these reagents were linked to the fluorophore Alexa Fluor 647, gave a signal-to-background ratio of up to 35 with TMDIBO as compared to ≈10 for DIFO3 and ≈5 for a phosphine reagent. TMDIBO-based probes should have applications in molecular imaging of cell surface glycans in vivo.

Inhibition of Hedgehog signaling enhances delivery of chemotherapy in a mouse model of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDA) is among the most lethal human cancers in part because it is insensitive to many chemotherapeutic drugs. Studying a mouse model of PDA that is refractory to the clinically used drug gemcitabine, we found that the tumors in this model were poorly perfused and poorly vascularized, properties that are shared with human PDA. We tested whether the delivery and efficacy of gemcitabine in the mice could be improved by coadministration of IPI-926, a drug that depletes tumor-associated stromal tissue by inhibition of the Hedgehog cellular signaling pathway. The combination therapy produced a transient increase in intratumoral vascular density and intratumoral concentration of gemcitabine, leading to transient stabilization of disease. Thus, inefficient drug delivery may be an important contributor to chemoresistance in pancreatic cancer.