Volume 68, Issue 2, Pages 228-235 (August 2015) Cabazitaxel Remains Active in Patients Progressing After Docetaxel Followed by Novel Androgen Receptor Pathway Targeted Therapies  Nader Al Nakouzi, Sylvestre Le Moulec, Laurence Albigès, Chris Wang, Philippe Beuzeboc, Marine Gross-Goupil, Thibault de La Motte Rouge, Aline Guillot, Dorota Gajda, Christophe Massard, Martin Gleave, Karim Fizazi, Yohann Loriot  European Urology  Volume 68, Issue 2, Pages 228-235 (August 2015) DOI: 10.1016/j.eururo.2014.04.015 Copyright © 2014 European Association of Urology Terms and Conditions

Fig. 1 Waterfall plots of maximal prostate-specific antigen (PSA) changes on cabazitaxel therapy. (A) Response for each patient is plotted (n=79); (B) overall survival of patients treated with cabazitaxel; (C) progression-free survival (PFS) of patients treated with cabazitaxel. European Urology 2015 68, 228-235DOI: (10.1016/j.eururo.2014.04.015) Copyright © 2014 European Association of Urology Terms and Conditions

Fig. 2 (A) LNCaP, PC3, enza-R MR49C, and CRPC-V16D cells were exposed to increasing concentrations of cabazitaxel (Caba) for 48h, and half maximal inhibitory concentration (IC50) was determined using WST-1 cell survival assay. (B) Whole cell extracts of CRPC-V16D and enza-R cells were subjected to immunoblotting after a 48-h exposure to 0, 2.5, or 10 nmol/l Caba with specific antibodies for androgen receptor (AR), prostate-specific antigen (PSA), or vinculin (Vinc) (loading control). (C) Parental and resistant 22RV1 cell lines were exposed to increasing concentrations of Caba, enzalutamide (ENZ), or abiraterone (Abi) for 48h, and IC50 ranges were determined using WST1 assay and GraphPad Prism for analysis. European Urology 2015 68, 228-235DOI: (10.1016/j.eururo.2014.04.015) Copyright © 2014 European Association of Urology Terms and Conditions

Fig. 3 (A) Enza-R (MR49C and MR49F) and enza-S cells (CRPC-V16D) (2.5×105) were plated on six-well plates and transfected with probasin luciferase reporter along with Renilla plasmid and then treated with cabazitaxel (Caba) or docetaxel (Doce) for 12h before exposure to R1881 (1 nmol/l) for 4h. All experiments were carried out in triplicate wells and repeated three times. (B) Inhibition of androgen receptor (AR) expression in CRPC-V16D and Enz-R49C cells after a 48-h transfection with small interfering RNA (siRNA) targeting AR (siAR). Cells transfected with a nontargeted siRNA (siSCR) were used as control. Western blot analysis was performed using specific antibodies for AR or vinculin. Half maximal inhibitory concentration ranges were calculated comparing AR-depleted cells (siAR) with control (siSCR). These data are representative of three independent experiments. European Urology 2015 68, 228-235DOI: (10.1016/j.eururo.2014.04.015) Copyright © 2014 European Association of Urology Terms and Conditions

Fig. 4 (A) Enza-R and enza-S cells (2.5×105) were plated on six-well plates and transfected with probasin luciferase reporter along with Renilla plasmid as in Figure 3A and then exposed to R1881 (1 nmol/l) for 4h before being treated with cabazitaxel (caba) for 12h. All experiments were carried out in triplicate wells and repeated three times. (B) CRPC-V16D cells were treated with cabazitaxel (10 nmol/l), R1881 (1 nmol/l), caba then R1881 or reverse sequence (R1881 then caba). Androgen receptor (AR) localisation was assessed by immunofluorescence as described in the Methods section. Cellular features of mitotic catastrophe (multiple nuclei cells) are shown by arrows. European Urology 2015 68, 228-235DOI: (10.1016/j.eururo.2014.04.015) Copyright © 2014 European Association of Urology Terms and Conditions