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. 2009 Oct 2;284(40):27701-11.
doi: 10.1074/jbc.M109.031849. Epub 2009 Jul 27.

p110 CUX1 homeodomain protein stimulates cell migration and invasion in part through a regulatory cascade culminating in the repression of E-cadherin and occludin

Valerie Kedinger  1 , Laurent Sansregret, Ryoko Harada, Charles Vadnais, Chantal Cadieux, Kelly Fathers, Morag Park, Alain Nepveu
Affiliations

p110 CUX1 homeodomain protein stimulates cell migration and invasion in part through a regulatory cascade culminating in the repression of E-cadherin and occludin

Valerie Kedinger et al. J Biol Chem. .

Abstract

In this study, we investigated the mechanism by which the CUX1 transcription factor can stimulate cell migration and invasion. The full-length p200 CUX1 had a weaker effect than the proteolytically processed p110 isoform; moreover, treatments that affect processing similarly impacted cell migration. We conclude that the stimulatory effect of p200 CUX1 is mediated in part, if not entirely, through the generation of p110 CUX1. We established a list of putative transcriptional targets with functions related to cell motility, and we then identified those targets whose expression was directly regulated by CUX1 in a cell line whose migratory potential was strongly stimulated by CUX1. We identified 18 genes whose expression was directly modulated by p110 CUX1, and its binding to all target promoters was validated in independent chromatin immunoprecipitation assays. These genes code for regulators of Rho-GTPases, cell-cell and cell-matrix adhesion proteins, cytoskeleton-associated proteins, and markers of epithelial-to-mesenchymal transition. Interestingly, p110 CUX1 activated the expression of genes that promote cell motility and at the same time repressed genes that inhibit this process. Therefore, the role of p110 CUX1 in cell motility involves its functions in both activation and repression of transcription. This was best exemplified in the regulation of the E-cadherin gene. Indeed, we uncovered a regulatory cascade whereby p110 CUX1 binds to the snail and slug gene promoters, activates their expression, and then cooperates with these transcription factors in the repression of the E-cadherin gene, thereby causing disorganization of cell-cell junctions.

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Figures

FIGURE 1.
FIGURE 1.
p200 CUX1 mediates its effects on migration, at least in part, through the generation of p110 CUX1. Populations of NMuMG-NYPD mouse mammary epithelial cells stably carrying a retroviral vector, either empty or expressing p110-HA or p200-HA CUX1, were established. Following the indicated treatment, CUX1 protein expression was analyzed by Western blotting using an anti-CUX1 or anti-HA antibody as indicated, and cell motility was measured in a two-chamber migration assay. The migration assays were all performed in triplicate and repeated at three independent times. A, no treatment. B, cells were incubated for 48 h in the presence of the cell-permeable cysteine protease inhibitor, E64d, or the carrier. C, cells were infected with a retroviral vector, either empty or expressing cathepsin L, and were analyzed 48 h later.
FIGURE 2.
FIGURE 2.
p110 CUX1 stimulates cell spreading and cell adhesion. A, adhesion ability of NMuMG-NYPD/vector and p110 CUX1-expressing cells was analyzed using plates that were either uncoated or coated with Matrigel or collagen. After 45 min, adherent cells were fixed in 10% formalin and stained with 0.1% crystal violet, and pixel counts were measured using Scion software. B, spreading of NMuMG-NYPD/vector or p110-expressing cells was analyzed at 45, 60, and 90 min after plating on collagen-coated slides. At the indicated time, cells were fixed, stained with phalloidin, and visualized by fluorescence microscopy. Nuclei were counterstained with DAPI. The proportion of cells that were spread was counted at 45 min.
FIGURE 3.
FIGURE 3.
Validation of p110 CUX1 targets. Chromatin from invasive human ductal carcinoma-derived cell line Hs578T was submitted to immunoprecipitation using anti-CUX1 antibodies (Ab) or IgG as a control, and analyzed by PCR using primers specific for the promoter of each potential CUX1 target gene. G6PDH and Dlx2 were used as negative and positive controls, respectively. Input DNA (0.02%) was used as control. Note that the region to be amplified was chosen to be approximately in the middle of the sequence spotted on the location array.
FIGURE 4.
FIGURE 4.
Protein expression of CUX1 target genes. A, total protein extract was prepared from logarithmic growing populations of NMuMG-NYPD cells stably carrying a p110 CUX1 or an empty retroviral vector. Equal amount of proteins was loaded on SDS-polyacrylamide gel for Western blot analysis using specific antibodies raised against different CUX1 target genes. HA antibody was used to verify the expression of p110 CUX1, and actin and γ-tubulin serve as loading controls. B, primary cell line established from a p110 transgenic mouse mammary gland tumor was transfected with scrambled or anti-CUX1 siRNA. 5 days after transfection, total protein extract was prepared. Equal amount of proteins was loaded on SDS-polyacrylamide gel for Western blot analysis using specific antibodies raised against different CUX1 target genes. Anti-CUX1 antibody was used to verify p110 CUX1 expression, and β-actin and γ-tubulin serve as a loading controls. In the histogram, bands from A (light gray) and B (dark gray) were quantified using the Scion software, and expression of targets in p110-expressing cells compared with control cells was calculated relative to actin.
FIGURE 5.
FIGURE 5.
Luciferase reporter assays. The promoter regions of vimentin, N-cadherin, E-cadherin, and occludin were cloned into a luciferase reporter plasmid. Hs578T cells were transfected with each reporter plasmid together with a vector expressing p110 CUX1 or with an empty vector. The experiments were done in triplicate and performed independently at least three times. RLU, relative luciferase unit.
FIGURE 6.
FIGURE 6.
p110 CUX activates Snail and Slug expression and cooperates with them in the repression of E-cadherin. A, luciferase reporter assays. NMuMG-NYPD cells were cotransfected with a reporter plasmid containing nucleotides −166 to + 62 of the E-cadherin promoter and a vector expressing p110 CUX1, either alone or together with Snail or Slug. As a control, cells were transfected with vector alone. Values are means of three measurements, and error bars represent standard deviation. B, snail and slug mRNA expression was measured by quantitative real time PCR in NMuMGNYPD/vector and NMuMG-NYPD/p110 CUX1 cells. The values are the mean of three measurements, and the error bars represent standard deviation. *, p < 0.05; ***, p < 0.001. C, scanning ChAP and ChIP within the snail and slug gene promoters. Chromatin from Hs578T/p110CUX1-Tag2 and from Hs578T cells was submitted to affinity purification or immunoprecipitation and analyzed by quantitative real time PCR using primer pairs specific for different regions of the promoters. Templates for the PCRs were 0.1% total input DNA (I), nonspecific DNA from Sepharose beads alone (S), and ChAP- (AP) or ChIP-purified DNA. The positions of amplified fragments are indicated over the maps, and primer sequences are given under “Experimental Procedures.” The respective fold enrichment of the different DNA fragments are indicated relative to the DNA obtained by purification on Sepharose beads without IgG (S). Enrichment was calculated using the G6PDH locus as a reference. D, replacement mutations were made to change the first and sixth nucleotide of each of the three E boxes (CANNTG) within a reporter plasmid containing nucleotides −166 to + 62 of the E-cadherin promoter as follows: box A, CAGGTG to AAGGTA; box B, CACCTG to AACCTA; and box C, CACCTG to AACCTA. NMuMG-NYPD cells were transfected with either the wild type or mutant E-cadherin reporter plasmid together with a vector expressing a FLAG-tagged Snail protein. The chromatin was immunoprecipitated with an anti-FLAG antibody and qRT-PCR was performed with the indicated primers to compare the recruitment of Snail to the wild type and mutated reporter. E, luciferase reporter assays. NMuMG-NYPD cells were cotransfected either the wild type or mutated (−166/+62) E-cadherin reporter plasmid together with expression vectors expressing either nothing (vector), p110 CUX1 or FLAG-tagged Snail. F, NMuMG-NYPD cells were infected with a Tet-On lentiviral vector expressing a CUX1-specific shRNA in the presence of doxycycline. Cell were either treated with doxycycline or left untreated, and CUX1 protein levels were assessed by Western blotting after 5 days. In parallel, doxycycline-treated or untreated cells were transfected with the wild type or mutated (−166/+62) E-cadherin reporter plasmid, and a luciferase assay was performed. RLU, relative luciferase unit.
FIGURE 7.
FIGURE 7.
Elevated expression of p110 CUX1 causes loss of E-cadherin and occludin from cell-cell junctions. Clones of MDCK epithelial cells stably expressing p110 CUX1 or carrying the empty retroviral vector were seeded on coverslips and analyzed by phase-contrast microscopy and indirect immunofluorescence. A, phase-contrast microscopy: note the larger size and elongated shape of p110 CUX1 cells and the loose contacts they form between each other, as compared with vector cells. B, cells were stained for E-cadherin (red) and HA (green), and nuclei were stained with DAPI. C, cells were stained for occludin (red) and HA (green), and nuclei were stained with DAPI. B and C, original magnification was ×100.
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