PACE4 cleaves PRR intracellularly in prostate cancer cells.

(A) Western blot analysis showing Prorenin Receptor (PRR), furin, and PACE4 expression, sPRR secretion, and cell lysate and conditioned media total lane protein (CLTLP, CMTLP) in LNCaP cells infected with a control non-target shRNA (NT), Furin shRNA (shfurin), or PACE4 shRNA (shPACE4). (B) Western Blot analysis demonstrating the expression of PRR and secretion of sPRR in LNCaP cells infected with an empty pLenti6 vector or with pLenti6-PACE4 to overexpress PACE4. (C) Quantification of sPRR levels in pLenti6 and pLenti-PACE4-infected LNCaP cells (*PD) Western blot showing the reduction in PRR processing resembled as a ratio of HA-tagged M8.9 (M8.9-HA) to HA-tagged full-length PRR (PRR-HA) in cellular extract of LNCaP cells after a 50 μM PACE4 inhibitor [33] LLLRVK-amidinobenzylamide (Amba) (C23) treatment. (E) Corresponding quantification of the ratio of M8.9-HA to PRR-HA standardized over total lane protein (TLP) (*PF) Analysis of PRR peptide cleavage by recombinant PACE4 (rPACE4) or recombinant furin (rfurin) monitored after a 2-hour incubation by high pressure liquid chromatography (HPLC). Mass spectrometry was done to confirm identity of peptide after cleavage. Cleavage site is underlined on the peptide sequence. Western blot analysis of PRR expression and sPRR secretion and quantification of sPRR secretion after DMSO (Vehicle), 50 μM multi-Leucine peptide (ML) PACE4 inhibitor, or 50 μM PEGylated cell-impermeable ML (PEG8-ML) treatment of DU145 (***PG, H) or LNCaP (I, J) (**P<0.01, n = 3) cells, respectively. Beta-Actin (β-actin) and TLP were used as loading controls. Data are presented as the mean ± SEM. Statistical tests were conducted using Student’s t test.</p

PACE4 inhibition, like PRR knockdown, reduces V-ATPase activity.

(A) Representative proliferation images of LNCaP exposed to both DMSO or a 100 μM of PACE4 inhibitor [33] LLLRVK-amidinobenzylamide (Amba) (C23) over 0 and 64 hours. (B) Western blot analysis of soluble prorenin receptor (sPRR) secretion in conditioned media of LNCaP exposed to DMSO or a 100 μM C23. (C) Representative LysoTracker images of LNCaP cells transfected with non-silencing control siRNA (NSC), PRR siRNA 1 and 2, PACE siRNA, or treated with 100 nM Bafilomycin A1. (D) Quantification of LysoTracker signal intensity relative to number of cells in brightfield images treated with 1% DMSO (Vehicle), 50 μM C23, 100 nM Bafilomycin A1 (BafA1), or transfected with PRR siPRR 1 or siPRR 2. (E) Schematic showing the PRR-HA, sPRR-HA, and M8.9-HA vectors used in panels F, G and H. (F) Representative LysoTracker images of LNCaP cells transfected with empty vector (EV) and treated with DMSO, treated with 100 μM C23, transfected with PRR-HA and treated with 100 μM C23, transfected with M8.9-HA and treated with 100 μM C23, transfected with sPRR-HA and treated with 100 μM C23, or treated with 100 nM BafA1. (G) Corresponding quantification of LysoTracker intensity relative to cell number in the same treatments (**P ≤ 0.01, ***P ≤ 0.001, **** P H) Western blot analysis of PRR and HA expression in the same treatments. Total lane protein (TLP) was used as loading control. Data are presented as the mean ±SEM. Statistical tests were conducted using Student’s t test. Scale bar measures 100.</p

PTEN controls PACE4 expression and sPRR secretion in human prostate cancer cells, and both PACE4 and PRR expressions increase in human prostate tumor tissue.

(A) Western blot analysis showing less PACE4 endogenous protein expression after adenoviral Phosphatase and tensin homolog (PTEN) infection (PTEN-Ad) in PC3 and LNCaP cells. The same cells also exhibited less Prorenin Receptor (PRR) processing and less soluble Prorenin Receptor (sPRR) secretion in conditioned media (CM). Total lane protein (TLP) was used as a loading control. Quantification of (B) PACE4 expression and (C) sPRR secretion relative to the corresponding TLP in PC3 and LNCaP prostate cancer cells (*P D) Representative IHC tissue microarray images of PTEN, PACE4, and PRR staining on prostatic intraepithelial neoplasia (PIN) tissue and tumor tissue. Scale bar measures 100 μm. Dot plots representing the mean of the qualitative score assigned to (E) PTEN, (F) PACE4, and (G) PRR staining on PIN (n = 68) and tumor (n = 105) patient tissue microarrays (*PH) The Cancer Genome Atlas (TCGA) mRNA expression data in Log2 scale of PTEN, PCSK6 (PACE4), and ATP6AP2 (PRR) in tumor (T) and normal (N) prostate tissue.</p

PTEN controls PRR processing and PACE4 protein expression in mouse.

(A) Western blot analysis of PTEN, PRR, and sPRR in homogenized WT, Pten-/-, Pten-/-/14-3-3σ-/-, and 14-3-3σ-/- mouse prostate tissue. Total lane protein (TLP) was used as a loading control. Dot plots of (B) Prorenin Receptor (PRR) and (C) PACE4 mRNA expression levels in wild type (WT), Pten-/-, Pten-/-/14-3-3σ -/-, and 14-3-3σ-/- prostate tissue (*P D) Corresponding representative IHC images of WT, Pten-/-, Pten-/-/14-3-3σ-/-, and 14-3-3σ-/- mouse prostate sections after H&E, PRR, and PACE4 staining. Scale bar measures 70 μm.</p

S1 Raw images -

Phosphatase and tensin homolog (PTEN) mutation is common in prostate cancer during progression to metastatic and castration resistant forms. We previously reported that loss of PTEN function in prostate cancer leads to increased expression and secretion of the Prorenin Receptor (PRR) and its soluble processed form, the soluble Prorenin Receptor (sPRR). PRR is an essential factor required for proper assembly and activity of the vacuolar-ATPase (V-ATPase). The V-ATPase is a rotary proton pump required for the acidification of intracellular vesicles including endosomes and lysosomes. Acidic vesicles are involved in a wide range of cancer related pathways such as receptor mediated endocytosis, autophagy, and cell signalling. Full-length PRR is cleaved at a conserved consensus motif (R-X-X-R↓) by a member of the proprotein convertase family to generate sPRR, and a smaller C-terminal fragment, designated M8.9. It is unclear which convertase processes PRR in prostate cancer cells and how processing affects V-ATPase activity. In the current study we show that PRR is predominantly cleaved by PACE4, a proprotein convertase that has been previously implicated in prostate cancer. We further demonstrate that PTEN controls PRR processing in mouse tissue and controls PACE4 expression in prostate cancer cells. Furthermore, we demonstrate that PACE4 cleavage of PRR is needed for efficient V-ATPase activity and prostate cancer cell growth. Overall, our data highlight the importance of PACE4-mediated PRR processing in normal physiology and prostate cancer tumorigenesis.</div

Full-length PRR expression increases and sPRR secretion decreases in the absence of PACE4 in mouse.

(A) Western blot analysis of prorenin receptor (PRR) and soluble prorenin receptor (sPRR) in prostate tissue extracted from wild type mice treated with saline, 2mg/Kg of PACE4 inhibitor [33] LLLRVK-amidinobenzylamide (Amba) (C23), and 4 mg/Kg C23. (B) Quantification of PRR processing in prostate tissue showing less PRR processing in prostate tissue extracted from mice treated with 2 mg/Kg and 4mg/Kg C23 (*PC) Quantitative PCR measurement of PACE4 expression in prostate, brain, and cerebellum tissue demonstrating the difference in PACE4 expression between wild type (WT) and Pace4-null (Pace4-/-) mice. (D) Western blot analysis of prostate, brain, and cerebellum tissue to analyze PRR expression in WT and Pace4-/- mice. (E) Corresponding quantification of PRR expression in the same tissues (*PF) ELISA quantification of plasma sPRR levels in WT (n = 4) and Pace4-/- (*P < 0.05, n = 8) mice. Beta-Actin (β-actin) and total lane protein were used as loading controls. Data are presented as the mean ±SEM. Statistical tests were conducted using Student’s t test.</p