Abstract 1344: Progranulin promotes ubiquitination, sorting and lysosomal degradation of sortilin in castration-resistant prostate cancer cells
Authors: Tanimoto Ryuta , Palladino Chiara , Buraschi Simone , Xu Shi-Qiong , Gomella Leonard G. , Iozzo Renato V. , Belfiore Antonino , Morrione Andrea 
Published: July 2017

Abstract
Introduction and Objective: Despite extensive clinical and experimental studies over the past decades, the pathogenesis of prostate cancer remains largely unknown. Furthermore, the mechanisms promoting the castration-resistant stage of prostate cancer are still very poorly understood. We recently demonstrated that progranulin acts as an autocrine growth factor and promotes castration-resistant prostate cancer cell motility, invasion and anchorage-independent growth. Progranulin was also overexpressed in prostate cancer tissues vis-à-vis non-neoplastic controls. Despite the strong connections with cancer, progranulin’s mode of action is not well understood. Furthermore, proteins that regulate early stages of progranulin signaling have not been identified. Sortilin, a transmembrane protein of the Vps10 family, binds progranulin and negatively regulates progranulin action by promoting progranulin uptake and degradation. Significantly, castration-resistant DU145 and PC3 cells express very low sortilin levels, which were associated with high progranulin production and enhanced motility, suggesting that mechanisms regulating sortilin/progranulin levels may contribute to prostate cancer progression.
Methods: Progranulin-mediated sortilin ubiquitination was assessed by transient ubiquitination assays in PC3 and DU145 cells either expressing or depleted of endogenous progranulin. Sortilin stability was assessed by immunoblotting with or without protein synthesis inhibitors and immunofluorescence microscopy with GFP-tagged sortilin. Progranulin-dependent sortilin endocytosis and sorting for degradation were evaluated by confocal microscopy and colocalization with specific intracellular markers.
Results: Prolonged progranulin stimulation induced sortilin ubiquitination in PC3 and DU145 cells, which was associated with enhanced sortilin internalization through a clathrin-dependent pathway. Upon progranulin stimulation, sortilin was then sorted into early endosomes and subsequently targeted for degradation in the lysosomal compartment as assessed by immunoblot and IF experiments in the presence or absence of lysosomal inhibitors. Significantly, sortilin ubiquitination was significantly inhibited in progranulin-depleted PC3 and DU145 cells, which showed enhanced sortilin stability when compared to progranulin-expressing control cells.
Conclusions: Our data demonstrate that progranulin activates a feedback loop by inducing sortilin downregulation via the lysosomal pathway. This novel regulatory mechanism would ensure the maintenance of progranulin signaling in castration-resistant prostate cancer cells by limiting sortilin-mediated progranulin uptake and degradation. Deciphering the cross-talk between sortilin and progranulin signaling could provide avenues to design novel therapeutic strategies in prostate tumors.
Citation Format: Ryuta Tanimoto, Chiara Palladino, Simone Buraschi, Shi-Qiong Xu, Leonard G. Gomella, Renato V. Iozzo, Antonino Belfiore, Andrea Morrione. Progranulin promotes ubiquitination, sorting and lysosomal degradation of sortilin in castration-resistant prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1344. doi:10.1158/1538-7445.AM2017-1344