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. We have now initiated retrospective and prospective proteomic studies of patient specimens using our recently optimized SOPs and qualified controls. Preliminary proteomic results from effusion cell pellets are under analysis currently to address the hypothesis that cell-cell signaling is linked to a viability signal in these cells in the absence of substratum. Clinical samples linked to two clinical trials were examined. Vandetanib, an inhibitor of both VEGFR2 and EGFR in vitro and by report, was found to inhibit EGFR activation in patient samples obtained 6 weeks into treatment compared with pretherapy. No modulation of VEGFR2 activation was observed although good signals indicating detection were present. Illustration of mechanism was observed in serial samples obtained in the bevacizumab/sorafenib phase I study. This is discussed further in the Clinical annual report. Other preclinical development has continued in our studies of progranulin (pgrn) and secretory leukocyte protease inhibitor (SLPI) in invasion and metastasis of ovarian cancer. Increased aggressiveness, invasive behavior, and metastatic dissemination was observed in cells overexpressing SLPI and/or its protease inhibitor mutants. Data indicate that matrix metalloproteinase-9 (MMP9) is involved with ovarian cancer, and its activity and production are downstream of SLPI. A direct relationship between SLPI and MMP9 (r2=0.9) was observed a tissue microarray of serous but not endometrioid ovarian cancer samples (Gynecologic Oncology Group array). We are now looking to advance clinical development of CR012, a humanized monoclonal antibody obtained collaboratively from our industry partner. Our studies of asparaginase have shown activity both against the ovarian cancer cell lines and also microvascular endothelium. This was evident in vascular remodeling, invasive behavior, and heterotypic cell adhesion. This is leading to a pilot clinical trial of pegylated asparaginase in women with ovarian cancer with translational endpoints evaluating the heterotypic interactions and vascular activity. We will continue our collaboration with Kohn lab alum, Prof. R. Alessandro at the Univ. Palermo in their studies, proteomic, biochemical, and biological, of CML. A new direction has been initiated to investigate interactive proteomic signaling between the endothelium and tumor cells in an in vitro model. Success with this model and signal characterization will lead to detailed dissection and in vivo modeling. It is anticipated that these studies will lead to new translational and/or clinical directions for the laboratory. 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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