Fig 2: Capicua (CIC) and ETS2 repressor factor (ERF) cooperatively bind an ETV1 regulatory element to suppress ETV1 expression and transcriptional activity.(A) Percentage of CIC and ERF peaks located in defined genomic regions. (B) Schematic algorithm to identify shared CIC and ERF target genes in prostate cells (top). Functional Clustering Analysis of the 91 shared CIC and ERF target genes using DAVID (bottom table). (C) ETV1 mRNA expression in prostate epithelial cells (PNT2) (CIC-ERF-replete) cells with ERF knockdown (KD), CIC knockout (KO), or ERF KD +CIC KO (n=3). (D) Schematic of CIC and ERF DNA-binding motifs in the ETV1 promoter. (E) Chromatin immunoprecipitation (ChIP)-PCR from PNT2 cells showing CIC occupancy on the ETV1 promoter. (F–G) ChIP-PCR with ERF occupancy on the ETV1 promoter. (H) ETV1 mRNA expression in DU-145 (ERF-deficient) cells with ERF rescue, CIC KO, or ERF rescue +CIC KO (n=3). ETV1 mRNA expression in PC-3 cells with (I) ERF KD (n=3) and (J) ERF overexpression (OE) (n=3). p Values were calculated using Student’s t test. *p<0.05, **p<0.01, and ****p<0.0001. Error bars represent SD. Performed in triplicate. (K) ETV1 luciferase promoter assay in 293T cells comparing Empty vector (EV) with ERF OE (n=6). Student’s t test, *p<0.05. Error bars represent SD. (L) Single sample gene set enrichment analysis (ssGSEA) alignments comparing gene expression patterns in PNT2 cells with ERF KD and CIC KO. IC = information coefficient. Figure 4—source data 1.Full-length PCR gel images of ETV1 after Capicua (CIC) pull down in prostate epithelial cells (PNT2).Cropped images and description shown in Figure 4E. Figure 4—source data 2.Full-length PCR gel images of ETV1 after ETS2 repressor factor (ERF) pull down in prostate epithelial cells (PNT2).Cropped images and description shown in Figure 4F. Figure 4—source data 3.Full-length PCR gel images of ETV1 after ETS2 repressor factor (ERF) pull down in DU-145 cells.Cropped images and description shown in Figure 4G.

Fig 3: Capicua (CIC) and ETS2 repressor factor (ERF) are co-deleted in aggressive prostate cancer (PCa) and associate with worse clinical outcomes.(A) CIC transcriptionally represses ETV1/4/5. (B) The 19q13.2 genomic locus demonstrating the physical location of ERF and CIC. (C) 15 PCa studies (cBioPortal) demonstrating the co-occurrence of ERF and CIC homozygous and heterozygous deletions. The co-occurrence of ERF and CIC alterations is highly significant (p<0.001 co-occurrence, Fisher exact test). (D) ERF-CIC co-deleted PCa stratified by Gleason score and tumor stage. (E) Frequency of ERF and CIC alterations in primary PCa (top) and metastatic castrate resistant prostate cancer (mCRPC; bottom), demonstrating enrichment in mCRPC. (F) Onco-print of known genetic drivers (ERG, ETV1, ETV4, SPOP, and FOXA1) of PCa aligned with CIC and ERF (cBioPortal). CIC-ERF co-deleted prostate tumors (red box) do not frequently co-occur with other known oncogenic events. (G) Survival analysis performed using 15 PCa datasets from cBioPortal. Disease-free survival (DFS) and progression-free survival (PFS) in patients harboring the ERF-CIC co-deletion (red) vs. no ERF-CIC co-deletion (blue). p=value, log-rank. Figure 1—source data 1.Prostate cancer studies identified in cBioPortal demonstrating the total number of patients, number of patients with shallow or deep deletions in Capicua (CIC)-ETS2 repressor factor (ERF), and the frequency of CIC-ERF alterations in each cohort.Studies that analyzed predominantly primary prostate cancers (green) and metastatic castrate resistant prostate cancer (mCRPC; yellow) are highlighted.