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Growth and development of the prostate is highly dependent on androgen, and aberrant androgen and androgen receptor (AR) signaling is the key driver in the pathology and progression of prostate cancer. Blocking androgen synthesis and inhibiting AR function is the first line of therapy for prostate cancer patients. Although this approach is initially effective in suppressing disease progression, castration resistant tumors eventually develop resulting from reactivation of AR activity. A novel approach to overcome resistance to therapy is to identify selective AR modulators (SARMs) that inhibit the expression of genes promoting tumor growth but enable the expression of genes for differentiation. We hypothesize that functionally distinct set of genes have different promoter signature marks that are recognized by AR. Re-testing and validation of several hits obtained from a high-throughput promoter-dependent compound screen of FDA approved drugs identified a lead compound that had a differential effect on AR promoter-element recognition in transient transfection assays and in chromatin precipitation assays, and in the expression of select AR target genes associated with proliferation and differentiation. Cell proliferation assays indicate that the compound is able to inhibit AR-dependent cell growth. Our discovery of this compound in an unbiased screen for SARMs provides proof-of-concept that drugs may be developed that differentially target promoter elements that distinguish pro-proliferation from pro-differentiation genes. Further optimization of transfection protocol and multiplexing of reporters for the high-throughput promoter screen was performed. A second high-throughput screen was done with a 1.6% hit rate that yielded 26 candidate compounds to be tested for secondary assay validation and mechanism of action analysis., The original document contains color images.

The overall objective of this proposal is to develop a durable cure for lethal prostate cancer through the elucidation of the role of cancer stem cells in the pathogenesis of the disease. During the past year, we have made the following significant findings/observations: i)3D culture of human prostate cancer cells with magnetic nanoparticles is not optimal for tumor initiation studies, ii) in vitro co-culture of human prostate cancer cells (established cell lines and primary patient samples) with human prostate fibroblasts hold promise as models of tumor initiation/cancer stem cell activity. We continue to optimize and validate our in vitro model of prostate cancer initiation to facilitate cancer stem cell discovery as well as drug targeting., The original document contains color images.

The overall objective of this proposal is to develop a durable cure for lethal prostate cancer through the elucidation of the role of cancer stem cells in the pathogenesis of the disease. During the past year, we have made the following significant findings/observations: i)3D culture of human prostate cancer cells with magnetic nanoparticles is not optimal for tumor initiation studies, ii) in vitro co-culture of human prostate cancer cells (established cell lines and primary patient samples) with human prostate fibroblasts hold promise as models of tumor initiation/cancer stem cell activity. We continue to optimize and validate our in vitro model of prostate cancer initiation to facilitate cancer stem cell discovery as well as drug targeting., The original document contains color images.

The current proposal aims to directly compare the psychotherapy and medication treatments for PTSD considered to have the most evidence for effectiveness. While both SSRI and PE have demonstrated efficacy, there are significant individual differences in clinical responses to both treatments. To achieve best clinical outcomes and to utilize available treatment most effectively, it is critical to examine how PTSD and related psychopathology and functional impairment change with these treatments alone and in combination. Further, in order to inform clinical practice, we plan to examine psychological and neurobiological predictors of response to treatment and mechanisms of change during treatment (pre to post treatment change) based on previously identified predictors, including emotion regulation and processing with fMRI in response to emotional challenge tasks, DNA and mRNA (pre and post treatment), and cortisol response to awakening. Start-up activities were completed in 2012Q1 and the primary activity and focus is this year has been on recruitment. To date, we have recruited and randomized 45 Veterans.

The androgen signaling pathway remains a key therapeutic target for prostate cancer. Despite recent advances in treatment, disease invariably recurs. Genes regulated by androgen receptor (AR) in recurrent prostate cancer differ from those in normal cells or early disease. A novel goal is to inhibit AR target genes involved in cancer growth but retain expression of genes for normal cell survival. These gene sets may differ in androgen response element (ARE) signatures. AREs include consensus inverted repeats (cARE) shared amongst receptors and more selective direct repeats (sAREs). Genes involved in differentiation appear to rely more on sAREs than genes driving proliferation. ARE preference can vary with ligand, cofactor interactions or AR mutation. Thus compounds altering receptor conformation via any domain may influence promoter choice. To identify such compounds, we developed a multiplexed assay for differential AR action in transfected cells using fluorescent reporters driven by multimerized cAREs, sAREs or a PSA promoter. We performed a high-throughput screen of a library of FDA-approved drugs, seeking candidates that suppressed cARE but not sARE reporters, and are currently narrowing hit compounds for validation. This promoter-dependent screen may identify compounds that elicit differential gene expression and lead to drugs generating fewer side effects and less resistance., The original document contains color images.

Acute lung injury in rats following airway delivery of CEES is associated with loss of distal lung barrier function (resulting in alveolar flooring) and an intense inflammatory response, which is lung-damaging. These acute lung injury parameters are attenuated by neutrophil depletion or complement blockade. Injuring the redox balance in lung after exposure to CEES by administration into lung of liposomes containing antioxidant compounds is highly protective even when delivery of liposomes is delayed by at least 1 hr. CEES-induced lung injury is progressive, as manifested by development of interstitial fibrosis which seems to peak at three weeks. Whether STIMAL will attenuate development of lung fibrosis is currently unknown.

Major research accomplishment during the first year of this research project includes 1) fabrication of a fast speed noninvasive PAT prostate imaging system by using a stand-alone commercial ultrasound system; 2) testing the performance of this imaging system through the experiments on phantoms and ex vivo canine prostates; and 3) fabrication and optimization of a specially designed PVDF array transducer for high sensitive broad bandwidth detection of photoacoustic signals., The original document contains color images.

Two demonstrations of the capabilities developed under Cooperative Agreement Contract W81XH-04-0012, "Core Development Integration and Demonstration of the DARPA Virtual Soldier" (The Virtual Soldier Project) were performed as final deliverables. The March 17th 2005 Demonstration targeted 5 areas: Statistical Reasoning, Multiscale Modeling, Casual Reasoning, P-Tag-CODEC-Homer Displays-Hotbox, and Autostereoscopic/ Holographic Display. The 14 June 2005 Demonstration targeted 3 areas: (1) An End-to-End Demonstration of Statistical Reasoning, (2) Multiscale Modeling and Anatomy from Anatomy Forecasts with Autopsy Results and a Report of Statistical Findings, and (3) a Casual Reasoning demonstration using the Virtual Soldier Knowledge Base, simulated physiology data, and ballistic modeling. The 7 Tasks of the project: (1) Global Architecture, (2) Organ-tissue systems, (3) Property-levels model, (4) Automatic segmentation of organ-tissue systems, (5) Holomer display and interface, (6) Holomer storage, retrieval and interface, and (7) Demonstration; have been met in fulfillment of the main contract demonstration goal of statistical prediction of outcomes to an accuracy greater than 0.80. Subcontractors developed the software and model systems to achieve the required degree of faithfulness to physiology, anatomy, physical properties, and anatomy information hierarchy to statistically predict wounding outcomes from minimal post-wound experimental data, and to separately describe post-wound effects from initial wound states., The original document contains color images. All DTIC reproductions will be in black and white.

The major goal in the first year of the project is to investigate the in vitro anti-tumor activity of (-)-grossypol using prostate cancer cell lines. We have finished the tasks proposed in the Statement of Work for the first year. Specifically, we have investigated the in vitro anti-tumor activity of (-)-gossypol and potential synergistic effects of (-)-gossypol in combination with chemotherapeutic drugs. (-)-gossypol showed potent anti-tumor activity in human prostate cancer PC-3, LnCaP, CL-1 cells, but only limited or minimal effect on DU-145 and human normal prostate epithelial cells (PrEC) with low Bcl-xL. (-)-gossypol potently enhanced apoptosis induction by CDDP and docetaxel, currently used chemotherapeutic agents for prostate cancer. In PC-3 and CL-1 cells, (-) gossypol showed either additive or more than additive effects in combination of CDDP and docetaxel using MTT-based WST-1 assay. (-)-gossypol potently enhanced X-ray irradiation induced growth inhibition in a clonogenic assay, and apoptosis induction in Annexin V and PI staining assays. The data obtained in the first year provide us a solid foundation to move the project to in vivo testing and further mechanism studies, to develop (-)-gossypol as a novel molecular targeted therapy for the treatment of prostate cancer with Bcl-xL overexpression.

An automated spot mammography technique was developed to improve the imaging of lesions within dense breast tissue. The initial goal was to build a device that would automatically detect suspicious regions in mammograms and immediately perform spot compression and collimation at that region to produce a second image that better distinguishes masses from overlapping tissues. Preliminary studies with a prototype device and breast simulating test objects showed promise, but spot compression didn't always separate the tissues as much as desired. The authors conceived an improved method that employs three-dimensional stereoscopic digital spot mammography acquisition and display for superior perception and characterization of masses. Automated detection of suspicious regions was evaluated by comparing regions in patients' mammograms selected by radiologists with those selected by a computer program. There was good agreement between the radiologists and the computer. A spot collimator to restrict the x-ray beam to the suspicious region for stereo spot image acquisition was designed, built, tested, and refined. Then preliminary studies were performed on a further evolution of the technique: utilizing tomosynthesis to reconstruct slices of the breast, with the eventual goal of developing automated spot tomosynthesis for enhanced sensitivity and specificity in breast cancer screening.

A major genetic event that occurs in the pathogenesis of breast carcinoma involves alterations in the Bcl-2 survival pathway. In the original proposal, we proposed studies to determine the mechanism involved in Bcl-xS-mediated apoptosis, to characterize cellular proteins that interact with Bcl-xS using biochemical and genetic approaches, and to use a transgenic model of Bcl-xS expression in the breast to assess the requirement for Bcl-2/Bcl-XL in the maintenance of normal breast epithelia and tumor growth. During the last year, we have further studied the apoptotic pathway induced by Bcl-xS. The analysis showed that Bcl-xS kills cells, at least in part, through an Apaf-1-caspase-9 pathway. Bcl-xS associates with Bcl-xL via its BH3 domain and with Apaf-1 possibly through Bcl-xL. We have cloned and characterized a novel Apaf-1 cDNA form that, in contrast to the originally described Apaf-1, activates procaspase-9 in a cytochrome c and dATP-dependent manner. Finally, transgenic mice expressing Bcl-XS in the breast have been crossed with oncogenic erb2-B transgenic mice to assess the effects of Bcl-xS on breast cancer development.

A major genetic event that occurs in the pathogenesis of breast carcinoma involves alterations in the Bcl-2 survival pathway. To determine the role of the Bcl-2 family in maintaining cancer cell viability, we constructed a recombinant adenovirus vector that expresses Bcl-xs, a dominant inhibitor of these proteins. In the original proposal, we proposed studies to determine the mechanism involved in Bcl-xs-mediated apoptosis, to characterize cellular proteins that interact with Bcl-xs using biochemical and genetic approaches, and to use a transgenic model of Bcl-xs expression in the breast to assess the requirement for Bcl-2/Bcl-xL in the maintenance of normal breast epithelia and tumor growth. During the last two years, we have characterized the interaction of Bcl-xs with Bcl-2 and Bcl-xL and identified through a genetic screen, HRK, a novel protein that interacts with Bcl-2 and Bcl-xL. Mutational analysis of Bcl-xs revealed a conserved region of this protein that is important for interaction with Bcl-xL and induction of apoptosis. Bcl-xL and Bcl-xs were found to associate with Apaf-1, a critical regulator of caspases and cell death, suggesting that Bcl-xs might antagonize Bcl-xL through Apaf-1. Finally, transgenic mice expressing Bcl-xs in the breast have been developed and partially characterized.

The gene frequency data have been collated, and the terminal report is being prepared on the descriptive aspects of the study. The fertility data have been analyzed in a preliminary fashion but set aside for the moment because they present special problems in analysis for which we have not as yet worked out the methodology.

Repeated stimulation of the vestibular endorgan of the inner ear produces a response decline which has been termed habituation and which we term vestibular suppression. Machinery was constructed to produce high rotary acceleration in a finely controlled manner to study this phenomenon in cats. Suppression was found to be long lasting and to reach about 60% of original response. It was most rapidly produced at just submaximal levels of stimulus. Postrotatory nystagmus duration was chosen as the response characteristic for reasons stated, after study of nystagmus parameters. Punishment slowed down the rate of acquisition of suppression in one cat. Intensity function studies, nerve block studies, and increment-decrement intensity function curves not only failed to show presence of functional endorgan damage due to high rates of deceleration but demonstrated the endorgan to be a good deal more rugged than is generally recognized. Transference of effects between the otolith organs and ampullae, using rotation and linear acceleration, was absent. The locus of suppression was shown to be most likely in the pontine reticular formation and not in the nucleus fastigius. The theory of efferent vestibular bundle causation was considered. All lesions were proven histologically. Linear acceleration studies demonstrated an otolithoocular reflex which can produce nystagmic eye movements. (Author)