HO-1 modulates the actin stress fiber architecture in prostate cancer cells: towards a less aggressive phenotype

ALEJANDRA PAEZ; CARLA PALLAVICINI; JIMENA GIUDICE; NOELIA CARABELOS; NICOLÁS ANSELMINO; EMILIANO G. ORTIZ; SCHUSTER FEDERICO; ESTEFANÍA LABANCA; MARCELO MARTI; MARIA BINAGHI; PIA VALACCO; LUCIANA BRUNO; VALERIA LEVI; NORA NAVONE; ELBA VAZQUEZ; GERALDINE GUERON

Philadelphia

Congreso; AMERICAN ASSOCIATION FOR CANCER RESEARCH annual meeting; 2015

AMERICAN ASSOCIATION FOR CANCER RESEARCH

P { margin-bottom: 0.08in; direction: ltr; color: rgb(0, 0, 0); widows: 2; orphans: 2; }Cellularmotility is the basis for cancer cell invasion and metastasis. Tumordevelopment and progression are thus partly a consequence of the lossor defect of the mechanisms that control cytoskeletal remodeling. Wehave previously shown that heme oxygenase-1 (HO-1), the rate-limitingenzyme in heme degradation, plays a critical role in prostate cancer(PCa) impairing cell proliferation, migration and invasion. HO-1 isalso capable of regulating the adhesive properties and morphology ofPCa cells. In an effort to understand the molecular mechanisms bywhich HO-1 regulates cell morphology, we used a vertical approach toidentify HO-1 molecular partners and effector genes; and tookadvantage of confocal microscopy to quantify and compare microtubuleand actin dynamics at the leading edge level in PCa cells.FLAGimmunoprecipitation assays were performed using lysates from PC3cells transfected with FLAG-tagged HO-1, and the isolated proteinswere subjected to LC/ESI-MSMS analysis.Protein interaction network and gene ontology analyses of HO-1interacting proteins (performed with Metacore,GeneMANIA and DAVID) showed enrichment of proteins associated withthe cytoskeleton organization, transportation and membrane bounding.In particular a cluster of HO-1 interacting proteins were associatedto the dynamics of the actin stress fibers, such as gelsolin, lasp1,SIPA1L1, testin, moesin, tropomodulin and vinculin. Effector geneswere analyzed by RT-qPCR Oligo GEArray human cell motility microarrayanalysis revealing HO-1 modulation of genes such as Actin alpha 3 andMMP14, intimately related to cell locomotion and motility. Toquantify contacts among cells, PC3 cells were exposed to hemin (80µM, 24 h), a pharmacological inducer of HO-1, fixed and stained withphalloidin-rhodamin. We selected regions in which the filopodia fromtwo neighboring cells touched each other, considered as ?contacts?,and divided these regions into segments where the distance betweenthe cells remained constant. An intensity profile for each of thesesectors was analyzed with a custom made algorithm to count contacts.A 'contact density' was defined for each region as the ratio betweenthe number of contacts and the length of the profile. Microtubuledynamics in PC3 cells was evaluated using confocal and stochasticoptical reconstruction microscopy (STORM). Although no variation ofthe persistence length of microtubules was found when cellsover-expressed HO-1, a significant higher proportion offilopodia-like protrusions among neighboring cells and increasedcellular contact were observed under HO-1 modulation. Altogether,these results show that HO-1 modulation in PCa induces the remodelingof the actin filament architecture at filopodia, altering cellularmorphology, yielding a more adhesive and less invasive phenotype,further supporting the anti-tumoral function of HO-1in PCa.