This project has focused on development and application of technology to evaluate signaling events in the laboratory and in clinical and preclinical samples, and preclinical investigation of microenvironmental targets, as related to invasion and angiogenesis. Concerted efforts were put into quality assurance of the protein lysate array technology. Buffer, loading, and total protein quantity optimization has been completed. Controls were developed, expanded, and aliquoted. Our findings of large tissue requirements of in order to measure CD31 by array has led to alternative angiogenesis endpoints of phospho eNOS, total and phospho-VEGF receptor. Good signal could be obtained with tissue quantities anticipated from the clinical trial. CD31 will be applied to tissue samples by immunohistochemistry. All endpoints to be applied to the clinical trial samples were validated and optimized; specific lots were examined and locked in. Archival anonymized clinical samples were used to confirm quality control then the system was applied to serial biopsies obtained for the sorafenib/bevacizumab study. New targets related to microenvironment signaling, including cadherins, FAK, and paxillin have been optimized to the protein array format. These are being applied collaboratively to a series of pleural and peritoneal ovarian cancer fluid samples to address the hypothesis that these proteins are differentially regulated during an anoikis-resistant phenotype as is seen in effusions. Further in vitro studies will accompany these results. We have been studying the role of progranulin (prgn) and secretory leukocyte protease inhibitor (SLPI) in invasion and metastasis of ovarian cancer xenografts. Following our demonstration that SLPI is a prosurvival protein for ovarian cancer and that this did not depend upon the protease inhibitor activity, we investigated the behavior of cells overexpressing SLPI and/or its protease inhibitor mutants. Increased aggressiveness, invasive behavior and metastatic dissemination were observed. This has led to investigation of the role of SLPI in the biology of invasion and metastasis with mechanistic studies ongoing. Data indicate that matrix metalloproteinase-9 (MMP9) is involved with ovarian cancer, and is downstream of SLPI regulation. Analysis of blinded clinical samples from a prior clinical trial of the Section and also using a tissue microarray generated by the Gynecologoic Oncology Group. Preliminary results indicate the relationship between SLPI and MMP9 observed in the xenograft experiments bears out in clinical samples. Studies with CAI remain collaborative with a prior member of the Section. Imatinib-resistant chronic myelogenous leukemia, recent results assessing CAI in imatinib-resistant CML cells is promising. CAI treatment inhibited activation of abl kinase and MAPK in these cells. Use of CAI for ophthalmologic disease has been licensed and proceeds with collaboration. CAI will be tested therapeutically for benefit in macular degeneration. This comprehensive approach to angiogenesis and microenvironment study in the laboratory and patient samples will provide opportunity for proof of principle of molecularly targeted agents in stromal therapy and will complement our continuing clinical work with signal transduction inhibition therapy.
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