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. 2011 Feb 4;286(5):3915-24.
doi: 10.1074/jbc.M110.162511. Epub 2010 Dec 2.

Role of DeltaNp63gamma in epithelial to mesenchymal transition

Jaime Lindsay  1 , Simon S McDade, Adam Pickard, Karen D McCloskey, Dennis J McCance
Affiliations

Role of DeltaNp63gamma in epithelial to mesenchymal transition

Jaime Lindsay et al. J Biol Chem. .

Abstract

Although members of the p63 family of transcription factors are known for their role in the development and differentiation of epithelial surfaces, their function in cancer is less clear. Here, we show that depletion of the ΔNp63α and β isoforms, leaving only ΔNp63γ, results in epithelial to mesenchymal transition (EMT) in the normal breast cell line MCF10A. EMT can be rescued by the expression of the ΔNp63α isoform. We also show that ΔNp63γ expressed in a background where all the other ΔNp63 are knocked down causes EMT with an increase in TGFβ-1, -2, and -3 and downstream effectors Smads2/3/4. In addition, a p63 binding site in intron 1 of TGFβ was identified. Inhibition of the TGFβ response with a specific inhibitor results in reversion of EMT in ΔNp63α- and β-depleted cells. In summary, we show that p63 is involved in inhibiting EMT and reduction of certain p63 isoforms may be important in the development of epithelial cancers.

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Figures

FIGURE 1.
FIGURE 1.
Knockdown of p63α and β isoforms results in a change of epithelial phenotype MCF10A retroviral cell lines. A, phase-contrast images of the control (SCR), p53-depleted (p53), all p63 isoforms depleted (DBD), and p63α and β isoform-depleted (UTR), showing EMT change in cells with UTR shRNA. B, p63 and p53 protein levels determined by Western blotting (β-actin detected using the mouse anti-actin antibody). C, qRT-PCR analysis of the p63α, β, and γ isoform mRNA expression levels. D, phase-contrast images of MCF10A cells, with knockdown of p63α and β isoforms, using a siRNA targeting a different sequence to the UTR (UTRsi) from that shown in A and B (β-actin detected using the mouse anti-actin antibody). E, confocal microscopy images of MCF10A retroviral cell lines. Blue DAPI nuclear staining and green Alexa Fluor 488 for laminin staining are shown. F, BrDU incorporation of MCF10A retroviral transduced cell lines, showing reduced proliferation of cells with DBD and UTR shRNA. All experiments were carried out in triplicate, and three independent studies were performed. Paired t tests were performed (Fig. 1, C and E), and significant differences with respect to the control, SCR, are shown (*, p < 0.05; **, p < 0.001).
FIGURE 2.
FIGURE 2.
Knockdown of p63α and β isoforms results in an increase in markers of EMT and invasion. A, protein levels of E-cadherin, vimentin, Slug, Snail, and Twist in p-Super retroviral MCF10A cells depleted of p53, all p63 isoforms (DBD) or p63α and β (UTR) using retroviral transduced shRNA (right panel) or depleted p63α and β using siRNA (UTRsi, right panel). Protein loading: mouse anti-actin antibody. B, qRT-PCR analysis of Slug, Snail, and Twist mRNA expression levels. C, invasion analysis of MCF10A retroviral transduced cell lines, showing significant increase in invasion in cells with UTR shRNA. All experiments were carried out in triplicate, and three independent studies were performed. Paired t tests were performed (B and C), and significant differences with respect to the control, SCR, are shown (*, p < 0.05; **, p < 0.001).
FIGURE 3.
FIGURE 3.
Snail is not involved in the EMT phenotype. A, phase-contrast images of SCR and UTR control cells and cells overexpressing Snail, showing that there is no change in phenotype. B, protein levels of pan-TGFβ, E-cadherin, vimentin, Slug, Snail, and Twist analyzed by Western blotting in cells from A. The levels showed no change in morphology when Snail was exogenously expressed. C, phase-contrast images of SCR and UTR control cells and cells with a knockdown in Snail, showing that there is no difference in phenotype. D, protein levels of pan-TGFβ, E-cadherin, vimentin, Slug, Snail, and Twist analyzed by Western blotting, in cells from C. There was no change in morphology when Snail was depleted (β-actin detected using the mouse anti-actin antibody). All experiments were carried out in triplicate, and three independent studies were performed.
FIGURE 4.
FIGURE 4.
TGFβ pathway is involved in the EMT phenotype in MCF10A retroviral cells. A, pan-TGFβ, total Smads2/3/4, and phospho-Smad2/3 protein levels were analyzed using Western blotting and showed that phospho-Smad2/3 is increased in UTR containing cell only (β-actin detected using the rabbit anti-actin antibody). B, immunohistochemistry of phospho-Smad2/3 complex in control cells and cells depleted of p63α and β (UTR) shows that only in UTR-containing cells is the Smad2/3 phosphorylated in the nucleus. C, phase-contrast images of MCF10A cells with SCR and UTR shRNA, with and without the addition of the TGFβ pathway inhibitor SB-431542, show a rescue of the epithelial cell phenotype. D, protein levels of total Smad2 and 3, phospho-Smad2 and 3, Slug, Snail, and Twist were analyzed by Western blotting, in SCR and UTR cells with and without TGFβ inhibitor and showed that inhibition reduced phospho-Smad2/3 levels as well as total levels of Slug and Snail. All experiments were carried out in triplicate, and three independent studies were performed.
FIGURE 5.
FIGURE 5.
Transcriptional control of TGFβ by the ΔNp63γ isoform. A, ChIP for p63 binding to TGFβ-1 intron 1. B, qRT-PCR analysis of p63 chromatin-immunoprecipitates from MCF10A cells. C, qRT-PCR analysis of TGFβ-1, TGFβ-2, and TGFβ-3 mRNA expression levels in MCF10A cells depleted of p53, all isoforms of p63 (DBD) or only p63α and β (UTR). D, Western blotting of H1299 cells transiently transfected with pΔN63 isoforms, showing expression of pΔN63 isoforms (protein loading, mouse anti-actin antibody). E, qRT-PCR analysis of TGFβ-1, TGFβ-2, and TGFβ-3 mRNA expression levels in H1299 cells transiently transfected with different ΔNp63 isoforms, showing that only ΔNp63γ transcriptionally activated the TGFβ promoters. All experiments were carried out in triplicate, and three independent studies were performed. Paired t tests were performed (C and E), and significant differences with respect to the control, SCR, are shown (*, p < 0.05; **, p < 0.001).
FIGURE 6.
FIGURE 6.
Rescue of epithelial phenotype by reexpression of p63α. A, phase-contrast images of SCR and DBD with and without addition of adenoviral expressing ΔNp63γ (ΔNγ) isoform, showing that ΔNp63γ can induce an EMT phenotype. B, protein levels of Slug, Snail, and Twist analyzed by Western blotting in cells from A. C, phase-contrast images of SCR and UTR cells with and without addition of adenoviral ΔNp63α (ΔNα) isoform, showing that ΔNp63α can rescue an epithelial phenotype. D, protein levels of Slug, Snail, and Twist analyzed by Western blotting, in cells from C. β-Actin was detected using the mouse anti-actin antibody. All experiments were carried out in triplicate, and three independent studies were performed.
FIGURE 7.
FIGURE 7.
p63 and TGFβ levels in basal and nonbasal breast cancers. A, p63 levels in normal breast, nonbasal-like, and basal-like breasts tumors, from publically available microarray data (30). There is a significant difference in p63 expression between normal and both basal and non-BLC, p < 0.001. B, TGFβ levels in normal breast, non-basal like and basal like breasts tumors, from the same data set. There is a significance difference between normal and non-BLC, p < 0.006, but no significant difference between normal and basal. Error bars, S.E.
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