Peroxiredoxin IV plays a critical role in cancer cell growth and radioresistance through the activation of the Akt/GSK3 signaling pathways

Abstract

High levels of redox enzymes have been commonly observed in various types of human cancer, although whether and how the enzymes contribute to cancer malignancy and therapeutic resistance have yet to be understood. Peroxiredoxin IV (Prx4) is an antioxidant with bona fide peroxidase and molecular chaperone functions. Here, we report that Prx4 is highly expressed in prostate cancer patient specimens, as well as established prostate cancer cell lines, and that its levels can be further stimulated through the activation of androgen receptor signaling. We used lentivirus-mediated shRNA knockdown and CRISPR-Cas9 based KO techniques to establish Prx4-depleted prostate cancer cells, which showed delayed cell cycle progression, reduced rate of cell proliferation, migration, and invasion compared to control cells. In addition, we used proteome profiler phosphokinase arrays to identify signaling changes in Prx4-depleted cells; we found that loss of Prx4 results in insufficient phosphorylation of both Akt and its downstream kinase GSK3α/β. Moreover, we demonstrate that Prx4-depleted cells are more sensitive to ionizing radiation as they display compromised ability to scavenge reactive oxygen species and increased accumulation of DNA damage. In mouse xenograft models, we show depletion of Prx4 leads to significant suppression of tumor growth, and tumors formed by Prx4-depleted cells respond more effectively to radiation therapy. Our findings suggest that increased levels of Prx4 contribute to the malignancy and radioresistance of prostate cancer through the activation of Akt/GSK3 signaling pathways. Therefore, strategies targeting Prx4 may be utilized to potentially inhibit tumor growth and overcome radioresistance in prostate cancer.

Keywords: antioxidant; peroxiredoxin; prostate cancer; radioresistance.

Figure 1

Evaluation of PRDX4 gene alterations, transcript levels, and their correlation with prognosis in patients with all types of tumors through meta-analysis of multiple existing datasets as of April 2022.A, top 10 studies with high frequency of PRDX4 gene alterations. Data were summarized from a total of 145,223 patients/150,839 samples in 285 studies (cBioPortal). ∗Prostate cancer studies and citation sources are #1: metastatic prostate adenocarcinoma (59); #2: prostate adenocarcinoma (23); #3: metastatic prostate adenocarcinoma (24); #5: metastatic prostate cancer (25); #6: the metastatic prostate cancer project (22). Other cancer studies and citations sources are #4: pan-cancer analysis of whole genomes (60); #7: esophageal carcinoma (TCGA, Firehose Legacy); #8: sarcoma (TCGA, Firehose Legacy); #9: esophageal carcinoma (61); #10: diffuse large B-cell lymphoma (TCGA, PanCancer Atlas). Amplification indicates a high-level gain of more than a few copies of the gene. Deep deletion indicates a deep loss, possibly a homozygous deletion. B, the levels of PRDX4 gene transcript in various stages of prostate cancer compared with normal prostate. Data were obtained from the TCGA RNA-Seq data (compared with control, ∗p < 0.05, two-way ANOVA). C the levels of PRDX4 gene transcript is higher in patients with AR amplification than those without AR amplification. Data were obtained from the TCGA RNA-Seq data (∗p < 0.05, unpaired Student’s t test). D and E, amplification of PRDX4 gene in prostate cancer patients correlates with shorter time in the duration of disease-free survival (D) and overall survival (E). Altered group in red indicates patients with PRDX4 amplification; unaltered group in blue indicates patients without PRDX4 amplification.

Figure 2

Examination of Prx4 protein levels in patient specimens of prostate cancer by tissue microarray or established prostate cancer cell lines by Western blotting.A, tissue microarray slides and representative images of anti-Prx4 staining of prostate normal and adenocarcinoma. Positive anti-Prx4 staining shows in dark brown and counter staining for nuclear shows light blue. The scale bars represent 5 mm (whole slide), 500 μm (individual tumor), and 100 μm (zoomed in). B and C, anti-Prx4 staining intensity were quantitatively scored by board-certified pathologist and data were compared between prostate normal (n = 50) and adenocarcinoma (n = 66) (B), as well as between prostate normal and cancer with different levels of Gleason grading (C). D, examination of Prx4 levels in prostate derived nontumorigenic or tumor cell lines by Western blotting. The bar graph on the right shows quantitative data from three independent blots. ∗ p < 0.05 by two-way ANOVA. Prx, peroxiredoxin.

Figure 3

The expression of Prx4 is activated by AR signaling in both cell culture and mouse models.A, indicated cell lines were treated with or without increasing doses of R1881 to activate AR signaling. PSA is a known positive control of AR activation. The bar graph on the right shows quantitative data from three independent blots. B, tumor growth curve and final tumor weight (C) in mice receiving LNCaP cells without (Sham) or with surgically removal of both testicles (Castration). D, lysates from mouse xenograft tumors were subjected to Western blot to examine the levels of Prx4, PSA, and AR. Results of duplicates from each lysate of individually numbered mouse were shown. The bar graph on the right shows quantitative results of (D). In all bar graphs, ∗ p < 0.05 (two-way ANOVA). AR, androgen receptor; Prx, peroxiredoxin; PSA, prostate-specific antigen.

Figure 4

Depletion of Prx4 in prostate cancer cell lines inhibits cell cycle progression, proliferation, migration, and invasion.A, depletion of endogenous Prx4 in LNCaP and DU145 cell lines by stably expressing of shRNA targeting PRDX4 coding region. Representative images of Western blots with duplicate samples are shown and quantitative results are shown on the right. B, depletion of endogenous Prx4 in LNCaP and DU145 cell lines using the CRISPR-Cas9 techniques and quantification are shown on the right. C and D, cell cycle analysis by flow cytometry. Cells cultured with serum starvation for 24 h and treated with serum-containing medium. After 16 or 24 h, the percentage of cell in G0/G1, S, and G2/M phased were determined using PI staining. The bar graph on the right shows quantitative results of triplicate samples. E, KO of Prx4 inhibits the cell proliferation. Same number of cells were seeded into 96-well plate and cell number is determined using CCK8 assay for consecutive 8 days. (F,) loss of Prx4 inhibits cell migration in wound healing assay. ∗p < 0.05 by two-way ANOVA. G and H, cell movement tracking analysis using CellTracker software. Data (velocity and moving distance) in bar graph were obtained from more than 50 cells tracked in each group. Examples of cell moving routes were shown in the right panel. I, Matrigel invasion assay. Cells were seeded in plain medium in upper chamber and medium containing 10% FBS was added into bottom chamber as chemoattractant. Invaded cells were stained using crystal violet and quantitated with ImageJ software. The scale bars in (F) and (I) represent 1 mm. ∗In all graphs, compared with control, p < 0.05 (one-way ANOVA). FBS, fetal bovine serum; PI, propidium iodide; Prx, peroxiredoxin.

Figure 5

Identification and validation of kinase signaling changes in Prx4-depleted cells.A, proteome profiler human phosphokinase arrays using LNCaP-shNT and LNCaP-shPrx4 cell lysates. Framed spots indicated the levels of phosphorylated GSK3α/β and phosphorylated Akt in original (top) and zoomed in images (bottom). Bar graph on the right shows quantification results of duplicate samples. B, validation of findings in (A) by Western blotting. Cells were serum starved overnight and replenished with fresh medium for different time period, and cell lysates were collected for Western blotting. Quantification results were shown in right. ∗In all bar graphs, compared with control, p < 0.05 (one-way ANOVA). Prx, peroxiredoxin.

Figure 6

Depletion of Prx4 sensitizes prostate cancer cell to radiation treatment.A and B, cells were treated with increasing doses of ionizing radiation and then clonogenic assay was performed. Quantification results were shown on the right. C, cells were exposed to increasing concentration of H₂O₂ for 30 min and then rinsed with culture medium. Cells ability to scavenge H₂O₂ was then measured by using the DCF-DA assay. D, Western blotting shows the restoration of Prx4 expression in Prx4 KO cells with stably expression of Prx4Flag. E, indicated cell lines were subjected to one-time, 6 Gy of radiation, and levels of intracellular ROS were measured using the DCF-DA assay. F, indicated cell lines were subjected to one-time, 6 Gy of radiation, and cell lysates were harvested at indicated time points for Western blotting. Bar graphs on the bottom show quantitative results of γH2AX from LNCaP or DU145 cells. G, indicated cells were subjected to one-time, 6 Gy of radiation. At 4 hr postradiation, the levels of γH2AX were examined by immunofluorescence imaging (cells without radiation treatment did not show positive γH2AX staining). Quantification results were shown on the right. IR, irradiation. ∗Compared with control, p < 0.05 (one-way/two-way ANOVA). DFC-DA, dichlorofluorescein-diacetate; Prx, peroxiredoxin.

Figure 7

Depletion of Prx4 significantly attenuates tumor growth in mouse xenograft model.A, tumor growth curve. NSG-SCID mice were subcutaneously injected with either control (DU145-V2, n = 6) or Prx4-depleted cells (DU145-Prx4KO, n = 5). B, comparison of final tumor weight. C, histological comparison using H&E, anti-Prx4, anti-cleaved caspase 3, and anti-Ki67 staining of tumor sections. The scale bars represent 1 mm (tumor) or 100 μm (zoomed in). In all graphs, ∗ p < 0.05 (t test). Prx, peroxiredoxin.

Figure 8

Depletion of Prx4 sensitizes mouse xenograft to ionizing radiation.A, tumor growth curve postradiation. NSG-SCID mice were subcutaneously injected with either control (DU145-V2, n = 8) or Prx4-depleted cells (DU145-Prx4KO, n = 6). After average volume of tumors reaches 100 mm³ in each group, tumors were treated with 2 Gy of ionizing radiation once every day for 3 days. B, comparison of final tumor weight. C, comparison of tumors using H&E, anti-Prx4, anti-cleaved caspase 3, and anti-Ki67. The scale bars represent 1 mm (tumor) or 100 μm (zoomed in). In all graphs, IR: irradiation. ∗ p < 0.05 (t test). Prx, peroxiredoxin.

Figure 9

A schematic model of Prx4 in prostate cancer malignancy and radiation resistance. Prx, peroxiredoxin.