Overexpression of EGFR is frequently linked to more aggressive tumor behavior, including increased proliferation, metastasis, and therapeutic resistance. Here, we identified a molecular linkage between IKK and EGFR signaling in breast cancer cells. Inhibition of IKKs activity elevates EGFR tyrosine phosphorylation. In addition, IKK forms a specific interaction with EGFR in Golgi apparatus and catalyzes EGFR S1026 phosphorylation. We found that EGFR S1026A possess a stronger tumorgenesis phenotype compare with wild type EGFR suggesting a negative regulation of IKK in EGFR signaling. In agreement with an earlier finding where conditional ablation of IKK in the mice keratinocytes elevates the autocrine loop of EGFR, our results further provide a potent role of IKK kinase activity in preservation of EGFR activity.

关键词：乳腺癌；肿瘤细胞（生物学）；炎症

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This is a basic research project designed to understand the role of mechanically sensitive excitatory ion channels (MSC) in the pathology of chronic pain, and the use of a small pepti de inhibitor of these channels called GsMTx4 to treatment peripheral pain. Our first goal is to develop an in vitro assay using single neurons to study the properties of MSCs and to determine the affinity of GsMTx4 and muta nt analogs for neuronal MSCs. We have learned to prepare dorsal root ganglion neurons (DRG) from mouse spinal cord and have determined the types of MSCs expressed and the frequency of their occurrence in the cell-attached and outside-out patch assay. We have also designed a second assay for MSC activity by pressing on DRG with a precision controlled motion glass probe while measuring whole cell currents as a independent measure of the MSC activity. Stress on the internal cytoskeleton may play a critical role in the activation of MSCs on the surface and so we will measure stress changes in the proteins that make up the cytoskeleton of DRGs using a new FRET based stress probe we have made which can be inserted into the sequence of cytoskeletal proteins to report stress. We have tried to reproduce the published findings of others showing GsMTx4 analgesic activity in whole animals by treating inflamed tissue in mice, but were unable to do so. However, we have learned new information about the pharmaco kinetics of GsMTx4 that will be used to redesign the study.

关键词：神经节；动力学；疼痛；激活

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This project is aimed at test the hypothesis that targeting androgen receptor (AR) expression by small-molecule agents represents a potentially successful strategy to block prostate tumor growth and to delay prostate progression. Based on our finding that the AR-ablative agent CG-12 blocked AR expression through the inhibition of glucose uptake, we embarked on the lead optimization of CG-12 to generate novel glucose transporter inhibitors with high potencies in suppressing AR signaling in LNCaP and VCaP cells. These agents inhibited AR expression through -TrCP-mediated Sp1 downregulation, leading to transcriptional repression of the AR gene. By using a structurally optimized derivative, CG-5, the present study demonstrates the therapeutic relevance of targeting the Warburg effect to prostate cancer therapy, in part, through the blockade of AR signaling. Oral CG-5 exhibits in vivo efficacy in suppressing LNCaP-abl xenograft tumor growth in nude mice, and in suppressing the progression of pre-neoplastic prostatic intraepithelial neoplasia progression in TRAMP mice, which correlated with the drug's ability to modulate biomarkers associated with AR signaling.

关键词：雄激素；肿瘤；前列腺癌；受体位点（生理学）

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Since this application was written, it has become increasingly appreciated that NF B activation can either promote cancer cell death or cancer cell survival the outcome being dependent on the context of parallel biological processes. Notably, the presence of tumor suppressors influences the outcome. Our bioinformatic approach to define a cancer promoting NF B gene activation signature is proving to be well suited for accounting for the varied and opposing roles of NF B activation. Specifically, our prostate cancer specific work has identified absence of a unique set of tumor suppressors which leads to cancer cell survival and ultimately lethal prostate cancer. Notably, PTEN was not one of these tumor suppressors. It is also now appreciated that indiscriminate inhibition of NF B activation may be problematic as this may block the anti-cancer effect of NF B activation. As such the increased understanding of NF B activation s context dependency adds further support for the work we are doing. In year one and two of the project we have found elevated cytokines and presence of T. Vaginalis at time of diagnosis of prostate cancer are not associated with higher grade disease nor risk of relapse after prostatectomy. We have also identified a 31 tumor gene signature which correlates with relapse with lethal disease post prostatectomy in our training set. We have also used the 31 gene signature and publically available data-bases to computationally create a refined network of cancer promoting NF B gene activation. This network is now being used to (i) further inform selection of genes to test in multiple independent data sets for association with lethal disease; (ii) inform and increase power for identification of new SNPs in GWAS datasets associated with lethal outcome, and (iii) help to interpret the mechanisms of action of genes associated with lethal disease identified separately in the tumor gene expression profiling and SNP analyses.

关键词：基因；前列腺癌；生物细胞（生物学）

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Transfer RNAs (tRNA) are small non-coding RNAs that read the genetic codes in protein synthesis. It is essential for the proliferation, fitness and adaptation of the cell that each tRNA is aminoacylated (charged) with its designated amino acid. The utilization of mischarged tRNAs (i.e. tRNAs with incompatibly charged amino acid and decoding capacity) leads to the synthesis of mutated proteins that can fold incorrectly. Accumulation of misfolded proteins in the cell activates an integrated cellular mechanism, the Unfolded Protein Response which dictates cell fate in response to the amount of misfolded proteins. High accumulation of misfolded proteins derived from the cellular presence of mischarged tRNAs can therefore induce apoptosis of the cell. We aim to engineer tRNAs that are always mischarged in a human cell and study their effects on breast tumor cell physiology and cell death. Protein demand in rapidly proliferating cells is extremely high and small defects during cellular protein synthesis caused by the presence of such tRNAs can have a strong impact on both tumor invasiveness and survival. Ultimately, we aim to demonstrate that these mischarged tRNAs can be developed as a novel class of RNA-based agents to treat breast tumors.

关键词：乳腺癌；化疗药物；氨基酸

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Prostate cancer poses a major public health problem in the United States and worldwide. It has the highest incidence and is the second most common cause of cancer deaths in North American men resulting in over 30,000 deaths per annum. Consequently, there is an urgent need to develop novel therapeutic approaches. We propose to use a novel cyclotide-based molecular scaffold for generating molecular libraries that will be screened and selected in vivo to identify antagonists of the RING-mediated Hdm2/Hdmx interaction. Our innovative approach will use cell-based E. coli libraries in which individual bacteria express a different cyclotide. This comprises a new single cell-single compound approach to identify protein-protein binding antagonists. These compounds will be subjected to a two-step screen, the first involving a high throughput FRET-based FACS screen in bacteria, and the second a bioluminescence complementation assay in cancer cells. Together, these assays will identify cyclotides that disrupt Hdm2-Hdmx interactions, activate p53, and elicit p53- dependent cytotoxicity in prostate cancer cells. During the second year of this project we have accomplished the following: (1) Developed a more efficient method for the biosynthesis of cyclotides in E. coli cells; (2) We have validated our FRET-based reporter to sort cell populations; (3) Developed and characterized a novel cyclotide with p53 activating properties.

关键词：分子；前列腺癌；治疗；细菌

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The 'Fracture Putty' project was initiated to develop better, faster and more efficient methods to treat difficult fractures. This contract supported study of hydrogel polymers, which are polymerized under direct control of the operating surgeon, and are also designed for biologic cleavage by osteoclast specific proteases. Processes in bone growth and repair were studied in cell cultures, mice, rats, sheep, pigs, and feasibility of lymphatic imaging was demonstrated in humans. We believe this system is safe and efficacious and will lead to profound enhancements in patient care.

关键词：骨头；缺陷（材料）；凝胶；基因工程

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Therapies designed to damage DNA (e.g. chemotherapy and irradiation) cure many primary prostate carcinomas (PCa) and produce significant responses in a subset of advanced metastatic cancers. However, a subset of localized cancers resist genotoxic treatments, and most advanced cancers treated with such therapies eventually progress to a lethal phenotype. Thus, therapy resistance is a major contributor to PCa morbidity and mortality. We want to explore the hypothesis that DNA damaging therapeutics generates responses in benign cell types comprising TME that promote tumor cell survival and enhance resistance. We have assessed the outcome of targeting individual mediators of this microenvironment-derived DDSP---specifically a member of the Wnt superfamily, WNT16B and demonstrated the highly effective neutralization of prostate cancer cell malignancy in vitro by purified anti-WNT16B. We anticipate that suppressing WNT16B will diminish treatment-initiated resistance, and improve in vivo tumor responses. We have established primary mouse prostate fibroblast cell lines and examined their responses including DDSP development upon DNA damage, and demonstrated the potential complication of regulatory mechanisms of DDSP program.

关键词：前列腺癌；脱氧核糖核酸；分泌

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We have a cohort of 200 untreated CFS cases and 400 matched controls that will undergo two novel tests (CyTOF-phosphoflow and HLA Typing through a breakthrough method discovered at Stanford) in order to help enhance our understanding of CFS and contribute to the elucidation of the pathogenesis of the disease. For CyTOF testing, we are exploring the immune responses by a novel flow cytometer that detects individual cell traits with time-of-flight mass spectrometry (CyTOF). We have completed the CyTOF phenotyping, phospho- flow panel, and gating schemes for testing. Also, the flow cytometry preparation robotics for CyTOF was optimized and antibodies were conjugated. The testing is ongoing and we hope to continue testing as permitted. For HLA typing testing, we are determining the human leukocyte antigens (HLA) types using a novel method that combines long-range polymerase chain reaction (PCR) with very high-throughput, multiplexed Illumina paired-end sequencing of genes permitting direct haplotyping. We have identified the DNA PAXgene tubes that will be used for testing. Also, we have identified the best possible conditions to amplify all the HLA gene of interest, namely for the class I genes HLA-A, -B, -C and for the class II genes: HLA DQA, DQB, DRB DPA and DPB. Lastly, we achieved the goal of robust and highly reproducible amplification of each In summary, for CyTOF testing we finalized the Phospho-CyTOF panel and have begun testing samples. To date we have tested 54 samples. These samples will continue to be tested for Year 2. The data is being received; no analysis has been done yet. The data will be analyzed by our statistical team. For HLA typing testing, all the DNA PAXgenes have been identified and separated into different batches. To date, two plates have been run and are currently being analyzed. To date we have tested 180 samples Year 1 has allowed us to ready the logistics and to begin CyTOF and HLA Typing testing.

关键词：抗体；疲劳（生理）；发病机制

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The project is based on the generation of metastatic brain tumors using a brain selective cell line, 231-BR, derived from human breast cancer. Therefore, the experimental model to be used must be immune compromised. The other requirement we have for this project is that the experimental animals express human HLA-A2 complexes (major histocompatibility complex class I). The antibody we want to evaluate as a potential therapeutic agent is a T-cell receptor mimic restricted to human HLA-A2. Therefore, we must use HLA-A2 transgenic mice. In experiments conducted to date the mouse strain, which fulfills both requirements, JAX 9617, and the corresponding non-transgenic control strain, 5557, were receptive for tumor growth not only in brain, but in a number of peripheral organs (e.g. lung, liver, spleen). To avoid the confounding influence of peripheral metastases, we currently explore alternative methods to generate brain tumors intracarotid injections, stereotaxic brain implantation. Regarding the analytical side, we have established an immunoradiometric assay for the RL6A antibody with high sensitivity. In saturation and in competition experiments, radioiodinated RL6A and unlabeled antibody showed a Kd value of 1.16 nM and a Ki of 1.26 nM, respectively.

关键词：抗体；乳腺癌细胞；转移；肿瘤

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