Nanotechnology has tremendous potential to contribute to cancer immunotherapy. The “in situ vaccination” immunotherapy strategy directly manipulates identified tumours to overcome local tumour-mediated immunosuppression and subsequently stimulates systemic anti-tumour immunity to treat metastases. We show that inhalation of self-assembling virus-like nanoparticles from Cowpea Mosaic Virus (CPMV) reduces established B16F10 lung melanoma and simultaneously generates potent systemic anti-tumour immunity against poorly immunogenic B16F10 in the skin. Full efficacy required Il-12, Ifn-γ, adaptive immunity, and neutrophils. Inhaled CPMV nanoparticles were rapidly taken up by and activated neutrophils in the tumour microenvironment as an important part of the anti-tumour immune response. CPMV also exhibited clear treatment efficacy and systemic anti-tumour immunity in ovarian, colon, and breast tumour models in multiple anatomic locations. CPMV nanoparticles are stable, nontoxic, modifiable with drugs and antigens, and their nanomanufacture is highly scalable. These properties, combined with their inherent immunogenicity and demonstrated efficacy against a poorly immunogenic tumour, make CPMV an attractive and novel immunotherapy against metastatic cancer.

Dendritic cells are transformed to become immunosuppressive during ovarian cancer progression.

No abstract

Deoxyribonuclease IIa (DNase IIa) is one of many endonucleases implicated in DNA digestion during apoptosis. We produced mice with targeted disruption of DNase IIa and defined its role in apoptosis. Mice deleted for DNase IIa die at birth with many tissues exhibiting large DNA-containing bodies that result from engulfed but undigested cell corpses. These DNA-containing bodies are pronounced in the liver where fetal definitive erythropoiesis occurs and extruded nuclei are degraded. They are found between the digits, where apoptosis occurs, and in many other regions of the embryo. Defects in the diaphragm appear to cause death of the mice due to asphyxiation. The DNA in these bodies contains 3'-hydroxyl ends and therefore stain positive in the TUNEL assay. In addition, numerous unengulfed TUNEL-positive cells are observed throughout the embryo. Apoptotic cells are normally cleared rapidly from a tissue; hence the persistence of the DNA-containing bodies and TUNEL-positive cells identifies sites where apoptosis occurs during development. These results demonstrate that DNase IIa is not required for the generation of the characterisitic DNA fragmentation that occurs during apoptosis but is required for degrading DNA of dying cells and this function is necessary for proper fetal development. Cell Death and Differentiation (2002) 9, 956 ± 962.

Purpose: Despite adjuvant endocrine therapy for patients with estrogen receptor alpha (ER)-positive breast cancer, dormant residual disease can persist for years and eventually cause tumor recurrence. We sought to deduce mechanisms underlying the persistence of dormant cancer cells to identify therapeutic strategies. Experimental Design: Mimicking the aromatase inhibitor-induced depletion of estrogen levels used to treat patients, we developed preclinical models of dormancy in ER+ breast cancer induced by estrogen withdrawal in mice. We analyzed tumor xenografts and cultured cancer cells for molecular and cellular responses to estrogen withdrawal and drug treatments. Publicly available clinical breast tumor gene expression datasets were analyzed for responses to neoadjuvant endocrine therapy. Results: Dormant breast cancer cells exhibited upregulated 5' adenosine monophosphate-activated protein kinase (AMPK) levels and activity, and upregulated fatty acid oxidation. While the anti-diabetes AMPKactivating drug metformin slowed the estrogen-driven growth of cells and tumors, metformin promoted the persistence of estrogen-deprived cells and tumors through increased mitochondrial respiration driven by fatty acid oxidation. Pharmacologic or genetic inhibition of AMPK or fatty acid oxidation promoted clearance of dormant residual disease, while dietary fat increased tumor cell survival.Conclusions: AMPK has context-dependent effects in cancer, cautioning against the widespread use of an AMPK activator across disease settings. The development of therapeutics targeting fat metabolism is warranted in ER+ breast cancer.

Estrogens have been shown to elicit anticancer effects against estrogen receptor α ( ER )‐positive breast cancer. We sought to determine the mechanism underlying the therapeutic response. Response to 17β‐estradiol was assessed in ER + breast cancer models with resistance to estrogen deprivation: WHIM 16 patient‐derived xenografts, C7‐2‐ HI and C4‐ HI murine mammary adenocarcinomas, and long‐term estrogen‐deprived MCF ‐7 cells. As another means to reactivate ER , the anti‐estrogen fulvestrant was withdrawn from fulvestrant‐resistant MCF ‐7 cells. Transcriptional, growth, apoptosis, and molecular alterations in response to ER reactivation were measured. 17β‐estradiol treatment and fulvestrant withdrawal induced transcriptional activation of ER , and cells adapted to estrogen deprivation or fulvestrant were hypersensitive to 17β‐estradiol. ER transcriptional response was followed by an unfolded protein response and apoptosis. Such apoptosis was dependent upon the unfolded protein response, p53, and JNK signaling. Anticancer effects were most pronounced in models exhibiting genomic amplification of the gene encoding ER ( ESR 1 ), suggesting that engagement of ER at high levels is cytotoxic. These data indicate that long‐term adaptation to estrogen deprivation or ER inhibition alters sensitivity to ER reactivation. In such adapted cells, 17β‐estradiol treatment and anti‐estrogen withdrawal hyperactivate ER , which drives an unfolded protein response and subsequent growth inhibition and apoptosis. 17β‐estradiol treatment should be considered as a therapeutic option for anti‐estrogen‐resistant disease, particularly in patients with tumors harboring ESR 1 amplification or ER overexpression. Furthermore, therapeutic strategies that enhance an unfolded protein response may increase the therapeutic effects of ER reactivation.

Active gene promoters are associated with covalent histone modifications, such as hyperacetylation, which can modulate chromatin structure and stabilize binding of transcription factors that recognize these modifications. At the ␤-globin locus and several other loci, however, histone hyperacetylation extends beyond the promoter, over tens of kilobases; we term such patterns of histone modifications "hyperacetylated domains." Little is known of either the mechanism by which these domains form or their function. Here, we show that domain formation within the murine ␤-globin locus occurs before either high-level gene expression or erythroid commitment. Analysis of ␤-globin alleles harboring deletions of promoters or the locus control region demonstrates that these sequences are not required for domain formation, suggesting the existence of additional regulatory sequences within the locus. Deletion of embryonic globin gene promoters, however, resulted in the formation of a hyperacetylated domain over these genes in definitive erythroid cells, where they are otherwise inactive. Finally, sequences within ␤-globin domains exhibit hyperacetylation in a context-dependent manner, and domains are maintained when transcriptional elongation is inhibited. These data narrow the range of possible mechanisms by which hyperacetylated domains form. (Blood. 2009;114: 3479-3488) IntroductionA crucial feature of gene activation is the interaction between transcription factors and chromatin. All eukaryotic genomic DNA, with limited exceptions, is packaged with core histones to form chromatin. The fundamental subunit of chromatin is the nucleosome, consisting of approximately 147 bp of DNA wrapped in approximately 1.75 turns about an octamer of core histones; a variable length of linker DNA extends between nucleosomes and can in turn be partially sequestered by interactions with core histone amino-terminal "tail" regions and/or linker histones, such as histone H1. The resulting structure, when observed on low-salt spreads by electron microscopy, has been termed "beads on a string" based on its appearance. 1,2 At physiologically relevant salt concentrations, however, this structure spontaneously condenses, first to a helical array of nucleosomes termed the 30-nm fiber, then through additional levels of higher-order structure, which are not well understood. 3 Nevertheless, this packaging renders the eukaryotic DNA relatively inaccessible to transcription factors. [4][5][6] The transcriptional machinery possesses mechanisms for modulating chromatin structure. One of these is covalent modification of core histones, including acetylation, methylation, phosphorylation, ubiquitylation, SUMOylation, and ADP ribosylation, in short, the gamut of modifications known to occur on cellular proteins. Different modifications can lead to different functional consequences. Histone acetylation is associated with transcriptional activation; indeed, core histones proximal to active gene promoters are universally hyperacetylated. 7,8 Histone methylation can...

Mammalian ␤-globin loci contain multiple genes that are activated at different developmental stages. Studies have suggested that the transcription of one gene in a locus can influence the expression of the other locus genes. The prevalent model to explain this transcriptional interference is that all potentially active genes compete for locus control region (LCR) activity. To investigate the influence of transcription by the murine embryonic genes on transcription of the other ␤-like genes, we generated mice with deletions of the promoter regions of Ey and ␤h1 and measured transcription of the remaining genes. Deletion of the Ey and ␤h1 promoters increased transcription of ␤ma-jor and ␤minor 2-fold to 3-fold during primitive erythropoiesis. Deletion of Ey did not affect ␤h1 nor did deletion of ␤h1 affect Ey, but Ey deletion uniquely activated transcription from ␤h0, a ␤-like globin gene immediately downstream of Ey. Protein analysis showed that ␤h0 encodes a translatable ␤-like globin protein that can pair with alpha globin. The lack of transcriptional interference between Ey and ␤h1 and the gene-specific repression of ␤h0 did not support LCR competition among the embryonic genes and suggested that direct transcriptional interference from Ey suppressed ␤h0. IntroductionThe mammalian ␤-globin loci consist of multiple genes that are activated at different developmental stages in a tissue-specific manner. In the mouse, 2 "embryonic" ␤-like globin genes, Ey and ␤h1, are transcribed at high levels only during primitive erythropoiesis in the embryonic yolk sac. The "adult" expressed ␤-type globin genes-␤-major and ␤-minor-are expressed at low levels in embryos and at high levels during fetal and adult definitive erythropoiesis. This developmental up-regulation of the adult ␤-like globin genes is coincident with the silencing of the embryonic ␤-like globin genes and is hypothesized to be mechanistically related to the silencing of the embryonic genes.Regulatory elements of each ␤-like globin gene include a promoter and associated gene proximal cis-regulatory elements bound by multiple-tissue specific or ubiquitous transcription factors. High-level expression of all the genes at the locus requires a gene distal cis-regulatory element, the locus control region (LCR), which is located 5 to 22 kb upstream of the embryonic Ey gene in the mouse locus (for a review, see Stamatoyannopoulos and Grosveld 1 ). The role, if any, of the LCR in the developmental regulation of individual genes within the locus is unclear.Previous studies of ␤-globin gene regulation in transgenic mice carrying portions of the human ␤-globin locus have suggested that developmental expression of the embryonic and adult genes is regulated through different mechanisms. For the embryonic genes, the developmental silencing is gene autonomous and is achieved through binding or dissociation of specific transcription factors to or from the gene proximal cis-regulatory elements. When directly linked to the LCR, the human embryonic ⑀ is expressed only at embryo...