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Prostaglandins E$ sb1$ and E$ sb1$ are bound with high affinity and specificity to particulate fractions from bovine, ovine and human adrenal medulla. An identical fraction from fetal bovine and ovine adrenals binds PGEs with similar kinetic properties and specificities. The ${ rm lbrack sp3H rbrack}$PGE$ sb2$-binding site complex dissociates in a biphasic manner, suggesting the presence of both a low and high affinity form of the complex. Gpp(NH)p, a stable GTP analogue, inhibits PGE$ sb2$ binding by promoting the conversion of the high affinity form of the complex to the lower affinity state. In subcellular fractionation studies, PGE$ sb2$ binding sites co-purity with acetylcholinesterase and Ca$ sp{2+}$-ATPase activities (marker enzymes for the chromaffin cell plasma membrane). Bovine adrenal medullary cells in culture, prelabeled with ${ rm lbrack sp3H rbrack}$arachidonic acid, metabolize the precursor fatty acid to PGE$ sb2,$ 6-oxoPGF$ sb{1 alpha}$ and other unidentified products, when stimulated with the calcium-ionophore A23187. PGE$ sb2$ (10$ sp{-9}$ M) inhibits both nicotine and high K$ sp+$-induced catecholamine secretion from chromaffin cells by a mechanism unrelated to alterations in intracellular levels of cyclic nucleotides. LTC$ sb4$ and LTB$ sb4$ stimulate spontaneous catecholamine release but this effect is not always reproducible. NDGA attenuates nicotine-induced secretion, while indomethacin has no effect. These data suggest that the inhibitory effect of PGE$ sp2$ on chromaffin cell secretion is initiated by the release of the prostaglandin during cellular activation, followed by the binding of PGE$ sb2$ to its receptor and the subsequent interaction of this complex with a guanine nucleotide-binding protein.

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Thesis (Ph.D.)--University of California, San Francisco, 2008. / Source: Dissertation Abstracts International, Volume: 68-11, Section: B, page: 7042. Adviser: Arthur Weiss.

Glioblastoma (GBM) is the most common and aggressive brain tumor in humans. Because GBM is highly angiogenic, the anti-vascular endothelial growth factor (VEGF) antibody bevacizumab has now become the standard of care for treatment of recurrent GBM. However, GBMs rapidly become refractory to standard anti-VEGF therapy. It was previously demonstrated that vessel normalization and subsequent reduction of intracranial vasogenic edema accounts for a majority of the benefit from inhibiting VEGF-signaling in GBM. Also, ectopic over-expression of Ang-2 compromises the benefits of anti-VEGF receptor (VEGFR) treatment in mice and showed that circulating levels of Ang-2 rebound after an initial decrease in GBM patients treated with anti-VEGFR agents. This thesis examines the hypothesis that combined inhibition of VEGF and Ang-2 signaling can improve survival in murine models of GBM, and investigates potential underlying mechanisms. The efficacy of cediranib, a pan-VEGFR tyrosine kinase inhibitor, combined with MEDI3617, an anti-Ang-2 neutralizing antibody, was tested in two orthotopic models of GBM (U87 and Gl261). Combination therapy improved survival in both models beyond that of the monotherapy arms and of control IgG by delaying Gl261 growth and increasing U87 necrosis. Combination therapy increased perivascular cell coverage in both tumor models and led to a more mature, normalized tumor vasculature than that in the tumors treated with cediranib alone. Importantly, combination therapy was as effective at controlling edema as cediranib. Vascular normalization with VEGF-blockade has been shown to convert an immunoinhibitory tumor microenvironment to an immunostimulatory one. Improved vessel normalization resulting from combined Ang-2 and VEGFR inhibition was associated with an increase in the number of M1-like (anti-tumor) tumor associated macrophages (TAMs) compared to the cediranib-treated tumors. Inhibition of TAM recruitment with an anti-colony stimulating factor-1 (CSF1) neutralizing antibody compromised the survival benefit of anti-Ang-2/VEGFR combination therapy. The work described here provides new insight into a potential therapeutic strategy for the treatment of GBM and should inform of the design of clinical trials on this therapeutic combination.

Uveal melanoma (UM) is a rare form of melanoma that is lethal once metastatic. Primary tumors in the iris, ciliary body, and choroid of the eye metastasize in 50% of patients, despite effective treatment of the initial tumor. The majority of uveal melanomas harbor activating mutations in GNAQ or GNA11, which relay signaling to downstream effectors including protein kinase C (PKC) and the mitogen-activated protein kinase (MAPK) signaling pathway (RAF-MEK-ERK). Both PKC and MEK inhibitors are currently in clinical trials, however, MEK inhibition alone is insufficient to improve overall survival. These observations highlight a need to identify new drug targets for the design of novel therapies including combinations. To uncover novel UM biology and nominate strategies for combination therapy, genomic and functional genomic approaches were applied. Whole exome sequencing of primary and metastatic tumors identified somatic genetic alterations that drive tumorigenesis. Recurrent mutations in GNAQ, GNA11, BAP1, SF3B1, and EIF1AX were confirmed. Mutations in potential drivers of metastasis, SMARCA4 and IQGAP1, were also identified. Furthermore, the function of N-terminal tail mutations in the translation initiation factor EIF1AX was probed using loss of function studies to assess both viability and mRNA regulation at the level of translation. Upon EIF1AX knockdown, the efficiency of ribosomal protein translation was reduced in wild type, but not mutant cells. Deregulated translation may play an important role in UM tumorigenesis. To identify putative co-targets of MEK and PKC inhibitors, genome-scale RNA interference drug enhancer screens were performed. These screens nominated several novel genes and pathways for further study in UM. In particular, the mitochondrial folate pathway enzyme MTHFD2 was identified as a novel PKC inhibitor sensitizer. The strongest MEK inhibitor enhancer was the MAPK pathway member, BRAF. Indeed, targeting multiple nodes of the MAPK signaling pathway achieved stronger pathway suppression and synergistic effects. Co-inhibition of RAF/MEK or MEK/ERK may warrant clinical investigation in patients. Overall, these studies provide a foundation for our understanding of UM genomics and combination therapy opportunities. Several novel avenues for future study of UM biology and co-dependencies are uncovered. Translation of these findings into clinical studies will be of the utmost importance. / Medical Sciences

Uveal melanoma is the most common primary intraocular tumor in adults, and is often characterized by poor prognosis and few effective therapeutic options. The typical site of metastasis for uveal melanoma is the liver, and over 80% of patients with metastatic disease will die within one year of metastasis diagnosis. The vast majority of human uveal melanomas contain activating somatic mutations in the GPCR alpha subunits GNAQ or GNA11. To directly observe the in vivo effects of GNA11 Q209L (constitutively active) overexpression, I used a zebrafish cancer model in which plasmids can be injected into transgenic fish and melanoma formation can be assessed. Surprisingly, zebrafish injected with a construct overexpressing mitfa:GNA11 Q209L developed a significant incidence of uveal melanomas. A mini-screen of HOX genes using this system revealed a novel role for HOXB7, which also functioned as an inducer of uveal melanomas in our zebrafish model. Other plasmids containing oncogenes do not lead to uveal tumors, suggesting a specificity for GNA11 and HOXB7. Cell lines were derived from GNA11 Q209L-overexpressing zebrafish tumors and uveal melanoma cells were sensitive to PKC inhibition, which has been observed in human uveal melanoma cells as well. Additionally, RNAseq analysis of zebrafish uveal melanoma cell lines revealed high expression of genes such as cyr61 and ctgf, YAP pathway genes that are known to be induced by GNAQ in human uveal melanomas. I found that overexpression of catalytically inactive BAP1, a chromatin factor whose function is frequently lost in metastatic uveal melanomas, led to a significant acceleration of overall melanoma onset but did not induce a uveal melanoma phenotype in our model. Morpholino experiments showed that knockdown of either zebrafish gna11 or bap1 affected the spatial expression of hoxb7a, with a combined knockdown of gna11 and bap1 suggesting that bap1 is epistatic to gna11. My studies demonstrate the first known instance of spontaneous uveal melanoma formation in response to overexpression of a human uveal melanoma oncogene, and that the resulting uveal melanomas share genetic features and drug response tendencies with human uveal melanomas. This model is a useful new tool for the study of uveal melanoma pathogenesis, genetics, and therapeutics. / Medical Sciences