Sustained induction of epithelial to mesenchymal
transition activates DNA methylation of genes
silenced in basal-like breast cancers
Nancy Dumont, Matthew B. Wilson, Yongping G. Crawford*, Paul A. Reynolds†, Mahvash Sigaroudinia,
and Thea D. Tlsty‡
Department of Pathology and Comprehensive Cancer Center, University of California, San Francisco, CA 94143-0511
Communicated by Joan S. Brugge, Harvard Medical School, Boston, MA, July 31, 2008 (received for review March 4, 2008)
The active acquisition of epigenetic changes is a poorly understood
but important process in development, differentiation, and dis-
ease. Our work has shown that repression of the p16/pRb pathway
in human epithelial cells, a condition common to stem cells and
many tumor cells, induces dynamic epigenetic remodeling result-
ing in the targeted methylation of a selected group of CpG islands.
We hypothesized that cells in this epigenetically plastic state could
be programmed by the microenvironment to acquire epigenetic
changes associated with tumorigenesis. Here, we describe an in
vitro model system where epigenetically plastic cells were placed
in an environment that induced epithelial to mesenchymal transi-
tion (EMT) and led to a program of acquired de novo DNA meth-
ylation at targeted sites. In this model, we found that repression of
E-cadherin transcription preceded the subsequent acquisition of
methylated CpG sites. Furthermore, the induction of EMT was
accompanied by de novo methylation of several other gene pro-
moters, including those of the estrogen receptor and Twist. These
data demonstrate that signals from the microenvironment can
induce phenotypic and gene expression changes associated with
targeted de novo epigenetic alterations important in tumor pro-
gression, and that these alterations occur through a deterministic,
rather than stochastic, mechanism. Given the dynamic epigenetic
observed in human tumors may reflect the history of environmen-
tal exposures during the genesis of a tumor.
epigenetic remodeling ? human mammary epithelial cells ?
microenvironment ? ras
controlled by epigenetic modifications of proteins and DNA
sequences. We recently reported that the repression of p16INK4A
in primary human mammary epithelial cells (HMEC) activates
an E2F-mediated increase in proteins that remodel chromatin
and causes targeted de novo DNA methylation at a non-random
collection of loci (1). These studies show that cells can acquire
epigenetic plasticity by altering the p16/pRb pathway, and that
this program of acquired de novo methylation has a deterministic
(predictable) rather than stochastic (random) pattern. Further-
more, the coordinated set of de novo DNA methylation events
are preceded by, and dependent upon, the repression of gene
expression. Thus, during cancer progression, one may envision
that tumor cells can acquire epigenetic plasticity through repres-
sion of the p16/pRb pathway via mutations, deletions, or methyl-
ation (2), which then provides the potential for programming
epigenetic events. These observations are reminiscent of studies
that show the acquisition of promoter hypermethylation upon
modulation of estrogen or retinoic acid signaling (3, 4). In these
cell population-based studies it is unclear whether the non-
random hypermethylation events observed are due to induction
or selection. To explore this question further, we chose a
clinically relevant malignant phenotype and determined if re-
he heritable regulation of gene expression changes that are
pression of gene expression induced subsequent DNA methyl-
ation events or whether these occurred by selection.
chymal transition (EMT), play a critical role in tumor progres-
sion (5), have been implicated in tumor recurrence (6), and are
often associated with a poor prognosis in women with breast
cancer (7). Consistent with this, there is now evidence demon-
strating a link between EMT, basal-like tumors, the stem-cell
phenotype, and the acquisition of tumorigenic and metastatic
potential (8, 9). EMT is characterized by several molecular
changes that include the loss of epithelial markers such as
E-cadherin, and the induction of mesenchymal markers such as
N-cadherin, fibronectin, and Snail (5). Though alterations in
E-cadherin expression can occur through multiple mechanisms,
including loss of heterozygosity and mutational inactivation,
E-cadherin is frequently silenced through aberrant DNA hyper-
methylation of its promoter (10). Interestingly, when E-cadherin
is silenced through promoter DNA hypermethylation, mammary
cell lines often exhibit a mesenchymal morphology through the
coordinated induction of a set of genes involved in EMT (11). In
contrast, when E-cadherin is inactivated by mutation, the cells
not induced (11). This suggests that a program of molecular
alterations leading to EMT, invasion, and metastasis can be
EMT has been shown to be induced in murine cells by
oncogenic ras in cooperation with factors in serum (12). There
is also evidence that exposing cells to serum induces a gene
expression pattern that resembles that of a wounding response.
This wound-response signature is strongly predictive of future
invasive and metastatic behavior, both of which require EMT
(13). To determine whether HMEC with repressed p16INK4A
(vHMEC) could be programmed by the microenvironment to
acquire epigenetic changes associated with tumorigenesis, im-
mortalized vHMEC-expressing oncogenic ras (vHMEC-ras)
were exposed to serum. When cultured in serum-rich media,
vHMEC-ras cells underwent phenotypic changes indicative of
EMT and became more motile. This morphological transition
was accompanied by a program of directed de novo DNA
methylation of genes such as E-cadherin. Thus, signals from the
Author contributions: N.D. and T.D.T. designed research; N.D., M.B.W., Y.G.C., P.A.R., and
M.S. performed research; N.D. analyzed data; and N.D. and T.D.T. wrote the paper.
The authors declare no conflict of interest.
*Present address: Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080.
†Present address: University of St. Andrews, Bute Medical School, St. Andrews KY16 9TS,
‡To whom correspondence should be addressed at: Department of Pathology, University
of California, San Francisco, 513 Parnassus Avenue, HSW 511, Box 0511, San Francisco,
CA 94143-0511. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
September 30, 2008 ?
vol. 105 ?
no. 39 ?
immortalization. Understanding these processes may provide
avenues to effective intervention and prevention.
Materials and Methods
pLXSP3 vector (N.D., et al., unpublished data) were grown in MEGM (Lonza)
supplemented with either 0.5% or 10% FBS and 2 ?g/ml puromycin, as
previously described (1). vHMEC-ras0.5 clones were isolated using standard
were conducted between 5 and 15 passages following exposure to serum;
experiments with late passage clones were conducted 40 or more passages
following exposure to serum. For 3D cultures, 5 ? 103cells were resuspended
in media containing 2% Matrigel (BD Biosciences) and seeded into eight-well
glass chamber slides on top of a thin layer of Matrigel.
Immunofluorescence Analysis. Cells were fixed with 100% methanol for 7 min
at ?20°C (for E-cadherin, ?1-integrin, and cytokeratin staining) or with 4%
paraformaldehyde/PBS for 20 min at room temperature (RT) (for F-actin
staining). Paraformaldehyde-fixed cells were permeabilized with 0.1% Triton
X-100/PBS for 10 min at RT and washed with PBS. Nonspecific binding sites
were blocked and the cells were then incubated with primary antibodies
RT, or incubated with Alexa Fluor 488-Phalloidin (Molecular Probes, A12379)
for 1 h at RT and appropriately washed. Images were captured using a Nikon
epifluorescence inverted microscope and a cooled CCD digital camera
Motility Assay. Motility assays were conducted using Chemicon’s QCM Fluoro-
Methylation-Specific PCR. Genomic DNA was isolated from cells using the
Wizard Genomic DNA Isolation Kit (Promega). Approximately 750 ng of DNA
was bisulfite treated with the EZ DNA Methylation-Gold Kit according to the
performed on bisulfite-modified DNA using previously described primer pairs
and PCR cycle conditions (see SI Text for additional details on this and other
TGF? and Dr. Stephen Baylin for GATA3 MSP primers. This research was
(to N.D.), the National Cancer Institute Institutional Training Grant T32
CA009043 (to M.B.W.), the Department of Defense Breast Cancer Research
Program Concept Award BC023982 (to T.D.T.), and National Institutes of
Health National Cancer Institute Grants CA097214–01A1 and CA122024–01
1. Reynolds PA, et al. (2006) Tumor suppressor p16INK4A regulates polycomb-mediated
DNA hypermethylation in human mammary epithelial cells. J Biol Chem 281:24790–
2. Sherr CJ, McCormick F (2002) The RB and p53 pathways in cancer. Cancer Cell 2:103–112.
3. Leu YW, et al. (2004) Loss of estrogen receptor signaling triggers epigenetic silencing
of downstream targets in breast cancer. Cancer Res 64:8184–8192.
4. Ren M, et al. (2005) Impaired retinoic acid (RA) signal leads to RARbeta2 epigenetic
silencing and RA resistance. Mol Cell Biol 25:10591–10603.
5. Lee JM, Dedhar S, Kalluri R, Thompson EW (2006) The epithelial-mesenchymal transi-
tion: New insights in signaling, development, and disease. J Cell Biol 172:973–981.
6. Moody SE, et al. (2005) The transcriptional repressor Snail promotes mammary tumor
recurrence. Cancer Cell 8:197–209.
in breast cancer. Anticancer Res 22:3415–3419.
8. Sarrio D, et al. (2008) Epithelial-mesenchymal transition in breast cancer relates to the
basal-like phenotype. Cancer Res 68:989–997.
9. Mani SA, et al. (2008) The epithelial-mesenchymal transition generates cells with
properties of stem cells. Cell 133:704–715.
10. Droufakou S, et al. (2001) Multiple ways of silencing E-cadherin gene expression in
lobular carcinoma of the breast. Int J Cancer 92:404–408.
11. Lombaerts M, et al. (2006) E-cadherin transcriptional downregulation by promoter
methylation but not mutation is related to epithelial-to-mesenchymal transition in
breast cancer cell lines. Br J Cancer 94:661–671.
12. Oft M, et al. (1996) TGF-beta1 and Ha-Ras collaborate in modulating the phenotypic
plasticity and invasiveness of epithelial tumor cells. Genes Dev 10:2462–2477.
13. Chang HY, et al. (2004) Gene expression signature of fibroblast serum response
predicts human cancer progression: Similarities between tumors and wounds. PLoS
14. Thiery JP, Sleeman JP (2006) Complex networks orchestrate epithelial-mesenchymal
transitions. Nat Rev Mol Cell Biol 7:131–142.
15. Shirakihara T, Saitoh M, Miyazono K (2007) Differential regulation of epithelial and
mesenchymal markers by deltaEF1 proteins in epithelial mesenchymal transition in-
duced by TGF-beta. Mol Biol Cell 18:3533–3544.
16. Peinado H, Quintanilla M, Cano A (2003) Transforming growth factor beta-1 induces
snail transcription factor in epithelial cell lines: Mechanisms for epithelial mesenchy-
mal transitions. J Biol Chem 278:21113–21123.
17. Davidson NE, Sukumar S (2005) Of Snail, mice, and women. Cancer Cell 8:173–174.
18. Dhasarathy A, Kajita M, Wade PA (2007) The transcription factor snail mediates
epithelial to mesenchymal transitions by repression of estrogen receptor-alpha. Mol
19. Oft M, Heider KH, Beug H (1998) TGFbeta signaling is necessary for carcinoma cell
invasiveness and metastasis. Curr Biol 8:1243–1252.
20. Moreira JM, Ohlsson G, Rank FE, Celis JE (2005) Down-regulation of the tumor sup-
pressor protein 14–3-3sigma is a sporadic event in cancer of the breast. Mol Cell
21. Peinado H, Ballestar E, Esteller M, Cano A (2004) Snail mediates E-cadherin repression
by the recruitment of the Sin3A/histone deacetylase 1 (HDAC1)/HDAC2 complex. Mol
Cell Biol 24:306–319.
22. Nass SJ, et al. (2000) Aberrant methylation of the estrogen receptor and E-cadherin 5?
CpG islands increases with malignant progression in human breast cancer. Cancer Res
23. Parrella P, et al. (2004) Nonrandom distribution of aberrant promoter methylation of
cancer-related genes in sporadic breast tumors. Clin Cancer Res 10:5349–5354.
24. Lehmann U, et al. (2002) Quantitative assessment of promoter hypermethylation
during breast cancer development. Am J Pathol 160:605–612.
25. Fackler MJ, et al. (2003) DNA methylation of RASSF1A, HIN-1, RAR-beta, Cyclin D2 and
Twist in in situ and invasive lobular breast carcinoma. Int J Cancer 107:970–975.
26. Roll JD, Rivenbark AG, Jones WD, Coleman WB (2008) DNMT3b overexpression con-
27. Li S, Rong M, Iacopetta B (2006) DNA hypermethylation in breast cancer and its
association with clinicopathological features. Cancer Lett 237:272–280.
in tumor metastasis. Cell 117:927–939.
beta-catenin expression during breast carcinogenesis and tumour progression: A
comparative study with CD44. Histopathology 34:25–34.
30. Park D, et al. (2007) Expression pattern of adhesion molecules (E-cadherin, alpha-,
beta-, gamma-catenin and claudin-7), their influence on survival in primary breast
carcinoma, and their corresponding axillary lymph node metastasis. Apmis 115:52–65.
mesenchymal transition is a rare event in vitro. Breast Cancer Res 6:R215–231.
www.pnas.org?cgi?doi?10.1073?pnas.0807146105 Dumont et al.