A RT I C L E S
p53 controls cancer cell invasion by inducing the MDM2-mediated degradation of Slug Shu-Ping Wang1, Wen-Lung Wang1, Yih-Leong Chang2, Chen-Tu Wu2, Yu-Chih Chao1, Shih-Han Kao3, Ang Yuan4, Chung-Wu Lin5, Shuenn-Chen Yang6, Wing-Kai Chan7, Ker-Chau Li8, Tse-Ming Hong9,11 and Pan-Chyr Yang4,6,10,11,12 The tumour suppressor p53 is known to prevent cancer progression by inhibiting proliferation and inducing apoptosis of tumour cells. Slug, an invasion promoter, exerts its effects by repressing E-cadherin transcription. Here we show that wild-type p53 (wtp53) suppresses cancer invasion by inducing Slug degradation, whereas mutant p53 may stabilize Slug protein. In non-smallcell lung cancer (NSCLC), mutation of p53 correlates with low MDM2, high Slug and low E-cadherin expression. This expression profile is associated with poor overall survival and short metastasis-free survival in patients with NSCLC. wtp53 upregulates MDM2 and forms a wtp53–MDM2–Slug complex that facilitates MDM2-mediated Slug degradation. Downregulation of Slug by wtp53 or MDM2 enhances E-cadherin expression and represses cancer cell invasiveness. In contrast, mutant p53 inactivates Slug degradation and leads to Slug accumulation and increased cancer cell invasiveness. Our findings indicate that wtp53 and p53 mutants may differentially control cancer invasion and metastasis through the p53–MDM2–Slug pathway. Cancer metastasis is the most common cause of treatment failure and death in cancer patients1. Although metastasis is a complicated process involving multiple factors and genetic events, increased migratory and invasive capabilities are critical to the initiation of the metastatic process1. Tumorigenesis and metastasis are two distinct aspects of cancer progression; the clinical significance of tumour suppressor activity is enhanced if both cancer invasion and metastasis are inhibited. The well-characterized tumour suppressor p53 is normally maintained at low levels by MDM2, which mediates the ubiquitylation and proteasomal degradation of p53 (ref. 2). Oncogene activation or genotoxic stress can activate p53, resulting in cell cycle arrest, apoptosis, DNA repair and cell differentiation3,4. Mutations in p53 occur frequently in cancers and are present in about 50% of non-small-cell lung cancers (NSCLCs)4–6. p53 mutations in NSCLC are associated with increased neoangiogenesis, higher proliferative index and decreased overall survival6. In addition, p53 mutations are associated with decreased E-cadherin expression7 and increased cancer invasiveness8. Recently, two groups have generated p53-mutant mouse models and found that the tumours developing in these mice metastasize9,10, indicating that mutant p53 may mediate a gain-of-function metastatic phenotype. Moreover, several reports have indicated that p53 is involved in cancer progression by specifically regulating cancer invasion11–13.
Downregulation of E-cadherin is a hallmark of epithelial–mesenchymal transition (EMT), a process that allows epithelial cells to separate from their neighbours and migrate to distant regions during development or metastasis14. E-cadherin is a suppressor of invasion and metastasis. Its downregulation is associated with the development of malignant epithelial cancers15–17. Forced expression of E-cadherin suppresses cancer metastasis, whereas functional knockout of E-cadherin confers a metastatic phenotype on benign tumour cells18,19. The predominant mechanism responsible for E-cadherin silencing in most carcinomas is transcriptional repression20,21. Slug (also known as SNAI2), a member of the Snail family of transcriptional repressors, is capable of repressing E-cadherin expression and triggering EMT22–24, suggesting that it may act as an invasion promoter. Recently, we have found that Slug promotes NSCLC cell invasion by downregulating E-cadherin and upregulating matrix metalloproteinase-2 (ref. 25). Consistent with the view that Slug can promote invasion, aberrant upregulation of Slug is correlated with reduced E-cadherin expression and is associated with malignant transformation and metastatic progression in numerous cancers25–30. Here we report an additional mechanism by which p53 regulates cancer invasion. We show that wild-type p53 is able to suppress cancer cell invasion by inducing MDM2-mediated Slug degradation. Furthermore, we
Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 11490, Taiwan. 2Department of Pathology and Graduate Institute of Pathology, National Taiwan University, Taipei 10043, Taiwan. 3Graduate Institute of Molecular Biology, College of Medicine, National Taiwan University, Taipei 10043, Taiwan. 4 Department of Internal Medicine, National Taiwan University Hospital, Taipei 10043, Taiwan. 5Department of Pathology, National Taiwan University Hospital, Taipei 10043, Taiwan. 6Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan. 7Department of Medical Research, National Taiwan University Hospital, Taipei 10043, Taiwan. 8Institute of Statistical Science, Academia Sinica, Taipei, 11529, Taiwan. 9Institute of Clinical Medicine, National Cheng Kung University, Tainan 70101, Taiwan. 10NTU Center for Genomic Medicine, College of Medicine, National Taiwan University, Taipei 10043, Taiwan. 11 These authors contributed equally to this work. 12 Correspondence should be addressed to P.-C Y. (e-mail:
[email protected]) 1
Received 29 October 2008; accepted 19 February 2009; published online 17 May 2009; corrected online 19 May 2009; DOI:10.1038/ncb1875
694
nature cell biology volume 11 | number 6 | JUNE 2009 © 2009 Macmillan Publishers Limited. All rights reserved.
A RT I C L E S a
b
p53mt–
0.7
E-cadherin+
(n = 46)
MDM2–
Slug+
E-cadherin–
0.6 0.5 0.4 0.3
p53 mt+ (n = 33)
0.2 P=0.039
0.1 0
0
10
20
30
40
50
60
70
80
p53 (mutant)
Probability of survival
0.9 0.8
Slug–
p53 (wild-type)
1.0
MDM2+
Time (months)
c
e
d 1.5
UV (J m–2)
0
10
20
30
40
Relative Slug mRNA levels
HFB (p53+/+) 50
p53
IB: DO-1 1
2.26
3.26 3.73 4.23 3.82
Slug
Anti-Slug 1
1
0.85 0.42 0.42 0.33
p53 1.0
UV (30 J m–2)
0.5
0 UV(J m–2) 0
β-Actin
Anti-β-actin
MEF
HFB (p53 ) +/+
f
–
p53–/–
+/+
+
–
+
IB: Ab-1
p53
Anti-Slug
Slug
Anti-β-actin 10
20 30
40 50
β-Actin 1
2
3
4
g HFB(p53+/+)
0
10
si-p53-2 25
0
10
25
IB: DO-1
p53 1
0.3
0.17
1
0.47
0.34
Anti-Slug
Slug 1
Anti-β-actin
nM
2.4
2.44
1
2.06
1.95
Relative mRNA levels
si-p53-1
β-Actin
1.5
1.5
1.0
1.0
0.5
0.5
0
0
10
25
si-p53-1 (nM)
0
Slug MDM2
0
10
25
si-p53-2 (nM)
Figure 1 p53 mutation is associated with poor clinical outcome in patients with NSCLC, and p53 can regulate Slug protein expression. (a) Kaplan– Meier plots of overall survival for patients with NSCLC with (p53mt+) or without (p53mt−) p53 mutation. (b) Immunohistochemistry of MDM2, Slug and E-cadherin in serial sections of NSCLC tumour specimens with wildtype or mutant p53. Scale bars represent 100 μm. (c–e) UV-induced p53 activation decreases the level of Slug protein but not that of Slug mRNA. Cell lysates were analysed by immunoblotting (IB) (c) or real-time PCR analysis (d) 12 h after different doses of UV radiation. β-Actin protein served as a control for equal loading. Data are shown as means and s.e.m. for three
independent experiments (n = 3). (e) p53+/+ and p53−/− MEFs were exposed to UV (30 J m−2) radiation or left untreated. Cell lysates were analysed by immunoblotting 12 h after UV irradiation. (f, g) HFBs transfected with different concentrations of p53-siRNA oligonucleotides (si-p53-1 or sip53-2) were lysed and analysed by immunoblotting (f) or real-time PCR analysis (g) 72 h after transfection. Data are shown as means and s.e.m. for three independent experiments (n = 3). Numbers below the panels of the immunoblots indicate the densitometric values normalized to the respective β-actin value. Uncropped images of the scans in c, e and f are shown in Supplementary Information, Fig. S4.
demonstrate that mutant p53 represses MDM2 expression, thereby stabilizing Slug protein in cancer cells and promoting cancer cell invasiveness.
specifically promoting cancer cell invasion11,13. To assess the role of mutant p53 in lung cancer progression, we collected a cohort of 79 patients with NSCLC and determined the p53 mutation status in lung tumour specimens by direct DNA sequencing, as well as p53 protein expression with immunohistochemistry (Table 1; Supplementary Information, Fig. S1a). The clinical characteristics of the patients with NSCLC are shown in Supplementary Information, Table S1. Among the 79 patients with NSCLC, 33 samples
Results Mutation of p53 correlates with outcomes of patients with NSCLC Mutations of p53 occur frequently in lung cancer5,6. Mutant p53 has recently been shown to regulate breast and oesophageal cancer progression by nature cell biology volume 11 | number 6 | JUNE 2009
© 2009 Macmillan Publishers Limited. All rights reserved.
695
A RT I C L E S Table 1 Relationship between p53 mutation status and the protein expression of p53, MDM2, Slug, and E-cadherin in tumour specimens from 79 NSCLC patients p53 mutation Protein expression*
Positive (n = 33)
Negative (n = 46)
p53
High
18 (54.5%)
13 (28.3%)
Low
15 (45.5%)
33 (71.7%)
MDM2
High
11 (33.3%)
36 (78.3%)
Low
22 (66.7%)
10 (21.7%)
High
25 (75.8%)
13 (28.3%)
Low
8 (24.2%)
33 (71.7%)
High
5 (15.2%)
39 (84.8%)
Low
28 (84.8%)
7 (15.2%)
Slug E-cadherin
P† 0.018
