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. 2018 Oct 23;9(83):35378-35393.
doi: 10.18632/oncotarget.26237.

NOX2 oxidase expressed in endosomes promotes cell proliferation and prostate tumour development

Ian P Harrison  1 , Antony Vinh  2 , Ian R D Johnson  3 , Raymond Luong  1 , Grant R Drummond  2 , Christopher G Sobey  2 , Tony Tiganis  4 , Elizabeth D Williams  5 , John J O' Leary  6   7 , Doug A Brooks  3 , Stavros Selemidis  1   8
Affiliations

NOX2 oxidase expressed in endosomes promotes cell proliferation and prostate tumour development

Ian P Harrison et al. Oncotarget. .

Abstract

Reactive oxygen species (ROS) promote growth factor signalling including for VEGF-A and have potent angiogenic and tumourigenic properties. However, the precise enzymatic source of ROS generation, the subcellular localization of ROS production and cellular targets in vivo that influence tumour-promoting processes, are largely undefined. Here, using mRNA microarrays, we show increased gene expression for NOX2, the catalytic subunit of the ROS-generating NADPH oxidase enzyme, in human primary prostate cancer compared to non-malignant tissue. In addition, NOX4 gene expression was markedly elevated in human metastatic prostate cancers, but not in primary prostate tumours. Using a syngeneic, orthotopic mouse model of prostate cancer the genetic deletion of NOX2 (i.e. NOX2 -/y mouse) resulted in reduced angiogenesis and an almost complete failure in tumour development. Furthermore, pharmacological inhibition of NOX2 oxidase suppressed established prostate tumours in mice. In isolated endothelial cells, and in human normal and prostate cancer cells, NOX2 co-located to varying degrees with early endosome markers including EEA1, Appl1 and Rab5A and the late endosome marker Rab7A, and this correlated with significant VEGF-A-dependent ROS production within acidified endosomal compartments and endothelial cell proliferation that was NOX2 oxidase- and hydrogen peroxide dependent. We concluded that NOX2 oxidase expression and endosomal ROS production were important for prostate cancer growth and that this was required to positively regulate the VEGF pathway. The research provides a paradigm for limiting tumour growth through a better understanding of NOX2 oxidase's effect on VEGF signalling and how controlling the development of tumour vasculature can limit prostate tumour development and metastasis.

Keywords: NADPH oxidase; NOX2; endosome; prostate cancer; reactive oxygen species.

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Conflict of interest statement

CONFLICTS OF INTEREST The authors declare no potential conflicts of interest.

Figures

Figure 1
Figure 1. NOX2 and NOX4 expression in microarray database analyses of normal, primary and metastatic human prostate cancers
Vertical scatter plot of NOX2 and NOX4 expression data from the Tomlins cohort (A and D respectively) consisting of 18 non-malignant tissues, 13 prostatic intraepithelial neoplasia’s, 30 primary prostate cancer and 19 metastatic cancer tissue samples. NOX2 and NOX4 expression displayed as percentage-change from the Taylor cohort (B and E respectively) consisting 29 non-malignant and 131 primary-cancer tissue samples. Log2 median-centred ratio of NOX2 and NOX4 from the Grasso cohort (C and F respectively) consisting 59 cancer 28 normal tissue samples. *P < 0.05, **P < 0.01 and ****P < 0.0001 for Students t test (C, E and F) or one-way ANOVA (A and D).
Figure 2
Figure 2. VEGFR2 and NOX2 activity are crucial for prostate tumour growth in mice
(A) The effect of the VEGFR2 inhibitor Ki8751 (25 mg/kg/day, i.p) administered from Day 10 on prostate tumour growth in mice after 14 days (n = 8). (B) The data and (C) representative images showing the growth of prostate tumours over 14 days in WT and NOX2-/y mice (n = 8–15). For (A) and (B) the prostate weights include the prostate and any associated tumour plus seminal vesicles. (D) Representative images of the density of CD31+ cells in prostate tumours in WT mice at Day 14. Note, the image in the NOX2-/y represents the CD31+ staining in one of the larger tumours formed in the NOX2-/y mice group. Horizontal black bar represents the scale of 200mm. The graph in (D) shows the average number of CD31+ cells per field in each group. (E) Representative images of the density of VEGFR2+ cells in prostate tumours in WT mice at Day 14. Horizontal black bar represents the scale of 200 mm. The graph in (E) shows the average number of VEGFR2+ cells per field in each group. (F) Group data showing effect of apocynin (50 mg/kg/day i.p. and 500 mg/L drinking water) on tumour development at Day 14 when administered in WT mice bearing tumours at Day 10 (n = 8). Data are mean ± SEM. Students unpaired t test (A, D, F) or one-way ANOVA (B and G). *P < 0.05, **P < 0.01.
Figure 3
Figure 3. Co-location of early endosomes and VEGFR2 in the presence of VEGF is endocytosis-dependent
(A) Confocal immunofluorescent images showing EEA1 and VEGFR2 co-location within HMEC-1 in and around the DAPI-stained nucleus after 30 min incubation with either PBS, VEGF-A (30 ng/mL), VEGF-A (30 ng/mL) plus dynasore (Dyna; 100 µM) or VEGF-A (30 ng/mL) plus Pitstop 2 (Pit; 30 µM). (B) Graphs show the degree of co-location within these cells in both groups expressed as percentage of total EEA1 positive staining. Data is representative of >50 cells imaged across 4 experiments and are shown as mean ± SEM. *P < 0.05 for one-way ANOVA.
Figure 4
Figure 4. Co-location of NOX2 with endosome markers in response to VEGF-A treatment
(A–B) Confocal fluorescent images showing co-located NOX2 (red) with endosome markers (green) (A) Rab5 and (B) Rab7 in non-malignant (PNT1A) and malignant (LNCaP) human prostate cancer cells and in LNCaP post-VEGF-A treatment. (C) Graphs show the degree of co-location of NOX2 with the endosome markers Rab5 and Rab7 in VEGF-A-treated and untreated PNT1a and LNCaP cells. Data is representative of 6 randomly selected cells and are shown as mean ± SEM. *P < 0.05, **P < 0.01 for two-way ANOVA.
Figure 5
Figure 5. Co-localisation of NOX2 with Rab5-positive endosomes reduces in the presence of VEGF
(A) Confocal immunofluorescent images showing NOX2 (red) with the endosome markers Appl1, Rab5A, EEA1 and Rab7A (green) co-location within HMEC-1 cells after 30 min incubation with either PBS or VEGF-A (30 ng/mL). (B) Graph shows the degree of co-localisation (Pearson’s correlation) within the cells. Data are mean ± SEM. **P < 0.01 for Mann–Whitney unpaired t test.
Figure 6
Figure 6. VEGF stimulates endosomal superoxide production in endothelial cells via a NOX2 oxidase-dependent mechanism
(A) Confocal fluorescence images of HMEC-1 incubated with OxyBURST green for 5 min before incubating for 30 min with either PBS, VEGF-A (30 ng/mL) or VEGF-A (100 ng/mL). (B) Graphs showing the area of fluorescence and mean fluorescence per cell detected in each group (n = 5). (C) Confocal images of time course of endosomal ROS generation in HMEC-1 cells. Cells were incubated with OxyBURST green for 5 min and then either PBS for 30 min (control) or VEGF-A (30 ng/mL) for between 5 and 30 min (n = 3). (D) Confocal images of endosomal ROS generation after 30 min incubation with VEGF-A (30 ng/mL) and 30 min of incubation with either SOD (100 U/mL), apocynin (300 µM) or bafilomycin A (10 nM) followed by 30 min incubation with VEGF-A (30 ng/mL). (E) Graphs show total number of ROS-producing cells expressed as percentages of the total number of cells per group plus the mean fluorescence per ROS-producing cell (n = 3-5). (F) Confocal images of endosomal ROS generation after 30 min incubation with VEGF-A (30 ng/mL) and (G) their corresponding graphs showing total number of ROS-producing cells expressed as percentages of the total number of cells per group plus the mean fluorescence per ROS-producing cell for each group in wild-type and NOX2 knockout (NOX2-/y) mouse lung endothelial cells (MLEC cells; n = 4). (H) Extracellular H2O2 production as assessed by Amplex Red fluorescence in the absence or presence of VEGF-A (n = 5). Data are mean ± SEM. *P < 0.05, **P < 0.01 for Students unpaired t test (E and G) or one-way ANOVA (B and E).
Figure 7
Figure 7. VEGF significantly increases endothelial cell proliferation in a H2O2- dependent manner and is further dependent on endocytosis, endosomal acidification and NOX2 activity
(A–F) The proportion of HMEC-1 per well after 24 hr treatment with either PBS or VEGF-A (30 ng/mL) in the absence or presence of either (A) Dynasore (Dyna; 100 µM), (B) pitstop 2 (Pit; 30 µM), (C) bafilomycin A (Baf; 10 nM), (D) catalase (Cat; 1000 U/mL), (E) SOD (100 U/mL) or (F) apocynin (Apo; 300 µM). (G) Graph shows the effects of VEGF-A (10 and 30 ng/mL) on WT and NOX2-/- mouse lung endothelial cell proliferation after 24 hr expressed as percentages of the PBS control. Data are mean ± SEM for n = 5-7 experiments. *P < 0.05, **P < 0.01, for one-way ANOVA.
Figure 8
Figure 8. VEGF-dependent endosomal ROS production and proliferation occurs independently of NOX1
(A) Confocal immunofluorescent images of endosomal ROS production within HMEC-1 in the presence of either PBS, VEGF-A alone or VEGF-A (30 ng/mL) with ML171 (either 0.25 or 0.5 µM). (B) Graphs show total number of ROS-producing cells expressed as percentages of the total number of cells plus the mean fluorescence per ROS-producing cell for each group (n = 6). (C) The proportion of HMEC-1 per well after 24 hr treatment with either PBS alone, VEGF-A (30 ng/mL) alone or VEGF-A (30 ng/mL) plus ML171 (0.5 µM). Data are expressed as a percentage of the PBS control. Data are mean ± SEM for n = 3–6 experiments.
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