Inflammation, Attention, and Processing Speed in Patients With Breast Cancer Before and After Chemotherapy.

BACKGROUND: Inflammation may contribute to cognitive difficulties in patients with breast cancer. We tested 2 hypotheses: inflammation is elevated in patients with breast cancer vs noncancer control participants and inflammation in patients is associated with worse attention and processing speed over the course of chemotherapy. METHODS: Serum cytokines (interleukin [IL]-4, 6, 8, 10; tumor necrosis factor [TNF]-α) and soluble receptors [sTNFRI, II]) were measured in 519 females with breast cancer before and after chemotherapy and 338 females without cancer serving as control participants. Attention and processing speed were measured by Rapid Visual Processing (RVP), Backward Counting (BCT), and Trail Making-A (TMT-A) tests. Linear regression models examined patient vs control cytokines and receptor levels, adjusting for covariates. Linear regression models also examined relationships between patient cytokines and receptor levels and test performance, adjusting for age, body mass index, anxiety, depression, cognitive reserve, and chemotherapy duration. Statistical tests were 2-sided (α = .05). RESULTS: sTNFRI and sTNFRII increased over time in patients relative to controls, whereas IL-4, IL-6, and IL-10 decreased. Prechemotherapy, higher IL-8 associated with worse BCT (ß = 0.610, SE = 0.241, P = .01); higher IL-4 (ß = -1.098, SE = 0.516, P = .03) and IL-10 (ß = -0.835, SE = 0.414, P = .04) associated with better TMT-A. Postchemotherapy, higher IL-8 (ß = 0.841, SE = 0.260, P = .001), sTNFRI (ß = 6.638, SE = 2.208, P = .003), and sTNFRII (ß = 0.913, SE = 0.455, P = .045) associated with worse BCT; higher sTNFRII also associated with worse RVP (ß = -1.316, SE = 0.587, P = .03). At prechemotherapy, higher IL-4 predicted RVP improvement over time (ß = 0.820, SE = 0.336, P = .02); higher sTNFRI predicted worse BCT over time (ß = 5.566, SE = 2.367, P = .02). Longitudinally, increases in IL-4 associated with BCT improvement (ß = -0.564, SE = 0.253, P = .03). CONCLUSIONS: Generally, worse attention and processing speed were associated with higher inflammatory cytokines and receptors and lower anti-inflammatory cytokines in patients; future confirmatory studies are needed.

Using Chemistry to Target Neuroblastoma.

Neuroblastoma is a cancer of the neural crest almost exclusively seen in childhood. While children with single, small primary tumors are often cured with surgery alone, the 65% of children with neuroblastoma whose disease has metastasized have less than a 50% chance of surviving five years after diagnosis. Innovative pharmacological strategies are critically needed for these children. Efforts to identify novel targets that afford ablation of neuroblastoma with minimal toxicity to normal tissues are underway. Developing approaches to neuroblastoma include those that target the catecholamine transporter, ubiquitin E3 ligase, the ganglioside GD2, the retinoic acid receptor, the protein kinases ALK and Aurora, and protein arginine N-methyltransferases. Here, as examples of the use of chemistry to combat neuroblastoma, we describe targeting of the protein arginine N-methyltransferases and their role in prolonging the half-life of the neuroblastoma oncoprotein N-Myc, redox signaling in neuroblastoma, and developmentally regulated proteins expressed in primitive neuroblastoma cells but not in mature neural crest elements.

Protein arginine methyltransferase 1 is a novel regulator of MYCN in neuroblastoma.

Amplification or overexpression of MYCN is associated with poor prognosis of human neuroblastoma. We have recently defined a MYCN-dependent transcriptional signature, including protein arginine methyltransferase 1 (PRMT1), which identifies a subgroup of patients with high-risk disease. Here we provide several lines of evidence demonstrating PRMT1 as a novel regulator of MYCN and implicating PRMT1 as a potential therapeutic target in neuroblastoma pathogenesis. First, we observed a strong correlation between MYCN and PRMT1 protein levels in primary neuroblastoma tumors. Second, MYCN physically associates with PRMT1 by direct protein-protein interaction. Third, depletion of PRMT1 through siRNA knockdown reduced neuroblastoma cell viability and MYCN expression. Fourth, we showed that PRMT1 regulates MYCN stability and identified MYCN as a novel substrate of PRMT1. Finally, we demonstrated that mutation of putatively methylated arginine R65 to alanine decreased MYCN stability by altering phosphorylation at residues serine 62 and threonine 58. These results provide mechanistic insights into the modulation of MYCN oncoprotein by PRMT1, and suggest that targeting PRMT1 may have a therapeutic impact on MYCN-driven oncogenesis.

EYA1 expression and subcellular localization in neuroblastoma and its association with prognostic markers.

Neuroblastoma, the most frequently occurring extracranial solid tumor of childhood, arises from neural crest-derived cells that are arrested at an early stage of differentiation in the developing sympathetic nervous system. There is an urgent need to identify clinically relevant biomarkers for better prognosis and treatment of this aggressive malignancy. Eyes Absent 1 (EYA1) is an essential transcriptional coactivator for neuronal developmental programs during organogenesis. Whether or not EYA1 is implicated in neuroblastoma and subcellular localization of EYA1 is relevant to clinical behaviour of neuroblastoma is not known. We studied EYA1 expression and subcellular localization by immunohistochemistry in tissue microarrays containing tumor specimens from 98 patients, 66 of which were characterized by known clinical prognostic markers of neuroblastoma. Immunostaining results were evaluated and statistically correlated with the degree of histologic differentiation and with neuroblastoma risk stratification group characteristics, including stage of disease, patient age, tumor histology and mitosis-karyorrhexis index (MKI), respectively. We found that EYA1 levels were significantly higher in neuroblastomas than in ganglioneuromas and ganglioneuroblastomas. EYA1 was more highly expressed in stage 1,2,3 or 4S tumors as compared to stage 4 tumors (P<0.01). Tumors with high levels of nuclear EYA1 were more frequently associated with high nuclear MYCN levels. These results suggest that modulation of expression and intracellular localization of EYA1 in neural crest cells may provide a novel direction for therapeutic strategies.

Virion-associated cofactor high-mobility group DNA-binding protein-1 facilitates transposition from the herpes simplex virus/Sleeping Beauty amplicon vector platform.

The development of the integration-competent, herpes simplex virus/Sleeping Beauty (HSV/SB) amplicon vector platform has created a means to efficiently and stably deliver therapeutic transcription units (termed "transgenons") to neurons within the mammalian brain. Furthermore, an investigation into the transposition capacity of the HSV/SB vector system revealed that the amplicon genome provides an optimal substrate for the transposition of transgenons at least 12 kb in length [de Silva, S., Mastrangelo, M.A., Lotta, L.T., Jr., Burris, C.A., Federoff, H.J., and Bowers, W.J. ( 2010 ). Gene Ther. 17, 424-431]. These results prompted an investigation into the factors that may contribute toward efficient transposition from the HSV/SB amplicon. One of the cellular cofactors known to play a key role during SB-mediated transposition is the high-mobility group DNA-binding protein-1 (HMGB1). Our present investigation into the role of HMGB1 during amplicon-based transposition revealed that transposition is not strictly dependent on the presence of cellular HMGB1, contrary to what had been previously demonstrated with plasmid-based SB transposition. We have shown for the first time that during amplicon preparation, biologically active HMGB1 derived from the packaging cell line is copackaged into amplicon vector particles. As a result, HSV/SB amplicon virions arrive prearmed with HMGB1 protein at levels sufficient for facilitating SB-mediated transposition in the transduced mammalian cell.

Herpes simplex virus/Sleeping Beauty vector-based embryonic gene transfer using the HSB5 mutant: loss of apparent transposition hyperactivity in vivo.

The Sleeping Beauty (SB) transposon system has been successfully used as a gene delivery tool in nonviral and viral vector platforms. Since its initial reconstruction, a series of hyperactive mutants of SB have been generated. Questions remain as to whether the enhanced in vitro activities of these SB transposase mutants translate to the in vivo setting, and whether such increased integration efficiencies will ultimately compromise the safety profile of the transposon platform by raising the risk of genomic insertional mutagenesis. Herein, we compared the in vivo impact of a herpes simplex virus (HSV) amplicon-vectored "wild-type" SB transposase (SB10) and a "hyperactive" SB mutant (HSB5), codelivered in utero with the HSVT-ßgeo transposable reporter amplicon vector to embryonic day 14.5 C57BL/6 mice. The SB10 and HSB5 transposases do not disparately affect the viability and development of injected mouse embryos. Quantitation of brain-resident ßgeo expression on postnatal day 21 revealed that mice receiving HSB5 exhibited only a trending increase in transgene expression compared with the SB10-infused group, an outcome that did not mirror the marked enhancement of HSB5-mediated transposition observed in vitro. These findings indicate that in vivo application of hyperactive SB mutants, although not differentially genotoxic to the developing mouse embryo, does not necessarily provide a significant therapeutic advantage over the employment of a lesser active SB when delivered in the context of the HSV/SB amplicon platform.

Hexamethylene bisacetamide leads to reduced helper virus-free HSV-1 amplicon expression titers via suppression of ICP0.

The herpes simplex virus (HSV)-derived amplicon vector has evolved into a promising gene transfer platform for widespread DNA delivery in gene replacement strategies and vaccine development given its ease of molecular manipulation, large transgene capacity, and transduction efficiencies of numerous cell types in vivo. The recent development of helper virus-free packaging methodologies bodes well for this vector system in its eventual implementation as a clinically viable therapeutic modality. For realization of clinical application, efforts have been made to enhance yields and quality of helper-free amplicon stocks. Hexamethylene bisacetamide (HMBA), a hybrid polar compound that exhibits stimulatory activity of HSV-1 immediate-early gene expression, has been employed as a standard reagent in helper virus-free packaging given its purported mode of action on virus gene expression kinetics. Unexpectedly, we have found that HMBA exhibits no titer-enhancing activity; in contrast, the compound enhances the proportion of amplicon virions that are non-expressive. Omission of HMBA during vector packaging led to a marked reduction in the ratios of vector genome-transducing to transgene-expressing virions. This effect was neither packaging-cell-specific nor amplicon-promoter-dependent. Analysis of resultant vector stocks indicated amplicon genome replication/concatenation was unaffected, but the level of particle-associated ICP0 was reduced in stocks packaged in the presence of HMBA. Inclusion of a co-transfected, ICP0-expressing plasmid into the packaging process led to significant rescue of amplicon expression titers, indicating that regulation of ICP0 concentrations is critical for maintenance of the amplicon genome expressive state.