TAp63 suppresses metastasis through coordinate regulation of Dicer and miRNAs

Abstract

Aberrant expression of microRNAs (miRNAs) and the enzymes that control their processing have been reported in multiple biological processes including primary and metastatic tumours, 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.

Figure 1. TAp63^−/− mice develop metastatic tumours

a, b, Metastatic mammary adenocarcinoma in lung (LU; a) and brain (BR; b). c, Tumour-free survival curves; n = 30 and P ≤ 0.05. WT, wild type. d, Tumour spectra of the indicated mice genotypes; n = 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 = 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 = 30. Asterisk, statistical difference between TAp63/p53 mutant and p53^+/− or p53^−/− 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.

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 TAp63^−/−;p53^+/− mouse. c, Percentage of SA-β-gal-positive sarcomas; n = 6 of each genotype. d, Quantitative real-time polymerase chain reaction (qRT-PCR) for markers of senescence (PML, p16^Ink4a and p19^Arf) in the indicated tumours; n = 6 of each genotype. e, Percentage of TAp63^−/− and TAp63^+/− tumours with chromosomal aberrations determined by metaphase spreads; n = 6 of each genotype. Frag/DM, fragmented, double-minute chromosome. f, Representative metaphase spread from a TAp63^−/− tumour. Coloured arrows indicate aberrations: rings (red arrows) and double minutes (blue arrow). g, Invasion assays in wild-type MEFs (n = 3) and TAp63^−/− MEFs (n = 3). h, qRT-PCR for TAp63 in the indicated human squamous cell carcinoma (HNSCC) cell lines; n = 5. i, Invasion assays in HNSCC cell lines; n = 5. Error bars indicate s.e.m. Asterisk, P ≤ 0.05.

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. d, qRT-PCR of ChIP assay using keratinocytes and indicating p63-binding site (p63BS) and nonspecific binding site (NSBS) on Dicer promoter; n = 3. e, Luciferase assay for Dicer; n = 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 = 3. i, qRT-PCR for Dicer in MEFs expressing TAp63γ; n = 3. j, Invasion assays of MEFs expressing TAp63γ; n = 3. k, qRTPCR for Dicer in wild-type (TAp63^fl/fl) and TAp63-ablated (TAp63^Δ/Δ) MEFs; n = 3. l, Invasion assays of TAp63^Δ/Δ MEFs; n = 3. m, Northern blot analysis for the indicated mature miRNAs in wild-type and TAp63^−/− MEFs. n, o, Northern blot analysis for miR-10b (n) or miR-130b (o) using MEFs of the indicated genotypes expressing vector (V) or TAp63γ. Error bars indicate s.e.m. Asterisk, P ≤ 0.05.

Figure 4. TAp63 regulates miR-130b in metastasis

a–c, TaqMan reverse-transcriptase-mediated PCR (RT–PCR) for miR-130b and miR-34a in MEFs (a), murine tumours (b) and HNSCCs (c); n = 5. d, qRT-PCR of ChIP assay for miR-130b indicating p63-binding site (p63BS) and non-specific binding site (NSBS); n = 3. e, Luciferase assay for miR-130b; n = 3. f, g, TaqMan RT–PCR for miR-130b in wild-type and TAp63^−/− MEFs expressing the indicated vectors (f) and wild-type (TAp63^fl/fl) and TAp63-ablated (TAp63^Δ/Δ) MEFs (g); n = 3. h, i, Invasion assays of MEFs infected with shRNA-miR-130b (sh-130b) (h) and wild-type and TAp63^−/− MEFs expressing the indicated vectors (i); n = 3. Scrambled sequence (sc) shRNA was used as a negative control. Error bars indicate s.e.m. Asterisk, P ≤ 0.05.