Morphologically normal-appearing mammary epithelial cells obtained from high-risk women exhibit methylation silencing of INK4a/ARF.
OBJECTIVE
p16(INK4a) has been appreciated as a key regulator of cell cycle progression and senescence. Cultured human mammary epithelial cells that lack p16(INK4a) activity have been shown to exhibit premalignant phenotypes, such as telomeric dysfunction, centrosomal dysfunction, a sustained stress response, and, most recently, a dysregulation of chromatin remodeling and DNA methylation. These data suggest that cells that lack p16(INK4a) activity would be at high risk for breast cancer development and may exhibit an increased frequency of DNA methylation events in early cancer.


METHODS
To test this hypothesis, the frequencies of INK4a/ARF promoter hypermethylation, as well as four additional selected loci, were tested in the initial random periareolar fine needle aspiration samples from 86 asymptomatic women at high risk for development of breast cancer, stratified using the Masood cytology index.


RESULTS
INK4a/ARF promoter hypermethylation was observed throughout all early stages of intraepithelial neoplasia and, importantly, in morphologically normal-appearing mammary epithelial cells; 29 of 86 subjects showed INK4a/ARF promoter hypermethylation in at least one breast. Importantly, INK4a/ARF promoter hypermethylation was not associated with atypia, and the frequency of hypermethylation did not increase with increasing Masood cytology score. The frequency of INK4a/ARF promoter hypermethylation was associated with the combined frequency of promoter hypermethylation of retinoic acid receptor-beta2, estrogen receptor-alpha, and breast cancer-associated 1 genes (P = 0.001).


CONCLUSIONS
Because INK4a/ARF promoter hypermethylation does not increase with age but increases with the frequency of other methylation events, we predict that INK4a/ARF promoter hypermethylation may serve as a marker of global methylation dysregulation.
Bean
Gregory R
GR
Duke University Medical Center, Durham, NC 27710, USA.
Bryson
Andrew D
AD
Pilie
Patrick G
PG
Goldenberg
Vanessa
V
Baker
Joseph C
JC
Ibarra
Catherine
C
Brander
Danielle M U
DM
Paisie
Carolyn
C
Case
Natalie R
NR
Gauthier
Mona
M
Reynolds
Paul A
PA
Dietze
Eric
E
Ostrander
Julie
J
Scott
Victoria
V
Wilke
Lee G
LG
Yee
Lisa
L
Kimler
Bruce F
BF
Fabian
Carol J
CJ
Zalles
Carola M
CM
Broadwater
Gloria
G
Tlsty
Thea D
TD
Seewaldt
Victoria L
VL
D000328:Adult; D000368:Aged; D001943:Breast Neoplasms / Q000235:genetics*; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics*; D019175:DNA Methylation; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D005260:Female; D020868:Gene Silencing; D006801:Humans; D042361:Mammary Glands, Human / Q000166:cytology / Q000378:metabolism*; D008875:Middle Aged; D011401:Promoter Regions, Genetic; D012306:Risk
D019941:Cyclin-Dependent Kinase Inhibitor p16