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

PAEZ ALEJANDRA, ; PALLAVICINI CARLA, ; GIUDICE JIMENA, ; CARABELOS NOELIA, ; ANSELMINO NICOLÁS, ; ORTIZ EMILIANO,; SCHUSTER FEDERICO,; LABANCA ESTEFANIA ; MARTI MARCELO,; BINAGHI MARIA,; VALACCO MARIA PIA, ; BRUNO LUCIANA,; LEVI VALERIA, .; NAVONE NORA ; VAZQUEZ ELBA, ; 1GUERON GERALDINE

Philadelphia

Congreso; AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA; 2015

American Association Cancer Research

Cellular motility is the basis for cancer cell invasion and metastasis. Tumor development and progression are thus partly a consequence of the loss or defect of the mechanisms that control cytoskeletal remodeling. We have previously shown that 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 invasion. HO-1 is also capable of regulating the adhesive properties and morphology of PCa cells. In an effort to understand the molecular mechanisms by which HO-1 regulates cell morphology, we used a vertical approach to identify HO-1 molecular partners and effector genes; and took advantage of confocal microscopy to quantify and compare microtubule and actin dynamics at the leading edge level in PCa cells.FLAG immunoprecipitation assays were performed using lysates from PC3 cells transfected with FLAG-tagged HO-1, and the isolated proteins were subjected to LC/ESI-MSMS analysis. Protein interaction network and gene ontology analyses of HO-1 interacting proteins (performed with Metacore, GeneMANIA and DAVID) showed enrichment of proteins associated with the cytoskeleton organization, transportation and membrane bounding. In particular a cluster of HO-1 interacting proteins were associated to the dynamics of the actin stress fibers, such as gelsolin, lasp1, SIPA1L1, testin, moesin, tropomodulin and vinculin. Effector genes were analyzed by RT-qPCR Oligo GEArray human cell motility microarray analysis revealing HO-1 modulation of genes such as Actin alpha 3 and MMP14, intimately related to cell locomotion and motility. To quantify contacts among cells, PC3 cells were exposed to hemin (80 µM, 24 h), a 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 analyzed 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. Microtubule dynamics in PC3 cells was evaluated using confocal and stochastic optical reconstruction microscopy (STORM). Although no variation of the persistence length of microtubules was found when cells over-expressed HO-1, a significant higher proportion of filopodia-like protrusions among neighboring cells and increased cellular contact were observed under HO-1 modulation. Altogether, these results show that HO-1 modulation in PCa induces the remodeling of the actin filament architecture at filopodia, altering cellular morphology, yielding a more adhesive and less invasive phenotype, further supporting the anti-tumoral function of HO-1in PCa.