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. Author manuscript; available in PMC: 2015 Mar 27.
Published in final edited form as: Oncogene. 2013 Apr 15;33(13):1680–1689. doi: 10.1038/onc.2013.114

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

H Rajabi 1,1, M Alam 1,1, H Takahashi 1,1, A Kharbanda 1, M Guha 1, R Ahmad 1,2, D Kufe 1
PMCID: PMC3783575  NIHMSID: NIHMS485966  PMID: 23584475

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 epithelial-mesenchymal transition (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.

Keywords: MUC1-C, ZEB1, miR-200c, breast cancer, EMT

INTRODUCTION

The epithelial–mesenchymal transition (EMT) is a fundamental process by which polarized epithelial cells acquire mesenchymal cell properties with an enhanced potential for migration.1 EMT is a reversible program during embryonic development that is directed by a group of transcription factors, which includes members of the zinc finger E-box binding homeobox, basic helix-loop-helix and Snail families.2 Aberrant activation of EMT by these embryonic transcription factors in cancer cells, however, contributes to the malignant phenotype with an increased capacity for invasion, metastases and resistance to apoptosis.1,2 EMT has also been associated with the acquisition of stem cell properties.3,4

ZEB1 is a member of the zinc finger E-box binding homeobox family of zinc finger/homeodomain transcriptional repressors that activates EMT.5 The transforming growth factor-β pathway, which is associated with EMT activation, induces ZEB1 expression.6 In turn, ZEB1 confers loss of cell polarity and cell–cell adhesion, and promotes cell motility and metastases.6,7 ZEB1 contributes to the induction of EMT by repressing the expression of cell polarity factors, basement membrane components and other proteins that are of importance for epithelial cell function.7–10 Other studies have demonstrated that ZEB1 suppresses transcription of microRNA-200 family members, such as miR-200c, which induce epithelial differentiation and thereby suppress EMT.5,11 In a negative feedback loop, miR-200c inhibits ZEB1 expression.11 The balance between ZEB1 and miR-200c expression therefore controls EMT and qthe progression of cancer cells with metastatic properties.6 These findings have supported the proposal that targeting the ZEB1–miR-200c feedback loop could be effective for the treatment of breast and other types of cancers that develop EMT properties.

Mucin 1 (MUC1) is a heterodimeric glycoprotein that is expressed at the apical border of polarized epithelial cells.12 With transformation and loss of polarity, MUC1 is overexpressed and repositioned over the entire cell membrane of carcinoma cells.12 Such an aberrant overexpression of MUC1 is found in about 90% of human breast tumors and has been linked to the malignant phenotype.13 Increased expression of MUC1 has also been associated with EMT in breast and other cancer cells by mechanisms that are not well understood.14–16 Of importance for understanding its function, MUC1 consists of an N-terminal ectodomain (MUC1-N) in a cell surface complex with a C-terminal transmembrane subunit (MUC1-C).12 With its overexpression as found in breast cancer cells, MUC1-C accumulates in the cytoplasm and is targeted to the nucleus, where it interacts with transcription factors, such as NF-κB (nuclear factor kappa B) p65, that activate genes involved in growth and survival.12,13,17 MUC1-C also localizes to the mitochondrial outer membrane, where it attenuates the apoptotic response to genotoxic and oxidative stress.12,18 In this way, overexpression of MUC1 is sufficient to induce transformation in vitro and in transgenic mouse tumor models.12,13

The present studies demonstrate that MUC1-C induces activation of the ZEB1 gene by a NF-κB-mediated mechanism in breast cancer cells. In turn, MUC1-C associates with ZEB1 and promotes the repression of miR-200c expression. By extension, we show that MUC1-C induces breast cancer cell EMT and invasion by a ZEB1-dependent pathway.

RESULTS

MUC1-C upregulates ZEB1 expression

To assess a potential role for MUC1-C in the regulation of ZEB1 expression, we first stably silenced MUC1-C in MDA-MB-231 breast cancer cells (Figure 1a). The downregulation of MUC1-C was associated with decreases in the abundance of ZEB1 protein (Figure 1a, left). Densitometric scanning of the ZEB1 protein signals demonstrated a decrease of 40±9% in response to MUC1-C silencing (Figure 1a, left, legend). As determined by quantitative reverse transcription–PCR (qRT–PCR), ZEB1 mRNA levels were also decreased in MDA-MB-231 cells silenced for MUC1-C expression (Figure 1a, right). To extend this analysis, similar studies were performed on BT-549 breast cancer cells. Stable silencing of MUC1-C was associated with a decrease of 53±4% in ZEB1 protein (Figure 1b, left). Moreover, the downregulation of ZEB1 expression in BT-549 cells was conferred, at least in part, by decreases in ZEB1 mRNA levels (Figure 1b, right). Studies were also performed using MCF-7 breast cancer cells, which constitutively express MUC1-C and low to undetectable levels of ZEB1 protein (Figure 1c, left). Notably, stable overexpression of MUC1-C resulted in a marked induction of ZEB1 protein (Figure 1c, left) and mRNA (Figure 1c, right), indicating that a threshold of MUC1-C is necessary for this response. Immunofluorescence studies of MDA-MB-231 cells further demonstrated that both MUC1-C and ZEB1 are detectable in the nucleus, and that there is colocalization of the MUC1-C and ZEB1 signals (Figure 1d). Similar findings were obtained with BT-549 (Supplementary Figure S1A), MCF-7/MUC1-C (Figure 1e) and primary breast cancer (Supplementary Figure S1B) cells.

Figure 1.

Figure 1

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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).

MUC1-C activates the ZEB1 gene by a NF-κB-dependent mechanism

MUC1-C associates with NF-κB p65 and promotes NF-κB-mediated gene transcription.17 Other studies have shown that NF-κB activation is associated with increases in ZEB1 expression in mammary epithelial cells.19 In concert with a role for NF-κB p65 in the regulation of ZEB1 expression, silencing of p65 in MDA-MB-231 cells was associated with decreases in ZEB1 abundance (Figure 2a, left). Densitometric scanning of the ZEB1 protein signals demonstrated a decrease of 79±5% in response to p65 silencing (Figure 2a, left, legend). Inhibition of NF-κB activity with the small molecule BAY11-708520 also suppressed ZEB1 levels (Figure 2a, right). Similar results were obtained in BT-549 cells silenced for p65 (Figure 2b, left and legend) or treated with BAY11-7085 (Figure 2b, right). To assess the involvement of NF-κB p65 in the induction of ZEB1 expression by MUC1-C, we silenced p65 in the MCF-7/MUC1-C cells (Figure 2c, left). Downregulation of p65 abrogated MUC1-C-induced increases in ZEB1 expression (Figure 2c, left). In addition, BAY11-7085 treatment was associated with the suppression of ZEB1 levels (Figure 2c, right), indicating that MUC1-C induces ZEB1 expression by a NF-κB p65-dependent mechanism. The ZEB1 promoter contains a consensus sequence for NF-κB binding (GGGAACTCCC; position −568 to −559) (Figure 2d); however, to our knowledge, there is no published evidence for NF-κB occupancy of that site. ChIP experiments performed on MDA-MB-231/control short hairpin RNA (CshRNA) and MDA-MB-231/MUC1shRNA cells showed binding of NF-κB p65 to the ZEB1 promoter (Figure 2e, left). The results further show that NF-κB occupancy is decreased by silencing MUC1-C expression (Figure 2e, left). In addition, re-ChIP studies demonstrated that MUC1-C occupies the ZEB1 promoter with NF-κB (Figure 2e, right). ChIP experiments performed on MCF-7/vector and MCF-7/MUC1-C cells further demonstrate that overexpression of MUC1-C increases NF-κB occupancy (Figure 2f, left) and that MUC1-C occupies the ZEB1 promoter with NF-κB (Figure 2f, right). The findings that MUC1-C (i) forms complexes with NF-κB and (ii) increases NF-κB promoter occupancy have been obtained with other NF-κB target genes, for example, Bcl-xL and MUC1 itself,17 indicating that this effect is not specific for the ZEB1 promoter.

Figure 2.

Figure 2

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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.

MUC1-C binds directly to ZEB1

MUC1-C interacts directly with certain transcription factors that include, among others, NF-κB and transcription factor 4 (TCF4).17,21 Coimmunoprecipitation studies using lysates prepared from MDA-MB-231 cells provided further support for an association between endogenous MUC1-C and ZEB1 (Figure 3a, left). Similar results were obtained with BT-549 (Supplementary Figure S2) and MCF-7/MUC1-C (Figure 3a, right) cells. To define the basis for the interaction, we generated three recombinant glutathione S-transferase (GST) fragments from the 1108-amino acid ZEB1 protein (Figure 3b). Incubation of the GST–ZEB1 fragments with MCF-7/MUC1-C cell lysates demonstrated binding of MUC1-C to GST–ZEB1(1–385) and, to a lesser extent, GST–ZEB1(386–711) (Figure 3b). By contrast, there was no detectable binding of MUC1-C to GST or GST–ZEB1(712–1108) (Figure 3b). To determine whether the interaction is direct, the GST–ZEB1 fragments were incubated with purified MUC1-C cytoplasmic domain (MUC1-CD). Binding of MUC1-CD was also detectable with GST–ZEB1(1–385) and GST–ZEB1(386–711), but not GST–ZEB1(712–1108) (Figure 3c). To further define the basis for the interaction, incubations were performed with fragments of the 72-amino acid MUC1-CD (Figure 3d). The results demonstrate that MUC1-CD(1–45), but not MUC1-CD(46–72), binds to ZEB1(1–385) and ZEB1(386–711) (Figure 3d, left and right). MUC1-CD(1–45) contains a CQC motif that confers the formation of MUC1-C homodimers.22,23 Mutation of the CQC motif to AQA abrogated the binding of MUC1-CD to both ZEB1(1–385) and ZEB1(386–711) (Figure 3e). These findings indicate that MUC1-C associates with ZEB1 in cells through a direct interaction involving the MUC1-CD.

Figure 3.

Figure 3

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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.

MUC1-C promotes ZEB1-mediated suppression of miR-200c

ZEB1 binds to Z-boxes in the miR-200c promoter and thereby suppresses miR-200c expression.5,11 The above observations that MUC1-C associates with ZEB1 invoked the possibility that MUC1-C could occupy the miR-200c promoter with ZEB1. To directly address this possibility, we performed ChIP studies on the miR-200c promoter in MCF-7 cells (Figure 4a). Consistent with the low to undetectable ZEB1 levels in MCF-7/vector cells, ZEB1 occupancy on the miR-200c promoter was similar to that obtained with the immunoglobulin G (IgG) control (Figure 4a, left). By contrast, ZEB1 occupancy was significantly increased in MCF-7/MUC1-C cells (Figure 4a, left). Re-ChIP studies further demonstrated that ZEB1 occupies the miR-200c promoter in a complex with MUC1-C (Figure 4a, right). In turn, the overexpression of MUC1-C in MCF-7 cells was associated with marked downregulation of miR-200c expression as determined by RT–PCR (Figure 4b, left). qRT–PCR analysis further demonstrated MUC1-C-dependent decreases in both pri-miR-200c and mature miR-200c levels (Figure 4b, right). As a control for these MCF-7 cell studies, we overexpressed MUC1-C, in which the critical CQC motif for homodimerization and nuclear localization22 was mutated to AQA (Figure 4c). In contrast to wild-type MUC1-C, MUC1-C(AQA) was inactive in inducing ZEB1 and suppressing miR-200c expression (Figure 4c, left and right). In concert with these results, silencing of MUC1-C in MDA-MB-231 cells resulted in upregulation of pri-miR-200c and miR-200c levels (Figure 4d, left and right). Similar results were obtained in BT-549 cells (Figure 4e, left and right), indicating that MUC1-C promotes ZEB1-mediated suppression of miR-200c expression. ZEB1 downregulates other miRNAs; for example, miR-203, which has been linked to stemness.6 As found for miR-200c, levels of miR-203 were (i) decreased by the overexpression of MUC1-C in MCF-7 cells (Supplementary Figure S3A), and increased by silencing MUC1 in MDA-MB-231 (Supplementary Figure S3B) and BT-549 (Supplementary Figure S3C) cells. Thus, MUC1-C-induced regulation of ZEB1 targets is not restricted to miR-200c.

Figure 4.

Figure 4

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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.

MUC1-C induces EMT and invasion

The co-ordinate upregulation of ZEB1 and suppression of miR-200c has been linked to the induction of EMT.6 In that regard, overexpression of MUC1-C in MCF-7 cells and thereby the upregulation of ZEB1 was associated with a decrease in E-cadherin abundance and an increase in vimentin, consistent with the induction of EMT (Figure 5a). Immunofluorescence studies confirmed loss of E-cadherin localization at the cell membrane of MCF-7/MUC1-C cells as compared with that found in MCF-7/vector cells (Figure 5b). To determine whether the overexpression of MUC1-C affects invasion, we analyzed the MCF-7/vector and MCF-7/MUC1-C cells in a transwell invasion assay (Figure 5c, left). The MCF-7/MUC1-C cells exhibited an enhanced ability to invade through matrigel as compared with that obtained with MCF-7/vector cells (Figure 5c, left). Quantitation of the number of invaded MCF-7 cells confirmed that MUC1-C significantly promotes invasion (Figure 5c, right). Consistent with these results, silencing MUC1-C in MDA-MB-231 cells was associated with an increase in E-cadherin expression (Figure 5d) and localization of E-cadherin at the cell membrane (Figure 5e). Moreover, invasion of MDA-MB-231 cells with MUC1-C silencing was decreased compared with that found for MDA-MB-231 cells expressing the control CshRNA (Figure 5f, left and right). Overexpression of MUC1-C in MCF-7 cells was also associated with a substantial increase in size of mammospheres (Supplementary Figure S4A). Additionally, silencing MUC1 in MDA-MB-231 cells resulted in a marked decrease in mammosphere size (Supplementary Figure S4B). These findings support a model, in which the MUC1-C subunit is sufficient to induce EMT, invasion and the formation of mammospheres.

Figure 5.

Figure 5

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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).

MUC1-C induces EMT and invasion by a ZEB1-dependent mechanism

To demonstrate that MUC1-C induces EMT and invasion by a ZEB1-dependent mechanism, we stably silenced ZEB1 in MCF-7/MUC1-C cells (Figure 6a). ZEB1 silencing was associated with upregulation of E-cadherin, consistent with induction of the mesenchymal-epithelial transition (Figure 6a). In concert with these results, ZEB1 silencing resulted in upregulation of miR-200c as determined by RT–PCR (Figure 6b, left) and qRT–PCR (Figure 6b, right). Silencing ZEB1 in MCF-7/MUC1-C cells was also associated with a significant decrease in invasion (Figure 6c, left and right). In addition and as found in MCF-7/MUC1-C cells, stable silencing of ZEB1 in MDA-MB-231 cells induced E-cadherin (Figure 6d, left) and miR-200c (Figure 6d, right) expression. Invasion of MDA-MB-231 cells with ZEB1 silencing was also significantly decreased compared with that obtained with cells infected to express the control CshRNA (Figure 6e). These findings collectively indicate that MUC1-C induces EMT and invasion by a ZEB1-dependent mechanism.

Figure 6.

Figure 6

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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).

DISCUSSION

MUC1-C oncoprotein induces ZEB1 expression

The transforming growth factor cytokines are well-characterized inducers of EMT in cancer cells.24 Pathways involving Notch, Wnt and receptor tyrosine kinase signaling have also been shown to contribute to the induction of EMT programs.1 The EMT phenotype, which contributes to invasion, metastatic disease and resistance to treatment, is conferred by an array of transcription factors that include, among others, ZEB1. The present studies demonstrate that the MUC1-C oncoprotein contributes to the induction of ZEB1 expression in breast cancer cells. Previous work had shown that (i) Notch induces EMT by activating NF-κB25 and (ii) TNF-induced NF-κB activation and EMT is associated with increases in ZEB1 expression.19 Given that MUC1-C binds directly to NF-κB p65 and promotes transcription of NF-κB target genes,17 we reasoned that MUC1-C-induced expression of ZEB1 might be conferred by a NF-κB-dependent mechanism. Indeed, studies in MCF-7/MUC1-C cells showed that silencing NF-κB p65 or treatment with a NF-κB inhibitor decreases MUC1-C-induced ZEB1 expression. Interrogation of the bcl-xL gene promoter previously showed that binding of MUC1-C to NF-κB increases NF-κB occupancy and its transactivation function.17 In concert with those observations,17 the ZEB1 promoter contains a consensus NF-κB binding sequence and NF-κB occupancy of that site was increased by MUC1-C overexpression. These results thus supported a model, in which MUC1-C induces ZEB1 expression by interacting with NF-κB and promoting NF-κB-mediated activation of the ZEB1 gene (Figure 7). Other work has demonstrated that Wnt signaling induces ZEB1 expression by binding of the β-catenin/TCF4 complex to the ZEB1 promoter.26 Notably, in this regard, MUC1-C interacts directly with β-catenin and thereby increases β-catenin levels.27 Moreover, MUC1-C promotes the formation of β-catenin/TCF4 complexes and thereby activation of Wnt target genes.21 Thus, the present findings that MUC1-C induces ZEB1 expression in certain breast cancer cells by a NF-κB-dependent mechanism do not exclude the possibility that MUC1-C could contribute to ZEB1 activation through the Wnt pathway in other cell types.

Figure 7.

Figure 7

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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 p6517 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.

MUC1-C interacts with ZEB1 in the suppression of miR-200c

The overexpression of MUC1-C as found in breast cancer cells is associated with targeting of MUC1-C to the nucleus, where it interacts with transcription factors, such as NF-κB and TCF4/TCF7L2.17,21 In the present work, coimmunoprecipitation studies demonstrated that MUC1-C associates with ZEB1, suggesting that MUC1-C may interact with ZEB1 on the promoters of ZEB1 target genes. To search for additional evidence in support of such an interaction, we found that the MUC1-CD binds directly to ZEB1 at the N-terminal region (aa 1–385) that contains zinc finger domains. In addition, direct binding of the MUC1-CD was detectable with the homeodomain-containing region (aa 386–711). To determine whether MUC1-C forms a complex with ZEB1 in the nucleus, we studied occupancy of the miR-200c promoter, which contains two Z-boxes (CAGGTA) that are consensus ZEB1 binding sites.11,28 As shown in colorectal cancer cells,11 our results demonstrated ZEB1 occupancy on the miR-200c promoter in MCF-7 breast cancer cells. Moreover and significantly, MUC1-C overexpression was associated with the formation of MUC1-C/ZEB1 complexes on the miR-200c promoter and increased ZEB1 occupancy. ZEB1 suppresses the miR-200c promoter and thereby decreases miR-200c expression.11 In concert with the MUC1-C-induced increases in ZEB1 occupancy on the miR-200c promoter, MUC1-C was also effective in suppressing miR-200c levels in breast cancer cells. A role for MUC1-C in contributing to the suppression of miR-200c expression is of potential importance to activation of the EMT program. In this respect, miR-200c downregulates ZEB1 abundance in a double-negative feedback loop and thereby blocks ZEB1-mediated induction of EMT.6,11 miR-200c and other miR-200 family members also target stem cell factors, such as SOX2, KLF4 and BMI1, in cancer cells.5,29 In this way, the contribution of MUC1-C to suppression of miR-200c could promote both EMT and stem cell properties. EMT has been linked to the generation of mammary stem-like cells;3,4 however, recent work indicates that reversion to the epithelial phenotype is associated with the acquisition of stem cell properties.30 Nonetheless, the present results demonstrate that MUC1-C also has a marked effect on mammosphere formation, suggesting that MUC1-C functions in the regulation of epithelial stem cell properties.

MUC1-C induces EMT by a ZEB1-dependent mechanism

Loss of E-cadherin is one of the hallmarks of EMT and cancer progression.1,2 Like Snail and Slug, ZEB1 is a transcriptional repressor of E-cadherin.31 In addition, ZEB1 suppresses the expression of multiple genes of importance to cell–cell adhesion and differentiation, indicating that ZEB1 regulates EMT and epithelial polarity.9 Consistent with these findings, the present studies demonstrate that MUC1-C-induced upregulation of ZEB1 is associated with the downregulation of E-cadherin. MUC1-C-induced ZEB1 expression was also associated with increases in breast cancer cell invasion. MUC1 has previously been associated with the induction of EMT in DA3 mouse mammary tumor cells14 and in human pancreatic cancer cells.15 However, to our knowledge, there has been no previous evidence to support a link between MUC1-C and the ZEB1/miR-200c feedback loop in activation of the EMT program. MUC1-C function is dependent on the transient loss of polarity associated with the epithelial stress response.12 In this regard, the repositioning of apical MUC1-C over the entire cell membrane promotes the interaction of MUC1-C with receptor tyrosine kinases that are normally expressed at the basolateral borders of polarized epithelial cells.12,13 In turn, MUC1-C contributes to the activation of downstream effectors of receptor tyrosine kinase signaling pathways, including the upregulation of MUC1-C expression.12,13 The available evidence indicates that breast cancer cells have subverted and appropriated this function of MUC1-C to support their growth and survival.13 The present results further indicate that MUC1-C could also promote sustained loss of polarity by upregulating ZEB1 in cancer cells and thereby activating the EMT program.

MATERIALS AND METHODS

Cell culture

Human MDA-MB-231 (ATCC HTB-26) and MCF-7 (ATCC HTB-22) breast cancer cells were cultured in Dulbecco's modified Eagle's medium with 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin and 2 mm l-glutamine. Human BT-549 breast cancer cells (ATCC HTB-122) were grown in Roswell Park Memorial Institute 1640 medium with heat-inactivated fetal bovine serum, antibiotics, l-glutamine and 10 μg/ml insulin. MDA-MB-231 and BT-549 cells were infected with a lentiviral vector expressing a MUC1shRNA (Sigma, St Louis, MO, USA) or with a scrambled control shRNA vector (CshRNA; Sigma) as described.32 MCF-7 cells were transfected to stably express a control pHR-CMV-GFP and vectors expressing MUC1-C or MUC1-C(AQA). Transfection of cells with small interfering RNA pools was performed in the presence of Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). For inhibition of NF-κB activity, cells were treated with the NF-κB inhibitor BAY11-7085 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Cells were also infected with lentiviral vectors expressing a p65 shRNA (Sigma), a ZEB1 shRNA (Sigma) or a scrambled control shRNA vector (CshRNA; Sigma).

Immunoprecipitation and immunoblotting

Total cell lysates were prepared in NP-40 lysis buffer as described.33 Soluble proteins were subjected to immunoprecipitation with anti-ZEB1 antibody (Cell Signaling Technology, Danvers, MA, USA). Precipitates and cell lysates were analyzed by immunoblotting with anti-MUC1-C (LabVision, Fremont, CA, USA), anti-NF-κB (Santa Cruz Biotechnology), anti-ZEB1 (Cell Signaling Technology), anti-E-cadherin (Cell Signaling Technology), anti-vimentin (Cell Signaling Technology) and anti-β-actin (Sigma). Detection of immune complexes was achieved using horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence (GE Healthcare, Piscataway, NJ, USA). Intensity of the signals was determined by densitometric scanning using the Alphaimager EC and Spot Denso analysis with auto-background control.

Quantitative RT–PCR

For qRT–PCR, complementary DNA (cDNA) synthesis was performed with 1 μg of total RNA using the Thermoscript RT–PCR system (Invitrogen). The SYBR green qPCR assay kit (Applied Biosystems, Carlsbad, CA, USA) was used with 1 μl of diluted cDNA for each sample. The samples were amplified with the ABI Prism 7000 Sequence Detector (Applied Biosystems). Primers used for qRT–PCR of ZEB1 and glyceraldehyde 3-phosphate dehydrogenase are listed in Supplementary Table SI. Statistical significance was determined by the Student's t-test.

Chromatin immunoprecipitation assays

Soluble chromatin was prepared as described16 and precipitated with anti-NF-κB, anti-MUC1-C (Ab5/CT2; LabVision), anti-ZEB1 (Cell Signaling Technology, no. 3396) or a control non-immune immunoglobulin G. For PCR, 2 μl from a 50 μl DNA extraction were used with 25–35 cycles of amplification. For real time ChIP qPCR, the SYBR green Champion ChIP qPCR assay kit (SABiosciences, Valencia, CA, USA) was used and the samples were amplified with the ABI Prism 7000 Sequence Detector (Applied Biosystems). The primers used for PCR and qPCR for the ZEB1 promoter and control region are listed in Supplementary Table SII. The primers used for the miR-200c promoter and glyceraldehyde 3-phosphate dehydrogenase are as described11 (Supplementary Table SIII). Relative fold enrichment was calculated as described.34

Immunofluorescence studies

Cells were fixed in 4% paraformaldehyde in 2% sucrose/phosphate-buffered saline (PBS) solution for 15 min and permeabilized in 0.5% Triton-X 100/PBS for 5 min. Following permeabilization, cells were washed two times with 1X PBS and incubated with anti-MUC1-C, anti-ZEB1 (Novus Biologicals, Littleton, CO, USA) or anti-E-cadherin diluted in 1% bovine serum albumin/PBS for 30 min at 37 °C. Cells were then washed three times with 1X PBS and incubated with appropriate conjugated secondary antibodies for 25 min at 37 °C. Following two washes with 1X PBS, coverslips were mounted on slides with Prolong Gold Antifade mounting reagent containing 4',6-diamidino-2-phenylindole (DAPI) (Invitrogen). Slides were visualized using a Leica SP5X: Laser scanning confocal microscope. Images were processed using ImageJ software program (National Institutes of Health).

Protein binding assays

GST-tagged fragments of ZEB1 were generated by PCR amplification of mRNA from MCF-7/MUC1-C cells and subcloning into pGEX-5X-1 (GE Healthcare). Purified MUC1-CD, MUC1-CD(1–45), MUC1-CD(46–72) and MUC1-CD(AQA) were prepared by expressing the appropriate GST-fusion protein and cleavage of the GST tag with thrombin as described.22 GST and GST-fusion proteins bound to glutathione beads were incubated with cell lysates or purified proteins. The adsorbates were analyzed by immunoblotting with anti-MUC1-CD antibodies CD135 and CT2 (Ab5; LabVision).

Analysis of miR-200c expression

Total RNA was isolated from cells using the RNeasy total RNA isolation kit (Qiagen, Valencia, CA, USA). cDNAs were prepared from 1 μg total RNA using the cDNA synthesis kit specific for small RNA (System Biosciences, Mountain View, CA, USA). Expression of miR-200c and miR-203 was assessed by end-point RT–PCR and by qRT–PCR with a universal reverse primer and forward primers specific for these miRNAs. Forward and reverse primers for the analysis of pri-miR-200c were derived from Ensembl (MIR200C; ENSG00000207713) and are included in Supplementary Table SIV. The end-point PCR products were assayed using 1 μl of diluted cDNA and Taq DNA polymerase (Qiagen). Human U6 small RNA was used as control.36 For qPCR, the SYBR green qPCR assay kit (Applied Biosystems) was used with 1 μl of diluted cDNA sample and analyzed with the ABI Prism 7000 Sequence Detector (Applied Biosystems). Fold enrichment was calculated as described.34

Invasion assays

Cell invasion assays were performed using 8 μm pore transwell chambers precoated with matrigel (BD BioScience) as described.16,37 Briefly, 25 000 cells in 500 μl medium were seeded into the upper chamber. After incubation for 24 h, membranes were fixed with methanol and stained with 1% toluidine blue.

Mammosphere assays

MCF-7 cells (10 000 cells/well) and MDA-MB-231 cells (5000 cells/well) were suspended in 2 ml of complete Mammocult Medium (Stem Cell Technologies, Vancouver, BC, Canada) and seeded in individual wells of ultra-low attachment 6-well culture plates (Costar, Tewksbury, MA, USA). Mammospheres were visualized after culture for 7 d using a Nikon TE2000 inverted live-cell imaging system.

Supplementary Material

01

ACKNOWLEDGEMENTS

Research reported in this publication was supported by the National Cancer Institute of the National Institutes of Health under award numbers CA97098 and CA166480.

Footnotes

CONFLICT OF INTEREST Dr D Kufe is a founder of Genus Oncology and holds equity in the company. The remaining authors disclosed no potential conflicts of interest.

Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)

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Supplementary Materials

01
NIHMS485966-supplement-01.pdf (16MB, pdf)

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