Hitting the breaks on the migratory capacity of tumoral cells: targeting key regulators of actin dynamics in prostate cancer

GERALDINE GUERON; ALEJANDRA PAEZ; EMILIANO G. ORTIZ; CARLA PALLAVICINI; JIMENA GIUDICE; PIA VALACCO; MARIA BINAGHI; MARCELO MARTI; LUCIANA BRUNO; VALERIA LEVI; MARCELO SALIERNO; JAVIER COTIGNOLA; NORA NAVONE; ELBA VAZQUEZ

Congreso; 22nd Annual PCF Scientific Retreat; 2015

PROSTATE CANCER FOUNDATION

P { margin-bottom: 0.08in; direction: ltr; color: rgb(0, 0, 0); widows: 2; orphans: 2; }Background:Metastatic cancer involves themovement of cancer cells from the primary site to distant homingorgans. The migratory capacity of cells, requires a dynamicremodeling of the cell cytoskeleton. While actin filaments,microtubules and intermediate filaments, coordinate their functionsin normal cells, tumoral cells display abnormal expression ofcytoskeletal proteins resulting in an augmented capacity to resistchemotherapy and colonize other organs. The induction of hemeoxygenase-1 (HO-1), the rate-limiting enzyme in heme degradation, hasa strong anti-tumoral effect in prostate cancer (PCa) and regulatesthe adhesive properties of PCa cells. Here we have extended ourstudies to assess the role of HO-1 on cell morphology and dynamics ofthe actin cytoskeleton at filopodia in PCa cells.Methods:Motility changes were assessed on fiber-like 1D and 2D migrationscenarios in cells after hemin treatmemt (HO-1 pharmacologicalinducer). Actin cytoskeleton visualization was performed on PC3 cellsexposed to hemin or siRNAHO-1, fixed and stained withphalloidin-rhodamin. Filopodia from neighboring cells were observedby confocal microscopy and analysed with a custom made algorithm tocount contacts. Isolatedproteins from PC3 cells transfected with FLAG-tagged HO-1 wereimmunoprecipitated and subjected to LC/ESI-MSMS analysis. Geneontology (GO) analyses of HO-1 interacting proteins were performedusing DAVID.For the bioinformatics screening of HO-1 interactingproteins, we used Oncomine and the public repository Gene ExpressionOmnibus (GEO) from the National Center for Biotechnology Informationto browse for gene expression microarrays data. Raw expression datawas processed using the limma R package from Bioconductor. Results:A reduced frequency in migration events and in migration speed wasdetected under hemin exposure. Nuclear shape was also altered. Asignificant higher proportion of filopodia-like protrusions amongneighboring cells and cellular contacts were observed under HO-1induction. HO-1 silencing reversed these effects. Theproteomics analysis of HO-1 interacting proteins yielded a 17% ofcytoskeletal-associated proteins regulating actin filament dynamics.Our bioinformatics screening of these proteins revealed that most ofthe genes studied lie within the 10 or less percent of the mostconsistently low-expressed genes in prostate adenocarcinoma comparedto normal tissue.Conclusion:Our experimental findings demonstrate that HO-1 modulation in PCainduces the remodeling of the actin filament architecture atfilopodia, alters the migratory patterns and cellular morphology,showcasing the relevance of thecytoskeleton and its interacting partnersas potential therapeutic targets againstaggressive and metastatic disease.