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. 2012 Oct 9;109(41):16654-9.
doi: 10.1073/pnas.1205822109. Epub 2012 Sep 24.

Canonical Wnt signaling regulates Slug activity and links epithelial-mesenchymal transition with epigenetic Breast Cancer 1, Early Onset (BRCA1) repression

Zhao-Qiu Wu  1 , Xiao-Yan Li, Casey Yuexian Hu, Michael Ford, Celina G Kleer, Stephen J Weiss
Affiliations

Canonical Wnt signaling regulates Slug activity and links epithelial-mesenchymal transition with epigenetic Breast Cancer 1, Early Onset (BRCA1) repression

Zhao-Qiu Wu et al. Proc Natl Acad Sci U S A. .

Abstract

Slug (Snail2) plays critical roles in regulating the epithelial-mesenchymal transition (EMT) programs operative during development and disease. However, the means by which Slug activity is controlled remain unclear. Herein we identify an unrecognized canonical Wnt/GSK3β/β-Trcp1 axis that controls Slug activity. In the absence of Wnt signaling, Slug is phosphorylated by GSK3β and subsequently undergoes β-Trcp1-dependent ubiquitination and proteosomal degradation. Alternatively, in the presence of canonical Wnt ligands, GSK3β kinase activity is inhibited, nuclear Slug levels increase, and EMT programs are initiated. Consistent with recent studies describing correlative associations in basal-like breast cancers between Wnt signaling, increased Slug levels, and reduced expression of the tumor suppressor Breast Cancer 1, Early Onset (BRCA1), further studies demonstrate that Slug-as well as Snail-directly represses BRCA1 expression by recruiting the chromatin-demethylase, LSD1, and binding to a series of E-boxes located within the BRCA1 promoter. Consonant with these findings, nuclear Slug and Snail expression are increased in association with BRCA1 repression in a cohort of triple-negative breast cancer patients. Together, these findings establish unique functional links between canonical Wnt signaling, Slug expression, EMT, and BRCA1 regulation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Slug is a GSK3β substrate. (A, Upper) MCF7 cells transiently transfected with FLAG-Slug-WT or -4A were treated with cycloheximide (CHX) for varying time intervals and analyzed by Western blotting (Left), and the protein levels in the indicated cells were quantified by densitometry as a function of time (Right). (Lower) Transfected MCF7 cells were treated with MG132 for the indicated time intervals and analyzed by Western blotting (Left), and the protein levels were quantified by densitometry as a function of time (Right). (B) Sequence alignment of human Snail with human, mouse, rat, chicken, and Xenopus Slug showing the relative positions of the two potential GSK3β consensus phosphorylation motifs. (C) Recombinant GSK3β was incubated with increasing amounts of bacterially expressed, purified GST-Slug or GST-Snail in the presence of [γ-32P]ATP. The reaction mixtures were resolved by SDS/PAGE with Snail/Slug proteins visualized by autoradiography. Arrow, phosphorylation of Snail/Slug; asterisk, autophosphorylation of GSK3β. Coomassie stains (Coom.) are shown for increasing amounts of Snail/Slug proteins. (D, Left) GSK3β was incubated with a series of purified GST-Slug peptides. (Right) GSK3β was incubated with GST-Slug-WT or the indicated serine-to-alanine mutants. Coomassie stains for the peptides/mutants are shown. Rectangle (red) outlines a nonspecific phosphorylation band that does not align with the Coomassie-stained peptide. (E) GSK3β was incubated with GST-Slug in the presence of cold ATP. The reaction mixtures were resolved by SDS/PAGE, subjected to mass spectrometry analysis, and the phosphorylation sites identified.
Fig. 2.
Fig. 2.
A canonical Wnt/GSK3β signaling pathway regulates Slug-dependent EMT. (A) MCF7 cells transiently transfected with FLAG-tagged Slug-WT or -4A were treated with or without recombinant Wnt3a (100 ng/mL). Total cell lysates were analyzed by Western blotting (Left) or quantitative RT-PCR (Right). Nuclear extracts were analyzed for GSK3β kinase activity (Right). **P < 0.01, Wnt3a vs. vehicle. (B) MCF7 cells stably expressing mock, Slug-WT, or Slug-4A expression vectors were analyzed by Western blotting (Left) or qRT-PCR (Right). Inset, relative Slug mRNA levels in -WT or -4A expressing cells. **P < 0.01, 4A vs. mock. (C) The morphology of MCF7 cells stably expressing mock, Slug-WT, or Slug-4A expression vectors as assessed by phase-contrast microscopy (Upper) or immunofluorescence following staining with anti–E-cadherin antibody (Lower). (D) (Left) MCF7 cells were cotransfected with increasing amounts of mock, Slug-WT, or -4A expression vectors in combination with an E-cadherin promoter reporter construct (25 ng) and analyzed by luciferase assay. (Right) Slug protein and mRNA levels in -WT or -4A expressing cells. (E) (Left) MCF7 cells stably expressing mock, Slug-WT, or Slug-4A expression vectors were labeled with fluorescent beads (green) and cultured atop the live chick CAM for 4 d. CAMs were fixed and stained with an anti-chicken type IV collagen antibody (red). Dashed lines mark the CAM basement membrane. Cell nuclei were stained with DAPI (blue). (Right) Relative invasion was determined. **P < 0.01, 4A vs. Mock; ##P < 0.01, 4A vs. WT.
Fig. 3.
Fig. 3.
A Wnt/GSK3β/Slug axis regulates BRCA1 expression. MDA-MB-231 cells were treated with the indicated GSK3 inhibitors (A) or recombinant Wnt3a (B) and analyzed by Western blotting or qRT-PCR. **P < 0.01. (C) Cell lysates from MDA-MB-231 cells stably expressing Scr-, Slug- or Snail-shRNAs were subjected to Western blotting or qRT-PCR analyses, respectively. **P < 0.01. (D) MCF7 cells stably expressing mock, Slug-WT, Slug-4A, or Snail-96A expression vectors were analyzed by Western blotting or qRT-PCR. **P < 0.01. (E) Snail/Slug-E-cadherin or Snail/Slug-BRCA1 promoter interactions in MCF7 cells stably expressing Slug-WT or Snail-WT expression vectors were assessed within the indicated promoter regions (marked with red lines) by ChIP/qPCR. **P < 0.01. (F) MCF7 cells were cotransfected with increasing amounts of mock, Slug-WT, Slug-4A, Snail-WT, or Snail-96A expression vectors in combination with a BRCA1 reporter construct (25 ng) and relative promoter activities determined by luciferase assay. (G) MDA-MB-231 cells stably expressing Scr-, Slug-, Snail-, or Slug/Snail-shRNAs were transfected with a BRCA1 promoter or control reporter construct and the cell lysates were subjected to luciferase assay (Upper) and Western blot analysis (Lower). **P < 0.01. (H) MCF7 cells were cotransfected with mock, Slug-4A, or Snail-96A expression vectors in combination with wild-type or mutated BRCA1 promoter reporter constructs, and relative promoter activity was determined by luciferase assay.
Fig. 4.
Fig. 4.
LSD1-dependent Slug-mediated BRCA1 repression. (A) Lysates from MDA-MB-231 cells stably expressing Scr-, Slug-, or Snail- shRNA were subjected to ChIP analysis using antibodies directed against H3K4me2, H3K4me3, or H3K9Ac and BRCA1 occupancy was determined by qPCR. **P < 0.01. (B) Cell lysates from MCF7 cells stably expressing mock, Snail-96A, or Slug-4A expression vectors were subjected to ChIP analysis using anti-H3K4me2 antibody and BRCA1 or E-cadherin occupancy was determined by qPCR. **P < 0.01. (C) MDA-MB-231 cells stably expressing Scr-shRNA, LSD1-shRNA-1 or LSD1-shRNA-2 were cotransfected with either a BRCA1 or E-cadherin promoter construct, and the cell lysates were subjected to luciferase assays. Inset, LSD1 protein levels in the indicated cell lysates. **P < 0.01. (D) 293T cells were cotransfected with mock, FLAG-tagged Snail, or Slug expression vectors, and whole cell extracts prepared, subjected to anti-FLAG immunoprecipitation, and analyzed by Western blot with the indicated antibodies. (E) MDA-MB-231 cells stably expressing Scr-shRNA or LSD1-shRNA-1 were transduced with lentiviruses expressing Scr-shRNA, Slug-shRNA, or Snail-shRNA. The cells were transfected with a BRCA1 promoter construct, and the cell lysates were subjected to luciferase assay (Left) and Western blotting analysis (Right). **P < 0.01, treated vs. control.
Fig. 5.
Fig. 5.
Snail/BRCA1 expression in human breast cancer tissues. Representative sections stained for Snail, Slug, or BRCA1 from normal breast tissues and three triple-negative breast cancer samples are shown.
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