PUMA mediates EGFR tyrosine kinase inhibitor-induced apoptosis in head and neck cancer cells - PubMed (original) (raw)

PUMA mediates EGFR tyrosine kinase inhibitor-induced apoptosis in head and neck cancer cells

Q Sun et al. Oncogene. 2009.

Abstract

Overexpression of epidermal growth factor receptor (EGFR) is found in over 80% of head and neck squamous cell carcinomas (HNSCC) and associated with poor clinical outcomes. EFGR selective tyrosine kinase inhibitors (TKIs) or antibodies have recently emerged as promising treatments for solid tumors, including HNSCC, though the response rate to these agents is low. p53 upregulated modulator of apoptosis (PUMA), a BH3-only Bcl-2 family protein, is required for apoptosis induced by p53 and various chemotherapeutic agents. In this study, we show that PUMA induction is correlated with EGFR-TKI sensitivity, and is mediated through the p53 family protein p73beta and inhibition of the PI3K/AKT pathway. In some HNSCC cells, the gefitinib-induced degradation of oncogenic Delta Np63 seems to facilitate p73-mediated PUMA transcription. Inhibiting PUMA expression by small hairpin RNA (shRNA) impairs gefitinib-induced apoptosis. Furthermore, PUMA or BH3 mimetics sensitize HNSCC cells to gefitinib-induced apoptosis. Our results suggest that PUMA induction through p73 represents a new mechanism of EGFR inhibitor-induced apoptosis, and provide potential ways for enhancing and predicting the sensitivity to EGFR-targeted therapies in HNSCC.

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Conflict of interest statement

Conflict of interest

The authors declare no conflict of interest.

Figures

Figure 1

Figure 1

PUMA was induced by EGFR inhibitors. (a) The indicated HNSCC cell lines were treated with gefitinib (10 μM) or left untreated for 72 h. *, 2 μM gefitinib for JHU-029. PUMA and α-tubulin (loading control) were analyzed by western blotting. The two major isoforms of PUMA (α-upper and β-lower band) were detected. (b) The indicated cell lines were treated with erlotinib (E, 20 μM) or cetuximab/C225 (C, 6 μg/ml) for 48 h. PUMA and α-tubulin (loading control) were analyzed by western blotting. (c) JHU-012 cells were treated with 15 μM gefitinib. PUMA expression was analyzed by quantitative RT–PCR (left) and western blotting (right) at indicated time points, respectively. (d) EGFR-targeting agents suppressed the growth of 1483 HNSCC xenograft tumors (left). The growth curve of 1483 tumors subjected to erlotinib (oral gavage daily 90 mg/kg), C225 (i.p. 0.8 mg/mouse thice a week) or vehicle (DMSO) treatments for 2 weeks. P = 0.08 (Erlotinib vs Vehicle), *P = 0.0107 (C225 vs Vehicle) PUMA mRNA levels in the xenografts were analyzed by quantitative RT-PCR (right). Values are means±s.d. (n = 3 in each group). *P = 0.0141, **P<0.0001.

Figure 2

Figure 2

PUMA mediates EGFR-TKI sensitivity and-induced apoptosis in HNSCC cells. (a) JHU-012 and JHU-029 cells were treated with different doses of gefitinib for 48 h. PUMA and active caspase-3 levels were analyzed by western blotting. The apoptosis was analyzed by nuclear fragmentation assay. (b) PUMA levels in the indicated 686LN cell lines were analyzed by western blotting at 48 h of the gefitinib treatment. (c) The Effects of gefitinib on EGFR phosphorylation in JHU-012 and JHU-029 cells. Cells were serum-starved for 72 h, incubated with various concentrations of gefitinib for 1 h, and then stimulated with EGF (50 ng/ml) for 5 min. The levels of phosphor (Y1068)- and total EGFR were analyzed by western blotting. (d) PUMA siRNA inhibited gefitinib-induced apoptosis. JHU-012 and JHU-029 cells were transfected with PUMA siRNA or scrambled siRNA for 24 h, and subjected to 10 or 2 μM gefitinib treatment for 72 h, respectively. Upper panel, PUMA and active caspase-3 expression were analyzed by western blotting. Lower panel, apoptosis was determined by sub-G1 population measured by flow cytometry. Apoptosis induced by gefitinib in the parental cells transfected with scrambled siRNA was set as 100%. *P<0.01, PUMA si RNA vs Scrambled.

Figure 3

Figure 3

p73 mediates PUMA transcription after EGFR inhibition in HNSCC cells. (a) Indicated HNSCC lines were left untreated (U), or treated with 15 μM gefitinib for 72 h, or with 20 μM erlotinib (E) or 6 μg/ml cetuximab (C) for 48 h. *, 2 μM gefitinib for JHU-029. p73, p53 and PUMA levels were analyzed by western blotting. (b) The recruitment of p73 to the PUMA promoter was analyzed by ChIP assay. HA-tagged p73β construct was transfected into the cells for 24 h followed by gefitinib treatment for 24 or 36 h. Inputs correspond to 1% of the total chromatin used for IP. (c) JHU-012 cells were transfected with the indicated reporters (Ming et al., 2008) for 16 h and subjected to 15 μM gefitinib treatment or left untreated for 48 h. Cont., empty reporter vector. The ratios of normalized relative luciferase activities (RLA) with or without treatment were plotted. (d) JHU-012 cells were transfected with the indicated siRNAs for 16 h, and subjected to 15 μM gefitinib treatment for 48 h. p73 and PUMA levels were analyzed by western blotting.

Figure 4

Figure 4

The PI3K/AKT pathway is involved in gefitinib-induced p73 and PUMA expression. (a) Indicated HNSCC lines were treated with 15 or 2 μM (JHU-029) gefitinib for 72 h. Phospho-AKT (S473) and total AKT levels were determined by western blotting. (b) The effects of AKT on PUMA induction by gefitinib were analyzed by western blotting. The AKT or empty vector (pUSE) was transfected into JHU-012 and JHU-029 cells for 24 h, followed by gefitinib treatment for 48 h. The expression of Akt (Myc) was confirmed by western blotting. (c) The effects of dominant negative (DN) PI3K on phospho-Akt (S473), p73 and PUMA levels in indicated cells were analyzed by western blotting 24 h after transfection.

Figure 5

Figure 5

ΔNp63 degradation induced by gefitinib enhances p73-mediated PUMA transcription. (a) The indicated cells were treated with 15 μM gefitinib and 20 μM erlotinib (E) for 72 and 48 h, respectively. p63 and α-tubulin were analyzed by western blotting. (b) Upper panel, the binding of endogenous p63 to the PUMA promoter was analyzed by ChIP assay in JHU-012 and JHU-029 cells at 0 or 36 h after gefitinib treatment. Lower panel, the effect of ΔNp63 on p73 binding to the PUMA promoter was analyzed by ChIP. JHU-012 cells were transfected with TAp73β alone or with ΔNp63 (1:5 molar ratio) followed by 15 μM gefitinib treatment for 36 h. Inputs correspond to 1% of the total chromatin used in IP. (c) PUMA reporter (Frag c) was transfected into p53 KO HCT 116 cells with TAp73β, along with either ΔNp63 (1:9 molar ratio), or the DNA binding domain-deleted ΔNp63, ΔDBD. The value from cells transfected with the reporter and the empty vector (pCDNA3.1, bar 1) was set as 1. Values are means±s.d. (n = 3 in each group). The levels of PUMA, p63, and p73 were analyzed by western blotting. (d) The indicated HNSCC cells were transfected with ΔNp63, ΔDBD or vector (pcDNA3.1) followed by gefitinib treatment for 48 h. PUMA levels were analyzed by western blotting.

Figure 6

Figure 6

PUMA and the BH3 mimetic gossypol-sensitized HNSCC cells to gefitinib-induced apoptosis. (a) Indicated HNSCC lines were infected with Ad-PUMA (40 MOI) for 24 h, then followed by 2 μM gefitinib treatment for 48 h. Apoptosis was analyzed by nuclear fragmentation assay. **P<0.001, combination vs single treatment. (b) Indicated TKI-resistant HNSCC lines were treated with 10 μM (5 μM for JHU-012) gossypol for 24 h followed by 10 μM (1 μM for JHU-012) gefitinib for 48 h. Apoptosis was analyzed by nuclear fragmentation assay. **P<0.001, *P<0.01, combination vs single treatment. (c) Bcl-2 rescues gefitinib-induced apoptosis. JHU-012 or JHU-029 cells were transfected with a Bcl-2 expression construct or empty vector followed by 10 or 2 μM gefitinib treatment for 48 h, respectively. Upper panel, the expression of Bcl-2 was confirmed by western blotting. Lower panel, apoptosis was analyzed by nuclear fragmentation assay. *P<0.01, Bcl-2 vs vector or mock (un) transfection group. (d) A model of the role of PUMA in EGFR inhibitors-induced apoptosis.

References

    1. Barbieri CE, Barton CE, Pietenpol JA. Delta Np63 alpha expression is regulated by the phosphoinositide 3-kinase pathway. J Biol Chem. 2003;278:51408–51414. - PubMed
    1. Barbieri CE, Pietenpol JA. p63 and epithelial biology. Exp Cell Res. 2006;312:695–706. - PubMed
    1. Bonner JA, Harari PM, Giralt J, Azarnia N, Shin DM, Cohen RB, et al. Radiotherapy plus cetuximab for squamous-cell carcinoma of the head and neck. N Engl J Med. 2006;354:567–578. - PubMed
    1. Bunz F, Hwang PM, Torrance C, Waldman T, Zhang Y, Dillehay L, et al. Disruption of p53 in human cancer cells alters the responses to therapeutic agents. J Clin Invest. 1999;104:263–269. - PMC - PubMed
    1. Choong NW, Cohen EE. Epidermal growth factor receptor directed therapy in head and neck cancer. Crit Rev Oncol Hematol. 2006;57:25–43. - PubMed

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