Figure 5: Targeting the CBX2 chromodomain in Neuroendocrine Prostate Cancer.

A). Neuroendocrine differentiation (NED) of prostate cancer induced by androgen deprivation. Representative pictures of LNCaP cells treated with normal media or charcoal-stripped serum (CSS) media for 6 days to induce neuroendocrine differentiation (LNCaP_NED). B). Transcriptional fold changes of KLK3/TMPRSS2/ENO2/CHGA were quantified normalized to control gene YWHAZ upon CSS media treatment for 6 or 9 days. C). Western blotting of CBX paralogs in LNCaP and LNCaP_NED cells generated from 14-day CSS treatment. D). Cell proliferation of LNCaP_NED following 5 days incubation with 2 or 10 μM SW2_152F. DMSO was used as negative control. E). LNCaP_NED cells were treated with 10 μM SW2_152F for 24h-96h. Cell morphology pictures were captured starting from 24h. Average cell size were plotted at 24h or 48h for both DMSO group and compound treated group. F). LNCaP_NED cells were treated with 10 μM or 50 μM SW2_152F for 48 hours. Transcriptional fold change of genes (KLK3/TMPRSS2/AR/CBX2) were normalized to YWHAZ in LNCaP_NED cells with SW2_152F treatment or DMSO. G). LNCaP cells were treated with DMSO. LNCaP_NED cells were treated with DMSO, 10 μM GSK343 or 10 μM SW2_152F for 48 hours. Whole cell lysates were extracted for western blotting. H). LNCaP_NED cells were treated with DMSO or 10 μM SW2_152F for 4 hours before harvest. Binding of H3K27me3 and CBX2 at specific genomic sites (KLK3/TMPRSS2, e: enhancer, p: promoter) were evaluated using chromatin immunoprecipitation-qPCR. For all experiments in the figure, error bars represent SEM n=3 biological replicates, p-values were calculated using two-tailed Student’s t-test, * = p < 0.05, ** = p <0.01, *** = p < 0.001, **** = p <0.0001.