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. Author manuscript; available in PMC: 2012 Mar 1.
Published in final edited form as: Cancer Res. 2011 Feb 22;71(5):1637–1646. doi: 10.1158/0008-5472.CAN-10-3674

Peroxiredoxin 1 Controls Prostate Cancer Growth through Toll-Like Receptor 4 Dependent Regulation of Tumor Vasculature†

Jonah R Riddell 1, Wiam Bshara 2, Michael T Moser 3, Joseph A Spernyak 4, Barbara A Foster 3, Sandra O Gollnick 1,‡
PMCID: PMC3076642  NIHMSID: NIHMS263538  PMID: 21343392

Abstract

In recent years a number of studies have implicated chronic inflammation in prostate carcinogenesis. However, mitigating factors of inflammation in the prostate are virtually unknown. Toll-like receptor 4 (TLR4) activity is associated with inflammation and is correlated with progression risk in prostate cancer (CaP). TLR4 ligands include bacterial cell wall proteins, danger signaling proteins, and intracellular proteins such as heat shock proteins and peroxiredoxin 1 (Prx1). Here we show that Prx1 is overexpressed in human CaP specimens and that it regulates prostate tumor growth through TLR4-dependent regulation of prostate tumor vasculature. Inhibiting Prx1 expression in prostate tumor cells reduced tumor vascular formation and function. Further, Prx1 inhibition reduced levels of angiogenic proteins such as VEGF within the tumor microenvironment. Lastly, Prx1 stimulated endothelial cell proliferation, migration and differentiation in a TLR4- and VEGF-dependent manner. Taken together, these results implicate Prx1 as a tumor-derived inducer of inflammation, providing a mechanistic link between inflammation and TLR4 in prostate carcinogenesis. Our findings implicate Prx1 as a novel therapeutic target for CaP.

INTRODUCTION

Prostate cancer (CaP) is a highly heterogeneous disease of unknown etiology (1, 2). In recent years a number of histopathological and epidemiological studies have implicated chronic inflammation in prostate carcinogenesis (3). Toll like receptor 4 (TLR4) can contribute to CaP cell proliferation and invasion (4, 5) and to CaP risk (6). Pre-clinical studies have shown that TLR4 expression is required for CaP growth (4), suggesting the presence of a tumor-derived TLR4 ligand that contributes to the maintenance of a chronic inflammatory and pro-tumorigenic environment and promotion of CaP growth.

Peroxiredoxin 1 (Prx1) is a multifunctional member of the 2-Cys subfamily of the evolutionarily conserved thiol-dependent antioxidant Prx family of enzymes (7) that is over-expressed by multiple cancers. Elevated Prx1 expression in lung and bladder cancer is associated with diminished overall survival and poor clinical outcome (8–10).

Prx1 is secreted in a non-classical fashion by stressed, activated and transformed cells, including prostate tumor cells (11–13). Extracellular Prx1 is a TLR4 ligand that stimulates the expression of pro-inflammatory cytokines from macrophages and dendritic cells (13). We hypothesize that secretion of Prx1 by prostate tumor cells leads to the generation of a pro-tumorigenic microenvironment through its interaction with TLR4. Activation of TLR4 increases VEGF expression in both cancer and normal cells (14, 15). We further predict that Prx1 enhances prostate carcinogenesis and CaP growth through TLR4 dependent induction of VEGF and augmentation of tumor vasculature. The results presented here support these hypotheses. Reduction of Prx1 expression by prostate tumor cells with shRNA inhibited tumor growth in subcutaneous CaP models. Delayed tumor growth in tumors with reduced Prx1 levels appears to be due to reduced tumor vasculature. These findings suggest that Prx1 plays a pivotal role in CaP progression by orchestrating VEGF expression and vascular network formation.

MATERIALS AND METHODS

Materials

Bovine serum albumin (BSA), insulin, and antibodies specific for β-actin were obtained from Sigma-Aldrich (St. Louis, MO). Antibodies specific for SV40 Large T Antigen, CD31 and all isotype control antibodies were purchased from PharMingen (Mountain View, CA). Antibodies against VEGF were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against NG2 were obtained from R&D Systems (Minneapolis, MN). Antibodies specific for Prx1 were obtained from Lab Frontier (Seoul, South Korea) (13). Sunitinib malate was purchased from Selleck Chemicals LLC (San Francisco, CA). The VEGF121 expression construct was a gift from Douglas Fraker, MD (University of Pennsylvania, PA) (16). The MyD88DN expression vector was a gift from Stuart Calderwood, PhD (Harvard Medical School, Boston, MA) (17).

TMA

Tissue microarrays (TMA) were constructed by the Pathology Resource Network at Roswell Park Cancer Institute. Tissue cores (0.6 mm) from 92 formalin-fixed paraffin embedded donor blocks of prostatic adenocarcinoma, each representing a different case. Controls include cores of normal prostate tissue taken from each case of CaP. The TMA contains an unequal number of samples of each tumor grade and reflects the patient population at RPCI.

The TMA was stained with antibodies specific for β-cytokeratin, p63, racemase and Prx1. Scoring of Prx1 expression was done in a non-continuous (semiquantitative) scale and was determined for both the percentage of cells positive for Prx1 and intensity of epithelial staining. Overall Prx1 expression was grouped as follows: 1, 0–5%; 2, 6–25%; 3, 26–50%; 4, 51–75%; 5, 75–100%. The epithelial staining intensity was ranked on a relative scale 1–4 with 4 being the greatest staining intensity. To overcome the intra-observer variability the score was done twice and compared.

Cell Lines

The murine CaP cell line C2H was maintained as described in the presence of 10−8 M dihydrotestosterone (Sigma Chemical Co., St. Louis MO) at 37oC and 10 % CO2 (18). The human CaP cell line, PC-3M, was obtained from ATCC (Rockville, MD) and cultured according to the company’s directions. Retroviral short hairpin RNA expression constructs specific for Prx1 expression (shPrx1) and non-specific (scramble) were used to reduce Prx1 expression in PC-3M and C2H cell lines (9, 19). Stable scramble/shPrx1 cell lines were maintained in the presence of G418 (CellGro, Manassas, VA). The VEGF cDNA was obtained from Dr. D. Fraker (Univ. of Penn., Philadelphia, PA; (16)) and sub-cloned into pcDNA3.1/hygro (Invitrogen). Stable shPrx1_VEGF PC-3M cell lines were maintained in G418 and hygromycin.

Human Umbilical Vein Endothelial Cells (HUVECs) were cultured in supplemented endothelial growth media (Promega, Madison, WI) at 37oC and 5 % CO2. Murine endothelial cells, 2H-11, were cultured in DMEM supplemented with 10% defined FBS at 37oC and 10 % CO2.

Cells were transfected with vectors encoding a MyD88 dominant negative (DN) cDNA (20) using FuGENE6 (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol.

Animals

C57BL/6J (TLR4+/+) and C57BL/6ScNtlr4lps−del/JthJ (TLR4−/−) pathogen-free mice were purchased from The Jackson Laboratory (Bar Harbor, ME). C57BL/6N (TLR4+/+) were purchased from the National Cancer Institute; no difference in C2H scramble tumor growth was observed in B6 mice purchased from either source (data not shown). Severe combined immunodeficiency (SCID) pathogen-free mice were purchased through the Laboratory Animal Resource of RPCI. Transgenic adenocarcinoma of mouse prostate (TRAMP) mice (21) were bred, maintained, and housed at RPCI. The RPCI Institutional Animal Care and Use Committee approved both animal care and experiments.

Immunohistochemistry

Immunohistochemical analyses of murine tissue included staining tissues with antibodies specific for Prx1 (10 μg/mL, Lab Frontier) and SV40 T Ag (1.25 μg/mL, Pharminogen) expression in TRAMP prostatic tissues (22). Briefly, prostatic tissues (collected at necropsy) were fixed in 10% (vol/vol) buffered formalin and sections (5.0 μm) were cut from paraffin-embedded tissues. Slides/tissues were incubated overnight with primary antibodies. Secondary antibodies were incubated with tissues for 2 hours. Products were visualized with the chromogen 3′3′-diaminobenzidine tetrahydrochloride (DAB; Sigma). The cell nuclei were counterstained with hematoxylin.

PC-3M scramble/shPrx1 tumors (150 mm3) were harvested, fixed in zinc-formalin (R&D Systems), and paraffin embedded. CD31 and Prx1 expression in PC-3M scramble/shPrx1 tumors was performed by the immunohistochemistry facility at Roswell Park Cancer Institute as previously described (23).

Assessment of Tumor Growth

Tumor growth was monitored by measuring the orthogonal diameters of tumors once every three days with calipers (24). The tumor volume, V, was calculated with the formula V=(lw2/2), were l is the longest axis of the tumor and w is the axis perpendicular to l.

Magnetic Resonance Imaging

Microvascular assessment of PC-3M scramble and shPrx1 tumors (150mm3) was carried out using dynamic contrast, magnetic resonance imaging (MRI). A multi-slice, T2-weighted fast spin echo (TE/TR=40/2500ms) scan was acquired in the axial orientation to provide an anatomical reference for tumor volume and localization. Three pre-contrast T1 relaxation rate measurements of the tumors were carried out using a saturation recovery, fast-spin echo scan with variable repetition times ranging from 360 to 6000 ms. Human serum albumin-(Gd-DTPA)35 (Center for Pharmaceutical and Molecular Imaging, University of California, San Francisco) was then injected intravenously via the tail vein at a dose of 0.1mmol/kg. Serial T1 measurements were then carried out for up to 45 minutes after injection.

MR datasets were converted into the Analyze 7.5 format and regions of interest (ROI) were defined for the tumor and kidneys using Analyze 7.0 (AnalyzeDirect, Overland Park, KS). Mean signal intensity at each repetition time was determined within each ROI and T1 relaxation rates (R1) were calculated by nonlinear fitting of the equation: S(TR) = SMAX (1−e −(R1 • TR)), using Matlab’s Curve Fitting Toolbox (Matlab 2008a, MathWorks Inc., Natick, MA). The increase in R1 (ΔR1) was calculated by subtracting the average baseline R1 from each post-injection value. All ΔR1 values were corrected by dividing by the ΔR1 value of the kidney to account for injection variability. Normalized tumor ΔR1 values were plotted against time after injection, and a linear regression curve was calculated to yield the relative permeability (slope) and fractional blood volume (intercept)(25).

Endothelial Cell Proliferation, Migration, and Differentiation

Endothelial cell proliferation assays were performed as described (26). In indicated experiments VEGFR inhibitor (sunitinib malate) or blocking antibodies for VEGF (4 μg/mL) were added 2 hours prior to stimulation with rPrx1 or cultured supernatants from PC-3M scramble/shPrx1 cells. MyD88DN transfection was performed 24 hours prior to stimulation. Proliferation was determined 24 hours after stimulation by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (27).

Endothelial cell migration assays were performed by coating 24-well plates with 50 μl of collagen per well (Roche, Nutley, NJ) mixed with rPrx1. Endothelial cell media was added to the bottom of the collagen coated well and HUVEC or 2H-11 cells (5 × 105) were placed in the top of a 3.0 μm pore transwell. The number of migrated cells was determined after 24 hours by trypan blue staining.

To assess the role of Prx1 in the recruitment of vessels in vivo, Matrigel (BD Bioscience) containing rPrx1 was injected subcutaneously into SCID mice. Eight days later mice were euthanized and the hemoglobulin content present in the Matrigel plugs was determined (SIGMA).

Endothelial differentiation assays were performed according to Godoy et al (26). Total tube length per image was quantified using Optimas 6.2 (Media Cybernetics, Inc., Bethesda, MD). Graphs represent a summation of the lengths of all individual tubular structures.

Molecular Analyses

Mouse and human ELISA kits specific for mouse and human VEGF were purchased from R&D systems (Minneapolis, MN). The mouse/human Prx1 ELISA kit was purchased from Northwest Life Science Specialties, LLC (Vancouver, WA). Assays were completed according to manufacturers’ instructions.

Statistical Analysis

Statistical analyses were performed using a standardized Student’s t-test with Welch’s correction. For tumor growth analysis, hours-to-400 mm3 tumor volume, was calculated for each animal by linearly interpolating between the times just before and after this volume was reached, using log (tumor volume) for the calculations (28). Tumor responses between groups were compared using the Kaplan-Meier analysis. Event curves were compared using the log-rank test, which calculated a two-tailed P value testing the null hypothesis that the curves were identical. Differences were considered significant when P values were ≤ 0.05.

RESULTS

Prx1 is Elevated in CaP

Peroxiredoxin 1 (Prx1) expression in human CaP was determined utilizing a human CaP tissue microarray (TMA). Tumor grade present in each core was determined by analysis of β-cytokeratin, p63 and racemace expression (29) (Triple stain; Figure 1A). Both overall Prx1 (Figure 1B) and epithelial cell expression of Prx1 (Figure 1C) increased in malignant prostatic cancer samples with respect to normal tissue. Prx1 expression was increased at the earliest tumor grade examined when compared to expression in normal tissue.

Figure 1. Prx1 is elevated in CaP.

Figure 1

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(A) Representative samples of benign/normal and malignant human prostate tissue samples from a human prostate TMA stained with antibodies specific for basal cells (high MW cytokeratin and p63–brown color), secretory epithelial cells (racemace–pink color), and Prx1 are shown. (B) Prx1 expression is plotted vs. tissue grade: N= normal (n=110), T3=hyperplasia (n=49), T4 = dysplasia accompanied by invasion basal layer (n=19), T5=fully invasive carcinoma (n=2). The numbers in parentheses indicate the number of normal samples and samples/tumor grade present in the TMA. (C) The epithelial cell staining intensity is plotted vs. tissue grade. Error bars represent mean expression ± SEM; ** = P≤0.01 *: P≤0.05.. (D) Representative samples of WT lateral prostate and TRAMP anterior/lateral prostate stained with H&E or antibodies specific for SV40 T antigen (Ag) or Prx1 are shown at 4x magnification; Prx1 expression is also shown at 14x magnification. WT, anterior prostate w/transformed foci and intermediate CaP tissues were harvested from 8–10 week old mice and the late stage TRAMP tumor tissue was taken from a tumor-bearing 15 week old TRAMP mouse.

Prx1 expression levels also increase during CaP progression in the TRAMP murine model of CaP. Normal prostates from (C57BL/6 x FVB)F1 (WT) mice express minimal levels of Prx1 (Figure 1D). Prx1 expression is increased in foci of transformed cells and is further increased in intermediate and late stage CaP tissues.

Prx1 Regulation of CaP Growth is TLR4 Dependent

The role of Prx1 in CaP was evaluated in two subcutaneous models of CaP, human PC-3M and murine C2H. PC-3M and C2H cell lines were transfected with vectors engineered to express Prx1 specific shRNA (shPrx1), which resulted in a 50% reduction in Prx1 expression as compared to cells expressing non-specific (scramble) shRNA (Figure 2A/B). PC-3M shPrx1 tumor growth was delayed and growing tumors exhibited slower growth rate when compared to PC-3M scramble tumors (1.90 ± 0.16 mm3/day vs. 21.2 ± 2.02 mm3/day; P 0.0001; Figure 2C). C2H shPrx1 tumors did not grow (Figure 2D). To confirm that Prx1 control of CaP tumor growth was specific, Prx1 expression was restored in shPrx1 tumor cells by expression of shRNA resistant Prx1. Expression of shRNA resistant Prx1 (sRP) restored Prx1 expression and tumor growth, indicating that Prx1 specifically regulates CaP growth (Figure 2A). Prx1 can regulate cell proliferation/apoptosis through its peroxidase/chaperone activities (7); however, PC-3M shPrx1 tumors did not demonstrate decreased numbers of proliferating cells or increases in apoptosis (data not shown).

Figure 2. Prx1 regulates prostate tumor growth in a TLR4 dependent manner.

Figure 2

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(A) Prx1 levels were measured by ELISA in supernatants of PC-3M prostate tumor cells expressing scramble or Prx1 specific shRNA (NT: not transfected) or in cells transfected with cDNA encoding shPrx1 resistant Prx1 (sRP) or a vector control (VC). Results are presented as the mean of three experiments ± SEM; **: P≤0.01. (B) Prx1 levels present in stable transfectants of C2H prostate tumor cells expressing nonspecific shRNA (scramble) or Prx1 specific shRNA (shPrx1) were determined by western analysis. Blots were analyzed, normalized to α-tubulin and are presented as relative expression of Prx1. Error bars represent SEM; n=5. **: P≤0.01 (C) The growth of PC-3M tumors expressing nonspecific shRNA (scramble), shRNA specific for Prx1 (shPrx1), or shRNA specific for Prx1 and shPrx1 resistant Prx1 (shPrx1_sRP) as a function of time is shown; each line represents tumor growth in an individual mouse; n=10. (D) The growth of C2H tumors expressing nonspecific shRNA (scramble) or shRNA specific for Prx1 (shPrx1) in C57BL/6 (TLR4+/+) and C57BL/6ScNtlr4lps−del (TLR4−/−) mice as a function of time is shown; each line represents tumor growth in an individual mouse, n=8.

To examine the role of TLR4 in mediating Prx1 control of tumor growth, C2H scramble cells were injected into TLR4 signaling deficient (TLR4−/−) mice. Tumor growth was delayed in TLR4−/− mice (Figure 2D), suggesting that Prx1 promotion of C2H tumor growth is TLR4 dependent. Cumulatively, these studies support the hypothesis that Prx1 regulation of CaP is an in vivo phenomenon that depends on host expression of TLR4.

Prx1 Regulates Tumor Vasculature Formation and Function

PC-3M shPrx1 tumors have decreased numbers of vessels and overall vascular area as compared to control (scramble) tumors (Figure 3A/C/D). The vascular area per vessel, or the average size of each vessel, is increased in shPrx1 tumors (Figure 3E), which correlates with the presence of large vessels and minimal microvasculature (Figure 3A).

Figure 3. Prx1 regulates tumor vasculature formation.

Figure 3

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PC-3M tumors expressing either scramble or Prx1 specific shRNA were harvested when tumors growth reached 150 mm3. (A) Tumors sections were stained with antibodies specific to Prx1 or CD31. A representative sample is shown for each tumor. (B) Prx1 overall staining intensity; (C) the number of vessels/field or (D) the overall vascular area (CD31+ area) was determined for each tumor. (E) Vessel size was extrapolated by measuring the MOD (mean optical density) of CD31 expression/vessel. Each symbol represents an individual tumor; lines represent the mean. **: P≤0.01.

Co-localization of pericytes and endothelial cells is a marker of mature vasculature and an indication of vessel function (30). The majority of endothelial cells (CD31+) and pericytes (NG2+) are co-localized in PC-3M scramble tumors (Figure 4A). Endothelial cells and pericytes are also co-localized in the limited number of vessels present in PC-3M shPrx1 tumors, suggesting that although vasculature is limited in shPrx1 tumors, the vasculature that is present is mature. Reduction of Prx1 expression in PC-3M tumors leads to reduced vascular permeability, which was determined using dynamic contrast MRI (Figure 4B/C). In summary, these results suggest that Prx1 expression contributes to tumor vasculature formation and function.

Figure 4. Prx1 regulates tumor vasculature function.

Figure 4

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(A) Scramble and shPrx1 PC-3M tumor sections from 150 mm3 tumors were stained with antibodies specific for CD31 or NG2. Representative individual or merged images from 5 sets of tumors are shown. (B) Tumor bearing mice were subjected to MRI as described in the Materials and Methods section. Representative scans of tumors at 10 min and 45 min are shown. Increased vascular permeability in scramble tumors is evidenced by the increase in T1 rates from 10 to 45 minutes in the scramble tumors (reduction in blue areas). Minimal changes in T1 relaxation rates are seen in shPrx tumors. (C) Relative vascular permeability was determined as described in Materials and Methods; results represent the mean ± SEM. Five tumors were analyzed for each group. * indicates P≤.05.

Prx1 Regulation of VEGF Expression is TLR Dependent

Analysis of angiogenic protein expression in PC-3M tumor lysates showed that shPrx1 tumors contained reduced levels of VEGF, TNFα, IL-6, TGFα and TGFβ (data not shown). Further analysis showed that total host (mouse) and tumor derived (human) VEGF levels were decreased in shPrx1 tumors (Figure 5A). Total VEGF consists of a number of isoforms, the predominant of which are VEGF121 and VEGF165. The ratio of VEGF121/VEGF165 is a prognostic factor of malignancy in clinical and preclinical models of CaP (31). The ratio of VEGF121/VEGF165 is decreased in shPrx1 tumors and cells (Figure 5B). Restoration of VEGF expression by PC-3M shPrx1 cells restores tumor growth (Figure 5C).

Figure 5. Prx1 regulation of VEGF expression is TLR dependent.

Figure 5

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(A) PC-3M scramble (n=5) and shPrx1 (n=5) tumors were harvested at 150 mm3. Tumor lysates were tested for the presence of either mouse (host) or human (tumor) VEGF by ELISA. Each symbol represents a separate tumor; lines represent the mean expression. ** indicates P≤0.01. (B) PC-3M scramble and shPrx1 tumor lysates (In vivo) and cell lysates (In vitro) were separated by electrophoresis and immunoblotted for VEGF or β-actin. Quantification of VEGF121 and VEGF165 expression levels is shown for both tumor (n=5) and cell (n=6) samples. In order to determine the ratio shown, quantization for VEGF165 was performed on a longer exposure blot. (C) The growth of PC-3M tumors expressing nonspecific shRNA (scramble) or shRNA specific for Prx1 (shPrx1) as a function of time is shown. Also shown is the growth of shPrx1 PC-3M tumors in which VEGF levels have been restored by transfection of shPrx1 PC-3M cells with a cDNA encoding for VEGF (shPrx1_VEGF); each line represents tumor growth in an individual mouse; n=10. (D) VEGF levels in supernatants from PC-3M scramble, shPrx1, and sRP cells, with (My88DN) or without (NT) simultaneous expression of MyD88DN, were determined by ELISA. sRP cells are shPrx1 cells expressing a shPrx1 resistant Prx1. Three independent trials were performed; error bars represent SEM. ** indicates P≤0.01. (E) VEGF levels in supernatants collected from peritoneal elicited macrophages isolated from either C57BL/6 (TLR4+/+) or C57BL/6ScNtlr4lps−del (TLR4−/−) and treated with rPrx1 were measured by ELISA. Three independent trials were performed; error bars represent SEM. ** indicates P≤0.01.

VEGF secretion is reduced in PC-3M scramble cells expressing MyD88DN (Figure 5D), suggesting that Prx1 induction of VEGF is mediated by interaction with TLR4 and dependent upon MyD88. This finding is supported by results showing that extracellular addition of recombinant Prx1 (rPrx1) or restoration of Prx1 by expression of a shPrx1 resistant Prx1 (sRP) stimulated VEGF expression in shPrx1 cells. The induction of VEGF by rPrx1 or sRP was abrogated by the expression of MyD88DN (Figure 5D). VEGF secretion from macrophages was also stimulated by incubation with rPrx1 and was abrogated in macrophages derived from TLR4−/− mice, confirming the TLR4 dependence of Prx1 regulation of VEGF (Figure 5E).

Prx1/TLR4 Interaction Regulates Endothelial Cell Proliferation, Migration, and Differentiation

VEGF mediates angiogenesis partly through its effects on endothelial cell proliferation, migration, and differentiation (32). To determine if Prx1 influences endothelial cell proliferation, endothelial cells were cultured in the presence or absence of rPrx1. Addition of rPrx1 to endothelial cells increased their proliferation (Figure 6A). Conditioned media (CM) from scramble PC-3M, but not CM from shPrx1 PC-3M cells also increased the proliferation of endothelial cells (Figure 6A). Inhibition of TLR4 signaling by expression of MyD88DN in endothelial cells ablated the proliferative effects of Prx1 (Figure 6A). Similar results were observed with C2H CM (data not shown).

Figure 6. Prx1/TLR4 interaction regulates endothelial cell proliferation, migration, and differentiation.

Figure 6

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(A) Proliferation of HUVEC cells transfected with either control vector (VC) or MyD88DN and treated with rPrx1, media (Media) or CM from PC-3M scramble or shPrx1 cells is presented as fold proliferation over proliferation observed in untreated cells. Results are representative of four individual experiments performed; error bars represent SEM. ** Indicates P≤0.01. (B) HUVEC cells were treated with Media, 20 nM rPrx1, peroxidase-null recombinant Prx1 (rC52S) or chaperone-null recombinant Prx1 (rC83S). Results are presented as fold proliferation over proliferation observed in untreated cells (Media). Error bars represent SEM; n=4. (C) Hemoglobin content per mg Matrigel isolated 8 days after s.c. injection into SCID hosts is shown. Error bars represent SEM; n=3. (D) Migration of HUVEC cells transfected with either control vector (VC) or MyD88DN and treated with 20 nM rPrx1 or CM from PC-3M scramble cells was measured as described in Materials and Methods. Results are representative of four individual experiments; error bars represent SEM. (E) Differentiation of HUVEC cells transfected with either control vector (VC) or MyD88DN and treated rPrx1 was determined as described in Materials and Methods. Results are presented as an average of total μm of tubules/field and are representative of four individual experiments; error bars represent SEM. *: P≤0.05; ** Indicates P≤0.01.

To test whether Prx1 control of endothelial cell proliferation is dependent on its chaperone or peroxidase activity, endothelial cells were incubated with wild type (Prx1), peroxidase-null (Prx1C52S), or chaperone-null (Prx1C83S) Prx1. Proliferative effects on endothelial cells were peroxidase independent and chaperone/structure dependent (Figure 6B), supporting the conclusion that Prx1-TLR4 interaction promotes endothelial cell proliferation.

A Matrigel model was utilized to address the effects of Prx1 on endothelial cell recruitment or migration in an in vivo setting. Prx1 augmented recruitment of endothelial cells into Matrigel in vivo (Figure 6C). Transwell assays were performed with rPrx1 or PC-3M scramble/shPrx1 CM to further evaluate regulation of endothelial cell recruitment and migration. Addition of rPrx1 and CM from scramble PC-3M cells but not CM from shPrx1 PC-3M cells, led to increased endothelial cell migration. Migration was inhibited by endothelial cell expression of MyD88DN (Figure 6D). Incubation of endothelial cells with Prx1 led to a dose dependent increase in tubule formation that was abrogated by endothelial cell expression of MyD88DN (Figure 6E).

Prx1 Regulation of Endothelial Cell Proliferation Depends on VEGF

To address if Prx1 regulation of endothelial cell proliferation was dependent on VEGF, endothelial cells were incubated with PC-3M scramble CM in the presence of blocking antibodies specific to VEGF or control antibodies. The proliferative effects of PC-3M scramble CM on HUVECs was reduced when VEGF activity was blocked (Figure 7A). This finding was confirmed by pre-incubation of endothelial cells with suntinib malate, a tyrosine kinase inhibitor that inhibits VEGF signaling (33)(Figure 7B).

Figure 7. Prx1 regulation of endothelial cells is VEGF dependent.

Figure 7

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(A) HUVEC cells were treated with CM from PC-3M scramble cell in the presence of either control antibodies (IgG) or antibodies specific to VEGF (VEGF); proliferation was measured 24h later. Results are presented as fold proliferation over proliferation observed in untreated cells (Media). Error bars represent SEM; n=3. (B) HUVEC cells were pre-incubated with sunitinib malate before stimulation with media or PC-3M scramble CM. Cell proliferation was determined by MTT. Results are presented as fold proliferation over proliferation observed in cells treated with PC-3M scramble CM; error bars represent SEM. (C) 2H-11 cells were treated with control (IgG) or Prx1 depleted PC-3M CM. Cell proliferation was determined by MTT. Results are presented as fold proliferation over proliferation observed in untreated cells (Media); error bars represent SEM; n=3. ** Indicates P≤0.01.

PC-3M scramble CM contains tumor-derived VEGF, which could mediate the proliferative effects on endothelial cells independent of Prx1 interaction with TLR4. To confirm PC-3M secreted Prx1 was directly inducing endothelial cell proliferation, Prx1 was depleted from scramble CM. Prx1-depleted scramble CM failed to stimulate endothelial cell proliferation (Figure 7C). Thus, tumor cell derived VEGF does not appear to be sufficient to drive endothelial cell proliferation.

DICUSSION

Our results show that Prx1 expression correlates with CaP progression and that Prx1 expression is critical for CaP growth. Reduction in tumor expression of Prx1 led to reduced tumor vasculature formation and function. Investigation into the mechanism of Prx1 control of CaP showed that Prx1 stimulates the expression of VEGF in both tumor and host cells in a TLR4 dependent manner. Tumor cell secreted Prx1 stimulates TLR4 and VEGF dependent endothelial cell proliferation, migration and differentiation. These findings suggest that tumor secreted Prx1 interacts with TLR4 to promote a pro-angiogenic tumor microenvironment that is required for tumor growth. TLR4 activation has previously been linked to angiogenesis (14, 15) and increased CaP progression (4, 34, 35), however to our knowledge this is the first identification of a tumor derived TLR4 ligand that promotes CaP growth by enhancing angiogenesis. Tumor derived high mobility group B1 (HMGB1), which interacts with TLRs 2 and 4, can stimulate angiogenesis, but these effects are attributed to its interaction with the receptor for advanced glycation end products (RAGE) and not TLR (36).

PC-3M tumor cells with reduced Prx1 appear to undergo a period of adaption in vivo that results in tumor growth delay and slower growth kinetics. Over-expression of VEGF in these tumor cells reduces the tumor growth delay, but does not completely eliminate the period of adaptation. These results suggest that reduction of VEGF is not the sole reason for the limited formation of tumor vasculature in the shPrx1 expressing tumors. Prx1 reduction also leads to lower expression of other angiogenic factors including IL-6 and TGF-β, which can also influence angiogenesis in CaP (37).

Numerous studies have implicated bacteria-associated immune responses as mediators of prostate carcinogenesis (3, 38). This hypothesis is supported by studies showing that TLR4 can contribute to CaP cell proliferation and invasion (4, 5) and to CaP risk (6). However not all incidences of CaP are associated with a prior pathogen infection, suggesting the presence of a host-derived TLR4 ligand that contributes to the maintenance of a chronic inflammatory and pro-tumorigenic environment, initiation of an angiogenic switch and promotion of CaP growth. Our findings indicate that Prx1 expression is increased at the time of transformation, suggesting that Prx1 may be a link between inflammation and prostate carcinogenesis.

The role of Prx1-TLR4 interaction in CaP in our experiments was largely demonstrated by inhibition of MyD88 signaling. All TLRs, except TLR3, signal through MyD88 (39) and genetic variations of TLR1, TLR6, and TLR10 resulting in increased activity are associated with increased prostate cancer risk (40). Therefore, the effects of Prx1 on CaP growth may also be due to interactions with these receptors. However, VEGF secretion in response to Prx1 was lost in the absence of TLR4 signaling and our previous study showed that Prx1 appears to interact with solely TLR4 (13).

Prx1 was first identified as a tumor suppressor (41, 42) because Prx1 deficient mice have increased susceptibility to sarcomas and blood malignancies (43). However, Prx1 expression is increased in numerous cancers and is correlated with poor prognosis (8, 12, 44–50). Our findings indicate that Prx1 is elevated during CaP progression.

The tumor suppressive activity of Prx1 was attributed to its intracellular interactions with the known tumor suppressors, c-myc and PTEN (41, 51) and is associated with its intracellular peroxidase activity. Our studies indicate that promotion of CaP by Prx1 is due to its extracellular chaperone activity and interaction with TLR4. Thus the role of Prx1 in carcinogenesis may depend upon its location and activity. In this scenario, Prx1 acts as a tumor suppressor by controlling the activity of PTEN and c-myc prior to transformation. Upon transformation Prx1 is secreted and interacts with TLR4 to promote a pro-tumorigenic microenvironment, stimulate angiogenesis and tumor growth.

Prx1 may provide a new therapeutic target for treatment of CaP. Prx1 interaction with TLR4 and subsequent regulation of angiogenesis and VEGF production is dependent on its chaperone activity and/or decameric structure (13). Targeted inhibition of Prx1 decameric structure/chaperone activity with small molecule inhibitors may reduce chronic inflammation, inhibit VEGF expression and limit angiogenesis in prostate carcinogenesis.

Acknowledgments

Drs. James Mohler and Saroop Sing (Department of Urology) graciously provided the analysis program used for quantization of CD31 expression. Dr. David Gold, Department of Bioinformatics and Biostatistics, University at Buffalo assisted in the statistical analysis of the TMA.

Footnotes

†

This work was supported by NIH Grants CA109480 (SOG), CA98156 (SOG), CA095367 (BAF), CA111846 (BAF) and in part by the Roswell Park Cancer Center Support Grant CA16056.

Conflict of Interest

No potential conflicts of interest

References

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