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• W2049063169 abstract "Aldehyde dehydrogenase 1A1 (ALDH1A1) activity is used as a marker of breast cancer stem cells; however, little is known about the regulation of ALDH1A1 expression. Mucin 1 (MUC1) is a heterodimeric protein that is aberrantly overexpressed in most human breast cancers. In studies of breast cancer cells stably silenced for MUC1 or overexpressing the oncogenic MUC1-C subunit, we demonstrate that MUC1-C is sufficient for induction of MEK→ERK signaling and that treatment with a MUC1-C inhibitor suppresses ERK activation. In turn, MUC1-C induces ERK-mediated phosphorylation and activation of the CCAAT/enhancer-binding protein β (C/EBPβ) transcription factor. The results further show that MUC1-C and C/EBPβ form a complex on the ALDH1A1 gene promoter and activate ALDH1A1 gene transcription. MUC1-C-induced up-regulation of ALDH1A1 expression is associated with increases in ALDH activity and is detectable in stem-like cells when expanded as mammospheres. These findings demonstrate that MUC1-C (i) activates a previously unrecognized ERK→C/EBPβ→ALDH1A1 pathway, and (ii) promotes the induction of ALDH activity in breast cancer cells.Background: Aldehyde dehydrogenase 1A1 (ALDH1A1) activity is up-regulated in breast cancer cells by mechanisms that are unclear.Results: The MUC1-C oncoprotein induces ERK-mediated activation of the C/EBPβ transcription factor and, thereby, ALDH1A1 expression.Conclusion: MUC1-C activates a novel ERK→C/EBPβ→ALDH1A1 pathway that up-regulates ALDH activity.Significance: Overexpression of MUC1-C in breast cancer cells contributes to an ALDH+ phenotype that is linked to stemness. Aldehyde dehydrogenase 1A1 (ALDH1A1) activity is used as a marker of breast cancer stem cells; however, little is known about the regulation of ALDH1A1 expression. Mucin 1 (MUC1) is a heterodimeric protein that is aberrantly overexpressed in most human breast cancers. In studies of breast cancer cells stably silenced for MUC1 or overexpressing the oncogenic MUC1-C subunit, we demonstrate that MUC1-C is sufficient for induction of MEK→ERK signaling and that treatment with a MUC1-C inhibitor suppresses ERK activation. In turn, MUC1-C induces ERK-mediated phosphorylation and activation of the CCAAT/enhancer-binding protein β (C/EBPβ) transcription factor. The results further show that MUC1-C and C/EBPβ form a complex on the ALDH1A1 gene promoter and activate ALDH1A1 gene transcription. MUC1-C-induced up-regulation of ALDH1A1 expression is associated with increases in ALDH activity and is detectable in stem-like cells when expanded as mammospheres. These findings demonstrate that MUC1-C (i) activates a previously unrecognized ERK→C/EBPβ→ALDH1A1 pathway, and (ii) promotes the induction of ALDH activity in breast cancer cells. Background: Aldehyde dehydrogenase 1A1 (ALDH1A1) activity is up-regulated in breast cancer cells by mechanisms that are unclear. Results: The MUC1-C oncoprotein induces ERK-mediated activation of the C/EBPβ transcription factor and, thereby, ALDH1A1 expression. Conclusion: MUC1-C activates a novel ERK→C/EBPβ→ALDH1A1 pathway that up-regulates ALDH activity. Significance: Overexpression of MUC1-C in breast cancer cells contributes to an ALDH+ phenotype that is linked to stemness. The CCAAT/enhancer-binding protein β (C/EBPβ) 4The abbreviations used are: C/EBPβCCAAT/enhancer-binding proteinMUC1mucin 1MUC1-CMUC1 C-terminal subunitMUC1-CDMUC1 cytoplasmic domainERKextracellular signal-regulated kinaseALDHaldehyde dehydrogenase. is a leucine-zipper transcription factor that regulates growth and differentiation of hematopoietic and epithelial cells (1.Ramji D.P. Foka P. CCAAT/enhancer-binding proteins: structure, function and regulation.Biochem. J. 2002; 365: 561-575Google Scholar). Multiple isoforms of human C/EBPβ (p55, p45/42, and p20) are expressed as a result of alternative sites for translation initiation (2.Eaton E.M. Hanlon M. Bundy L. Sealy L. Characterization of C/EBPβ isoforms in normal versus neoplastic mammary epithelial cells.J. Cell. Physiol. 2001; 189: 91-105Google Scholar). The C/EBPβ p55 and p45/42 isoforms (LAP1 and LAP2) function as transcriptional activators, whereas the p20 isoform (LIP) acts as a repressor (3.Descombes P. Schibler U. A liver-enriched transcriptional activator protein, LAP, and a transcriptional inhibitory protein, LIP, are translated from the same mRNA.Cell. 1991; 67: 569-579Google Scholar). C/EBPβ plays an important role in mammary gland development by regulating stem cell repopulation activity and specifying luminal cell fate (4.Grimm S.L. Rosen J.M. The role of C/EBPbeta in mammary gland development and breast cancer.J. Mammary Gland Biol. Neoplasia. 2003; 8: 191-204Google Scholar, 5.LaMarca H.L. Visbal A.P. Creighton C.J. Liu H. Zhang Y. Behbod F. Rosen J.M. CCAAT/enhancer-binding protein beta regulates stem cell activity and specifies luminal cell fate in the mammary gland.Stem Cells. 2010; 28: 535-544Google Scholar). Dysregulation of C/EBPβ has also been associated with the development and metastatic progression of breast cancer (6.Bundy L.M. Sealy L. CCAAT/enhancer binding protein beta (C/EBPβ)-2 transforms normal mammary epithelial cells and induces epithelial to mesenchymal transition in culture.Oncogene. 2003; 22: 869-883Google Scholar, 7.Atwood A.A. Jerrell R. Sealy L. Negative regulation of C/EBPβ1 by sumoylation in breast cancer cells.PLoS One. 2011; 6: e25205Google Scholar, 8.Zahnow C.A. Younes P. Laucirica R. Rosen J.M. Overexpression of C/EBPβ-LIP, a naturally occurring, dominant-negative transcription factor, in human breast cancer.J. Natl. Cancer Inst. 1997; 89: 1887-1891Google Scholar, 9.Gomis R.R. Alarcón C. Nadal C. Van Poznak C. Massagué J. C/EBPβ at the core of the TGFbeta cytostatic response and its evasion in metastatic breast cancer cells.Cancer Cell. 2006; 10: 203-214Google Scholar). Inactive C/EBPβ is maintained in a closed conformational state that interferes with binding of the basic region to DNA (10.Kowenz-Leutz E. Twamley G. Ansieau S. Leutz A. Novel mechanism of C/EBPβ(NF-M) transcriptional control: activation through derepression.Genes Dev. 1994; 8: 2781-2791Google Scholar, 11.Williams S.C. Baer M. Dillner A.J. Johnson P.F. CRP2 (C/EBPβ) contains a bipartite regulatory domain that controls transcriptional activation, DNA binding and cell specificity.EMBO J. 1995; 14: 3170-3183Google Scholar). In contrast, ERK-mediated phosphorylation of C/EBPβ on Thr-235 results in an open conformation with induction of its transactivation function (10.Kowenz-Leutz E. Twamley G. Ansieau S. Leutz A. Novel mechanism of C/EBPβ(NF-M) transcriptional control: activation through derepression.Genes Dev. 1994; 8: 2781-2791Google Scholar, 11.Williams S.C. Baer M. Dillner A.J. Johnson P.F. CRP2 (C/EBPβ) contains a bipartite regulatory domain that controls transcriptional activation, DNA binding and cell specificity.EMBO J. 1995; 14: 3170-3183Google Scholar, 12.Nakajima T. Kinoshita S. Sasagawa T. Sasaki K. Naruto M. Kishimoto T. Akira S. Phosphorylation at threonine 235 by a RAS-dependent mitogen-activated protein kinase cascade is essential for transcription factor NF-IL6.Proc. Natl. Acad. Sci. U.S.A. 1993; 90: 2207-2211Google Scholar). These findings have linked activation of C/EBPβ to the MEK→ERK signaling pathway. CCAAT/enhancer-binding protein mucin 1 MUC1 C-terminal subunit MUC1 cytoplasmic domain extracellular signal-regulated kinase aldehyde dehydrogenase. The aldehyde dehydrogenase (ALDH) superfamily of enzymes plays an important role in cellular signaling and protection by catalyzing the oxidation of aldehydes (13.Marchitti S.A. Brocker C. Stagos D. Vasiliou V. Non-P450 aldehyde oxidizing enzymes: the aldehyde dehydrogenase superfamily.Expert Opin. Drug Metab. Toxicol. 2008; 4: 697-720Google Scholar). The ALDH1A1 isoform has been a particular focus of study as a marker for both normal and cancer stem cells (14.Ma I. Allan A.L. The role of human aldehyde dehydrogenase in normal and cancer stem cells.Stem Cell Rev. 2011; 7: 292-306Google Scholar). ALDH1A1 functions in part by oxidizing retinol to retinoic acid, a regulator of gene transcription and inducer of cellular differentiation (15.Douville J. Beaulieu R. Balicki D. ALDH1 as a functional marker of cancer stem and progenitor cells.Stem Cells Dev. 2009; 18: 17-25Google Scholar). In this respect, high expression of ALDH1A1 in normal hematopoietic stem cells has been linked to retinoid metabolism and control of self-renewal capacity (16.Chute J.P. Muramoto G.G. Whitesides J. Colvin M. Safi R. Chao N.J. McDonnell D.P. Inhibition of aldehyde dehydrogenase and retinoid signaling induces the expansion of human hematopoietic stem cells.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 11707-11712Google Scholar). ALDH1A1 has also been identified as a marker of normal and malignant mammary stem cells that regulates retinoid signaling and breast cancer stem cell differentiation (17.Ginestier C. Hur M.H. Charafe-Jauffret E. Monville F. Dutcher J. Brown M. Jacquemier J. Viens P. Kleer C.G. Liu S. Schott A. Hayes D. Birnbaum D. Wicha M.S. Dontu G. ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome.Cell Stem Cell. 2007; 1: 555-567Google Scholar, 18.Ginestier C. Wicinski J. Cervera N. Monville F. Finetti P. Bertucci F. Wicha M.S. Birnbaum D. Charafe-Jauffret E. Retinoid signaling regulates breast cancer stem cell differentiation.Cell Cycle. 2009; 8: 3297-3302Google Scholar). Despite the potential role of ALDH1 in self-renewal and differentiation, little is known about the regulation of ALDH1A1 expression in cancer cells. Studies in human K562 erythroleukemia and Hep3B hepatoma cells demonstrated that the ALDH1A1 promoter contains a positive regulatory region (−91 to +53 bp to the transcription start site) with a CCAAT box as the major cis-acting element (19.Yanagawa Y. Chen J.C. Hsu L.C. Yoshida A. The transcriptional regulation of human aldehyde dehydrogenase I gene. The structural and functional analysis of the promoter.J. Biol. Chem. 1995; 270: 17521-17527Google Scholar). Other work in mouse hepatoma cells showed that the retinoic acid receptor α (RARα) transactivates the ALDH1A1 promoter by binding to a RA response-like element (RARE) located at positions −91 to −75 bp (20.Elizondo G. Corchero J. Sterneck E. Gonzalez F.J. Feedback inhibition of the retinaldehyde dehydrogenase gene ALDH1 by retinoic acid through retinoic acid receptor alpha and CCAAT/enhancer-binding protein beta.J. Biol. Chem. 2000; 275: 39747-39753Google Scholar). In addition, C/EBPβ has been shown to transactivate the ALDH1A1 promoter by interacting with the CCAAT box that resides at −75 to −71 bp adjacent to the RARE (20.Elizondo G. Corchero J. Sterneck E. Gonzalez F.J. Feedback inhibition of the retinaldehyde dehydrogenase gene ALDH1 by retinoic acid through retinoic acid receptor alpha and CCAAT/enhancer-binding protein beta.J. Biol. Chem. 2000; 275: 39747-39753Google Scholar). These findings and the demonstration that ERK activates C/EBPβ invoked the possibility that ERK→C/EBPβ signaling may contribute to induction of ALDH1A1 expression in cancer cells. Mucin 1 (MUC1) is a transmembrane protein that is aberrantly overexpressed in most human breast cancers (21.Kufe D. Mucins in cancer: function, prognosis and therapy.Nature Reviews Cancer. 2009; 9: 874-885Google Scholar, 22.Kufe D. MUC1-C oncoprotein as a target in breast cancer: activation of signaling pathways and therapeutic approaches.Oncogene. 2013; 32: 1073-1081Google Scholar). Studies of MUC1 function have been directed by the findings that MUC1 undergoes auto-cleavage into two subunits that, in turn, form a stable non-covalent complex at the cell membrane (22.Kufe D. MUC1-C oncoprotein as a target in breast cancer: activation of signaling pathways and therapeutic approaches.Oncogene. 2013; 32: 1073-1081Google Scholar). The MUC1 N-terminal subunit (MUC1-N) contains glycosylated tandem repeats that are a physical characteristic of the mucin family members. MUC1-N is positioned extracellularly in a heterodimeric complex with the MUC1 C-terminal subunit (MUC1-C) that spans the cell membrane (21.Kufe D. Mucins in cancer: function, prognosis and therapy.Nature Reviews Cancer. 2009; 9: 874-885Google Scholar, 22.Kufe D. MUC1-C oncoprotein as a target in breast cancer: activation of signaling pathways and therapeutic approaches.Oncogene. 2013; 32: 1073-1081Google Scholar). The MUC1-N/MUC1-C heterodimer resides at the apical membrane of non-transformed epithelial cells. However, with stress or transformation and thereby loss of polarity, the MUC1-N/MUC1-C complex is expressed over the entire cell membrane, where it interacts with receptor tyrosine kinases (RTKs), such as EGFR, HER2, and others (23.Li Y. Ren J. Yu W. Li Q. Kuwahara H. Yin L. Carraway 3rd, K.L. Kufe D. The EGF receptor regulates interaction of the human DF3/MUC1 carcinoma antigen with c-Src and β-catenin.J. Biol. Chem. 2001; 276: 35239-35242Google Scholar, 24.Li Y. Yu W.-H. Ren J. Chen W. Huang L. Kharbanda S. Loda M. Kufe D. Heregulin targets γ-catenin to the nucleolus by a mechanism dependent on the DF3/MUC1 protein.Mol. Cancer Res. 2003; 1: 765-775Google Scholar, 25.Ramasamy S. Duraisamy S. Barbashov S. Kawano T. Kharbanda S. Kufe D. The MUC1 and galectin-3 oncoproteins function in a microRNA-dependent regulatory loop.Mol. Cell. 2007; 27: 992-1004Google Scholar). The weight of evidence further supports a role for the MUC1-C subunit in conferring RTK activation. In this regard, targeting MUC1-C down-regulates HER2 activation and its downstream signals in breast cancer cells (26.Raina D. Uchida Y. Kharbanda A. Rajabi H. Panchamoorthy G. Jin C. Kharbanda S. Scaltriti M. Baselga J. Kufe D. Targeting the MUC1-C oncoprotein downregulates HER2 activation and abrogates trastuzumab resistance in breast cancer cells.Oncogene. 2013; (in press)Google Scholar). The MUC1-C cytoplasmic domain also contains a YHPM site that, when phosphorylated on tyrosine, interacts with PI3K and contributes to activation of the AKT pathway (27.Raina D. Kharbanda S. Kufe D. The MUC1 oncoprotein activates the anti-apoptotic PI3K/Akt and Bcl-xL pathways in rat 3Y1 fibroblasts.J. Biol. Chem. 2004; 279: 20607-20612Google Scholar, 28.Raina D. Kosugi M. Ahmad R. Panchamoorthy G. Rajabi H. Alam M. Shimamura T. Shapiro G. Supko J. Kharbanda S. Kufe D. Dependence on the MUC1-C oncoprotein in non-small cell lung cancer cells.Mol. Cancer Therapeutics. 2011; 10: 806-816Google Scholar). In addition, the MUC1-C cytoplasmic domain contains a pYTNP site that interacts with GRB2, linking MUC1-C to SOS and the RAS pathway (29.Pandey P. Kharbanda S. Kufe D. Association of the DF3/MUC1 breast cancer antigen with Grb2 and the Sos/Ras exchange protein.Cancer Res. 1995; 55: 4000-4003Google Scholar). Moreover, the MUC1-C subunit localizes to the nucleus, where it interacts with transcription factors, such as NF-κB, and promotes expression of genes involved in growth and survival (30.Ahmad R. Raina D. Joshi M.D. Kawano T. Ren J. Kharbanda S. Kufe D. MUC1-C oncoprotein functions as a direct activator of the NF-κB p65 transcription factor.Cancer Res. 2009; 69: 7013-7021Google Scholar, 31.Ahmad R. Rajabi H. Kosugi M. Joshi M. Alam M. Vasir B. Kawano T. Kharbanda S. Kufe D. MUC1-C oncoprotein promotes STAT3 activation in an auto-inductive regulatory loop.Science Signaling. 2011; 4: ra9Google Scholar, 32.Rajabi H. Ahmad R. Jin C. Joshi M. Guha M. Alam M. Kharbanda S. Kufe D. MUC1-C oncoprotein confers androgen-independent growth of human prostate cancer cells.Prostate. 2012; 72: 1659-1668Google Scholar). The present studies demonstrate that MUC1-C induces ERK signaling and thereby phosphorylation and activation of C/EBPβ. We also show that a complex of MUC1-C and C/EBPβ occupies the ALDH1A1 promoter and induces ALDH1A1 expression. These findings support a novel MUC1-C→ERK→C/EBPβ pathway that up-regulates ALDH activity in breast cancer cells. Human MDA-MB-468 and MCF-7 breast cancer cells were cultured in DMEM with 10% heat-inactivated fetal bovine serum (FBS), 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mm l-glutamine. SKBR3 breast cancer cells were grown in McCoy's 5A medium containing FBS, antibiotics, and l-glutamine. MDA-MB-468 and SKBR3 cells were infected with a lentiviral vector expressing a MUC1 shRNA or, as a control, with a scrambled shRNA vector (CshRNA) as described (33.Jin C. Rajabi H. Pitroda S. Kharbanda A. Li A. Weichselbaum R. Kufe D. Cooperative interaction between the MUC1 oncoprotein and the Rab31 GTPase in human breast cancer cells.PLoS One. 2012; 7: e39432Google Scholar). Cells were also infected with a lentiviral vector expressing C/EBPβ shRNA (Sigma). MCF-7 cells were stably transfected with a control pHR-CMV-GFP vector or one expressing MUC1-C. Cells were treated with the (i) MUC1-C inhibitor GO-203 (28.Raina D. Kosugi M. Ahmad R. Panchamoorthy G. Rajabi H. Alam M. Shimamura T. Shapiro G. Supko J. Kharbanda S. Kufe D. Dependence on the MUC1-C oncoprotein in non-small cell lung cancer cells.Mol. Cancer Therapeutics. 2011; 10: 806-816Google Scholar, 34.Raina D. Ahmad R. Rajabi H. Panchamoorthy G. Kharbanda S. Kufe D. Targeting cysteine-mediated dimerization of the MUC1-C oncoprotein in human cancer cells.Int. J. Oncol. 2012; 40: 1643-1649Google Scholar), (ii) MEK inhibitor U0126 (Sigma-Aldrich) (35.Favata M.F. Horiuchi K.Y. Manos E.J. Daulerio A.J. Stradley D.A. Feeser W.S. Van Dyk D.E. Pitts W.J. Earl R.A. Hobbs F. Copeland R.A. Magolda R.L. Scherle P.A. Trzaskos J.M. Identification of a novel inhibitor of mitogen-activated protein kinase kinase.J. Biol. Chem. 1998; 273: 18623-18632Google Scholar) or (iii) NF-κB pathway inhibitor BAY11-7085 (Santa Cruz Biotechnology) (36.Pierce J.W. Schoenleber R. Jesmok G. Best J. Moore S.A. Collins T. Gerritsen M.E. Novel inhibitors of cytokine-induced IκBα phosphorylation and endothelial cell adhesion molecule expression show anti-inflammatory effects in vivo.J. Biol. Chem. 1997; 272: 21096-21103Google Scholar). Whole cell lysates were prepared in Nonidet P-40 lysis buffer as described (31.Ahmad R. Rajabi H. Kosugi M. Joshi M. Alam M. Vasir B. Kawano T. Kharbanda S. Kufe D. MUC1-C oncoprotein promotes STAT3 activation in an auto-inductive regulatory loop.Science Signaling. 2011; 4: ra9Google Scholar). Immunoprecipitation of soluble proteins was performed with anti-C/EBPβ (Santa Cruz Biotechnology) or anti-MUC1-C (CT2; LabVision). Precipitates and cell lysates not subjected to precipitation were analyzed by immunoblotting with anti-MUC1-C (LabVision), anti-phospho-MEK, anti-MEK, anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho-C/EBPβ (Cell Signaling Technology), anti-C/EBPβ, anti-NF-κB p65 (Santa Cruz Biotechnology), and anti-β-actin (Sigma). Detection of immune complexes was achieved using horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence (GE Healthcare). For qRT-PCR analysis of C/EBPβ and ALDH1A1 mRNA levels, cDNA synthesis was performed with 1 μg of total RNA using the Thermoscript RT-PCR system (Invitrogen). cDNA samples were amplified using the SYBR green qPCR assay kit (Applied Biosystems) and the ABI Prism 7000 Sequence Detector (Applied Biosystems). Primers used for qRT-PCR detection of C/EBPβ and ALDH1A1 are listed in supplemental Table S1. Statistical significance was determined by the Student's t test. Cells plated on cover slips were fixed in 4% paraformaldehyde/2% sucrose solution for 15 min and permeabilized in 0.5% Triton X-100/PBS for 5 min. The cells were then washed twice with PBS, incubated with anti-MUC1-C or anti-p-C/EBPβ (Abcam) antibodies diluted in 1% BSA/PBS for 30 min at 37 °C, washed three times with PBS and incubated with appropriate conjugated secondary antibodies diluted in PBS for 25 min at 37 °C. Following two washes with PBS, the coverslips were mounted on slides with Prolong Gold Antifade mounting reagent containing DAPI (Invitrogen) and stored in the dark at 4 °C overnight to cure before visualization. The slides were visualized using a Leica SP5X: Laser Scanning Confocal microscope. Images were processed using ImageJ software program (NIH). GST-tagged human C/EBPβ was generated by subcloning C/EBPβ from pBABE-puro-LAP2 plasmid vector (Addgene) into the pGEX-5X-1 plasmid (GE Healthcare). GST-C/EBPβ(C35A) and GST-C/EBPβ(C184A) were generated from GST-C/EBPβ by site-directed mutagenesis (Stratagene). 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 (37.Leng Y. Cao C. Ren J. Huang L. Chen D. Ito M. Kufe D. Nuclear import of the MUC1-C oncoprotein is mediated by nucleoporin Nup62.J. Biol. Chem. 2007; 282: 19321-19330Google Scholar). GST and GST fusion proteins bound to glutathione beads were incubated with purified proteins. The adsorbates were analyzed by immunoblotting with anti-C/EBPβ or anti-MUC1-C cytoplasmic domain antibodies CD1 (38.Panchamoorthy G. Rehan H. Kharbanda A. Ahmad R. Kufe D. A monoclonal antibody against the oncogenic mucin 1 cytoplasmic domain.Hybridoma. 2011; 30: 531-535Google Scholar) or CT2 (Ab5; LabVision). Cells were transfected with pGL3, pGL3-pALDH1A1-Luc or pGL3-pALDH1A1(CCAAT→GAGTC; mut)-Luc and, as an internal control, SV-40-Renilla-Luc (Promega) in the presence of Superfect (Qiagen). After 48 h, the cells were lysed in passive lysis buffer. The lysates were analyzed using the dual luciferase assay kit (Promega). Soluble chromatin was prepared as described (32.Rajabi H. Ahmad R. Jin C. Joshi M. Guha M. Alam M. Kharbanda S. Kufe D. MUC1-C oncoprotein confers androgen-independent growth of human prostate cancer cells.Prostate. 2012; 72: 1659-1668Google Scholar) and precipitated with anti-C/EBPβ or a control nonimmune IgG. For re-ChIP assays, C/EBPβ complexes from the primary ChIP were eluted and reimmunoprecipitated with anti-MUC1-C as described (39.Rajabi H. Joshi M.D. Jin C. Ahmad R. Kufe D. Androgen receptor regulates expression of the MUC1-C oncoprotein in human prostate cancer cells.Prostate. 2011; 71: 1299-1308Google Scholar). The SYBR green qPCR assay kit was used for real time ChIP qPCR with the ABI Prism 7000 Sequence Detector (Applied Biosystems). The primers used for qPCR of the ALDH1A1 promoter and control region are listed in supplemental Table S2. Relative fold enrichment was calculated as described (40.Wang Q. Carroll J.S. Brown M. Spatial and temporal recruitment of androgen receptor and its coactivators involves chromosomal looping and polymerase tracking.Mol. Cell. 2005; 19: 631-642Google Scholar). ALDH enzymatic activity was measured using the Aldefluor kit (Stem Cell Technologies). Cells suspended in aldefluor assay buffer were incubated with ALDH enzyme substrate, BODIPY-aminoacetaldehyde (BAAA), for 40 min at 37 °C. As a control for baseline fluorescence, cells were also treated with the ALDH inhibitor, diethylaminobenzaldehyde (DEAB). Fluorescence was detected using a BD Biosciences LSRFortessa flow cytometer and analyzed using FACSDiva software (BD Biosciences). Statistical significance was determined by the Student's t test. Single-cell suspensions were cultured in MammoCult™ Human Medium Kit (StemCell Technologies) at a density of 40,000 cells per well of a 6-well ultra-low attachment culture plate (Corning CoStar). For first generation M1 culturing, cells were grown with replenishment of the medium twice over 7 days. For second M2 generation culturing, M1 mammospheres were harvested, incubated with trypsin for 3 min at 37 °C, and mechanically dispersed by gentle pipetting. Single cells were confirmed under a microscope, counted, and resuspended in fresh MammoCult™ medium. Mammospheres were imaged using a Nikon inverted TE2000 microscope. C/EBPβ is an auto-repressed transcription factor that is activated by ERK-mediated phosphorylation on Thr-235 (10.Kowenz-Leutz E. Twamley G. Ansieau S. Leutz A. Novel mechanism of C/EBPβ(NF-M) transcriptional control: activation through derepression.Genes Dev. 1994; 8: 2781-2791Google Scholar, 12.Nakajima T. Kinoshita S. Sasagawa T. Sasaki K. Naruto M. Kishimoto T. Akira S. Phosphorylation at threonine 235 by a RAS-dependent mitogen-activated protein kinase cascade is essential for transcription factor NF-IL6.Proc. Natl. Acad. Sci. U.S.A. 1993; 90: 2207-2211Google Scholar, 41.Lee S. Shuman J.D. Guszczynski T. Sakchaisri K. Sebastian T. Copeland T.D. Miller M. Cohen M.S. Taunton J. Smart R.C. Xiao Z. Yu L.R. Veenstra T.D. Johnson P.F. RSK-mediated phosphorylation in the C/EBP{β} leucine zipper regulates DNA binding, dimerization, and growth arrest activity.Mol. Cell. Biol. 2010; 30: 2621-2635Google Scholar). MUC1-C has been linked to activation of the RAS→MEK→ERK pathway (29.Pandey P. Kharbanda S. Kufe D. Association of the DF3/MUC1 breast cancer antigen with Grb2 and the Sos/Ras exchange protein.Cancer Res. 1995; 55: 4000-4003Google Scholar, 42.Hattrup C.L. Gendler S.J. MUC1 alters oncogenic events and transcription in human breast cancer cells.Breast Cancer Res. 2006; 8: R37Google Scholar, 43.Yao M. Zhang W. Zhang Q. Xing L. Xu A. Liu Q. Cui B. Overexpression of MUC1 enhances proangiogenic activity of non-small-cell lung cancer cells through activation of Akt and extracellular signal-regulated kinase pathways.Lung. 2011; 189: 453-460Google Scholar). To determine whether MUC1-C plays a role in the regulation of C/EBPβ, we stably silenced MUC1 in triple-negative MDA-MB-468 breast cancer cells (Fig. 1A). Down-regulation of MUC1-C was associated with a marked decrease in p-MEK and p-ERK levels (Fig. 1A). In concert with ERK-mediated phosphorylation of C/EBPβ on Thr-235, abundance of p-C/EBPβ (p45/42; LAP2) was also decreased by MUC1-C silencing (Fig. 1A). By contrast, there was no detectable phosphorylation of the C/EBPβ p20 isoform (LIP) in MDA-MB-468/CshRNA or MDA-MB-468/MUC1shRNA cells (data not shown). Treatment with the MUC1-C inhibitor, GO-203, was similarly associated with down-regulation of MEK and ERK phosphorylation (Fig. 1B). In addition, GO-203 treatment resulted in a 55 ± 2% decrease in p-C/EBPβ levels (Fig. 1B, legend). In HER2-overexpressing SKBR3 breast cancer cells, targeting MUC1-C with silencing or treatment with GO-203 is associated with down-regulation of HER2 activation (26.Raina D. Uchida Y. Kharbanda A. Rajabi H. Panchamoorthy G. Jin C. Kharbanda S. Scaltriti M. Baselga J. Kufe D. Targeting the MUC1-C oncoprotein downregulates HER2 activation and abrogates trastuzumab resistance in breast cancer cells.Oncogene. 2013; (in press)Google Scholar). Silencing MUC1 in SKBR3 cells also resulted in decreased activation of MEK, ERK and C/EBPβ (Fig. 1C). In addition, treatment of SKBR3 cells with GO-203 was associated with suppression of p-MEK and p-ERK levels and a 54 ± 5% decrease in the abundance of p-C/EBPβ (Fig. 1D). As a control for involvement of ERK, treatment of the MDA-MB-468 and SKBR3 cells with the MEK inhibitor U0126 decreased both ERK and C/EBPβ phosphorylation (Fig. 1, E and F). Collectively, these findings demonstrate that MUC1-C promotes ERK-dependent phosphorylation of C/EBPβ on Thr-235. Studies were also performed on MCF-7 breast cancer cells that express MUC1-C and C/EBPβ at levels lower than those found in MDA-MB-468 and SKBR3 cells (Fig. 2A). Here, stable overexpression of MUC1-C in MCF-7 cells was associated with increases in C/EBPβ protein (Fig. 2B, left). qRT-PCR further demonstrated that MUC1-C increases C/EBPβ mRNA levels (Fig. 2B, right). Similar results have been obtained with a separate set of MCF-7 cells that were transduced with lentiviruses expressing an empty vector or MUC1-C, indicating that the increase in C/EBPβ expression is not due to clonal selection (data not shown). Previous studies have linked NF-κB to activation of the C/EBPβ gene (44.Niehof M. Kubicka S. Zender L. Manns M.P. Trautwein C. Autoregulation enables different pathways to control CCAAT/enhancer binding protein beta (C/EBPβ) transcription.J. Mol. Biol. 2001; 309: 855-868Google Scholar). In addition, MUC1-C interacts with NF-κB p65 and promotes NF-κB-mediated gene transcription (30.Ahmad R. Raina D. Joshi M.D. Kawano T. Ren J. Kharbanda S. Kufe D. MUC1-C oncoprotein functions as a direct activator of the NF-κB p65 transcription factor.Cancer Res. 2009; 69: 7013-7021Google Scholar). To determine whether MUC1-C induces C/EBPβ expression by a NF-κB-dependent mechanism, we silenced NF-κB p65 in the MCF-7/MUC1-C cells (Fig. 2C). Silencing p65 was associated with down-regulation of C/EBPβ mRNA and protein (Fig. 2C, left and right). Treatment of MCF-7/MUC1-C cells with the NF-κB inhibitor BAY11–7085 also resulted in decreases in abundance of C/EBPβ protein (Fig. 2D, left). Diverse cell types respond to BAY11–7085 with increases in ERK phosphorylation (45.Relic B. Benoit V. Franchimont N. Ribbens C. Kaiser M.J. Gillet P. Merville M.P. Bours V. Malaise M.G. 15-deoxy-Δ12,14-prostaglandin J2 inhibits Bay 11–7085-induced sustained extracellular signal-regulated kinase phosphorylation and apoptosis in human articular chondrocytes and synovial fibroblasts.J. Biol. Chem. 2004; 279: 22399-22403Google Scholar). Similar results were obtained in the MCF-7/MUC1-C cells (Fig. 2D, right), indicating that MUC1-C induces C/EBPβ protein levels by an NF-κB-dependent, ERK-independent pathway. With regard to MUC1-C-induced activation of the MEK→ERK pathway, overexpression of MUC1-C was associated with increases in p-MEK and p-ERK levels (Fig. 2E, left). In addition, we found that the MUC1-C-induced C/EBPβ protein is phosphorylated on Thr-235 (Fig. 2E, left) and that treatment with the MEK inhibitor U0126 blocks this modification (Fig. 2E, right). These results demonstrate that overexpression of MUC1-C in MCF-7 cells induces both C/EBPβ abundance and ERK-mediated phosphorylation of C/EBPβ on Thr-2" @default.
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• W2049063169 date "2013-10-01" @default.
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• W2049063169 title "MUC1-C Oncoprotein Activates ERK→C/EBPβ Signaling and Induction of Aldehyde Dehydrogenase 1A1 in Breast Cancer Cells" @default.
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