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• W2170025905 abstract "The mucin-like glycoprotein episialin (MUC1) is highly overproduced by a number of human carcinomas. We have shown previously in a variety of mammalian cell lines that overexpression of this very large transmembrane molecule diminishes cellular adhesion, suggesting that episialin/MUC1 overexpression may play an important role in tumor invasion and metastasis. By using in situhybridization, we show here that episialin/MUC1 mRNA expression can be increased more than 10-fold in breast carcinoma cells relative to the expression in adjacent normal breast epithelium. In search of the molecular mechanism of this overexpression, we observed that the episialin/MUC1 promoter contains a candidate binding site for transcription factors of the STAT family ∼500 base pairs upstream of the transcription start site. Cytokines and/or growth factors such as interleukin-6 or interferon-γ can activate STATs. In the human breast carcinoma cell line T47D, both compounds are able to stimulate transcription of a luciferase reporter gene under the control of a 750-base pair MUC1 promoter fragment proximal to the transcription start site. The observed increase is entirely mediated by the single STAT-binding site, since mutation of this site abolishes stimulation of the reporter by interleukin-6 and interferon-γ. In addition, mutation of the STAT site also decreased the promoter activity in nonstimulated T47D cells, suggesting that the STAT-binding site is among the elements that are involved in the overexpression of MUC1 in tumor cells. The mucin-like glycoprotein episialin (MUC1) is highly overproduced by a number of human carcinomas. We have shown previously in a variety of mammalian cell lines that overexpression of this very large transmembrane molecule diminishes cellular adhesion, suggesting that episialin/MUC1 overexpression may play an important role in tumor invasion and metastasis. By using in situhybridization, we show here that episialin/MUC1 mRNA expression can be increased more than 10-fold in breast carcinoma cells relative to the expression in adjacent normal breast epithelium. In search of the molecular mechanism of this overexpression, we observed that the episialin/MUC1 promoter contains a candidate binding site for transcription factors of the STAT family ∼500 base pairs upstream of the transcription start site. Cytokines and/or growth factors such as interleukin-6 or interferon-γ can activate STATs. In the human breast carcinoma cell line T47D, both compounds are able to stimulate transcription of a luciferase reporter gene under the control of a 750-base pair MUC1 promoter fragment proximal to the transcription start site. The observed increase is entirely mediated by the single STAT-binding site, since mutation of this site abolishes stimulation of the reporter by interleukin-6 and interferon-γ. In addition, mutation of the STAT site also decreased the promoter activity in nonstimulated T47D cells, suggesting that the STAT-binding site is among the elements that are involved in the overexpression of MUC1 in tumor cells. mucin-1 electrophoretic mobility shift assay fetal bovine serum intercellular adhesion molecule-1 interferon interleukin-6 signal transducer and activator of transcription Dulbecco's modified Eagle's medium base pair phosphate-buffered saline Episialin/MUC11 (also known as MUC1, PEM, CA 15-3 antigen, or EMA) is a transmembrane molecule with a large extracellular mucin-like domain. In normal cells episialin/MUC1 is exclusively present at the apical side of the cell, but in carcinoma cells normal polarization is lost and episialin/MUC1 co-localizes with adhesion molecules such as integrins and cadherins. The long and relatively rigid extracellular domain of episialin/MUC1 can shield these adhesion molecules and diminish cellular adhesion, if present at a sufficiently high density on the cell surface (1Ligtenberg M.J. Buijs F. Vos H.L. Hilkens J. Cancer Res. 1992; 52: 2318-2324PubMed Google Scholar, 2Wesseling J. van der Valk S.W. Vos H.L. Sonnenberg A. Hilkens J. J. Cell Biol. 1995; 129: 255-265Crossref PubMed Scopus (442) Google Scholar, 3Wesseling J. van der Valk S.W. Hilkens J. Mol. Biol. Cell. 1996; 7: 565-577Crossref PubMed Scopus (358) Google Scholar). Overexpression of episialin/MUC1 in carcinoma cells has been frequently reported (4Hilkens J. Buijs F. Hilgers J. Hageman P. Calafat J. Sonnenberg A. van der Valk M. Int. J. Cancer. 1984; 34: 197-206Crossref PubMed Scopus (402) Google Scholar, 5Zotter S. Lossnitzer A. Hageman P.C. Delemarre J.F. Hilkens J. Hilgers J. Lab. Invest. 1987; 57: 193-199PubMed Google Scholar, 6Zaretsky J.Z. Weiss M. Tsarfaty I. Hareuveni M. Wreschner D.H. Keydar I. FEBS Lett. 1990; 265: 46-50Crossref PubMed Scopus (45) Google Scholar) and is expected to have a similar effect on cellular behavior as loss of E-cadherin, the major epithelial cell-cell adhesion molecule, which has been shown to promote invasion and metastasis of carcinoma cells (for review see Refs. 7Behrens J. Invasion Metastasis. 1994; 14: 61-70PubMed Google Scholar, 8Shiozaki H. Oka H. Inoue M. Tamura S. Monden M. Cancer (Phila.). 1996; 8: 133-1605Google Scholar, 9Bracke M.E. Van Roy F.M. Mareel M.M. Curr. Top. Microbiol. Immunol. 1996; 213: 123-161Crossref PubMed Scopus (214) Google Scholar). Therefore, we have proposed that episialin/MUC1 also plays an important role in invasion and metastasis in vivo (10Hilkens J. Vos H.L. Wesseling J. Peterse J. Storm J. Boer M. van der Valk S.W. Maas A.M. Calvo F. Crepin M. Magdelenat H. Breast Cancer: Advances in Biology and Therapeutics. John Libbey Eurotext, Montrouge, France1996: 281-288Google Scholar). Indeed, transgenic mice overexpressing episialin/MUC1 develop more aggressive lung tumors than nontransgenic mice, 2J. Wesseling and J. Hilkens, unpublished data. 2J. Wesseling and J. Hilkens, unpublished data.whereas episialin/MUC1 null mice show a slower rate of tumor progression (11Spicer A.P. Rowse G.J. Lidner T.K. Gendler S.J. J. Biol. Chem. 1995; 270: 30093-30101Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar).Recent reports have shown that episialin overexpression in various types of neoplasia correlates with poor survival (12Nakamori S. Ota D.M. Cleary K.R. Shirotani K. Irimura T. Gastroenterology. 1994; 106: 353-361Abstract Full Text PDF PubMed Google Scholar, 13Takao S. Uchikura K. Yonezawa S. Shinchi H. Aikou T. Cancer (Phila.). 1999; 86: 1966-1975Crossref PubMed Scopus (52) Google Scholar, 14Guddo F. Giatromanolaki A. Koukourakis M.I. Reina C. Vignola A.M. Chlouverakis G. Hilkens J. Gatter K.C. Harris A.L. Bonsignore G. J. Clin. Pathol. 1998; 51: 667-671Crossref PubMed Scopus (103) Google Scholar). Episialin/MUC1 is also the antigen that is measured in the CA 15-3 assay (the main blood marker to detect recurrence of breast cancer), and it is a molecule that is widely considered as one of the most promising molecules to be used for vaccination against breast cancer. Therefore, knowledge about the regulation of episialin/MUC1 overexpression in tumor cells is of utmost clinical importance. In B cell lymphomas it has been shown that episialin/MUC1 overexpression is frequently the result of a t(1;14) (q21;q32) translocation, which brings the episialin/MUC1 gene under the control of an immunoglobulin γ heavy chain enhancer (15Dyomin V.G. Palanisamy N. Lloyd K.O. Dyomina K. Jhanwar S.C. Houldsworth J. Chaganti R.S. Blood. 2000; 95: 2666-2671Crossref PubMed Google Scholar, 16Gilles F. Goy A. Remache Y. Shue P. Zelenetz A.D. Blood. 2000; 95: 2930-2936Crossref PubMed Google Scholar) and is consistent with a role in tumorigenesis of these lymphomas. However, the cause(s) of episialin/MUC1 overexpression in carcinoma cells have not been established yet. Previous studies (6Zaretsky J.Z. Weiss M. Tsarfaty I. Hareuveni M. Wreschner D.H. Keydar I. FEBS Lett. 1990; 265: 46-50Crossref PubMed Scopus (45) Google Scholar, 17Bièche I. Lidereau R. Cancer Genet. Cytogenet. 1997; 98: 75-80Abstract Full Text PDF PubMed Scopus (35) Google Scholar) have shown that the episialin/MUC1 mRNA level is severalfold increased in primary breast carcinoma specimens relative to adjacent normal epithelium. However, these results, which were obtained using mRNA blots of tissue homogenates, are inherently imprecise, since both breast carcinoma and normal breast tissue specimens usually contain significant amounts of other nonepithelial cell types. Even so, these results indicate that overexpression of episialin/MUC1 coincides with increased mRNA levels. Episialin/MUC1 mRNA levels in breast carcinoma cell lines also are increased in comparison to immortalized nontransformed epithelial cells (18Ligtenberg M.J. Vos H.L. Gennissen A.M. Hilkens J. J. Biol. Chem. 1990; 265: 5573-5578Abstract Full Text PDF PubMed Google Scholar). The study of Bièche and Lidereau (17Bièche I. Lidereau R. Cancer Genet. Cytogenet. 1997; 98: 75-80Abstract Full Text PDF PubMed Scopus (35) Google Scholar) showed that the higher episialin/MUC1 mRNA levels to some degree may be caused by amplification of theMUC1 gene, but additional mechanisms of overexpression clearly are operative as well, since the level of overexpression often is much higher than the level of amplification.We hypothesized that overexpression of episialin/MUC1 in carcinoma cells is the result of constitutive activation of a transcription factor or inactivation of a repressor. Constitutively activated signaling pathways are common in carcinoma cells and strongly contribute to the malignant properties of these cells. As an initial step to elucidate such a putative factor or pathway, we studied the normal transcriptional regulation of the episialin/MUC1 gene first. As a lead we considered exogenous factors that have been shown to up-regulate episialin biosynthesis.IFN-γ is known to up-regulate episialin/MUC1 expression in cell lines derived from breast carcinomas (19Clark S. McGuckin M.A. Hurst T. Ward B.G. Dis. Markers. 1994; 12: 43-50Crossref PubMed Scopus (35) Google Scholar). 3C. F. M. Molthoff, personal communication. 3C. F. M. Molthoff, personal communication.Similarly, mouse episialin/MUC1 expression is positively regulated by prolactin, when cells are grown on matrigel-coated membrane filters (20Parry G. Li J. Stubbs J. Bissell M.J. Schmidhauser C. Spicer A.P. Gendler S.J. J. Cell Sci. 1992; 101: 191-199PubMed Google Scholar). Induction of expression by prolactin is in line with the increased expression of human episialin during lactation (4Hilkens J. Buijs F. Hilgers J. Hageman P. Calafat J. Sonnenberg A. van der Valk M. Int. J. Cancer. 1984; 34: 197-206Crossref PubMed Scopus (402) Google Scholar), when prolactin levels are strongly increased. Notably, breast carcinoma cells may synthesize prolactin (21Clevenger C.V. Chang W.P. Ngo W. Pasha T.L. Montone K.T. Tomaszewski J.E. Am. J. Pathol. 1995; 146: 695-705PubMed Google Scholar, 22Ginsburg E. Vonderhaar B.K. Cancer Res. 1995; 55: 2591-2595PubMed Google Scholar) that could lead to autocrine stimulation of episialin expression. Both IFN-γ and prolactin act via signaling transducers and activators of transcription (STATs). Different members of this protein family function during inflammatory processes and during mammary gland development and lactation to up-regulate the transcription of a large number of genes (23Liu X. Robinson G.W. Wagner K.U. Garrett L. Wynshaw-Boris A. Hennighausen L. Genes Dev. 1997; 11: 179-186Crossref PubMed Scopus (909) Google Scholar). In addition to interferons and prolactin, STATs may also be activated by cytokines of the interleukin family, such as interleukin-6 (IL-6). IL-6, which signals via STAT3, is produced by a variety of cells among which are epithelial cells (for a review see Klein (24Klein B. Klein B. Cytokines in Human Multiple Myeloma. R. G. Landes Co., Austin, TX1994Google Scholar)). It is noteworthy that enhanced levels of IL-6 are found in many solid tumors (25Krueger J. Ray A. Tamm I. Sehgal P.B. J. Cell. Biochem. 1991; 45: 327-334Crossref PubMed Scopus (88) Google Scholar), whereas in the breast carcinoma cell lines T47D and ZR-75-1 IL-6 cause an enhanced motility and decreased adhesion (26Tamm I. Cardinale I. Murphy J.S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4414-4418Crossref PubMed Scopus (34) Google Scholar, 27Tamm I. Kikuchi T. Cardinale I. Krueger J.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3329-3333Crossref PubMed Scopus (45) Google Scholar), properties that could involve overexpression of episialin/MUC1.From the considerations presented above, we concluded that STAT proteins might be one of the important regulators of episialin/MUC1 transcription. Further evidence that STATs might be involved in episialin/MUC1 (over)expression is as follows. (i) A putative STAT-binding element, which is conserved in human and mouse, is present around 500 bp upstream of the transcription start site (28Spicer A.P. Parry G. Patton S. Gendler S.J. J. Biol. Chem. 1991; 266: 15099-15109Abstract Full Text PDF PubMed Google Scholar, 29Vos H.L. De Vries Y. Hilkens J. Biochem. Biophys. Res. Commun. 1991; 181: 121-130Crossref PubMed Scopus (55) Google Scholar, 30Kovarik A. Peat N. Wilson D. Gendler S.J. Taylor-Papadimitriou J. J. Biol. Chem. 1993; 268: 9917-9926Abstract Full Text PDF PubMed Google Scholar) and is located well within the 750-bp proximal region that has been identified to contain the regulatory sequences for episialin/MUC1 expression in various cancer cell lines (30Kovarik A. Peat N. Wilson D. Gendler S.J. Taylor-Papadimitriou J. J. Biol. Chem. 1993; 268: 9917-9926Abstract Full Text PDF PubMed Google Scholar,31Abe M. Kufe D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 282-286Crossref PubMed Scopus (108) Google Scholar). 4H. L. Vos, S. Van der Valk, and J. Hilkens, unpublished data. 4H. L. Vos, S. Van der Valk, and J. Hilkens, unpublished data.(ii) STAT proteins are known to be constitutively activated in a number of cancer types including breast carcinoma (32Watson C.J. Miller W.R. Br. J. Cancer. 1995; 71: 840-844Crossref PubMed Scopus (208) Google Scholar, 33Garcia R., Yu, C.L. Hudnall A. Catlett R. Nelson K.L. Smithgall T. Fujita D.J. Ethier S.P. Jove R. Cell Growth Differ. 1997; 8: 1267-1276PubMed Google Scholar). In addition, a recent publication (34Bromberg J.F. Wrzeszczynska M.H. Devgan G. Zhao Y. Pestell R.G. Albanese C. Darnell J.E.J. Cell. 1999; 98: 295-303Abstract Full Text Full Text PDF PubMed Scopus (2471) Google Scholar) shows that STAT3 can act as an oncogene. Our results unambiguously show for the first time that the expression of the episialin/MUC1 gene indeed is highly up-regulated in breast cancer and that STAT transcription factors contribute to this overexpression of episialin/MUC1.DISCUSSIONImmunohistological studies of tissue sections with monoclonal antibodies against peptide epitopes in the mucin domain of episialin suggested that this molecule is strongly overexpressed in breast cancer (4Hilkens J. Buijs F. Hilgers J. Hageman P. Calafat J. Sonnenberg A. van der Valk M. Int. J. Cancer. 1984; 34: 197-206Crossref PubMed Scopus (402) Google Scholar, 5Zotter S. Lossnitzer A. Hageman P.C. Delemarre J.F. Hilkens J. Hilgers J. Lab. Invest. 1987; 57: 193-199PubMed Google Scholar). However, the mucin domain of episialin on normal breast epithelial cells carries numerous branched O-linked carbohydrates that will block or hamper binding of the antibodies, whereas episialin molecules derived from carcinomas mainly contain shorter nonbranched glycans, more easily allowing the access of the antibodies to their epitopes (49Girling A. Bartkova J. Burchell J. Gendler S. Gillett C. Taylor-Papadimitriou J. Int. J. Cancer. 1989; 43: 1072-1076Crossref PubMed Scopus (301) Google Scholar, 50Burchell J. Taylor-Papadimitriou J. Epithelial Cell Biol. 1993; 2: 155-162PubMed Google Scholar, 51Hanisch F.G. Stadie T.R. Deutzmann F. Peter-Katalinic J. Eur. J. Biochem. 1996; 236: 318-327Crossref PubMed Scopus (121) Google Scholar, 52Lloyd K.O. Burchell J. Kudryashov V. Yin B.W.T. Taylor- Papadimitriou J. J. Biol. Chem. 1996; 271: 33325-33334Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar). Thus, the increased reactivity of most monoclonal antibodies against episialin with tumor cells as reported in the literature could well be explained by differences in post-translational modifications in normal and tumor cells and not necessarily by overexpression of the episialin/MUC1 gene. In this report, our immunohistological studies, employing monoclonal antibodies that are hardly, if at all, affected byO-glycosylation, indicate that overexpression of episialin/MUC1 protein in breast carcinoma cells indeed occurs. The reports of Zaretsky et al. (6Zaretsky J.Z. Weiss M. Tsarfaty I. Hareuveni M. Wreschner D.H. Keydar I. FEBS Lett. 1990; 265: 46-50Crossref PubMed Scopus (45) Google Scholar) and Bièche and Lidereau (17Bièche I. Lidereau R. Cancer Genet. Cytogenet. 1997; 98: 75-80Abstract Full Text PDF PubMed Scopus (35) Google Scholar) indicate that increased episialin mRNA levels are responsible for the overexpression of episialin/MUC1 protein in breast carcinoma cells. However, their studies were performed on whole tissue homogenates and therefore are difficult to interpret. Our in situ hybridization results unequivocally show for the first time that the episialin/MUC1 gene is indeed strongly overexpressed in breast carcinomas relative to normal breast epithelium.One possible explanation for the increased episialin/MUC1 mRNA levels in carcinoma cells is stabilization of the mRNA. However, the half-life in the normal breast epithelial cell line HBL-100 (Fig.2) and in the tumor cell lines MCF-7 (41Abe M. Kufe D. J. Cell. Physiol. 1990; 143: 226-231Crossref PubMed Scopus (27) Google Scholar) and T47D (Fig. 8) is not significantly different. In fact, episialin/MUC1 mRNA is very stable, which is compatible with a role of episialin as a structural protein. Other ways to overproduce an mRNA are amplification of the gene and, more rarely, up-regulation of the promoter by mutation. The first mechanism is indeed operative in the case of episialin/MUC1, as shown by Bièche and Lidereau (17Bièche I. Lidereau R. Cancer Genet. Cytogenet. 1997; 98: 75-80Abstract Full Text PDF PubMed Scopus (35) Google Scholar), but it is neither able to explain all cases of overexpression nor it can explain the extent of mRNA overexpression in carcinomas and cell lines. A promoter mutation is expected to affect only one of the two alleles. Northern data provided by Ligtenberg et al. (18Ligtenberg M.J. Vos H.L. Gennissen A.M. Hilkens J. J. Biol. Chem. 1990; 265: 5573-5578Abstract Full Text PDF PubMed Google Scholar) and Bièche and Lidereau (17Bièche I. Lidereau R. Cancer Genet. Cytogenet. 1997; 98: 75-80Abstract Full Text PDF PubMed Scopus (35) Google Scholar) clearly indicate that the expression of both alleles, which can be easily distinguished from one another on the basis of differences in the number of repeats, is directly proportional to the number of gene copies as determined on Southern blots. These observations exclude mutation(s) in the promoter as a mechanism of overexpression and leave changes in transcriptional regulation of theMUC1 gene as the most important mechanism of episialin/MUC1 mRNA overexpression.Our study shows that the episialin/MUC1 promoter can be stimulated by STAT transcription factors via a STAT-binding site in its promoter. The position and sequence of this site is completely conserved in the promoters of mouse (29Vos H.L. De Vries Y. Hilkens J. Biochem. Biophys. Res. Commun. 1991; 181: 121-130Crossref PubMed Scopus (55) Google Scholar) and gibbon (28Spicer A.P. Parry G. Patton S. Gendler S.J. J. Biol. Chem. 1991; 266: 15099-15109Abstract Full Text PDF PubMed Google Scholar), and the 9-nucleotide core sequence is identical to that of the proven binding site for STAT1 and STAT3 in the ICAM1 promoter (44Look D.C. Pelletier M.R. Holtzman M.J. J. Biol. Chem. 1994; 269: 8952-8958Abstract Full Text PDF PubMed Google Scholar, 45Caldenhoven E. Coffer P. Yuan J. Van de Stolpe A. Horn F. Kruijer W. Van der Saag P.T. J. Biol. Chem. 1994; 269: 21146-21154Abstract Full Text PDF PubMed Google Scholar). We identified the proteins that bind to the STAT site in the episialin/MUC1 promoter as STAT1 in the case of induction by IFN-γ and STAT3 in the case of IL-6. This is completely in line with the current model of IFN-γ and IL-6 signaling (42Darnell Jr., J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3345) Google Scholar, 53Ihle J.N. Cell. 1996; 84: 331-334Abstract Full Text Full Text PDF PubMed Scopus (1262) Google Scholar).Even without the addition of any growth factor or serum the W3B construct, which contains a firefly luciferase reporter gene driven by 720 bp of the episialin/MUC1 promoter (including the STAT-responsive element), already showed a significant activity in reporter assays in the mammary carcinoma cell line T47D, which decreases 3-fold upon mutation of the STAT site. This observation indicates that a constitutively activated (STAT) factor is present in this cell line, which may contribute to overexpression of episialin/MUC1. Indeed constitutively activated STATs have been reported in breast cancers (32Watson C.J. Miller W.R. Br. J. Cancer. 1995; 71: 840-844Crossref PubMed Scopus (208) Google Scholar, 33Garcia R., Yu, C.L. Hudnall A. Catlett R. Nelson K.L. Smithgall T. Fujita D.J. Ethier S.P. Jove R. Cell Growth Differ. 1997; 8: 1267-1276PubMed Google Scholar). Moreover, our bandshift experiments do show a specific, STAT response element binding complex that is unaffected by treatment of the cells with IL-6 or IFN-γ. This complex also binds to the ICAM-1 oligonucleotide but is absent in bandshift experiments using an oligonucleotide with a mutated STAT site. The identity of this constitutively activated protein (complex) has not been established yet, but it may affect the overexpression of episialin/MUC1 in carcinoma cells. The latter notion is in line with the observation that neither the non-identified protein complex is present in the low episialin expressing A549 cells nor is there a difference between the basal activity of the W3B and M3B constructs transfected into these cells.Although a strong induction of the promoter activity by IL-6 occurs in reporter assays with T47D and ZR-75-1 cells, episialin/MUC1 protein levels do not increase to the same extent when the same cells are treated with IL-6. Since we have found no evidence for post-transcriptional regulatory mechanisms affecting the episialin/MUC1 levels, as for instance has been reported for the rat sialomucin complex (54Price-Schiavi S.A. Carraway C.A. Fregien N. Carraway K.L. J. Biol. Chem. 1998; 273: 3537-35228Abstract Full Text Full Text PDF Scopus (52) Google Scholar), this discrepancy must be attributed to either the inability of the promoter to be further stimulated, because it is already fully activated in these cells, or to the presence of the promoter in a truncated form in a plasmid instead of its normal genomic surroundings. Indeed, the level of episialin/MUC1 in the two cell lines showing the highest expression, T47D and ZR-75-1, can barely be induced any further, whereas in cell lines with an intermediate or low expression level, episialin/MUC1 can be up-regulated by either IL-6 or IFN-γ, suggesting that the promoter activity in the former cells has reached its limits. Alternatively, an additional regulatory element located outside the promoter fragment used in the W3B construct may counteract the STAT-induced expression in T47D and ZR-75-1 cells. Preliminary results show that increasing the size of the 5′ promoter fragment in the reporter construct to 2.9 kb (i.e. into the 3′-untranslated sequence of the trombospondin 3 gene) does not diminish the ratio between the noninduced and the IL-6-induced luciferase levels. 6I. C. Gaemers, H. H. Volders, and J. Hilkens, manuscript in preparation. Another explanation for the unresponsiveness of the episialin protein levels to induction by IL-6 in some of the IL-6 receptor-positive carcinoma cells may be the inability of the STAT3 protein to bind to its binding site in the episialin/MUC1 promoter, because the site is already occupied, for instance by the previously mentioned unidentified protein in the bandshifts. Preliminary experiments suggest that this band has a similar or even higher affinity for the oligonucleotide than STAT3. The fact that a STAT3-specific band can be observed in the EMSAs should then be attributed to the presence of excess target oligonucleotide. Induction of the promoter activity in the reporter construct by IL-6 can be similarly explained by the abundance of reporter plasmid in the transiently transfected cells.Finally, it can be envisaged that by transfecting multiple copies of the promoter plasmid, a repressor of the STAT site is titered out, resulting in an IL-6-induced expression of the MUC1 promoter in transfected cells only. However, a concentration series of W3B, while keeping the total amount of DNA constant, did not show any evidence for such an effect. Induction of the promoter could be observed with a broad range of W3B DNA concentrations (0.05–2.0 μg of added plasmid, results not shown).Several other regulatory elements have been identified in the episialin/MUC1 promoter. For instance, Kovarik et al. (30Kovarik A. Peat N. Wilson D. Gendler S.J. Taylor-Papadimitriou J. J. Biol. Chem. 1993; 268: 9917-9926Abstract Full Text PDF PubMed Google Scholar, 55Kovarik A. Lu P.J. Peat N. Morris J. Taylor-Papadimitriou J. J. Biol. Chem. 1996; 271: 18140-18147Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) identified an E box, termed E-MUC1, at position −84 to −74 and a Sp1-like binding site at position −101 to −89. The latter site can bind an inhibitory factor, SpA, or in the absence of SpA the stimulatory factor Sp-1. Since SpA and E-MUC1 are expected to be expressed in a tissue-specific fashion, they may confer tissue-specific expression of episialin/MUC1. In line with these results, we found that the presence of activated STAT1, capable of binding to the STAT-binding site in the episialin/MUC1promoter, does not lead to episialin/MUC1 expression in the promyelocytic HL-60 cells.Episialin is expressed in activated T cells (56Agrawal B. Krantz M.J. Parker J. Longenecker B.M. Cancer Res. 1998; 58: 4079-4081PubMed Google Scholar) and plasma cells and not or at very low levels during earlier stages of B cell development. 7J. Hilkens, unpublished data. In addition, STATs are known to regulate various stages of lymphocyte development. Therefore, STAT-dependent regulation of episialin/MUC1 during development of hematopoietic cells can be expected. In this respect it is important to note that IL-6 is an autocrine growth factor in various leukemia cells, including myeloma cells and other plasma cell malignancies, which frequently express high levels of episialin/MUC1 (57Akira S. Kishimoto T. Semin. Cancer Biol. 1992; 3: 17-26PubMed Google Scholar, 58Hawley R.G. Berger L.C. Leuk. & Lymphoma. 1998; 29: 465-475Crossref PubMed Scopus (28) Google Scholar, 59Gouilleux-Gruart V. Gouilleux F. Desaint C. Claisse J.F. Capiod J.C. Delobel J. Weber-Nordt R. Dusanter-Fourt I. Dreyfus F. Groner B. Prin L. Blood. 1996; 87: 1692-1697Crossref PubMed Google Scholar).Induction of episialin expression has important consequences for cellular adhesion in cancer cells and certain normal cells. Tight regulation of the expression levels seems therefore of utmost importance. Regulation of episialin/MUC1 via cytokines and the JAK-STAT pathway seems functional in several cell types. Constitutive activation of STATs, as has been reported in breast carcinomas and in various lymphomas and leukemias, is probably one of the factors responsible for the overexpression of episialin in these cells. Episialin/MUC11 (also known as MUC1, PEM, CA 15-3 antigen, or EMA) is a transmembrane molecule with a large extracellular mucin-like domain. In normal cells episialin/MUC1 is exclusively present at the apical side of the cell, but in carcinoma cells normal polarization is lost and episialin/MUC1 co-localizes with adhesion molecules such as integrins and cadherins. The long and relatively rigid extracellular domain of episialin/MUC1 can shield these adhesion molecules and diminish cellular adhesion, if present at a sufficiently high density on the cell surface (1Ligtenberg M.J. Buijs F. Vos H.L. Hilkens J. Cancer Res. 1992; 52: 2318-2324PubMed Google Scholar, 2Wesseling J. van der Valk S.W. Vos H.L. Sonnenberg A. Hilkens J. J. Cell Biol. 1995; 129: 255-265Crossref PubMed Scopus (442) Google Scholar, 3Wesseling J. van der Valk S.W. Hilkens J. Mol. Biol. Cell. 1996; 7: 565-577Crossref PubMed Scopus (358) Google Scholar). Overexpression of episialin/MUC1 in carcinoma cells has been frequently reported (4Hilkens J. Buijs F. Hilgers J. Hageman P. Calafat J. Sonnenberg A. van der Valk M. Int. J. Cancer. 1984; 34: 197-206Crossref PubMed Scopus (402) Google Scholar, 5Zotter S. Lossnitzer A. Hageman P.C. Delemarre J.F. Hilkens J. Hilgers J. Lab. Invest. 1987; 57: 193-199PubMed Google Scholar, 6Zaretsky J.Z. Weiss M. Tsarfaty I. Hareuveni M. Wreschner D.H. Keydar I. FEBS Lett. 1990; 265: 46-50Crossref PubMed Scopus (45) Google Scholar) and is expected to have a similar effect on cellular behavior as loss of E-cadherin, the major epithelial cell-cell adhesion molecule, which has been shown to promote invasion and metastasis of carcinoma cells (for review see Refs. 7Behrens J. Invasion Metastasis. 1994; 14: 61-70PubMed Google Scholar, 8Shiozaki H. Oka H. Inoue M. Tamura S. Monden M. Cancer (Phila.). 1996; 8: 133-1605Google Scholar, 9Bracke M.E. Van Roy F.M. Mareel M.M. Curr. Top. Microbiol. Immunol. 1996; 213: 123-161Crossref PubMed Scopus (214) Google Scholar). Therefore, we have proposed that episialin/MUC1 al" @default.
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• W2170025905 title "A STAT-responsive Element in the Promoter of the Episialin/MUC1 Gene Is Involved in Its Overexpression in Carcinoma Cells" @default.
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