Very high frequency of hypermethylated genes in breast cancer metastasis to the bone, brain, and lung.
OBJECTIVE
Most often it is not the primary tumor, but metastasis to distant organs that results in the death of breast cancer patients. To characterize molecular alterations in breast cancer metastasis, we investigated the frequency of hypermethylation of five genes (Cyclin D2, RAR-beta, Twist, RASSF1A, and HIN-1) in metastasis to four common sites: lymph node, bone, brain, and lung.


METHODS
Methylation-specific PCR for the five genes was performed on DNA extracted from archival paraffin-embedded specimens of paired primary breast cancer and its lymph nodes (LN) metastasis (n = 25 each); in independent samples of metastasis to the bone (n = 12), brain (n = 8), and lung (n = 10); and in normal bone, brain, and lung (n = 22).


RESULTS
No hypermethylation was detected in the five genes in the normal host tissues. In paired samples, LN metastasis had a trend of higher prevalence of methylation compared with the primary breast carcinoma for all five genes with significance for HIN-1 (P = 0.04). Compared with the primary breast carcinomas, all five genes had higher methylation frequencies in the bone, brain, and lung metastasis, with HIN-1 and RAR-beta methylation being significantly higher (P < 0.01) in each group. Loss of expression of all five genes correlated, with a few exceptions, to hypermethylation of their promoter sequences in metastatic carcinoma cells microdissected from LNs.


CONCLUSIONS
The frequent presence of hypermethylated genes in locoregional and distant metastasis could render them particularly susceptible to therapy targeted toward gene reactivation combining demethylating agents, histone deacetylase inhibitors, and/or differentiating agents.
Mehrotra
Jyoti
J
Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21231, USA.
Vali
Mustafa
M
McVeigh
Megan
M
Kominsky
Scott L
SL
Fackler
Mary Jo
MJ
Lahti-Domenici
Jaana
J
Polyak
Kornelia
K
Sacchi
Nicoletta
N
Garrett-Mayer
Elizabeth
E
Argani
Pedram
P
Sukumar
Saraswati
S
D001859:Bone Neoplasms / Q000556:secondary; D001932:Brain Neoplasms / Q000556:secondary; D001943:Breast Neoplasms / Q000235:genetics / Q000473:pathology*; D045744:Cell Line, Tumor; D056742:Cyclin D2; D016213:Cyclins / Q000235:genetics; D016207:Cytokines / Q000235:genetics; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D017403:In Situ Hybridization; D008175:Lung Neoplasms / Q000556:secondary; D008207:Lymphatic Metastasis; D008875:Middle Aged; D009363:Neoplasm Proteins / Q000235:genetics*; D009687:Nuclear Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D018168:Receptors, Retinoic Acid / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics; D025521:Tumor Suppressor Proteins / Q000235:genetics; D051879:Twist-Related Protein 1
C080336:CCND2 protein, human; D056742:Cyclin D2; D016213:Cyclins; D016207:Cytokines; D004273:DNA, Neoplasm; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; C411762:RASSF1 protein, human; D012333:RNA, Messenger; D018168:Receptors, Retinoic Acid; C434732:SCGB3A1 protein, human; C103173:TWIST1 protein, human; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D051879:Twist-Related Protein 1; C082925:retinoic acid receptor beta