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The current study will identify D-methionine (D-met) dose and delayed time otoprotective response from steady state and impulse noise-induced hearing loss (NIHL). The third year was dedicated to completing data analysis from Year 2 and to further define maximum time delay for D-met post-noise rescue protection from permanent NIHL and to determine if additional D-met dosing for another 48 hours can improve post-noise rescue protection at the first rescue time with suboptimal protection. Groups of Chinchillas laniger (n = 10) received five intraperitoneal D-met (200 mg/kg/dose) every 12 hours beginning 28 or 32 hours after steady state or impulse. D-met otoprotection was assessed by auditory brainstem response (ABR) analysis and outer hair cell (OHC) quantification. ABR analyses identified 28 hour rescue as the first sub-optimal rescue dose time. Additional D-met administration at 28 hour rescue significantly recovered the lost protection in steady state noise-exposed animals. Histological cytocochleogram analysis is still in progress to confirm electrophysiological assessment., The original document contains color images.

Identification of genes driving prostate carcinogenesis will lead to new cancer treatment. The human chromosome 8q24.21 region has been linked with increased risk for prostatic carcinoma but the how this region contributes to prostate carcinogenesis is unknown. We cloned a candidate gene, POU5F1B (also called POU5F1P1), in this gene desert of 1.2Mb between FAM84B and the c-MYC oncogene. POU5F1B is a pseudogene of embryonic Oct4 (POU5F1). A recent study found that tumor Oct4 found in prostate cancer cells is due to the gene expression of POU5F1B, not embryonic Oct4 (POU5F1). In a dataset of 171 patients, it was found that tumor Oct4 was significantly increased in primary tumors and markedly increased in metastatic tumors, when compared to normal prostate or adjacent normal tissues. Based on the analyses and our preliminary data, we think, tumor Oct4, expressed from POU5F1B in the prostate cancer susceptibility loci 8q24, is a driver of prostate tumor formation and progression, and therefore, this driver is a novel target of intervention to eliminate prostate cancer. We propose to further determine the roles of tumor Oct4 in prostate tumor formation and metastasis. We hope we can validate whether tumor Oct4 can be targeted to inhibit prostate cancer progression and metastasis. In addition, we will map out the regions critical for Oct4 to promote prostate carcinogenesis so that we can target this region to develop therapeutics for cancer treatment in the future., The original document contains color images.

The current study will identify D-methionine (D-met) dose and delayed time otoprotective response from steady state and impulse noise-induced hearing loss (NIHL). The second year was dedicated to determining the latest time D-met may be administered for efficient protection. Groups of Chinchillas laniger (n = 10) received five intraperitoneal D-met (200 mg/kg/dose) every 12 hours beginning 7, 9, 12, 18, 34, 36, or 48 hours after steady state or impulse. D-met otoprotection was assessed by auditory brainstem response (ABR) analysis and outer hair cell (OHC) quantification. ABR analyses identified time-dependent D-met protection from steady state and impulse noise exposures when D-met administration began as late as 24 hours post-noise exposure. Histological cytocochleogram analysis is still in progress to confirm electrophysiological assessment. Statistical analysis is currently in progress., The original document contains color images.

Hearing loss can render a soldier less able to detect and identify the enemy, less able to understand commands, particularly in background noise typical on the battlefield, and may permanently reduce quality of life. Currently, no FDA approved pharmacological prevention exists for noise-induced hearing loss (NIHL). We have documented in animal studies that administration of D-methionine (D-met) can reduce or prevent NIHL. We now need to determine if it has similar efficacy in humans. This prospective study is a randomized, double-blind, placebo-controlled Phase 3 clinical trial of oral D-met to reduce noise-induced hearing loss (NIHL) and tinnitus. The goal of the study is to develop a safe, oral pharmacological agent to augment physical hearing protectors for noise exposures that exceed the protective capabilities of ear plugs and/or muffs. The study population is a cohort of Drill Sergeant (DS) instructor trainees during and 22 days after their 11 day weapons training. The primary objective of this study is to determine the efficacy of D-Met in preventing NIHL or reducing tinnitus secondary to a minimum of 500 rounds of M-16 weapons training occurring over an 11 day period..

Resistance to chemotherapy presents challenges to prostate cancer management. Preliminary data suggest that Nanog is associated with prostate cancer stem cells and Nanog may cause resistance toward chemotherapy. The objective of this proposal is to define the role of Nanog in resistance of prostate cancer cells toward chemotherapy, to determine whether Nanog can be targeted to sensitize tumor cells toward chemotherapy, and to identify the downstream effectors,especially a family of efflux transporters, called, ATP-binding cassette transporters, in Nanog-mediated chemoresistance. Various molecular, cellular, and pharmacological approaches will be employed to achieve the goals of the proposed studies. The proposed studies will validate whether we can target tumor Nanog to sensitize prostate cancer stem cells toward chemotherapy so that tumor recurrence or drug resistance can be eliminated. By performing and achieving the goals of the proposed research, the principal investigator will learn how to conduct cutting edge research that can be of high significance in the translational cancer research.

The goal of my career is to become an independent and productive scientist in the area of prostate cancer research. I am especially committed to improve the efficacy of current treatment modalities for advanced prostate cancer patients through translating my research into clinical applications., The original document contains color images.

Hearing loss can render a soldier less able to detect and identify the enemy, less able to understand commands, particularly in background noise typical on the battlefield, and may permanently reduce quality of life. Currently, no FDA approved pharmacological prevention exists for noise-induced hearing loss (NIHL). We have documented in animal studies that administration of D-methionine (D-met) can reduce or prevent NIHL. We now need to determine if it has similar efficacy in humans. This prospective study is a randomized, double-blind, placebo controlled Phase 3 clinical trial of oral D-met to reduce noise-induced hearing loss (NIHL) and tinnitus. The goal of the study is to develop a safe, oral pharmacological agent to augment physical hearing protectors for noise exposures that exceed the protective capabilities of ear plugs and/or muffs. The study population is a cohort of Drill Sergeant (DS) instructor trainees during and 22 days after their 11 day weapons training. The primary objective of this study is to determine the efficacy of DMet in preventing NIHL or reducing tinnitus secondary to a minimum of 500 rounds of M-16 weapons training occurring over an 11 day period., The original document contains color images.

Tasks include ABR data collection, solution preparation, animal injection, animal noise exposure, histological microdissection, ABR analysis, SEM analysis, and animal protocol development. ABR threshold analysis is conducted daily. Microdissection and SEM work are conducted daily. SEM analysis is conducted as samples are prepared. Preliminary outer hair cell analyses support statistically significant otoprotection for impulse and steady state noise-exposed animals that were observed by ABR analysis.

Bone is the most common metastatic site for prostate cancer. The growth of the tumor cells in the bone is generally slow and they often become dormant until an appropriate microenviroment is established for their re-growth. The recent stem cell theory predicts that the metastatic cells are a small population of stem-like cells in the primary tumor. However, the precise mechanism of dormancy is virtually unknown, and identifying the responsible factors and understanding their underlining mechanism are crucial for developing a novel therapeutic approach. Our preliminary data indicate that (i) bone morphogenetic protein 7 (BMP7) which is secreted from bone marrow stromal cells is able to induce senescence to prostate tumor cell and (ii) this induction is mediated by activation of the tumor metastasis suppressor gene, N-myc downstream regulated gene 1 (NDRG1). These results strongly suggest that the BMP7-NDRG1axis plays a critical role in dormancy of prostate tumor cells in the bone. The overall goal of this proposal is to elucidate the mechanism of BMP7-induced dormancy in tumor stem cells and explore a possibility of using BMP7 as an anti-metastatic drug for prostate cancer., The original document contains color images.

According to the recent cancer stem cell (CSC) theory, recurrent tumor must arise from a dormant tumor stem cell whose re- growth is triggered by shifting of microenvironment. This project aims at clarifying the roles of TAM in recurrent growth of dormant stem cell in breast cancer. We hypothesize that the balance of dormancy and recurrence is determined by the ability of the tumor stem cells to recruit TAM which in turn promotes self-renewal of the stem cell. We have established necessary mouse colonies and also developed the method to generate TAM. We have also shown that TAM indeed promoted the growth of CSCs in our animal model. Due to the relocation of our entire lab, Task 2 which involves extensive animal breeding has been delayed; however, we have compensated the lack of progress in Task 2 by further extending our mechanistic study in Task 1. We have shown that the interaction of TAM with CSCs lead to enhanced secretion of PDGF-BB from TAMs which then activated stromal cells and enhanced CSC self-renewal. We believe that the outcome of our results set a new paradigm in our understanding of metastatic niche of CSCs in the bone, which may provide novel targets such as TAM, PDGF and FGF, for the treatment of recurrent disease., The original document contains color images.

Dissemination and metastasis of tumor cells are major causes of morbidity and mortality in breast cancer patients. Therefore it is of vital importance to identify druggable targets to inhibit breast tumor invasion and metastasis. A critical component in the invasive growth, dissemination, and metastasis of cancer is acquisition of motility by tumor cells. Our preliminary studies suggest a novel role for the thromboxane A2 receptor (TP) in controlling breast tumor cell motility via regulating cytoskeleton reorganization. The objective of this proposal is to define the function of TP in tumor cell motility and to validate TP as a target for anti-metastasis therapy of breast cancer. In the first aim, the role of TP in breast tumor cell motility will be determined in the presence or absence of TP activation or inhibition. The isoform(s) of TP involved in tumor cell contraction and motility will be identified in the second specific aim. In the third aim, an orthotopic mouse model will be used to assess whether TP can be targeted to reduce breast cancer metastasis. The proposed studies will be highly significant toward the goals of developing mechanism-based interventions for cancers., The original document contains color images.

The purpose of this Phase 2 clinical trial is to determine if an oral, orange flavored suspension of D-methionine can prevent noise-induced hearing loss (NIHL) and tinnitus in our troops.

Recurrent disease is the most daunting aspect of cancer therapy; however, how tumor cells become dormant and later recur is poorly understood. It is of paramount importance to decipher the underlining molecular mechanism of dormancy in order to define specific targets for the treatment of recurrent metastatic disease. The overall goal of this project is to identify key factor(s) and signaling that control tumor stem cell dormancy. In this fiscal year, we have established a unique model of tumor dormancy and recurrence in vivo using CSCs of prostate cancer cell line. This model provides us with an excellent tool to explore the molecular mechanism of tumor cell dormancy of CSCs in prostate cancer in bone. In the next fiscal year, we plan to define the roles of SPARC and Noggin in controlling bone microenvironment for dormancy and recurrence and we have already obtained approval of no-cost extension of this grant. We believe that the results of our experiments will provide important clues to identify specific targets for recurrent prostate tumor so that we can confine metastasized tumor to dormant state., The original document contains color images.

Dissemination and metastasis of tumor cells are major causes of morbidity and mortality in breast cancer patients. Therefore it is of vital importance to identify druggable targets to inhibit breast tumor invasion and metastasis. A critical component in the invasive growth, dissemination, and metastasis of cancer is acquisition of motility by tumor cells. Our preliminary studies suggest a novel role for the thromboxane A2 receptor (TP) in controlling breast tumor cell motility via regulating cytoskeleton reorganization. The objective of this proposal is to define the function of TP in tumor cell motility and to validate TP as a target for anti-metastasis therapy of breast cancer. In the first aim, the role of TP in breast tumor cell motility will be determined in the presence or absence of TP activation or inhibition. The isoform(s) of TP involved in tumor cell contraction and motility will be identified in the second specific aim. In the third aim, an orthotopic mouse model will be used to assess whether TP can be targeted to reduce breast cancer metastasis. The proposed studies will be highly significant toward the goals of developing mechanism-based interventions for cancers., The original document contains color images.

The pro-inflammatory microenvironment in the tumor is established by first recruiting various leukocytes including tumor-associated macrophage (TAM) which is believed to promote tumor growth and angiogenesis, suppress adaptive immunity, and hence remodel tumor microenvironment. According to the recent cancer stem cell (CSC) theory, recurrent tumor must arise from a dormant tumor stem cell whose re-growth is triggered by shifting of microenvironment. This project aims at clarifying the roles of TAM in recurrent growth of dormant stem cell in breast cancer. We hypothesize that the balance of dormancy and recurrence is determined by the ability of the tumor stem cells to recruit TAM which in turn promotes self-renewal of the stem cell. We have established necessary mouse colonies and also developed the method to generate TAM. We have also shown that TAM indeed promotes the growth of CSCs in our animal model. In the next fiscal year, we will address two important points. First, we will clarify whether TAM indeed plays a critical role in recurrent growth of tumor stem cells, which we believe will bring in a paradigm shift into our research field for the understanding of the mechanism of recurrent tumor growth. Secondly, the results of this research will provide us with strong rationale for targeting TAM for chemo-prevention of recurrent breast cancer., The original document contains color images.

Bone is the most common metastatic site for prostate cancer. The growth of the tumor cells in the bone is generally slow and they often become dormant until an appropriate microenviroment is established for their re-growth. The recent stem cell theory predicts that the metastatic cells are a small population of stem-like cells in the primary tumor. However, the precise mechanism of dormancy is virtually unknown, and identifying the responsible factors and understanding their underlining mechanism are crucial for developing a novel therapeutic approach. Our preliminary data indicate that (i) bone morphogenetic protein 7 (BMP7) which is secreted from bone marrow stromal cells is able to induce senescence to prostate tumor cell and (ii) this induction is mediated by activation of the tumor metastasis suppressor gene, N-myc downstream regulated gene 1 (NDRG1). These results strongly suggest that the BMP7-NDRG1axis plays a critical role in dormancy of prostate tumor cells in the bone. The overall goal of this proposal is to elucidate the mechanism of BMP7-induced dormancy in tumor stem cells and explore a possibility of using BMP7 as an anti-metastatic drug for prostate cancer., The original document contains color images.

It is now increasingly accepted that cancer stem cells (CSCs, or tumor initiating cells) are responsible for tumor initiation. If cancer treatment kills most of cancer cells in the stage of transit amplifying and differentiation without killing the stem cells, the surviving cancer stem cells will eventually lead to recurrence of tumors. To eradicate cancer, we must learn more about the biology of cancer stem cells, their responses to treatments, and their role in tumor recurrence after treatment. In the preliminary studies, I found that Nanog, a transcription factor essential for self-renewal of embryonic stem cells, was expressed in prostate cancer cells, and further its expression was associated with tumor cells positive for stem/progenitor markers. Knockdown of Nanog reduced the ability of cancer cell to form tumors in an animal model. I further found that tumor cells with endogenous Nanog expression were particular resistant to chemotherapy. The data suggest that Nanog is associated with prostate cancer stem cells and Nanog may cause resistance toward chemotherapy., The original document contains color images.

More than 20% of breast cancer patients detected by mammography are DCIS and this number keeps on increasing (1). Although DCIS is a non-invasive benign tumor, it is considered a precursor of malignant cancer. Therefore, understanding the molecular changes from normal cell to DCIS is of paramount importance and it is under intensive study. However, the exact mechanism of normal-DCIS transition is still not well understood. The purpose of this project is to identify specific microRNAs in tumor stem cells that are responsible for the formation of DCIS. The results of our microRNA array analysis for CSCs from normal and DCIS cells revealed that the expression of a series of microRNA are changed during the transition from normal to DCIS in CSCs. Particularly, we found that miR29a and miR29c function as suppressors of DCIS by blocking the expression of SREBP1, suggesting that modulation of these microRNAs in CSCs triggers the initiation of DCIS. Because the major target of miR29a and miR29c is SREBP, it is possible that this gene can be a potential candidate as a preventive drug for DCIS. Alternatively, the expression of miR29a and miR29C can be restored by a small molecule. It would be ideal if this can be accomplished with specific nutrition. However, this requires further investigation., The original document contains color images.

More than 90% of deaths caused by breast cancer are attributed to metastatic disease. However, the exact molecular mechanism of tumor metastasis is still poorly understood. It has been well recognized that only a fraction of cells in the primary tumor eventually metastasizes to the distant organs; however, the origin and nature of these cells are still unclear. The purpose of this project is to test our novel hypothesis that metastatic cells originate from a distinct tumor cell population which has both stem-like properties and an invasive ability. We have successfully isolated the cell population (CD24-/ CD44+/ ESA+) that has tumor initiating ability as well as metastatic capability. The expression profile analysis revealed that the HAS2 gene plays a critical role in the process of bone metastasis of CSCs, which was also strongly supported by our results of in vivo experiment. Interestingly, small molecule HAS2 inhibitor, 4MU, was shown to significantly suppress the CSC-induced bone metastasis. Therefore, our results open a possibility of using 4MU for the treatment of metastatic bone disease., The original document contains color images.

Adenocarcinoma of the prostate is currently the most prevalent cancer in men in the United States and represents 36% of all cancers among men (1). It is estimated that more than 210,000 new cases are diagnosed and 32,000 patients succumb to this disease every year (1). Although patients with localized lesions can be cured by radical prostatectomy or radiotherapy, more than 90% of cancer deaths are attributed to metastatic disease (2). Even those patients who have localized cancer and have been "successfully" treated with surgery often experience "recurrent" disease after many years. In the last two decades, numerous chemotherapeutic agents have been studied. However, the overall results have been quite disappointing. The failure of the current approach to develop an anti-cancer drug for prostate cancer suggests that we need essentially a new approach by defining a specific target molecule in this cancer (3). Traditional screening of anti-cancer drugs has been mostly dependent on growth inhibition assay for cancer cells. However, targeting a specific gene with well-defined clinical rationale will provide a better chance of developing a more effective therapeutic agent., The original document contains color images.

Tumor metastasis is an extremely inefficient process and only a fraction of cells in the primary tumor can successfully establish metastatic colonization. These cells by definition have a stem-like ability, but they also need to have an ability of metastasizing to other organs. Therefore, in addition to a tumor stem cell, an existence of a metastatic stem cell is predicted. Despite the critical importance of the concept, this idea has not been rigorously tested due to a lack of an appropriate experimental system. We propose to take an innovative approach to challenge this question by isolating stem cell population from a unique set of breast tumor cell lines and by examining their metastatic behavior in an animal model. The overall objective of our project is to identify metastatic stem cells of breast cancer and define basic characteristics of these cells. To test our hypothesis, we will (i) isolate stem-cell population from non-metastatic and metastatic cells of a pair of syngenic breast tumor cell lines, and test their metastatic ability in an animal model, and (ii) examine their gene expression profiles by microarray analysis and verify the results in tumor stem cells of human breast cancer specimens., The original document contains color images.

Dissemination and metastasis of tumor cells are major causes of morbidity and mortality in breast cancer patients. Therefore it is of vital importance to identify druggable targets to inhibit breast tumor invasion and metastasis. A critical component in the invasive growth, dissemination, and metastasis of cancer is acquisition of motility by tumor cells. Our preliminary studies suggest a novel role for a classical G protein-coupled receptor (GPCR), the thromboxane A2 receptor (TP), in controlling breast tumor cell motility via regulating cytoskeleton reorganization. The objective of this proposal is to define the function of TP in tumor cell motility and to validate TP as a target for anti-metastasis therapy of breast cancer. In the first aim, the role of TP in breast tumor cell motility will be determined in the presence or absence of TP activation or inhibition. The isoform(s) of TP involved in tumor cell contraction and motility will be identified in the second specific aim. In the third aim, an orthotopic mouse model will be used to assess whether TP can be targeted to reduce breast cancer metastasis. The proposed studies will provide significant insights into how the tumor cells enlist TP GPCR to regulate cell motility, and will be highly significant toward the goals of developing mechanism-based interventions for cancers., The original document contains color images.

Obesity increases the risk of breast cancer in post-menopausal women. The degree of risk increases proportionally with an increase in adiposity. There is mounting evidence that stromal cells in the tumor microenvironment make pivotal contributions to tumor progression. Adipocytes have been shown to exert their influence on breast cancer cells via two secreted adipocytokines, leptin and adiponectin. Leptin stimulates proliferation and invasiveness of breast cancer cell lines. Conversely, adiponectin is down-regulated in obese individuals, and there is an inverse relationship between adiponectin levels and risk of breast cancer. Therefore, in breast cancer development, it is important to know if there are significant interactions or tumorigenic effects of excess adipose tissue on mammary epithelium. The question of how adipocytes exert their influence on pre-malignant mammary epithelial cells had not been resolved until now. Adiponectin was found to suppress the effects of oncogenic ras or Myc in p53-/- MMECs. These cells slowed their rate of growth in response to adiponectin, and were better able to maintain genomic stability. This lead to reduced transformation potential in a soft agar assay. Conversely, the effects of leptin were very striking. The presence of p53 was able to suppress the oncogenic effects of ras and Myc. However, when leptin was present, oncogene-expressing WT-MMECs behaved just like p53-/- MMECs. These cells had a greatly enhanced rate of growth and became highly aneuploid. Furthermore, p53 was no longer able to function as a tumor suppressor in the presence of leptin, allowing oncogenic transformation., The original document contains color images.

Elevation of VEGFR-3, the primary mediator of lymphangiogenesis (i.e., new lymphatic vessel formation), is frequently associated with inflammation related to chronic disease and cancer. In the latter case, VEGFR-3 dependent lymphangiogenesis induced by inflamed tumors increases the incidence of distant metastasis, leading to decreased patient survival. However, the molecular mechanisms underlying inflammation-induced VEGFR-3 elevation and lymphangiogenesis are currently unknown. Two potential candidate genes that may regulate expression of VEGFR-3 are Prox1, the primary mediator of embryonic lymphangiogenesis, and NF-kB, the key intracellular regulator of inflammation-induced transcription. We hypothesized that the key inflammatory mediator, NF-kB, regulates transcription of key mediators of lymphangiogenesis, VEGFR-3 and Prox1. We further hypothesized that inflammation-induced elevation of VEGFR-3 and Prox1 are essential steps required for robust lymphangiogenesis in response to inflammation. The three primary goals of this study were to (1) delineate the time-course of events leading to inflammation-induced lymphangiogenesis in vivo; (2) clone and characterize the VEGFR-3 promoter and identify factors regulating VEGFR-3 expression in vitro; and (3) characterize the lymphatic phenotype of NF-kB p50 knockout mice. To begin testing these hypotheses, we used a mouse model of peritonitis to characterize induction of lymphangiogenesis and expression kinetics of NF-kB, Prox1 and VEGFR-3. In vivo time-course analysis of inflammationinduced lymphangiogenesis showed activation of NF-kB followed by sequential upregulation of Prox1 and VEGFR-3 that preceded lymphangiogenesis by 4 and 2 days, respectively. Characterization of the VEGFR-3 promoter by luciferase-reporter and ChIP assays showed direct activation by Prox1, NF-kB p50 and p65 transcription factors. This also revealed that Prox1 and NF-kB p50 bind in close proximity and synergistically activate the VEGFR-3 promoter., The original document contains color images.

Elevation of VEGFR-3, the primary mediator of lymphangiogenesis (i.e., new lymphatic vessel formation), is frequently associated with inflammation related to chronic disease and cancer. In the latter case, VEGFR-3 dependent lymphangiogenesis induced by inflamed tumors increases the incidence of distant metastasis, leading to decreased patient survival. However, the molecular mechanisms underlying inflammation-induced VEGFR-3 elevation and lymphangiogenesis are currently unknown. Two potential candidate genes that may regulate expression of VEGFR-3 are Prox1, the primary mediator of embryonic lymphangiogenesis, and NF-kB, the key intracellular regulator of inflammation-induced transcription. We hypothesized that the key inflammatory mediator, NF-kB, regulates transcription of key mediators of lymphangiogenesis, VEGFR-3 and Prox1. We further hypothesized that inflammation-induced elevation of VEGFR-3 and Prox1 are essential steps required for robust lymphangiogenesis in response to inflammation. The three primary goals of this study were to (1) delineate the time-course of events leading to inflammation-induced lymphangiogenesis in vivo; (2) clone and characterize the VEGFR-3 promoter and identify factors regulating VEGFR-3 expression in vitro; and (3) characterize the lymphatic phenotype of NF-kB p50 knockout mice.

Breast cancer is the most common carcinoma of women in the United States, accounting for more than 40,000 American women's deaths annually. Targeted therapies, such as tamoxifen in estrogen receptor-positive breast cancer, have led to major important improvements in the outcomes of this cancer, but effective chemotherapy targets are lacking and the mortality rate of advanced breast cancer is still high. Aldo-keto reductase 1 B10 (AKR1B10) is overexpressed in tested human breast cancer tissues and mediates acetyl-CoA carboxylase-alpha (ACCA) stability, affecting fatty acid de novo synthesis and cell growth. This study is aimed to identify and evaluate AKR1B10 as a now target for the treatment of breast cancer. In this study, we will determine AKR1B10 expression in breast cancer, define the role of AKR1B10 in lipid metabolism, proliferation, and tumorigenicity of breast cancer cells using AKR1B10-targeted breast cancer cells and animal tumor models, and elucidate the regulatory mechanisms of AKR1B10 on lipid metabolism of breast cancer cells via identifying the functional domain(s)., The original document contains color images.

Prostate cancer is one of the most resistant tumors to chemotherapy among all adenocarcinomas, and there is virtually no effective therapeutic regimen available for this cancer. Hormonal treatment is the most effective therapy in advanced cancer, however, almost all the patients who undergo hormonal therapy inevitably develop hormone-resistant tumors. Therefore, developing a better therapeutic agent by targeting a specific gene or pathway with well-defined clinical rationale is needed. We chose a target called Fatty acid synthase (FAS) because we found that FAS is strongly expressed in prostate cancer cells but not in normal cells and that inhibiting the FAS expression causes specific tumor cell death. In this project, we plan to test the hypothesis that an active component of Solidago virgaurea specifically inhibits the FAS activity and induces apoptosis in prostate tumor cells. Our specific aims are (i) to elucidate the molecular mechanism of growth inhibitory effect of S. virgaurea by defining the signal pathway and factors responsible for apoptosis, and (ii) to examine the effect of the active component of Solidago virgaurea on tumorigenesis in an animal model., The original document contains color images.

Once breast cancer is diagnosed, the most critical question is whether the disease is localized or has it already metastasized to other organs. However, the molecular basis of tumor metastasis is poorly understood as yet. The major goal of this project was to elucidate the function of the tumor metastasis suppressor gene, Drg-1/NDRG1. Toward this goal, we have set three specific aims; (i) to clarify the role of the NDRG1 gene in the progression of breast cancer(Task 1), (ii) to clarify the control mechanism of the NDRG1 gene by PTN (Task 2), and (iii) to evaluate diagnostic/prognostic value of NDRG1 in breast cancer (Task 3). We have successfully accomplished Task 1 and Task 3. Task 1 especially generated exciting data that provide several promising future directions to further study the function of NDRG1. Task 2 is still ongoing and we plan to continue our effort on this task even after completion of this grant., The original document contains color images.

Obesity increases the risk of breast cancer in post-menopausal women. The degree of risk increases proportionally with an increase in adiposity. There is mounting evidence that stromal cells in the tumor microenvironment make pivotal contributions to tumor progression. Stromal adipocytes have been shown to exert their influence on breast cancer cells via two secreted adipocytokines, leptin and adiponectin. Leptin stimulates proliferation and invasiveness of breast cancer cell lines. Conversely, adiponectin is down-regulated in obese individuals, and there is an inverse relationship between adiponectin levels and risk of breast cancer. Therefore, in breast cancer development, it is important to know if there are significant interactions or tumorigenic effects of excess adipose tissue on mammary epithelium. The question of how adipocytes exert their influence on pre-malignant mammary epithelial cells has not been resolved. Adiponectin was found to suppress the effects of oncogenic ras or Myc in p53-/- MMECs. These cells slowed their rate of growth in response to adiponectin, and was better able to maintain genomic stability. This lead to reduced transformation potential in a soft agar assay. Conversely, the effects of leptin was very striking. The presence of p53 was able to suppress the oncogenic effects of ras and Myc. However, when leptin was present, oncogene-expressing WT-MMECs behaved just like p53-/- MMECs. These cells had a greatly enhanced rate of growth and became highly aneuploid. Furthermore, p53 was no longer able to function as a tumor suppressor in the presence of leptin, allowing oncogenic transformation., The original document contains color images.

Double oxide phases that combine a transition metal and a noble metal have recently become a subject of investigation as potential lubricious materials for high temperature tribological applications. Two selected double metal oxide phases, namely silver molybdates and tungstates, were produced in thin film and powder forms using magnetron sputtering and hydrothermal methods, respectively. The lowest frictional properties of these materials were measured at 600 degrees C to be 0.1 and 0.4 for the Ag2Mo2O7 and alpha-Ag2WO4 phases, respectively. Changes in the chemistry and crystal structure of these materials were investigated with increasing the temperature up to 600 degrees C using in-situ Raman spectroscopy, X-Ray diffraction and a differential scanning calorimeter. These tools revealed that the coatings went through a phase transition accompanied by a phase segregation of silver. Simulations performed to investigate the structural and thermal properties of these materials using ab-initio molecular dynamics (AIMD) method were in agreement with the experimental results. The structural and chemical information obtained using computational and experimental studies was correlated to their high temperature tribological performance. Moreover, a new double metal oxide based on the Ag-V-O system was created and tested at very high temperatures. This system was found to be lubricious (friction coefficient of 0.1) at temperatures as high as 1000 degrees C. This is a significant result given that this is the first solid lubricant that was found to perform well at these extreme temperatures. This finding will enable the design of new military machinery that operates at these high temperatures. Potential applications include solid lubrication for small caliber barrel and weapon action components, EM Gun rail materials, and helicopter engines., The original document contains color images.

The concept of inflammation-induced lymphangiogenesis (i.e., formation of new lymphatic vessels) has long been recognized, but the molecular mechanisms remained largely unknown. The two primary mediators of lymphangiogenesis are vascular endothelial growth factor receptor-3 (VEGFR-3) and Prox1. The key factors that regulate inflammation-induced transcription are members of the NF-kB family; however, the role of NF-kB in regulation of lymphatic-specific genes has not been defined. Here, we identified VEGFR-3 and Prox1 as downstream targets of the NF-kB pathway. In vivo time-course analysis of inflammationinduced lymphangiogenesis showed activation of NF-?B followed by sequential upregulation of Prox1 and VEGFR-3 that preceded lymphangiogenesis by 4 and 2 days, respectively. Activation of NF-kB by inflammatory stimuli also elevated Prox1 and VEGFR-3 expression in cultured lymphatic endothelial cells resulting in increased proliferation and migration. We also show that Prox1 synergizes with the p50 of NF-kB to control VEGFR-3 expression. Collectively, our findings suggest that induction of the NF-kB pathway by inflammatory stimuli activates Prox1, and both NF-kB and Prox1 activate the VEGFR-3 promoter leading to increased receptor expression in lymphatic endothelial cells. This, in turn, enhances the responsiveness of pre-existing lymphatic endothelium to VEGFR-3 binding factors, VEGF-C and VEGF-D, ultimately resulting in robust lymphangiogenesis., The original document contains color images.

Prior studies have found that VNS is effective in accelerating and enhancing recovery of function following traumatic brain injury (TBI). The main limitation to these earlier results is that VNS treatment was initiated at either 2 hr or 24 hr after TBI. Present findings indicate that VNS treatment is effective for recovery of function after TBI even when the initiation of VNS treatment is delayed until two weeks after injury. In addition, present evidence demonstrates that, in general, practice, or a rehabilitation regimen, that is conducted across consecutive days is more effective when done in conjunction with VNS treatment. In conjunction with VNS, a regimen of delayed practice (every two days) was found to be less effective than consecutive practice for functional recovery. These results provide heretofore unknown answers regarding the potential utility of VNS as a clinical treatment for human TBI.

Participants in a video game environment were required to make a series of decisions in which they must identify which of three targets was causing a distal explosion. The potential targets were firing weapons which could produce an explosion after a constant or variable delay, a delay that was filled or unfilled with an auditory event, and may have produced explosions probabilistically. Delays had profound effects on accuracy and decision latencies, decreasing weapon effectiveness from 100% to 50% had little effect on accuracy and modest effects on latencies (men only), filling a delay helped under very limited conditions, and varying the delay actually improved performance for longer (average) delays. Furthermore, men's decision accuracy was higher but not when prior video game experience was controlled for. In contrast, women observed their targets for much longer before making a choice regardless of prior experience. The results disconfirmed the proposed forward inference model and instead supported the operation of a backward inference model of causal choice., The original document contains color images. All DTIC reproductions will be in black and white.

Prostate cancer is one of the most resistant tumors to chemotherapy among all adenocarcinomas, and there is virtually no effective therapeutic regimen available for this cancer. Hormonal treatment is the most effective therapy in advanced cancer, however, almost all the patients who undergo hormonal therapy inevitably develop hormone-resistant tumors. Therefore, developing a better therapeutic agent by targeting a specific gene or pathway with well-defined clinical rationale is needed. We chose a target called Fatty acid synthase (FAS) because we found that FAS is strongly expressed in prostate cancer cells but not in normal cells and that inhibiting the FAS expression causes specific tumor cell death. In this project, we plan to test the hypothesis that an active component of Solidago virgaurea specifically inhibits the FAS activity and induces apoptosis in prostate tumor cells. Our specific aims are (i) to elucidate the molecular mechanism of growth inhibitory effect of S. virgaurea by defining the signal pathway and factors responsible for apoptosis, and (ii) to examine the effect of the active component of Solidago virgaurea on tumorigenesis in an animal model., The original document contains color images.

The objective was to perform a "proof of principle" assessment of genetic diversity and gene flow of gopher tortoises on Camp Shelby, MS. The specific aims were to determine if there is evidence that military activity affects genetic diversity and gene flow. DNA was extracted from the blood of gopher tortoises collected from 22 colonies in and around Camp Shelby. The amount of genetic diversity in each colony and treatment group was determined from microsatellite DNA markers and mitochondrial DNA. The data indicate that the amount of genetic diversity may be affected by a combination of military activity and habitat quality. Data on gene flow indicated that military activity may have affected the rates of dispersal among the tortoise colonies. Genetic distances did not correlate well with geographic distance. This may indicate that gene flow is affected by factors other than geographic distance in these tortoises, possible by military activities on Camp Shelby. Overall, the data indicate that there is enough genetic diversity among these colonies to use genetic approaches in delimiting critical habitat needs for conservation of gopher tortoises, which was the main goal of the research., The original document contains color images.

Fatty acid synthase (FAS) is significantly over-expressed in prostate tumor cells and inhibition of FAS results in apoptosis, suggesting that FAS is an ideal target for drug development. The overarching hypothesis of this project is that a specific inhibitor for FAS dimerization will block the function of this enzyme and cause apoptosis of the tumor cell. Our specific aims are (1) to characterize the apoptotic pathway induced by FAS inhibition, and (2) to identify small chemical compounds that specifically inhibit dimer formation of FAS enzyme. During the last four months (Apr. 1-July 31, 2008) we mainly focused our effort on the second specific aim and screened a compound library provided by Developmental Therapeutics Program of NCI. We also screened a library of natural products that particularly focused on marine lives and found that one of the natural products in the library showed strong activity of inhibiting FAS. We also found that the purified compound, Cacalol, significantly blocked the activity and expression of FAS. Our results suggest that this compound has a potential utility for the treatment of prostate cancer.

Breast cancer is the most frequently diagnosed cancer and the second leading cause of cancer death among women in the US. Because metastatic disease is the major cause of death it is crucial to understand the mechanism by which tumor cells metastasize to the distant organs so that we can identify a better therapeutic target. During this funding period we had a breakthrough finding that the metastasis suppressor gene NDRG1 can directly interact with the Wnt receptor LRP6 and block the Wnt signaling. This inhibition leads to down-regulation of the ATF3 gene and thus suppressing metastases. We also found that a combination of NDRGI, PTEN and ATF3 is a good prognostic marker for breast cancer patients. These results suggest that the Wnt and ATF3 pathways are a potential therapeutic target for patients with metastatic disease. We will focus our next year's effort on further clarification of the NDRG1 pathway., The original document contains color images.

The treatment options for prostate cancer are currently quite limited. Hormonal treatment is the most effective therapy in advanced cancer, however, virtually all the patients who undergo hormonal therapy inevitably develop hormone-resistant tumor. Traditional screening of anti-cancer drugs has been mostly dependent on growth inhibition assay for cancer cells. However, targeting a specific gene with well-defined clinical rationale will provide a better chance of developing a more effective therapeutic agent. We chose a target called Fatty acid synthase (FAS) which we found to be strongly expressed in prostate cancer cells but not in normal cells. Importantly, an inhibition of the FAS expression causes specific tumor cell death. In this project, we plan to test the hypothesis that an active component of Solidago virgaurea specifically inhibits the FAS activity and induces apoptosis in prostate tumor cells. Our specific aims are (i) to elucidate the molecular mechanism of growth inhibitory effect of S. virgaurea by defining the signal pathway and factors responsible for apoptosis, and (ii) to examine the effect of the active component of Solidago virgaurea on tumorigenesis in an animal model.

Radiotherapy is a prevalent modality for the treatment of prostate tumor. Although radiation is capable of eradicating localized prostate tumors, nearly 30% of patients treated with potentially curative doses relapse at the sites of irradiation. Therefore, there is an imperative need to improve the success rate of radiotherapy for PCa. This proposal is focused on a role of 12-lipoxygenase (LOX) in modulating the radiation response of PCa cells. 12-LOX catalyzes the formation of 12(S)-hydroxyeicosatetraenoic acid (HETE). Our studies suggest an involvement of 12-LOX in radioresistance of PCa cells. It is our hypothesis that an increase in 12-LOX expression/activity may lead to an increased resistance in tumors to radiation treatment. We also hypothesize that VEGF is an important intermediary for 12-LOX mediated radioresistance in PCa. We intend to define the role of 12-LOX in radioresponse in PCa. 12- LOX will be overexpressed in LNCaP and DU145 cells. Then we will study whether an increase in 12-LOX expression in LNCaP and DU145 cells can enhance their resistance to radiotherapy. We also propose to study whether VEGF is required by 12-LOX to enhance PCa radioresistance through blockade of VEGF activity with a neutralizing antibody. Finally, we will evaluate whether BHPP, a 12-LOX inhibitor, can be used to sensitize prostate tumors to radiotherapy., The original document contains color images.

KAl1 is a tumor metastases suppressor gene which is capable of blocking the metastatic process without affecting the primary tumorigenesis. In this project we tested the hypothesize that the KAl1 protein on tumor cells interacts with gp-Fy (DARC) on the endothelial cells which activates a signal pathway of the KAl1 molecule and that this activation eventually leads to cell growth arrest of tumor cells. We originally planned to examined whether the interaction of KAl1 and DARC leads to suppression of tumor metastasis in vivo (Task 1) and identify specific peptide sequences that activate KAl1 and to assess the efficacy of the peptides on tumor growth in an animal model (Task 2). We have successfully completed Task 1 and published the results in Nature Medicine. Task 2 has been partly accomplished but there are some tasks remained. However we consider that our project has been overall quite successful and our original idea has been fully developed. The results of this project have led to successful RO1 funding and we will continue to develop and expand our research., The original document contains color images.

KAl1 is a tumor metastases suppressor gene which is capable of blocking the metastatic process without affecting the primary tumorigenesis. In this project we tested the hypothesize that the KAl1 protein on tumor cells interacts with gp-Fy (DARC) on the endothelial cells which activates a signal pathway of the KAl1 molecule and that this activation eventually leads to cell growth arrest of tumor cells. We originally planned to examined whether the interaction of KAl1 and DARC leads to suppression of tumor metastasis in vivo (Task 1) and identify specific peptide sequences that activate KAl1 and to assess the efficacy of the peptides on tumor growth in an animal model (Task 2). We have successfully completed Task 1 and published the results in Nature Medicine. Task 2 has been partly accomplished but there are some tasks remained. However we consider that our project has been overall quite successful and our original idea has been fully developed. The results of this project have led to successful ROl funding and we will continue to develop and expand our research., The original document contains color images.

In a study of the United States military's current methods of message communication, it is important to understand its long and constantly evolving relationship with the press in the U.S., and its traditional means of disseminating information. In addition, an understanding of public relations practices and methods of communicating is necessary. This chapter will examine the history and evolution of the relationship between the U.S. military and the press, and provide a brief overview of public relations practices and use of evolving technology as a means of information dissemination., The original document contains color images. All DTIC reproductions will be in black and white.

The newly discovered RNA interference is a novel type of gene regulation mechanism, which is required for normal expression of genes. This study tests the hypothesis that breast tumor carries dysregulated RNA interference pathways, and thus, some tumor suppressor genes will be down-regulated while other genes (e.g., oncogenes) will be up-regulated, leading to tumor cell proliferation and survival. Using real time RT-PCR, we demonstrate that microRNA-21 is overexpressed in breast tumors compared to the matched normal breast tissue. Furthermore, we show that antisense oligonucleotide against microRNA-21 can suppress the endogenous microRNA-21 and causes tumor cell growth inhibition. Experiments with a xenograft carcinoma mouse model reveal that the antisense microRNA-21 oligonucleotide also inhibits tumor growth. Therefore, microRNA-21 is a potential therapeutic target for breast cancer therapy., The original document contains color images.

Breast cancer is the most frequently diagnosed cancer and the second leading cause of cancer death among women in the US. Because metastatic disease is the major cause of death, it is crucial to understand the mechanism by which tumor cells metastasize to the distant organs so that we can identify a better therapeutic target. During this funding period, we found that the metastasis suppressor gene, Drg-1, was capable of down-regulating the ATF3 gene and thus suppressing metastases. We also found that Drg-1 is a good prognostic marker for patient outcome. Our results also suggest that the ATF3 pathway is a potential therapeutic target for patients with metastatic disease. We will focus our next year's effort on further clarification of the Drg-1 pathway and its relation to PTEN., The original document contains color images.

KAI1 is a tumor metastases suppressor gene which is capable of blocking the metastatic process without affecting the primary tumorigenesis. Based on our preliminary data, we hypothesize that the KAI1 protein on tumor cells interacts with gp-Fy on the endothelial cells, which activates a signal pathway of the KAI1 molecule, and that this activation eventually leads to cell growth arrest of tumor cells. To test this hypothesis, we will examine whether the interaction of KAI1 and gp-Fy leads to suppression of tumor metastasis in vivo (Task 1), and identify specific peptide sequences that activate KAI1 and to assess the efficacy of the peptides on tumor growth in an animal model (Task 2). We have successfully completed Task 1 and published the results during this cycle. Task 2 is currently underway and one year non-cost extension was requested. Our long-term goal is to elucidate the molecular mechanism of tumor suppression by the KAI1 gene and to develop an effective therapeutic method which restores the function of the KAI1 gene in the metastatic tumor cells., The original document contains color images.

Radiotherapy is a prevalent modality for the treatment of prostate tumor. Although radiation is capable of eradicating localized prostate tumors, nearly 30% of patients treated with potentially curative doses relapse at the sites of irradiation. Therefore, there is an imperative need to improve the success rate of radiotherapy for PCa. This proposal is focused on a role of 12-lipoxygenase (LOX) in modulating radiation response of PCa cells. 12-LOX catalyzes the formation of 12(S)-hydroxyeicosatetraenoic acid (HETE). Our studies suggest an involvement of 12-LOX in radioresistance of PCa cells. It is our hypothesize that VEGF is an important intermediary for 12-LOX mediated radioresistance in PCa. We intend to define the role of 12-LOX in radioresponse in PCa. 12-LOX will be overexpressed in LNCaP and DU145 cells. Then we will study whether an increase in 12-LOX expression LNCaP and DU145 cells can enhance their resistance to radiotherapy. We also propose to study whether VEGF is required by 12-LOX to enhance PCa radioresistance through blockade of VEGF activity with a neutralizing antibody. Finally, we will evaluate whether BHPP, a 12-LOX inhibitor, can be used to sensitize prostate tumors to radiotherapy., The original document contains color images.

The newly discovered RNA interference is a novel type of gene regulation mechanism, which is required for normal expression of genes. This study tests the hypothesis that breast tumor carries dysregulated RNA interference pathways, and thus, some tumor suppressor genes will be down-regulated while other genes (e.g., oncogenes) will be up-regulated, leading to tumor cell proliferation and survival. Using real time RT-PCR, we demonstrate that microRNA-21 is overexpressed in breast tumors compared to the matched normal breast tissue. Furthermore, we show that antisense oligonucleotide against microRNA-21 can suppress the endogenous microRNA-21 and causes tumor cell growth inhibition. Experiments with a xenograft carcinoma mouse model reveal that the antisense microRNA-21 oligonucleotide also inhibits tumor growth. Therefore, microRNA-21 is a potential therapeutic target for breast cancer therapy., The original document contains color images.

Final report of funded work. Summarize problems studied; activities carried out; and results found. Describe the significance of these results for quantum computing and physics. Describe training and development of scientific personnel that have contributed to the activities associated with this grant.

In this study, we performed linear and nonlinear FE (Finite Element) analyses to compute J-integrals for a centrally perforated star-shaped disc, which was made of an elastomeric material, under crack surface pressures and isothermal loads. Deformations of the disc were constrained by a circular steel ring enclosing the disc. Different crack sizes were assumed to exist in the front of the star-shaped notches. For the linear analysis, material compressibility was modeled with Poisson's varying form 0.48 to 0.4999. In addition, with the presence of the crack surface pressure, the J-integral was modified by including an additional line integral. Numerical studies show that the value of the J-integral increases with the increase of the crack length, reaches a maximum value at 1 inch of crack length, and then decreases gradually. Both linear and nonlinear analyses agree qualitatively but differ quantitatively. It is also found that values of the J-integral strongly depend upon the material compressibility., Presented at the International Mechanical Engineering Congress and Exposition (IMECE) held in Anaheim, CA on 13-19 November 2004.

A process model that includes the coupled phenomenon of resin flow and preform compaction was developed and used to simulate resin infiltration of a fibrous preform using the vacuum-assisted resin transfer molding (VARTM) process. Flow of resin through the distribution medium and preform were modeled as flow through porous media. The finite element/control volume method was used to calculate the infiltrating fluid pressure distribution and track the progression of the flow front. The simulation results were compared with data obtained during infiltration of a carbon fiber preform with an epoxy resin. The parameters measured include the flow front location, resin pressure, and preform thickness change. With accurate inputs, the flow front locations and resin pressure distribution can be accurately predicted. The predicted transverse displacements do not agree well with the experimental measurements. The reasons for the differences are discussed, and further investigations are recommended to develop a more accurate compaction model., Presented at the International Conference on Flow Processes in Composite Materials (7th) held in Newark, DE on 7-9 July 2004. Pub. in Proceedings of the International Conference on Flow Processes in Composite Materials (FPCM7), p333-337, Jul 2004. Prepared in cooperation with the Dept. of Mechanical Engineering, Michigan State University, East Lansing, MI; NASA Langley Research Center, Hampton, VA; and the Dept. of Mechanical Engineering, McGill University, Montreal, Quebec, Canada. This article is from ADA433173 International Conference on Flow Processes in Composite Materials Held in Newark