Linda J. Sealy, Ph.D.
Associate Professor of Cancer Biology, Cell and Developmental Biology, Molecular Physiology and Biophysics
Vanderbilt University Medical Center
752 Preston Building
Nashville, TN 37232-0615
identifying the transcription factors that control the conversion to EMT and metastasis in breast cancer
It is the ability to metastasize that ultimately makes breast cancer a fatal disease. Metastatic cells are often characterized as having undergone an epithelial to mesenchymal transition (EMT). EMT is a common feature of both embryonic development and invasive tumors where epithelial cells dedifferentiate to a more fibroblast-like state and regain the ability to invade, migrate, and/or proliferate in an uncontrolled fashion. Mayny studies have sought to define the genes and signaling pathways that underlie the conversion to EMT and metastasis in breast cancer. Much less emphasis has been placed on identifying the transcription factors that ultimately control this process.
We have recently developed a new model for EMT in human breast cancer involving the transcription factor CCAAT/Enhancer Binding Protein (C/EBP)beta. C/EBPbeta is critical for growth and differentiation of the mammary gland. Increased mammary epithelial cell proliferation, migration, and branching during puberty or early pregnancy and differentiation at late pregnancy are severely impaired in C/EBPbeta null mice which fail to lactate. 3 isoforms of C/EBPbeta can be produced in cells via alternative translation initiation at 3 in-frame methionines. C/EBPbeta-1 and beta-2 are transactivators, and differ by only 23 N-terminal amino acids present in beta-1 but not beta-2. C/EBPbeta-3, lacks the N-terminal half of C/EBPbeta including the transactivation domain, and therefore represses transcription. C/EBPbeta-1 is the only isoform present in normal tissue from reduction mammoplasty. However, 70% of invasive surgical primary breast tumor samples have acquired a high level of C/EBPbeta-2 expression, and C/EBPbeta-2 is the only transactivator isoform expressed in breast cancer cell lines.
Although it was first assumed that C/EBPbeta-1 and ¿2 would be functionally redundant transactivators because of their extensive similarity, their different expression patterns suggest otherwise. In fact, MCF10A normal human mammary epithelial cells overexpressing C/EBPbeta-2, but not C/EBPbeta-1, undergo EMT and acquire an invasive phenotype. MCF10A C/EBPbeta-2 cells are anchorage-independent, form foci in soft agar, show loss of junctional E cadherin localization, exhibit cytoskeletal reorganization with actin stress fibers typical of motile fibroblasts, express vimentin, and are invasive in vitro.
From these and other studies we propose that C/EBPbeta-1 and -2 govern different phases of mammary gland development. C/EBPbeta-1 may be required for terminal differentiation during late pregnancy and lactation (likely activating milk protein genes), whereas ductal epithelial outgrowth and invasion through the stromal fat pad during puberty is the dominion of C/EBPbeta-2. Abberant C/EBPbeta-2 expression during cancer progression may activate a genetic program of motility and invasion in breast tumor cells.
Currently, we are extending our studies into animal models. We are evaluating if expression of C/EBPbeta-2 in breast cancer cell lines that are not invasive (MCF7, BT20) will cause the cells to undergo EMT and metastasize once implanted as xenografts in the mammary gland. We have also generated mice carrying an MMTV-driven C/EBPbeta -2 transgene; virgin females exhibit precocious, hyperplastic mammary gland development whereas multiparous females develop tumors. We will continue to study these animals to determine if females show accelerated development of metastatic carcinoma when crossed with other mouse models of breast cancer. Understanding the transcription factors responsible for metastatic capability, of which C/EBPbeta-2 is an enticing candidate, will accelerate the design of molecularly-targeted therapies capable of slowing or halting the often fatal spread of breast cancer to secondary sites.
- Bundy, L, Wells, S, Sealy, L C/EBPbeta-2 confers EGF-independent growth and disrupts the normal acinar architecture of human mammary epithelial cells. Mol Cancer, 443, 2005.
- Tripathi, MK, Misra, S, Khedkar, SV, Hamilton, N, Irvin-Wilson, C, Sharan, C, Sealy, L, Chaudhuri, G Regulation of BRCA2 gene expression by the SLUG repressor protein in human breast cells. J Biol Chem, 280(17), 17163-71, 2005.
- Qiao, Liang, Han, Song Iy, Fang, Youwen, Park, Jong Sung, Gupta, Seema, Gilfor, Donna, Amorino, George, Valerie, Kristoffer, Sealy, Linda, Engelhardt, John F, Grant, Steven, Hylemon, Philip B, Dent, Paul Bile acid regulation of C/EBPbeta, CREB, and c-Jun function, via the extracellular signal-regulated kinase and c-Jun NH2-terminal kinase pathways, modulates the apoptotic response of hepatocytes. Mol Cell Biol, 23(9), 3052-66, 2003.
- Bundy, L M, Sealy, L CCAAT/enhancer binding protein beta (C/EBPbeta)-2 transforms normal mammary epithelial cells and induces epithelial to mesenchymal transition in culture. Oncogene, 22(6), 869-83, 2003.
- Eaton, Erin M., Sealy, Linda Modification of CCAAT/Enhancer binding protein beta by the small ubiquitin-like modifier (SUMO) family members, SUMO-2 and SUMO-3. J Biol Chem, 27833416-21, 2003.
- Ireton, Renee C, Davis, Michael A, van Hengel, Jolanda, Mariner, Deborah J, Barnes, Kirk, Thoreson, Molly A, Anastasiadis, Panos Z, Matrisian, Linsey, Bundy, Linda M, Sealy, Linda, Gilbert, Barbara, van Roy, Frans, Reynolds, Albert B A novel role for p120 catenin in E-cadherin function. J Cell Biol, 159(3), 465-76, 2002.
- Duong, David T, Waltner-Law, Mary E, Sears, Rosalie, Sealy, Linda, Granner, Daryl K Insulin inhibits hepatocellular glucose production by utilizing liver-enriched transcriptional inhibitory protein to disrupt the association of CREB-binding protein and RNA polymerase II with the phosphoenolpyruvate carboxykinase gene promoter. J Biol Chem, 277(35), 32234-42, 2002.
- Eaton, E M, Hanlon, M, Bundy, L, Sealy, L Characterization of C/EBPbeta isoforms in normal versus neoplastic mammary epithelial cells. J Cell Physiol, 189(1), 91-105, 2001.
- Hanlon, M, Sturgill, T W, Sealy, L ERK2- and p90(Rsk2)-dependent pathways regulate the CCAAT/enhancer-binding protein-beta interaction with serum response factor. J Biol Chem, 276(42), 38449-56, 2001.
- Hanlon, M, Bundy, L M, Sealy, L C/EBP beta and Elk-1 synergistically transactivate the c-fos serum response element. BMC Cell Biol, 1(1), 2, 2000.
- Mobley, C M, Sealy, L The Rous sarcoma virus long terminal repeat promoter is regulated by TFII-I. J Virol, 74(14), 6511-9, 2000.
- Liao, J, Piwien-Pilipuk, G, Ross, S E, Hodge, C L, Sealy, L, MacDougald, O A, Schwartz, J CCAAT/enhancer-binding protein beta (C/EBPbeta) and C/EBPdelta contribute to growth hormone-regulated transcription of c-fos. J Biol Chem, 274(44), 31597-604, 1999.
- Hanlon, M, Sealy, L Ras regulates the association of serum response factor and CCAAT/enhancer-binding protein beta. J Biol Chem, 274(20), 14224-8, 1999.
- Mobley, C M, Sealy, L Role of the transcription start site core region and transcription factor YY1 in Rous sarcoma virus long terminal repeat promoter activity. J Virol, 72(8), 6592-601, 1998.
- Sealy, L, Malone, D, Pawlak, M Regulation of the cfos serum response element by C/EBPbeta. Mol Cell Biol, 17(3), 1744-55, 1997.
- Sealy, L, Mota, F, Rayment, N, Tatnell, P, Kay, J, Chain, B Regulation of cathepsin E expression during human B cell differentiation in vitro. Eur J Immunol, 26(8), 1838-43, 1996.
- Svaren, J, Klebanow, E, Sealy, L, Chalkley, R Analysis of the competition between nucleosome formation and transcription factor binding. J Biol Chem, 269(12), 9335-44, 1994.
- Sears, R C, Sealy, L Multiple forms of C/EBP beta bind the EFII enhancer sequence in the Rous sarcoma virus long terminal repeat. Mol Cell Biol, 14(7), 4855-71, 1994.
- Hann, S R, Dixit, M, Sears, R C, Sealy, L The alternatively initiated c-Myc proteins differentially regulate transcription through a noncanonical DNA-binding site. Genes Dev, 8(20), 2441-52, 1994.
- Briggs, R C, Briggs, J A, Ozer, J, Sealy, L, Dworkin, L L, Kingsmore, S F, Seldin, M F, Kaur, G P, Athwal, R S, Dessypris, E N The human myeloid cell nuclear differentiation antigen gene is one of at least two related interferon-inducible genes located on chromosome 1q that are expressed specifically in hematopoietic cells. Blood, 83(8), 2153-62, 1994.
- Ozer, J, Chalkley, R, Sealy, L Characterization of rat pseudogenes for enhancer factor I subunit A: ripping provides clues to the evolution of the EFIA/dbpB/YB-1 multigene family. Gene, 133(2), 187-95, 1993.
- Ozer, J, Chalkley, R, Sealy, L Isolation of the CCAAT transcription factor subunit EFIA cDNA and a potentially functional EFIA processed pseudogene from Bos taurus: insights into the evolution of the EFIA/dbpB/YB-1 gene family. Gene, 124(2), 223-30, 1993.
- Sears, R C, Sealy, L Characterization of nuclear proteins that bind the EFII enhancer sequence in the Rous sarcoma virus long terminal repeat. J Virol, 66(11), 6338-52, 1992.
- Boulden, A M, Sealy, L J Maximal serum stimulation of the c-fos serum response element requires both the serum response factor and a novel binding factor, SRE-binding protein. Mol Cell Biol, 124769-83, 1992.
- Boulden, A, Sealy, L Identification of a third protein factor which binds to the Rous sarcoma virus LTR enhancer: possible homology with the serum response factor. Virology, 174(1), 204-16, 1990.
- Ozer, J, Faber, M, Chalkley, R, Sealy, L Isolation and characterization of a cDNA clone for the CCAAT transcription factor EFIA reveals a novel structural motif. J Biol Chem, 265(36), 22143-52, 1990.
- Faber, M, Sealy, L Rous sarcoma virus enhancer factor I is a ubiquitous CCAAT transcription factor highly related to CBF and NF-Y. J Biol Chem, 265(36), 22243-54, 1990.
- Greuel, B T, Sealy, L, Majors, J E Transcriptional activity of the Rous sarcoma virus long terminal repeat correlates with binding of a factor to an upstream CCAAT box in vitro. Virology, 177(1), 33-43, 1990.
- Sealy, L., Cotten, M., Burgess, R., and Chalkley, R. Wassarman, P. and Kornberg, R. (Eds.) "Preparation of Nucleoplasmin and Reconstitution of Nucleosomes. In Methods in Enzymology: Nucleosomes.." Academic Press, Inc., 612- 629, 1989 .
- Sealy, L, Burgess, R R, Cotten, M, Chalkley, R Purification of Xenopus egg nucleoplasmin and its use in chromatin assembly in vitro. Methods Enzymol, 170612-30, 1989.
- Sealy, L. and Chalkley, R "At Least Two Nuclear Proteins Bind Specifically to the Rous Sarcoma Virus Long Terminal Repeat Enhancer." Mol. Cell. Biol. , 7787-798, 1987.
- Cotten, M, Sealy, L, Chalkley, R Massive phosphorylation distinguishes Xenopus laevis nucleoplasmin isolated from oocytes or unfertilized eggs. Biochemistry, 25(18), 5063-9, 1986.
- Sealy, L, Cotten, M, Chalkley, R Novobiocin inhibits passive chromatin assembly in vitro. EMBO J, 5(12), 3305-11, 1986.
- Cotten, M, Bresnahan, D, Thompson, S, Sealy, L, Chalkley, R Novobiocin precipitates histones at concentrations normally used to inhibit eukaryotic type II topoisomerase. Nucleic Acids Res, 14(9), 3671-86, 1986.
- Sealy, L, Cotten, M, Chalkley, R Xenopus nucleoplasmin: egg vs. oocyte. Biochemistry, 25(10), 3064-72, 1986.
- Sealy, L, Privalsky, M L, Moscovici, G, Moscovici, C, Bishop, J M Site-specific mutagenesis of avian erythroblastosis virus: erb-B is required for oncogenicity. Virology, 130(1), 155-78, 1983.
- Sealy, L, Moscovici, G, Moscovici, C, Bishop, J M Site-specific mutagenesis of avian erythroblastosis virus: v-erb-A is not required for transformation of fibroblasts. Virology, 130(1), 179-94, 1983.
- Privalsky, M L, Sealy, L, Bishop, J M, McGrath, J P, Levinson, A D The product of the avian erythroblastosis virus erbB locus is a glycoprotein. Cell, 32(4), 1257-67, 1983.
- Covault, J, Sealy, L, Schnell, R, Shires, A, Chalkley, R Histone hypoacetylation following release of HTC cells from butyrate. J Biol Chem, 257(10), 5809-15, 1982.
- Sealy, L, Hartley, J, Donelson, J, Chalkley, R, Hutchison, N, Hamkalo, B Characterization of a highly repetitive sequence DNA family in rat. J Mol Biol, 145(2), 291-318, 1981.
- Rubenstein, P., Sealy, L., Marshall, S. and Chalkley, R "Cellular Protein Synthesis and Inhibition of Cell Division Appear to be Independent of Butyrate- Induced Histone Hyperacetylation." Nature, 280692-693, 1979 .
- Rubenstein, P, Sealy, L, Marshall, S, Chalkley, R Cellular protein synthesis and inhibition of cell division are independent of butyrate-induced histone hyperacetylation. Nature, 280(5724), 692-3, 1979.
- Moore, M, Jackson, V, Sealy, L, Chalkley, R Comparative studies on highly metabolically active histone acetylation. Biochim Biophys Acta, 561(1), 248-60, 1979.
- Sealy, L, Chalkley, R Modification of histones immediately following synthesis. Arch Biochem Biophys, 197(1), 78-82, 1979.
- Sealy, L, Chalkley, R DNA associated with hyperacetylated histone is preferentially digested by DNase I. Nucleic Acids Res, 5(6), 1863-76, 1978.
- Nelson, D A, Perry, M, Sealy, L, Chalkley, R DNAse I preferentially digests chromatin containing hyperacetylated histones. Biochem Biophys Res Commun, 82(4), 1346-53, 1978.
- Sealy, L, Chalkley, R The effect of sodium butyrate on histone modification. Cell, 14(1), 115-21, 1978.