Abstract 5311: Proteomic characterization of zoledronic acid-resistant prostate cancer cells identified key proteins in cytoskeleton organization and cancer stem cell markers associated with a very aggressive phenotype
Authors: Milone Maria Rita , Pucci Biagio , Iannelli Federica , Lombardi Rita , Bifulco Katia , Bruzzese Francesca , Gennaro Elena D. , Avallone Antonio , Carriero Maria Vincenza , Budillon Alfredo 
Published: May 2015

The aminobiphosphonate Zoledronic acid (ZOL) inhibit osteoclast-mediated bone resorption and it is used to prevent skeletal complications from bone metastases. Accumulating evidences in both preclinical and clinical studies indicated that ZOL might also have anticancer activity. We have recently selected for the first time the ZOL-resistant DU145R80 prostate cancer (PCa) cell line that demonstrated an antiapoptotic and proangiogenic phenotype with increased invasive capability and epithelial to mesenchymal transition (EMT), compared to parental DU145 cells. Interestingly, we also demonstrated that both the acquired resistance to ZOL and the aggressive phenotype are mediated by p38-MAPK (Milone et al. Cell Death Dis. 2013, 4:e641). To further investigate the mechanism of ZOL-resistance and of the parallel acquisition of an aggressive phenotype in this novel syngeneic model of PCa, we took advantage of a two-dimensional difference gel electrophoresis (2D-DIGE)/mass spectrometry (MS) proteomic approach, to investigate differential expressed proteins between DU145R80 and DU145 cell lines. We found 21 statistically differentially expressed protein spots between the two cell lines (average volume ratio threshold of ±1.4-fold and 95% level of significance, p&lt;0.05) and 15 of these were identified by MS and validated by real-time PCR, 1D or 2D immunoblots. The identified proteins were involved in cell metabolism, cytoskeleton, nuclear lamina organization, protein fate and RNA processing, such as ALDH7A1, ANXA1, Lamin A/C, Filamin A, Lamin B2, PSMA6 and eEF1γ. Several of these proteins are related with the acquisition of a cancer stem cells (CSC) phenotype and indeed we showed in DU145R80 also the overexpression of additional prostate CSC markers such as Nanog, Oct-4, Snail and Integrin-aV. Furthermore, in DU145R80 compared to DU145 cells, we observed the increased presence of numerous podosomes/invadopodia and membrane bleb protrusions where FLNA clearly co-localize with actin. Interestingly, by using publicly available genomic data on PCa (http://www.oncomine.org) we demonstrated statistically significant higher expression of the genes codifying the identified proteins, in PCa vs normal tissues. Finally, proteomic results were further processed by interactome pathway analysis demonstrating a strong relationships between the identified proteins and additional effectors in both direct/indirect analysis, including p38-MAPK, and generating a relevant statistically significant network associated with cancer disease as well as with cellular movement and organization. Overall we confirmed that ZOL-resistant PCa cells developed an aggressive phenotype related with stemness feature and identified novel potential prognostic markers and/or therapeutic targets for PCa.

Citation Format: Maria Rita Milone, Biagio Pucci, Federica Iannelli, Rita Lombardi, Katia Bifulco, Francesca Bruzzese, Elena Di Gennaro, Antonio Avallone, Maria Vincenza Carriero, Alfredo Budillon. Proteomic characterization of zoledronic acid-resistant prostate cancer cells identified key proteins in cytoskeleton organization and cancer stem cell markers associated with a very aggressive phenotype. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5311. doi:10.1158/1538-7445.AM2014-5311