Abstract
Dr. Ute Reuning, Clinical Research Unit, Department of Obstetrics & Gynecology, Technische Universitaet Muenchen, Munich, Germany.
Ovarian cancer is characterized by local intraperitoneal tumor cell spread and proli-feration, in part mediated by adhesion receptors of the integrin superfamily. Among those, integrin αvβ3 was shown to be significantly elevated in human ovarian cancer correlating with cancer progression. Upon overexpression of αvβ3 and engagement by its major extracellular matrix ligand vitronectin (VN), we previously showed enhan-ced adhesion, proliferation, and motility of human ovarian cancer cells. By studying differential expression of genes possibly related to these tumor biological events, we identified among other candidates, the epidermal growth-factor receptor (EGF-R) to be under control of αvβ3. Thus, we characterized αvβ3-dependent changes of EGF-R and found significant upregulation of its expression and activity. This was reflected by obvious changes in EGF-R promoter activity. Upon disruption of DNA-binding mo-tifs for several prominent transcription factors, we sought to identify DNA elements contributing to αvβ3-mediated EGF-R promoter induction. Hereby, we showed that an EGF-R promoter mutant displaying a disrupted DNA binding motif for rel trans-cription factors did not only display prominent reduction of it activity but, most interestingly, was not longer responsive to enhanced αvβ3, strongly implicating rel proteins in αvβ3-dependent EGF-R regulation. Furthermore, we identified the inte-grin-linked kinase (ILK), an important membrane-proximal kinase in integrin signaling routes, to be regulated by αvβ3. ILK was also significantly increased and co-localized with αvβ3 at the surface of human ovarian cancer cells in the presence of VN. Aug-mented ILK protein expression was traced back to enhanced ILK promoter activity, an effect, which we also further characterized with regard to transcriptional response elements involved. Herewith, we noticed that disruption of an ets DNA consensus sequence did not only result in slightly diminished promoter activity but also, abrogated ILK promoter induction by αvβ3. These data suggested that ets factors contribute to αvβ3-mediated ILK upregulation. Thus, enhancement of EGF-R and ILK by αvβ3, all of these molecules being well-defined with respect to their funtions in cancer progression and metastasis, might represent an additional mechanism to adapt synergistic receptor signaling and crosstalk in response to an altered tumor cell microenvironment within the metastatic cascade.
Since many years, a plethora of scientific evidence points to an important role of con-formational changes in integrin transmembrane domains (TMD) during integrin acti-vation, even the molecular/structural nature is still rather unclear. Computational se-quence alignments indicated for most integrin subtypes a GXXXG-like association motif analogous to that found in the well characterized homodimeric protein glyco-phorin A (GpA). In order to test the impact of a GpA TMD conformation on avβ3 activation, we generated human ovarian cancer cell transfectants expressing eleva-ted levels of avβ3 wild type as well as an αvβ3 TMD mutant carrying the complete TMD of GpA. As control, a TMD-GpA variant was produced which encompasses instead of a GXXXG- a GXXXI-sequence known to not longer support dimerization and thus presumably maintaining integrin legs in a separated, open, and inactive state. Using these αvβ3 TMD variants, we studied effects of integrin TMD conforma-tion on αvβ3 activation, by cell adhesion, proliferation, and migration assays. Moreover, we characterized initiation of integrin-mediated signaling cascades by studying the activation of important kinases for integrin signaling, such as the focal adhesion kinase (FAK), the mitogen-activated protein kinases (MAPK) p44/p42, and the protein kinase B/Akt, respectively, as a function of αvβ3 TMD conformation. Elucidation of these complex mechanisms will hold great potential for the identifi-cation of promising leads in order to develop novel therapeutical agents capable of allosterically stabilizing particular integrin conformations or competing with binding of certain integrin-related effector molecules.
Host: Prof. Walter Hunziker
