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. 2014 Jan 9;33(2):212-24.
doi: 10.1038/onc.2012.564. Epub 2012 Dec 17.

ΔNp63α regulates Erk signaling via MKP3 to inhibit cancer metastasis

J Bergholz  1 , Y Zhang  2 , J Wu  3 , L Meng  3 , E M Walsh  3 , A Rai  3 , M Y Sherman  3 , Z-X Jim Xiao  1
Affiliations

ΔNp63α regulates Erk signaling via MKP3 to inhibit cancer metastasis

J Bergholz et al. Oncogene. .

Abstract

Reduced expression of the p53 family member p63 has been suggested to play a causative role in cancer metastasis. Here, we show that ΔNp63α, the predominant p63 isoform, plays a major role in regulation of cell migration, invasion and cancer metastasis. We identified mitogen-activated protein (MAP) kinase phosphatase 3 (MKP3) as a downstream target of ΔNp63α that is required for mediating these effects. We show that ΔNp63α regulates extracellular signal-regulated protein kinases 1 and 2 (Erk1/2) activity via MKP3 in both cancer and non-transformed cells. We further show that exogenous ΔNp63α inhibits cell invasion and is dependent on MKP3 upregulation for repression. Conversely, endogenous pan-p63 ablation results in increased cell migration and invasion, which can be reverted by reintroducing the ΔNp63α isoform alone, but not by other isoforms. Interestingly, these effects require Erk2, but not Erk1 expression, and can be rescued by enforced MKP3 expression. Moreover, MKP3 expression is reduced in invasive cancers, and reduced p63 expression increases metastatic frequency in vivo. Taken together, these results suggest an important role for ΔNp63α in preventing cancer metastasis by inhibition of Erk2 signaling via MKP3.

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Conflict of interest statement

Conflict of interest

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Wild type ΔNp63α, but not disease-associated mutants, up-regulates MKP3 expression. Hs-578T cells were transduced with a recombinant retrovirus encoding wild type murine ΔNp63α (WT), a mutant derivative (C306R or C526W), or a vector control (V). Puromycin-resistant cells were subjected to gene expression profiling by Affymetrix array in two independent experiments performed in triplicate (a), to Q-PCR analysis for MKP3 mRNA levels (b), or to western blotting for MKP3 protein expression (c). The Q-PCR data are presented as means and standard errors (SE) from three independent experiments performed in triplicate. *: P < 0.05 compared to V; **: P < 0.01 compared to V; #: P < 0.05 compared to WT; ##: P < 0.01 compared to WT. (d and e) Box plots representing p63 and MKP3 expression from breast tumor samples at different stages. Oncomine™ (Compendia Bioscience, Ann Arbor, MI) was used for analysis and visualization. (d) Zhao Breast dataset. (e) TCGA Breast dataset.
Figure 2
Figure 2
ΔNp63 reduces phosphorylated Erk1/2 in nuclei and attenuates Erk1/2 signaling. MDA-MB-231 cells stably expressing ΔNp63α (p63) or an empty vector control (V) were subjected to Q-PCR analysis for MKP3 mRNA levels (a) and protein expression (b). The Q-PCR data are presented as means and SE from three experiments performed in triplicate (P = 0.032) (c) MDA-MB-231 cells were transduced with retrovirus encoding ΔNp63α. A mixed population of ΔNp63α-positive [p63 (+)] and ΔNp63α-negative [p63 (−)] cells was assessed by immunofluorescence for p63 (green) expression and phosphorylated Erk1/2 (red) levels, and the nuclei were counterstained with DAPI (blue). Arrows denote cells with high levels of nuclear phosphorylated Erk1/2. Scale bar = 20 µm. (d) Cells with high levels of phosphorylated Erk1/2 were scored for both p63 (−) and p63 (+) cells. Data are presented as percentage of cells with high phosphorylated Erk1/2 over total p63 (−) or p63 (+) cells. At least 100 cells in total from five random fields were scored for each of six experiments. Results are presented as means and SE. (e) MDA-MB-231 cells stably expressing ΔNp63α (p63) or vector control (V) were subjected to cellular fractionation to separate the nuclear from the cytosolic fractions, which were then subjected to western blotting as shown. (f) MDA-MB-231 cells stably expressing ΔNp63α (p63) or vector control (V) were subjected to Q-PCR analysis for mRNA levels of MMP1 and MMP9. Results are presented as means and SE from three experiments performed in triplicate.
Figure 3
Figure 3
ΔNp63α inhibits cell invasion in an MKP3-dependent manner. (a) MDA-MB-231 cells stably expressing ΔNp63α (p63) or a vector control (V) were subjected to transwell assays for cell invasion. Twenty-four hours after plating, invading cells were fixed and stained with crystal violet and photographed under a light microscope. Representative pictures are shown. (b) Results from invasion assay were quantitated as described in the Materials and Methods section and presented as means and SE from three independent experiments. (c) Stable MDA-MB-231 cells were transduced with recombinant lentivirus encoding an shRNA specific for MKP3 (shMKP3) or for GFP as a control (shC) and subjected to western blotting. (d and e) Stable MDA-MB-231 cells were subjected to transwell assay for invasiveness. Results are presented as means and SE from three independent experiments. (f) Stable MDA-MB-231 cells were subjected to Q-PCR analysis for mRNA levels of MMP1 and MMP9.
Figure 4
Figure 4
ΔNp63α regulates MKP3 expression and Erk1/2 activity. (a) MCF-10A and FaDu cells were infected with recombinant lentivirus encoding shRNA specific for pan-p63 (shp63) or for GFP as a control (shC). ΔNp63 and MKP3 mRNA levels were analyzed by Q-PCR. Results are presented as means and SE from three independent experiments performed in triplicate. (b) Whole-cell lysates were subjected to western blotting as indicated. (c) Diagram of the MKP3 gene and promoter locus highlighting a putative p63 binding site, and the primers (arrows) used for PCR amplification of the immunoprecipitated DNA. (d) Binding of p63 to this putative binding site was assessed in FaDu cells by chromatin immunoprecipitation (ChIP) using a specific p63 antibody (4A4) or a control mouse IgG, followed by PCR amplification. (e) MCF-10A cells infected with recombinant lentivirus encoding shRNA specific for p63 (shp63) or GFP as a control (shC) were subjected to Q-PCR analyses for MMP1 and MMP9 mRNA levels. Results are presented as means and SE from three experiments performed in triplicate. *: P < 0.05; **: P < 0.01.
Figure 5
Figure 5
ΔNp63α, but not other p63 isoforms, inhibits cell migration and invasion. (a and b) MCF-10A and FaDu cells stably expressing shRNA specific for pan-p63 (shp63) or GFP (shC) were subjected to cell migration (a) or invasion assays (b), using transwell systems. Migratory and invasive cells were fixed and stained with crystal violet and quantitated as described in the Materials and Methods section. Results are presented as means and SE from three experiments. (c) MCF-10A cells infected with recombinant lentivirus encoding shRNA specific for p63 (shp63) or GFP as a control (shC) were grown in fresh normal growth media (see Materials and Methods) for two hours prior to fixation. Cells were then immunostained for phospho-paxillin (pTyr118; green) and counterstained with DAPI (blue). A representative figure from two independent experiments performed in triplicate is shown. Note the punctuated staining of focal adhesion complexes in p63-ablated cells in contrast to the organized peripheral staining in control cells (insets). Scale bars = 20 µm. (d and e) MCF-10A cells infected with recombinant lentivirus encoding shRNA specific for p63 (shp63) or GFP as a control (shC) were transiently transfected with mouse Myc-tagged ΔNp63α, ΔNp63γ, TAp63α, TAp63γ, or a vector control (V) as shown. Cells were subjected to western blotting (d) or to invasion assays using transwell systems (e). Results are presented as means and SE from two independent experiments. *: P < 0.05; **: P < 0.01.
Figure 6
Figure 6
p63 ablation-induced cell motility and invasion requires Erk2, but not Erk1. (a) MCF-10A cells infected with lentivirus encoding an shRNA specific for pan-p63 (shp63) or GFP as a control (shC) were subjected to cell invasion assays using transwell systems in the presence of 10 µM U0126 (+) or DMSO as a vehicle control (−). Results are presented as means and SE from three independent experiments. (b) MCF-10A cells expressing shRNA against p63 (shp63) or GFP as a control (shC) were infected with a lentivirus encoding shRNA against Erk1 and/or Erk2, or a control shRNA against GFP (shC). Cells were subjected to western blotting (b) or to cell invasion assays (c). Results from invasion assays are presented as means and SE from three independent experiments. *: P < 0.05; **: P < 0.01; #: P < 0.05 compared to shC-C (first column). (d) MCF-10A cells expressing shRNAs as indicated above were grown in fresh normal growth media for two hours prior to fixation. Cells were then immunostained for phospho-paxillin (pTyr118; green) and counterstained with DAPI (blue). Representative figures from two independent experiments performed in triplicate are shown. A magnified view of the respective boxed area is shown below each image. Note the organized peripheral staining of focal adhesion complexes in p63-ablated cells with reduced Erk2 expression. Scale bar = 20 µm.
Figure 7
Figure 7
MKP3 expression overcomes p63 ablation-induced cell migration and invasion. MCF-10A cells expressing shRNA against p63 (shp63) or a control shRNA against GFP (shC) were transiently transfected with MKP3 or a vector control (Ctrl). Cells were subjected to western blotting (a), or cell migration (b) and invasion assays (c), using transwell systems. Migratory and invasive cells were fixed and stained with crystal violet and quantitated as described in the Materials and Methods section. Results are presented as means and SE from three independent experiments. (d) MCF-10A cells expressing shRNAs and expression vectors as indicated were grown in fresh normal growth media for two hours prior to fixation. Cells were then immunostained for phospho-paxillin (pTyr118; green) and counterstained with DAPI (blue). Two independent experiments were performed in triplicate. Representative pictures are shown. A magnified view of the respective boxed area is shown below each image. Scale bar = 20 µm.
Figure 8
Figure 8
Reduced p63 expression enhances metastasis in vivo. MCF-10A cells transformed with NeuT were infected with lentivirus expressing shRNA against p63 (NeuT-shp63) or GFP (NeuT-shC). NCr nude mice were injected intravenously with 1.0×106 NeuT-shC or NeuT-shp63 cells. Mice were observed daily and euthanized after 45 days. (a) Lungs were dissected, fixed, and inspected for metastatic nodules on their surface. Arrows point to metastatic nodules. Scale bar = 1.0 cm. (b) Graph represents the number of metastatic nodules in the lungs per mouse. A horizontal line indicates the mean for each group. (c) Lungs were fixed, embedded in paraffin, sectioned, and stained by H&E for histological analysis. Metastatic nodules are indicated by arrows on the top panel and by a T on the bottom panel. Scale bars = 200 µm.
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References

    1. Serber Z, Lai HC, Yang A, Ou HD, Sigal MS, Kelly AE, et al. A C-terminal inhibitory domain controls the activity of p63 by an intramolecular mechanism. Mol Cell Biol. 2002 Dec 1;22(24):8601–8611. - PMC - PubMed
    1. Thanos CD, Bowie JU. p53 Family members p63 and p73 are SAM domain-containing proteins. Protein Sci. 1999 Aug 1;8(8):1708–1710. - PMC - PubMed
    1. Yang A, Kaghad M, Wang Y, Gillett E, Fleming MD, Dötsch V, et al. p63, a p53 homolog at 3q27-29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities. Mol Cell. 1998 Sep 1;2(3):305–316. - PubMed
    1. Candi E, Cipollone R, Rivetti di Val Cervo P, Gonfloni S, Melino G, Knight R. p63 in epithelial development. Cell Mol Life Sci. 2008 Oct 1;65(20):3126–3133. - PMC - PubMed
    1. Melino G. p63 is a suppressor of tumorigenesis and metastasis interacting with mutant p53. Cell Death Differ. 2011 Jul 15; - PMC - PubMed

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