Aberrant Methylation of the Maspin Promoter Is an Early Event in Human Breast Cancer

Futscher, Bernard W.; O’Meara, Megan M.; Kim, Christina J.; Rennels, Margaret A.; Lü, Di; Gruman, Lynn M.; Seftor, Richard E.B.; Hendrix, Mary J.C.; Domann, Frederick E.

doi:10.1593/neo.04115

Cited by 66 publications

(43 citation statements)

References 33 publications

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“…The phenotype of these P0 cells cultured in the presence of FBS retained some of the characteristics of keratocytes in that they have not yet assumed the characteristic elongated shape of fibroblasts (Ngamkitidechakul et al, 2001). Hypermethylation of the maspin promoter also occurred early in the conversion of mammary epithelial cells to carcinoma cells (Futscher et al, 2004). The maspin promoter in ductal carcinoma in situ cells (DCIS), an intermediate cell type between normal mammary epithelial cells and carcinoma cells, is often hypermethylated when maspin mRNA and protein are still observed.…”

Section: Discussionmentioning

confidence: 99%

Epigenetic silencing of maspin expression occurs early in the conversion of keratocytes to fibroblasts

Horswill

Narayan

Warejcka

et al. 2008

Experimental Eye Research

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Maspin, a 42 kDa non-classical serpin (serine protease inhibitor) that controls cell migration and invasion, is mainly expressed by epithelial-derived cells but is also expressed in corneal stromal keratocytes. Upon culture of stromal keratocytes in the presence of FBS, maspin is down regulated to nearly undetectable levels by passage two. DNA methylation is one of several processes that controls gene expression during cell differentiation, development, genetic imprinting, and carcinogenesis but has not been studied in corneal stromal cells. The purpose of this study was to determine whether DNA methylation of the maspin promoter and histone H3 dimethylation are involved in the mechanism of down regulation of maspin synthesis in human corneal stromal fibroblasts and myofibroblasts. Human donor corneal stroma cells were immediately placed into serum-free defined medium or cultured in the presence of FBS and passed into serum-free medium or medium containing FBS or FGF-2 to induce the fibroblast phenotype or TGF-β1 for the myofibroblast phenotype. These cell types are found in wounded corneas. The cells were used to prepare RNA for semi-quantitative or quantitative RT-PCR or to extract protein for western analysis. In addition, P4 FBS cultured fibroblasts were treated with the DNA demethylating agent, 5-aza-2′-deoxycytidine (5-Aza-dC), and the histone deacetylase inhibitor, trichostatin A (TSA). Cells with and without treatment were harvested and assayed for DNA methylation using sodium bisulfite sequencing. The methylation state of histone H3 associated with the maspin gene in the P4 fibroblast cells was determined using a ChIP assay. Freshly harvested corneal stromal cells expressed maspin but upon phenotypic differentiation, maspin mRNA and protein were dramatically down-regulated. Sodium bisulfite sequencing revealed that the maspin promoter in the freshly isolated stromal keratocytes was hypomethylated while both the P0 stromal cells and the P1 cells cultured in the presence of serum free defined medium, FGF-2 and TGF-β1 were hypermethylated. Down regulation of maspin synthesis was also associated with histone H3 dimethylation at Lysine 9. Both maspin mRNA and protein were reexpressed at low levels with 5-Aza-dC but not TSA treatment. Addition of TSA to 5-Aza-dC treated cells did not increase maspin expression. Treatment with 5-Aza-dC did not significantly alter demethylation of the maspin promoter but did demethylate histone H3. These results show maspin promoter hypermethylation and histone methylation occur with down regulation *Corresponding Author: Sally S. Twining, PhD, Department of Biochemistry, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, Phone: 414-456-8431, Fax: 414-456-6510, e-mail: stwining@mcw.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of...

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Section: Discussionmentioning

confidence: 99%

Epigenetic silencing of maspin expression occurs early in the conversion of keratocytes to fibroblasts

Horswill

Narayan

Warejcka

et al. 2008

Experimental Eye Research

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“…To this end, we analysed the effects of 5-aza-CdR on DNA and histone H3 K9 di-methylation in the promoter regions of two antimetastatic, tumor suppressor genes, DSC3 and MASPIN, which are aberrantly silenced in a significant fraction of breast tumors via epigenetic mechanisms (Domann et al, 2000;Futscher et al, 2004;Oshiro et al, 2005). We show here that the ability of 5-aza-CdR to reactivate this class of genes in MDA-MB-231 and UACC 1179 breast tumor cells is consistent with reductions in H3 K9 di-methylation to the levels seen in DSC3-and MASPIN-positive normal mammary epithelial cells (HMECs), even in the absence of significant DNA demethylation in some cases.…”

Section: Introductionmentioning

confidence: 99%

5-Aza-2′-deoxycytidine-mediated reductions in G9A histone methyltransferase and histone H3 K9 di-methylation levels are linked to tumor suppressor gene reactivation

et al. 2006

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The epigenetic silencing of tumor suppressor genes is a common event during carcinogenesis, and often involves aberrant DNA methylation and histone modification of gene regulatory regions, resulting in the formation of a transcriptionally repressive chromatin state. Two examples include the antimetastatic, tumor suppressor genes, desmocollin 3 (DSC3) and MASPIN, which are frequently silenced in this manner in human breast cancer. Treatment of the breast tumor cell lines MDA-MB-231 and UACC 1179 with 5-aza-2 0 -deoxycytidine (5-azaCdR) induced transcriptional reactivation of both genes in a dose-dependent manner. Importantly, DSC3 and MASPIN reactivation was closely and consistently linked with significant decreases in promoter H3 K9 di-methylation. Moreover, 5-aza-CdR treatment also resulted in global decreases in H3 K9 di-methylation, an effect that was linked to its ability to mediate dose-dependent, posttranscriptional decreases in the key enzyme responsible for this epigenetic modification, G9A. Finally, small interfering RNA (siRNA)-mediated knockdown of G9A and DNMT1 led to increased MASPIN expression in MDA-MB-231 cells, to levels that were supra-additive, verifying the importance of these enzymes in maintaining multiple layers of epigenetic repression in breast tumor cells. These results highlight an additional, complimentary mechanism of action for 5-aza-CdR in the reactivation of epigenetically silenced genes, in a manner that is independent of its effects on DNA methylation, further supporting an important role for H3 K9 methylation in the aberrant repression of tumor suppressor genes in human cancer.

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“…Loss of imprinting due to hypomethylation has been reported for insulin-like growth factor 2 (IGF2) gene in breast, liver, lung and colon cancers (Ito et al, 2008). Repetitive sequences are hypomethylated and reactivated in many cancers (Goel et al, 1985;Gaudet et al, 2003;Futscher et al, 2004), as documented for lung, breast, bladder, and liver cancers (Wilson et al, 2007). Lastly, hypomethylation of microsatellite regions at the pericentromeric region leads to genomic instability (Kuismanen et al, 1999).…”

Section: Dna Methylationmentioning

confidence: 99%

Epigenetic Therapies for Cancer

Bojang¹,

.²,

Kenneth³

2011

Current Cancer Treatment - Novel Beyond Conventional Approaches

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Aberrant Methylation of the Maspin Promoter Is an Early Event in Human Breast Cancer

Cited by 66 publications

References 33 publications

Epigenetic silencing of maspin expression occurs early in the conversion of keratocytes to fibroblasts

Epigenetic silencing of maspin expression occurs early in the conversion of keratocytes to fibroblasts

5-Aza-2′-deoxycytidine-mediated reductions in G9A histone methyltransferase and histone H3 K9 di-methylation levels are linked to tumor suppressor gene reactivation

Epigenetic Therapies for Cancer

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