Our working hypothesis focuses on the convergence of prosurvival, angiogenesis and motility signals at common pathways in the local tumor microenvironment for therapeutic targeting and monitoring. We continue to examine two pathways we identified: the BAG-3 stress co-chaperone protein and the ovarian cancer growth and survival factor and its partner, progranulin (pgrn) and secretory leukocyte protease inhibitor (SLPI). Element 1) BAG3 causes a gain-of-function action in survival and proliferation. We have identified a functional role for the WW domain of BAG3 in the proper regulation of actin organization during G2/M;these findings put BAG3 in the cell cycle as both a regulator and a subject of cell cycle regulation. Disruption of WW domain-specific events results in polynucleation and dysfunctional actin nucleation. New partner protein interactions necessary for proper cytokinesis have been identified and their effects on cell cycle are under study. Further, this domain, wholly encoded in exon 1, is genomically 18MB upstream of subsequent exons in a gene poor region of chromosome 10. We are investigating if there is disequilibrium between exon expression in ovarian cancer in that there is an amplicon in this genomic region. Dr. McCollum, a senior postdoctoral fellow in our group received peer-reviewed funding from the Kaleidoscope of Hope Foundation with which to prosecute this hypothesis. In addition, new studies under that funding are ongoing to dissect the genetic and proteomic effects of BAG3. Functional effects of forced huBAG3 expression in MMTV-BAG3 transgenic mice have uncovered gain-of-function effects with excessive events during mammary gland maturation and improper involution upon pup withdrawl from nursing females. Characterization is ongoing to determine potential underlying mechanism. Hyperplastic nodules (HAN) are seen both developing transgenic mice and in those exposed to DMBA where we also see some malignant tumor development selectively in the virgin transgenic mice. Malignant lesions are ductal and ER+. Cytokeratin and hormone receptor staining are ongoing to classify the HAN and developmental changes. Repeat pregnancy experiments are ongoing to examine the role of the MMTV activation of huBAG3 expression and its carcinogenic potential alone, and in crosses to WAP/INT3 mice with Dr. Smith MBTL, CCR. 2) The progranulin/SLPI axis promotes EMT and invasion. Our studies of pgrn and its partner protein, SLPI, have continued. Increased dissemination in vivo of HEY-A8 cells overexpressing SLPI and SLPI mutants was observed and is independent of loss of protease inhibition function. The requirement for PGRN interaction is also found in HaCaT keratinocyte EMT experiments. These cells were selected in order to remove the proliferative drive of SLPI after we demonstrated that SLPI did not promote anchorage-independent colony formation. This allowed the separation of the pro-invasive and EMT-like behavior of SLPI and mutants. SLPI collaborates with TGFbeta to augment invasive potential of HaCaT cells;this is altered in the mutants that cannot bind pgrn and completely abrogated when pgrn is silenced. Plans to advance these findings for clinical translation are now on hold due to across the board resource reduction. We continue to collaborate with Dr. Annunziata in her investigations of the NKFB pathways in womens cancers. MMP-9, shown by us to be regulated and activated by SLPI, is in the NFKB IKKB gene signature and there is relationships to SLPI as well. Planning for these projects is underway. 3) Recent findings indicating that TNFR1/2 have higher affinity for pgrn than TNF are provocative given the expression of TNFR1 has been long known to be present and active in ovarian cancer. Further, TNFR1/2 are preferred BAG domain binding partners for SODD, silencer of death domain, also recognized to be BAG4, another member of the pro-survival end of the BAG domain. Thus, it is logical for us to meld our interest in pgrn and the BAG family and further delve into this, especially in the context of ovarian cancer.
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