TAp63 suppresses metastasis through coordinate regulation ... - Nature

LETTER

doi:10.1038/nature09459

TAp63 suppresses metastasis through coordinate regulation of Dicer and miRNAs Xiaohua Su1*, Deepavali Chakravarti1,2*, Min Soon Cho1,2, Lingzhi Liu1, Young Jin Gi1, Yu-Li Lin1, Marco L. Leung1, Adel El-Naggar3, Chad J. Creighton4, Milind B. Suraokar3, Ignacio Wistuba3 & Elsa R. Flores1,2

Aberrant expression of microRNAs (miRNAs) and the enzymes that control their processing have been reported in multiple biological processes including primary and metastatic tumours1–6, but the mechanisms governing this are not clearly understood. Here we show that TAp63, a p53 family member, suppresses tumorigenesis and metastasis, and coordinately regulates Dicer and miR-130b to suppress metastasis. Metastatic mouse and human tumours deficient in TAp63 express Dicer at very low levels, and we found that modulation of expression of Dicer and miR-130b markedly affected the metastatic potential of cells lacking TAp63. TAp63 binds to and transactivates the Dicer promoter, demonstrating direct transcriptional regulation of Dicer by TAp63. These data provide a novel understanding of the roles of TAp63 in tumour and metastasis suppression through the coordinate transcriptional regulation of Dicer and miR-130b and may have implications for the many processes regulated by miRNAs. The precise roles of p63 in tumour suppression have been hotly debated7–10. Although some studies show p63 overexpression in human cancer7,8,11, some demonstrate a loss of p63 associated with tumour progression and metastasis8,12–14. Much of this controversy is due to the existence of multiple isoforms15. The full-length TA isoform of p63 bears structural and functional similarity to p53, whereas the DN isoforms of p63, which lack the transactivation (TA) domain, act primarily in dominant-negative fashion against p53, TAp63 and TAp73 (ref. 15). These activities suggest that TAp63 is a tumour suppressor gene and DNp63 is an oncogene; however, this has not been directly tested in vivo. To examine whether TAp63 is a tumour suppressor gene, we generated a cohort of 30 mice of each of the following genotypes: TAp632/2, TAp631/2 and wild-type, and aged them for 2.5 years (Fig. 1 and Supplementary Fig. 1a, b). We found that both TAp631/2 mice and TAp632/2 mice developed spontaneous carcinomas and sarcomas (Fig. 1a–d, Supplementary Fig. 1a, b, and Supplementary Table 1) and had a significantly shorter lifespan than the wild-type cohort (Fig. 1c). Paradoxically, we noted that a larger proportion of the TAp632/2 mice (24%) were tumour free compared with TAp631/2 mice (15%) (Fig. 1c, d). These data suggest that TAp63 is a haploinsufficient tumour suppressor gene. Consistent with this finding, sarcomas (n 5 10) and carcinomas (n 5 10) from TAp631/2 mice retained the wild-type allele of TAp63 (Fig. 1e). TAp631/2 and TAp632/2 mice developed highly metastatic tumours (Fig. 1a, b, f, and Supplementary Fig. 1a, b), and 10% of these metastases were found in the brain (Fig. 1b), a rare finding in spontaneous mouse tumour models. Although equivalent numbers of carcinomas metastasized in the TAp632/2 and TAp631/2 mice, a greater number of sarcomas metastasized in TAp631/2 mice than in TAp632/2 mice (Fig. 1f, g). These data again indicate that heterozygosity for TAp63 results in a more severe phenotype in specific tissues. To further understand the mechanisms employed by TAp63 as a tumour suppressor gene, we studied the effects of TAp63 on a p531/2

or p532/2 background8,16–18. Some human tumours show alterations in both p53 and p63 (refs 12, 13), and point mutant p53 binds and functionally inactivates p63 (refs 17, 18). To study these interactions, we generated six cohorts of 30 mice each: TAp632/2;p531/2 and TAp631/2;p531/2 for comparison with p531/2 mice, and TAp632/2;p532/2 and TAp631/2;p532/2 for comparison with p532/2 mice. Unlike p531/2 and p532/2 mice, TAp63/p53 compound mutant mice developed a remarkable number of metastatic tumours (Fig. 1h–k, Supplementary Fig. 1c–f and Supplementary Table 1)8,16. Whereas the TAp631/2;p531/2 mice had a similar tumour spectrum to that of TAp632/2;p531/2 mice (Supplementary Table 1), the sarcomas of the TAp631/2;p531/2 mice were significantly more invasive and metastatic (88%) than those in the TAp632/2;p531/2 cohort (14%) (Fig. 1k). In contrast, the carcinomas in the TAp632/2;p531/2 cohort were more metastatic (44%) than those in the TAp631/2;p531/2 group (29%) (Fig. 1k). We next examined sarcomas (n 5 10) and carcinomas (n 5 10) from TAp631/2;p531/2 mice and found that the wild-type allele of TAp63 was retained (Fig. 1l). We also examined TAp631/2;p531/2 tumours and TAp632/2;p531/2 tumours for p53 loss of heterozygosity (LOH) and found that one out of ten sarcomas showed LOH of p53 in TAp631/2;p531/2 tumours (Fig. 1m). A slightly higher frequency was noted in tumours from TAp632/2;p531/2 sarcomas (one out of eight) and carcinomas (two out of eight), indicating that there is increased selective pressure to lose the wildtype p53 allele in the complete absence of TAp63 (Fig. 1m). However, the frequency of p53 LOH was lower than the previously reported frequency (ranging from 75–90%) of sarcomas from p531/2 mice8,16 and was more reminiscent of the frequency of p53 LOH in mouse models of Li–Fraumeni syndrome17, further supporting the concept that p53 and p63 interact in tumour suppression. We also analysed TAp632/2;p532/2 and TAp631/2;p532/2 mice and found that highly metastatic carcinomas and sarcomas developed in addition to the thymic lymphomas found in the p532/2 mice (Fig. 1j and Supplementary Table 1). Sarcomas (n 5 5) and carcinomas (n 5 5) from these mice retained the wild-type allele of TAp63 (Fig. 1l), providing further evidence that TAp63 is a haploinsufficient tumour suppressor gene. To understand how heterozygosity of TAp63 led to a more severe tumour phenotype than a complete loss of TAp63, we examined whether senescence in the TAp632/2 mice, which age prematurely19, inhibited tumorigenesis or metastasis. We found several examples of TAp632/2;p531/2 mice that developed non-metastatic osteosarcomas together with metastatic carcinomas (Fig. 2a, b and Supplementary Fig. 1e, f). Tumours from these mice, along with sarcomas and carcinomas from TAp631/2;p531/2 mice, were assayed for senescenceassociated markers, namely SA-b-gal (Fig. 2c and Supplementary Fig. 2a, b) or PML (encoding promyelocytic leukemia protein), p16Ink4a and p19Arf (Fig. 2d). We found that osteosarcomas and rhabdomyosarcomas in the TAp632/2;p531/2 mice expressed high

1

Department of Molecular and Cellular Oncology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030, USA. 2Graduate School of Biomedical Sciences, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030, USA. 3Department of Pathology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030, USA. 4Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, Texas 77030, USA. *These authors contributed equally to this work. 9 8 6 | N AT U R E | VO L 4 6 7 | 2 1 O C T O B E R 2 0 1 0

©2010 Macmillan Publishers Limited. All rights reserved

LETTER RESEARCH a

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Figure 2 | TAp63-deficient tumours show high levels of senescence and genomic instability. a, b, Non-metastatic osteosarcoma (a) and metastatic lung adenocarcinoma (LA) in kidney (KI) (b) from TAp632/2;p531/2 mouse. c, Percentage of SA-b-gal-positive sarcomas; n 5 6 of each genotype. d, Quantitative real-time polymerase chain reaction (qRT-PCR) for markers of senescence (PML, p16Ink4a and p19Arf) in the indicated tumours; n 5 6 of each genotype. e, Percentage of TAp632/2 and TAp631/2 tumours with chromosomal aberrations determined by metaphase spreads; n 5 6 of each genotype. Frag/ DM, fragmented, double-minute chromosome. f, Representative metaphase spread from a TAp632/2 tumour. Coloured arrows indicate aberrations: rings (red arrows) and double minutes (blue arrow). g, Invasion assays in wild-type MEFs (n 5 3) and TAp632/2 MEFs (n 5 3). h, qRT-PCR for TAp63 in the indicated human squamous cell carcinoma (HNSCC) cell lines; n 5 5. i, Invasion assays in HNSCC cell lines; n 5 5. Error bars indicate s.e.m. Asterisk, P # 0.05.

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Figure 1 | TAp632/2 mice develop metastatic tumours. a, b, Metastatic mammary adenocarcinoma in lung (LU; a) and brain (BR; b). c, Tumour-free survival curves; n 5 30 and P # 0.05. WT, wild type. d, Tumour spectra of the indicated mice genotypes; n 5 30. e, LOH analysis of TAp63 in sarcomas (S) and carcinomas (C) from the indicated genotypes. f, g, Percentage of metastatic sarcomas and carcinomas (f) and multiple malignancies (g); n 5 30. h, i, Metastatic squamous cell carcinoma (SCC) on skin (h) and in lung (LU; i). j, k, Tumour spectrum (j) and metastases (k) of the indicated mice genotypes; n 5 30. Asterisk, statistical difference between TAp63/p53 mutant and p531/2 or p532/2 mice; two asterisks, statistical difference between two double-mutant genotypes; P # 0.05. l, m, PCR analysis for LOH of TAp63 (l) or p53 (m) in carcinomas (C) and sarcomas (S) of the indicated genotypes.

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RESEARCH LETTER result of the acquisition of further genetic alterations. Although a decrease in TAp63 expression promotes metastasis in both tumour types, senescence is triggered in sarcomas, thus inhibiting progression, whereas in carcinomas, genomic instability ensues, permitting further genetic events favouring tumour progression. Given the high level of metastasis in TAp632/2 mice, we examined whether TAp632/2 mouse embryonic fibroblasts (MEFs) had an enhanced invasive ability. TAp632/2 MEFs showed a 1.8-fold increase in invasion (Fig. 2g). To assess whether this finding could be generalized to human tumours, we examined primary human head and neck squamous-cell carcinomas (HNSCCs) (cell lines 10A, 17A and 22A) and matched metastatic lesions with markedly low levels of TAp63 (Fig. 2h) (cell lines 10B, 17B and 22B)20. Tumours with low levels of TAp63 showed an increased invasive ability (Fig. 2i), indicating that TAp63 is a critical regulator of cancer metastasis.

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Figure 3 | TAp63 regulates metastasis by transcriptional activation of Dicer. a–c, qRT-PCR of Dicer mRNA in murine tumours (a), MEFs (b) and HNSCC cell lines (c); n 5 5. d, qRT-PCR of ChIP assay using keratinocytes and indicating p63-binding site (p63BS) and nonspecific binding site (NSBS) on Dicer promoter; n 5 3. e, Luciferase assay for Dicer; n 5 5. V, pcDNA3 vector. f, Western blot analysis for Dicer expression in MEFs infected with the indicated vectors. g, h, Invasion assays of MEFs expressing Dicer (g) and MEFs expressing shRNA-Dicer (h); n 5 3. i, qRT-PCR for Dicer in MEFs expressing

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levels of senescence markers, whereas the same tumour types in the TAp631/2;p531/2 mice did not (Fig. 2c, d). None of the carcinomas from TAp632/2;p531/2 or TAp631/2;p531/2 mice was positive for these markers, indicating that TAp63 deficiency has a tissue-specific function in the induction of senescence. These results correlated with the level of aggressiveness and metastatic potential of these tumours. Genomic instability in tissues of TAp632/2 mice has been shown to be high19. To examine whether there is a similarly high level of genomic instability in tumours from TAp632/2;p531/2 mice and whether this is correlated with tumour aggressiveness and metastasis, we performed c-H2AX immunostaining and metaphase spreads on tumours from TAp63 mutant mice (Fig. 2e, f and Supplementary Fig. 2c–g). We did indeed find that TAp632/2 carcinomas showed high levels of c-H2AX, polyploid cells and chromosomal aberrations, suggesting that carcinomas lacking TAp63 overcome senescence in epithelial tissues as a

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TAp63c; n 5 3. j, Invasion assays of MEFs expressing TAp63c; n 5 3. k, qRTPCR for Dicer in wild-type (TAp63fl/fl) and TAp63-ablated (TAp63D/D) MEFs; n 5 3. l, Invasion assays of TAp63D/D MEFs; n 5 3. m, Northern blot analysis for the indicated mature miRNAs in wild-type and TAp632/2 MEFs. n, o, Northern blot analysis for miR-10b (n) or miR-130b (o) using MEFs of the indicated genotypes expressing vector (V) or TAp63c. Error bars indicate s.e.m. Asterisk, P # 0.05.

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LETTER RESEARCH Our findings that TAp631/2 tumours are more aggressive than those from TAp632/2 mice are reminiscent of the phenotype of Dicer conditional knock out (Dicer1fl/1) mice intercrossed to the KrasLSL-G12D lung cancer model21, which indicate that Dicer is haploinsufficient for tumour suppression4, in a similar manner to our previous observations for TAp63. Additionally, Dicer12/2 mice show embryonic lethality (before embryonic day E8.5) similar to that in TAp632/2 mice on an enriched C57BL/6 background19; 35% of TAp632/2 embryos die between E6.5 and E8.5. Given these similarities, we examined whether messenger RNA levels of Dicer are dysregulated in tumours and cells lacking TAp63. TAp632/2 osteosarcomas, lung adenocarcinomas and mammary adenocarcinomas (n 5 6) and MEFs expressed significantly lower levels of Dicer than p532/2 tumours and MEFs (Fig. 3a, b and Supplementary Fig. 3), suggesting that TAp63 is required for the transcription of Dicer. Similarly, we found that metastatic human HNSCCs (n 5 46), lung adenocarcinomas and squamous cell carcinomas (n 5 92) and mammary adenocarcinomas (n 5 43) with low levels of TAp63 had low Dicer expression (Fig. 3c, Supplementary Fig. 4 and Supplementary Tables 2 and 3). These data indicate a critical role for TAp63 in the regulation of Dicer in metastasis in human cancer. Consistent with this is the observation that Dicer and Drosha levels have been shown to be low in human cancer20. To determine whether Dicer is a transcriptional target of TAp63, we performed chromatin immunoprecipitation (ChIP) analysis for a site matching the p63 consensus binding site (Supplementary Table 4). We found that p63, and not p53, bound to the Dicer promoter (Fig. 3d and Supplementary Fig. 5a), suggesting that Dicer is a direct transcriptional target of p63. In addition, all isoforms of TAp63 (a, b and c) were able to transactivate a Dicer–luciferase reporter gene with the p63-binding site (Fig. 3e), further indicating that TAp63 regulates Dicer transcriptionally. To understand whether TAp63 controls metastasis through transcriptional regulation of Dicer, we re-expressed Dicer in TAp632/2

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MEFs (Fig. 3f). These cells lost their ability to invade (Fig. 3g). Conversely, wild-type MEFs expressing a Dicer short hairpin RNA (shRNA)22 (Fig. 3f) showed increased invasiveness (Fig. 3h). These data indicate that low levels of Dicer lead to increased cell invasion, similar to that observed in TAp63-deficient cells. To further explore the connection between TAp63 and Dicer in metastasis, we modulated levels of TAp63 in MEFs by expressing TAp63c in TAp632/2 MEFs and human HNSCCs (Supplementary Figs 5b and 6a) and measured Dicer expression and invasion potential. Levels of Dicer increased to wild-type levels in TAp632/2 cells expressing TAp63c (Fig. 3i and Supplementary Fig. 6b), which resulted in decreased invasion potential (Fig. 3j and Supplementary Fig. 6c). We also acutely deleted TAp63 after the introduction of adenovirus-Cre in TAp63fl/fl MEFs (Supplementary Fig. 5c). These TAp63D/D MEFs showed concomitant downregulation of Dicer (Fig. 3k) and a twofold increase in cellular invasion (Fig. 3l), indicating that regulation of Dicer by TAp63 is critical in the suppression of invasion. Because Dicer expression is low in the absence of TAp63, we assessed the processing of miRNAs that have a recognized role in lung and mammary adenocarcinoma metastasis1–3,23, namely miR-10b, miR-200b, miR200c, miR34a and miR-130b, in TAp632/2 MEFs and found that their processing is defective (Fig. 3m). To examine whether modulating levels of TAp63 in cells would affect miRNA processing, we scored for processing of miR-10b in TAp632/2 MEFs reconstituted with TAp63c and in TAp63-ablated (TAp63D/D) MEFs. When TAp63 was re-expressed, we observed a rescue of miRNA processing, and when TAp63 was acutely deleted, we observed a diminution of miRNA processing (Fig. 3n). The precursor of miR-130b (pre-miR-130b) was present at low levels in TAp632/2 MEFs (Fig. 3o), indicating that the primary transcript of miR-130b may be expressed at low levels in the absence of TAp63. We found that levels of miR-130b were downregulated in TAp632/2 MEFs, suggesting a role for TAp63 in its regulation (Fig. 4a). We

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Figure 4 | TAp63 regulates miR-130b in metastasis. a–c, TaqMan reversetranscriptase-mediated PCR (RT–PCR) for miR-130b and miR-34a in MEFs (a), murine tumours (b) and HNSCCs (c); n 5 5. d, qRT-PCR of ChIP assay for miR-130b indicating p63-binding site (p63BS) and non-specific binding site (NSBS); n 5 3. e, Luciferase assay for miR-130b; n 5 3. f, g, TaqMan RT–PCR for miR-130b in wild-type and TAp632/2 MEFs expressing the indicated

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vectors (f) and wild-type (TAp63 ) and TAp63-ablated (TAp63D/D) MEFs (g); n 5 3. h, i, Invasion assays of MEFs infected with shRNA-miR-130b (sh130b) (h) and wild-type and TAp632/2 MEFs expressing the indicated vectors (i); n 5 3. Scrambled sequence (sc) shRNA was used as a negative control. Error bars indicate s.e.m. Asterisk, P # 0.05. fl/fl

2 1 O C T O B E R 2 0 1 0 | VO L 4 6 7 | N AT U R E | 9 8 9


RESEARCH LETTER reasoned that other miRNAs, such as miR-34a, which have been shown to be a transcriptional target of p53 (ref. 23), may also be a target of TAp63. Indeed, miR-130b and miR-34a were downregulated in the absence of TAp63 (Fig. 4a–c and Supplementary Fig. 7a). miR130b was also low in high-grade lung adenocarcinomas and HNSCCs with low levels of TAp63 and Dicer (Supplementary Table 3). To assess whether p63 binds to the promoters of these miRNAs, we performed a ChIP analysis with identified putative p63-binding sites (Supplementary Table 4). We found a significant level of p63 binding at both the miR-130b and miR-34a promoters (Fig. 4d and Supplementary Fig. 7b–d). To determine whether p63 can directly transactivate miR-130b and miR-34a, we performed luciferase assays (Fig. 4e and Supplementary Fig. 7e) and found that TAp63 transactivated the miR130b reporter. We modulated the levels of TAp63 by overexpression or acute deletion in MEFs and found that levels of miR-130b correlated with levels of TAp63 (Fig. 4f, g). Taken together, these data indicate that miR-130b is a direct target of TAp63. To understand whether regulation of miR-130b by TAp63 has a function in the metastatic phenotype, we modulated levels of miR130b in MEFs (Supplementary Fig. 7f–h) and assessed invasion (Fig. 4h, i). Wild-type MEFs with low levels of miR-130b showed increased invasion (Fig. 4h), and TAp632/2 MEFs expressing both miR-130b and Dicer showed decreased invasion (Fig. 4i) comparable to that of wild-type MEFs. This suggests that coordinate regulation of both Dicer and miR-130b by TAp63 is critical in suppressing metastasis. We have shown that TAp63 is a suppressor of tumorigenesis and metastasis and that its complete inactivation results in tissue-specific antagonistic pleiotropy with respect to tumour progression including genomic instability and activation of senescence. Both the spectrum and the highly metastatic nature of tumours in TAp63-deficient mice are consistent with a role for TAp63 as a master regulator of metastasis. Furthermore, we have shown a role for transcriptional regulation by TAp63 of Dicer and miR-130b in metastasis. Given the diverse biological roles of both Dicer and TAp63, a complete understanding of the regulation of Dicer by TAp63 in other processes is a key question for the future.

METHODS SUMMARY

Generation of mice and tumour analysis. TAp632/2 mice19 were intercrossed with p532/2 mice16 to generate compound mutant TAp63/p53 mice on an enriched C57BL/6 background ((C57BL/6 5 95%) and (129/SvJae 5 5%)). For analysis of tumour formation and metastatic disease, 30 mice of each genotype were aged for 2.5 years. Moribund mice were killed, following the approved guidelines of the IACUC at the University of Texas M. D. Anderson Cancer Center and analysed histologically by haematoxylin/eosin (H&E) staining as described previously8. Tumour-free survival of mice was plotted with PRISM5 software (GraphPad). Migration and invasion assay. MEFs or mouse and human tumour cell lines were resuspended in 500 ml of DMEM with 10% FBS and 1 3 penicillin/streptomycin (5 3 104 cells) and added to the top of each chamber containing BD BioCoat cell culture inserts (354578; BD Biosciences) or Matrigel Invasion Chamber (354480; BD Biosciences)19,24. Non-invasive cells were removed from the upper chamber. Cells remaining on the membrane were fixed, stained with Diff-Quik (Dade Behring, Inc.), and counted with a Zeiss AxioObserver A1 inverted microscope. Full Methods and any associated references are available in the online version of the paper at www.nature.com/nature. Received 27 February; accepted 24 August 2010. 1.

Baffa, R. et al. MicroRNA expression profiling of human metastatic cancers identifies cancer gene targets. J. Pathol. 219, 214–221 (2009).

2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.

Gibbons, D. L. et al. Contextual extracellular cues promote tumor cell EMT and metastasis by regulating miR-200 family expression. Genes Dev. 23, 2140–2151 (2009). Ma, L., Teruya-Feldstein, J. & Weinberg, R. A. Tumour invasion and metastasis initiated by microRNA-10b in breast cancer. Nature 449, 682–628 (2007). Kumar, M. S. et al. Dicer1 functions as a haploinsufficient tumor suppressor. Genes Dev. 23, 2700–2704 (2009). Lu, J. et al. MicroRNA expression profiles classify human cancers. Nature 435, 834–8 (2005). Merritt, W. M. et al. Dicer, Drosha, and outcomes in patients with ovarian cancer. N. Engl. J. Med. 359, 2641–2650 (2008). Flores, E. R. The roles of p63 in cancer. Cell Cycle 6, 300–304 (2007). Flores, E. R. et al. Tumor predisposition in mice mutant for p63 and p73: evidence for broader tumor suppressor functions for the p53 family. Cancer Cell 7, 363–373 (2005). Keyes, W. M. et al. p63 heterozygous mutant mice are not prone to spontaneous or chemically induced tumors. Proc. Natl Acad. Sci. USA 103, 8435–8440 (2006). Yang, A., Kaghad, M., Caput, D. & McKeon, F. On the shoulders of giants: p63, p73 and the rise of p53. Trends Genet. 18, 90–95 (2002). Hibi, K. et al. AIS is an oncogene amplified in squamous cell carcinoma. Proc. Natl Acad. Sci. USA 97, 5462–5467 (2000). Park, B. J. et al. Frequent alteration of p63 expression in human primary bladder carcinomas. Cancer Res. 60, 3370–3374 (2000). Urist, M. J. et al. Loss of p63 expression is associated with tumor progression in bladder cancer. Am. J. Pathol. 161, 1199–1206 (2002). Park, H. R. et al. Low expression of p63 and p73 in osteosarcoma. Tumori 90, 239–243 (2004). Yang, A. et al. p63, a p53 homolog at 3q27–29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities. Mol. Cell 2, 305–316 (1998). Jacks, T. et al. Tumor spectrum analysis in p53-mutant mice. Curr. Biol. 4, 1–7 (1994). Lang, G. A. et al. Gain of function of a p53 hot spot mutation in a mouse model of Li– Fraumeni syndrome. Cell 119, 861–872 (2004). Olive, K. P. et al. Mutant p53 gain of function in two mouse models of Li–Fraumeni syndrome. Cell 119, 847–860 (2004). Su, X. et al. TAp63 prevents premature aging by promoting adult stem cell maintenance. Cell Stem Cell 5, 64–75 (2009). Maruya, S. et al. Differential methylation status of tumor-associated genes in head and neck squamous carcinoma: incidence and potential implications. Clin. Cancer Res. 10, 3825–3830 (2004). Jackson, E. L. et al. Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras. Genes Dev. 15, 3243–3248 (2001). Kumar, M. S. et al. Impaired microRNA processing enhances cellular transformation and tumorigenesis. Nature Genet. 39, 673–677 (2007). He, L. et al. A microRNA component of the p53 tumour suppressor network. Nature 447, 1130–1134 (2007). Flores, E. R. et al. p63 and p73 are required for p53-dependent apoptosis in response to DNA damage. Nature 416, 560–564 (2002).

Supplementary Information is linked to the online version of the paper at www.nature.com/nature. Acknowledgements We thank C.-g. Liu for technical assistance with miRNA analyses and A. Multani for metaphase analyses (funded by NCI CA16672); K. Y. Tsai for scientific discussions and comments on the manuscript; and D. Yu, J. Jackson and S. Rehman for reagents and advice. This work was supported by grants to E.R.F. from the American Cancer Society (RSG-07-082-01-MGO), the Susan G. Komen Foundation (BCTR600208) and the Hildegardo E. and Olga M. Flores Foundation. This work was supported in part by an NCI-Cancer Center Core Grant (CA-16672) (University of Texas M. D. Anderson Cancer Center), a Career Development Award from the Genitourinary Cancer SPORE (P50CA091846) to E.R.F., Lung Cancer SPORE (P50CA070907) to I.W. and U01DE019765 to A.E.-N. E.R.F. is a scholar of the Rita Allen Foundation and the V Foundation for Cancer Research. Author Contributions X.S., D.C. and E.R.F. designed the experiments and analysed the data. X.S., D.C., M.S.C., L.L., Y.J.G., Y.-L.L. and M.L.L. performed the experiments. A.E.-N. performed pathology analysis and provided human HNSCC samples. M.B.S. and I.W. provided human lung adenocarcinoma pathology information and RNA samples. C.J.C. performed statistical analysis on miRNA data. X.S., D.C. and E.R.F. wrote the manuscript. All authors discussed the results and commented on the manuscript. Author Information Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests. Readers are welcome to comment on the online version of this article at www.nature.com/nature. Correspondence and requests for materials should be addressed to E.R.F. ([email protected]).

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Generation of mice and tumour analysis. TAp63 mice were intercrossed with p532/2 mice16 to generate compound mutant TAp63/p53 mice on an enriched C57BL/6 background ((C57BL/6 5 95%) and (129/SvJae 5 5%)). For analysis of tumour formation and metastatic disease, 30 mice of each genotype were aged for 2.5 years. Moribund mice were killed, following the approved guidelines of the IACUC at the University of Texas M. D. Anderson Cancer Center and analysed histologically by haematoxylin/eosin (H&E) staining as described previously8. Tumour-free survival of mice was plotted with PRISM5 software (GraphPad). Migration and invasion assay. MEFs or mouse and human tumour cell lines were resuspended in 500 ml of DMEM with 10% FBS and 1 3 penicillin/streptomycin (5 3 104 cells) and added to the top of each chamber containing BD BioCoat cell culture inserts (354578; BD Biosciences) or Matrigel Invasion Chamber (354480; BD Biosciences)19,24. Non-invasive cells were removed from the upper chamber. Cells remaining on the membrane were fixed, stained with Diff-Quik (Dade Behring, Inc.), and counted with a Zeiss AxioObserver A1 inverted microscope. Mouse and human tumour cell lines. Tumours were excised from p53 and TAp63 mutant mice at the time of necropsy. Half of the tumour was fixed in 10% formalin and analysed histologically by H&E staining. The other half of the tumour was minced and treated for 20 min with 0.25% trypsin-EDTA solution at 37 uC. Cells were suspended in DMEM medium containing 10% fetal bovine serum (FBS) and 1 3 penicillin/streptomycin. Human primary and metastatic squamous cell carcinomas (cell lines 10A, 10B, 17A, 17B, 22A and 22B) were cultured similarly20. Immunocytochemistry. Cells were fixed on chamber slides for 30 min at room temperature (25 uC) with 4% paraformaldehyde in PBS and incubated in blocking buffer (8% FBS, 0.3% Triton X-100 in PBS) for 30 min at room temperature as described previously19. Samples were incubated overnight in anti-cH2AX (dilution 1:100; Upstate Biotechnology, Inc.) at 4 uC under humidified conditions, followed by incubation for 1 h with secondary antibodies (goat anti-mouse Texas Red, dilution 1:500; Jackson ImmunoResearch Laboratories) at room temperature. 49,6-Diamidino-2-phenylindole (DAPI; Pierce Biotechnology) was used as a nuclear counterstain. Photomicrographs were taken with a Zeiss AxioObserver A1 inverted fluorescence microscope. Immunofluorescence and immunohistochemistry. Paraffin-embedded sections of tumours were prepared as described previously8. Tissue microarrays containing primary and metastatic HNSCCa (HN242), lung adenocarcinomas and squamous cell carcinomas (LC1005) and mammary adenocarcinomas (BR480) were obtained from US Biomax. For detection of Dicer, sections were incubated overnight with anti-Dicer (dilution 1:100; Abcam) at 4 uC in a humidified chamber followed by incubation for 1 h with goat anti-mouse-fluorescein isothiocyanate (dilution 1:500; Jackson ImmunoResearch Laboratories) at room temperature for immunofluorescence or using the Vectastain universal ABC Kit (Vector Labs), followed by the DAB kit (Vector Labs) for immunohistochemistry. For detection of TAp63, sections were incubated with anti-TAp63 (dilution 1:100; gift from C. Prives) followed by detection for immunohistochemistry as described above. For detection of cH2AX in frozen tumour sections, tumours were fixed in 4% paraformaldehyde, frozen in OCT medium (Tissue Tek) and detected by incubation in anti-cH2AX (dilution 1:100; Upstate Biotechnology, Inc.) followed by incubation for 1 h with goat anti-mouse Texas Red (dilution 1:500; Jackson ImmunoResearch Laboratories) at room temperature. DAPI (Pierce Biotechnology) was used as a nuclear counterstain for immunofluorescence or haematoxylin (Vector) for immunohistochemistry. Photomicrographs were taken with a Ziess Axioplan2 imaging fluorescence microscope. Human HNSCCs and lung adenocarcinomas. Total RNA was isolated from 25 human HNSCCs and 19 lung adenocarcinomas of various grades (well differentiated (grade I), moderately differentiated (grade II) and poorly differentiated and invasive (grade III)) for qRT-PCR using primers for TAp63, Dicer and miR-130b as described below. SA-b-gal staining. SA-b-gal staining was performed on frozen sections of tumours as described previously19. qRT-PCR. Total RNA was isolated from mouse MEFs, human squamous cell carcinomas and cell lines, or mouse tumours and tumour cell lines by using TRIzol LS Reagent (Invitrogen). qRT-PCR was performed with a StepOnePlus real-time PCR system (Applied Biosystems), a SuperScript First-Strand Synthesis System (Invitrogen) and Power SYBR Green PCR Master Mix (Applied Biosystems) in accordance with the manufacturers’ protocols. The following primers were used: human TAp63 (ref. 25), human DNp63 (ref. 25) and murine or human Dicer (ref. 26). Murine or human glyceraldehyde-3-phosphate dehydrogenase was used as an internal control19,25. Average cycle threshold (Ct) values were calculated from triplicate reactions of three biological replicates.

Cytogenetics. Tumour cells at 70% confluence were treated for 3 h with 0.02 mg ml21 colcemid and prepared as described previously19. Images were captured and processed with MetaMorph Premier (Molecular Devices) using a Nikon Eclipse E400 microscope. Northern blot analysis. Total RNA was fractionated on a denaturing 15% polyacrylamide gel containing 8 M urea, and transferred to Hybond-N1 membrane (Amersham Biosciences) by a semi-dry gel transfer method. Membranes were hybridized with end-labelled [c-32P]ATP miRCURY LNA detection probes (Exiqon) in ULTRA hyb-Oligo Hybridization Buffer (Ambion) at 37 uC. Membranes were washed twice for 5 min in 2 3 SSC, 0.1% SDS at 37 uC followed by a prolonged wash for 15 min at 25 uC, and then exposed to X-ray film overnight. miRNA TaqMan assays. Applied Biosystems TaqMan miRNA assays were used to detect and quantify mature miRNAs using looped-primer real-time PCR. Total RNA samples were prepared from tumour cell lines and MEFs with TRIzol LS Reagent (Invitrogen). Total RNA (1 mg) was used to synthesize complementary DNA with the Two-Step TaqMan MicroRNA Assay kit (Applied Biosystems) in accordance with the protocol. qRT-PCR was performed with the Step One Real Time PCR System, TaqMan PCR master mix, and TaqMan primers for the specific miRNAs (Applied Biosystems). Each sample was run in triplicate. Ct values for miRNAs were calculated and normalized to Ct values for RNU6B. ChIP assay. ChIP was performed with nuclear extracts from wild-type and p632/2;ArfG/G keratinocytes as described previously19,24,27. qRT-PCR was performed with the following primer sequences specific for p63-binding sites: murine Dicer, 21433, 59-AGGCTGGCCTTGATCTGTGA-39 (forward) and 21333, 59-CACAT CCTCGGCTGTCTTTCA-39 (reverse); mmu-miR-34a, 1367, 59-TAGCCAAACA GCCACCATCTT-39 (forward) and 1457, 59-CCCCAGCCCTCCACAAG-39 (reverse); and mmu-miR-130b, 2850, 59-CACGTGAGTAACTGGTCTGGGA TA-39 (forward) and 2763, 59-TCCTAACAGATTCTCCTGCCTAGAA-39 (reverse). Primer sequences for non-specific binding sites were as follows: murine Dicer promoter, 22560, 59-CGAACCCAGAGAGTCCACAAG-39 (forward) and 22499, 59-CCCCATCCCCGACACTTAC-39 (reverse); mmu-miR-34a promoter, 1732, 59-AAGCGGGTTTCAAGTGCATCTCAG-39 (forward) and 1796, 59-TC AGGCTACTAAACCAGTTGCCCT-39 (reverse); and mmu-miR-130b promoter, 21431, 59-AAATGTCCCATCCTGGAGGAGCAA-39 (forward) and 21323, 59-TCACCAAATTAGCGAGGGCTCTGA-39 (reverse). Primers for the p53binding site for miR-34a were used as described previously23. Lentiviral infection. Lentivirus-based vectors (2 mg) containing shRNAs (SBI System Biosciences) for mmu-miR-34a, mmu-miR-126, mmu-miR-130b or scramble sequence (as control) and tagged with green fluorescent protein (GFP) or pDESTmycDICER28 were transfected into 293T cells along with 2 mg of each vector required for lentivirus packaging (pCMV-VSVG, pRSV-REV or pMDLg/pRRE) using Fugene HD (Roche) in accordance with the manufacturer’s protocol. After transfection of 293T cells, supernatants containing the lentivirus were collected, filtered and added to target MEF or tumour cells for 24 h in the presence of 8 mg ml21 Polybrene. At 24 h after infection, puromycin was added to the media at 3 mg ml21 for 7 days. Infection efficiency of the cells was calculated by dividing the number of GFP-expressing cells by the total number of cells, with the use of a Zeiss AxioObserver A1 inverted fluorescence microscope. The infection efficiency was calculated to be 100% in all experiments. Infected cells were analysed by TaqMan assay to determine the level of miRNA silencing. These cells were further analysed by migration and invasion assays. All experiments were performed in triplicate. Retroviral infection (pSuper mouse Dicer1). Wild-type and TAp632/2 MEFs (106) were infected with pSuper mouse Dicer1 (Addgene) as described previously22. Adenovirus-Cre infection. TAp63fl/fl MEFs (106) were plated on 10-cm dishes and infected with 5 3 103 Ad-Cre–GFP (Vector Development Laboratory) particles per cell. Infection efficiency was calculated on the basis of expression of GFP and found to be 98%. To verify recombination, qRT-PCR was performed for TAp63 mRNA. Dual luciferase reporter assay. Luciferase assays were performed with p532/2;p632/2 MEFs as described previously29. To generate the luciferase reporter genes pGL3-Dicer, pGL3-miR-130b and pGL3-miR-34a, the DNA fragment containing the p63-binding site identified by ChIP was amplified from C57BL/6 genomic DNA by PCR with the following primers containing 59 NheI and 39 BglII cloning restriction enzyme sites: DICER1, 59-GCTAGCATGTGCCAGG GCTTTGGCATGTA-39 (forward) and 59-AGATCTTCCTGGAACTTGCTCTG TACACCA-39 (reverse); mmu-miR-34a, 59-GCTAGCTGGAGTGTGAGCACT TCTGGCTAA-39 (forward) and 59-AGATCTTGGACATTCAGGTGAGGGT CTTGT-39 (reverse); and mmu-miR-130b, 59-GCTAGCATGGTTAAAGATG GAGCCGAGGGA-39 (forward) and 59-AGATCTTCTCCTGCCTAGAAGAG CAGAACT-39 (reverse).


RESEARCH LETTER Statistics. Data are represented as means and s.e.m. All experiments were performed in triplicate. Student’s t-test was used for comparison between two groups or one-way analysis of variance test. P 5 0.05 was considered significant. 25. Pruneri, G. et al. The transactivating isoforms of p63 are overexpressed in highgrade follicular lymphomas independent of the occurrence of p63 gene amplification. J. Pathol. 206, 337–345 (2005).

26. Mudhasani, R. et al. Loss of miRNA biogenesis induces p19Arf-p53 signaling and senescence in primary cells. J. Cell Biol. 181, 1055–1063 (2008). 27. Su, X. et al. Rescue of key features of the p63-null epithelial phenotype by inactivation of Ink4a and Arf. EMBO J. 28, 1904–1915 (2009). 28. Landthaler, M. et al. Molecular characterization of human Argonaute-containing ribonucleoprotein complexes and their bound target mRNAs. RNA 14, 2580–2596 (2008). 29. Lin, Y. L. et al. p63 and p73 transcriptionally regulate genes involved in DNA repair. PLoS Genet. 5, e1000680 (2009).
