EGFR and HER2 are overexpressed in 20% and 30% of invasive breast cancer, respectively, and are associated with aggressive tumor subtypes and shortened patient survival. Both receptors are important targets of breast cancer therapy. However, despite the apparent promise of some of these therapies, EGFR- and HER2-based monotherapy and combination regimens have serious limitations and need improvement. The goal of this study is, thus, to gain insights into the biology of EGFR- and HER2-expressing invasive breast cancer in order to provide rationales for more effective EGFR- and HER2-based combination therapy for women with breast cancer. Our proposal is built on novel significant findings made from the initial Idea Award. We discovered that proapoptotic PUMA protein is highly expressed in the breast cancer cell lines and patient tumors that overexpress HER2 and/or EGFR. In addition to co- expression, we found HER2 and EGFR to interact with PUMA constitutively and under the treatment of apoptosis inducers. The HER2-PUMA and EGFR-PUMA interactions are not disrupted when breast cancer cells are treated with the EGFR kinase inhibitors, indicating a kinase-independent interaction. Despite the fact that PUMA has been reported to be primarily located on the mitochondrial membranes and initiate apoptosis upon appropriate stress, our results showed PUMA to be sequestered in the cytoplasm of EGFR-expressing breast cancer cells. Although, the BH3-only proapoptotic proteins can be functionally redundant, we observed PUMA to be essential for apoptotic induction in breast cancer cells. Interestingly, while no reports have investigated PUMA phosphorylation, our preliminary results show that PUMA undergoes tyrosine phosphorylation mediated by HER2 and EGFR.

关键词：乳腺癌；凋亡细胞；生物学；细胞质

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Prostate cancer (PCa) is the second leading cause of cancer death (approximately 30,000/year) in men in the USA. Surgery and radiotherapy are the most effective therapies to treat PCa patients. However, both these forms of treatment, show significant tumor recurrence with locally aggressive disease, metastasis and the morbidity in patients. Several biological disorders are thought to underlie the cause of prostate cancer. One such factor is a tumor suppressor gene DAB2IP which encodes a member of the Ras-GAP protein family. Genome wide Single Nucleotide Polymorphism (SNP) association studies in a large number of patients indicated that DAB2IP is linked with the risk of aggressive prostate cancer. DAB2IP deficient PCa cells are resistant to radiation treatment. Therefore, to improve radiation killing of these aggressive PCa cells, this proposal will explore the radiosensitizing property of NU7441, a specific kinase inhibitor of DNA-PKcs.

关键词：脱氧核糖核酸；前列腺癌；辐射

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关键词：激素；军事人员；神经科学；类固醇

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Background: Mild traumatic brain injury (mTBI) secondary to blast exposure is the most common battlefield injury in Southwest Asia. There has been little prospective work in the combat setting to test the efficacy of new countermeasures. The goal of this study was to compare the efficacy of N-acetyl cysteine (NAC) versus placebo on the symptoms associated with blast exposure mTBI in a combat setting. Methods: This study was a randomized double blind, placebo-controlled study that was conducted on active duty service members at a forward deployed field hospital in Iraq. All symptomatic U.S. service members who were exposed to significant ordnance blast and who met the criteria for mTBI were offered participation in the study and 81 individuals agreed to participate. Individuals underwent a baseline evaluation and then were randomly assigned to receive either Nacetyl cysteine (NAC) or placebo for seven days. Each subject was re-evaluated at 3 and 7 days. Outcome measures were the presence of the following sequelae of mTBI: dizziness, hearing loss, headache, memory loss, sleep disturbances, and neurocognitive dysfunction. The resolution of these symptoms seven days after the blast exposure was the main outcome measure in this study. Logistic regression on the outcome of no day 7 symptoms indicated that NAC treatment was significantly better than placebo (OR = 3.6, p = 0.006). Secondary analysis revealed subjects receiving NAC within 24 hours of blast had an 86% chance of symptom resolution with no reported side effects versus 42% for those seen early who received placebo. Conclusion: This study, conducted in an active theatre of war, demonstrates that NAC, a safe pharmaceutical countermeasure, has beneficial effects on the severity and resolution of sequelae of blast induced mTBI. This is the first demonstration of an effective short term countermeasure for mTBI. Further work on long term outcomes and the potential use of NAC in civilian mTBI is warranted.

关键词：半胱氨酸；安慰剂；创伤性脑损伤

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In this project we set out to establish conditions for differentiation of human neonatal foreskin skin fibroblast-derived iPSCs into prostate epithelium-like cells and identify differences in gene expression between prostate epithelial cells derived from iPSCs of Caucasian (white) and African-American (black) foreskin fibroblasts. We identified and optimized culture conditions that promote prostate epithelial cell-like differentiation of humaniPS clone, IMAR90-4. Our data show that a feeder layer of urogential mesenchymal(UGSM) cells from neonatal mouse of either gender in combination with neonatal human dermal fibroblasts induced a striking morphological changes that resembles epithelila differentiation with formation of lumen-like structures. We show requirement of components of the extracellular matrix that promote epithelial-type differentaition. Immunofluorescence and biochemivcal analyses showed expression of androgen receptor and markers of epithelial differentiation. Analyses of pluripotency marker expression by RT-PCR showed that while human dermal fibroblasts have higher constitutive expression of Nanog, Oct4 and Sox2 compared to UGSM and IMP90 cells. Preliminary studies also showed that black black fibrobalst population has higher constitutive expression of pluripotency markers than cells from white individuals.

关键词：前列腺癌；干细胞；血细胞

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In year 1 of this project, we determined whether Compound 49b, a novel beta-adrenergic receptor agonist, could prevent increased inflammatory and apoptosis proteins in mice after exposure to ocular blast. Eyes from C57/BL6 mice were exposed to a blast of air from a paintball gun at 26psi. Eyes were collected at 4, 24, and 72 hours after blast exposure. In a subset of mice, Compound 49b eye drops (1mM dose) were applied within 4 hours, 24 hours, or 72 hours after blast. Three days after exposure to blast, all mice were sacrificed. One eye was used for protein analyses of TNF, IL-1, Bax, Bcl-xL, caspase 3, and cytochrome C. The other eye was used for TUNEL labeling of apoptotic cells, which were co-labeled with NeuN to stain for retinal ganglion cells. We found that ocular exposure to 26psi of air pressure led to a significant increase in both inflammatory and apoptotic proteins. When Compound 49b was applied within 4 or 24 hours after blast, it mitigated the increase in inflammatory and apoptotic proteins. Ocular blast produces a significant increase in inflammatory and apoptotic proteins in the retina, specifically in retinal ganglion cells. These proteins are reduced after treatment with a topical betaadrenergic receptor agonist. This data suggests local application of beta-adrenergic receptor agonists may be protective in ocular blast.

关键词：爆炸；眼；创伤和损伤；细胞凋亡

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The altered balance in the expression of PKC isozymes is a distinguished feature of cancer. One of the most notable alterations in epithelial cancers is the upregulation of PKC . This kinase has emerged as a potential oncogene and tumor biomarker, however, little is known regarding a potential causality between its upregulation and cancer development. In this research we wished to understand the role played by PKC in prostate cancer cells and establishes the proof of principle of PKC inhibition as a putative therapeutic strategy. We already found that PKC inhibition decreases size of tumor generated by PC3-ML cells in athymic/balb-c mice. Therefore, during this second year we have focus in the study of the metastasic properties of PKCe. Using an RNAi approach we found that PKC depletion markedly impaired the ability of PC3-ML metastasize in bone marrow of athymic/balb-c mice. In addition we found that this kinase is relevant in attachment, MMPs activity, cytokines expression, and anchorage-dependent and anchorage- independent growth of PC3-ML cells. Moreover, we have determined that pharmacological inhibition of PKCe significantly reduced invasiveness phenotype of PC3-ML. In summary, our results argue for a role of PKC in prostate cancer development and metastasis, highlighting its potential as a therapeutic target.

关键词：转移；磷转移；前列腺癌；蛋白质

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The objective of this research is to design and develop a nanocarrier that is able to evade the immune system, circulate in the blood stream, find its target prostate cancer cells, and transfer therapeutic genes into prostate cancer cells efficiently. The gene carrier is composed of: a) histone H2A peptide to condense pDNA into nano-size particles, b) a PC-3 specific targeting motif (TM) to target prostate cancer cells, c) an endosomolytic motif to disrupt endosome membrane, and d) a nuclear localization signal (NLS) to actively translocate pDNA towards the nucleus of cancer cells. The gene delivery system was synthesized in E.coli. The vector was then complexed with plasmid DNA (pDNA) to form stable nanoparticles with sizes below 100nm. The nanoparticles were used to deliver reporter genes (pEGFP) to target PC-3 prostate cancer cells and RWPE-1 normal epithelial prostate cells. The induction of immune response by the vector was studied in BALB/c immune- competent mice. The results demonstrated that the gene delivery system is able to target and efficiently transfect PC-3 cancer cells with minimum cross- reactivity with normal epithelial prostate cells. An animal protocol was prepared and approved by IACUC and DOD ACURO. The immunogenicity studies showed that the vector does not induce production IgG or IgM after repeated systemic injection.

关键词：基因治疗；纳米技术；前列腺癌

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While we initially found that T effector cell responses against breast cancers can be augmented by increasing C3a and C5a receptor (C3aR/C5aR) signaling by antigen presenting dendritic cell dendritic (DCs) by blocking the function of decay accelerating factor (DAF), we have found that in breast cancers themselves and vascular endothelial cells C3aR/C5aR signaling promotes breast cancer growth. In the past years, we have found that blockade of C3aR/C5aR signaling in CD4+ cells enables endogenous TGF-b1 production and Foxp3 T regulatory cells induction. We are developing ways to control these effects and have prepared specialized mice for testing the clinical efficacy of our findings.

关键词：临床医学；T淋巴细胞；乳腺癌

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Acute lung injury (ALI) is a complex condition associated with diffuse injury to the distal alveolar epithelial gas exchange surface, resulting in marked impairment in the ability to oxygenate blood. Treatment is generally supportive. This condition commonly afflicts patients with cancer. In this regard, cancer patients are especially susceptible to developing acute lung injury (ALI) due to the immunosuppressive and toxic effects of chemotherapy and the debilitating effects of cancer. This condition also afflicts military personnel that experience severe trauma or receive multiple transfusions, and also commonly accompanies severe infections. The over-arching goal of our original application was to derive new approaches to treat ALI with an emphasis on developing new modes of mechanical ventilation and developing cell based strategies to directly repair the injured lung. In Project 1, we proposed to optimize so-called variable ventilation with the intent of minimizing the known injurious effects of conventional mechanical ventilation in patients with ALI. In this past year, where this grant has been in a limited no-cost extension, we have not been using DOD funds for Project 1. Based on data generated from this proposal, however we have been able to secure funds from the Coulter foundation to continue this work by performing a limited clinical study on the safety and efficacy of variable ventilation in patients with ALI. Using the funds that have been available in this no-cost extension has permitted us to continue work on Project 2, which is the cell therapy component. For Project 2, we have continued to develop protocols for the identification and derivation of lung stem cells that can be used for therapeutic purposes in ALI. We also have been identifying other cell populations that can be manipulated to hasten lung repair.

关键词：肺癌；肺癌；干细胞；输血细胞（生物学）

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