Fig 2: Androgen signaling negatively regulates PlexinD1 expression in PCa.(A) qPCR of PLXND1, SEMA3C, SEMA3E, KLK3, and NCAM1 in LNCaP, LAPC4, and VCaP cells upon culture under CSS condition for 24 h followed by R1881 stimulation (10 nM, 24 h) (n = 3 biological replicates). (B, C) Western blot of PlexinD1, AR, and PSA in LNCaP and LAPC4 cells upon treatment with 10 nM R1881 (B) or 10 µM ENZ (C) for the indicated times. (D) Genomic browser representation of AR binding at PLXND1 promoter encompassing three AREs, with the nucleotides identical to the canonical ARE highlighted in red, by interrogating AR ChIP-seq dataset GSE125245. (E) ChIP-qPCR of AR and H3K9ac occupancy at an ARE-centric PLXND1 promoter sequence as well as an AR-bound KLK3 promoter region upon R1881 stimulation (10 nM, 6 h) in LNCaP cells. Data represent the percent of input (n = 3 technical replicates). (F) Schematic diagrams of WT and mutated forms of individual AREs in PLXND1 ARE-Luc constructs. (G) Determination of WT and mutated PLXND1 ARE-Luc activities upon R1881 stimulation (10 nM, 6 h) in LNCaP cells (n = 3 biological replicates). (H) Pearson correlation analysis of PLXND1 mRNA expression with AR score in the indicated datasets from cBioPortal. (I) Pearson correlation analysis of mRNA co-expression between PLXND1 vs. different AR target genes in the indicated datasets from cBioPortal. Data information: In (A, E, G), data are presented as mean ± SEM. In (A), P values were determined by one-way ANOVA with Tukey’s multiple comparisons test. In (E), P values were determined by two-way ANOVA with Tukey’s multiple comparisons test. In (G), P values were determined by unpaired two-tailed Student’s t-test. In (H), P values were determined by Pearson’s correlation coefficient t-test. *P < 0.05, **P < 0.01; ns, not significant. Exact P values are listed in Appendix Table S4. Source data are available online for this figure.

Fig 3: PlexinD1 promotes cellular plasticity, stemness, and NE differentiation in PCa.(A) GSEA of cell stemness and lineage-related gene sets enriched in PlexinD1-overexpressing vs. control LNCaP cells. (B) Representative images and quantification of number and size of tumorspheres formed by control or PlexinD1-manipulated PCa cells (n = 3 biological replicates). Scale bars: 100 µm. (C) Flow cytometric analysis of % of ALDH+ cells in control and PlexinD1-manipulated PCa cells, with DEAB-treated groups as negative controls (n = 3 biological replicates). (D) Western blot of stemness, luminal, basal, and NE markers as indicated in control and PlexinD1-manipulated PCa cells. (E) Representative images of control and PlexinD1-knockdown C4-2BENZR cell morphology and quantification of per-cell number of neurites and neurite length in each group (n = 50 cells per group). A representative of 3 independent experiments is shown. Scale bars: 20 µm. (F) Cell proliferation assays of control and PlexinD1-overexpressing LNCaP cells upon ENZ treatment (20 μM, 5 days). Data represent the fold changes of cell proliferation on day 5 relative to cell seeding day (day 0) (n = 4 biological replicates), with fold changes in non-treated groups set as 1 for normalization of paired treated groups. (G) Representative PlexinD1 and SYP IHC staining in serial sections of tumor tissue from a CRPC patient cohort and corresponding Pearson correlation analysis of protein co-expression. Scale bars: 100 µm. (H) Comparisons of PLXND1 mRNA levels in NEPC vs. Adeno or CRPC Adeno patient samples from multiple PCa clinical datasets as indicated. The n of each group indicates the number of patient tumor samples included for comparisons. (I) GSEA plots of Hallmark Androgen Response and NEPC gene sets enriched in PLXND1-high vs. -low PCa patient samples from TCGA and SU2C/PCF 2019 cohorts in cBioPortal. (J) GSEA plots of PlexinD1 targets and semaphorin/plexin signaling-related gene sets enriched in NE score-high vs. -low cells of a CRPC patient tumor as in the scRNA-seq dataset GSE137829. Data information: In (B, C, E, F, H), data are represented as mean ± SEM. In (A, I, J), P values were determined by permutation test. In (B, E), P values were determined by one-way ANOVA with Dunnett’s multiple comparisons test [for C4-2BENZR and 22Rv1 in (B)] and unpaired two-tailed Student’s t-test [for LNCaP in (B)]. In (C, H), P values were determined by unpaired two-tailed Student’s t-test. In (F), P values were determined by one-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01; ns, not significant. Exact P values are listed in Appendix Table S4. Source data are available online for this figure.

Fig 4: PlexinD1 induces noncanonical Gli1-dictated Hedgehog signaling in PCa.(A) IPA upstream regulator analysis of RNA-seq data from PlexinD1-knockdown vs. control C4-2BENZR cells, with significantly downregulated hits shown (absolute value of z-score of ≥2, p < 0.05). (B) STRING analysis of Gli1 linked to multiple stemness, NE, and EMT marker and/or driver genes. (C) Western blot of Gli1 in control and PlexinD1-manipulated PCa cells. (D) Determination of Gli-Luc reporter activity in control and PlexinD1-manipulated PCa cells (n = 3 biological replicates). (E) qPCR of Gli1 target genes in control and PlexinD1-knockdown C4-2BENZR cells (n = 3 biologial replicates). (F) qPCR of Hh ligands in control and PlexinD1-knockdown C4-2BENZR and 22Rv1 cells (n = 3 biological replicates). (G) Determination of Gli-Luc reporter activity in 22Rv1 cells receiving an ErbB3 neutralizing antibody (100 ng/ml, 24 h), SGX-523 (5 µM, 24 h), or transient transfection of ErbB3/cMet expression plasmids followed by PLXND1 siRNA treatment for another 48 h (n = 3 biological replicates), and in control and PlexinD1-overexpressing LNCaP cells upon treatment with 100 ng/ml ErbB3 neutralizing antibody or 5 µM SGX-523 for 24 h (n = 3 biological replicates). (H) Representative images of Gli1 (red color) and α-tublin (a cytoplasm marker, green color) co-IF images and quantification of Gli1+ cells in the nuclei of control and PlexinD1-manipulated 22Rv1 and LNCaP cells under the conditions as in (G) (n = 3 biological replicates). Scale bars: 10 µm. (I) Western blot of Gli1 in nuclear and cytoplasmic fractions of control and PlexinD1-manipulated 22Rv1 and LNCaP cells under the conditions as in (G). (J) Cell proliferation assays of control and PlexinD1-overexpressing LNCaP cells upon treatment with 10 µM cyclopamine or 5 µM GANT61 for 5 days (n = 3 biological replicates). (K) Cell proliferation assays of C4-2BENZR and 22Rv1 cells upon treatment with 10 µM cyclopamine or 5 µM GANT61 for 5 days (n = 3 biological replicates). (L) qPCR of stemness, NE and EMT markers in control and PlexinD1-overexpressing LNCaP cells under treatment with GANT61 (5 µM, 24 h) (n = 3 biological replicates). (M) GSEA plots of two Hh signaling-related gene sets enriched in PLXND1-high vs. -low PCa patient samples from TCGA cohort. (N) Chi-square analysis of distribution of select Gli1 target gene mRNA expression (L, low; H, high) in PLXND1-low and -high PCa patient samples from TCGA cohort. Data information: In (D–H, J–L), data are presented as mean ± SEM. In (A), P values were determined by Fisher’s exact test. In (D–F, K), P values were determined by one-way ANOVA with Dunnett’s multiple comparisons test [for C4-2BENZR and 22Rv1 in (D)] and unpaired two-tailed Student’s t-test [for LNCaP in (D)]. In (G, H, J, L), P values were determined by one-way ANOVA with Tukey’s multiple comparisons test. In (M), P values were determined by permutation test. In (N), P values were determined by chi-square test. *P < 0.05, **P < 0.01; ns, not significant. Exact P values are listed in Appendix Table S4. Source data are available online for this figure.

Fig 5: PlexinD1 activates ErbB3 and cMet in PCa.(A) GSEA plots of transmembrane RTK-related gene sets enriched in PlexinD1-knockdown vs. control 22Rv1 cells, and PLXND1-high vs. -low PCa patient samples in TCGA cohort. (B) Representative images of a RTK phosphorylation antibody array and quantification of p-ErbB3 levels in control and PlexinD1-knockdown C4-2BENZR cells (n = 2 technical replicates). The raw values from measurement of p-ErbB3 spot intensity after background subtraction are presented. (C) Western blot of p-ErbB3, p-ErbB2, p-cMet, p-ERK, p-AKT, and their total protein forms in control and PlexinD1-knockdown C4-2BENZR and 22Rv1 cells. (D) qPCR of ErbB3 (NRG1 and NRG2) and cMet (HGF) ligand mRNA levels in control and PlexinD1-knockdown C4-2BENZR and 22Rv1 cells (n = 3 biological replicates). (E) Representative PLA images and quantification of PlexinD1-ErbB3/cMet interaction by per-cell cytoplasmic fluorescence intensity in C4-2BENZR cells treated with recombinant Sema3E/Sema3C proteins (200–500 ng/ml, 4 h) or SEMA3E/SEMA3C siRNA (10 μM, 48 h). PlexinD1 antibody incubation alone served as negative controls. The n of each group indicates the number of cells examined for quantification. Scale bars: 5 μm. (F) Co-IP assays of PlexinD1-ErbB3/cMet interaction in 22Rv1 cells. IgG was used in the IP step as negative control. Five percent of input was blotted as positive control. (G) Cell proliferation assays of 22Rv1 cells pre-treated with an ErbB3 neutralizing antibody (100 ng/ml, 24 h) or SGX-523 (5 µM, 24 h) and then subjected to PLXND1 siRNA addition followed by a 5-day observation period, and control and PlexinD1-overexpressing LNCaP cells upon treatment with an ErbB3 neutralizing antibody or SGX-523 during a 5-day observation period. Data represent the fold changes of cell proliferation on day 5 relative to siRNA treatment day (day 1) for 22Rv1 cells or treatment day (day 1) for LNCaP cells (n = 3 biological replicates), with fold changes in non-treated groups of control cells set as 1 for normalization of other groups. (H) qPCR of stemness, basal, and NE markers as indicated in 22Rv1 cells receiving an ErbB3 neutralizing antibody (100 ng/ml, 24 h), SGX-523 (5 µM, 24 h), or transient transfection of ErbB3/cMet expression plasmids followed by PLXND1 siRNA treatment for another 48 h (n = 3 biological replicates). (I) qPCR of stemness, basal, NE, and EMT markers as indicated in control and PlexinD1-overexpressing LNCaP cells upon treatment with 100 ng/ml ErbB3 neutralizing antibody or 5 µM SGX-523 for 24 h (n = 3 biological replicates). Data information: In (B, D, E, G, H, I), data are presented as mean ± SEM. In (A), P values were determined by permutation test. In (B, D), P values were determined by unpaired two-tailed Student’s t-test. In (E), P values were determined by one-way ANOVA with Dunnett’s multiple comparisons test. In (G, H, I), P values were determined by two-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01; ns, not significant. Exact P values are listed in Appendix Table S4. Source data are available online for this figure.