Genetic and epigenetic defects in Wnt/beta-catenin signaling play important roles in colorectal cancer progression. Here we identify DACT3, a member of the DACT (Dpr/Frodo) gene family, as a negative regulator of Wnt/beta-catenin signaling that is transcriptionally repressed in colorectal cancer. Unlike other Wnt signaling inhibitors that are silenced by DNA methylation, DACT3 repression is associated with bivalent histone modifications. Remarkably, DACT3 expression can be robustly derepressed by a pharmacological combination that simultaneously targets both histone methylation and deacetylation, leading to strong inhibition of Dishevelled (Dvl)-mediated Wnt/beta-catenin signaling and massive apoptosis of colorectal cancer cells. Our study identifies DACT3 as an important regulator of Wnt/beta-catenin signaling in colorectal cancer and suggests a potential strategy for therapeutic control of Wnt/beta-catenin signaling in colorectal cancer.

SUMMARY Post-translational modifications play central roles in myriad biological pathways including circadian regulation. We employed a circadian proteomic approach to demonstrate that circadian timing of phosphorylation is a critical factor in regulating complex GSK3β dependent pathways and identified O-GlcNAc transferase (OGT) as a substrate of GSK3β. Interestingly, OGT activity is regulated by GSK3β, hence OGT and GSK3β exhibit reciprocal regulation. Modulating OGlcNAcylation levels alter circadian period length in both mice and Drosophila, and conversely protein O-GlcNAcylation is circadianly regulated. Central clock proteins, Clock and Period, are reversibly modified by O-GlcNAcylation to regulate their transcriptional activities. In addition, O-GlcNAcylation of a region in PER2 known to regulate human sleep phase (S662–S674) competes with phosphorylation of this region, and this interplay is at least partly mediated by glucose levels. Together, these results indicate that O-GlcNAcylation serves as a metabolic sensor for clock regulation and works coordinately with phosphorylation to fine tune circadian clock.

In the quest for better medicines, attention is increasingly turning to cell-based therapies. The rationale is that infused cells can provide a targeted therapy to precisely correct a complex disease phenotype. Between 1987 and 2010, autologous macrophages (MΦs) were used in clinical trials to treat a variety of human tumors; this approach provided a modest therapeutic benefit in some patients but no lasting remissions. These trials were initiated prior to an understanding of: the complexity of MΦ phenotypes, their ability to alter their phenotype in response to various cytokines and/or the environment, and the extent of survival of the re-infused MΦs. It is now known that while inflammatory MΦs can kill tumor cells, the tumor environment is able to reprogram MΦs into a tumorigenic phenotype; inducing blood vessel formation and contributing to a cancer cell growth-promoting milieu. We review how new information enables the development of large numbers of ex vivo generated MΦs, and how conditioning and gene engineering strategies are used to restrict the MΦ to an appropriate phenotype or to enable production of therapeutic proteins. We survey applications in which the MΦ is loaded with nanomedicines, such as liposomes ex vivo, so when the drug-loaded MΦs are infused into an animal, the drug is released at the disease site. Finally, we also review the current status of MΦ biodistribution and survival after transplantation into an animal. The combination of these recent advances opens the way for improved MΦ cell therapies.

Mice homozygous for mutations in Dact1 (Dpr/Frodo) phenocopy human malformations involving the spine, genitourinary system, and distal digestive tract. We trace this phenotype to disrupted germ layer morphogenesis at the primitive streak (PS). Remarkably, heterozygous mutation of Vangl2, a transmembrane component of the Planar Cell Polarity (PCP) pathway, rescues recessive Dact1 phenotypes, whereas loss of Dact1 reciprocally rescues semidominant Vangl2 phenotypes. We show that Dact1, an intracellular protein, forms a complex with Vangl2. In Dact1 mutants, Vangl2 is increased at the PS where cells ordinarily undergo an epithelial-mesenchymal transition. This is associated with abnormal E-cadherin distribution and changes in biochemical measures of the PCP pathway. We conclude that Dact1 contributes to morphogenesis at the PS by regulating Vangl2 upstream of cell adhesion and the PCP pathway.

Circadian rhythms have been observed in diverse organisms, including plants, animals, bacteria, and fungi. In such organisms, the circadian clock is primarily composed of a cell-autonomous transcriptional feedback loop. In addition to transcriptional regulation, the modification of core clock transcripts and proteins can dramatically affect the circadian clock. In this review, the authors discuss some of the posttranscriptional and posttranslational modifications and their effects on the circadian clock. The combined outcome of these modifications is to adjust the timing of the clock to produce a circadian oscillator that takes approximately 24 h.

Protein phosphorylation plays an essential role in the generation of circadian rhythms, regulating the stability, activity, and subcellular localization of certain proteins that constitute the biological clock. This study examines the role of the protein kinase Doubletime (DBT), a Drosophila ortholog of human casein kinase I (CKI)ɛ/δ. An enzymatically active DBT protein is shown to directly phosphorylate the Drosophila clock protein Period (PER). DBT-dependent phosphorylation sites are identified within PER, and their functional significance is assessed in a cultured cell system and in vivo. The per S mutation, which is associated with short-period (19-h) circadian rhythms, alters a key phosphorylation target within PER. Inspection of this and neighboring sequence variants indicates that several DBT-directed phosphorylations regulate PER activity in an integrated fashion: Alternative phosphorylations of two adjoining sequence motifs appear to be associated with switch-like changes in PER stability and repressor function.

A variety of water-soluble polymers, when attached to a liposome, substantially increase liposome circulation half-life in animals. However, in certain conditions, liposomes modified with the most widely used polymer, polyethylene glycol (PEG), induce an IgM response resulting in an accelerated blood clearance (ABC) of the liposome upon the second injection. Modification of liposomes with other water-soluble polymers: HPMA (poly[N-(2-hydroxypropyl) methacrylamide]), PVP (poly(vinylpyrrolidone)), PMOX (poly(2-methyl-2-oxazoline)), PDMA (poly(N,N-dimethyl acrylamide)), and PAcM (poly(N-acryloyl morpholine)), increase circulation times of liposomes; but a precise comparison of their ability to promote long circulation or induce the ABC effect has not been reported. To obtain a more nuanced understanding of the role of polymer structure/MW to promote long circulation, we synthesized a library of polymer diacyl chain lipids with low polydispersity (1.04–1.09), similar polymer molecular weights (2.1–2.5 kDa) and incorporated them into 100 nm liposomes of a narrow polydispersity (0.25–1.3) composed of polymer-lipid/hydrogenated soy phosphatidylcholine/cholesterol/diD: 5.0/54.5/40/0.5. We confirm that HPMA, PVP, PMOX, PDMA and PAcM modified liposome have increased circulation times in rodents and that PVP, PDMA, PAcM do not induce the ABC effect. We demonstrate for the first time, that HPMA does not cause an ABC effect whereas PMOX induces a pronounced ABC effect in rats. We find that a single dose of liposomes coated with PEG and PMOX generate an IgM response in rats towards the respective polymer. Finally, in this homologous polymer series, we observe a positive correlation (R = 0.84 in rats, R = 0.92 in mice) between the circulation time of polymer-modified liposomes and polymer viscosity; PEG and PMOX, the polymers that can initiate an ABC response were the two most viscous polymers. Our findings suggest that that polymers that do not cause an ABC effect such as, HPMA or PVP, deserve further consideration as polymer coatings to improve the circulation of liposomes and other nanoparticles.

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