HO-1 INTERACTOME IN PROSTATE CANCER CELLS REPROGRAMS THE CYTOSKELETON AND REMODELS THE CELL ARCHITECTURE TOWARDS A LESS AGGRESSIVE PHENOTYPE.

GUERON GERALDINE; PAEZ ALEJANDRA; GIUDICE JIMENA; CARABELOS NOELIA; MARTI MARCELO; BINAGHI MARIA; VALACCO PIA; PALLAVICINI CARLA; BRUNO LUCIANA; LEVI VALERIA; NAVONE NORA; ELBA VAZQUEZ

Maryland

Congreso; 21ST ANNUAL PCF SCIENTIFIC RETREAT; 2014

Prostate Cancer Foundation

Cellular motility is the basis for cancer cell invasion and metastasis1. Tumor development and progression are partly a consequence of the loss or defect of the mechanisms that control cytoskeletal remodeling. Heme Oxygenase-1 (HO-1), the rate-limiting enzyme in heme degradation plays a critical role in prostate cancer (PCa), impairing cell proliferation, migration and invasion2. HO-1 is also capable of regulating the adhesive properties and the morphology of PCa cells3. In an effort to understand the molecular mechanisms by which HO-1 may be implicated in cell morphology regulation we used a vertical approach to identify HO-1 molecular partners and effector genes associated to the integrity of the cellular architecture; and took advantage of confocal microscopy to quantify and compare actin dynamics at the leading edge level in PCa cells.  Methods: GeneMANIA4 and DAVID5 were used as bioinformatics tools. FLAG immunoprecipitation assays were performed using lysates from PC3 cells transfected with either FLAG-tagged HO-1 or the empty vector, and the isolated proteins were subjected to LC/ESI-MSMS analyses. Effector genes were analysed by RT-qPCR Oligo GEArray human cell motility microarray analysis. To quantify contacts among cells, PC3 cells were exposed to hemin, the pharmacological inducer of HO-1, fixed and stained with phalloidin-rhodamin. We selected regions in which the filopodia from two neighboring cells touched each other, considered as ?contacts?, and divided these regions into segments where the distance between the cells remained constant. An intensity profile for each of these sectors was analysed with a custom made algorithm to count contacts. A ´contact density´ was defined for each region as the ratio between the number of contacts and the length of the profile.   Results: Protein interaction network analysis and gene ontology analyses of HO-1 interacting proteins showed enrichment of proteins associated with the cytoskeleton organization, transportation and membrane bounding. Microarray analysis revealed HO-1 modulation of genes such as Actin alpha 3 and MMP14, intimately related to cell locomotion and motility. Confocal microscopy analyses of PC3 cells overexpressing HO-1 revealed a high proportion of filopodia-like protrusions among neighboring cells and increased cellular contact.  Conclusion: These results show that HO-1 modulation in PCa induces the remodeling of the actin filament architecture at filopodia, altering cellular morphology, towards a more adhesive and less invasive phenotype.