Fig 2: Celltiter-Glo assay showed that HA-tagged MET overexpression was able to partially rescue the cell growth of PC3 prostate cancer cells stably expressing FOXP2 shRNA (two clones, #21 and #23).P values calculated by the Mann-Whitney U test, mean ± s.d.; n = 4. Immunoblot showing ectopic expression of HA-tagged MET protein in PC3 cells stably expressing FOXP2 shRNA.

Fig 3: Methylation status of FOXP2 gene in tumors from COSMIC datasets.

Fig 4: Oncogenic roles of FOXP2 in prostate cancer.(A) FOXP2-CPED1 fusion gene identified in the prostate tumor of patient (PC_1) by RNA-seq. Top: schematic of chromosome 7 with the position and strand orientation indicated for the FOXP2 and CPED1 gene. Bottom: schematic representation of the paired-end reads covering the junction between FOXP2 and CPED1. (B) FOXP2-CPED1-specific PCR from cDNA derived from the prostate tumors (PC_1 and PC_11). Sanger sequencing chromatogram of the PCR products showing the reading frame encompassing the breakpoint in the two fusion-positive tumors (PC_1 and PC_11). The fusion transcript sequence was identical in the two cases at the fusion junction (exon 14 of FOXP2 fused to exon 4 of CPED1), resulting in a stop-gain mutation right after the fusion breakpoint. (C) Schematic of protein structures of wild-type FOXP2 and truncated FOXP2 encoded by the FOXP2-CPED1 fusion transcript. FOXP2-CPED1 fusion protein has an aberrant C-terminus but it retains the complete FOXP2 functional domains. (D) Expression of natural FOXP2 and FOXP2-CPED1 fusion in the tumor (PC_1) and its matched normal tissue (NT_1) by qPCR. Mean ± SD. (E) Expression of FOXP2 mRNA in 92 primary prostatic adenocarcinoma tissues and 23 matched normal tissues analyzed by qPCR. (F) Expression of FOXP2 mRNA in normal prostate samples from the GTEx dataset (n = 106) and metastatic prostate tumors from the SU2C dataset (n = 117). The horizontal bar indicates the mean in each group. p-Values were calculated by two-tailed Mann–Whitney U-test. (G) Left: representative images of FOXP2 protein expression in benign prostatic hyperplasia (BPH) (n = 25) or primary prostatic adenocarcinoma (PCA) (n = 45) by immunohistochemistry. Scale bars, 100 μm. Right: the bar chart indicates the number of PCA and BPH with negative, low, medium, and high FOXP2 expression, respectively. (H) Expression of FOXP2 mRNA in two types of human benign prostate epithelial cells, RWPE-1 and NPrEC, and in two prostate cancer cell lines LNCaP and DU145 from the GEO database (1555516_at). p-Values were calculated by two-tailed Student’s t-test. (I) Western blot measuring FOXP2 protein in one normal human prostate epithelial cell HPrEC and one benign prostate epithelial cell RWPE-1, and in three human prostate cancer cell lines, PC3, VCaP, and LNCaP. The experiment was repeated twice with similar results. (J) Cell transformation and anchorage-independent growth were measured by a soft agar assay in parental NIH3T3 cells and NIH3T3 cells stably expressing the empty vector, FOXP2 and FOXP2-CPED1. Repeated 4-week assays were performed. Scale bars, 100 μm. See Figure 1—figure supplement 3D for details. Table indicating the number of total NIH3T3 cell lines overexpressing FOXP2 (n = 32) or FOXP2-CPED1 (n = 42) tested by soft agar colony formation assay (left column) and the number of transformed NIH3T3 cell lines induced by FOXP2 (n = 10) or FOXP2-CPED1 (n = 21) (right column). See Figure 1—figure supplement 3D for details. (K) Quantification of the tumor volume in NOD-SCID mice injected with NIH3T3 cells stably expressing vector, FOXP2 (two cell lines) or FOXP2-CPED1 (three cell lines) for 8 wk. Inset: representative images of xenografted tumors derived from the corresponding cells. See Figure 1—figure supplement 3E and F for details. (L, M). (L) Soft agar assay showing that RWPE-1 cells stably expressing FOXP2 (two cell lines) or FOXP2-CPED1 (two cell lines) were able to form colonies. Scale bars, 100 μm; (M) Quantitative analysis of the colony formation ability of the corresponding RWPE-1 cells. Mean ± SD; n = 4. (N) Cell growth of prostate cancer cell lines (LNCaP and PC3) stably expressing control vector or FOXP2 shRNA (two clones, #1 and #2). *p<0.05, **p<0.005 by two-tailed Student’s t-test, mean ± SD; n = 4. Inset: immunoblot showing knockdown of FOXP2 protein in LNCaP or PC3 cells. (O) Left: focus formation assay performed with PC3 or LNCaP cells stably expressing control vector or FOXP2 shRNA (two clones, #1 and #2). Right: bar graph showing the number of colonies formed by PC3 or LNCaP cells after FOXP2 silencing. **p<0.005 by two-tailed Student’s t-test; mean ± SD; n = 4. Figure 1—source data 1.Uncropped blot for Figure 1I. Figure 1—source data 2.Uncropped blot for Figure 1N.

Fig 5: FOXP2 activates oncogenic MET signaling in FOXP2-overexpressing cells and prostate tumors.(A) Schematic indicating the analysis of the TCGA dataset (Prostate Adenocarcinoma, Provisional, n = 495). In total, n = 255, top 25%, high expression (n = 120), bottom 25%, low expression (n = 135). (B) In the 255 primary prostate tumors shown in (A), the expression of 3206 genes was significantly correlated with the expression of FOXP2 (|Spearman’s ρ ≥ 0.5|; 1884 significantly positively correlated genes; 1322 significantly negatively correlated genes). See also Supplementary file 2. (C) The significant enrichment of the 3206 FOXP2 expression-correlated genes (FECGs) in known prostate cancer gene sets from the Molecular Signatures database by gene set enrichment analysis (GSEA). p-Values were calculated by two-tailed Fisher’s exact test. See also Supplementary file 1c. (D) Analysis of the biological pathways of 3206 FOXP2 expression-correlated genes using gene sets from MSigDB and KEGG. The number in parentheses corresponds to FOXP2 expression-correlated genes that are enriched in the corresponding pathway. The Spearman correlation coefficients between the expression of FOXP2 and the expression of four core components of MET signaling (HGF, MET, PIK3R1, and PIK3CA) are indicated in parentheses. Two-tailed p-values by Fisher’s exact test and adjusted by Bonferroni correlation. See also Supplementary file 1d. (E) The correlation analysis of gene expression between FOXP2 and MET signalling members in our primary prostate tumors (n = 92) and two other human primary prostate cancer datasets (GSE54460, n = 100; Taylor, n = 162) by qPCR. HGF, MET, PIK3R1, and PIK3CA expression levels (normalized to EIF4H) classified by FOXP2 expression level (bottom 25%, low expression; top 25%, high expression). p-Values were calculated by two-tailed Mann–Whitney U-test. (F) In FOXP2- or FOXP2-CPED1-transformed RWPE-1 cells and control cells (parental cells and empty vector-expressing cells), the relative mRNA expression levels of HGF, MET, PIK3R1, and PIK3CA (normalized to EIF4H) were examined by qPCR. p-Values calculated by two-tailed Student’s t-test, mean ± SD; n = 3. *p<0.05, **p<0.005.