MUC1-C oncoprotein activates the ZEB1/miR-200c regulatory loop and epithelial-mesenchymal transition

Abstract

The epithelial-mesenchymal transition (EMT) is activated in cancer cells by ZEB1, a member of the zinc finger/homeodomain family of transcriptional repressors. The mucin 1 (MUC1) heterodimeric protein is aberrantly overexpressed in human carcinoma cells. The present studies in breast cancer cells demonstrate that the oncogenic MUC1-C subunit induces expression of ZEB1 by a NF-κB (nuclear factor kappa B) p65-dependent mechanism. MUC1-C occupies the ZEB1 promoter with NF-κB p65 and thereby promotes ZEB1 transcription. In turn, ZEB1 associates with MUC1-C and the ZEB1/MUC1-C complex contributes to the transcriptional suppression of miR-200c, an inducer of epithelial differentiation. The co-ordinate upregulation of ZEB1 and suppression of miR-200c has been linked to the induction of EMT. In concert with the effects of MUC1-C on ZEB1 and miR-200c, we show that MUC1-C induces EMT and cellular invasion by a ZEB1-mediated mechanism. These findings indicate that (i) MUC1-C activates ZEB1 and suppresses miR-200c with the induction of EMT and (ii) targeting MUC1-C could be an effective approach for the treatment of breast and possibly other types of cancers that develop EMT properties.

Figure 1

MUC1-C increases ZEB1 protein and mRNA levels. (a–c) Lysates from (a) MDA-MB-231/CshRNA and MDA-MB-231/MUC1shRNA cells, (b) BT-549/CshRNA and BT-549/MUC1shRNA cells, and (c) MCF-7/vector and MCF-7/MUC1-C cells were immunoblotted with the indicated antibodies (left). Densitometric scanning of the ZEB1 signals in MDA-MB-231 (a) and BT-549 (b) cells demonstrated that silencing MUC1-C is associated with a 40±9 and 53±4% decrease in ZEB1 protein (mean±s.d. of three determinations), respectively. ZEB1 mRNA levels were determined by qRT–PCR (right). The results are expressed as relative ZEB1 mRNA levels (mean±s.d. of three determinations) as compared with that obtained for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a control. (d,e) Immunofluorescence images for MUC1-C (green) and ZEB1 (red) staining of the indicated MDA-MB-231 (d) and MCF-7 (e) cells. 4',6-diamidino-2-phenylindole (DAPI) (blue) counterstain was used to visualize nuclei. The first four panels represent the same fields (magnification ×63). The red box highlights the section taken for zoom (magnification ×252).

Figure 2

MUC1-C induces ZEB1 expression by a NF-κB-dependent mechanism. (a–b) Lysates from (a) MDA-MB-231 and (b) BT-549 cells transduced with lentiviruses expressing a control CshRNA or a p65 shRNA were immunoblotted with the indicated antibodies (left). Densitometric scanning of the ZEB1 signals in MDA-MB-231 (a) and BT-549 (b) cells demonstrated that silencing p65 is associated with a 79±5 and 68±2% decrease in ZEB1 protein (mean±s.d. of three determinations), respectively. Lysates from (a) MDA-MB-231 and (b) BT-549 cells left untreated or treated with 5 μm BAY11-7085 for 12 h were immunoblotted with the indicated antibodies (right). (c) Lysates from MCF-7/MUC1-C cells transiently transfected with control or NF-κB p65 siRNAs were immunoblotted with the indicated antibodies (left). Lysates from MCF-7/MUC1-C cells left untreated or treated with 5 μm BAY11-7085 for 12 h were immunoblotted with the indicated antibodies (right). (d) Schema of the ZEB1 promoter with localization of the NF-κB binding site. (e,f) Soluble chromatin from MDA-MB-231/CshRNA and MDA-MB-231/MUC1shRNA (e) and MCF-7/vector and MCF-7/MUC1-C (f) cells were precipitated with anti-NF-κB or a control IgG (left). In the re-ChIP studies, the anti-NF-κB precipitates were released and reimmunoprecipitated with anti-MUC1-C (right). The final DNA samples were amplified by qPCR with pairs of primers for the NF-κB binding region (−952 to −771) or a control region (−4766 to −4656). The results (mean±s.d. of three determinations) are expressed as the relative fold enrichment compared with that obtained with the IgG control.

Figure 3

Binding of MUC1-C and ZEB1. (a) Lysates from MDA-MB-231 (left) and MCF-7/MUC1-C (right) cells were precipitated with anti-ZEB1 or a control IgG. The precipitates were immunoblotted with the indicated antibodies. (b) Schema of the ZEB1 protein with localization of the zinc finger domains and the homeobox domain. GST, GST–ZEB1(1–385), GST–ZEB1(386–711) and GST–ZEB1(712–1108) were incubated with MCF-7/MUC1-C cell lysate. The adsorbates were immunoblotted with anti-MUC1-C. Input of the GST proteins was assessed by Coomassie blue staining. (c) GST, GST–ZEB1(1–385), GST–ZEB1(386–711) and GST–ZEB1(712–1108) were incubated with purified MUC1-CD. The adsorbates were immunoblotted with anti-MUC1-C. Input of the GST proteins was assessed by Coomassie blue staining. (d) Amino acid sequence of MUC1-CD with positioning of the regions detected with monoclonal antibodies CD1 and CT2. GST–ZEB1(1–385) and GST–ZEB1(386–711) were incubated with purified MUC1-CD(1–45) (left) or MUC1-CD(46–72) (right). The adsorbates and purified MUC1-CD proteins were immunoblotted with anti-MUC1-CD (CD1, left; CT2, right) antibodies. Input of the GST proteins was assessed by Coomassie blue staining. (e) GST–ZEB1(1–385) and GST–ZEB1(386–711) were incubated with purified MUC1-CD or MUC1-CD(AQA). The adsorbates were immunoblotted with anti-MUC1-CD. Input of the GST proteins was assessed by Coomassie blue staining.

Figure 4

MUC1-C occupies the miR-200c promoter with ZEB1. (a) Schema of the miR-200c promoter with localization of the two Z-boxes. Soluble chromatin from MCF-7/vector and MCF-7/MUC1-C cells was precipitated with anti-ZEB1 or a control IgG (left). In the re-ChIP studies, the anti-ZEB1 precipitates were released and reimmunoprecipitated with anti-MUC1-C (right). The final DNA samples were amplified by qPCR with pairs of primers for the ZEB1 binding region or GAPDH. The results (mean±s.d. of three determinations) are expressed as the relative fold enrichment compared with that obtained with the IgG control. (b) miR-200c levels in MCF-7/vector and MCF-7/MUC1-C cells were determined by RT–PCR (left). U6 was included as a control (left). Relative pri-miR-200c and miR-200c levels were determined by qRT–PCR (right). The results are expressed as relative levels (mean±s.d. of three determinations) as compared with that obtained for U6 as a control. (c) Lysates from MCF-7 cells expressing the empty vector, MUC1-C or MUC1-C(AQA) were immunoblotted with the indicated antibodies (left). Relative miR-200c levels were determined by qRT–PCR (right). The results are expressed as relative levels (mean±s.d. of three determinations) as compared with that obtained for U6 as a control. (d,e) miR-200c and U6 levels in MDA-MB-231/CshRNA and MDA-MB-231/MUC1shRNA (d) and BT-549/CshRNA and BT-549/MUC1shRNA (e) cells were determined by RT–PCR (left). Relative pri-miR-200c and miR-200c levels were determined by qRT–PCR (right). The results are expressed as relative levels (mean±s.d. of three determinations) as compared with that obtained for U6 as a control.

Figure 5

MUC1-C induces EMT and cell invasion. (a) Lysates from MCF-7/vector and MCF-7/MUC1-C cells were immunoblotted with the indicated antibodies. (b) Immunofluorescence images for E-cadherin (green) staining of MCF-7/vector and MCF-7/MUC1-C cells. DAPI (blue) counterstain was used to visualize nuclei (magnification × 252). Arrow denotes localization of E-cadherin at the cell membrane. (c) MCF-7/vector and MCF-7/MUC1-C cells were seeded in matrigel coated transwell chambers for 24 h. Photomicrographs of representative fields are shown (left). Results of the invasion cell assays are expressed as the number of cells invaded per field (mean±s.d. of 5 fields) (right). (d) Lysates from MDA-MB-231/CshRNA and MDA-MB-231/MUC1shRNA cells were immunoblotted with the indicated antibodies. (e) Immunofluorescence images for E-cadherin (green) staining of MDA-MB-231/CshRNA and MDA-MB-231/MUC1shRNA cells. DAPI (blue) counterstain was used to visualize nuclei (magnification × 252). Arrow denotes localization of E-cadherin at the cell membrane. (f) MDA-MB-231/CshRNA and MDA-MB-231/MUC1shRNA cells were seeded in matrigel coated transwell chambers for 24 h. Photomicrographs of representative fields are shown (left). Results of the invasion cell assays are expressed as the number of cells invaded per field (mean±s.d. of 5 fields) (right).

Figure 6

MUC1-C induces EMT and invasion by a ZEB1-dependent mechanism. (a) MCF-7/MUC1-C cells were infected with lentiviruses to stably express a scrambled control CshRNA or a ZEB1shRNA. Lysates from the MCF-7/MUC1-C/CshRNA and MCF-7/MUC1-C/ZEB1shRNA cells were immunoblotted with the indicated antibodies. (b) miR-200c levels in MCF-7/MUC1-C/CshRNA and MCF-7/MUC1-C/ZEB1shRNA cells were determined by RT–PCR (left). U6 was included as a control (left). Relative miR-200c levels were determined by qRT–PCR (right). (c) MCF-7/MUC1-C/CshRNA and MCF-7/MUC1-C/ZEB1shRNA cells were seeded in matrigel coated transwell chambers for 24 h. Photomicrographs of representative fields are shown (left). Results of the invasion cell assays are expressed as the number of cells invaded per field (mean±s.d. of 5 fields) (right). (d) MDA-MB-231 cells were infected with lentiviruses to stably express a scrambled CshRNA or a ZEB1shRNA. Lysates from the MDA-MB-231/CshRNA and MDA-MB-231/ZEB1shRNA cells were immunoblotted with the indicated antibodies (left). Relative miR-200c levels were determined by qRT–PCR (right). (e) MDA-MB-231/CshRNA and MDA-MB-231/ZEB1shRNA cells were seeded in matrigel coated transwell chambers for 24 h. Photomicrographs of representative fields are shown (left). Results of the invasion cell assays are expressed as the number of cells invaded per field (mean±s.d. of five fields) (right).

Figure 7

Proposed model for the effects of MUC1-C on the ZEB1/miR-200c regulatory loop and thereby EMT and invasion. (a) MUC1-C forms a complex with NF-κB p65 that occupies the ZEB1 promoter and activates ZEB1 expression. In turn, MUC1-C associates with ZEB1, increases ZEB1 occupancy of the miR-200c promoter and contributes to the suppression of miR-200c expression. (b) MUC1-C-mediated (i) induction of ZEB1 abundance and (ii) decreases in miR-200c levels promote EMT and cell invasion.