IMPLICATIONS OF THE HEME-OXYGENASE 1 (HO-1) INTERACTOME IN THE CYTOSKELETON REPROGRAMMING OF PROSTATE CANCER CELLS: TOWARDS AN EPITHELIAL-LIKE MORPHOLOGY

ALEJANDRA PAEZ; CARLA PALLAVICINI; JIMENA GIUDICE; ESTEFANÍA LABANCA; NICOLÁS ANSELMINO; EMILIANO G. ORTIZ; SCHUSTER FEDERICO; LUCIANA BRUNO; VALERIA LEVI; NORA NAVONE; ELBA VAZQUEZ; GERALDINE GUERON

Bariloche

Simposio; Symposium
 
in
 Signal
Transduction
 and 
Molecular
 Medicine; 2015

P { margin-bottom: 0.08in; direction: ltr; color: rgb(0, 0, 0); line-height: 100%; widows: 2; orphans: 2; }P.western { font-size: 12pt; }P.cjk { font-size: 12pt; }P.ctl { font-size: 12pt; }Cellular motility is a coordinated process that involvesvariations in the dynamics of the actin cytoskeleton and itsinterplay with focal adhesions. It is the re-arrangement of actin andits attachment to focal adhesions at the leading edge of a migratingcell, which generates the driving force necessary for movement. Theloss of cell-cell adhesion enables cancer cells to dissociate fromthe primary tumor mass and changes in cell-matrix interaction allowsthe cells to invade the surrounding stroma. Heme oxygenase-1 (HO-1),the rate-limiting enzyme in heme degradation, plays a critical rolein prostate cancer (PCa), impairing cell proliferation, migration,invasion and modulates cellular adhesion. We have began to screenHO-1 molecular partners through a proteomics and bioinformaticsapproach, to determine the molecular mechanisms by which HO-1regulates cell morphology, and at the same time quantify and comparemicrotubule and actin dynamics at the leading edge level in PCacells. FLAG immunoprecipitation assays were performed usinglysates from PC3 cells transfected with FLAG-tagged HO-1, and theisolated proteins were subjected to LC/ESI-MSMS analysis. Proteininteraction network and gene ontology analyses of HO-1 interactingproteins (performed with Metacore, GeneMANIA and DAVID) showedenrichment of proteins associated with the cytoskeleton organization,transportation and membrane bounding. Of note, gelsolin, lasp1,SIPA1L1, testin, moesin, tropomodulin and vinculin, all HO-1interacting proteins were associated to the dynamics of the actinstress fibers. Effector genes were analyzed by RT-qPCR Oligo GEArrayhuman cell motility microarray analysis revealing HO-1 modulation ofgenes such as Actin alpha 3 and MMP14, intimately related to celllocomotion and motility. To quantify contacts among cells, PC3 cellswere exposed to hemin (80μM, 24h), a pharmacological inducer ofHO-1, fixed and stained with phalloidin-rhodamin. We selected regionsin which the filopodia from two neighboring cells touched each other,considered as ?contacts?, and divided these regions into segmentswhere the distance between the cells remained constant. An intensityprofile for each of these sectors was analyzed with a custom madealgorithm to count contacts. A 'contact density' was defined for eachregion as the ratio between the number of contacts and the length ofthe profile. Microtubule dynamics in PC3 cells was evaluated usingconfocal and stochastic optical reconstruction microscopy (STORM).Although no variation of the persistence length of microtubules wasfound when cells over-expressed HO-1, a significant higher proportionof filopodia-like protrusions among neighboring cells and increasedcellular contact were observed under HO-1 modulation. These resultsshow that HO-1 modulation in PCa induces the remodeling of the actinfilament architecture at filopodia, altering cellular morphology,towards a less aggressive phenotype.