Research ArticleDRUG DEVELOPMENT

The nuclear translocation of the kinases p38 and JNK promotes inflammation-induced cancer

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Sci. Signal.  10 Apr 2018:
Vol. 11, Issue 525, eaao3428
DOI: 10.1126/scisignal.aao3428
  • Fig. 1 Identification of the nuclear translocation signal in p38α.

    (A) Schematic representation of truncation mutants of p38α. N, N terminus; KD, kinase domain; C, C terminus. (B and C) Representative images (B) and quantification (C) of fluorescence microscopy of p38 staining in HeLa cells transfected with green fluorescent protein (GFP)–tagged wild-type (WT) or truncated (ΔC40, ΔN20, or ΔN30) p38α, serum-starved for 16 hours, and then untreated (NT) or stimulated with anisomycin (Anis; 1 μg/ml) for 15 min. Scale bar, 15 μm. Data are the means ± SE. Percentage of cells with mostly nuclear (N, gray), all over [nuclear and cytoplasmic (NC); black], or mostly cytoplasmic (C, white) staining in at least three fields with >100 cells per field. **P < 0.01 by two-sample test for equality of proportions. (D and E) Representative blots (D) and quantification (E) of the CoIP of Imp7 or Imp9 with antibody to GFP in lysates from HeLa cells transfected with GFP-tagged WT or truncation-mutant (ΔN7, ΔN20, or ΔN30) p38α and treated as described in (B). Data are means ± SE. **P < 0.01 by two-way analysis of variance (ANOVA) followed by Tukey post-tests. (F) Sequence alignment of the PERY peptide with p38α/β and JNK1/2. “*”, identical amino acids (red); “:”, similar amino acids (green). (G and H) Representative blots (G) and quantification (H) of the CoIP of Imp7 with p38α antibody in lysates from HeLa cells that were serum-starved and preincubated with SCR peptide or peptides composed of residues 21 to 29, 15 to 29, or 21 to 34 of p38α (10 μM for 2 hours), stimulated with anisomycin (1 μg/ml for 15 min) or untreated (NT). Data are means ± SE; **P < 0.01 by two-way ANOVA followed by Dunnett’s post-tests. All data are representative of at least three independent experiments. A.U., arbitrary units.

  • Fig. 2 The PERY peptide inhibits JNK1/2 and p38α/β interaction with Imp7/9.

    (A and B) Representative blots (A) and quantification (B) of the CoIP of Imp7 or Imp9 with antibody to p38α, p38β, JNK1, or JNK2 in lysates from HeLa cells that were serum-starved [0.1% fetal bovine serum (FBS), 16 hours], preincubated with PERY or SCR peptides or DMSO (10 μM, 12 hours), and then stimulated with anisomycin (1 μg/ml, 15 min) or left untreated (NT). Data are means ± SE; *P < 0.05 and **P < 0.01 by one-way ANOVA. (C) Representative images (left) and quantification (right) of PLA analysis using anti–general p38 and Imp7 antibodies of HeLa cells that were serum-starved (0.1% FBS, 16 hours), preincubated with PERY or SCR peptides (10 μM each for 2 hours), and then stimulated with anisomycin (1 μg/ml, 15 min) or left untreated (NT). Scale bar, 10 μm. Data are means ± SE. **P < 0.01 by paired t test. (D) Representative blots (left) and quantification (right) of p38 in vitro kinase assay using MEF2A as a substrate. HeLa cells were transfected with GFP-p38α followed by starvation (16 hours, 0.1% FBS) and subjected to anisomycin stimulation (1 μg/ml, 15 min, +) or left untreated (NT, −). The cells were lysed and the extracts were subjected to IP using anti-GFP antibody. The immunoprecipitated kinase was then subjected to in vitro kinase assay using MEF2A in the presence of PERY or SCR peptide (10 μM). Data are means ± SE. All data are representative of at least three independent experiments.

  • Fig. 3 The PERY peptide inhibits the stimulation-dependent nuclear translocation of JNKs and p38s but not ERK or AKT.

    (A to C) Representative images (A and B) and quantification (C) of fluorescence microscopy of p38α and JNK1 antibodies (A) or ERK and AKT antibodies (B) in HeLa cells that were preincubated with the PERY or SCR peptides (10 μM each, 2 hours) and then treated with anisomycin (1 μg/ml, 15 min) or left untreated (NT). The nuclei were detected using 4′,6-diamino-2-phenylindole (DAPI). Scale bar, 20 μm. Data are means ± SE. Percentage of cells with mostly nuclear (N, gray), all over [nuclear and cytoplasmic (NC); black], or mostly cytoplasmic (C, white) staining in at least three fields with >100 cells per field. **P < 0.01 by two-sample test for equality of proportions. (D) Representative blots (upper) and quantification (bottom) of p38 nuclear fractions of HeLa cells, which were treated as described in (A) and were further subjected to subcellular fractionation as described in Materials and Methods. A control cytoplasmic fraction (C) and treated nuclear fractions (N) were subjected to Western blot analysis. Data are means ± SE. *P < 0.05 by one-way ANOVA. All data are representative of at least three independent experiments.

  • Fig. 4 The effect of the PERY peptide on downstream targets, apoptosis, and MAPK activation.

    (A) Representative blots (left) and quantification (right) of phosphorylation levels of nuclear and cytoplasmic p38 targets of HeLa cells that were serum-starved (0.1% FBS, 16 hours), preincubated with PERY or SCR peptides (10 μM each for 2 hours), and then stimulated with anisomycin (1 μg/ml) for the indicated times (15, 30, or 60 min) or left untreated (−). p, phosphorylated; g, general. Data are means ± SE; *P < 0.05 and **P < 0.01 by one-way ANOVA. (B) Representative blots (left) and quantification (right) of cleaved caspase 3 (cl-Casp3) of PC3 or αT3-1 cells that were serum-starved (0.1% FBS, 16 hours), pretreated with PERY or SCR peptides (10 μM for 2 hours), and stimulated with TPA (250 nM, +) or left untreated (NT, −) for 48 hours. Data are means ± SE; *P < 0.05 by one-way ANOVA. (C) Representative blots (left) and quantification (right) of MAPK and AKT phosphorylation levels of HeLa cells that were serum-starved (0.1% FBS, 16 hours), preincubated with PERY or SCR peptides (10 μM each for 2 hours), and then stimulated with anisomycin (1 μg/ml) and TPA (250 nM) for the indicated times (15, 30, or 60 min) or left untreated (−). Data are means ± SE. All data are representative of at least three independent experiments.

  • Fig. 5 The PERY peptide reduces proliferation of breast and melanoma cancer cells and prevents the growth of human breast cancer xenografts.

    (A) Proliferation of various cancer cell lines in the presence of PERY peptide, SCR peptides, or p38 inhibitor. Twenty-two cancer cell lines were treated with either SCR peptide, PERY peptide, SB203580 inhibitor (MDA-MB-231, AU565, and A2185), or PH797804 inhibitor (p38 inhibitor, all other cell lines), all at a concentration of 10 μM. Viable cells were quantified as the fold change of the initial cell number by methylene blue at 72 hours (BT549, HCC70, MIAPaca, ASPC1, PC3, and LNCaP) or 96 hours (all other cell lines) after cell seeding. Data are means ± SEM. *P < 0.05 and **P < 0.01 by two-way ANOVA followed by Dunnett’s post-tests. (B) Representatives time course of cell proliferation of MDA-MB-231, A2352, T47D, and MCF7 cells that were treated with the PERY peptide, SCR peptide, or p38 commercial inhibitor (SB203580 or PH797804) all at a concentration of 10 μM, and their proliferation was compared to DMSO control (0.1%) or no treatment at 1% fetal calf serum (FCS). Quantification of viable cells was detected as above, and the graphs present the kinetic of cell growth at the indicated times. All experiments were repeated three times in triplicate. The results were presented as a fold change of the initial cell number obtained from three independent experiments and represent means ± SEM. *P < 0.05 and **P < 0.01 by two-way ANOVA followed by Dunnett’s post-tests. (C) Growth of human breast cancer xenografts of severe combined immunodeficient mice that were inoculated subcutaneously with MDA-MB-231 cells. Upon establishment of tumors, mice were treated intravenously with PERY or SCR peptides (15 mg/kg) or by gavage administration with PH797804 (10 mg/kg), three times a week. Tumor size was recorded at the same time using a caliper, and the volumes were calculated accordingly. Data are means ± SEM, and experiments were reproduced two times; n = 7 mice per group. **P < 0.01 by one-way ANOVA followed by Tukey post-tests.

  • Fig. 6 The PERY peptide protects mice from DSS-induced colitis.

    C57BL/6 male mice (8 weeks, Harlan) were pretreated by intravenous injections with PERY or SCR peptides (15 mg/kg) or DMSO. The following day, mice were treated with 1.5% dextran sodium sulfate (DSS) in drinking water for 7 days followed by 3 days of normal drinking water. During DSS administration, mice were treated every other day (total of five intravenous injections). Control groups included nontreated mice (no DSS, no treatment) and mice receiving PERY peptide with normal drinking water (PERY only, no DSS). Mice were then euthanized and analyzed as described in Materials and Methods. Colitis was evaluated using (A) weight loss measurements, (B) in vivo endoscopy, and (C) hematoxylin and eosin (H&E) histological sections of the colons (top) and their magnifications (bottom). Scale bar, 2 mm. (D) Inflammation score based on the histological sections. (E) Measurements of colon length. n = 8 mice per group for all groups. The experiment was repeated three times. Data are means ± SEM; *P < 0.05 and **P < 0.01 by one-way ANOVA followed by Tukey post-tests.

  • Fig. 7 PERY peptide reduces the incidence of AOM/DSS-induced colon cancer.

    Ten-week-old ICR mice (Harlan) were injected once intraperitoneally with AOM (10 mg/kg) followed by DSS administration as described in Materials and Methods. Mice were treated with PERY or SCR peptides (15 mg/kg intravenously for each group) or with the commercial p38 inflammatory inhibitor SB203580 (15 mg/kg, gavage). Additional group was treated with the PERY peptide (15 mg/kg intravenously), but starting only from the second DSS cycle [PERY(2nd cycle)]. The abovementioned groups received AOM/DSS treatment (A/D). Control groups included nontreated mice (no DSS, no treatment) and mice receiving PERY peptide with normal drinking water (PERY only, no DSS). Mice were then euthanized and analyzed as described in Materials and Methods. Colitis and cancer severity were evaluated using (A) weight loss measurements and (B) in vivo endoscopy and (C) by determining tumor load by adding up the average diameters of all tumors for each animal. (D) Counting the average tumor number in each treatment based on tumor size. (E) H&E histological section analysis of the entire colons’ field (×1), magnification of the squared field in the upper panel (×4), or other colons’ magnifications (×10). Scale bar, 2 mm. n = 10 per group. The results were reproduced twice and are means ± SEM; *P < 0.05 and **P < 0.01 by one-way ANOVA followed by Tukey post-tests.

  • Fig. 8 The PERY peptide inhibits the stimulation-dependent nuclear translocation of p38 after DSS administration in AOM/DSS-induced colons.

    (A) Representative images of AOM/DSS-induced colon paraffin sections obtained from SCR peptide– or PERY peptide–treated mice and stained for p38 and DAPI. The upper panels (I) show the entire colon field containing the colon crypts (CC) and the lamina propria (LP). The lower panels (II) contain magnification of the lamina propria highlighted in the upper panel by a square. Scale bars, 2 mm (A, B, and D). (B) The same enlarged sections from p38-stained PERY peptide– and SCR peptide–treated mice from (A) where the nucleus is indicated. Arrows are white (PERY) or black (SCR) to emphasize the nature of p38 staining in these sections. (C) Quantification of (A) counting at least three fields with >50 cells per field. Bars represent the average percentage of cells with mostly nuclear (N, gray), all over [nuclear and cytoplasmic (NC); black], or mostly cytoplasmic (C, white) staining. Data are means ± SE; **P < 0.01 by two-sample test for equality of proportions. (D) Representative images of AOM/DSS-induced colon paraffin sections obtained from PERY peptide– or SCR peptide–treated mice and co-stained for p38, MAC2, and Hoechst. The upper panels (I) show the entire colon field containing the colon crypts (CC) and the lamina propria (LP). The lower panels (II) contain magnification of the lamina propria highlighted in the upper panel by a square to enlarge the co-stained area. The results are representative of two repeat experiments.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/525/eaao3428/DC1

    Fig. S1. Characterization of the N-terminal p38α domain.

    Fig. S2. The intracellular distribution of the PERY peptide and its ability to inhibit the stimulation-dependent nuclear translocation of p38β and JNK2.

    Fig. S3. The PERY peptide inhibits the stimulation-dependent nuclear translocation of p38 in several cancer cell lines.

    Fig. S4. The PERY peptide does not affect MAPK phosphorylation upon stimulation in several cancer cell lines.

    Fig. S5. Experimental design for the AOM/DSS-induced cancer model.

    Fig. S6. The PERY peptide reduces the incidence of AOM/DSS-induced colon cancer.

    Fig. S7. The PERY peptide reduces tumor load in the AOM/DSS colon cancer model.

    Fig. S8. The PERY peptide inhibits the stimulation-dependent nuclear translocation of p38α in macrophages.

  • Supplementary Materials for:

    The nuclear translocation of the kinases p38 and JNK promotes inflammation-induced cancer

    Galia Maik-Rachline, Elder Zehorai, Tamar Hanoch, John Blenis, Rony Seger*

    *Corresponding author. Email: rony.seger{at}weizmann.ac.il

    This PDF file includes:

    • Fig. S1. Characterization of the N-terminal p38α domain.
    • Fig. S2. The intracellular distribution of the PERY peptide and its ability to inhibit the stimulation-dependent nuclear translocation of p38β and JNK2.
    • Fig. S3. The PERY peptide inhibits the stimulation-dependent nuclear translocation of p38 in several cancer cell lines.
    • Fig. S4. The PERY peptide does not affect MAPK phosphorylation upon stimulation in several cancer cell lines.
    • Fig. S5. Experimental design for the AOM/DSS-induced cancer model.
    • Fig. S6. The PERY peptide reduces the incidence of AOM/DSS-induced colon cancer.
    • Fig. S7. The PERY peptide reduces tumor load in the AOM/DSS colon cancer model.
    • Fig. S8. The PERY peptide inhibits the stimulation-dependent nuclear translocation of p38α in macrophages.

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