Metastasis-associated protein 1 and its short form variant stimulates Wnt1 transcription through promoting its derepression from Six3 corepressor

Abstract

Although Wnt1 downstream signaling components have been well studied and activated in human cancer, the pathways that regulate Wnt1 itself have not been explored in depth. Here, we provide gain-of-function, loss-of function, and molecular evidence supporting functional interactions between metastasis-associated protein 1 short-form (MTA1s), metastasis-associated protein 1 (MTA1), and Wnt1 signaling components during mammary gland development and tumorigenesis. Using multiple model systems involving overexpression or knockdown of MTA1s or MTA1, we discovered that MTA1s and MTA1 hyperactivate the Wnt1 pathway due to increased expression of Wnt1 transcription. MTA1s and MTA1 physically interact with Six3 chromatin, a protein product of which is a direct histone deacetylase inhibitor-dependent repressor of Wnt1 transcription. Deletion of the MTA1s and MTA1 allele in murine embryonic fibroblasts resulted in the upregulation of Six3 and downregulation of Wnt signaling. In addition, mammary glands from the MTA1s/MTA1(-/-) mice exhibited increased recruitment of Six3 corepressor complex to the Wnt1 promoter and inhibition of Wnt1 pathway in mammary glands. These findings identify MTA1s and MTA1 as important upstream modifiers of the Wnt1 transcription, and consequently its functions, by directly inhibiting the transcription of Six3, allowing derepression of Wnt1 transcription.

Figure 1. MTA1s overexpression upregulates Wnt1 signaling pathway in mammary epithelial cells

(A) Expression of T7-MTA1s by confocal microscopy, T7-MTA1s (red). Scale bar, 10 µm. (B) RT-PCR and confocal analysis of Wnt1 in HC11/pcDNA and HC11/MTA1s cells. (C) RT-PCR and confocal analysis of Wnt1 in HC11/pcDNA and HC11/MTA1 cells. (D) Western blot analysis of β-catenin, Phospho-GSK3-β, GSK3-β, Wnt1 and T7 expression in HC11/pcDNA and HC11/MTA1 clones. (E) Transcription status of Top-flash, Fop-flash in HC11/pcDNA and HC11/MTA1 clones. F Western blot analysis of β-catenin, Wnt1, GSK-3β, Phospho-GSK3-β and MTA1 expression in MCF-7 cell line following transfection with MTA1 or control siRNA for 48h.

Figure 2. MTA1s and MTA1 targets Six3 expression in the mammary epithelial and breast cancer cells

(A) RT-PCR and Western blot analysis for Six3 in HC11/pcDNA, HC11/MTA1 and HC11/MTA1s cells. (B) MTA1s and MTA1 downregulates Six3 promoter-luc activity in HC11/MTA1s or HC11/MTA1 cells. (C) Six3 promoter luc activity in HC11/pcDNA and HC11/MTA1 or HC11/MTA1s cells following treatment with HDAC inhibitor, TSA.

Figure 3

(A) Western blot analysis of Six3, MTA1s and MTA1 and in MEFs from WT (+/+), heterozygous (+/−) and homozygous (−/−) mice for MTA1s/MTA1. (B) Top-Flash luciferase activity in MEFs from WT (+/+) and homozygous (−/−) mice for MTA1s/MTA1. (C) Western blot analysis of Six3 expression in MDA-MB-435 cell line following transfection with MTA1s or control siRNA for 48h. (D) Western blot analysis of Six3 in MCF-7 cell line following transfection with MTA1 or control siRNA for 48h. (E) Western blot analysis and Top-Flash luciferase activity in HC11 cells following transfection with myc-tagged MTA1s or MTA1 and Six3-siRNA.

Figure 4. Status of Wnt signaling components

(A) Western blot analysis of phospho-GSK-3β, GSK-3β, phospho-ERK, ERK and β-catenin and RT-PCR analysis of Wnt1 in MEFs from WT (+/+), heterozygous (+/−) and homozygous (−/−) mice for MTA1s/MTA1. (B) Western blot analysis of phospho-GSK-3β, GSK-3β, Six3 and β-catenin and RT-PCR analysis of Wnt1 in MTA1s/MTA1^−/−MEFs transiently transfected with pcDNA, myc-MTA1s and T7-MTA1. (C) Western blot analysis of phospho-GSK-3β, GSK-3β, Six3 and β-catenin and RT-PCR analysis of Wnt1 in MTA1s/MTA1^−/− MEFs stably expressing pcDNA, V5-MTA1 and V5-MTA1s.

Figure 5. MTA1s/MTA1/NuRD complex targets Six3 regulatory chromatin

(A) Schematic diagram of 5.5 kb regulatory chromatin of mouse Six3 gene. (B) ChIP showing differential recruitment of T7-MTA1s to regions 1–3 of Six3 chromatin, and T7-MTA1 to regions 1, 2 and 9 of Six3 chromatin in HC11-pcDNA, HC11-MTA1s and HC11-MTA1 cells. (C, D) ChIP showing recruitment of HDAC2 and Mi-2 to regions Six3 chromatin in HC11/pcDNA, HC11/MTA1s and HC11/MTA1 cells.

Figure 6. Transcriptional regulation of Six3 by MTA1 and MTA1s

(A) A double ChIP assay, first with T7-Ab followed by the HDAC2 Ab for the regions 1–3, 6 and 9 regions of the Six3 chromatin in HC11/pcDNA, HC11/MTA1s and HC11/MTA1 cells. (B) Status of the acetylated histone H3 in the MTA1s/MTA1-NuRD complex bound Six3 chromatin in the HC11/pcDNA, HC11/MTA1s and HC11/MTA1 by ChIP analysis. (C) Six3 promoter-luc and ΔSix3-promoter-luc activity in HC11/pcDNA and HC11/MTA1s cells. Schematic representation of the 1.36 kb Six3 promoter-luciferase construct and the three Six3 recognition (ATTA) sequences are presented as boxes. Schematic representation of the 1.36 kb ΔSix3 promoter-luciferase construct without the three Six3 recognition (ATTA) sequences. (D) Endogenous Six3 interacts with transiently transfected T7-MTA1s in the HC11 cells. (E) Transiently transfected T7-MTA1 interacts with endogenous Six3 in the HC11 cells. (F) Endogenous interaction of MTA1, MTA1s with Six3 in MDA-MB-435 cells.

Figure 7. Mammary gland development and status of Wnt pathway in MTA1s/MTA1^−/− mice

(A) Carmine Red-stained whole mounts of inguinal mammary glands obtained from MTA1s/MTA1^+/+ mice and MTA1s/MTA1^−/− mice at 12 weeks of age. The images in the bottom panels are the images of the top panels at increased magnification. (B) Immunohistochemical analysis of Six3 and β-catenin expression in mammary glands of 12-week old virgin MTA1s/MTA1^+/+ and MTA1s/MTA1^−/− mice. (C) Schematic representation of the Six3 binding sites in Wnt1 promoter. (D) ChIP analysis showing differential recruitment of Six3 to the Wnt1 proximal promoter region in MTA1s/MTA1^−/− mice and its corresponding MTA1s/MTA1^+/+mice. Bars below represents the relative quantitation of Six3 recruitment to the Wnt1 promoter using ChIP- q PCR assay. (E) ChIP analysis showing differential recruitment of Six3 to the Wnt1 enhancer region in MTA1s/MTA1^−/− mice and its corresponding MTA1s/MTA1^+/+ mice. Bars below represents the quantitation of Six3 recruitment to the Wnt1 enhancer using ChIP-qPCR assay. (F) Western blot analysis of Six3 expression and RT-PCR analysis of Wnt1 expression in MTA1s/MTA1^−/− mice and its corresponding MTA1s/MTA1^+/+ mice. (G) ChIP analysis showing enhanced recruitment of Six3 to both Wnt1 promoter (top panel) and Wnt1 enhancer region in MTA1s-specific siRNA transfected MDA-MB-435 cells as compared to control siRNA transfected cells for 48h. (H) Schematic representation of Wnt1 expression and signaling by MTA1s or MTA1. Dotted lines going to transformation represents other possible mechanisms not investigated here.