Fig 2: Downregulation of PRR results in a decrease in cell growth and V-ATPase activity.(A) Growth curves and Western blot analysis of LNCaP cells infected with empty vector (PLKO), Prorenin Receptor (PRR) shRNA 1 (* P = 0.034), shRNA 2, or shRNA 3 (Left Panel) (n = 3). (B) Growth curves and Western blot analysis of PC3 cells infected with PLKO, PRR shRNA 1 (* P = 0.017), shRNA 2 (* P = 0.042), or shRNA 3 (* P = 0.021) (Left Panel) (n = 3). Total lane protein (TLP) was used as the loading control. (C) LysoTracker DND-99 and corresponding brightfield images (20×) of NSC, PRR siRNA1, or siRNA 2-transfected, and Bafilomycin A1(Baf A1)-treated LNCaP C4-2 cells. (D) Quantification of Mean LysoTracker signal per cell showing a reduction in LysoTracker signal after transfection with PRR siRNA 1 (* P = 0.037), siRNA 2 (* P = 0.048), or Baf A1 (100 nM) (** P = 0.006) treatment in LNCaP C4-2 cells (n = 5). Chemical inhibition with Baf A1 serves as a positive control. Western blot analysis validating PRR knockdown in siRNA1 and siRNA2-transfected LNCaP C4-2 cells. TLP was used as a control (Top Panel). (E) Western blot analysis showing less expression of the V-ATPase B1/2 subunits and less localization in the membranous fraction of LNCaP cells following PRR knockdown. Chemical inhibition with Baf A1 (100 nM) increases cytosolic availability of the V1 domain, which is indicative of less V-ATPase assembly at the membranous fraction. Insulin Receptor (IR) and Actin localization were used to validate fractionation. TLP was used as a loading control. (F) Corresponding quantification of V-ATPase B1/2 membranous localization in NSC, PRR siRNA 1 (** P = 0.0015), siRNA 2 (*** P = 0.0008), or in Baf A1 (100 nM) treated cells (n = 3). Scale bar measures 100 µm. Data are presented as the mean ± SEM. Statistical tests conducted using Student’s t test.

Fig 3: Analysis of p-Akt and PRR expression in prostate cancer tissue microarrays.Box and Whisker plots demonstrating the distribution of (A) phosphorylated Akt (p-Akt) (** P < 0.01) (benign adjacent, upper extreme (UE)=2.33, upper quartile (UQ)=1.0, median (M)=0.67, lower quartile (LQ)=0.00, lower extreme (LE)=0.00; tumor, outlier (O)=2.82, UE=2.67, UQ=1.45, M=1.33, LQ=0.67, LE=0.00) and (B) Prorenin Receptor (PRR) (** P < 0.01) (benign adjacent, O=2.1, 1.98, 1.84, UE=1.75, UQ=1.00, M=0.75, LQ=0.50, LE=0.00; tumor, UE=2.15, UQ=1.75, M=1.25, LQ=0.75, LE=0.00) staining intensity scores (0-3) evaluated by IHC in Benign adjacent and tumor tissues from 285 radical prostatectomy specimens arrayed on Tissue microarrays. (C) Representative whole and IHC images (20×) of p-Akt and PRR stained benign and tumor tissue microarrays showing the increased expression of PRR and p-Akt in tumor tissue microarrays relative to benign tissue. Scale bar measures 200 µm. Statistical tests conducted using Student’s t test.

Fig 4: PTEN downregulates PRR expression and sPRR secretion.(A) Western blot analysis showing more endogenous Prorenin Receptor (PRR) expression in whole cell extracts and soluble Prorenin Receptor (sPRR) secretion in conditioned media (CM) of Phosphatase and tensin homolog (PTEN)-null prostate cancer lines (PC3, LNCaP, LNCaP C4, and LNCaP C4-2) than in PTEN-expressing lines (22RV1 and DU145). Total lane protein (TLP) was used as a loading control. Dot plots of the quantification of (B) PRR expression and (C) sPRR secretion relative to total lane protein in the same prostate cancer lines. (D) Western blot analysis showing that PTEN reintroduction in PTEN-null lines via PTEN adenovirus infection (Ad-PTEN) reduces sPRR secretion when compared to their LacZ-infected counterparts. PTEN reintroduction also reduces Akt phosphorylation as expected. Total Akt was used as a control for phosphorylation of Akt (p-Akt) and TLP was used as a loading control. (E) Dot plots of the quantification of PRR expression in the Ad-PTEN-WT-infected prostate cancer lines relative to the LacZ-infected counterparts and to TLP, demonstrating largely unchanged levels of PRR expression after Ad-PTEN infection. (F) Dot plots of the quantification of sPRR secretion shows significant reductions in the Ad-PTEN-WT-infected PC3 (* P = 0.015), LNCaP (*** P = 0.0002), LNCaP C4 (** P = 0.005), and LNCaP C4-2 relative to the LacZ-infected counterparts and to TLP (n = 3). (G) Representative IHC images of Wild type (WT) and Pten-null (Pten-/-) mouse prostate sections after H&E staining, and p-Akt and PRR IHC staining. (H) Western blot analysis demonstrating increased PRR expression in whole cell extracts, and sPRR secretion in CM of Pten-/- mouse embryonic fibroblasts compared to wild type (WT) fibroblasts. Actin was used as a loading control in whole cell extracts and Ponceau was used as loading control for CM. Dot plots of the quantification of (I) PRR expression in Pten-/- (*** P = 0.0001, n = 6) and of (J) sPRR secretion in Pten-/- (* P = 0.025, n = 4) in mouse embryonic fibroblasts relative to wild type counterparts. Scale bar measures 200 µm. Data are presented as the mean ± SEM. Statistical tests conducted using Student’s t test.

Fig 5: PRR expression increases in tumor tissue, correlates with Gleason score, and sPRR is elevated in urine of patients with aggressive prostate cancer.(A) Representative Western blot analysis demonstrating dampened Phosphatase and tensin homolog (PTEN) expression in tumor (T) tissue of patient prostates with Gleason grade 3+4 and 4+3 but not in 3+3 Gleason grade compared to normal adjacent tissue (NA) from the same prostate of each patient. Also, Prorenin Receptor (PRR) expression is increased in the tumor tissue in which PTEN expression is decreased (3+4, and 4+3 Gleason grade patients). Total lane Protein (TLP) was used as a loading control. (B) Quantification of PRR expression shows an increase in PRR expression in the tumor tissue compared to normal adjacent across all the Gleason grades (** P = 0.0075) (n = 21). (C) Stratification of PRR expression over Gleason grades 6 (n = 6) and 7 (n = 13) from the same patients shows a significant increase in PRR expression in Gleason 7 (* P = 0.042) but not in Gleason 6 patients. (D) ELISA on Plasma samples from prostate cancer patients shows no significant difference in soluble Prorenin Receptor (sPRR) concentration (ng/mL) between Gleason grade groups =7 (n = 190) and >7 (n = 53) (P = 0.186). ELISA analysis shows a significant increase in sPRR concentration (* P = 0.013) (E) but not in prostate specific antigen (PSA) concentration (P = 0.112) (F) (ng/mL) in urine samples from Gleason score >7 (n = 60) compared to =7 (n = 83) patients. Data are presented as the mean ± SEM. Statistical tests conducted using Student’s t test. (G) Heat map showing the mRNA expression profile of PTEN and PRR/ATP6AP2 genes across 551 genomic data commons (GDC) of the cancer genome atlas (TCGA) prostate adenocarcinoma (PRAD) tumors, classified based on unsupervised hierarchical clustering into Gleason scores 6 (n = 51), 7 (n = 287), 8 (n = 67), 9 (n = 142), and 10 (n = 4). (H) Box and Whisker plots showing the log 2-fold differential expression of the gene PRR/ATP6AP2 across TCGA samples grouped by 6 (upper extreme = 2.30, upper quartile = 1.16, median = 0.04, lower quartile = –0.90, lower extreme = –3.09), 7 (2.26, 1.07, 0.07, –0.70, –2.71), 8 (3.07, 1.31, 0.32, –0.65, 3.15) (* P = 0.02), 9 (3.43, 1.38, 0.70, –0.29, –2.81) (** P = 0.002), and 10 (2.51, 1.29, 0.88, 0.36, –0.51) (P = 0.054) Gleason scores. Significance was determined by the Wilcoxon rank T-Test as compared to Gleason-6. (I) Kaplan-Meier plot showing less progression-free survival of TCGA prostate adenocarcinoma patients with an amplified PRR/ATP6AP2 gene set (n = 321) compared to patients with an unamplified PRR/ATP6AP2 gene set (n = 170) (** P < 0.0086). Statistical analysis was conducted using the Logrank test to compare the survival distributions of the two patient populations.