Paracrine apoptotic effect of p53 mediated by tumor suppressor Par-4

Abstract

The guardian of the genome, p53, is often mutated in cancer and may contribute to therapeutic resistance. Given that p53 is intact and functional in normal tissues, we harnessed its potential to inhibit the growth of p53-deficient cancer cells. Specific activation of p53 in normal fibroblasts selectively induced apoptosis in p53-deficient cancer cells. This paracrine effect was mediated by p53-dependent secretion of the tumor suppressor Par-4. Accordingly, the activation of p53 in normal mice, but not p53(-)/(-) or Par-4(-)/(-) mice, caused systemic elevation of Par-4, which induced apoptosis of p53-deficient tumor cells. Mechanistically, p53 induced Par-4 secretion by suppressing the expression of its binding partner, UACA, which sequesters Par-4. Thus, normal cells can be empowered by p53 activation to induce Par-4 secretion for the inhibition of therapy-resistant tumors.

Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1. P53 activation in normal cells produces paracrine apoptosis in p53-deficient cancer cells

(A) Activation of p53 in MEFs induces apoptosis of co-cultured p53-deficient cancer cells. Co-cultures of p53+/+ or p53−/− MEFs with the indicated cancer cells, were treated with vehicle (v), Nutlin-3a (N, 10 μM) and/or PS-1145 (P, 10 μM) for 24 h. The cells were subjected to ICC for cytokeratins to detect epithelial cancer cells and for active caspase-3 to determine apoptotic cells. (B) Apoptosis of cancer cells by CM from p53-activated MEFs. P53+/+ or p53−/− MEFs were treated with vehicle (v), Nutlin-3a (N, 10 μM) and/or PS-1145 (P, 10 μM) for 24 h, and their CM was transferred to normal (HEL) or p53-deficient cancer (H1299) cells. Apoptotic cells were scored after 24 h by ICC for active caspase-3. (C) Co-parallel activation of p53 and inhibition of NF-κB activity additively increases Par-4 secretion in fibroblasts. MEFs were treated with vehicle (v), Nutlin-3a (N) and/or PS-1145 (P) for 24 h, and their CM, as well as whole-cell lysates were subjected to Western blot analysis for Par-4. β-actin was used to normalize loading of lysate. Coomassie blue-stained albumin in the serum was used to normalize loading of CM. Par-4 secretion, but not Col1A1 secretion, was dependent on the p53 status of the cells. Fold change in secreted Par-4 is shown. (D) Par-4 secretion is critical for the paracrine effect resulting from simultaneous p53-activation and NF-κB-inhibition. The CM from p53+/+ cells treated with Nutlin-3a + PS-1145 was incubated with polyclonal antibody for Par-4 or PTEN control, and then added to H1299 cells. Apoptosis of H1299 cells was scored after 24 h by ICC for active caspase 3 (left panel). Par-4+/+ and Par-4−/− MEFs were treated with Nutlin-3a + PS-1145 (N+P) or vehicle, then the CM was applied to the indicated cell lines, and apoptotic cells were scored by ICC for active caspase-3 (middle panel). Expression of Par-4 in CM was verified on Western blots (right panel). (A,B,D) Asterisk (*) indicates statistical significance (P < 0.001) by the Student t test; (**) indicates that N+P is significantly (P < 0.001) more effective than individual treatments based on two-way ANOVA.

Figure 2. Activation of p53 in mice induces systemic expression of Par-4 pro-apoptotic activity

(A) P53 function is essential for induction of systemic Par-4 in mice. Serum samples from p53+/+ mice (4 μl per lane) and p53−/− mice (8 μl per lane) (left panel); and also from p53+/+, p53−/−, and Par-4−/− mice (right panel) injected via i.p. route with either vehicle (v) or with Nutlin-3a + PS-1145 (N+P) were examined for Par-4 expression by Western blot analysis using serum albumin to normalize loading. Data are representative of 4 mice per treatment. (B) Serum from Nutlin-3a plus PS-1145-treated p53+/+ mice induces ex vivo apoptosis of cancer cells. Serum samples collected from p53+/+, p53−/−, and Par-4−/− mice injected i.p. with either vehicle (lower panel) or with Nutlin-3a + PS-1145 (N+P) (upper panel) was applied at a final concentration of 10% to the indicated normal or cancer cell lines. The cells were scored for apoptosis after 24 h. Fetal bovine serum (FBS, 10%) was used as an additional control. (C) Secreted Par-4 is essential for the paracrine effect of p53 activation. Serum samples collected from p53+/+ mice treated with vehicle or Nutlin-3a + PS-1145 (N+P) were pre-incubated with Par-4 antibody, control PTEN antibody or no antibody (no Ab), and then applied on lung cancer cells. After 24 hours of treatment, the cells were scored for apoptosis. (A,B) Asterisk (*) indicates statistical significance (P< 0.001) by the Student t test.

Figure 3. P53 stimulates Par-4 secretion by suppressing the expression of UACA

(A) P53 down-regulates UACA. Whole-cell lysates from p53+/+ and p53−/− MEFs that were either untreated or treated with vehicle (v), Nutlin-3a (N, 10μM), or PS-1145 (P, 10 μM) for 24 hours (left 3 panels) ; or whole-tissue lysates of highly vascular organs obtained from p53+/+ and p53−/− mice (right 3 panels), were examined for UACA by Western blot analysis. (B) Restoration of p53 activity inhibits UACA expression and promotes Par-4 secretion. P53−/− MEFs were infected with GFP-tagged p53- or GFP-producing adenoviral constructs (left panel). Also, the mouse fibroblasts 10(1), which do not express any p53, and 10(1) derived Val5 cells, which are engineered to stably over-express wild-type p53 at 32 °C or mutant p53 at 37 °C, were grown at 37 °C or shifted to 32°C to activate p53 (right panel). Expression of the indicated proteins in the CM or whole-cell lysate was examined by Western blot analysis. (C) UACA inhibits Par-4 secretion. UACA expression was knocked down in mouse (p53+/+ or p53−/− MEF) and human (HEL) cells with distinct siRNA pools from two different sources- Dharmacon (D), and SantaCruz Biotechnology, Inc. (SC), and the CM, as well as the whole cell lysates, were subjected to Western blot analysis. C, control (scrambled) siRNA. (D) P53 activation and UACA inhibition promotes secretion by a BFA-sensitive pathway. 10(1)/Val5 fibroblasts grown at 32°C were treated with BFA (1 μg/ml) or vehicle (v) for 3 h (left panel). UACA expression was inhibited in MEFs (p53+/+) with Nutlin-3a plus PS-1145 (N+P; 10 μM each) (middle panel), or with an siRNA pool (from Dharmacon) (right panel), and then the cells were further placed in the presence of BFA or vehicle (v) for 3 h. The CM, as well as the whole-cell lysates, were subjected to Western blot analysis.

Figure 4. P53 directly binds to UACA and inhibits its expression

(A) P53 binds to its consensus binding motif in UACA. HEL cells were treated with Nutlin-3a (N) or vehicle (v) for 24 h and subjected to ChIP analysis with p53 antibody (Ab) or control rabbit IgG Ab, and immunoprecipitated DNA fragments were analyzed by PCR with primers flanking the p53-binding site in UACA. Primers flanking the p53-binding motif #1 in p21 (see Figure S3A) or for GAPDH (which does not contain a p53-binding site), were used as positive or negative control, respectively. Input samples for each set of primers are shown. (B) Nutlin-3a causes inhibition of endogenous UACA expression in an NF-κB independent manner. IKKβ−/− MEFs or HEL cells were treated with Nutlin-3a or vehicle for 24 h, and whole-cell lysates were subjected to Western blot analysis for UACA, p53 or actin.