Prdx1 inhibits tumorigenesis via regulating PTEN/AKT activity

Abstract

It is widely accepted that reactive oxygen species (ROS) promote tumorigenesis. However, the exact mechanisms are still unclear. As mice lacking the peroxidase peroxiredoxin1 (Prdx1) produce more cellular ROS and die prematurely of cancer, they offer an ideal model system to study ROS-induced tumorigenesis. Prdx1 ablation increased the susceptibility to Ras-induced breast cancer. We, therefore, investigated the role of Prdx1 in regulating oncogenic Ras effector pathways. We found Akt hyperactive in fibroblasts and mammary epithelial cells lacking Prdx1. Investigating the nature of such elevated Akt activation established a novel role for Prdx1 as a safeguard for the lipid phosphatase activity of PTEN, which is essential for its tumour suppressive function. We found binding of the peroxidase Prdx1 to PTEN essential for protecting PTEN from oxidation-induced inactivation. Along those lines, Prdx1 tumour suppression of Ras- or ErbB-2-induced transformation was mediated mainly via PTEN.

Figure 1

(A) Left panel: rate of H₂O₂ release per minute was analysed by calculating the linear slope of data obtained in Supplementary Figure S1A. H₂O₂ release is expressed per 1 × 10⁶ cells per minute. P-values were calculated using an unpaired Student's t-test. Rate of H₂O₂ release in Prdx1^−/−MEFs compared with either Prdx1^{−/−Prdx1WT}MEFs (^*P=0.04) or Prdx1^{−/−Prdx1Cys51/172S} (^**P=0.001). Right panel: protein levels and oxidation status of expressed Prdx1 in MEFs used in left panel. Protein lysates were prepared as described under Materials and methods and analysed under non-reducing conditions. Status of over-oxidized Prdx1 was tested by using an antibody recognizing Prdx1 (Prdx1-SO₂/SO₃) (Woo et al, 2003). (B) Left panel: Prdx1^−/−MEFs and Prdx1^+/+MEFs were infected with retroviral constructs carrying genes for Prdx1^WT or Prdx1^C51/172S. MEFs were further infected with retrovirus expressing either H-Ras^V12 or puromycin resistance gene only (EV) and plated in duplicates in soft agar. Colonies were counted after 18–20 days. Right panel: proteins of clones used in soft agar experiment were analysed for expression levels of Prdx1 and Ras. (C) Rate of H₂O₂ release per minute was analysed by calculating the linear slope of MEFs used in (B, left panel). For each clone, six wells were plated and analysed. The experiment shown here is representative of three independent studies from two different sets of MEF clones obtained from Prdx1 littermates. P-values were calculated using an unpaired Student's t-test. ^*P=0.045; ^**P=0.004. Differences in Prdx1^+/+MEFs were not statistically significant.

Figure 2

(A) Prdx1^−/−MEFs and Prdx1^+/+MEFs were stimulated with H₂O₂ as indicated. Protein lysates were collected under argonized conditions by scraping cells into argon-purged lysis buffer (see Materials and methods) and analysed under non-reducing conditions on SDS–PAGE. Akt phosphorylation was detected on Ser473 and Thr308. Akt protein as loading control. (B) Prdx1^−/−MEFs and Prdx1^+/+MEFs protein lysates were treated as described under (A) and analysed for oxidized PTEN proteins. Actin as loading control. (C) Levels of oxidized PTEN proteins were evaluated by quantifying oxidized and reduced PTEN using a Fuji imaging system (LAS 3000) and related software (ImageGuage). Quantifications of staining intensities were obtained by analysing protein bands from the same ECL obtained film exposure. The y-axis represents staining intensities in arbitrary units of oxidized PTEN. Curves represent data from three independent experiments. (D) Prdx1^−/−MEFs and Prdx1^+/+MEFs were stimulated with PDGF as indicated. Protein lysates were collected and analysed as described under (A). (E) Prdx1^−/−MEFs and Prdx1^+/+MEFs protein lysates were treated as described under (D) and analysed for oxidized PTEN proteins. Actin as loading control. (F) Data analysis was done as described under (C). All experiments shown are representative of at least three independent studies including two sets of MEF clones from Prdx1 littermates. (G) Serum starved MEFs were stimulated with H₂O₂ as indicated. Protein lysates were collected and analysed as described under (A). Akt substrates were detected by western blotting using an phospho-Akt substrate antibody (RXRXXS/T).

Figure 3

(A) Epitope-tagged PTEN was expressed in 293T cells. Cell lysate was prepared under anaerobic conditions and immunoprecipitates were prepared as described in Materials and methods. A measure of 1000 μg of protein was immunoprecipitated (anti-PTEN antibody; Santa Cruz). Proteins were analysed by SDS–PAGE. PTEN and Prdx1 proteins were detected by staining membranes with anti-PTEN (138G6, Cell Signaling) and anti-Prdx1 (Abcam) antibodies, respectively. (B) Epitope-tagged PTEN and Prdx1 wild type were co-expressed in 293T cells. Before lysis, cells were treated with increasing dosages of H₂O₂ as indicated for 15 min. Cell lysates were prepared under anaerobic conditions, and precipitated over night using HA-conjugated agarose beads. IPs were washed four times with argon-purged lysis buffer and analysed by western blotting. Proteins were detected with anti-PTEN, anti-Prdx1-SO₂/-SO₃ and anti-Prdx1 antibodies. Epitope-tagged Prdx1 migrates slower electrophoretically than endogenous Prdx1, labelled as HA–Prdx1. Epitope-tagged PTEN is labelled as Myc–PTEN. Anti-Prdx1-SO₂/-SO₃ can cross-react with Prdx2-4 and stains more intense in the IP proteins due to over night incubation and longer exposure to atmospheric oxygen. In contrast, lysates were immediately frozen (Supplementary Figure S3A and B). (C) Myc–PTEN and HA–Prdx1^C51/172S were analysed as described under (B). Anti-Prdx1-SO₂/-SO₃ does not bind Prdx1^C51/172S. (D) Recombinant His–PTEN (100 nM) was applied to equimolar GST or GST–Prdx1 and incubated over night. Glutathione sepharose (GST) beads were added for GST-pull down and analysed by western blotting. Input represents 1/50 of the total. (E) HA–Prdx1 wild type and cysteine to serine mutants of catalytically active Prdx1 cysteines were co-expressed with Myc-tagged PTEN wild type. HA–IPs were processed as under (B). Left side shows co-immunoprecipitations, right side shows expression of PTEN and Prdx1 cysteine to serine mutants. (F) Schematic domain structure of potential interaction sites of Prdx1 with PTEN (upper schematic) and PTEN with Prdx (lower schematic). (G) Identification of potential interaction sites using computer modelling (SwissPdb Viewer version 4; http://www.expasy.org/spdbv) (Guex and Peitsch, 1997). ^*Prdx1 N terminus.

Figure 4

(A) Epitope-tagged Myc–PTEN and HA–Prdx1 were co-expressed in 293T cells, which were treated with H₂O₂ for 15 min, as indicated. Cell lysates were prepared under anaerobic conditions, and precipitated over night as described above by using anti-PTEN antibody. PTEN-immunoprecipitates were analysed for PI(3,4,5)P₃-utilization in a 96-well plate assay as described in Materials and methods (triplicate samples). All results are presented as mean±s.e. (or difference) for at least two independent experiments. Equal PTEN protein amount was confirmed by western blotting. (B) 500 μM of H₂O₂ was added to either 20 pmoles of recombinant His-purified PTEN in the presence of different amounts of recombinant purified Prdx1 as indicated and described in Materials and methods. Experiment shown is representative of three independent studies. (C) PTEN activity was measured as described in (B) in the presence of peptides interfering with Prdx1 binding to PTEN. P1 (peptide 1) is replica of first N-terminal 21 amino acids (AS) of Prdx1; P1 Sc, scrambled sequence of P1; P2 (peptide 2), replica of last C-terminal 26 AS of Prdx1; P2 Sc, scrambled sequence of P2. The results are presented as mean±s.e. (or difference) for at least two independent experiments. (D) Prdx1 peroxidase activity was measured by a standard thioredoxin (Trx)/thioredoxin reductase (TrxR)/NADPH-coupled spectrophotometric assay (see Materials and methods for details). A measure of 20 pmoles of purified Prdx1 (Sigma) was incubated with 500 μM H₂O₂ and increasing molar amounts of purified His–PTEN as indicated. Prdx1 peroxidase activity (without PTEN) was about 68 nmol/mg protein/min under those conditions (extinction coefficient 6.62 mM⁻¹ cm⁻¹ for NADPH at 340 nm). Experiment shown is representative of three to four independent studies. (E) Relative reaction velocity of Prdx1 peroxidase activity from (D) was determined by plotting linear regression coefficient K (y=a−Kx, where y is the absorbency at 340 nm and x is time) versus various PTEN/Prdx1 (mol/mol) ratios. The results are presented as mean±s.e. (or difference) for at least two independent experiments.

Figure 5

(A) PTEN^−/−MEFs were serum starved for 48 h and treated with H₂O₂ as indicated for 10 min. Protein lysates were analysed for pAkt^Ser473 as described before. (B) PTEN^−/−MEFs were infected with retrovirus expressing GFP–PTEN. After 5-day selection in puromycin (2 μg/ml), MEFs were serum starved, exposed for 10 min to H₂O₂ and analysed for Prdx1 oxidation and formation of dimeric structures by non-reducing SDS–PAGE. ^*Prdx1 hetero-dimer formation with other Prdxs. (C) PTEN^−/−MEFs were infected with retrovirus expressing shPrdx1, GFP–PTEN, H-Ras^V12 or various vectors carrying resistance gene only (pBabe, pMKO1). MEFs were plated in soft agar. Colonies were counted after 21 days. ^*P<0.001 (Student's t-test). (D) As under C, except MEFs express ErbB-2/neuT instead of H-ras^V12. ^**P<0.039 (Student's t-test). (E) Immunoblotting of MEFs from (C and D).

Figure 6

(A) The percentage of mice free of H-Ras-induced mammary tumours is shown in MMTV-v-H-Ras mice carrying only one, none, or both copies of the Prdx1 gene. (B) MECs (n=3) were isolated from MMTV-v-H-Ras mice/Prdx1^−/− or MMTV-v-H-Ras mice/Prdx1^+/+ and analysed on a non-reducing SDS–PAGE for PTEN oxidation as described above. Statistical analysis of PTEN oxidation was done using a χ² test. (C) The same protein lysates were also analysed for Akt phosphorylation on Ser473, as described above. ^*Non-statistically significant.