Negative regulation of the expressions of cytokeratins 8 and 19 by SLUG repressor protein in human breast cells

Abstract

Invasiveness of tumor cells is often determined by the profile of their expressed genes. To determine the gene expression differences between an invasive and a non-invasive human breast tumor cells, we selected BT-549 (invasive) and MDA-MB-468 (non-invasive) cells, and compared their transcriptomes by cDNA microarray analysis. Among the significant differences in gene expressions, notable are the up-regulation of cytokeratins 8 and 19, and down-regulation of metallothioneins 1G and IL in MDA-MB-468 cells. Since MDA-MB-468 cells do not express SLUG, a member of a small family of E2-box-binding zinc finger silencer proteins, we studied whether the cytokeratin gene overexpressions in these cells are due to the absence of SLUG. Inducible expression of SLUG in MDA-MB-468 cells inhibited the expressions of the cytokeratin 8 and 19 but not others as was revealed by microarray analysis. Similarly, siRNA knock down of SLUG in BT-549 cells increased the expressions of those cytokeratin mRNAs. SLUG levels in the cell regulated the function of cytokeratins 8 and 19 gene promoters. We conclude that the expressions of cytokeratins and metallothioneins may be associated with the differential invasive behaviors of these breast tumor cells and SLUG may have regulatory roles in this process. KeywordsInvasiveness; Breast tumor; SLUG; Cytokeratin 8; Cytokeratin 19; Metallothionein 1G; Metallothionein 1L; siRNA; Microarray A serious gap in our understanding of cancer concerns malignancy and metastasis. We have yet to clearly identify gene defects that specifically permit cells to invade surrounding tissues, spread through the body, and form metastases. Local invasiveness requires a break down of the mechanisms that normally hold epithelial cells together. To determine the gene expression differences between an invasive and a non-invasive human breast tumor cells, we selected BT-549 (invasive) and MDA-MB-468 (non-invasive) cells, and compared their transcriptomes by cDNA microarray analysis. One of the known genotypic differences between these two cells is that BT-549 expresses the zinc finger repressor protein SLUG but the MDA-MB-468 cells do not [1]. Hence, we tested whether any gene overexpressed in the MDA-MB-468 cells is regulated by SLUG.We paid particular attention to SLUG because it is implicated as one of the determinants of tumor cell invasiveness [2][3][4][5][6]. This protein is a member of a superfamily of transcriptional regulators that are implicated in the formation of mesoderm and neural crest, as well as in We then overexpressed SLUG in MDA-MB-468 cells from a doxycycline-inducible promoter and compared the gene expression profile in the presence or absence of the inducer. We identified that cytokeratins 8 and 19 genes are repressed when SLUG expression is induced. We have verified the role of SLUG in regulating these cytokeratins by siRNA-mediated knock down of SLUG in BT-549 cells. We also report here our preliminary characterization of the promoters of these cytokeratin genes as it is regulated by SLUG exp...

Snail2, a mediator of epithelial-mesenchymal transitions, expressed in progenitor cells of the developing endocrine pancreas

“…They regulate cell migration, left-right symmetry, stem cell survival, and epithelial-to-mesenchymal transitions (EMT) during metazoan development (Barrallo-Gimeno and Nieto, 2005;Boyer et al, 2000;Nieto, 2002), as well as dedifferentiation and regeneration in the mature adult (Savagner et al, 2005;Zhao et al, 2002). Members of this family, particularly Snail1 and Snail2, function primarily as repressors of gene transcription, regulating a variety of epithelial-specific genes involved in cell adhesion and epithelial cell identity (Bolos et al, 2003;Kajita et al, 2004;Tripathi et al, 2005). Expression of members of the Snail family have been identified in various in vitro cell systems for the expansion of pancreatic endocrine cells (Choi et al, 2004;Gershengorn et al, 2004 Rukstalis and Habener, unpublished observation), and therefore we sought to examine the expression of one member of the Snail family, Snail2, during normal pancreas development…”

Nuclear receptors agonists exert opposing effects on the inflammation dependent survival of breast cancer stem cells

“…6,7,34 SLUG, an NF-kB controlled pivot of the stem cell phenotype, 9,10 also takes part to such a regulatory activity of NRs agonists. Interestingly, SLUG is a repressor of differentiation markers, 35,36 and its regulation helps to explain the upregulation of KRT18 and ERa expression upon NRs agonists administration.…”

In vivo binding to and functional repression of the VDR gene promoter by SLUG in human breast cells

“…Human breast cancer cells MCF-7, MDA-MB-468, MDA-MB-231 and BT549 were obtained from ATCC (Manassas, VA) and were cultured in ATCC-recommended media [10,11]. FLAG M2 antibody was purchased from Sigma Chemical Co. (St. Louis, MO).…”

“…It is involved in the epithelial-mesenchymal transition during development [5], acts as an inhibitor of apoptosis [7], and causes tubulogenesis during breast and kidney developments [4,5]. The genes inhibited by SLUG include E-cadherin [8], claudins [9], BRCA2 [10], and cytokeratins [11]. Our ChIP-DSL analysis of 20,000 human gene promoter array revealed that more than 150 promoters bind to SLUG at their promoters (Mittal, M.K.…”