Figure 6.

Effects of RAR/RXR signaling on the differentiation of myoepithelial-like cells into ductal-like cells and the survival of ductal-like cells. A) Schematic workflow of experiments aimed at elucidating the population-specific effects of pharmacological manipulations of RAR/RXR signaling. Paired sets of CD49f^high/KIT^neg and CD49f^low/KIT⁺ cells were sorted in parallel from the same tumor (ACCX5M1) and cultured for 1 week as 2D monolayers, in the presence of either ATRA (10 µM) or BMS493 (10 µM), respectively. B-D) Evaluation of the effects of ATRA on sorted CD49f^high/KIT^neg cells. Treatment with ATRA did not affect the viability of CD49f^high/KIT^neg cells ([B] alamarBlue assay) but caused CD49f^high/KIT^neg cells to change phenotype and become CD49f^low/KIT⁺ ([C-D] FACS). E-F) Evaluation of the effects of BMS493 on sorted CD49f^low/KIT⁺ cells. Treatment with BMS493 caused the death of the majority CD49f^low/KIT⁺ cells ([E] alamarBlue assay). On visual inspection by conventional microscopy, CD49f^low/KIT⁺ cells treated with BMS493 appeared fragmented as compared with control cells treated with DMSO alone (F). Scale bar: 100 µm. G) Schematic workflow of the experiment aimed at testing the effects of a DNhRARα construct on the capacity of CD49f^high/KIT^neg cells to undergo myoepithelial-to-ductal differentiation. CD49f^high/KIT^neg cells were sorted from ACCX5M1 tumors, cultured for 6 days as 2D monolayers, and infected with lentivirus vectors encoding for either a DNhRARα-EGFP construct or a control EGFP reporter. Infected cells were then cultured for 1 additional week and analyzed by FACS for CD49f and KIT expression, restricting the analysis to infected (EGFP⁺) cells. H-J) Analysis by FACS of CD49f^high/KIT^neg cells purified form ACCX5M1 tumors and infected with lentivirus vectors encoding for either a DNhRARα-EGFP construct or a control EGFP reporter. Forced DNhRARα expression completely abrogated the capacity of CD49f^high/KIT^neg cells to produce a CD49f^low/KIT⁺ progeny, whereas forced expression of EGFP alone did not. K-N) Evaluation of the role of DNhRARα as a suppressor of myoepithelial-to-ductal differentiation in a second, independent PDX model (SGTX6). Error bars: mean +/- standard deviation; P values: Student’s t test, 2-tailed (n.s. = not significant; *P < .05; ***P < .001). 2D = 2-dimensional; APC = allophycocyanin; ATRA = all-trans retinoic acid; DMSO = dimethyl-sulfoxide; DNhRARα = dominant negative human retinoic acid receptor alpha; EGFP = enhanced green fluorescent protein; FACS = fluorescence-activated cell sorting; PDX = patient-derived xenograft; PE = phycoerythrin; RAR = retinoic acid receptor; RXR = retinoid X receptor.